surafce and bulk modification of poly(lactic acid)
TRANSCRIPT
SURAFCE AND BULK MODIFICATION OF POLY(LACTIC ACID)
A Thesis Presented to
the Graduate School of Clemson University
In Partial Fulfillment of the Requirements for the Degree
Doctor of Philosophy Chemical Engineering
by Rahul M. Rasal
May 2009
Accepted by: Dr. Douglas Hirt, Committee Chair
Dr. Anthony Guiseppi-Elie Dr. David Bruce Dr. Ken Webb
ABSTRACT
The major drawbacks of PLA are its poor toughness and lack of readily reactable
groups. Unfortunately, typical methods of PLA toughening are associated with significant
modulus and/or ultimate tensile strength (UTS) loss. The main objective of this research
was to toughen PLA, with minimal modulus and/or UTS loss, and introduce reactive
groups into the PLA matrix in one step. Initially, this objective was divided into two
separate parts: PLA surface modification followed by toughening.
PLA film was solvent cast from chloroform solution and was surface modified
using a sequential two-step photografting approach. Benzophenone was photografted
onto the film surface in Step 1 followed by photopolymerization of hydrophilic
monomers, acrylic acid and acrylamide, from the film surface. The resultant films were
characterized using ATR-FTIR spectroscopy, water contact angle goniometry,
transmission FTIR microspectroscopy, and tensile testing. The effect of the reaction
solvent (ethanol and water) in Step 2 on PLA film surface and bulk properties was also
studied. There was significant penetration of monomers into the films when ethanol was
used as the reaction solvent, resulting in significant toughness loss. This monomer
penetration into the films was successfully reduced by using water instead of ethanol as
the reaction solvent in Step 2 and resultant films showed higher toughness than films
surface-modified using ethanol as the reaction solvent in Step 2. It was also observed that
solvent cast PLA film retained approximately 13 wt% chloroform, as characterized using
thermogravimetric analyses (TGA). The presence of residual chloroform in the film
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specimens is undesirable from a biocompatibility standpoint. Therefore, further work was
conducted on melt-processed films where residual solvent from the film-formation
method would not be an issue.
Addition of a small amount of poly[(3-hydroxybutyrate)-co-(3-
hydroxyhexanoate)] (PHBHHx) to PLA improved the toughness of the resultant melt-
processed blend from 4 ± 2 MPa for neat PLA to 175 ± 35 MPa for PLA-PHBHHx blend
(90 wt% PLA). PLA-PHBHHx blend films were melt-processed using a single screw
extruder. These polyblend films appeared to be non-compatible as characterized using
dynamic mechanical analyses (DMA). PLA-PHBHHx blend films underwent rapid
physical aging losing their toughness from 175 ± 35 MPa (right after extrusion) to 68 ±
34 MPa (day 3). The blend films were surface modified using the sequential two-step
photografting protocol using water as the reaction solvent in Step 2. PLA-PHBHHx blend
films lost approximately 95% of their toughness on surface modification due to UV-
assisted solvent induced crystallization as characterized using wide angle X-ray
diffraction (WAXD) analyses.
A novel reactive blending approach was developed to toughen PLA with minimal
modulus and UTS loss and introduce reactive groups into the PLA matrix. PLA was
reactive blended with a stiffening polymer, poly(acrylic acid) (PAA), followed by
physical blending with a toughening polymer, poly(ethylene glycol) (PEG), in solution.
The modified PLA was extruded into films using a co-rotating twin-screw extruder and
characterized using tensile testing, differential scanning calorimetry (DSC), DMA, and
toluidine-blue-surface-staining. This material exhibited, for the first time, approximately
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10 fold increase in PLA’s toughness without significant modulus and/or UTS loss and
also introduced a controlled concentration of surface modifiable reactive acid groups into
the PLA matrix in one step.
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DEDICATION
I dedicate this work to my beloved Guru, H H Swami Shivkrupanandji, parents,
Mrs. and Mr. Maruti Rasal, and sister, Mrs. Shambhavi Kadam. Without their
unconditional support, love, and motivation, it would not have been possible.
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ACKNOWLEDGMENTS
I am grateful to my parents and sister for their motivation and encouragement.
Thank you very much for being there whenever I needed you.
I am very thankful to Dr. Douglas Hirt for his precious guidance, flexibility, and
support. A fantastic friend who has not only helped me through my Ph.D. dissertation
research but also played a vital role in shaping-up my personality as a researcher. I am
also thankful to my dissertation committee members (Dr. Guiseppi-Elie, Dr. Bruce, and
Dr. Webb) for their time and energy. I would like to sincerely acknowledge Dr. Drews
and Ms. Kim Ivey for their expert opinions on several analytical techniques.
I am thankful to the Center for Advanced Engineering Fibers and Films (CAEFF)
for the financial support. This work was supported by the Engineering Research Centers
Program of the National Science Foundation under NSF Award Number EEC-9731680.
Special thanks to my close friends at Clemson (Amol, Shamik, Sourabh, Carrie,
Halil, and Ward) for making Clemson my second home. I express my sincere thanks to
Nripen for being an excellent friend and making Clemson-life a fun experience. Past and
present Hirt group graduate and undergraduate students: Amol, Keisha, Jeffery, Richard,
Mary, Irl, and Courtney, thank you very much for your time on this research. All past and
present office-mates: Amol, Keisha, Jeffery, Richard, Greg, Esteban, and Fiaz, thank you
for making our office a fun-place to work. I must thank my friends in the Surabhi group,
Dr. Ogale and Mrs. Ogale for their enthusiasm, support, and guidance.
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Finally I would like to acknowledge all my friends who have not been mentioned
here. Thank you very much!!!!
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TABLE OF CONTENTS
Page
TITLE PAGE....................................................................................................................i ABSTRACT.....................................................................................................................ii DEDICATION.................................................................................................................v ACKNOWLEDGMENTS ..............................................................................................vi LIST OF TABLES...........................................................................................................x LIST OF SCHEMES.......................................................................................................xi LIST OF FIGURES .......................................................................................................xii CHAPTER I. INTRODUCTION .........................................................................................1 PLA Production and Applications ...........................................................3 PLA Materials Sceince ............................................................................6 PLA Modifications.................................................................................10 Summary of Dissertation Research........................................................23 References..............................................................................................25 II. ANALYTICAL TECHNIQUES..................................................................34 References..............................................................................................40 III. EFFECT OF THE PHOTOREACTION SOLVENT ON SURFACE AND BULK PROPERTIES OF PLA AND PHBHHx FILMS...................41 Introduction............................................................................................41 Materials ................................................................................................43 Methods..................................................................................................44 Results and Discussion ..........................................................................46 Conclusions............................................................................................65 References..............................................................................................66
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Table of Contents (Continued) IV. TOUGHNESS DECREASE OF PLA-PHBHHx BLEND FILMS
UPON SURFACE-CONFINED PHOTOPOLYMERIZATION ................69 Introduction............................................................................................69 Methods..................................................................................................70 Results and Discussion ..........................................................................72 Conclusions............................................................................................88 References..............................................................................................90 V. POLY(LACTIC ACID) TOUGHENING WITH A BETTER
BALANCE OF PROPERTIES....................................................................94 Introduction............................................................................................94 Materials ................................................................................................95 Methods..................................................................................................96 Results and Discussion ..........................................................................97 Conclusions..........................................................................................104 References............................................................................................106
VI. CONCLUSIONS AND FUTURE RESEARCH RECOMMENDATIONS ..........................................................................108
Conclusions..........................................................................................108 Recommendations for Future Research ...............................................110 References............................................................................................113
APPENDICES .............................................................................................................114 A: MICROPATTERNING OF COVALENTLY ATTACHED BIOTIN
ON POLY(LACTIC ACID) FILM SURFACES.......................................115 B: NOVEL TOUGHER POLY(LACTIC ACID)-POLY(ETHYLENE
GLYCOL) METHACRYLATE REACTIVE BLENDS .........................142 C: PACLITAXEL ATTACHMENT TO PLA-PHBHHx BLEND FILMS ...153
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LIST OF TABLES
Table Page 1.1 PLA in-vivo biocompatibility testing.............................................................2 1.2 PLA 2002D properties ...................................................................................8 1.3 Solubility parameters for PLA.....................................................................10 3.1 Water contact angles of neat and surface modified PLA and PHBHHx after 3 h UV-exposure time and using water or ethanol as the reaction solvent in step 2 ..........................................................................................52 3.2 Design of the photografting experiments to evaluate the effect of surface modification on mechanical properties of the PLA and PHBHHx films after 3 h UV-exposure time in step 2 ...........................................................54 3.3 Solubility parameters for PLA, PHBHHx, ethanol, and water. Solubility
parameters for PLA and PHBHHx were calculated based on Fedors cohesive energy and molar volume values using group contribution method..........................................................................................................57 4.1 Water contact angles of unmodified and surface-modified PLA and PLA
-PHBHHx blend (90 weight percent PLA) films after 3 h UV-exposure time in Step 2 ..............................................................................................79
x
LIST OF SCHEMES
Scheme Page 5.1 Reactive blending approach consisting of thermal polymerization of acrylic acid from PLA chains followed by PEG blending...........................96 A.1 Photolithography [4] approach to micropattern PAA on PLA ..................119 A.2 Scheme of the EDC/NHS mediated PAA conjugation with amine- terminated biotin ........................................................................................127 B.1 Reactive blending approach consisting of thermal polymerization of PEGMA......................................................................................................144 C.1 Reaction scheme used to attach Paclitaxel to PLA-PHBHHx blend
films ...........................................................................................................156
xi
LIST OF FIGURES
Figure Page 1.1 Reaction schemes to produce PLA (reproduced with permission from ref.
[22])................................................................................................................4 1.2 Rheology of linear and branched NatureWorks PLA (reproduced with
permission from ref. [25])..............................................................................7 1.3 Percent transmission versus wavelength for PLA (98% L-lactide), PS,
LDPE, PET, and cellophane films. (PLA samples were obtained from Cargill Dow LLC) (reproduced with permission from ref. [6]).....................9
1.4 Generalized reaction scheme for carboxylic acid activation using PCl5,
SOCl2, or water soluble carbodiimides followed by chemical conjugation with amine (-NH2) or hydroxyl (-OH) functionalities .............21
2.1 The DMA supplies a sinusoidal stress to the sample, which generates a
sinusoidal strain. Properties such as modulus, viscosity, and dampening can be calculated by measuring the deformation amplitude at the peak and the lag between stress and strain sine waves [1] ...................................34
3.1 Chemical structures of (a) PLA and (b) PHBHHx repeat units...................43 3.2 Reaction scheme for the sequential two-step photografting reaction of
acrylic acid onto a PLA film surface [24]....................................................45 3.3 The effect of the UV exposure time on the static water contact angle of
PLA (○) and PHBHHx (■) films grafted with acrylic acid with ethanol as the solvent in Step 2. The error bars represent 95% confidence intervals........................................................................................................48
3.4 The effect of the UV exposure time on the static water contact angle of
PLA (○) and PHBHHx (■) films grafted with the acrylamide with ethanol as the solvent in Step 2. The error bars represent 95% confidence intervals .....................................................................................49
xii
List of Figures (Continued) Figure Page 3.5 Representative ATR-FTIR spectra of the (a) neat PLA, (b) neat
PHBHHx, (c) PLA-g-PAA, (d) PHBHHx-g-PAA, (e) PLA-g-PAAm, and (f) PHBHHx-g-PAAm. Spectrum (a) shows the –C=O peak for the PLA ester at 1756 cm-1 (♦). Spectrum (b) shows the –C=O peak for the PHBHHx ester at 1721 cm-1 (). Spectrum (c) shows –C=O acid peak at 1720 cm-1 (), which is a shoulder to the PLA ester peak. Spectrum (d) shows the widening of the PHBHHx ester peak at 1721 cm-1 (), due to the overlapping of the –C=O peak for the PHBHHx ester and the –C=O acid peak. Spectra (e-f) show the –C=O amide peak at 1670 cm-1 (■) ...................................................................................51
3.6 ATR-FTIR peak area ratio, viz. 1670 cm-1 –C=O amide peak area
normalized by 1756 cm-1 –C=O ester peak area for PLA or 1720 cm-1 –C=O ester peak area for PHBHHx. Black bars represent films grafted with acrylamide after sonication for 5 min in the respective solvents and white bars represent films grafted with acrylamide after sonication for 5 min in the respective solvents plus microwave assisted extraction in water at 95 °C for 1 h. The error bars represent 95% confidence intervals........................................................................................................53
3.7 Effect of the photografting reaction solvent on the (a) modulus, (b)
crystallinity, and (c) toughness of PLA films. Black bars indicate films prepared by carrying out the reaction in ethanol and white bars indicate films prepared by carrying out the reaction in water. The neat PLA film had very low % crystallinity such that the DSC spectrum did not show a definite melting peak. The error bars represent 95% confidence intervals........................................................................................................56
3.8 TGA curves of neat and surface modified (a) PLA and (b) PHBHHx.
Note that the y-axis scales are different on the two plots ............................59 3.9 Effect of the photografting reaction solvent on the (a) modulus, (b)
crystallinity, and (c) toughness of PHBHHx films. Black bars indicate films prepared by carrying out the reaction in ethanol and white bars indicate films prepared by carrying out the reaction in water. The error bars represent 95% confidence intervals......................................................61
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List of Figures (Continued) Figure Page 3.10 Transmission FTIR peak area ratio, viz. 1670cm-1 –C=O amide peak
area normalized by 1452 cm-1 asymmetric –CH3 band peak area, as a function of analysis position into the bulk of PLA or PHBHHx films grafted with benzophenone in step 1 and then immersed in 10% v/v acrylamide solution and exposed to UV for 3 h (○), or dip coated in ethanol and exposed each side for 5 min in step 1 then immersed in 10% v/v acrylamide solution and exposed to UV for 3 h (■). The IR beam width at each analysis position was 25 microns.................................63
4.1 Engineering stress-strain curve of extruded PLA (♦) and PLA-PHBHHx
(90 weight percent PLA) blend (■) films ....................................................72 4.2 (A) Tan δ as a function of temperature for extruded PLA-PHBHHx
blend film (90 weight percent PLA). (B) Tan δ as function of temperature of (a) unmodified blend, (b) water control, (c) Blend-g- PAAm, and (d) Blend-g-PAA......................................................................73
4.3 WAXD patterns of (a) unmodified blend, (b) blend-g-PAA, (c) blend-
g-PAAm, and (d) blend films prepared by water-control experiments .......75 4.4 Effect of the physical aging on toughness of extruded and annealed (at
60 °C for 30 min) blend films. The error bars represent 95% confidence intervals........................................................................................................76
4.5 DSC thermograms of PLA-PHBHHx (90 weight percent PLA) blend
films (a) right after extrusion, (b) aged for 1 day, (c) after annealing at 60 °C for 30 min, and (d) 1 day post annealing...........................................78
4.6 Engineering stress-strain curve of unmodified (■) and surface-modified
PLA-PHBHHx (90 weight percent PLA) blend (♦) films (after annealing).....................................................................................................81
4.7 Effect of the photografting on (a) modulus, (b) ultimate tensile strength,
and (c) toughness of PLA-PHBHHx (90 weight percent PLA) blend films. The error bars represent 95% confidence intervals ...........................82
4.8 Effect of the photografting reaction on % crystallinity of PLA-
PHBHHx (90 weight percent PLA) blend films. The error bars represent 95% confidence intervals .............................................................84
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List of Figures (Continued) Figure Page 4.9 Effect of the UV irradiation on temperature of reaction solvent (water)
with time. The error bars represent 95% confidence intervals ....................86 4.10 Tan δ as function of temperature for the unmodified blend and water
control ..........................................................................................................87 5.1 (A) Tan δ as a function of temperature of (a) neat PLA, (b) PLA-g-
PAA(3%)/PEG(10%), (c) PLA-g-PAA(10%)/PEG(10%), and (d) PLA/PEG(10%). (B) DSC scans of melt quenched (a) neat PLA, (b) PLA-g-PAA(3%)/PEG(10%), (c) PLA-g-PAA(10%)/PEG(10%), and (d) PLA/PEG(10%)......................................................................................99
5.2 (a) Toughness and (b) representative stress-strain curves of neat PLA
and its reactive blends. Error bars represent 95% confidence intervals ....101 5.3 (a) Young’s modulus and (b) ultimate tensile strength of neat PLA and
its reactive blends. Error bars represent 95% confidence intervals ...........103 5.4 Toluidine-blue-stained images of (a) neat PLA, (b) PLA-g-
PAA(3%)/PEG(10%), and (c) PLA-g-PAA(10%)/PEG(10%) revealing the presence of reactive acid groups on the film surfaces .........................104
A.1 Representative ATR-FTIR spectra revealing the progression of PAA
micropatterning on PLA. The –C=O “ester stretch” of PLA is represented by a peak at 1747 cm-1 (♦) and the –C=O “acid stretch” of PAA is represented by a peak at 1720 cm-1(●). A 2 mm wide stripe on PLA was characterized to monitor the progression of PAA micropatterning ..........................................................................................122
A.2 Effect of UV irradiation time on static water contact angle of 2 mm
wide PAA stripe on PLA. The error bars represent 95% confidence intervals......................................................................................................123
A.3 Effect of UV irradiation time on PAA microstripe height and surface
topography of PAA micropatterned PLA ..................................................125 A.4 Optical micrographs of toluidine-blue-stained PAA micropatterned
PLA (UV irradiation time 15 min) revealing micropatterns of different shapes and sizes .........................................................................................126
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List of Figures (Continued) Figure Page A.5 XPS survey scans for (a) neat PLA and (b) PLA-g-biotin.........................129 A.6 XPS high-resolution C 1s spectra for PLA-g-biotin ..................................131 A.7 Fluorescent micrographs of (a) Alexa488-strepatavidin micropatterned
PLA surface and (b) PAA micropatterned PLA surface (without biotin) exposed to Alexa488-streptavidin..............................................................134
A.8 N concentration (as calculated from XPS survey scans) of streptavidin
adsorbed on biotin modified PAA stripe and neat PLA ............................136 B.1 The effect of PEGMA composition on the glass transition temperatures
of the reactive blend constituents...............................................................146 B.2 (a) Toughness and (b) % Elongation at Break of neat PLA and its
reactive blends. Error bars represent 95% confidence intervals ................148 B.3 Young’s modulii of neat PLA and its reactive blends. Error bars
represent 95% confidence intervals ...........................................................149 C.1 Representative ATR-FTIR spectra of (a) unmodified blend film, (b)
blend-g-PAA, and (c) blend-g-Paclitaxel. Spectrum (a) shows the “ester peak of PLA” at 1756 cm-1 (♦). Spectrum (b) shows the “acid peak of acrylic acid” at 1720 cm-1 (●). Spectrum (c) shows “the amide peak of Paclitaxel” at 1650 cm-1 (■) ..........................................................157
C.2 TGA curves of (a) unmodified blend film and (b) blend-g-Paclitaxel ......158
xvi
1
CHAPTER ONE
INTRODUCTION
Environmental concerns and sustainability issues associated with petrochemical-
based polymers have driven considerable engineering and scientific efforts devoted to the
discovery, development, and modifications of biodegradable and renewably-derived
polymers over the past several decades [1-2]. One such polymer is poly(lactic acid) or
poly(lactide) (PLA), a thermoplastic polyester that is renewably-derived (from corn,
starch, sugar, etc.), biodegradable, recyclable, and compostable [3]. PLA is
biocompatible with non-toxic degradation products (at low concentrations), making it a
natural choice for many biomedical applications [4]. Table 1.1 provides a chronological
list of PLA in-vivo studies conducted over last four decades, demonstrating its
satisfactory biocompatibility. The Food and Drug Administration (FDA) has also
approved PLA for direct contacting with biological fluids [5]. In addition to this, PLA
has excellent stiffness, comparable to that of poly(ethylene terphthalate) (PET) [6]. These
attractive properties serve to make PLA a suitable substitute for many petrochemical-
based polymers.
Table 1.1 PLA in-vivo biocompatibility testing (adapted, in part, from ref [17])
Application Results Reference
Sutures in guinea-pigs and rats
Non-toxic and non-tissue reactive
[7]
Sutures in rat muscle
Degraded suture induced giant cell
Reaction
[8]
Bone repair of rat tibia
No adverse tissue host responses
[9]
Fracture fixation in dogs and
sheep
Uneventful bone healing that proceeded
without callus formation or inflammation
signs
[10]
Subcutaneous implants in rats
Mild foreign body reaction
[11]
Drug release in rat soft tissue
PLA is tissue compatible
[12]
Bone fixation in rat
No inflammation or foreign body
reaction
[13]
Soft tissue/rabbit cornea
Non-toxic and safe
[15]
Fracture fixation of rabbit
femur
Insignificant inflammatory response
[16]
2
Ankle fracture fixation in human
Found safe and effective, no complications
[17]
Implants in the repair of goat
osteochondral defects
No obvious histological abnormalities
[18]
Fracture fixation of dog femur No inflammatory reaction [19]
Fixation of osteochondral
fractures of the femoral condyle
Complete bony healing without clinically
relevant complications
[20]
Bone defect coverage in sheep Good biocompatibily [21]
However, the main drawback of PLA is its brittleness (poor toughness), which
limits its use in many applications. Another drawback of PLA is lack of readily reactable
side-chain groups. This makes PLA’s surface modification a challenging task. PLA needs
to be surface modified in many applications such as friction modification, anti-fogging,
adhesion, implantable biomaterials, and biopolymer-based drug delivery.
PLA PRODUCTION AND APPLICATIONS
Figure 1.1 illustrates the various reactions involved in PLA production.
Carbohydrates (primarily sucrose and starch) derived from renewable resources are
bacterially fermented to produce lactic acid. All the carbon, hydrogen, and oxygen atoms
in carbohydrates and final PLA product have their origin in carbon dioxide and water,
photosynthesis reactants.
3
Figure 1.1 Reaction schemes to produce PLA (reproduced with permission from ref.
[22]).
4
There are two primary routes to produce PLA from lactic acid: direct
condensation polymerization of lactic acid and ring opening polymerization through a
lactide intermediate. The first approach involves the removal of water, the use of solvent
under high vacuum and temperature, and can produce only low to intermediate molecular
weight polymers. In addition to this, it requires relatively large reactors and leads to
increased color and racemization. Because of these disadvantages, the ring opening
polymerization has been more favored. In this approach, a low molecular weight
prepolymer is first produced by removing water under mild conditions and without the
use of a solvent. A cyclic intermediate dimer, lactide, is then produced by catalytically
depolymerizing this prepolymer. The lactide monomer is further subjected to a solvent
free ring opening polymerization to produce PLA [22].
Due to PLA’s bioresorbability and biocompatibility in the human body, it has
been used for resorbable sutures and prosthetic devices [20]. PLA has been finding
increasing consumer applications mainly due to its renewability, biodegradability,
transparency, processibility, and mechanical properties. Although PLA has been shown
to be a practically feasible packaging material, its higher cost has confined its use to
limited packaging application only [6]. Dannon and McDonald’s (Germany) pioneered
the use of PLA as a packaging material in yogurt cups and cutlery [6]. NatureWorks LLC
polymers have been used for a range of packaging applications such as high-value films,
rigid thermoformed containers, and coated papers [23]. IngeoTM, a PLA-based fiber, has
been designed for apparel, furnishings, and nonwovens applications [24]. BASF’s
Ecovio®, which is a derivative of petrochemical-based biodegradable Ecoflex® and
5
contains 45 wt% PLA, has been used to make carrier bags, compostable can liners, mulch
film, and food wrapping. Commercially available PLA films and packages have been
found to provide mechanical properties better than polystyrene (PS) and comparable to
PET [6]. The extensive utilization of PLA in consumer and biomedical applications will
be dictated mainly by cost reductions as well as fine control over PLA bulk and surface
properties.
PLA MATERIALS SCIENCE
Lactide has three stereoisomers: L-lactide, D-lactide, and meso-lactide. The
stereochemical composition of the PLA has a significant effect upon its melting point,
crystallization rate, extent of crystallization, and mechanical properties [25]. Pure
poly(D-lactide) or poly(L-lactide) have an equilibrium crystalline melting point of 207 °C
[26, 27]. However, due to small and imperfect crystallites, slight racemization, and
impurities, typical PLA melting points are 170-180 °C [28]. A 1:1 mixture of pure
poly(L-lactide) and poly(D-lactide) exhibited a higher melting temperature (230 °C) and
better mechanical properties than either pure polymer (the UTS for the 1:1 stereocomplex
was 50 MPa while that for pure poly(L-lactide) was 31 MPa [28-30]). Although
stereochemical composition had a significant effect on melting point, glass transition
temperature was not as significantly affected (e.g., glass transition temperature of pure
poly(L-lactide) was found to be 55-60 °C for Mv ~ 23-66 kDa and that of poly(D,L-
lactide) was found to be 49-52 °C for Mv ~ 47.5-114 kDa) [31].
6
Rheological characteristics of PLA make it suitable for cast and blown film
extrusion and fiber spinning. PLA has relatively poor melt strength and its melt viscosity
is not very shear-sensitive. This has been overcome by employing branching by treatment
of PLA with peroxide or by introduction of multifunctional initiators or monomers.
Branched PLA displays high viscosity (melt-strength) at low shear rates, making it more
suitable for applications such as extrusion coating, extrusion blow-molding, and foaming
(Figure 1.2) [25]. Typical properties of NatureWorks PLA 2002D resin (designed for
extrusion/thermoforming applications) are shown in Table 1.2. NatureWorks PLA grades
differ in steriochemical composition, molecular weight, and additive packages [25].
Figure 1.2 Rheology of linear and branched NatureWorks PLA (reproduced with
permission from ref. [25]).
7
PLA optical properties, more specifically transmission of UV and visible
wavelengths, are very important in designing the right packaging material to protect and
preserve products. Figure 1.3 shows the optical properties of PLA compared to standard
packaging materials. PLA shows significant UV light transmission at 225 nm. At 250 nm,
85% of the UV light is transmitted, while at 300 nm, 95% of UV light is transmitted.
Effective UV stabilizers are able to absorb UV and thus prevent damage to the UV
sensitive packaged products [6].
Table 1.2 PLA 2002D properties [32]
Physical/Mechanical Property PLA 2002D ASTM
Method
Specific Gravity
1.24
D792
Melt Index (210 °C/2.16 kg)
5-7
D1238
Tensile Strength @ Break, psi (MPa)
7700 (53)
D882
Tensile Yield Strength, psi (MPa)
8700 (60)
D882
Tensile Modulus, kpsi (GPa)
500 (3.5)
D882
Tensile Elongation, %
6.0
D882
8
PLA dissolves in many common organic solvents such as acetone, benzene,
chloroform, dichloromethane, dioxane, dimethylformamide, ethyl acetate,
tetrahydrofuran, toluene, trichloromethane, and p-xylene. PLA does not dissolve in water,
alcohols, and unsubstituted hydrocarbons [33]. Solubility parameters for polylactides
from the literature are reported in Table 1.3.
Figure 1.3 Percent transmission versus wavelength for PLA (98% L-lactide), PS, LDPE,
PET and cellophane films. (PLA samples were obtained from Cargill Dow LLC)
(reproduced with permission from ref. [6]).
9
Table 1.3 Solubility parameters for PLA [6]
Determination Method Solubility Parameter (cal0.5cm-1.5)
Density in Solution
10.25 ± 0.16
Limiting Viscosity Number
10.00 ± 0.20
Group Contribution Methods
Small
9.7
Hoy
9.9
Van Krevelen
9.4
PLA MODIFICATIONS
In order to overcome main drawbacks associated with PLA, mainly brittleness
and lack of readily reactable groups, PLA has been bulk and surface modified. Each of
these modifications has been aimed at either modifying mechanical properties or surface
properties.
10
Bulk modifications
Several bulk modification methods have been employed to improve mechanical
properties (mainly toughness), degradation behavior, processibility, and crystallinity of
PLA. With respect to structure-property relationships, crystallinity is an important
characteristic that affects PLA degradation rate [34] and mechanical properties [31].
Kolstad [35] observed approximately 40% increase in the crystallization half time for
every 1 wt% increase in the meso-lactide content in poly(L-co-meso-lactide). He also
observed that the addition of 15 wt% or more meso-lactide rendered the resulting
polymer significantly non-crystallizable. Perego et al. [31] studied the effect of molecular
weight and crystallinity on the mechanical properties of PLA. Poly(L-lactide) (Mv ~ 23-
66 kDa) and poly(D,L-lactide) (Mv ~ 47.5-114 kDa) exhibited small changes in the
tensile strength at break, which varied from 55 to 59 MPa for poly(L-lactide) and from 40
to 44 MPa for poly(D,L-lactide) in the given molecular weight range. It was also
observed that the tensional and flexural modulii of elasticity, Izod impact strength, and
heat resistance (the measure of polymer’s resistance to distortion under a given load at
elevated temperature) increased with crystallintiy. Crystallinity not only affects the bulk
properties but also the surface roughness. Washburn et al. [36] applied a linear
temperature gradient to produce a crystallinity gradient across a PLA film and observed
that MC3T3-E1 osteoblasts proliferated faster on the smoother regions than on the
rougher regions. The critical rms roughness, above which a statistically significant
reduction in proliferation rate occurred, was found to be approximately 1.1 nm.
11
Different processing methodologies have been applied to control orientation and,
hence, bulk properties of polymers. These approaches influence the bulk properties
without altering the PLA chemistry or introducing additives. Injection molded samples of
amorphous PLA showed higher tensile strength at break and notched Izod impact
strength upon drawing [37]. An injection molding process that applied an oscillating
shear flow to orient the semi-solid melt improved the Charpy impact strength [37]. Bigg
[38] observed a substantial increase in % elongation and tensile strength at break of PLA
with different ratios of L-lactide to D,L-lactide upon biaxial orientation. For L-lactide to
D,L-lactide copolymer ratio of 80/20, % elongation at break increased from 5.7 to 18.2%
and tensile strength at break increased from 51.7 to 84.1 MPa upon biaxial orientation at
85 °C. The literature on stereochemical and processing manipulations of PLA indicates
that these bulk modifications have not been very effective in toughening PLA.
PLA has been copolymerized with a range of polyesters and other monomers
either through polycondensation of lactic acid with other monomers, producing low
molecular weight copolymers, or ring opening copolymerization of lactide with cyclic
monomers like glycolide, ε-caprolactone, δ-valerolactone, trimethylene carbonate, etc. as
well as linear monomers like ethylene glycol [33] producing high molecular weight
copolymers. Fukuzaki et al. [39] copolymerized L-lactic acid and ε-caprolactone without
any catalyst to produce low molecular weight (Mw ~ 6.8-8.8 kDa) copolymers for
biomedical applications. These copolymers showed excellent in-vitro (enzymatic) and in-
vivo degradation properties. A key advantage that condensation copolymerization offers
is control over polymer end groups. Lactic acid has been condensation copolymerized
12
with diols or diacids in such a way that the resulting copolymer has either hydroxyl or
acid end groups and a particular molecular weight. Although polycondensation produces
low molecular weight polymers (Mw < 10 kDa), this control over the end groups is a
valuable tool in addition-type chemistry [40]. Ring opening copolymerization (ROC) of
L-lactide is a common approach for PLA copolymer synthesis, initiated with hydroxyl
groups, such as alcohol or polyol [41]. The ring opening lactide copolymerization route
has been used extensively due to its precise chemistry control and resulting favorable
copolymer properties [33]. The polymerization mechanism can be ionic, co-ordination, or
free radical depending on the type of catalyst system involved [33, 42]. The transition
metal compounds of tin [43, 44], aluminum [45], lead [46], zinc [47], bismuth [46], iron
[48], and yttrium [49] have been reported to catalyze lactide ROC. Haynes et al. [50]
copolymerized lactide with another commercially available biodegradable and renewably
derived thermoplastic polyester, poly(hydroxyalkanoate) (PHA). The resulting copolymer
was found to have a lower complex viscosity compared to neat PLA. Also, the storage
and loss modulii of this copolymer underwent less change with frequency (0.1-100
radians/sec) compared to neat PLA. PLA has been copolymerized extensively with PEG
due to PEG’s biocompatibility and hydrophilicity. An alternating copolymer of lactic acid
and ethylene oxide produced from the ring opening of the cyclic ester monomer 3-
methyl-1,4-dioxan-2-one has been used to plasticize PLA [51]. Diblock and triblock
PLA-PEG copolymers were also synthesized to improve hydrophilicity and drug-delivery
properties of PLA. However, PLA and PEG underwent phase separation leading to poor
mechanical properties of the copolymers [52]. To improve the compatibility between
13
PLA and PEG components, PLA-PEG copolymers were produced by copolycondensation
of PLA-diols and PEG-diacids using carbodiimide-based wet chemistry. The resultant
copolymer did not phase separate and exhibited improved mechanical properties [53].
In addition to copolymerization, PLA has been extensively bulk modified using
blending. Blending is probably the most widely used methodology to improve PLA
mechanical properties. PLA has been blended with different plasticizers and polymers
(biodegradable and non-biodegradable) to achieve desired mechanical properties. PLA is
a glassy polymer that has poor elongation at break (< 10%) [54]. Different biodegradable
as well as non-biodegradable plasticizers have been used to lower the glass transition
temperature, increase ductility, and improve processibility [55]. Martin and Avérous [56]
used glycerol, citrate ester, PEG, PEG monolaurate, and oligomeric lactic acid to
plasticize PLA and found that oligomeric lactic acid and low molecular weight PEG (Mw
~ 400 Da) gave the best results while glycerol was found to be the least efficient
plasticizer. Citrate esters (molecular weight 276-402 Da) derived from naturally
occurring citric acid were found to be miscible with PLA at all compositions. For these
blends with citrate esters, elongation at break was significantly improved accompanied
with considerable loss of tensile yield strength [57]. Baiardo et al. [58] used acetyl tri-n-
butyl citrate and PEGs with different molecular weights (Mw ~ 0.4-10 kDa) to plasticize
PLA. Acetyl tri-n-butyl citrate miscibility limit was found to be 50 wt% while PEG
miscibility decreased with increasing molecular weight. These researchers also observed
a significant increase in elongation at break at the expense of strength and tensile
modulus. Hillmyer et al. [59, 60] blended PLA with low density poly(ethylene) (LDPE)
14
to improve the toughness. Recently, DuPont has commercialized Biomax®Strong non
degradable PLA additives to improve toughness without significant transparency loss.
These additives are designed to have “special chemistry” for PLA, so even small amounts
(1-5 wt %) provide significant toughness benefits [61]. NatureWorks LLC studied
different commercial toughening agents for PLA [3]. In their work, BlendexTM 338, an
acrylonitrile-butadiene-styrene terpolymer containing 70% butadiene rubber, was found
to significantly improve notched Izod impact strength and elongation at break of PLA.
Another additive, PellethaneTM 2102-75A (a commercial grade polyurethane from Dow
Chemical Company), was also found to significantly improve these properties [3]. PLA
blends with biodegradable polymers have been extensively investigated because they
offer property improvements without compromising biodegradability. For example, PHA
is a bacterially produced family of biodegradable aliphatic homo or copolyesters with
more than 150 different types consisting of different monomers [62]. Addition of a small
amount (typically < 20 wt %) of Nodax copolymer to PLA remarkably improved the
toughness of the resultant blend without significantly affecting the optical clarity [63].
PLA/PCL is another extensively studied biodegradable PLA blend system. PCL is a
rubbery polymer with low Tg and degrades by hydrolytic or enzymatic pathways. Broz et
al. [64] tuned modulus, strain at break, and ultimate tensile strength through the blend
composition. Jiang et al. [65] blended PLA with a biodegradable thermoplastic
poly(butylene adipate-co-terephthalate) (PBAT) to improve toughness and processibility
of PLA. PLA has also been blended with chitosan, a naturally occurring biodegradable,
biocompatible, edible, and nontoxic biopolymer, to improve wettability [66, 67].
15
Although PLA/collagen blends had reduced tensile and bending strengths compared to
neat PLA, they underwent faster degradation under enzymatic conditions. The weight
decreased to half the original weight of a PLA/collagen blend (30 wt% collagen) after
five weeks, but neat PLA and PLA/collagen blends (10 wt% collagen) did so after eight
and six weeks, respectively [68]. PLA has been toughened by physically blending with a
variety of rubbery polymers, which was associated with significant modulus and/or UTS
loss [69-71].
In summary, efforts to improve PLA’s toughness have resulted in a decrease of
other important mechanical properties. The work presented in Chapter 5 addresses this
issue.
Surface modifications
PLA surface interactions with other materials play an important role in numerous
consumer and biomedical applications. Special surface chemical functionalities,
hydrophilicity, roughness, and topography are often required and need to be controlled.
While, undoubtedly, there has been work done to surface modify PLA for commodity
applications (e.g., packaging films), there is a scarcity of data in the literature related to
such things as friction modification, adhesion, and anti-fogging. However, there is
abundant research reported in the literature on PLA surface modification for biomedical
applications, so this portion of the introduction will focus on those investigations with the
notion that some of the approaches could also be suitable for other commodity
16
applications. PLA has been surface modified using coating, entrapment, migratory
additives, plasma treatment, chemical conjugation using wet chemistry, and
photografting. The first four methods are non-permanent (non-covalent attachment of
functional groups) while the later two are permanent (covalent attachment) surface
modification methods.
Surface coating involves the deposition/adsorption of the modifying species onto
the polymer surface. Typically, PLA has been coated with biomimetic apatite [72]; extra
cellular matrix (ECM) proteins like fibronectin, collagen, vitronectin, thrombospondin,
tenascin, laminin, and entactin [52, 73]; and RGD peptides [74] to control PLA-cell
interactions. Although coating is a simple and convenient surface modification protocol,
passive adsorption could induce competitive adsorption of other materials in the system
and change the configuration of adsorbed species [52].
Entrapment is another surface modification methodology that can be used to
incorporate molecules that do not adsorb onto PLA and does not require readily reactable
side chain groups. Biomacromolecules such as alginate [75], chitosan [75], gelatin [75],
poly(L-lysine) (PLL) [76], PEG [76-78], and poly(aspartic acid) [79] have been
entrapped during the reversible swelling of the PLA surface region upon exposure to a
solvent/nonsolvent mixture. This method typically requires a miscible mixture of a
solvent and nonsolvent for PLA, with the surface-modifying molecules being soluble in
the mixture and the nonsolvent [76]. Cai et al. [79] modified PLA surfaces by entrapping
poly(aspartic acid) (PASP) in order to enhance their cell affinity. Rat osteoblasts were
seeded onto the modified surfaces to examine their effects on cell adhesion and
17
proliferation. The findings showed that PASP-modified PLA surfaces may enhance the
cell-surface interactions. The solvent-nonsolvent mixtures used in these entrapment
protocols consisted of acetone or 2,2,2-trifluroethanol as a solvent for PLA. Typically,
most of the PLA solvents are not biocompatible. These studies have not reported on the
amounts of the residual solvent left behind in surface-modified films. From a
biocompatibility standpoint, surface-modification protocols should involve more benign
solvents or removal of non-biocomptible solvents from the film bulk without affecting
surface properties.
Migratory additives, carrying specific functional groups, are blended with PLA as
a way to tailor PLA surface properties. Yu et al. [80] blended poly(D,L-lactic acid)-
block-poly(ethylene glycol) (PLE) copolymer and RGD derivatives with PLA to engineer
the surface properties of the resultant blend to promote chondrocyte attachment and
growth. The blends prepared by this methodology showed enhanced hydrophilicity
compared to neat PLA. The water contact angle decreased from 76° for neat PLA to 50°
for PLA/PLE blends (75 wt % PLA). The chondrocyte cultures showed significant
improvement of chondrocyte attachment and viability on the PLA films modified with
PLE and RGD derivatives.
Plasma surface treatment of polymers began in the 1960s [81] and, within the last
decade, has been applied to improve PLA surface hydrophilicity and cell affinity. The
term “plasma” refers to a mixture of positive ions and electrons produced by ionization
[82]. Yang et al. [83] used anhydrous ammonia (NH3) plasma treatment to improve
hydrophilicity and cell (human skin fibroblast) affinity of complex shapes like porous
18
PLA scaffolds prepared using a particulate leaching technique. The NH3 plasma
generated reactive amine groups on PLA scaffolds that anchored collagen through polar
and hydrogen bonding interactions. These surface-modified scaffolds showed enhanced
cell adhesion [84]. The main disadvantage of this technique is that the effectiveness of
the surface modification is partially lost due to surface rearrangement [85]. The surface-
modifying species rearrange by thermally activated macromolecular motions to minimize
the interfacial energy, making the effect of plasma treatment non-permanent [83, 85-87].
Yang et al. [83] found that the modifying effects could be maintained by preserving
samples at a low temperature (0-4 °C). The mobility of surface molecular chains was
significantly decreased at temperatures much less than the Tg of PLA (55 °C). Since this
temperature range (0-4 °C) is much lower than physiological as well as room
temperature, this stabilization approach might not be practical. Apart from the
rearrangement tendency of the modifying species introduced using plasma treatment, the
treatment can also affect degradation of PLA. The NH3 plasma-modification depth
increased with treatment time, while the plasma power (20 to 80 W) influenced the depth
only slightly. It was observed that the PLA degradation increased with an increase of
plasma power and treatment time [88]. Although plasma treatment has been used to
improve wettability and cell affinity of PLA, the issues related to non-permanent surface
modification potentially make it unsuitable for certain biomedical and consumer
applications.
Chemical conjugation using wet chemistry has been used to surface modify PLA
extensively. Alkaline surface hydrolysis is a simple way to create reactive functional
19
groups, e.g., carboxylic acids (-COOH) and hydroxyls (-OH), on PLA [52]. The resulting
carboxylic acid groups on PLA can readily be conjugated with surface-modifying species
containing amine (-NH2) or hydroxyl (-OH) groups. Typically acid groups are first
activated with phosphorous pentachloride (PCl5) [89], thionyl chloride (SOCl2) [90], or
water soluble carbodiimides [91] and subsequently conjugated with amines or hydroxyls
(see Figure 1.4). Aminolysis is another way to introduce reactive amine groups onto PLA
surfaces. 1,6-hexanediamine has been used for aminolysis followed by conjugation with
biocompatible macromolecules like gelatin, chitosan, or collagen [92]. The aminolysis
reaction was performed by immersing PLA in hexanediamine-propanol solution (0.06
g/mL) at 50 °C (below PLA’s Tg) for 8 min. PLA surface hydrophilicity (as measured
using a sessile drop method) decreased slightly after aminolysis and further after
biomacromolecule immobilization.
Janorkar et al. [93] introduced amine groups on the PLA film surface by
photoinduced grafting of 4,4’-diaminobenzophenone followed by wet chemistry to create
branched arichitectures containing amine functionalities on the periphery of the grafted
layers. The grafted branched architectures were created by subsequent carbodiimide
mediated reactions with succinic acid and tris(2-aminoethyl) amine. MC3T3 fibroblast
attachment and viability improved with the grafting of amine terminated branched
architectures.
20
Figure 1.4 Generalized reaction scheme for carboxylic acid activation using PCl5, SOCl2,
or water soluble carbodiimides followed by chemical conjugation with amine (-NH2) or
hydroxyl (-OH) functionalities.
Photografting has been used extensively to tailor PLA surface properties primarily
due to the advantages it offers: low cost of operation, mild reaction conditions, selectivity
of UV light, and permanent alteration of surface chemistry [94]. This approach relies on
21
PLA photoactivation to create reactive groups associated with or followed by grafting of
selected functionalities. Since PLA does not have any readily reactable side chain groups,
this approach is useful for PLA surface modification. Typically, these methods are
classified as “grafting to” or “grafting from” approaches. Polymer chains of known
molecular weight, composition, and architecture are covalently attached to the surface in
a “grafting to” approach, which is convenient for preliminary studies [95]. However, it is
difficult to achieve high grafting densities with a “grafting to” approach because of steric
hindrance and diffusion limitations [96]. The “grafting from” approach, which involves
growing polymer chains from the surface, overcomes the limitations of the “grafting to”
approach. In “grafting from”, photoinitiators are immobilized onto the substrate to initiate
subsequent polymerization of vinyl or acrylic monomers from the surface. Photografting
reactions have been carried out either in liquid or vapor phases. Zhu et al. [97] used a
“grafting to” approach to immobilize chitosan chains onto PLA film surfaces using a
hetero-bifunctional crosslinking reagent, 4-azidobenzoic acid. The “grafting from”
approach has been used more extensively than the “grafting to” approach for PLA surface
modification. Typically, either plasma treatment or photoinitiator is used to activate the
PLA surface followed by photopolymerization of vinyl or acrylic monomers from the
surface. Janorkar et al. [34] successfully used a “grafting from” approach to create
bioactive PLA surfaces. The PLA film grafted with poly(acrylic acid) (PAA) and
poly(acrylamide) (PAAm) exhibited improved wettability. Another positive outcome of
this research was that PLA films grafted with PAA underwent faster in-vitro degradation,
which was attributed to acrylic acid monomer migrating into the film bulk and
22
polymerizing. Janorkar et al. [98] have also used single-monomer and mixed-monomer
systems of AA, AAm, and vinyl acetate (VAc) to produce surface-confined
homopolymers and copolymers to yield a spectrum of hydrophilicities, ranging from 82°
for unmodified PLA to 12° for PLA grafted with PAAm. In order to avoid detrimental
solvent effects on PLA, Edlund et al. [99] used a single-step vapor phase photografting
route to covalently attach poly(acrylamide), poly(maleic anhydride), and poly(N-
vinylpyrrolidone) to PLA-film surfaces. PLA film was exposed to the vapor phase
mixture of monomer and benzophenone (photoinitiator) under UV irradiation at 50 °C.
These reactions were carried out below PLA’s glass transition temperature to avoid any
significant bulk changes. The extent of grafting and wettability increased with UV
irradiation time. The static water contact angle values of PLA changed from 80° to 50°
for poly(maleic anhydride) grafting, to 35° for poly(acrylamide) grafting, and to 25° for
poly(N-vinylpyrrolidone) grafting for 30 min. Källrot et al. [100] observed that PLA
films grafted with poly(N-vinylpyrrolidone) using the single-step vapor phase
photografting protocol provided a good substrate for normal human cells of two types,
keratinocytes and skin fibroblasts, to adhere and proliferate.
SUMMARY OF DISSERTATION RESEARCH
In the first part of this dissertation research, PLA films were successfully surface
modified using a sequential two-step photografting method (Chapter 3). PLA was then
melt-blended with poly[(3-hydroxybutyrate)-co-(3-hydroxyhexanoate)] (PHBHHx) with
23
an ultimate aim of making it tougher. PLA-PHBHHx blend films were further surface
modified using a sequential two-step photgrafting method. It was observed that the blend
films lost their toughness on surface modification due to UV-assisted solvent induced
crystallization (UVasic) during photografting reactions (Chapter 4). In the final part of
this work, a novel reactive blending technology was developed to toughen PLA with
minimal modulus and ultimate tensile strength loss associated with introduction of a
controlled concentration of reactive acid groups into the PLA matrix (Chapter 5).
24
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65. Jiang L, Wolcott MP, Zhang J. Study of biodegradable polylactide/poly(butylene adipate-co-terephthalate) blends. Biomacromolecules 2006;7:199-207.
66. Wan Y, Wu H, Yu A, Wen D. Biodegradable polylactide/chitosan blend membranes. Biomacromolecules 2006;7:1362-72.
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68. Yang X, Yuan M, Li W, Zhang G. Synthesis and properties of collagen/ polylactic acid blends. J Appl Polym Sci 2004;94:1670-75.
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71. Topolkaraev VA, Soerens DA. Methods of making biodegradable films having enhanced ductility and breathability. US Pat 20030015826.
72. Chen Y, Mak AFT, Wang M, Li J, Wong MS. PLLA scaffolds with biomimetic apatite coating and biomimetic apatite/collagen composite coating to enhance osteoblast-like cells attachment and activity. Surface & Coatings Technology 2006;201:575–80.
73. Atthof B, Hilborn J. Protein adsorption onto polyester surfaces: is there a need for surface activation? J Biomed Mater Res Part B: Appl Biomater 2007;80B:121–30.
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75. Zhu H, Ji J, Shen J. Surface engineering of poly(DL-Lactic Acid) by entrapment of biomacromolecules. Macromol Rapid Commun 2002;23:819-23.
76. Quirk RA, Davies MC, Tendler SJB, Shakesheff KM. Surface engineering of poly(lactic acid) by entrapment of modifying species. Macromolecules 2000;33:258-60.
77. Quirk RA, Davies MC, Tendler SJB, Chan WC, Shakesheff KM. Controlling biological interactions with poly(lactic acid) by surface entrapment modification. Langmuir 2001;17:2817-20.
78. Zhang J, Roberts CJ, Shakesheff KM, Davies MC, Tendler SJB. Micro- and macrothermal analysis of a bioactive surface-engineered polymer formed by physical entrapment of poly(ethylene glycol) into poly(lactic acid). Macromolecules 2003;36:1215-21.
79. Cai K, Yao K, Hou X, Wang Y, Hou Y, Yang Z, Li X, Xie H. Improvement of the functions of osteoblasts seeded on modified poly(D,L-lactic acid) with poly(aspartic acid). J Biomed Mater Res 2002;62:283–91.
80. Yu G, Ji J, Zhu H, Shen J. Poly(D,L-lactic acid)-block-(ligand-tethered poly(ethylene glycol)) copolymers as surface additives for promoting chondrocyte attachment and growth. J Biomed Mater Res Part B: Appl Biomater 2006;76B:64 –75.
81. Wang CX, Ren Y, Qiu YP. Penetration depth of atmospheric pressure plasma surface modification into multiple layers of polyester fabrics. Surface & Coatings Technology 2007;202:77–83.
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83. Yang J, Shi GX, Wang SG, Bei JZ, Cao YL, Shang QX, Yang GH, Wang W J. Fabrication and surface modification of macroporous poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds for human skin fibroblast cell culture. J Biomed Mater Res 2002;62:438-46.
84. Yang J, Bei JZ, Wang SG. Enhanced Cell Affinity of Poly(D,L-Lactide) by Combining Plasma Treatment with Collagen Anchorage. Biomaterials 2002;23:2607-14.
85. Ximing X, Gengenbach TR, Griesser HJ. Changes in wettability with time of plasma modified perfluorinated polymers. J Adhes Sci Tech 1992;6:1411–31.
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87. Safinia L, Datan N, Höhse M, Mantalaris A, Bismarck A. Towards a methodology for the effective surface modification of porous polymer scaffolds. Biomaterials 2005;26:7537–47.
88. Wan Y, Tu C, Yang J, Bei J, Wang S. Influences of ammonia plasma treatment on modifying depth and degradation of poly(L-lactide) scaffolds. Biomaterials 2006;27:2699–704.
89. Luo N, Stewart MJ, Hirt DE, Husson SM, Schwark DW. Surface modification of ethylene-co-acrylic acid copolymer films: addition of amide groups by covalently bonded amino acid intermediates. J Appl Polym Sci 2004;92:1688-94.
90. Zhang P, He C, Craven RD, Evans JA, Fawcett NC, Wu Y, Timmons RB. Subsurface formation of amide in polyethylene-co-acrylic acid film: a potentially useful reaction for tethering biomolecules to a solid support. Macromolecules 1999;32:2149-55.
91. Janorkar AV, Luo N, Hirt DE. Surface modification of an ethylene-acrylic acid copolymer film: grafting amine-terminated linear and branched architectures. Langmuir 2003;20:7151-58.
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93. Janorkar AV, Fritz EW, Burg KJL, Metters AT, Hirt DE. Grafting amine-terminated branched architectures from poly(L-lactide) film surfaces for improved cell attachment. J Biomed Mater Res Part B: Appl Biomater 2007;81B:142–52.
94. Ma H, Davis RH, Bowman CN. A Novel Sequential Photoinduced Living Graft Polymerization. Macromolecules 2000;33:331-35.
95. Rahane SB, Kilbey SM II, Metters AT. Kinetics of surface-initiated photoiniferter-mediated photopolymerization. Macromolecules 2005;38:8202-10.
96. Zhao B, Brittain WJ. Polymer brushes: surface-immobilized macromolecules. Prog Polym Sci 2000;25:677-710.
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97. Zhu A, Zhang M, Wu J, Shen J. Covalent immobilization of chitosan/heparin complex with a photosensitive hetero-bifunctional crosslinking reagent on PLA surface. Biomaterials 2002;23:4657-65.
98. Janorkar AV, Proulx SE, Metters AT, Hirt DE. Surface-confined photopolymerization of single- and mixed-monomer systems to tailor the wettability of poly(L-lactide) film. J Polym Sci Part A: Polym Chem 2006;44:6534–43.
99. Edlund U, Källrot M, Albertsson AC. Single-step covalent functionalization of polylactide surfaces. J Am Chem Soc 2005;127:8865-71.
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33
CHAPTER TWO
ANALYTICAL TECHNIQUES
DYNAMIC MECHANICAL ANALYSES (DMA)
DMA is useful for characterizing the viscoelastic properties of polymers. DMA
primarily measures the stiffness and dampening properties of a material. It is one of the
most sensitive techniques to study relaxation events, such as glass transitions. DMA
applies an oscillating force to the sample and analyzes the material’s response to it
(Figure 2.1) [1].
Figure 2.1 The DMA supplies a sinusoidal stress to the sample, which generates a
sinusoidal strain. Properties such as modulus, viscosity, and dampening can be calculated
by measuring the deformation amplitude at the peak and the lag between stress and strain
sine waves [1].
34
A SEIKO INSTRUMENTS DMS210U dynamic mechanical analyzer was used to
monitor changes in the viscoelastic response of the materials as a function of temperature.
A specimen (2 cm x 1 cm) was placed in mechanical oscillation at a frequency of 1 Hz
and the test was conducted at a heating rate of 2 °C/min. Calibration was performed using
poly(methyl methacrylate) and steel standards and polycarbonate was used to check the
calibration.
WIDE-ANGLE X-RAY DIFFRACTION (WAXD)
WAXD patterns were obtained at room temperature in the scattering angle (2Ө)
range of 10 – 35° by using an XDS 2000, SCITAG INC., USA, instrument. The X-ray
generator produced Cu Kα radiation (wavelength = 1.5418 nm), which was used as an X-
ray source (40 kV, 30 mA). WAXD analyses were performed to monitor the
crystallization of PLA-PHBHHx blend films occurring during photografting reactions.
THERMOGRAVIMETRIC ANALYSIS (TGA)
Thermogravimetric analyses were performed using a TA Instruments TGA 2950
operated with Thermal Advantage software version 1.1. TGA enabled us to characterize
the amount of residual solvent in various samples. A film sample was loaded into a
platinum pan and scanned under a nitrogen purge at a flow rate 40 ml/min. The samples
35
were heated from room temperature to 200 °C at a heating rate 10 °C/min to monitor
weight loss.
DIFFERENTIAL SCANNING CALORIMETRY (DSC)
DSC was used to monitor crystallization and physical aging of PLA-based films.
A Pyris 1 PerkinElmer Instrument was used to obtain DSC data from 30 to 190 °C at a
heating rate 10 °C/min. The degree of crystallinity was calculated from the measured heat
of fusion relative to an estimated 93 J/g [3] for a 100% crystalline PLA and 164 J/g [4]
for a 100% crystalline PHB.
ATTENUATED TOTAL REFLECTANCE (ATR) FTIR SPECTROSCOPY
Attenuated total reflectance (ATR) FTIR spectroscopy was used to characterize
the modified film surfaces. The characterization was performed using a Thermo Nicolet
Magna 550 single bounce spectrometer equipped with a Thermo-Spectra-Tech
Foundation Series Diamond ATR with DTGS detector. 32 scans at a resolution of 4 cm-1
were collected and averaged. OMNIC software version 6.2 was used to process spectra
for ATR and baseline corrections.
36
CONTACT ANGLE GONIOMETRY
A Krüss G10 static contact angle apparatus was used to perform static water
contact angle measurements using a sessile drop method. Using a syringe, an
approximately 1 μL water drop volume was placed on the film surface and allowed to
stabilize for 2 min. Water contact angle values were measured using Drop Shape
Analysis software. The reported water contact angles are an average of 10 readings with
±95% confidence intervals.
X-RAY PHOTELECTRON SPECTROSCOPY (XPS)
XPS spectra (appendix A) were obtained using a Kratos Axis Ultra Photoelectron
Spectrometer with Al Kα radiation (15 kV, 225 W) and an overall instrument resolution
of 1.1 eV. All spectra were collected at an electron take-off angle of 90° to the sample
surface. Survey spectra were collected over the 0-1200 eV range, using a pass energy of
40 eV. High resolution spectra of the C 1s core levels were also collected using a pass
energy of 40 eV. CasaXPS software was used for spectral analysis. The binding energies
were corrected by referencing the C 1s binding energy to 285 eV.
37
OPTICAL AND FLUORESCENCE MICROSCOPY
A PAA micropatterned PLA specimen was immersed in toluidine blue (0.1
mg/ml) solution in water and observed with a Zeiss Axiovert 135 optical microscope
(Appendix A). An Insight color digital camera with SPOT image acquisition software
(Diagnostics Instruments, Sterling Heights, MI) was used to capture images. The optical
micrographs were analyzed using Image-Pro 4.1 software. Images of streptavidin
adsorbed on biotin modified PAA patterns on PLA were recorded with the aid of a
fluorescence microscopy filter at 515 nm.
MICROWAVE ASSISTED EXTRACTION (MAE)
A select few surface-modified films were subjected to microwave assisted
extraction in a MARS 5 microwave accelerated reaction system from CEM Corporation.
These surface-modified films were subjected to the extraction in water at 95 °C for 1 h.
TRANSMISSION-FTIR MICROSPECTROSCOPY WITH DIAMOND
COMPRESSION CELL
Transmission-FTIR microspectroscopic analyses were conducted using a Thermo
Nicolet Magna 550 FTIR spectrometer equipped with a Thermo Nicolet Ni-Plan FTIR
microscope. The microtomed sections (typically 50 μm thick) were compressed using a
38
Thermo-Spectra-Tech micro sample plan with diamond windows. 32 scans at a resolution
of 4 cm-1 with a KBr background were collected for each sample. An adjustable aperture
was used to form an analysis area approximately 25 microns wide and 100 microns long
with the long dimension parallel to the long axis of the microtomed section (i.e., the 25-
micron-wide beam was used for analysis at five discrete locations across the 125 microns
thickness of the film) [5].
STATISTICAL ANALYSIS
Statistical evaluation of the toughness data was performed using t-test. All results
are reported as mean ±95% confidence intervals (level of significance = 0.025 and n=5).
39
REFERENCES
1. http://www.triton-technology.co.uk/pdf/TTInf_DMA.pdf (Accessed November 25, 2008).
2. Furukawa T, Sato H, Murakami R, Zhang J, Noda I, Ochiai S, Ozaki Y. Comparison of miscibility and structure of poly(3-hydroxybutyrateco-3-hydroxyhexanoate)/poly(L-lactic acid) blends with those of poly(3-hydroxybutyrate)/poly(L-lactic acid) blends studied by wide angle X-ray diffraction, differential scanning calorimetry, and FTIR microspectroscopy. Polymer 2007;48:1749-55.
3. Janorkar AV, Metters AT, Hirt DE. Modification of poly(lactic acid) films: enhanced wettability from surface-confined photografting and increased degradation rate due to an artifact of the photografting process. Macromolecules 2004;37:9151-59.
4. Rasal RM, Metters AT, Hirt DE. Surface modification and bulk properties of PLA, PHA, and their blend films. ANTEC 2006 – proceedings of the 64th annual technical conference & exhibition, Charlotte, society of plastics engineers 2006; 52:1809-1813.
5. Joshi NB, Hirt DE. Evaluating bulk-to-surface partitioning of erucamide in LLDPE films using FT-IR microspectroscopy. Appl Spectrosc 1999;53:11-16.
40
CHAPTER THREE
EFFECT OF THE PHOTOREACTION SOLVENT ON SURFACE AND BULK
PROPERTIES OF PLA AND PHBHHx FILMS
[As published in Journal of Biomedical Materials Research Part B: Applied Biomaterials,
85B, 564-572 with minor changes]
INTRODUCTION
Poly(lactic acid) (PLA) and poly(hydroxyalkanoate) (PHA) are biodegradable
thermoplastics that show great potential in consumer and biomedical applications. PLA is
a well known biomaterial [1-11]. PHA is a bacterially produced homopolymer or
copolymer family with more than 150 different types consisting of different monomers
[12]. Poly[(3-hydroxybutyrate)-co-(3-hydroxyhexanoate)] (PHBHHx) is a member of the
PHA family and has been studied extensively for biomedical applications [13-21]. PLA is
relatively brittle but shows improved toughness when blended with a small amount of
PHBHHx [22].
Even though PLA-PHBHHx blends exhibit enhanced toughness over neat PLA,
the two polymers are hydrophobic and do not contain readily reactable groups, which
limit their use in many consumer and biomedical applications. These polymers need to be
surface modified to introduce readily reactable functional groups and to control their
hydrophilicity. Solvent cast PLA and PHBHHx films were surface modified using
photoinduced grafting because of the following advantages: low cost of operation, mild
41
reaction conditions, selectivity of UV light, and irreversible covalent grafting [23].
Acrylic acid and acrylamide were chosen as monomers because of their ability to make
surfaces hydrophilic on photopolymerization from the film surface [24]. Moreover,
acrylic acid could be subsequently conjugated to various synthetic and biomolecules. The
method consisted of two steps. Step 1 involved benzophenone (photoinitiator)
photografted on the film surface and Step 2 involved photopolymerization of hydrophilic
monomers from the film surface. In both steps, ethanol was used as the reaction solvent
and the procedure resulted in increased hydrophilicity of the films using acrylic acid and
acrylamide as the monomers [24-25].
PLA and PHBHHx films used in this study had very low crystallinity initially. It
was observed that the films underwent solvent-induced crystallization during the surface
reactions. We have previously surmised that monomer penetrated into the film,
photopolymerized, and subsequently influenced the degradation rate when ethanol was
used as the reaction solvent in Step 2 [24]. In this work, the extent of monomer
penetration was evaluated using FTIR microspectroscopy. We have also used water as
well as ethanol to investigate the effect of reaction solvent in Step 2 on surface
photografting, monomer penetration, solvent-induced crystallization, and resultant
mechanical properties of solvent-cast PLA and PHBHHx films.
42
MATERIALS
The chemical structures of PLA and PHBHHx repeat units are shown in Figure
3.1. PLA pellets (Mn ~ 110,000 g/mol) were supplied by NatureWorks LLC. PHBHHx
comprising 6.9 mol% 3HHx units was supplied by Procter & Gamble Company. Acrylic
acid (99.5% w/w) was obtained from Acros Organics. Acrylamide (99% w/w) and
chloroform (CHCl3) were obtained from Aldrich Chemicals. Ethanol, HPLC water,
benzophenone, and H2O2 (30% w/w) were obtained from Fisher Scientific. All chemicals
were used as received.
O
H
O
CH3
n
O
HCH2
CH3
O
O
HCH3
O
(a) (b)
n 1-n
2
Figure 3.1 Chemical structures of (a) PLA and (b) PHBHHx repeat units.
43
METHODS
Film Solvent Casting: Approximately 1.1 g polymer was dissolved in 60 ml of
chloroform. The films were cast in a glass petri dish, which was cleaned by exposing it to
Pirahna solution (concentrated H2SO4 and 30% H2O2 75:25 v/v) for 1 h. Extreme care
must be taken when using Pirahna solution. The dish was then washed using copious
amounts of distilled water and dried using nitrogen. The cleaned petri dish was aligned
perfectly horizontal to ensure that the resultant film had uniform thickness. The polymer-
chloroform solution was poured in the petri dish and kept in a chemical hood for 24 h to
allow the chloroform to evaporate. The resultant film was removed from the petri dish
using a razor blade [24].
Sequential two step photografting: Figure 3.1 shows the reaction scheme for the
sequential two-step photografting reaction of acrylic acid onto a PLA film surface. A film
specimen (approximately 3 cm x 1 cm x 125 μm) was dip coated in 5% w/w
benzophenone solution in ethanol. The film was allowed to stand at room temperature for
30 min to ensure that ethanol was evaporated. The benzophenone dip-coated film was
sealed in a quartz cuvette using parafilm in a glove box with a nitrogen atmosphere. Each
side of the film was exposed to UV irradiation for 5 min in a UV processor (OAI Model
No. 200; table top series, mask aligner and UV exposure system). The processor was
equipped with a 350 W bulb having a wavelength range of 290-500 nm and intensity of
25 mW/cm2 at 365 nm. After UV exposure, the resultant film was sonicated in ethanol for
44
5 min to remove unreacted benzophenone. Benzophenone-grafted film was put in a Pyrex
test tube containing 10% v/v of the chosen monomer in ethanol or water. The test tube
was purged with nitrogen for 30 min and exposed to UV for 3 h. The resultant film was
sonicated in the solvent used for Step 2 for 5 min to remove physically adsorbed polymer
from the surface [24].
H + C O C OH C OHUV irradiation
Step 1
C OHUV irradiation
monomer solutionStep 2
O
OH
O
OH
O
OH
H + C O C OH C OHUV irradiation
Step 1
C OHUV irradiation
monomer solutionStep 2
O
OH
O
OH
O
OH
Figure 3.2 Reaction scheme for the sequential two-step photografting reaction of acrylic
acid onto a PLA film surface [24].
Mechanical Testing: Mechanical properties were measured using an Applied Test System
Inc. (ATS) mechanical tester on the film samples (3 cm x 1 cm x 125 μm) according to
American Society for Testing and Materials Standard (ASTM D882) specifications. The
measurement values averaged for five specimens are reported.
45
RESULTS AND DISCUSSION
The neat, solvent cast PLA and PHBHHx films are hydrophobic, with water
contact angles ~ 82 ± 0.2°. Hydrophilic monomers, acrylamide and acrylic acid, were
successfully polymerized from the film surface using the sequential, two-step
photografting procedure discussed in detail elsewhere [24]. As mentioned above, surface-
modified films were sonicated in ethanol for 5 min to remove any physically adsorbed
species from the film surfaces. To ensure that sonication was sufficient, a few films were
also subjected to a more aggressive microwave assisted extraction in water at 95 °C for 1
h to make sure that unreacted monomer was removed and that the grafted layers were
covalently attached and not just physically adsorbed. The reason we chose water as a
solvent for the microwave assisted extraction is because free poly(acrylamide) (PAAm)
and poly(acrylic acid) (PAA) chains dissolve in water, while PAAm and PAA grafted to
PLA or PHBHHx do not dissolve in water. So the microwave assisted extraction
promoted the removal of physically adsorbed monomer, PAAm, or PAA from the
surfaces. Comparing sonicated with sonicated plus microwave-extracted films, there were
no significant changes in water contact angle or ATR-FTIR spectra, which confirmed the
removal of unbound species as well as the covalent grafting of PAAm and PAA from the
film surfaces. For all subsequent experiments, sonication was used but the use of
microwave assisted extraction was discontinued.
PLA and PHBHHx films reacted with benzophenone (Step 1) did not show any
significant change in the water contact angle values. A control experiment was also
46
performed where films were dip coated in ethanol (no benzophenone) in Step 1 and
subjected to UV exposure in the chosen monomer solution in Step 2. The reacted films
did not show any grafting from the film surfaces as evidenced from contact angle and
ATR-FTIR measurements. Figures 3.3 and 3.4 show the effect of UV exposure time on
water contact angle of the PLA and PHBHHx films grafted with benzophenone and
exposed to UV irradiation in 10% v/v monomer solution in ethanol. It was observed that
the water contact angle values plateaued after 3 h exposure in Step 2, so for all
subsequent experiments, UV exposure time in Step 2 was maintained at 3 h. The surface-
modified films are hereafter referred to as PLA-g-PAA, PLA-g-PAAm, PHBHHx-g-
PAA, and PHBHHx-g-PAAm, where g = grafted. It was observed that the static water
contact angle for modified PHBHHx was ultimately greater than that for modified PLA.
This behavior is in agreement with the fact that the benzophenone preferentially abstracts
tertiary hydrogens on the polymer [28], and the concentration of tertiary H-atoms in PLA
is greater than that in PHBHHx (see Figure 3.1).
47
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4
UV exposure time (h)
wat
er c
onta
ct a
ngle
(°)
Figure 3.3 The effect of the UV exposure time on the static water contact angle of PLA
(○) and PHBHHx (■) films grafted with acrylic acid with ethanol as the solvent in Step 2.
The error bars represent 95% confidence intervals.
48
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4
UV exposure time (h)
wat
er c
onta
ct a
ngle
(°)
Figure 3.4 The effect of the UV exposure time on the static water contact angle of PLA
(○) and PHBHHx (■) films grafted with the acrylamide with ethanol as the solvent in
Step 2. The error bars represent 95% confidence intervals.
49
Typical ATR-FTIR spectra for neat and surface-modified films are shown in
Figure 3.5. Figure 3.5a represents the neat PLA film with the –C=O peak for the PLA
ester at wave number 1756 cm-1. Figure 3.5b represents the neat PHBHHx film with the –
C=O peak for the PHBHHx ester at 1721 cm-1. Figure 3.5c represents the PLA film
grafted with PAA, showing a shoulder at 1720 cm-1, which is the –C=O acid peak. Figure
3.5d represents the PHBHHx film grafted with PAA, showing the broadening of the 1721
cm-1 peak. The amide I –C=O peak of PAAm was observed at 1670 cm-1 and an amide II
peak was observed at 1550 cm-1 in Figures 3.5e and 3.5f, representing PLA-g-PAAm and
PHBHHx-g-PAAm, respectively.
50
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4A
bsor
banc
e
15001600170018001900
Wavenumbers (cm-1)
(a)
(b)
(c)
(d)
(e)
(f)
♦
■
■
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4A
bsor
banc
e
15001600170018001900
Wavenumbers (cm-1)
(a)
(b)
(c)
(d)
(e)
(f)
♦
■
■
Figure 3.5 Representative ATR-FTIR spectra of the (a) neat PLA, (b) neat PHBHHx, (c)
PLA-g-PAA, (d) PHBHHx-g-PAA, (e) PLA-g-PAAm, and (f) PHBHHx-g-PAAm.
Spectrum (a) shows the –C=O peak for the PLA ester at 1756 cm-1 (♦). Spectrum (b)
shows the –C=O peak for the PHBHHx ester at 1721 cm-1 (). Spectrum (c) shows –C=O
acid peak at 1720 cm-1 (), which is a shoulder to the PLA ester peak. Spectrum (d)
shows the widening of the PHBHHx ester peak at 1721 cm-1 (), due to the overlapping
of the –C=O peak for the PHBHHx ester and the –C=O acid peak. Spectra (e-f) show the
–C=O amide peak at 1670 cm-1 (■).
51
Effect of reaction solvent on surface properties
Table 3.1 summarizes the static water contact angle values for surface modified
PLA and PHBHHx films using water and ethanol as the reaction solvent in Step 2. With
PAA, no significant difference in the water contact angle was observed when water or
ethanol was used as the reaction solvent. For PAAm, surface-modified films showed a
slightly lower water contact angle when ethanol was used as the reaction solvent. Figure
3.6 shows ATR-FTIR peak area ratio (PAR), viz. 1670 cm-1 –C=O amide peak area
normalized by 1756 cm-1 –C=O ester peak area for PLA or 1720 cm-1 –C=O ester peak
area for PHBHHx. Films surface-modified using ethanol as the reaction solvent in Step 2
showed slightly higher PAR values than the corresponding films surface-modified in
water. But this difference was not significant (some of the error bars overlap). In short,
the reaction solvent used in Step 2 had only a slight effect on the surface properties (static
water contact angle and ATR-FTIR peak area ratio).
Table 3.1 Water contact angles of neat and surface modified PLA and PHBHHx after 3 h
UV-exposure time and using water or ethanol as the reaction solvent in step 2
PLA (Ethanol) PLA (Water) PHBHHx (Ethanol) PHBHHx (Water)
Neat 82 ± 1 82 ± 1 76 ± 1 76 ± 1
Film-g-PAA 38 ± 2 41 ± 3 51 ± 3 48 ± 3
Film-g-PAAm 12 ± 2 17 ± 1 23 ± 2 28 ± 2
52
0
1
2
3
4
5
6
PLA-g-PAAmEthanol
PLA-g-PAAmWater
PHBHHx-g-PAAm Ethanol
PHBHHx-g-PAAm Water
PA
R
Figure 3.6 ATR-FTIR peak area ratio, viz. 1670 cm-1 –C=O amide peak area normalized
by 1756 cm-1 –C=O ester peak area for PLA or 1720 cm-1 –C=O ester peak area for
PHBHHx. Black bars represent films grafted with acrylamide after sonication for 5 min
in the respective solvents and white bars represent films grafted with acrylamide after
sonication for 5 min in the respective solvents plus microwave assisted extraction in
water at 95 °C for 1 h. The error bars represent 95% confidence intervals.
Effect of reaction solvent on bulk properties
Table 3.2 shows the various experiments designed to compare the effect of
surface modification on the bulk properties. Experiment 1 is the typical sequential, two-
step photografting process. Experiment 2 consisted of the same protocol as that of
53
experiment 1 except a film specimen was dip coated in ethanol instead of 5% w/w
benzophenone solution in ethanol in Step 1. The specimens prepared by experiment 2
were designated as “PAA Control” and “PAAm Control” in the subsequent figures.
Experiment 2 enabled us to study the effect of surface modification on bulk properties in
the absence of surface-confined photografting, since it omitted the benzophenone
photoinitiator. Experiment 3 involved the films subjected to the same UV treatment but
soaked only in the chosen solvent with specimens designated as “Solvent Control” in the
subsequent figures. Experiment 3 allowed us to study the effect of photografting solvent
on bulk properties in the absence of benzophenone and monomer, since the films were
subjected to the same UV treatment but soaked only in the chosen solvent.
Table 3.2 Design of the photografting experiments to evaluate the effect of surface
modification on mechanical properties of the PLA and PHBHHx films after 3 h UV-
exposure time in step 2
Expt. No. Step 1 Step 2 Nomenclature
1 + + Film-g-PAA/Film-g-PAAm
2 -α + PAA Control/PAAm Control
3 -α -γ Solvent Control
α Pure ethanol was used instead of 5% benzophenone solution γ Pure solvent was used instead of 10% monomer solution
54
PLA films
Figure 3.7a shows the Young’s modulii values for PLA films. The Young’s
modulii for the PLA-g-PAA and PLA-g-PAAm films were greater than that for the neat
film (experiment 1), particularly when ethanol was used as the solvent in Step 2 (black
bars). In order to investigate this behavior further, experiments 2 and 3 were designed to
study the effects of the chosen monomers and reaction solvent on the bulk properties.
PLA films subjected to control experiment 2 show Young’s modulii the same as that of
the corresponding PLA films subjected to experiment 1. This implied that omission of
benzophenone in Step 1 did not have a significant impact on the modulii. Morever, PLA
films subjected to the Solvent Control experiment (experiment 3) showed modulii
approximately the same as that of PLA films subjected to experiment 1. This result
indicated that the reaction solvent in Step 2 was the main variable to affect modulus.
55
(a) Modulus
0
50
100
150
200
250
300
Neat Film PLA-g-PAA
PLA-g-PAAm
SolventControl
PAAControl
PAAmControl
Mod
ulus
(M
Pa)
(b) Crystallinity
0
5
10
15
20
25
Neat Film PLA-g-PAA
PLA-g-PAAm
SolventControl
PAAControl
PAAmControl
% C
ryst
allin
ity
(c) Toughness
0102030405060708090
100
Neat Film PLA-g-PAA
PLA-g-PAAm
SolventControl
PAAControl
PAAmControl
Tou
ghne
ss (
MP
a)
(a) Modulus
0
50
100
150
200
250
300
Neat Film PLA-g-PAA
PLA-g-PAAm
SolventControl
PAAControl
PAAmControl
Mod
ulus
(M
Pa)
(b) Crystallinity
0
5
10
15
20
25
Neat Film PLA-g-PAA
PLA-g-PAAm
SolventControl
PAAControl
PAAmControl
% C
ryst
allin
ity
(c) Toughness
0102030405060708090
100
Neat Film PLA-g-PAA
PLA-g-PAAm
SolventControl
PAAControl
PAAmControl
Tou
ghne
ss (
MP
a)
Figure 3.7 Effect of the photografting reaction solvent on the (a) modulus, (b)
crystallinity, and (c) toughness of PLA films. Black bars indicate films prepared by
carrying out the reaction in ethanol and white bars indicate films prepared by carrying out
the reaction in water. The neat PLA film had very low % crystallinity such that the DSC
spectrum did not show a definite melting peak. The error bars represent 95% confidence
intervals. The toughness of PLA films grafted with PAA and PAAm using ethanol and
water as the reaction solvents showed statistically significant difference from the
toughness of neat PLA, as determined by a t-test.
56
Figure 3.7b shows the effect of the reaction variables on the % crystallinity of
PLA films. The neat PLA film had very low % crystallinity such that the DSC spectrum
did not show a definite melting peak. Compared to the neat PLA films, films prepared by
experiments 1, 2, and 3 all showed an increase in crystallinity to around 20% when
ethanol was used as the reaction solvent in Step 2 and 12% when water was used. These
experiments indicated that solvent-induced crystallinity occurred and, for a given solvent,
the presence of the monomer (acrylic acid or acrylamide) in the reaction solvent had little
influence on the crystallinity. Solvent-induced crystallization of PLA films was more
prevalent when ethanol was used as the reaction solvent in Step 2 than when water was
used. Compared to water, ethanol’s solubility parameter is closer to that of PLA (Table
3.3) and therefore exhibits a greater extent of penetration to promote PLA crystallization.
Table 3.3 Solubility parameters for PLA, PHBHHx, ethanol, and water. Solubility
parameters for PLA and PHBHHx were calculated based on Fedors cohesive energy and
molar volume values using group contribution method [30]
Chemical
Solubility parameter (MPa)0.5
PLA
22.8
PHBHHx
21.5
Ethanol
26.0 [31]
Water
47.9 [31]
57
The modulus of neat PLA film indicated in Figure 3.7a was much lower than
expected (typically reported as 1151 ± 10 MPa) [1], and we suspected that residual
solvent remained in the film. TGA analysis of the PLA film solvent cast from a
chloroform solution showed that the film retained around 13 weight percent of
chloroform. PLA film solvent cast in chloroform and then modified by experiments 1, 2,
or 3 showed an increased total solvent (chloroform or ethanol or both) content of around
3 to 6 weight percent when ethanol was used as the reaction solvent in Step 2.
Complementary experiments using water as the reaction solvent in Step 2 exhibited a
total weight increase of 9 to 11 weight percent (Figure 3.8a). These results indicated that
modified PLA films lost residual chloroform on crystallization, particularly with ethanol
that promoted a greater extent of PLA crystallization. Attempts were made to remove the
residual solvent by heating specimens at various temperatures under vacuum. That
methodology reduced the amount of residual solvent but significantly affected the
sample’s surface roughness, even at temperatures as low as 50 °C. The rough surfaces
were not suitable for surface modification and subsequent analysis. Our objective in this
study was to assess the effect of photografting steps on the film’s mechanical properties.
Even though the films contained residual solvent, the mechanical and surface properties
of surface-modified films were compared to those of neat (unmodified) film.
Figure 3.7c shows the toughness (as reflected by area under engineering stress -
strain curve) values for PLA films. The toughness shown by solvent cast neat PLA film is
the toughness of PLA with 13 weight percent residual chloroform. PLA films lost their
toughness significantly on surface modification when ethanol was used as the reaction
58
solvent in Step 2 (black bars). We attributed this toughness reduction to solvent-induced
crystallization and the loss of the residual chloroform in the modified films. When water
was used as the reaction solvent in Step 2, the toughness reduction was not as great. This
results from the fact that solvent-induced crystallization was less prevalent in water than
in ethanol and the solvent content in the modified films was higher when water was used
as the reaction solvent in Step 2.
86
88
90
92
94
96
98
100
25 50 75 100 125 150 175 200
Temperature (°C)
Wei
ght %
PLA pellets as received
PLA film surface modified in water
Solvent cast PLA film
PLA film surface modified in ethanol
(a)
96
97
98
99
100
25 50 75 100 125 150 175 200
Temperature (°C)
Wei
ght %
PHBHHx as received
Solvent cast PHBHHx film
PHBHHx film surface modified in water
PHBHHx film surface modified in ethanol
(b)
86
88
90
92
94
96
98
100
25 50 75 100 125 150 175 200
Temperature (°C)
Wei
ght %
PLA pellets as received
PLA film surface modified in water
Solvent cast PLA film
PLA film surface modified in ethanol
(a)86
88
90
92
94
96
98
100
25 50 75 100 125 150 175 200
Temperature (°C)
Wei
ght %
PLA pellets as received
PLA film surface modified in water
Solvent cast PLA film
PLA film surface modified in ethanol
(a)
96
97
98
99
100
25 50 75 100 125 150 175 200
Temperature (°C)
Wei
ght %
PHBHHx as received
Solvent cast PHBHHx film
PHBHHx film surface modified in water
PHBHHx film surface modified in ethanol
(b)
Figure 3.8 TGA curves of neat and surface modified (a) PLA and (b) PHBHHx. Note
that the y-axis scales are different on the two plots.
59
PHBHHx films
TGA analysis of the PHBHHx film solvent cast in chloroform showed that the
film retained around 1 weight percent of the chloroform. PHBHHx film (solvent cast in
chloroform) prepared by experiments 1, 2, and 3 showed the solvent (chloroform or
ethanol or both) content around 0.25 to 3 weight percent, when ethanol was used as the
reaction solvent in step 2. Complementary experiments using water as the reaction
solvent in Step 2 exhibited a weight gain of 0.25 to 2 weight percent (Figure 3.8b). In
either case, the PHBHHx films retained significantly less solvent than the PLA films.
For PHBHHx-film results in Figure 3.9a using ethanol as the reaction solvent
(black bars), a modulus increase was observed for PHBHHx-g-PAA compared to neat
film and an even greater increase for PHBHHx-g-PAAm. Referring to the crystallinity
data in Figure 3.9b, the % crystallinity for those same specimens remained the same, so
an increase in modulus must be a result of something other than crystallinity. We
speculated that monomer penetrated into the film and polymerized. PAA (Tg = 126 °C)
and PAAm (Tg = 188 °C) have high glass transition temperatures and hence they are
glassy at room temperature [29]. If there was significant penetration of these monomers
and subsequent polymerization within the films, we speculate that those chains could
contribute to an increase in stiffness. PHBHHx films subjected to experiments 1, 2, and 3
using water as the reaction solvent in Step 2 exhibited solvent-induced crystallization,
and those specimens also exhibited an increase in modulus (Figure 3.9a, white bars).
Most noteworthy, PHBHHx film immersed only in water (Solvent Control) showed
60
approximately same modulus as those for modified PHBHHx films, inferring that the
observed modulus increases were due to solvent-induced crystallization during these
experiments.
(a) Modulus
0
50
100
150
200
250
300
Neat Film PHBHHx-g-PAA
PHBHHx-g-PAAm
SolventControl
PAAControl
PAAmControl
Mod
ulus
(M
Pa)
(b) Crystallinity
0
5
10
15
20
25
Neat Film PHBHHx-g-PAA
PHBHHx-g-PAAm
SolventControl
PAAControl
PAAmControl
% C
ryst
allin
ity
(c) Toughness
0
20
40
60
80
100
120
Neat Film PHBHHx-g-PAA
PHBHHx-g-PAAm
SolventControl
PAAControl
PAAmControl
Tou
ghne
ss (
MPa
)
(a) Modulus
0
50
100
150
200
250
300
Neat Film PHBHHx-g-PAA
PHBHHx-g-PAAm
SolventControl
PAAControl
PAAmControl
Mod
ulus
(M
Pa)
(b) Crystallinity
0
5
10
15
20
25
Neat Film PHBHHx-g-PAA
PHBHHx-g-PAAm
SolventControl
PAAControl
PAAmControl
% C
ryst
allin
ity
(c) Toughness
0
20
40
60
80
100
120
Neat Film PHBHHx-g-PAA
PHBHHx-g-PAAm
SolventControl
PAAControl
PAAmControl
Tou
ghne
ss (
MPa
)
Figure 3.9 Effect of the photografting reaction solvent on the (a) modulus, (b)
crystallinity, and (c) toughness of PHBHHx films. Black bars indicate films prepared by
carrying out the reaction in ethanol and white bars indicate films prepared by carrying out
the reaction in water. The error bars represent 95% confidence intervals. The toughness
of PHBHHx films grafted with PAA and PAAm using ethanol as the reaction solvent
showed statistically significant difference from the toughness of neat PHBHHx, as
determined by a t-test.
61
The penetration of acrylamide into the bulk of the surface-modified films was
characterized by first microtoming the films to expose their cross-sections, which were
then analyzed using transmission FTIR microspectroscopy. Acrylamide was used because
of its distinct –C=O amide I peak, whereas acrylic acid did not show a distinct peak.
Figure 3.10 represents the transmission FTIR peak area ratio, viz. 1670 cm-1 –C=O amide
peak area normalized by 1452 cm-1 asymmetric –CH3 band peak area. Based on their
chemical structures, the concentration of CH3 groups (number of CH3 groups per unit
volume) is greater in PLA than PHBHHx, so we multiplied the denominator of
PHBHHx’s PAR by 1.36 to be able to compare results as a common basis (see Figure
3.1). In Figure 3.10, the circles represent the standard 2-step photografting process in
ethanol, while the squares represent the case where the benzophenone was omitted from
Step 1 (PAAm Control). A 25-micron-wide beam was used for analysis at five discrete
locations across the 125 micron thickness of the film. Comparing Figures 3.10a and
3.10b, there was significantly more acrylamide penetration into PHBHHx than PLA,
likely due to the lower crystallinity of the PHBHHx coupled with its low Tg (about -4 °C
for PHBHHx vs. 16 °C for PLA as determined by DMA for these solvent-cast films). The
PAR profiles in PLA are equivalent for PLA-g-PAAm (circles) and PAAm Control
(squares). The same is true for the modified PHBHHx films except at analysis positions 1
and 5, where the PARs for PHBHHx-g-PAAm are greater than that for PAAm Control in
Figure 3.10b. The reason for this accentuated PAR in the near-surface regions at
positions 1 and 5 is unknown at this point, but it appears to be statistically different
compared to the results for PAAm Control.
62
(a) PLA
0
1
2
3
1 2 3 4
Analysis Position
PA
R
5
(b) PHBHHx
0
1
2
3
1 2 3 4 5
Analysis Position
PA
R
(a) PLA
0
1
2
3
1 2 3 4
Analysis Position
PA
R
5
(b) PHBHHx
0
1
2
3
1 2 3 4 5
Analysis Position
PA
R
(a) PLA
0
1
2
3
1 2 3 4
Analysis Position
PA
R
5
(b) PHBHHx
0
1
2
3
1 2 3 4 5
Analysis Position
PA
R
Figure 3.10 Transmission FTIR peak area ratio, viz. 1670cm-1 –C=O amide peak area
normalized by 1452 cm-1 asymmetric –CH3 band peak area, as a function of analysis
position into the bulk of PLA or PHBHHx films grafted with benzophenone in step 1 and
then immersed in 10% v/v acrylamide solution and exposed to UV for 3 h (○), or dip
coated in ethanol and exposed each side for 5 min in step 1 then immersed in 10% v/v
acrylamide solution and exposed to UV for 3 h (■). The IR beam width at each analysis
position was 25 microns.
63
Referring back to Figure 3.9b, the crystallinities of neat PHBHHx, PHBHHx-g-
PAAm, and PAAm Control were nearly identical, yet the modulus of neat PHBHHx was
dramatically lower than PHBHHx-g-PAAm and PAAm Control (Figure 3.9a). This
behavior is consistent with the hypothesis that acrylamide penetrated into the PHBHHx,
as shown in Figure 3.10b, and polymerized to yield glassy PAAm chains that increased
the stiffness of the modified PHBHHx films. In the case of PLA, there was only slight
acrylamide penetration, but more substantial change in crystallinity to lead to increased
stiffness for those modified PLA films. Finally, complementary experiments were
conducted in water in Step 2 and very little amide was detected through the cross-section
of the films.
These results are also reflected in the toughness data for PHBHHx films (Figure
3.9c). Films modified using ethanol in Step 2 (black bars) showed low toughness values
coinciding with increased stiffness. PHBHHx films showed a greater toughness retention
when reactions were carried out in water because there was little monomer penetration as
detected by transmission FTIR microspectroscopy into the bulk of the films. There was
also no significant change in the amount of residual solvent in these PHBHHx films,
leading to a less significant impact on film toughness.
Finally, we did not observe significant changes in the molecular weights of PLA
or PHBHHx films upon surface modification as evidenced by gel permeation
chromatography (GPC).
64
CONCLUSIONS
PLA and PHBHHx were successfully surface modified using sequential, two step
photografting. Although the reaction solvent used in Step 2 did not have any significant
effect on surface properties, bulk properties were significantly affected. When ethanol
was used as the reaction solvent in Step 2, PLA and PHBHHx films drastically lost their
toughness and became stiffer. Morever, there was significant acrylamide penetration into
PLA and PHBHHx films when reactions were carried out in ethanol and the extent was
greater into PHBHHx films. When water was used as the reaction solvent, transmission
FTIR microspectroscopic analyses revealed very little acrylamide penetration into the
bulk of these films. Solvent-induced crystallization was more prevalent when ethanol was
used as the reaction solvent than when water was used as the reaction solvent for PLA
films. The observed toughness loss and modulus gain of PLA films on surface
modification was attributed to solvent-induced crystallization and loss of residual
chloroform. The presence of residual chloroform in the film specimens is undesirable
from a biocompatibility standpoint. Additionally, this work showed that the photoreaction
solvent affected the bulk properties of PLA and PHBHHx films cast from a chloroform
solution. Therefore, further work is being conducted on melt-processed films where
residual solvent from the film-formation method will not be an issue.
65
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12. Steinbüchel A, Lütke-Eversloh T. Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms. Biochem Eng J 2003;16:81-96.
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24. Janorkar AV, Metters AT, Hirt DE. Modification of poly(lactic acid) films: enhanced wettability from surface-confined photografting and increased degradation rate due to an artifact of the photografting process. Macromolecules 2004;37:9151-59.
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68
CHAPTER FOUR
TOUGHNESS DECREASE OF PLA-PHBHHX BLEND FILMS UPON
SURFACE-CONFINED PHOTOPOLYMERIZATION
[As published in Journal of Biomedical Materials Research Part A, DOI:
10.1002/jbm.a.32009 with minor changes]
INTRODUCTION
PLA is an extensively studied biodegradable thermoplastic polyester derived from
renewable resources, showing great potential in consumer and biomedical fields [1-9].
However, wide applicability of PLA is limited by several factors, including its brittleness
and therefore poor toughness and, like many polymers, its hydrophobicity and lack of
modifiable side chain groups. PLA has been blended with other polymers to improve the
toughness of the resultant blend [9-17]. For example, addition of a small amount of
PHBHHx to PLA has yielded a much tougher material compared with neat PLA [15].
PHBHHx belongs to a bacterially produced homopolymer or copolymer family referred
to as polyhydroxyalkanoates (PHAs) [18]. PHBHHx is a biodegradable aliphatic
polyester that tends not to crystallize when a small amount (typically less than 20 weight
%) is blended with PLA, markedly improving the toughness of the resultant blend
without losing optical clarity [15, 19]. PHAs have been investigated recently as potential
biomaterials [20-24]. However, their comparatively higher production costs, thermal
69
degradation during melt processing, and complexities involved in film casting by
extrusion limit their wide applicability [25-26].
One of the major emphases of our research is to modify the surface properties of
PLA-PHBHHx films using photografting [27-29]. In the surface modification process, the
films are typically immersed in various solvents. Therefore, one goal was to determine
the influence of solvent immersion on film toughness. Morever, we investigated blend
films that were not subjected to the surface-modification process (i.e., no immersion in
solvents) to assess the extent of physical aging on toughness reduction. Similar physical
aging has been reported for PLA and its blends [30-32]. In this chapter we report, for the
first time, toughness changes of PLA-PHBHHx blend films that are specifically
associated with physical aging and more importantly for this work, UV-assisted solvent
induced crystallization.
METHODS
Film Casting: PLA pellets were vaccum heated at 70 °C for 24 h and cooled in the
vaccum oven to remove any residual moisture. PHBHHx powder was used as received.
PLA, PHBHHx, and PLA-PHBHHx blend films (90 weight percent PLA) were formed
using a single screw extruder. The temperature profile of the extruder for PLA and PLA-
PHBHHx blend film casting was as follows: Zone 1 (located near the hopper) – 180 °C,
zone 2 – 190 °C, zone 3 – 200 °C, pump – 200 °C, and die – 190 °C. The temperature
profile of the extruder for PHBHHx film casting was as fallows: Zone 1 – 135 °C, zone 2
70
– 155 °C, zone 3 – 155 °C, pump – 165 °C, and die – 155 °C. Molten polymer exiting the
die was cooled on a chill roll.
Sequential two-step photografting: PLA-PHBHHx blend films were surface modified
using a sequential two-step photografting approach discussed in detail in Chapter 3.
Briefly, this method consists of photografting benzophenone which preferentially
abstracts tertiary hydrogen atoms in Step 1 [33], and photopolymerizing acrylic acid or
acrylamide from the film surface in Step 2. The reaction was carried out at room
temperature. We chose acrylic acid and acrylamide as monomers because of their ability
to improve hydrophilicity upon photopolymerization [28] and acrylic acid could be
subsequently conjugated with various biomolecules depending on the intended
application of the surface modified films.
Mechanical Testing: The film samples with a nominal thickness of 125 ± 10 μm were
kept in a vaccum oven at room temperature for 24 h and subsequently annealed at 60 °C
for 30 min before mechanical testing. An Applied Test System Inc. (ATS) mechanical
tester was used to measure mechanical properties of the film samples (3 cm x 1 cm x 125
μm) according to American Society for Testing and Materials Standard (ASTM D882)
specifications. A cross-head speed of 2.5 cm/min was used. The measurement values
averaged for five specimens with ±95% confidence intervals are reported.
71
RESULTS AND DISCUSSION
This research focused on adding a small amount of PHBHHx (10 weight percent)
to PLA with an ultimate aim of making tougher films and surface-modifying the blend
films to increase their wettability. Figure 4.1 shows an engineering stress-strain curve for
neat PLA and PLA-PHBHHx blend films, tested within 1 h after extrusion. The greater
toughness for PLA-PHBHHx blend film, as reflected by the area under the curve, was
governed by improvement in % elongation at break, from 11 ± 3 % for neat PLA to 360 ±
75 % for the blend.
0
10
20
30
40
50
60
70
80
0 1 2 3 4 5
Strain
Stre
ss (
MPa
)
Figure 4.1 Engineering stress-strain curve of extruded PLA (♦) and PLA-PHBHHx (90
weight percent PLA) blend (■) films.
72
Temperature (°C)
60 -40 -20 0 20 40 60 80 100 120
tan
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Temperature (°C)
60 -50 -40 -30 -20 -10 0 10
tan
0.000
0.005
0.010
0.015
0.020
0.025
(B)(a)
(b)
(c)
(d)
Temperature (°C)
60 -40 -20 0 20 40 60 80 100 120
tan
0.0
0.2
0.4
0.6
0.8
1.0
1.2(A)
Temperature (°C)
60 -40 -20 0 20 40 60 80 100 120
tan
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Temperature (°C)
60 -50 -40 -30 -20 -10 0 10
tan
0.000
0.005
0.010
0.015
0.020
0.025
(B)(a)
(b)
(c)
(d)
Temperature (°C)
60 -40 -20 0 20 40 60 80 100 120
tan
0.0
0.2
0.4
0.6
0.8
1.0
1.2(A)
Figure 4.2 (A) Tan δ as a function of temperature for extruded PLA-PHBHHx blend film
(90 weight percent PLA). (B) Tan δ as function of temperature of (a) unmodified blend,
(b) water control, (c) Blend-g-PAAm, and (d) Blend-g-PAA.
Figure 4.2A shows tan δ as a function of temperature of the blend film as
characterized using DMA. The peak temperature of tan δ is used to denote the Tg. DMA
revealed two well-defined tan δ peaks, denoting two different glass transition
73
temperatures at 60 and -18 °C, corresponding to the PLA and PHBHHx blend
components, respectively. From this result, the PLA-PHBHHx blend appeared to be non-
compatible, which is consistent with results reported previously by Furukawa and
coworkers [34].
Toughening mechanism
Optical clarity of extruded PLA-PHBHHx blend films suggested that either
PHBHHx (lower Tg component) crystallites were sufficiently small that they could not
scatter light, or they were not present in the PLA matrix. This inability of PHBHHx to
fully crystallize when melt blended with PLA at a low level was suggested as an
explanation for the increase in toughness of the PLA-PHBHHx blend by Noda and
coworkers [15]. WAXD patterns of blend films used in this study showed a reflection
peak corresponding to PHBHHx at 13.4° [34], implying that PHBHHx crystallized to
some extent, but not sufficient enough to scatter light (Figure 4.3).
74
2
10 15 20 25 30 35
Inte
nsity
(C
PS)
0
200
400
600
800
1000
1200
1400
(a)
(b)
(c)
(d)
2
10 15 20 25 30 35
Inte
nsity
(C
PS)
0
200
400
600
800
1000
1200
1400
(a)
(b)
(c)
(d)
Figure 4.3 WAXD patterns of (a) unmodified blend, (b) blend-g-PAA, (c) blend-g-
PAAm, and (d) blend films prepared by water-control experiments.
However, one of the serious drawbacks of PLA-based blends is that the PLA
phase tends to undergo physical aging, affecting their mechanical properties significantly
[31]. Figure 4.4 shows the effect of physical aging on the toughness of our extruded
blend films, where the toughness decreased as the room-temperature aging time
increased. Typically, the amorphous phase in glassy or partially glassy polymers
undergoes physical aging that usually occurs around its glass transition temperature [35-
75
36]. Physical aging involves completely reversible spontaneous changes in the
thermodynamic state of a polymer [31]. Blend films used here were cooled on a chill roll
from the melt. Since the temperature was below PLA’s Tg (major component), its
molecular chains became frozen. The polymer was in a non-equilibrium state, having
large volume, enthalpy, and entropy [31, 35]. Since room temperature was below Tg, the
free volume would tend to reduce spontaneously towards a thermodynamic equilibrium,
resulting in an enthalpic relaxation [31, 35].
0
50
100
150
200
250
Day 0 Day 1 Day 3 Annealed Day 1
Tou
ghne
ss (
MP
a)
Figure 4.4 Effect of the physical aging on toughness of extruded and annealed (at 60 °C
for 30 min) blend films. The error bars represent 95% confidence intervals.
Since physical aging was an enthalpic relaxation process, occurring around Tg,
DSC was used to monitor physical aging in the blend films. Figure 4.5 shows the effect
76
of physical aging on the glass transition event in DSC scans. A peak corresponding to the
excess enthalpy of relaxation of the blend can be observed at its Tg (Figure 4.5b). For
samples annealed at 60 °C for 30 min, this excess enthapic relaxation peak disappeared
(Figure 4.5c). Samples tested 1 day post annealing again showed the same enthapic
relaxation peak (Figure 4.5d). These results indicated that the PLA component underwent
rapid physical aging after extrusion. On annealing slightly above Tg, there was not any
significant physical aging evidence as characterized by DSC. Annealed blend samples
also underwent a rapid physical aging. The endothermic enthalpic relaxation peak around
Tg in DSC scans for physically aged samples (Figure 4.5b and 4.5d) indicated a reduction
in free volume. This reduction in free volume with physical aging would tend to reduce
molecular mobility, and hence the toughness. Wang and coworkers reported a similar
observation for loss of mechanical properties of PLA-starch blends due to physical aging
and attributed it to the loss of interfacial interaction between two phases [31]. The loss of
interfacial attraction between two phases was assigned to the shrinking of the PLA phase
on physical aging [31]. For our case, the similar interfacial attraction loss might be the
reason for blend toughness loss on physical aging, but that hypothesis could not be
supported based on morphological studies as the PHBHHx (minor component)
composition was low (10 weight percent).
77
Temperature (°C)
56 58 60 62 64
Hea
t flo
w (
w/g
)
2
3
4
5
6
7
8
9
(a)
(b)
(c)
(d)Endo
Temperature (°C)
56 58 60 62 64
Hea
t flo
w (
w/g
)
2
3
4
5
6
7
8
9
(a)
(b)
(c)
(d)Endo
Figure 4.5 DSC thermograms of PLA-PHBHHx (90 weight percent PLA) blend films (a)
right after extrusion, (b) aged for 1 day, (c) after annealing at 60 °C for 30 min, and (d) 1
day post annealing.
As shown in Figure 4.4, physically aged blend samples regained the original
toughness temporarily on annealing slightly above Tg (60 °C for 30 min). Physical aging
being a thermodynamically reversible process, annealing at 60 °C for 30 min led the
blend films to regain their original toughness. Since the main objective of this research
78
was to determine whether our surface modification process influenced the film’s
mechanical properties, all samples were annealed at 60 °C for 30 min just prior to testing
to minimize effects of physical aging. Annealing conditions were set in such a way that
there was not any significant crystallization of the blend films as characterized using
WAXD patterns. This enabled us to study solely the effect of photografting reactions on
blend mechanical properties.
Surface modification
Table 4.1 shows the water contact angle values for unmodified and surface
modified PLA and blend films after 3 h UV exposure in Step 2 of the surface-
modification process. Unmodified PLA and blend films were hydrophobic, with water
contact angle ~ 80°. Films grafted with poly(acrylic acid) (PAA) showed water contact
angle ~ 38° and grafted with poly(acrylamide) (PAAm) showed water contact angle ~
23°.
Table 4.1 Water contact angles of unmodified and surface-modified PLA and PLA-
PHBHHx blend (90 weight percent PLA) films after 3 h UV-exposure time in Step 2
Water Contact Angle (°) PLA Blend
Unmodified 81 ± 2 77 ± 1 Film-g-PAA 37 ± 4 38 ± 3
Film-g-PAAm 22 ± 2 23 ± 3
79
This surface chemistry was further investigated using ATR-FTIR spectroscopy.
The unmodified blend film showed the –C=O peak at wave number 1747 cm-1
corresponding to the PLA ester. The blend film grafted with PAA showed a shoulder at
wave number 1722 cm-1 corresponding to the –C=O acid peak. The blend film grafted
with PAAm showed the –C=O peak at wave number 1670 cm-1 corresponding to the
amide functionality of PAAm and an amide II peak at wave number 1550 cm-1, which is
the combination of N-H bending and C-N stretching vibrational modes (spectra not
shown). These peaks confirm the presence of the expected functional groups consistent
with the photografting chemistry.
Effect of the photografting reaction on bulk properties
Figure 4.6 shows the engineering stress-strain results for unmodified and surface-
modified blend films (after annealing). These films lost their toughness significantly on
surface modification due to the reduction in % elongation at break upon surface
modification (Figure 4.6). Figure 4.7c summarizes the toughness data for unmodified and
surface modified blend films. To investigate this toughness loss further, a control
experiment was performed where unmodified blend films were subjected to the same
sequential two-step photografting method, except in Step 1 the films were dip coated in
pure ethanol (omitting the benzophenone), and in Step 2, the films were immersed in pure
water (omitting the monomer) and the samples were exposed to the same UV irradiation.
These samples are referred to as “Water Control” in Figure 4.7. The water-control
80
specimens showed the same toughness loss as blend-g-PAA and blend-g-PAAm (where g
denotes grafted). Since all films were annealed at 60 °C for 30 min before tensile testing,
the observed toughness loss was attributed primarily to the surface-modification process
and not due to physical aging. DMA analyses of unmodified and surface-modified films
showed that that tan δ peak height decreased upon surface modification (Figure 4.2B).
These DMA results suggested that the blend films might be undergoing crystallization
during the photografting reaction.
0
10
20
30
40
50
60
0 1 2 3 4 5
Strain
Str
ess
(MP
a)
Figure 4.6 Engineering stress-strain curve of unmodified (■) and surface-modified PLA-
PHBHHx (90 weight percent PLA) blend (♦) films (after annealing).
81
Unmodified
Blend
You
ng's
mod
ulus
(M
Pa)
0
200
400
600
800
1000
1200
1400
Blend-g-PAA Blend-g-PAAm Water Control Unmodified Blend
UT
S (M
Pa)
0
10
20
30
40
50
60
70
Blend-g-PAA Blend-g-PAAm Water Control
Unmodified Blend
Tou
ghne
ss (
MP
a)
0
50
100
150
200
250
Blend-g-PAA Blend-g-PAAm Water Control
(a) (b)
(c)
Unmodified
Blend
You
ng's
mod
ulus
(M
Pa)
0
200
400
600
800
1000
1200
1400
Blend-g-PAA Blend-g-PAAm Water Control Unmodified Blend
UT
S (M
Pa)
0
10
20
30
40
50
60
70
Blend-g-PAA Blend-g-PAAm Water Control
Unmodified Blend
Tou
ghne
ss (
MP
a)
0
50
100
150
200
250
Blend-g-PAA Blend-g-PAAm Water Control
(a) (b)
(c)
Figure 4.7 Effect of the photografting on (a) modulus, (b) ultimate tensile strength, and
(c) toughness of PLA-PHBHHx (90 weight percent PLA) blend films. The error bars
represent 95% confidence intervals. The toughness of blend films grafted with PAA and
PAAm showed statistically significant difference from the toughness of unmodified
blend, as determined by a t-test.
82
To analyze this behavior in detail, % crystallinity of unmodified and surface-
modified blend films was measured using WAXD (Figure 4.3). Unmodified blend
(Figure 4.3a) showed a reflection peak at 13.5°. Blend-g-PAA, blend-g-PAAm, and blend
films prepared by water-control experiments (Figure 4.3b, 4.3c, and 4.3d) showed a new
reflection peak at 16.7°, indicating crystallization during photografting reactions. Figure
4.8 summarizes the % crystallinity values for unmodified and surface-modified blend
films as calculated from WAXD patterns. It was observed that % crystallinity of blend
films almost doubled during photografting reactions. To investigate the cause of the
observed crystallization during photografting, two other control experiments were
performed. In the first, blend films were immersed in water for 3 h without UV exposure
and WAXD analyses of resultant films did not show a significant crystallinity increase
(labeled “Only Water” in Figure 4.8). In another control experiment, films were exposed
to 3 h of UV treatment without solvent, monomer, and initiator (labeled “Only UV”) and
no significant crystallinity increase was observed. These observations led us to infer that
the crystallinity increase was a combined effect of reaction solvent (water) and UV
irradiation, hereafter referred to as “UV-assisted solvent induced crystallization”
(UVasic).
83
0
1
2
3
4
5
6
7
8
UnmodifiedBlend
Blend-g-PAA Blend-g-PAAm Water Control Only Water Only UV
% C
ryst
alli
nity
Figure 4.8 Effect of the photografting reaction on % crystallinity of PLA-PHBHHx (90
weight percent PLA) blend films. The error bars represent 95% confidence intervals.
To investigate this behavior further, the temperature of reaction solvent (water)
being exposed to UV irradiation was measured with time. It was observed that the UV
irradiation heated the water from room temperature (25 °C) to 35 °C (Figure 4.9). In
addition to this, DMA analyses indicated that water plasticized the blend films and
increased the chain mobility. The tan δ vs. temperature curve for the water control
showed the onset of chain mobility around -25 °C and significant chain mobility at 35 °C
84
(Figure 4.10). Blend films immersed in water for 3 h without UV exposure did not
undergo any significant crystallization even if there was some extent of chain mobility
detected by DMA. DMA did not reveal the onset of significant chain mobility of
unmodified blend films in the temperature range investigated. This may be the reason
blend films exposed to UV irradiation for 3 h (no water) did not undergo any significant
crystallization. The water plasticization in conjunction with the UV heating was more
likely the mechanism behind UVasic. Blend films did not show any significant change in
molecular weight on surface modification as characterized using gel permeation
chromatography (GPC). So the observed reduction in the toughness of the blend films on
surface modification was likely due to UVasic. WAXD patterns of unmodified PLA film
did not show any reflection peaks, a typical characteristic of the amorphous matrix.
WAXD patterns of surface-modified PLA films also did not show any reflection peaks,
indicating little, if any, UVasic during photografting (spectra not shown). Since
unmodified blend films underwent UVasic during photografting and PLA films did not,
PHBHHx may have acted as nucleating sites for PLA-phase crystallization. It is also
possible that the PHBHHx crystallizes. A WAXD spectrum for unmodified extruded
PHBHHX film showed reflection peaks at 13.5° and 16.9° (spectrum not shown). A
reflection peak for PLA cast from a hot chloroform solution was observed at 16.7° [34].
In short, both PLA and PHBHHx show a reflection peak at approximately 16.7°, so the
new peak at 16.7° may result from PLA-phase crystallization, PHBHHx-phase
crystallization, or both.
85
25
27
29
31
33
35
37
39
0 0.5 1 1.5 2 2.5 3
Time (h)
Tem
pera
ture
(°C
)
Figure 4.9 Effect of the UV irradiation on temperature of reaction solvent (water) with
time. The error bars represent 95% confidence intervals.
Crystallinity developed during photografting reactions may have important
ramifications with respect to biomedical applications of these films. First, the surface-
reacted films lose their toughness significantly. Moreover, PLA (major phase) degrades
through the hydrolysis of backbone ester groups [28], and it has been reported that the
hydrolysis selectively occurs in the amorphous regions of PLA [37-38]. So the
crystallinity developed may affect the PLA degradation rate.
86
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
-50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35
Temperature (°C)
Tan
δ
Unmodified Blend
Water Control
Figure 4.10 Tan δ as function of temperature for the unmodified blend and water control.
For blend films undergoing UVasic during photografting, one would expect
surface-modified films to be more brittle, reflected by an increase in modulus.
Surprisingly, modulus did not change significantly upon surface modification (Figure
4.7a). A similar effect was observed for ultimate tensile strength of unmodified and
surface-modified films (Figure 4.7b). To investigate this behavior further,
87
thermogravimetric analyses were performed. The mass retention increased from 0.21 ±
0.18 weight percent for unmodified blend films to around 1.70 ± 0.30 weight percent for
surface-modified films. Blend films prepared by water control experiments showed the
mass retention of 0.70 ± 0.29 weight percent. This mass retention on surface modification
was more likely due to retention of the reaction solvent (water), which could act as a
plasticizer. In light of these two counteracting effects, UVasic tending to increase
modulus and ultimate tensile strength and “water retention” tending to reduce modulus
and ultimate tensile strength, there was not any significant change in these mechanical
properties on surface modification.
CONCLUSIONS
The PLA and PHBHHx components of melt processed blend films appeared to
be non-compatible based on DMA analyses. Unmodified blend films were tougher (tested
right after extrusion), but lost their toughness significantly due to physical aging.
Physically aged films regained their toughness temporarily on annealing at 60 °C for 30
min.
The PLA-PHBHHx blend films were successfully surface modified using a
sequential two-step photografting approach, and the effect of photografting on
mechanical properties of blend films was studied extensively. Contribution of physical
aging to the change in mechanical properties was minimized by annealing films at 60 °C
for 30 min before testing. It was observed that blend films lost their toughness
88
significantly due to UV-assisted solvent induced crystallization. TGA data showed the
presence of reaction solvent in surface-modified films. The combination of “UV-assisted
solvent induced crystallization” and the plasticizing effect of residual water resulted in
insignificant changes in Young’s modulus and tensile strength on surface modification.
This study showed that PLA-PHBHHx blend films lost their toughness due to 1) physical
aging and 2) UV-assisted solvent induced crystallization occurring during the
photografting process.
89
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93
CHAPTER FIVE
POLY(LACTIC ACID) TOUGHENING WITH A
BETTER BALANCE OF PROPERTIES
INTRODUCTION
The market for renewable-resource-derived, biodegradable polymers is growing
due to environmental concerns and sustainability issues associated with petroleum-based
polymers [1-2]. PLA is a renewably derived (from corn starch, sugar, etc.),
biodegradable, and bioabsorbable thermoplastic polyester that exhibits excellent
processibility and biocompatibility and requires 25-55% less energy to produce than
petroleum-based polymers [3-5]. However, its use in certain applications has been limited
by its poor toughness (less than 10% elongation at break) and lack of readily reactable
functional groups [6].
PLA has been toughened using a variety of plasticizers, stereochemical and
processing manipulations, and biodegradable as well as nonbiodegradable rubbery (low
Tg) polymers [7]. These approaches often lead to significant stiffness (modulus) loss,
making resultant formulations unsuitable for certain applications. Reactive groups have
also been introduced onto PLA to create bioactive surfaces for biomedical applications
and tailored surfaces for commodity applications (e.g., friction modification, anti-
fogging, and adhesion). However, the solvents and reagents involved in these surface-
modification protocols often affect PLA bulk properties, especially toughness [6, 8].
94
Here we report a novel reactive-blending approach that involves a combination of
polymers with complementary properties, PAA and PEG, to achieve PLA toughening
without significant modulus or ultimate tensile strength (UTS) losses. In addition, this
technology introduces into the PLA matrix a controlled concentration of reactive acid
groups that can be readily conjugated with a variety of biomolecules containing amine or
alcohol groups using carbodiimide [9-10], thionyl chloride [11], or phosphorous
pentachloride [12] chemistry. Morever, PAA is known to accelerate PLA hydrolytic
degradation rate [13], which may be an advantage in certain applications. PAA was
chosen as a stiffening agent due to its glassy nature. PEG was chosen as a toughening
agent due to its rubbery nature. The resultant reactive blends were extruded into films and
analyzed using tensile testing, DSC, DMA, and toluidine-blue-dye staining.
MATERIALS
PLA pellets (Mn ~ 110 kDa) were supplied by NatureWorks LLC. Acrylic acid
(99.5% w/w) was obtained from Acros Organics and used as received without further
purification. PEG (Mn ~ 1500 Da) was obtained from Sigma. Chloroform was purchased
from VWR. Benzoyl peroxide (BPO) was obtained from Fluka.
95
METHODS
PLA reactive blending: As shown in Scheme 5.1, a predetermined amount of PLA was
dissolved in 140 mL CHCl3 at 100 °C for 1 h followed by addition of predetermined
amounts of BPO and acrylic acid. The solution was allowed to stand at 100 °C for 10 min
while the acrylic acid polymerized off the PLA backbone (PLA-g-PAA). PEG was then
added to the solution and kept at 100 °C for an additional hour. The solution was then
cooled to room temperature and poured in a glass dish. The solution was kept at room
temperature overnight and then transferred to a vacuum oven at 70 °C for 24 h and cooled
in the vacuum oven to remove any residual chloroform.
+BPO
Chloroform, 100 °C, 10 minPLA Acrylic acid PLA-g-PAA
PEG1500
Chloroform, 100 °C, 1hPLA-g-PAA-g/PEG Extrusion
Vacuum
70 °C, 24h+
BPO
Chloroform, 100 °C, 10 minPLA Acrylic acid PLA-g-PAA
PEG1500
Chloroform, 100 °C, 1hPLA-g-PAA-g/PEG Extrusion
Vacuum
70 °C, 24h
Scheme 5.1 Reactive blending approach consisting of thermal polymerization of acrylic
acid from PLA chains followed by PEG blending.
Film Extrusion: The polymer blend was immediately transferred to an extruder after
drying. A twin-screw microextruder (DSM Xplore) operating in a co-rotating mode was
used to cast films at 190 °C. The tapered screws were 170 mm long and the barrel
volume was 15 cm3. The polymer melt exiting the die was cooled by a stream of nitrogen
gas and collected on a chill roll. The resultant films had a nominal thickness 80 ± 10 μm.
96
Mechanical Testing: The film samples were stored at room temperature after extrusion
for 24 h before mechanical testing. The mechanical properties of the film samples (7.5
cm x 1.5 cm x 80 μm) were measured using an Applied Test System Inc. (ATS)
mechanical tester according to American Society for Testing and Materials Standard
(ASTM D882) specifications. A cross-head speed of 1.25 cm/min was used. The
measured values averaged for five specimens with ±95% confidence intervals are
reported.
RESULTS AND DISCUSSION
Scheme 5.1 represents the PLA reactive blending approach consisting of thermal
polymerization of acrylic acid from PLA chains followed by PEG blending. This
technology offers PLA toughening with a better balance of properties associated with
introduction of reactive acid groups into the PLA matrix. Briefly, PLA was
thermopolymerized with acrylic acid using benzoyl peroxide (BPO) thermal initiator
followed by blending with PEG in chloroform. The resultant blend was dried and
extruded using a twin-screw extruder operated in a co-rotating mode. Miscibility and
crystallization behavior of the films prepared using this chemistry were evaluated using
DMA and DSC, respectively (Figure 5.1). We first examined the miscibility of PLA/PEG
blends. Blend miscibility is governed mainly by molecular weight and composition of the
constituents. Higher molecular weight and concentration of PEG showed a tendency for it
to phase separate, so relatively lower molecular weight PEG (Mn ~ 1500 Da) at a
97
composition of 10% was used to blend with PLA, hereafter referred to as
PLA/PEG(10%). PLA/PEG(10%) blends did not undergo any significant phase
separation as characterized using DMA (Figure 5.1A (d)). Tan δ vs. temperature for
PLA/PEG(10%) showed only one peak corresponding to PLA’s Tg. When PLA was
thermopolymerized with 3 or 10 wt% acrylic acid prior to blending with PEG, hereafter
referred to as PLA-g-PAA(3%)/PEG(10%) (Figure 5.1A (b)) or PLA-g-
PAA(10%)/PEG(10%) (Figure 5.1A (c)), a tan δ peak corresponding to the PEG phase
was observed. This observation indicated that the PEG phase showed a phase separation
tendency when blended with PLA-g-PAA (‘g’ denotes grafted). When the PAA
concentration was increased from 3 to 10 wt%, the tan δ peak (Tg) corresponding to the
PEG phase shifted from -47 ± 2.6 °C to -32 ± 2.6 °C. Additionally, Tg corresponding to
the PLA phase increased from 43 ± 2.1 °C to 48 ± 1.7 °C. These Tg shifts with
composition indicated the partial miscibility of blend constituents. PLA is hydrophobic
while PAA and PEG are hydrophilic. These observations also showed the possibility of
favorable intermolecular interactions between PAA and PEG (as indicated by PEG’s Tg
shift with PAA concentration associated with phase separation) and between PAA and
PLA (as indicated by PLA’s Tg shift with PAA concentration).
98
0
4
8
12
16
20
0 50 100 150 200
Temperature (°C)
Hea
tflo
w(m
W)
(d)
(c)
(b)
(a)
Exo up
Tc = 129 ± 1
Tc = 104 ± 3
Tc = 108 ± 1
Tc = 93 ± 2
(B)
0
0.5
1
1.5
2
2.5
3
-100 -50 0 50 100
Temperature (°C)
Tan
δ
0
0 . 0 0 5
0 . 0 1
0 . 0 15
0 . 0 2
0 . 0 2 5
0 . 0 3
0 . 0 3 5
0 . 0 4
0 . 0 4 5
0 . 0 5
- 10 0 - 5 0 0
(A)
(a)
(b)
(c)
(d)(b)
(a)
(d)
(c)
0
4
8
12
16
20
0 50 100 150 200
Temperature (°C)
Hea
tflo
w(m
W)
(d)
(c)
(b)
(a)
Exo up
Tc = 129 ± 1
Tc = 104 ± 3
Tc = 108 ± 1
Tc = 93 ± 2
(B)
0
0.5
1
1.5
2
2.5
3
-100 -50 0 50 100
Temperature (°C)
Tan
δ
0
0 . 0 0 5
0 . 0 1
0 . 0 15
0 . 0 2
0 . 0 2 5
0 . 0 3
0 . 0 3 5
0 . 0 4
0 . 0 4 5
0 . 0 5
- 10 0 - 5 0 0
(A)
(a)
(b)
(c)
(d)(b)
(a)
(d)
(c)
(d)
(c)
(b)
(a)
Exo up
Tc = 129 ± 1
Tc = 104 ± 3
Tc = 108 ± 1
Tc = 93 ± 2
(B)
0
0.5
1
1.5
2
2.5
3
-100 -50 0 50 100
Temperature (°C)
Tan
δ
0
0 . 0 0 5
0 . 0 1
0 . 0 15
0 . 0 2
0 . 0 2 5
0 . 0 3
0 . 0 3 5
0 . 0 4
0 . 0 4 5
0 . 0 5
- 10 0 - 5 0 0
(A)
(a)
(b)
(c)
(d)(b)
(a)
(d)
(c)
Figure 5.1 (A) Tan δ as a function of temperature of (a) neat PLA, (b) PLA-g-
PAA(3%)/PEG(10%), (c) PLA-g-PAA(10%)/PEG(10%), and (d) PLA/PEG(10%).
(B) DSC scans of melt quenched (a) neat PLA, (b) PLA-g-PAA(3%)/PEG(10%), (c)
PLA-g-PAA(10%)/PEG(10%), and (d) PLA/PEG(10%).
99
The crystallization temperature (Tc) of PLA decreased from 129 ± 1 °C (Figure
5.1B (a)) for neat PLA to 93 ± 2 °C (Figure 5.1B (d)) for the PLA/PEG(10%) physical
blend. The thermopolymerization of PAA with PLA, prior to blending with PEG,
increased the Tc to 104 ± 3 °C (Figure 5.1B (b)) for PLA-g-PAA(3%)/PEG(10%) and to
108 ± 1 °C (Figure 5.1B (c)) for PLA-g-PAA(10%)/PEG(10%). This increase in Tc with
PAA concentration supported the possibility of favorable intermolecular interactions
between PAA and PLA in these blends. PLA-g-PAA(3%)/PEG(10%) and PLA-g-
PAA(10%)/PEG(10%) blends dissolved in chloroform but could not be filtered through
0.2 μm teflon filter smoothly. This may have been a result of either a small extent of
crosslinking during thermal polymerization or PAA homopolymerization (PAA does not
dissolve in chloroform) or both. Even small extents of crosslinking could affect glass
transition and crystallization events. In order to study the effect of crosslinking, if any,
during PAA thermal polymerization, films were prepared using the same chemistry but
excluding the PEG blending step, hereafter referred to as PLA-g-PAA(10%). It was
observed that there was not any significant effect of PAA thermal polymerization on
PLA’s Tg (as characterized using DMA). However, PLA’s Tc decreased from 129 ± 1 °C
for neat PLA to 104 ± 1 °C for PLA-g-PAA(10%). This reduction was likely a result of
the nucleating effect of homopolymerized PAA domains since crosslinking and favorable
intermolecular interactions would tend to increase Tc. These observations confirmed the
possibility of favorable intermolecular interactions affecting glass transition and
crystallization events in PLA-g-PAA(3%)/PEG(10%) and PLA-g-PAA(10%)/PEG(10%)
blends and not the crosslinking, if any, occurring during PAA thermal polymerization.
100
There was only a slight increase in the toughness of the PLA/PEG(10%) physical
blend over neat PLA, as represented by the area under engineering stress-strain curves
(Figure 5.2). However, thermopolymerization of 3 wt% acrylic acid, prior to PEG
blending, resulted in significant toughness improvement (Figure 5.2a). Figure 5.2b shows
the engineering stress-strain curves of these reactive blends. It was clearly evident that
the toughness improvement was due to an increase in % elongation at break from less
than 10% for neat PLA to 150 ± 20% for PLA-g-PAA(3%)/PEG(10%). This could have
been an outcome of favorable intermolecular interactions between PLA-g-PAA and PEG.
Tou
ghne
ss (
MP
a)
0
10
20
30
40
50
60
Nea
t PL
A
PL
A-g
-PA
A(3
%)/
PE
G(1
0%)
PL
A-g
-PA
A(1
0%)/
PE
G(1
0%)
PL
A/P
EG
(10%
)
0
10
20
30
40
50
60
0 0.5 1 1.5 2
Strain
Str
ess
(MP
a)
Neat PLA
PLA-g-PAA(10%)/PEG(10%)
PLA/PEG(10%)
(a) (b)
Tou
ghne
ss (
MP
a)
0
10
20
30
40
50
60
Nea
t PL
A
PL
A-g
-PA
A(3
%)/
PE
G(1
0%)
PL
A-g
-PA
A(1
0%)/
PE
G(1
0%)
PL
A/P
EG
(10%
)
0
10
20
30
40
50
60
0 0.5 1 1.5 2
Strain
Str
ess
(MP
a)
Neat PLA
PLA-g-PAA(10%)/PEG(10%)
PLA/PEG(10%)
(a) (b)
Figure 5.2 (a) Toughness and (b) representative stress-strain curves of neat PLA and its
reactive blends. Error bars represent 95% confidence intervals.
101
Other methods to improve toughness result in a substantial reduction in tensile
strength and/or modulus. For this reactive-blended material, as shown in Figure 5.3,
Young’s modulus and ultimate tensile strength decreased slightly from 1370 ± 130 MPa
for neat PLA to 990 ± 100 MPa for PLA-g-PAA(3%)/PEG(10%) and from 42 ± 3 MPa to
35 ± 3 MPa, respectively (Figure 5.3). Increase in acrylic acid content from 3 wt% to 10
wt% retained the toughness of the films with minimal Young’s modulus (1235 ± 70 MPa)
and ultimate tensile strength (37 ± 3 MPa) loss compared to neat PLA. This modulus and
ultimate tensile strength retention was attributed to glassy (Tg ~ 125 °C) PAA chains. In
addition to this, increase in Tg from 43 ± 2.1 °C of PLA phase in PLA-g-
PAA(3%)/PEG(10%) to 48 ± 1.7 °C of PLA phase in PLA-g-PAA(10%)/PEG(10%),
indicated the possibility of favorable intermolecular interactions between PLA and PAA.
102
You
ng's
mod
ulus
(M
Pa)
0
200
400
600
800
1000
1200
1400
1600
Nea
t PL
A
PL
A-g
-PA
A(3
%)/
PEG
(10%
)
PL
A-g
-PA
A(1
0%)/
PE
G(1
0%)
PL
A/P
EG
(10%
)
Ult
imat
e te
nsile
str
engt
h (M
Pa)
0
10
20
30
40
50
60
Nea
t PL
A
PLA
-g-P
AA
(3%
)/PE
G(1
0%)
PL
A-g
-PA
A(1
0%)/
PE
G(1
0%)
PL
A/P
EG
(10%
)
(a) (b)
You
ng's
mod
ulus
(M
Pa)
0
200
400
600
800
1000
1200
1400
1600
Nea
t PL
A
PL
A-g
-PA
A(3
%)/
PEG
(10%
)
PL
A-g
-PA
A(1
0%)/
PE
G(1
0%)
PL
A/P
EG
(10%
)
Ult
imat
e te
nsile
str
engt
h (M
Pa)
0
10
20
30
40
50
60
Nea
t PL
A
PLA
-g-P
AA
(3%
)/PE
G(1
0%)
PL
A-g
-PA
A(1
0%)/
PE
G(1
0%)
PL
A/P
EG
(10%
)
(a) (b)
Figure 5.3 (a) Young’s modulus and (b) ultimate tensile strength of neat PLA and its
reactive blends. Error bars represent 95% confidence intervals.
Another key advantage this technology offers is the introduction of reactive acid
groups into the PLA matrix for further modifications. As a proof-of-concept, these film
surfaces were stained with toluidine blue dye. Toluidine blue is a cationic dye that readily
binds with acid groups and not with PLA. Neat PLA did not show any significant staining
(Figure 5.4a). The color intensity increased with acid concentration (Figures 5.4b and
5.4c), indicating the presence of acid groups available for subsequent binding or
conjugation.
103
(a) (b) (c)(a) (b) (c)
Figure 5.4 Toluidine-blue-stained images of (a) neat PLA, (b) PLA-g-
PAA(3%)/PEG(10%), and (c) PLA-g-PAA(10%)/PEG(10%) revealing the presence of
reactive acid groups on the film surfaces.
CONCLUSIONS
This simple reactive blending technology offers PLA toughening with only
minimal modulus and ultimate tensile strength loss associated with the introduction of a
controlled concentration of reactive acid groups into the PLA matrix. This was achieved
by using a reactive-blending approach that relied on the choice of two complementary
polymers, PAA (stiffening and reactive agent) and PEG (toughening agent). PLA surface
and bulk properties could be controlled by varying the concentrations of PAA and PEG.
PLA toughening was attributed to an increase in PLA chain mobility due to a rubbery
104
PEG phase and modulus and ultimate tensile strength retention was attributed to the
glassy nature of PAA and favorable intermolecular interactions between PLA, PEG, and
PAA phases.
105
REFERENCES
1. Eling B, Gogolewski S, Pennings AJ. Biodegadable materials of poly(L-lactic acid): 1. Melt-spun and solution spun fibers. Polymer 1982;23:1587-93.
2. Schmack G, Tändler B, Vogel R, Beyreuther R, Jacobsen S, Fritz H-G. Biodegradable fibers of poly (L-lactide) produced by high-speed melt spinning and spin drawing. J Appl Polym Sci 1999;73:2785-97.
3. Ray SS, Okamoto M. Biodegradable polylactide and its nanocomposites: opening a new dimension for plastics and composites. Macromol Rapid Commun 2003;24:815-40.
4. Gottschalk C, Frey H. Hyperbranched polylactide copolymers. Macromolecules 2006;39:1719-23.
5. Vink ETH, Rabago KR, Glassner DA, Gruber PR. Application of life cycle assessment to NatureWorksTM polylactide (PLA) production. Polym Degrad Stab 2003;80:403-19.
6. Rasal RM, Hirt DE. Toughness decrease of PLA-PHBHHx blend films upon surface-confined photopolymerization. J Biomed Mater Res Part A DOI: 10.1002/jbm.a.32009.
7. Anderson KS, Schreck KM, Hillmyer MA. Toughening polylactide. Polymer Reviews 2008;48:85–108.
8. Rasal RM, Bohannon BG, Hirt DE. Effect of the photoreaction solvent on surface and bulk properties of poly(lactic acid) and poly(hydroxyalkanoate) films. J Biomed Mater Res Part B: Appl 2008;85B:564-72.
9. Janorkar AV, Fritz EW, Burg KJL, Metters AT, Hirt DE. Grafting amine-terminated branched architectures from poly(L-lactide) film surfaces for improved cell attachment. J Biomed Mater Res Part B: Appl Biomater 2007;81B:142–52.
10. Zhang C, Luo N, Hirt DE. Surface grafting poly(ethylene glycol) (PEG) onto poly(ethylene-co-acrylic acid) films. Langmuir 2006;22:6851-57.
11. Zhang R, He CB, Craven RD, Evans JA, Fawcett NC, Wu YL, Timmons RB. Subsurface formation of amide in polyethylene-co-acrylic acid film: a potentially useful reaction for tethering biomolecules to a solid support. Macromolecules 1999;32:2149-55.
12. Luo N, Stewart MJ, Hirt DE, Husson SM, Schwark DW. J. Surface modification of ethylene-co-acrylic acid copolymer films: addition of amide groups by covalently bonded amino acid intermediates J Appl Polym Sci 2004;92:1688-94.
106
13. Janorkar AV, Metters AT, Hirt DE. Modification of poly(lactic acid) films: enhanced wettability from surface-confined photografting and increased degradation rate due to an artifact of the photografting process. Macromolecules 2004;37:9151-59.
107
CHAPTER SIX
CONCLUSIONS AND FUTURE RESEARCH RECOMMENDATIONS
CONCLUSIONS
In the first part of this research, solvent cast PLA and PHBHHx films were
successfully surface modified using a sequential two-step photografting method. Acrylic
acid and acrylamide were photopolymerized from the film surface with ultimate aims of
improving wettability and introducing readily reactable groups onto the surface.
Although the reaction solvent used in Step 2, water or ethanol, had an insignificant effect
on surface properties, bulk properties were significantly affected. PLA and PHBHHx
films lost their toughness and became stiffer on surface modification, with the effect
being more prevalent when ethanol was used as the reaction solvent. Likewise, solvent-
induced crystallization was more prevalent when ethanol was used as the reaction solvent
than when water was used as the reaction solvent for PLA films. The observed toughness
loss and modulus gain of PLA films on surface modification was attributed to solvent-
induced crystallization and loss of residual chloroform. The presence of residual
chloroform in the film specimens was undesirable from a biocompatibility standpoint.
Additionally, this work showed that the photoreaction solvent affected the bulk properties
of PLA and PHBHHx films cast from a chloroform solution. Therefore, further work was
conducted on melt-processed films where residual solvent from the film-formation
method was not an issue.
108
Addition of a small amount of PHBHHx (10 wt %) to PLA successfully improved
the toughness of the resulting melt-processed blends. PLA-PHBHHx blend films were
surface modified using the same sequential two-step photografting method using water as
the reaction solvent in Step 2. The PLA and PHBHHx components of melt processed
blend films appeared to be non-compatible based on DMA analyses. The PLA-PHBHHx
blend films lost their toughness significantly due to physical aging. It was also observed
that physically aged films regained their toughness temporarily on annealing at 60 °C for
30 min. The contribution of physical aging to the change in mechanical properties was
minimized by annealing films at 60 °C for 30 min before testing. This enabled us to study
the effect of photografting on mechanical properties of blend films. It was observed that
the blend films underwent UV-assisted solvent induced crystallization on suface
modification and lost their toughness significantly. TGA data showed the presence of
reaction solvent in surface-modified films. The combination of solvent heating during
UV-irradiation and the plasticizing effect of residual water resulted in insignificant
changes in Young’s modulus and UTS on surface modification.
Finally, a novel reactive blending approach was designed to toughen PLA with
minimal modulus and/or UTS losses and to introduce a controlled concentration of
reactive acid groups into the matrix in one step. This approach relied on the choice of two
complementary polymers, PAA (stiffening and reactive agent) and PEG (toughening
agent). PLA surface and bulk properties were controlled by varying the concentrations of
PAA and PEG. PLA toughening was attributed to an increase in PLA chain mobility due
109
to PEG and modulus and UTS retention was attributed to the glassy nature of PAA and
favorable intermolecular interactions between PLA, PEG, and PAA phases.
RECOMMENDATIONS FOR FUTURE RESEARCH
Physical aging is a major concern for PLA and PLA-based formulations. Results
in Chapter 4 showed that melt-processed PLA-PHBHHx (90 wt % PLA) blend films lost
their toughness significantly due to physical aging. The effect of physical aging can be
minimized by stretching these films uniaxially and/or biaxially. These extruded films can
be stretched above PLA’s glass transition temperature to induce orientation and minimize
the physical aging effect. However, care should be taken to minimize PLA thermal
degradation while stretching. Stretching these films slightly above PLA’s glass transition
temperature may help minimize the thermal degradation. A more economical way would
be to stretch these films online during extrusion.
As a proof-of-concept, PLA was successfully toughened with only minimal
modulus and UTS losses and introduction of a controlled concentration of reactive acid
groups into the PLA matrix. This was achieved by using a novel reactive blending
approach. Following are different potential modifications that may render this technology
more attractive and industrially relevant:
1. These blending reactions should be carried out in the melt-phase in an extruder.
This could be performed in CAEFF’s microextruder (DSM Xplore) that enables
110
2. One of the major drawbacks of PLA is its slow degradation rate. This technology
introduces controlled concentrations of hydrophilic PAA and PEG into the PLA
matrix. PAA and PEG have the potential to increase the hydrolytic degradation
rate of PLA. The effect of PAA and PEG concentrations on the rate of PLA’s
hydrolytic degradation should be studied. This work should be performed at
different pH and temperature conditions including physiological pH (7.4) and
temperature (37 °C).
3. PLA-based formulations reported in Chapter 5 should be prepared using
thermopolymerizable hyaluronic acid instead of acrylic acid (a more
biocompatible version for biomedical applications). This may retain all the
advantages of these formulations and make it more biocompatible.
4. A detailed study should be conducted to evaluate the effect of PAA, PEG, and
benzoyl peroxide concentrations, reaction time, and temperature on surface and
bulk properties of the resultant films. An ultimate aim of this study could be to
create a library of these variables and resultant surface and bulk properties. This
library may be used to produce several formulations depending on the need of the
applications.
5. A detailed rheological study should be performed for these PLA-based
formulations.
111
6. PLA-based composites have exhibited excellent shape memory properties.
PLA/hydroxyapatite composite shape-memory properties have been studied
above 70 °C [1]. It would be very interesting with respect to biomedical
applications (such as sutures, implants, etc.) to study the dual and triple shape
memory properties of these PLA-based formulations. Increasing the composition
of PEG may lead to desirable shape-memory properties at physiological
temperature (37 °C). Langer et al. [2-3] and Mather et al. [4] have done
significant research on polymer shape-memory properties and their work should
serve as a guide for further studies with our materials.
112
REFERENCES
1. Zheng X, Zhou S, Li X, Weng J. Shape memory properties of poly(D,L-lactide)/hydroxyapatite composites. Biomaterials 2006;27:4288-95.
2. Lendlein A, Schmidt AM, Langer R. AB-polymer networks based on oligo(ε-caprolactone) segments showing shape-memory properties. Proc Natl Acad Sci USA 2001;98:842-47.
3. Lendlein A, Langer R. Biodegradable, elastic shape-memory polymers for potential biomedical applications. Science 2002;296:1673-76.
4. Liu C, Qin H, Mather PT. Review of progress in shape-memory polymers. J Mater Chem 2007;17:1543–58.
113
APPENDICES
114
APPENDIX A
MICROPATTERNING OF COVALENTLY ATTACHED BIOTIN ON
POLY(LACTIC ACID) FILM SURFACES
INTRODUCTION
Micropatterning biomolecules on surfaces has applications ranging from
biosensors [1-2], medical implants [3], bioassays [4], and lab-on-a-chip [5]. Spatially
controlled organization of biological ligands and proteins on surfaces is very important
with respect to these applications. The commercial development of specific patterns relies
mainly on the fabrication ease, repeatability, stability, and cost. Polymers have
progressively shown the potential to be a viable alternative to the conventional
microfabrication materials such as glass, silicon, or gold [4]. Environmental concerns
associated with petroleum-based polymers make biodegradable polymers more attractive
microfabrication candidates. Aliphatic poly(hydroxyacid) type biodegradable polymers,
especially poly(lactic acid) (PLA), are of growing importance. PLA is a biodegradable
and bioabsorbable thermoplastic polyester derived from renewable resources like corn,
starch, or rice and exhibits excellent biocompatibility [6-12]. PLA films are commercially
produced and their mechanical properties can be significantly improved by blending with
other biodegradable polymers like poly(hydroxyalkanoates) (PHAs) [11]. PLA
production requires 25-55% less energy compared to petroleum-based polymers and
estimates are that this can be further reduced to less than 10% in the future [8]. This
115
makes the use of PLA films in microfabrication technology potentially advantageous
with respect to cost as well as degradability.
PLA has been widely researched to improve its surface [13-15] and bulk
properties [16-20]. However, methods to spatially control the organization of
biomolecules on PLA, which are important with respect to specific biomedical
applications, are still very limited. Lin et al. [21] have applied soft lithography techniques
to micropattern proteins on PLA. Briefly, poly(oligoethyleneglycol methacrylate) (poly-
OEGMA) was printed on PLA to create micron-size, protein-resistant areas. Proteins
adsorbed on unprinted regions leaving printed regions intact. Since the poly-OEGMA
was not covalently attached to the PLA surface, these protein micropatterns might not be
suitable for certain biomedical applications in which devices would require permanent
surface patterns.
Micropatterning methods often consist first of a surface modification process.
PLA is chemically inert with no readily reactable side chain groups making its surface
modification a challenging task. PLA has been surface modified using a variety of
techniques such as coating [22], migratory additives [23], plasma treatment [24-25], and
entrapment [26-27]. Each has its own advantages and disadvantages, but most of these
surface modifications are not permanent. In earlier reports, we have used a photoinduced
grafting approach to surface modify PLA [15, 19, 20]. This approach resulted in the
covalent attachment of various molecules to PLA film surfaces.
In this research, photoinduced grafting in conjuction with photolithography [4]
was used to micropattern reactive poly(acrylic acid) (PAA) on PLA. These micropatterns
116
were confirmed by staining with toluidine blue dye, as well as characterization of surface
topography using AFM. The PAA micropatterns were subsequently conjugated to amine-
terminated biotin using water soluble carbodiimide chemistry. The conjugation reaction
was investigated using XPS. These biotin modified PAA patterned PLA films were then
subjected to fluorescent proteins to demonstrate their patterning efficiency. To our
knowledge, there have not been any attempts to covalently micropattern biological
ligands, such as biotin, on PLA surfaces. Therefore, the overall objective was to
covalently micropattern biotin on PLA surfaces, evaluate the robustness of the
micropattern, and study subsequent streptavidin adsorption.
MATERIALS
Acrylic acid (99.5% w/w) was obtained from Acros Organics and used as
received without further purification. Ethanol, glass slides, HPLC water, and
benzophenone were purchased from Fisher Scientific. N-hydroxysuccinimide (NHS) and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased
from Aldrich. (+)-Biotinyl-3,6,9-trioxaundecanediamine and Alexa488 labeled
streptavidin were purchased from Pierce. The Alexa488 labeled streptavidin was supplied
as a yellow colored liquid at a concentration of 1 mg/ml in 0.1 M sodium phosphate, 0.15
M sodium chloride, pH 7.2, containing 1% BSA and 0.02% sodium azide. Dulbecco’s
phosphate buffered saline (PBS) solution was purchased from Gibco.
117
METHODS
PLA Film Extrusion: Prior to extrusion, PLA pellets were dried in a vaccum oven at 70
°C for 24 h and cooled in the vaccum oven to remove any residual moisture. A single
screw extruder (HAAKE INC.) was used to cast PLA films. The temperature profile of
the extruder was as follows: Zone 1 (located near the hopper) – 180 °C, zone 2 – 190 °C,
zone 3 – 200 °C, pump – 200 °C, and die – 190 °C. The polymer melt exiting the die was
cooled on a chill roll. The resultant films had a nominal thickness 125 μm.
Micropatterning PAA on PLA: Scheme A.1 shows the PAA micropatterning process
flow diagram. A PLA specimen was sonicated in ethanol for 5 min to clean the surface. It
was subsequently dipped into benzophenone solution in ethanol (5 wt %) for 1 min and
washed with copious amounts of ethanol. The benzophenone-dip-coated PLA specimen
was dried using a nitrogen gas stream. This specimen was fixed on a glass slide. A couple
drops of aqueous acrylic acid solution (10 wt %) were placed on the film surface. When a
photomask was lowered on top of the specimen, aqueous acrylic acid solution spread
uniformly. Another glass slide was placed on top to ensure that there was no air gap
between photomask and aqueous acrylic acid solution. This assembly was subsequently
exposed to UV irradiation for predetermined time in a UV processor (EXFO 100 W
Acticure ultraviolet/visible spot-curing system). The processor had a wavelength range of
250-650 nm and intensity of 40 mW/cm2 at 365 nm measured using an OAI 306 UV
powermeter. The resulting PAA micropatterned PLA specimen was sonicated in water
118
for 5 min and washed with copious amounts of water. The acid group surface density, γ
(acid groups/nm2), was estimated using the following equation [28]
M
h
M
Nh dAd 3.60221
(1)
where ρ is density of PAA (1.22 g/cm3) [29], hd is the dry layer thickness (nm) of the
surface grafted PAA layer, NA is Avogadro’s number, and M is the molecular weight
(g/mol) of acrylic acid.
PLA
5 wt% Benzophenone in Ethanol
UV Light
10 wt% Acrylic acid in Water
Photomask
Poly(acrylic acid)
PLA
5 wt% Benzophenone in Ethanol
UV Light
10 wt% Acrylic acid in Water
Photomask
Poly(acrylic acid)
Scheme A.1 Photolithography [4] approach to micropattern PAA on PLA.
119
PAA-Biotin Conjugation and Subsequent Streptavidin Adsorption: The PAA
micropatterned PLA specimen was stirred with EDC (6 wt %) and NHS (3.6 wt %)
solution in PBS buffer for 3 h at room temperature. EDC and NHS concentrations were
selected to give a stochiometric molar ratio of 1:1. The specimen was then washed with
copious amounts of PBS buffer solution to remove any unreacted EDC or NHS. This
procedure was used to activate the acid groups. The EDC/NHS activated film was then
stirred in amine-terminated biotin ligand solution in ethanol (0.42 wt %) for 3 h and
rinsed with ethanol and PBS buffer solution. The specimen was subsequently immersed
in a solution of Alexa488 streptavidin (35 μl/ml) in PBS buffer for 3 h. It was then
washed with copious amounts of PBS buffer solution and dried in a vacuum oven before
examining under a fluorescence microscope. Neat PLA film was immersed in biotin
solution for 3 h and then washed with copious amounts of ethanol to examine the extent
of biotin adsorption on neat PLA. Neat PLA film was immersed in streptavidin solution
for 3 h and then washed with copious amounts of buffer to examine the extent of
streptavidin adsorption on neat PLA.
120
RESULTS AND DISCUSSION
Micropatterning PAA on PLA
Scheme A.1 represents the process flow diagram of PAA micropatterning on PLA
using photolithography. This approach offers permanent micropatterning with a range of
microfeature shapes and large patterned areas. The progression of the PAA
micropatterning was characterized using ATR-FTIR spectroscopy (Figure A.1). It was
observed that the –C=O acid stretch of PAA at 1720 cm-1, which is a shoulder to the –
C=O backbone PLA ester stretch at 1747 cm-1, became stronger with UV irradiation time.
This observation was supported by water contact angle goniometry data as shown in
Figure A.2. Neat PLA is hydrophobic with a water contact angle ~ 80°, and the water
contact angle decreased with UV irradiation time. PAA is hydrophilic.
121
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70A
bsor
banc
e
1500 1600 1700 1800 1900 2000 2100 2200
Wavenumbers (cm-1)
Neat
10 min
15 min
20 min
♦
●
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70A
bsor
banc
e
1500 1600 1700 1800 1900 2000 2100 2200
Wavenumbers (cm-1)
Neat
10 min
15 min
20 min
♦
●
Figure A.1 Representative ATR-FTIR spectra revealing the progression of PAA
micropatterning on PLA. The –C=O “ester stretch” of PLA is represented by a peak at
1747 cm-1 (♦) and the –C=O “acid stretch” of PAA is represented by a peak at 1720 cm-
1(●). A 2 mm wide stripe on PLA was characterized to monitor the progression of PAA
micropatterning.
The decrease in water contact angle suggested that a water drop placed on a PLA-
g-PAA (where g denotes grafted) region encountered a greater fraction of hydrophilic
PAA than relatively hydrophobic PLA as UV irradiation time increased. For a given light
intensity (40 mW/cm2 at 365 nm) and monomer concentration (10% w/w acrylic acid in
122
water), the surface grafted PAA molecular weight and number of PAA graft
polymerization initiation sites were expected to increase with UV irradiation time. Either
(or both) of those effects would cause a decrease in contact angle with increased UV
irradiation time. This contact-angle decrease was the same trend observed in prior work
on non-patterned PLA films (direct UV exposure without photomask) photografted with
PAA [15, 19, 20].
0
10
20
30
40
50
60
70
80
90
0 min (Neat PLA) 10 min 15 min 20 min
UV Exposure T ime
Wat
er C
onta
ct A
ngle
(°)
Figure A.2 Effect of UV irradiation time on static water contact angle of 2 mm wide
PAA stripe on PLA. The error bars represent 95% confidence intervals.
This behavior was further investigated by monitoring the PAA micropatterned
PLA surface topography using AFM (Figure A.3). A well-defined contrast in these phase
123
images indicated that the AFM tip experienced distinct interactions with PLA and PAA.
As shown in the upper plots in Figure A.3, the dry thickness of the PAA stripe on PLA
increased with irradiation time from 60 to 170 to 360 nm, corresponding to 600 to 1700
to 3700 acid groups/nm2 from equation (1). This thickness increase with UV irradiation
time confirmed the increase in surface grafted PAA molecular weight, consistent with the
increase in the ATR-FTIR –C=O acid stretch of PAA at 1720 cm-1, which is a shoulder to
the –C=O backbone PLA ester stretch at 1747 cm-1 (Figure A.1), and the decrease in
water contact angle (FigureA.2) with UV irradiation time. This surface-topography
control could be an important consideration for biomaterial design since it is one of the
cell response governing factors [30-31]. The surface topography images in Figure A.3
revealed that, for a UV irradiation time of 10 min, the PAA stripe was blunt with
significant edge defects. For UV irradiation times of 15 and 20 min, the PAA stripe was
sharper, with minimal edge defects. However, it was observed that for a UV irradiation
time of 20 min, there was significant PAA bulk polymerization in the supernatant, which
led to significant non-specific adsorption of PAA chains from the supernatant onto the
PLA surface. This non-specific PAA adsorption was not acceptable for subsequent biotin
conjugation. The non-specific PAA adsorption on PLA, for UV irradiation times longer
than 15 min, was minimized significantly by immersing PAA micropatterned films in hot
water or sonicating them in water at room temperature for 1 h. However, these methods
significantly damaged the entire PAA micropatterned PLA surface topography making it
unsuitable for subsequent reactions and characterization. Since a UV irradiation time of
15 min produced the best pattern quality with minimal edge defects and insignificant
124
non-specific PAA adsorption on PLA, the UV irradiation time was set to 15 min for
subsequent experiments.
60 nm
170 nm 360 nm
UV irradiation time = 10 min UV irradiation time = 15 min UV irradiation time = 20 min
60 nm
170 nm 360 nm60 nm60 nm
170 nm170 nm 360 nm360 nm
UV irradiation time = 10 min UV irradiation time = 15 min UV irradiation time = 20 min
Figure A.3 Effect of UV irradiation time on PAA microstripe height and surface
topography of PAA micropatterned PLA.
The PAA pattern quality and the extent of non-specific PAA adsorption from the
supernatant onto PLA were also monitored by staining the grafted surface with toluidine
blue dye and observing under an optical microscope. Toluidine blue is a cationic dye that
125
readily binds to the carboxylate groups of PAA, but not to inert PLA. The best pattern
quality was achieved for a UV-irradiation time of 15 min (Figure A.4). For a UV-
irradiation time of 10 min, the boundary between PLA-g-PAA and base PLA was not
sharp, revealing some edge defects. For a UV-irradiation time of 15 min, the boundary
between PLA-g-PAA and base PLA was sharper, giving the best pattern quality. These
observations were consistent with the AFM results.
100 μm500 μm
100 μm100 μm100 μm100 μm500 μm500 μm500 μm
Figure A.4 Optical micrographs of toluidine-blue-stained PAA micropatterned PLA (UV
irradiation time 15 min) revealing micropatterns of different shapes and sizes.
126
PAA-Biotin conjugation
PAA has carboxylic acid (-COOH) groups that can be conjugated to -NH2 groups
of amine-terminated biotin using standard water soluble carbodiimide chemistry (Scheme
A.2). EDC and NHS were used to activate the acid groups, which were subsequently
reacted with amine-terminated biotin.
EDC/NHS
PBS Buffer
Amine-terminated Biotin
Ethanol
CH2 CH
COOH
n
O
O N
O
O
O
NHO
NH
O
3 S
NHNH
O
CH2 CH
COOH
n
O
O N
O
O
O
NHO
NH
O
3 S
NHNH
O
EDC/NHS
PBS Buffer
Amine-terminated Biotin
Ethanol
EDC/NHS
PBS Buffer
Amine-terminated Biotin
Ethanol
CH2 CH
COOH
n
O
O N
O
O
O
NHO
NH
O
3 S
NHNH
O
CH2 CH
COOH
n
O
O N
O
O
O
NHO
NH
O
3 S
NHNH
O
Scheme A.2 Scheme of the EDC/NHS mediated PAA conjugation with amine-terminated
biotin.
127
The surface chemistry was investigated using XPS. Figures A.5a and A.5b show
XPS survey scans for neat PLA and PLA-g-biotin respectively. Figure A.5a showed two
peaks located at binding energies 531 and 284 eV corresponding to the O 1s and C 1s
signals. Based on XPS survey scans for neat PLA, the C/O ratio was 1.49 ± 0.34, close to
the theoretical value of 1.5 based on the chemical structure of PLA (see Figure A.6). XPS
survey scans for PLA-g-biotin showed two additional elements: N 1s at 398 eV and S 2p
at 165 eV. The presence of these two elements, especially S, confirmed successful PAA-
biotin conjugation.
128
(a)
(b)
Figure A.5 XPS survey scans for (a) neat PLA and (b) PLA-g-biotin.
129
The biotin immobilization surface chemistry was further investigated using a high
resolution XPS scan of C 1s. The high resolution scan of neat PLA was deconvoluted into
three C 1s component peaks (283.8, 285.7, and 288.0 eV) of approximately equal
composition corresponding to the three types of carbon atoms present in PLA [32]
(spectrum not shown). These results were expected based on the PLA chemical structure
and agree well with previous reports [32-33]. The high resolution C 1s scan of PLA-g-
biotin showed two additional peaks (Figure A.6). Peak 4 at 284.6 eV was assigned to C
atoms bonded to secondary N atoms (C-NH-CO) and peak 5 at 288.5 eV was assigned to
the carbonyl C atoms in the amide linkages (C-NH-CO). These peaks confirmed the
carbodiimide mediated reaction of biotin’s –NH2 with acid groups to form amide
linkages. An increase in peak 2 at 285.7 eV (C-O) likely resulted from C-O groups of
poly(ethylene glycol) (PEG) chains contained within the biotin. An increase in the peak 3
at 283.8 eV (CH2) likely resulted from biotin CH2 groups. These peak assignments were
based on literature reports. [32-36]
130
5 4
1
2
3
5 4
1
2
3
HCH3
O
O
n
3
2 1
HCH3
O
O
n
3
2 1
NHHN
O
S
NH
O
ONH
3
23
333
3
4 4
4
5
5
4
NHHN
O
S
NH
O
ONH
3
23
333
3
4 4
4
5
5
4
Peak B.E. (eV) Atom 1 288.0 O=C-O 2 285.7 C-O 3 283.8 CH2, CH3, 4 284.6 C-NH-CO 5 288.5 C-NH-CO
Figure A.6 XPS high-resolution C 1s spectra for PLA-g-biotin.
131
The N/S ratio of a biotin modified PAA stripe on PLA was 9.2 ± 2.7, while the
theoretical N/S ratio based on the biotin structure is 4.0. This indicated that the
EDC/NHS activated PAA-biotin conjugation reaction conversion was only 16% (16 out
of 100 activated acid groups were successfully conjugated with biotin), and the excess N
concentration resulted from EDC/NHS activated acid groups that were not conjugated to
biotin (Scheme A.2). This lower conversion was the same outcome for the carbodiimide
based chemistry, involving poly(methacrylic acid) (PMAA) and RGD peptide
conjugation, with acid-peptide conversion of only 12% as reported by others [37]. The
lower EDC/NHS activated PAA-biotin conjugation conversion is thought to be a result of
minimized mass transfer of biotin into the EDC/NHS activated PAA layer, due to the
large acid group density (1700 acid groups/nm2) and the relatively long (23 Å [38])
spacer arm attached to biotin. Attempts were made to increase activated acid-biotin
conjugation conversion by increasing the reaction time. Since this methodology
significantly affected the resultant film topography (longer reaction time resulted in
curled films with rough surfaces), biotin modified micropatterned films with the 16%
PAA-biotin conjugation were used for the subsequent streptavidin adsorption
experiments.
Streptavidin adsorption on biotin modified PAA micropatterned PLA
The biotin modified PAA micropatterns were then immersed in fluorescent
streptavidin solution in PBS buffer to demonstrate their patterning efficiency. Figure
132
A.7a demonstrated that streptavidin adsorption was primarily confined to the biotin
modified PAA regions. A control experiment was performed where a PAA
micropatterned PLA surface (without biotin) was exposed to the fluorescent streptavidin
solution and did not reveal any significant streptavidin adsorption on PAA stripes (Figure
A.7b). This confirmed that the streptavidin adsorption resulted from the biotin
modification of the PAA stripes. As shown in Figure A.8, the N concentration of biotin
modified PAA micropatterned PLA film exposed to streptavidin solution was
approximately the same as the N concentration of the fluorescent streptavidin solution
used as received (the latter was calculated from an XPS survey scan of a gold-coated
silicon wafer dipped in fluorescent streptavidin solution). XPS probes the uppermost 2-3
nm. High resolution X-ray crystallographic studies of streptavidin showed it to be
approximately 5.4 nm X 5.8 nm X 4.8 nm in size with the two pairs of biotin binding
sites on the opposite faces separated by the shortest dimension [39]. This implied that
XPS may not detect significant biotin once streptavidin was adsorbed on it, and was
confirmed by an XPS spectrum of biotin modified PAA patterns exposed to streptavidin
solution, which did not reveal any S atoms (coming exclusively from biotin). The cross-
sectional area of a streptavidin molecule is about 30 nm2 while that of a biotin ligand is
no more than 0.3 nm2 [40]. Theoretically two biotin molecules bind to each streptavidin
molecule. Assuming all biotin molecules were equispaced on the surface and all of them
were bound to streptavidin, the surface coverage of streptavidin molecules would likely
make very few unreacted activated acid groups detectable by XPS. This indicated that the
N concentration of biotin modified micropatterns exposed to streptavidin resulted mainly
133
from the streptavidin N atoms (and not from unreacted activated acid groups or
underlying biotin molecules). Hence it was inferred that even if the biotin immobilization
conversion was only 16%, there was significant streptavidin adsorption on the biotin
modified PAA micropatterns on PLA. This significant streptavidin adsorption could be a
result of extraordinarily high streptavidin affinity for biotin.
100 μm
(a) (b)
100 μm100 μm100 μm100 μm
(a) (b)
Figure A.7 Fluorescent micrographs of (a) Alexa488-strepatavidin micropatterned PLA
surface and (b) PAA micropatterned PLA surface (without biotin) exposed to Alexa488-
streptavidin.
134
Non-specific adsorption
PLA is hydrophobic with a water contact angle ~ 80°. There is likely to be some
degree of non-specific protein adsorption on PLA, some of which is evident in Figure
A.7a. Control experiments were performed to investigate the extent of non-specific
adsorption on PLA. In control experiment 1, neat PLA film was immersed in biotin
solution for 3 h and then washed with copious amounts of ethanol to examine the extent
of biotin adsorption on neat PLA. An XPS survey scan of this film did not detect the
presence of N or S (elements coming exclusively from biotin), and confirmed that the
biotin adsorption on neat PLA was not significant. Biotin used in this research was
attached to hydrophilic PEG chains, while the neat PLA is relatively hydrophobic,
resulting in insignificant biotin adsorption on neat PLA.
135
0
1
2
3
4
5
6
7
8
Streptavidin Adsorbed onBiotin Modified PAA Stripe
Streptavidin Adsorbed on NeatPLA
Streptavidin as Received
N C
once
ntra
tion
(%)
Figure A.8 N concentration (as calculated from XPS survey scans) of streptavidin
adsorbed on biotin modified PAA stripe and neat PLA.
In control experiment 2, neat PLA film was immersed in streptavidin solution for
3 h and then washed with copious amounts of buffer to examine the extent of streptavidin
adsorption on neat PLA. The N atomic concentration was monitored to examine the
extent of streptavidin non-specific adsorption on PLA (Figure A.8). The N concentration
of neat PLA film exposed to streptavidin solution (middle bar) was lower than that of
biotin modified micropatterns exposed to streptavidin solution (left-most bar). Therefore,
the streptavidin adsorption was preferentially confined to the biotin modified PAA
regions. In future work, the non-specific streptavidin adsorption on the unmodified PLA
136
regions can be significantly reduced by grafting a non-fouling polymer like PEG to PLA
prior to micropatterning.
CONCLUSIONS
Biotin was successfully covalently micropatterned on PLA using a two-step
approach. Reactive PAA groups were micropatterned on PLA using photolithography in
Step 1. The PAA grafted layer thickness increased with UV irradiation time. The PAA
micropatterned PLA films analyzed by ATR-FTIR spectroscopy, water contact angle
goniometry, and AFM indicated that “the optimum UV irradiation time” required to
achieve the best pattern quality, with minimal edge defects and non-specific PAA
adsorption from supernatant onto PLA, was 15 min. PAA was successfully conjugated to
amine-terminated biotin using water soluble carbodiimide chemistry in Step 2. XPS
analyses confirmed the amide linkages formed by reaction of biotin’s amine with acid
groups. Even if the EDC/NHS activated PAA-biotin conjugation reaction conversion was
only 16%, there was significant streptavidin adsorption on biotin modified micropatterns,
likely due to high streptavidin affinity for biotin. The non-specific biotin adsorption on
PLA was minimal and this was attributed to hydrophilic PEG chain contained within the
biotin. Although streptavidin adsorption was primarily confined to biotin modified PAA
regions, XPS analyses revealed some degree non-specific streptavidin adsorption on
unmodified PLA.
137
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APPENDIX B
NOVEL TOUGHER POLY(LACTIC ACID)-POLY(ETHYLENE GLYCOL)
METHACRYLATE REACTIVE BLENDS
INTRODUCTION
Poly(lactic acid) or poly(lactide) (PLA) is a renewably derived biodegradable and
bioabsorbable thermoplastic polyester that has exhibited excellent biocompatibility and
thermal processibility [1-8]. In addition to this, PLA is recyclable, compostable, and
requires 25-55% less energy to produce than petroleum-based polymers [9-10]. These
attractive characteristics make PLA a potential replacement for many petroleum-based
polymers. However, the major drawback of PLA is its poor toughness with % elongation
at break less than 10% [11]. This limits its use in many consumer and biomedical
applications.
Poly(ethylene glycol) methacrylate (PEGMA) is a thermopolymerizable
macromer with excellent biocompatibility and hydrophilicity. PEGMA itself has poor
mechanical properties and thermal processibility but has a potential to toughen PLA.
PLA has been copolymerized with poly(ethylene glycols) (PEGs) to improve its
mechanical and biomaterial properties. PLA’s drug-delivery properties have been
improved by synthesizing diblock and triblock PLA-PEG copolymers. However, PLA
and PEG underwent phase separation leading to poor mechanical properties of the
copolymers [12]. PLA-PEG block copolymers produced by copolycondensation of PLA-
142
diols and PEG-diacids using carbodiimide-based wet chemistry showed better
compatibility. These copolymers did not phase separate and exhibited improved
mechanical properties [13].
Compared to PLA-PEG copolymerization, blending is a more simple and
convenient methodology to improve PLA’s mechanical and biomaterial properties. Pillin
et al. [14] have reported PEG as the most efficient for glass transition temperature
reduction when compared with poly(1,3-butanediol), dibutyl sebacate, and acetyl glycerol
monolaurate. PEG (Mn ~ 20 kDa)-PLA solvent cast blends (40 wt% PEG) were found to
be very ductile [15]. Blend miscibility and mechanical properties are governed mainly by
the composition of the constituents. Melt processed PLA-PEG bends (PEG Mn ~ 20 kDa)
were found to be miscible, showed improved ductility, and reduced tensile strength for
concentrations up to 50 wt% PEG. However, above 50 wt% PEG, blend crystallinity was
found to increase significantly and resulted in an increased modulus and decreased
ductility [16].
In this publication, we report the synthesis and mechanical properties of novel
tougher PLA-PEGMA reactive blends. The mechanical properties of these reactive
blends were found to be composition dependent. PLA-PEGMA blends were
characterized using dynamic mechanical analysis (DMA) and tensile testing.
143
MATERIALS
PLA pellets (Mn ~ 110 kDa) were supplied by NatureWorks LLC. PEGMA (Mn
~ 360 Da) was obtained from Sigma and was purified by passing through a neutral
alumina column to remove the monomethyl ether hydroquinone inhibitor. Chloroform
was purchased from VWR. Benzoyl peroxide (BPO) was obtained from Fluka.
METHODS
PLA reactive blending: As shown in Scheme B.1, a predetermined amount of PLA was
dissolved in 100 mL CHCl3 at 100 °C followed by addition of predetermined amounts of
PEGMA and BPO (10 wt% of PEGMA) predissolved in 20 mL chloroform at room
temperature. The solution was allowed to stand at 100 °C for 1 h. The solution was then
cooled to room temperature and poured in a glass dish. The solution was kept at room
temperature overnight and then transferred to a vacuum oven at 70 °C for 24 h and cooled
in the vacuum oven to remove any residual chloroform.
+BPO
Chloroform, 100 °C, 1 hPLA PEGMA PLA-PEGMA Reactive Blend Extrusion
Vacuum
70 °C, 24h+
BPO
Chloroform, 100 °C, 1 hPLA PEGMA PLA-PEGMA Reactive Blend Extrusion
Vacuum
70 °C, 24h
Scheme B.1 Reactive blending approach consisting of thermal polymerization of
PEGMA.
144
Film Extrusion: The polymer blend was immediately transferred to an extruder after
drying. A twin-screw microextruder (DSM Xplore) operating in a co-rotating mode at
190 °C was used to cast films. The tapered screws were 170 mm long and the barrel
volume was 15 cm3. The polymer melt exiting the die was cooled by a stream of nitrogen
gas and collected on a chill roll. The resultant films had a nominal thickness 80 ± 10 μm.
Mechanical Testing: The film samples were stored at room temperature after extrusion
for 24 h before mechanical testing. The mechanical properties of the film samples (7.5
cm x 1.5 cm x 80 μm) were measured using an Applied Test System Inc. (ATS)
mechanical tester according to American Society for Testing and Materials Standard
(ASTM D882) specifications. A cross-head speed of 1.25 cm/min was used. The
measured values averaged for five specimens with ±95% confidence intervals are
reported.
RESULTS AND DISCUSSION
Scheme B.1 represents the PLA reactive blending approach consisting of thermal
polymerization of PEGMA. Briefly, PEGMA was thermopolymerized in the presence of
PLA using BPO thermal initiator. The resultant blend was dried and extruded using a
twin-screw extruder operated in a co-rotating mode. Miscibility of the reactive blend
films prepared was studied using DMA. Tan δ vs. temperature for these reactive blend
films showed two well defined peaks corresponding to the constituent PLA and PEGMA
145
phases. This confirmed the blend constituents to be non compatible. In addition to this,
PLA’s glass transition temperature decreased with an increase in PEGMA composition
(i.e., at 20 wt% and 40 wt% PEGMA). However, the glass transition temperature of the
PEGMA phase did not change as significantly (Figure B.1).
40
45
50
55
60
65
0 5 10 15 20 25 30 35 40
PEGMA Wt%
Tg
(°C
)
PLA Tg
-PEGMA Tg
Figure B.1 The effect of PEGMA composition on the glass transition temperatures of the
reactive blend constituents.
It was observed that the reactive blends containing 10 wt% and 20 wt% PEGMA
were tougher than neat PLA with the effect being more prominent for the reactive blends
containg 20 wt% PEGMA (Figure B.2a). This was attributed to the increase in chain
mobility as indicated by the reduction in PLA’s glass transition temperature from 59 °C
for neat PLA to 53 °C for the reactive blend containing 20 wt% PEGMA. Further
146
increase in PEGMA concentration had only minimal toughness improvements. The
observed increase in the toughness of PLA was a result of increase in % elongation at
break (Figure B.2b). Although PLA was toughened successfully using this chemistry, the
toughness improvements were associated with stiffness loss. It was observed that the
reactive blends containing 10 wt%, 20 wt%, and 40 wt% PEGMA lost their modulus
compared to neat PLA (Figure B.3). To minimize this deficiency, the PLA modification
detailed in Chapter 5 was developed.
147
PEGMA Wt%
Tou
ghne
ss (
MP
a)
0
10
20
30
40
0 5 10 20 40
(a)
PEGMA Wt%
% E
long
atio
n at
Bre
ak
0
20
40
60
80
100
120
140
0 5 10 4020
(b)
Figure B.2 (a) Toughness and (b) % Elongation at Break of neat PLA and its reactive
blends. Error bars represent 95% confidence intervals.
148
PEGMA Wt%
You
ng's
Mod
ulus
(M
Pa)
0
200
400
600
800
1000
1200
1400
1600
1800
0 5 10 20 40
Figure B.3 Young’s modulii of neat PLA and its reactive blends. Error bars represent
95% confidence intervals.
CONCLUSIONS
PLA was successfully toughened using a novel reactive blending technology that
relies on the thermal polymerization of PEGMA in the presence of PLA. PLA bulk
properties could be controlled by varying the concentrations of the blend constituents.
PLA toughening (particularly the reactive blend containing 20 wt% PEGMA) was
attributed to an increase in PLA chain mobility due to a rubbery PEGMA as indicated by
the reduction in the glass transition temperature of the PLA phase. However, PLA
149
toughening was associated with stiffness loss, and further work to alleviate this problem
is provided in Chapter 5.
150
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new dimension for plastics and composites. Macromol Rapid Commun 2003;24:815-40.
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2006;39:1719-23. 5. Zhu KJ, Xiangzhou L, Shilin Y. Preparation, characterization, and properties of
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and bulk properties of poly(lactic acid) and poly(hydroxyalkanoate) films. J Biomed Mater Res Part B: Appl 2008;85B:564-72.
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surface-confined photopolymerization. J Biomed Mater Res Part A DOI: 10.1002/jbm.a.32009.
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151
13. Luo WJ, Li SM, Wang SG, Bei JZ. Synthesis and characterization of poly(L-
lactide)-poly(ethylene glycol) multiblock copolymers. J Appl Polym Sci 2002;84:1729-36.
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plasticized PLA: Is the miscibility the only significant factor? Polymer 2006;47:4676–82.
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152
APPENDIX C
PACLITAXEL ATTACHMENT TO PLA-PHBHHx BLEND FILMS
INTRODUCTION
Poly(lactic acid) PLA is an attractive biodegradable polymer since its mechanical
properties can be improved by blending with other biodegradable polymers like poly[(3-
hydroxybutyrate)-co-(3-hydroxyhexanoate)] (PHBHHx) [1-2]. However, surface
modification of the blend is extremely difficult due to the lack of any modifiable side
chain groups on PLA or PHBHHx. Hence a sequential two-step photografting approach
was used to graft poly(acrylic acid) (PAA) to blend films [3].
Paclitaxel is a widely used anti-cancer drug. It is favored due to its high efficacy
against a variety of cancers including: small and non-small cell lung cancer, ovarian
cancer, breast cancer, head and neck cancer, colon cancer, melanonma, and Kaposi’s
sarcoma [4]. There are several drug-delivery techniques, such as poly(ethylene glycol)
(PEG) based hydrogels [5-6], microspheres [7], and nanocarriers [8].
The overall objective of this small part of my research was to prepare Paclitaxel-
delivering PLA-PHBHHx blend films, focusing on drug attachment to the films. In this
research, Paclitaxel was covalently attached to PLA-PHBHHx blend films through an
easily hydolyzable ester bond using water soluble carbodiimide chemistry. ATR-FTIR
spectroscopy and thermogravimetric analyses (TGA) were used to characterize the drug-
attached films.
153
MATERIALS
Acrylic acid (99.5% w/w) and dimethyl sulfoxide (DMSO) were obtained from
Acros Organics. Benzophenone and HPLC water were obtained from Fisher Scientific.
Buffer solution of pH 7.2 was obtained from Invitrogen Corporation. Paclitaxel (> 99%
purity) was obtained from NATLAND International Corporation. N-hydroxysuccinimide
(NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were
purchased from Aldrich. All chemicals were used as received.
METHODS
The blend films (10 wt% PHBHHx) with a nominal thickness of 125 μm were
extruded using a single screw extruder. The acrylic acid monomers were photografted
using a previously designed sequential two-step photografting approach [3]. Briefly, a
film specimen was dip coated in a 5% w/w benzophenone solution in ethanol for one
minute. The film was then allowed to dry at room temperature to ensure that the ethanol
was evaporated. The film was subsequently exposed to UV irradiation in an inert
atmosphere for 5 min on each side. The resultant film was sonicated in ethanol to remove
unreacted benzophenone. Following UV exposure and sonication, the benzophenone-
grafted film was placed in a 10% v/v acrylic acid solution in water and exposed to UV
irradiation for 1.5 h. The film was then sonicated in water for 5 min in order to remove
154
excess monomer or physisorbed polymer. To ensure attachment of PAA chains, the films
were characterized using ATR-FTIR spectroscopy. The characterization was conducted
using a Nicolet Avatar 360 with a horizontal, multibounce ATR attachment.
The resulting PLA-g-PAA film was stirred with a 1% w/w NHS and 7.5% EDC
solution in water for 2 h. The film was then sonicated in water for 5 min to remove any
unreacted chemicals. The resulting film was stirred with a 0.25% w/w Paclitaxel solution
in DMSO for 3 h. The resulting film was sonicated in DMSO for 5 min to remove any
extraneous Paclitaxel.
RESULTS AND DISCUSSION
A sequential two-step photografting method was successfully employed to create
reactive PAA groups on film surface. These acid groups were subsequently linked to
Paclitaxel –OH groups as shown in Scheme C.1. Theoretically, Paclitaxel would be
attached to the film surfaces through an easily hydrolyzable ester bond.
Typical ATR-FTIR spectra of unmodified blend film, blend-g-PAA, and blend-g-
Paclitaxel are shown in Figure C.1. The unmodified blend film spectrum showed a peak
at 1756 cm-1 (spectrum C.1a) corresponding to the –C=O peak for the backbone ester in
PLA. Blend-g-PAA film spectrum showed a peak corresponding to the –C=O acid stretch
at 1720 cm-1 (spectrum C.1b). Blend-g-Paclitaxel film spectrum showed a peak at 1650
cm-1 corresponding to the –C=O amide (tertiary) stretch (spectrum C.1c). This confirmed
the covalent attachment of Paclitaxel to the PLA film.
155
EDC/NHS
2h 3h
PaclitaxelPoly(acrylic acid) Activated Acid Paclitaxel
EDC/NHS
2h 3h
PaclitaxelPoly(acrylic acid) Activated Acid Paclitaxel
Ph
O
NH
Ph
O
O
O
AcO O OH
HOAc
OCOPh
O
OH
C O
CH
CH2H2C
n
Ph
O
NH
Ph
O
O
O
AcO O OH
HOAc
OCOPh
O
OH
C O
CH
CH2H2C
n
Scheme C.1 Reaction scheme used to attach Paclitaxel to PLA-PHBHHx blend films.
156
0.00
0.04
0.08
Ab
sorb
ance
1400 1600 1800 2000 2200 2400 2600 2800
Wavenumbers (cm-1)
♦
●
■
(a)
(b)
(c)
0.00
0.04
0.08
Ab
sorb
ance
1400 1600 1800 2000 2200 2400 2600 2800
Wavenumbers (cm-1)
♦
●
■
(a)
(b)
(c)
Figure C.1 Representative ATR-FTIR spectra of (a) unmodified blend film, (b) blend-g-
PAA, and (c) blend-g-Paclitaxel. Spectrum (a) shows the “ester peak of PLA” at 1756
cm-1 (♦).Spectrum (b) shows the “acid peak of acrylic acid” at 1720 cm-1 (●). Spectrum
(c) shows “the amide peak of Paclitaxel” at 1650 cm-1 (■).
One crucial step towards biocompatibility of blend-g-Paclitaxel films would be to
determine the residual DMSO. TGA was employed to determine the amount of solvent
that remained in the film (Figure C.2). Paclitaxel-attached blend showed residual mass
around 20 wt%. This could primarily be DMSO. The presence of residual solvent is not
157
acceptable in terms of biocompatibility; as a result, it is essential to modify Paclitaxel
attachment chemistry using more benign solvents.
70
75
80
85
90
95
100
35 85 135 185
Temperature (°C)
Wei
gh
t %
(a)
(b)
Figure C.2 TGA curves of (a) unmodified blend film and (b) blend-g-Paclitaxel.
CONCLUSIONS
Reactive acid groups were successfully created on PLA-PHBHHx blend films
using a sequential two-step photografting method. Water soluble carbodiimide chemistry
was then used to attach the Paclitaxel. Paclitaxel was successfully attached to the blend
film surface; however, the chemistry used needs modification to employ more benign
solvents.
158
159
REFERENCES
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3. Janorkar AV, Metters AT, Hirt DE. Modification of poly(lactic acid) films: enhanced wettability from surface-confined photografting and increased degradation rate due to an artifact of the photografting process. Macromolecules 2004;37:9151-59.
4. Feng SS, Mu L, Win KY, Huang G. Nanoparticles of biodegradable polymers for clinical administration of paclitaxel. Current Medicinal Chemistry 2004;11:413-24.
5. Huynh DP, Shim WS, Kim JH, Lee DS. pH/temperature sensitive poly(ethylene glycol)-based biodegradable polyester block copolymer hydrogels. Polymer 2006;47:7918-26.
6. Iwasaki Y, Akiyoshi K. Synthesis and characterization of amphiphilic polyphosphates with hydrophilic graft chains and cholesteryl groups as nanocarriers. Biomacromolecules 2006;7:1433-38.
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