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DEVELOPMENT OF ANTIMICROBIAL COPOLYMERS FOR POLYMERS AND
FIBERS
by
VIKRAM P. DHENDE
(Under the Direction of Ian R. Hardin)
ABSTRACT
Commercial applications of antimicrobial agents include textiles, food packaging
and storage, the shoe industry, water purification, medical devices, and dental surgery
equipment. There are ranges of antimicrobial agents with differing chemistries available
commercially today and many more are being studied in academic laboratories.
Quaternary polymeric compounds have proven to be very effective antimicrobial agents
because of their unique structural properties. The scientific literature is replete with
reports of surface modifications of different substrates by covalent attachment of these
biocidal polymers. However, covalent surface attachment of biocidal polymers on
common inert plastic materials such as polypropylene, polyethylene and polyvinyl
chloride is very challenging with very few examples in the literature. In our study, we
have successfully synthesized quaternary polyethylenimine based copolymers with a
photoactive benzophenone pendant group that allows covalent attachment of copolymer
on any surface with C-H bonds upon irradiation with mild UV light. The coating showed
impressive antimicrobial activity against both Gram positive and Gram negative bacteria.
A simple spray application technique was used to coat the substrates uniformly with
copolymer to create permanent ultrathin biocidal coating.
The other section of the work includes modification of natural fiber such as cotton
with biocidal polymers. The main aim of the work was to design and optimize novel
reactive copolymers, which can be applied on cellulosic textile materials using a simple
application method such as the exhaustion. In this research, the goal was to incorporate
hydroxy reactive groups on the backbone of quaternary polyethyleneimine (PEI)
polymers. The covalent attachment of antimicrobial agent with fiber would improve its
durability and avoid its release in the environment. To this end, three new copolymers
were synthesized namely sulfated quaternary based PEI (SQ-PEI), monochlorotriazine
based quaternary PEI (MCT-PEI) and dichlorotriazine based (DCT-PEI) which contain
both fiber reactive vinyl sulfone, monochloro and dichlorotriazine side chains,
respectively, and hydrophobic side chains (dodecane, C12). The polymeric antimicrobial
agents were chosen because they have the advantages of being non-permeable through
the skin, non-volatile, stable, efficient and selective. These polymers were tested for their
antimicrobial properties and durability to accelerated laundering.
INDEX WORDS: Antimicrobial, polyethylenimine, photo-crosslinking, vinyl
sulfone, triazine, plastics, textiles
DEVELOPMENT OF ANTIMICROBIAL COPOLYMERS FOR POLYMERS AND
FIBERS
by
VIKRAM P. DHENDE
B. Tech., University Institute of Chemical Technology, Mumbai, India, 2003
M. Tech., University Institute of Chemical Technology, Mumbai, India, 2005
A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial
Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
ATHENS, GEORGIA
2011
© 2011
Vikram P. Dhende
All Rights Reserved
DEVELOPMENT OF ANTIMICROBIAL COPOLYMERS FOR POLYMERS AND
FIBERS
by
VIKRAM P. DHENDE
Co-Major Professor: Ian R. Hardin
Jason Locklin
Committee: Suraj Sharma
Wendy Dustman
Charles Yang
Electronic Version Approved:
Maureen Grasso
Dean of the Graduate School
The University of Georgia
August 2011
iv
To
Pratap and Malti Dhende, my beloved parents
v
ACKNOWLEDGEMENTS
First and foremost, I would like to extent my deepest gratitude to my major
advisor Dr. Ian Hardin, for his invaluable support, constant encouragement and guidance
throughout my doctoral work. He stood by me through thick and thin. As a mentor, he
has motivated me to see life and science in their full depth. I have thoroughly enjoyed
working with him. Thank you for believing in me.
I would like to thank my co-advisor, Dr. Jason Locklin for giving me the
opportunity to work in his group and relentless help. He has been inspirational to me for
developing and applying the scientific knowledge.
Special thanks to my committee members, Dr. Suraj Sharma, Dr. Wendy
Dustman, and Dr. Charles Yang for providing valuable suggestions, ideas and support
during my work. Thanks for their time and willingness to assist me in the past three
years. I am grateful to the faculty, staff and friends in the department of Textiles,
Merchandising and Interiors, Susan, Mary, Renuka, Diane, and Barbara.
I want to thank all my group members in Dr. Locklin’s lab, Sara, Kristen, Nick,
Gareth, Kyle, Shameem, Joe, Evan, Rachelle, and Jenna. It has been wonderful
experience working with you guys!! I want to thank Dr. Samanta for all the help and
teaching in my initial days at Dr. Locklin’s group.
A big thank you to Kamal, Vijay and Sujit for their support and all the help. I also
want to thank all my friends in Athens, Shaku, Sonu, Kishor, Anand, Finto, and Reben
for making my time in Athens memorable.
vi
Last, but not least, my special thanks to my family for their unconditional love,
blessings and support.
vii
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS .................................................................................................v
LIST OF TABLES ........................................................................................................... viii
LIST OF FIGURES ........................................................................................................... ix
LIST OF SCHEMES........................................................................................................ xiv
CHAPTER
1 INTRODUCTION AND LITERATURE REVIEW .........................................1
References ..................................................................................................41
2 PHOTOCROSSLINKABLE ANTIMCROBIAL COPOLYMER ..................62
Materials and Methods ...............................................................................66
Results and Discussion ..............................................................................72
References ..................................................................................................84
3 REACTIVE ANTIMICROBIAL COPOLYMERS FOR TEXTIL FIBERS...90
Material and Methods ................................................................................93
Results and Discussion ............................................................................104
References ................................................................................................113
4 SUMMARY ...................................................................................................114
viii
LIST OF TABLES
Page
Table 2.1: Antimicrobial test with S. aureus along with percent bacterial
reduction. There were four sets of samples tested: (1) Control
glass substrate with OTS coated SAM, (2) spin coated glass
substrate with 5 mg/mL polymer concentration, (3) spin coated
glass substrate with 10 mg/mL polymer, and (4) spin coated glass
substrate with 15 mg/mL concentration. ..........................................................77
Table 2.2: Antimicrobial test with E. coli along with percent bacterial
reduction. There were four sets of samples tested: (1) Control
glass substrate with OTS coated SAM, (2) spin coated glass
substrate with 5 mg/mL polymer concentration, (3) spin coated
glass substrate with 10 mg/mL polymer, and (4) spin coated
glass substrate with 15 mg/mL concentration. .................................................79
Table 3.1: Percentage nitrogen content on the treated and untreated
fabrics before wash and after wash by elemental analysis ............................107
ix
LIST OF FIGURES
Page
Figure 1.1: Structure of poly (tributyl-4-vinyl benzyl phosphonium chloride) ..................4
Figure 1.2: Quaternary N-hexyl PVP ..................................................................................5
Figure 1.3: Derivatives of DMAEMA ................................................................................6
Figure 1.4: Structure of poly (2-methyl-1, 3-oxazoline)s (PMOX) polymer .....................7
Figure 1.5: Synthetic mimics of antimicrobial peptides .....................................................8
Figure 1.6: Structure of 3-(trimethoxysilyl)-propyldimethylalkyl ammonium
chlorides .......................................................................................................9
Figure 1.7: Immobilization of quaternary PEI on NH2 glass ............................................10
Figure 1.8: Surface modification of textile fibers with quaternary PEI ............................10
Figure 1.9: Modification of polyolefin film with quaternary PEI .....................................11
Figure 1.10: Quaternary PEI nanoparticle .........................................................................12
Figure 1.11: PEI-bound anthraquinone derivative .............................................................13
Figure 1.12: Modified surface with quaternized DMAEMA .............................................14
Figure 1.13: Modified surface with SMAMPs ..................................................................15
Figure 1.14: Functionalization with quaternary groups on filter paper .............................15
Figure 1.15: Fluorinated pyridinium block copolymers ....................................................16
Figure 1.16: Quaternary N-[3-(methacryloylamino)propyl]-N,N-
dimethyldodecylammonium bromide based antibacterial monomer ............16
Figure 1.17: Vapor crosslinking method ...........................................................................17
x
Figure 1.18: Poly (DMA-MEA-DMAEMAC12) random copolymer ................................18
Figure 1.19: Structures of a) cetylpyridinium chloride (CPC) and b)
benzyldimethyl-hexadecylammonium chloride (BDHAC) .........................22
Figure 1.20: Modification of PP with cationic derivative of
2N-morpholino ethyl methacrylate (MEMA) ..............................................23
Figure 1.21: Structure of N-dodecyl-N, N-dimethyl glycine
cystearnine hydrochloride (DABM) ...........................................................24
Figure 1.22: Structure of 3-trimethoxysilylpropyldimethyloctadecyl
ammonium chloride .....................................................................................24
Figure 1.23: Structure of polycationic imidazolium-modified polysiloxane ....................25
Figure 1.24: Deacetylation of chitin .................................................................................26
Figure 1.25: Structure of N-(2-hydroxy) propyl-3-trimethylammonium
chitosan chloride ............................................................................................27
Figure 1.26: Methylated N - (4-N, N -dimethylaminobenzyl) chitosan chloride
(MDMBzCh) ...................................................................................................27
Figure 1.27: Methylated N - (4-pyridylmethyl) chitosan chloride (MPyMeCh) .............27
Figure 1.28: Structure of NMA-HTCC ..............................................................................28
Figure 1.29: Structure of PHMB ........................................................................................29
Figure 1.30: Structure of chlorhexidine .............................................................................30
Figure 1.31: Regenerable antimicrobial activity of N-halamine ........................................31
Figure 1.32: Polymerizable N-halamine derivatives, a) 3-(4'-vinylbenzyl)-5,5-
dimethylhydantoin, b) N-chloro-2,2,6,6-tetramethyl- 4-piperidinyl
methacrylate ..................................................................................................32
xi
Figure 1.33: Regenerable antimicrobial activity of peroxyacids .......................................33
Figure 1.34: Structure of triclosan (2, 4, 4’-trichloro-2’-hydroxydiphenyl ether) ............34
Figure 1.35: Structure of synthetic cationic aminoanthraquinone dye ..............................36
Figure 1.36: Structure of cationic reactive dye ..................................................................36
Figure 1.37: Structure of cationic monoazo dye ................................................................36
Figure 1.38: Structure of Berberine chloride ....................................................................37
Figure 1.39: Structure of curcumin ....................................................................................37
Figure 2.1: Photoreaction of benzophenone (BP) .............................................................65
Figure 2.2: Change in UV spectra of benzophenone in polymer 2 with UV exposure
with time (365 nm)........................................................................................74
Figure 2.3: FTIR spectra of a thin film of copolymer 2, before (A) and after (B) UV
exposure ........................................................................................................75
Figure 2.4: Tapping mode AFM image for the film of copolymer 2 (A) as cast before
sonication (thickness 93 nm, RMS roughness 0.48 nm) and (B) after
sonication (thickness 77 nm, RMS roughness 0.83 nm) ..............................76
Figure 2.5: Digital pictures of the glass substrates sprayed with S. aureus and
incubated for 24 hours at 37°C (A) Control glass slide and
(B) polymer coated glass slide ......................................................................78
Figure 2.6: Digital pictures of the glass substrates sprayed with E.coli and incubated
for 24 hours at 37°C (A) Control glass slide and (B) polymer coated glass
slide ...............................................................................................................80
Figure 2.7: Digital pictures of the textiles and plastic substrates sprayed with S.
aureus. (A) untreated cotton, (B) cotton sprayed coated with 15 mg/mL
xii
polymer 2, (C) untreated polypropylene (nonwoven geotextile fabric), (D)
polypropylene spray-coated with 15 mg/mL polymer 2, (E) untreated
poly(vinyl chloride) substrate, (F) poly(vinyl chloride) substrate spray
coated with 15 mg/mL polymer 2, (G) untreated polyethylene substrate, and
(H) polyethylene substrate spray coated with 15 mg/mL polymer 2 ...........81
Figure 2.8: Biofouling testing in the ocean water off the coast of Chile
for 50 days ....................................................................................................83
Figure 2.9: Biofouling testing in the ocean water off the coast
of Canada for 40 days ...................................................................................83
Figure 3.1: FTIR spectra (a) Quaternary PEI (b) Sulfated quaternary
PEI (SQ-PEI) .............................................................................................105
Figure 3.2: Finishing of SQ-PEI with cotton fabric under alkaline conditions ..............106
Figure 3.3: Finishing of MCT-PEI with cotton fabric under alkaline conditions ............106
Figure 3.4: Untreated cotton fabrics (a) control S. aureus and (b) control E. coli ..........109
Figure 3.5: Evaluation against S. aureus, (a) treatment with SQ-PEI before washing
(b) treatment with SQ-PEI after washing ....................................................109
Figure 3.6: Evaluation against E.coli, (a) treatment with SQ-PEI before washing
(b) treatment with SQ-PEI after washing ....................................................110
Figure 3.7: Evaluation against S. aureus, (a) treatment with MCT-PEI before washing
(b) treatment with MCT-PEI after washing ................................................110
Figure 3.8: Evaluation against E. coli, (a) treatment with MCT-PEI before washing
(b) treatment with MCT-PEI after washing ................................................111
Figure 3.9: Evaluation against S. aureus, (a) treatment with DCT-PEI before washing
xiii
(b) treatment with DCT-PEI after washing .................................................111
Figure 3.10: Evaluation against E. coli, (a) treatment with DCT-PEI before washing
(b) treatment with DCT-PEI after washing .................................................112
xiv
LIST OF SCHEMES
Page
Scheme 2.1: Synthesis of 4-[(6-Bromohexyl) oxy] benzophenone ..................................69
Scheme 2.2: Synthesis of linear copolymer N, N-dodecyl methyl and
N, N-[(6-hexyl)oxy] benzophenone methyl PEI ...........................................70
Scheme 2.3: Surface attachment of benzophenone-PEI copolymer ..................................73
Scheme 3.1: Synthesis of linear PEI .................................................................................96
Scheme 3.2: Synthesis of 4-(2-hydroxyethansulfonyl) phenol .........................................98
Scheme 3.3: Synthesis of 2-(4-(6-bromohexyloxy) phenylsulfonyl) ethanol ....................99
Scheme 3.4: Synthesis of quaternary PEI copolymer ........................................................99
Scheme 3.5: Sulfation of quaternary PEI .........................................................................100
Scheme 3.6: Synthesis of 6-bromohexan-1-ol .................................................................101
Scheme 3.7: Synthesis of 4-(4, 6-dichloro-1,3,5-triazin-2-ylamino)
benzenesulfonic acid ...................................................................................101
Scheme 3.8: Synthesis of hydroxy based quaternary PEI copolymer .............................102
Scheme 3.9: Synthesis of monochlorotriazine based quaternary PEI copolymer ...........103
Scheme 3.10: Synthesis of dichlorotriazine based quaternary PEI copolymer ...............103
1
CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW
2
Microbes are ubiquitous, present everywhere, on the surface of the skin and in the
gut of human beings to deep inside the earth’s crust and ocean floor. Microbes play major
role in the proper function of the biosphere. They are diverse in nature, and include
bacteria, fungi, algae, protists and archaea. The harmful effect of microbes on textiles and
food has been known for a long time, but it was Anton van Leeuwenhoek (1632-1723)
who observed and described microorganisms for the first time. He used his self-made
microscope to establish the existence of the life forms which were not visible to the
naked eye. Later in the eighteenth century, Lazzaro Spallanzani, Louis Pasteur and
Robert Koch established through their experiments that microbes can cause diseases. In
the same century, some chemical agents were developed to act against bacteria. In 1895,
8-hydroxyquinoline was sold as an antiseptic and was later used on cellulosic materials as
a microbiocide. [1]
An antimicrobial agent is defined as a substance which kills or inhibits the growth
of microbial cells. There are two general types of antimicrobial agents; one that kills a
microbe is called a microbiocide and one that stops the growth of microbes is called a
microbiostat. Antimicrobial agents play a vital role in areas such as health care, hospitals,
food packaging and storage, water purification, dental care, and household sanitation. [2]
Recent survey by Global Industry Analysts, Inc. (GIA) predicted that the US
antimicrobial coatings market would reach US $978.7 million by 2015. [3]
There are several strategies used to create polymeric antimicrobial materials. One
approach is to dope the polymers with organic or inorganic biocides during processing.
Doping the polymers such as polyethylene or polypropylene with silver nanoparticles by
a melt blending process is an example of this strategy.[4] The second approach is to treat
3
the polymer with biocides after polymer processing. The third approach is polymerizing
monomers which contain biocide groups or copolymerizing with another monomer. The
final approach is to use a grafting technique to endow the polymer with antimicrobial
properties. In this technique antimicrobial polymers can be immobilized on natural or
synthetic polymers.
In the last few years there have been many efforts to understand the effects of
structural parameters on antimicrobial activity. Factors such as molecular weight, alkyl
chain length, hydrophobic/hydrophilic balance and counterions have been studied
extensively in cationic polymer systems.
Molecular weight
Molecular weight has been shown to alter the antimicrobial performance of the
polymer. Ikeda and co-workers explored the effect of molecular weight of
polymethacrylate containing biguanide and poly(vinylbenzyl ammonium chloride)
polymer systems. They found that the optimum molecular weight range for treatment
against S. aureus was between 5×104 to 10×10
4 Da.[5] Cooper and his group carried out
structure activity studies of the quaternary poly(propylene imine) and demonstrated
molecular weight between 5×104 to 1.2×10
5 Da to be optimum for effective antimicrobial
activity against both Gram positive (S. aureus) and Gram negative (E. coli) bacteria.[6]
Klibanov et al. demonstrated the effect of molecular weight on antimicrobial
activity in N-alkylated PEI polymer systems.[7] The N-alkylated PEI polymers of 750
kDa and 25 kDa molecular weights immobilized on amino-glass slides were found to be
4
lethal against S. aureus. Similar polymer systems with the lower molecular weights of
2kDa and 0.8 kDa were found to be less effective.
Matyajaszewski and co-workers showed that poly(2-(dimethylamino) ethyl
methacrylate) (PDMAEMA) polymers with molecular weight of 10 kDa have excellent
antimicrobial performance. However, polymers with a molecular weight of 1.5kDa with
the same graft density showed poor antimicrobial activity. [8]
Kanazawa et al. demonstrated that poly (tributyl-4-vinyl benzyl phosphonium
chloride) (Figure 1.1) in a saline solution had effective antimicrobial efficacy between the
molecular weight range of 1.6×104 to 9.4×10
4 Da.[9]
Figure 1.1: Structure of poly (tributyl-4-vinyl benzyl phosphonium chloride)
It is generally hypothesized that polycations, due to their increased hydrophobic
mass and greater net charge per molecule compared to monomers result in greater
binding interaction with the microbial cell membrane/wall which enhances the
antimicrobial performance. [10] However, it is reasonable to state that molecular weight
effect on antimicrobial efficacy also depends on the particular polymer system used.
5
Alkyl chain
The alkyl chain attached to the active site (e.g. quaternary nitrogen or
phosphorous) is believed to alter the antimicrobial performance of biocidal polymers.
Ikeda et al. reported that poly(trialkylvinylbenzylammonium chloride) polymer with a
linear alkyl chain of twelve carbons (C12) attached to nitrogen had enhanced
antimicrobial activity compared to other chain lengths such as -(CH2)3CH3, -CH(CH3)2,
and -CH2CH3.[11] Klibanov and co-workers showed that hydrophobic alkyl chains have
an important role in biocidal activity, however very long alkyl chains can have a
detrimental effect on the performance of polymer. The loss in performance was attributed
to aggregation of long alkyl chains within the polymer molecules, altering its interaction
with the microbial membrane. In their work, PVP polymers with alky chains of C6
(Figure 1.2) were found to be effective. [12]
Figure 1.2: Quaternary N-hexyl PVP
The study of the quaternized poly (propylene imine) dendrimer system by Cooper
and co-workers showed that the C10 hydrophobic alkyl chain had the best performance
among C8, C10, C12, C14, and C16 chains. The antimicrobial activity of the alkyl chains in
the quaternary amine groups followed a parabolic relationship. [6] Fu and co-workers
four examined quaternary dimethylaminoethyl methacrylate (DMAEMA) (Figure 1.3)
based monomers and their homopolymers. The alkyl substituent attached to the nitrogen
6
in each of these polymers contained benzyl, butyl (C4), dodecyl (C12), and hexadecyl
(C16) units. The homopolymers had higher antibacterial efficacy than their respective
monomers. In the case of homopolymers the antibacterial efficacy increased with
increase in the chain length of the substituents, following the order of poly (DMAEMA-
BC) > poly (DMAEMA-BB) > poly (DMAEMA-DB) and poly (DMAEMA-HB). [13]
Figure 1.3: Derivatives of DMAEMA
The effect of the hydrophobic side chain in systematically synthesized poly (2-methyl-1,
3-oxazoline)s (PMOX) polymer systems was studied by Tiller and co-workers. The
PMOX polymers with varying lengths of satellite (non-bactericidal groups) groups
(methyl, decyl, and hexadecyl) and N,N-dimethyldodecyl ammonium (DDA)
(bactericidal group) end groups were synthesized as shown in the Figure 1.4. It was found
that the antibacterial performance of polymer (A) was greater than (B) against E. coli;
polymer (C) had very low antibacterial activity. The experimental results on aggregation
behavior of these polymers in solution and their interactions with model liposomes
indicated that aggregation of the polymers was not responsible for their varying
antibacterial performances. The study also suggested that satellite groups had control
over the antibacterial function of DDA, and the low activity of polymer (C) was
attributed to prevention of penetration of DDA by long hexadecyl satellite groups. [14]
7
Figure 1.4: Structure of poly (2-methyl-1, 3-oxazoline)s (PMOX) polymer
It should be noted that the alkyl chain length also has an effect on the overall
hydrophilic/hydrophobic balance and charge density of the polymer, which determines its
interaction with the microbial cell membrane and can lead to variation in antimicrobial
performance. [10, 15]
Counterion
The earlier work done by Panarin et al. reported that counterions such as chloride,
bromide or iodide had no significant effect on antibacterial efficacy of homopolymers of
vinylamine and aminoalkyl methacrylates with pendant quaternary ammonium salts.[16]
Kanazawa et al. in the study of the poly(tributyl-4-vinyl benzyl phosphonium
chloride) polymer system demonstrated the effect of a counterion, along with molecular
weight (Figure 1.1). Four different counterions Cl, BF4, ClO4, and PF6 were analyzed.
8
The antibacterial activity against S. aureus was influenced by the tightness of the ion pair
with the phosphonium ion. Antibacterial activity followed a trend of Cl > BF4 > ClO4 >
PF6 and indicated that tighter ion pairs were less effective. This could be due to change in
their polymer’s solubility.[9]
The structure-activity study of quaternized poly (propylene imine) dendrimer
systems conducted by Cooper et al. reported that bromide counterion was more potent
than the chloride counterion. [6]
Recent work by Tew and Nusslein on poly (oxanorbornene) based synthetic
mimics of antimicrobial peptides (SMAMPs) (Figure 1.5) demonstrated the effect of
organic counterions on antibacterial efficacy. Organic counterions such as
trifluoroacetate, benzonate, tosylate, hexanoate and dodecanoate were analyzed for their
membrane activity towards model vesicles (S. aureus mimics). It was observed that
hydrophobic counterions form a tight ion pair which led to reduced antibacterial activity,
as observed by Kanazawa et al. in the case of inorganic counterions.[17]
Figure 1.5: Synthetic mimics of antimicrobial peptides
Non-leachable antimicrobial coatings on surfaces
A contact active non-leachable type of biocidal coating was developed as early as
the 1970s. In 1972, Walters and co-workers from Dow Corning Corporation synthesized
3-(trimethoxysilyl)-propyldimethylalkyl ammonium chlorides (Figure 1.6) with alkyl
9
chains containing 6 to 22 carbons. The organiosilicon quaternary ammonium salt was
attached to glass surfaces and to cotton cloth. [18]
Figure 1.6: Structure of 3-(trimethoxysilyl)-propyldimethylalkyl ammonium
chlorides
In the last few years, there has been increasing interest in non-leachable
polymeric antimicrobial coatings due to their wide applications, unique properties and
environmental benefits. [15, 19] Pioneering work was done by Klibanov and co-workers
on creating antimicrobial surfaces through hydrophobic polycations. Klibanov et al.
showed immobilization of quaternary PEI on NH2-glass slides and Fe3O4 nanoparticles.
Glass slides were treated with 4-bromobutyryl chloride, followed by treatment with PEI.
The immobilized PEI was further treated with alkyl bromide and finally quaternized with
iodomethane (Figure 1.7). The NH2 containing Fe3O4 nanoparticles were modified with a
similar procedure. The derivatized substrates demonstrated antibacterial performance
against both Gram positive and Gram negative bacteria.[20]
10
Figure 1.7: Immobilization of quaternary PEI on NH2 glass. Figure from reference [20].
A similar synthetic strategy was employed to modify textile fibers such as cotton,
polyester, nylon and wool (Figure 1.8). The treated fibers showed biocidal activity
against bacteria as well as fungi.
Figure 1.8: Surface modification of textile fibers with quaternary PEI. Figure from
reference [21].
In the case of polyolefin, quaternary PEI was immobilized using a free radical
grafting technique. The polyolefin film/fabric was treated with an ethyl acetate solution
11
which contained maleic anhydride and the free-radical polymerization initiator 2, 2 -
azobisisobutyronitrile (AIBN) at 60°C overnight.
Figure 1.9: Modification of polyolefin film with quaternary PEI. Figure from reference
[22].
The film/fabric was washed with ethyl acetate and further treated with a
dimethylformamide (DMF) solution of PEI at 90°C for overnight. The grafted PEI was
finally treated with bromohexyl and iodomethane, respectively to create antimicrobial
coating (Figure 1.9).
Park et al. created antimicrobial coatings of glass and polyethylene substrates by
simple dip coating method in organic solution of the N-alkyl-PEI polycations. The
procedure did not use an elaborate multistep synthesis or surface modification steps, but
involved very practical approach such as painting. The coating showed excellent
antimicrobial efficacy but was not covalently attached to the surface and therefore the
durability of coating was questionable.[23] Further work done by Haldar et al. and
Larson et al. on N-alkylated-PEI polycations showed that the glass slides or polyethylene
substrates coated with the same were capable of significantly eliminating influenza A
virus, (non-enveloped) poliovirus and rotavirus.[24-26]
Quaternary PEI nanoparticles used in the application of dental restorative resins
were reported to have good antibacterial efficacy that lasted at least one month.[27-29]
Recently, Yudovin-Farber et al. showed a synthetic strategy to create quaternary PEI
12
nanoparticles by using two synthetic methods, namely reductive amination and N-
alkylation, followed by N-alkylation with iodomethane. Among the synthesized QA-PEIs
nanoparticles (Figure 1.10) with varying ratios of primary amine of PEI and alkylating
agents, a 1:1 molar ratio of octyl alkylated QA-PEI was found to be the most potent. [30]
Figure 1.10: Quaternary PEI nanoparticle. Figure from reference [73].
Quaternary PEIs have been utilized in biofilm reduction applications. According
to Nelis and co-workers [31], quaternary PEIs may find future applications in coating of
medical devices such as catheters and prostheses. In their work, quaternized
dimethylaminoethylmethacrylate (DMAEMA) and PEI were grafted on to
polydimethylsiloxane (PDMS) disks. The PDMS disks were treated with oxygen plasma
and subsequently oxidized chemically to attach quaternary DMAEMA and PEI on to the
surface. The modified disks showed reduction in accumulation of Candida albicans cells.
These cells are known to form biofilms on medical prostheses.
Klibanov and Hammond et al. followed a layer by layer (LBL) approach to create
ultrathin films on silicon substrates. The polycations were synthesized by N-alkylation of
PEI and quaternization with iodomethane. Alkyl chains of varying lengths were used,
namely, dodecyl, hexyl, butyl, and methyl. Poly (acrylic acid) was used as a polyanion.
13
The LBL films of 10nm thickness showed excellent antimicrobial activity and were lethal
against a strain of influenza virus, Gram positive and Gram negative bacteria. [32]
Bilyk et al. synthesized poly (ethylene imine) (PEI) polymers with anthraquinone
(AQ) moieties as pendant groups (Figure 1.11). The copolymer was applied through a
methoxyethanol solution to a corona activated low density polyethylene (LDPE), and air
dried for 1 hour. The anthraquinone pendant moiety underwent photoreduction on
exposure to low energy UV light to generate hydrogen peroxide on the coatings on
exposure to air.
Figure 1.11: PEI-bound anthraquinone derivative. Figure from reference [33].
The authors expected the hydrogen peroxide produced from the coatings to work
as a biocidal.[33]
Matyjaszewski and Russell described the polymerization of 2-(-
dimethylaminoethyl methacrylate) (DMAEMA) using a living polymerization technique
such as atom transfer radical polymerization (ATRP). The low polydispersity polymers
were developed on Whatman #1 filter paper and on amino reacted glass slides. The
14
tertiary amino group from the DMAEMA was quaternized with ethyl bromide (Figure
1.12). The modified substrates showed excellent antibacterial performance. [34]
Figure 1.12: Modified surface with quaternized DMAEMA. Figure from reference [6].
Murata and co-workers synthesized poly (quaternary ammonium) compounds
which can be used as antimicrobial polymeric brushes on inorganic surfaces. In their
work, N, N- dimethylaminoethyl methacrylate (DMAEMA) monomer was polymerized
using atom transfer radical polymerization (ATRP) technique to create polymer brushes
of precise molecular weights and densites. They also found out that short chains with a
high grafting density and long chains with low grafting density were similar in their
performance against E. coli.[35]
Tew and co-workers used ATRP technique to synthesize new synthetic mimics of
antimicrobial peptides (SMAMPs) based on poly (butylmethacrylate)-co-poly(Boc-
aminoethyl methacrylate) (Figure 1.13). The co-polymers were developed on silicon
wafers and glass slides.
15
Figure 1.13: Modified surface with SMAMPs. Figure from reference [4].
The authors reported that the synthesized SMAMPs can kill bacteria in less than 5
mins upon contact and the antimicrobial activity of these polymers is independent of
polymer chain length and grafting density. [36]
Jampala and co-workers used cold plasma technology to activate stainless steel
and cellulose surfaces. The cellulose substrate surface was first activated by an ethylene
diamine plasma deposited film; its reaction with hexyl bromide and subsequently
quaternization with methyl iodide created a quaternary amine group (Figure 1.14).[37]
Figure 1.14: Functionalization with quaternary groups on filter paper. Figure from
reference [37].
Ober and co-workers developed pyridinium block copolymers with fluorinated
side chains. The copolymers were immobilized on polystyrene-b-poly (ethylene-ran-
butylene)-b-polystyrene (Figure 1.15) coated glass slides by spray coating and heating at
80°C. The study demonstrated that fluorinated copolymers had enhanced antimicrobial
activity compared to their corresponding non-fluorinated copolymers.[38] The higher
16
antimicrobial efficiency was attributed to fluorinated groups because of their inherent
rigidity and hydrophobicity.
Figure 1.15: Fluorinated pyridinium block copolymers [38]
An acrylate based contact active polymeric network (Figure 1.16) was
synthesized by Tiller and co-workers. The polymer was attached to a methacrylate
modified glass slide by photopolymerization. The coated material was tested against S.
aureus with excellent results. [39]
Figure 1.16: Quaternary N-[3-(methacryloylamino)propyl]-N,N-
dimethyldodecylammonium bromide based antibacterial monomer [39]
Chemical vapor deposition technique has been utilized to create antimicrobial
surfaces for fragile substrates. Gleason and co-workers used the technique to coat
quaternary poly(dimethylaminomethyl styrene) on fabric. [40] The same technique was
17
followed by Mao and co-workers to fabricate non-leaching antibacterial surfaces in a
single-step vapor crosslinking method (Figure 1.17). The vaporized monomers of
quaternary dimethylaminomethylstyrene (DMAMS) and ethylene glycol diacrylate
(EGDA) at 75 and 60°C respectively, along with vaporized tert-butyl peroxide (TBP)
initiator at room temperature were fed into a reactor in a controlled fashion in the
proximity (25 cm) of the substrates. The substrates (nylon fabric) were kept at 45-48°C
and a deposition of 800 nm ± 20 nm was achieved which was monitored by comparison
to deposition on an adjacent on silicon wafer.[41]
Figure 1.17: Vapor crosslinking method. Figure from reference [41].
Recently, an amphiphilic random copolymer from monomers containing dodecyl
quaternary ammonium, methoxyethyl and catechol groups (Figure 1.18) was synthesized
by Kuroda and co-workers. The polymer was applied on a glass substrate by a simple dip
coating method, followed by drying and heat fixing. The catechol group acted as an
adhesive or cross-linking moiety for the copolymer, preventing its leaching from the
surface. [42]
18
Figure 1.18: Poly (DMA-MEA-DMAEMAC12) random copolymer
It has been demonstrated that the polymer structural parameters such as molecular
weight, alkyl chain length, charge density and counterions have an impact on
antimicrobial performance of the polymers and it is also important to note that efficacy is
also largely dependent upon the specific polymer system used. In our work, we chose PEI
based system as it has been demonstrated in the literature that they have excellent
antimicrobial performance against wide range of microbes and kill bacteria upon contact.
Recently, the quaternary PEI with linear side chain of twelve carbons (-C12H25) have been
found to be one of the potent quaternary PEI polymers [23, 43] which was utilized in our
synthetic approach. The synthetic approach also included modification of linear PEI
polymer instead of branched PEI because of its ease of structural analysis and
characterization. The current scientific literature reports many non-leachable surface
attached biocidal polymer systems but almost all require elaborate synthetic strategies
and substrate modifications for covalent immobilization of biocidal polymers.
Antimicrobial agents in textile:
An ideal antimicrobial agent used in textile finishing should have following
properties:
19
1. Be effective against a broad spectrum of microbes and microbes should not
become immune to the agent
2. Be durable to washing, dry cleaning, and hot pressing
3. Possess low toxicity to humans; should not cause allergic reactions
4. Be compatible with other textile finishes, e.g. flame-retardant, dyes, and water
repellents
5. Ease of application; agent can be applied through standard textile machinery
6. Not be harmful to the environment
7. Not affect quality or physical properties of the textile
8. Be cost-effective
The following section includes a brief review of traditional antimicrobial agents
used in textiles as well as new agents which are being explored at an academic level.
Metal ions
The antimicrobial actions of many heavy metals and their ions are still not clearly
understood. The proposed mechanisms for this antimicrobial activity include denaturing
of deoxyribonucleic acid (DNA) through binding with metal ions, damaging adenosine
triphosphate (ATP) synthesis by binding action on the ATP synthesis enzyme in the cell
wall, disruption of important physical structures in the cell and interruption of the
respiratory functions of micro-organisms [44, 45]. A variety of metals and their oxides
have been explored for antimicrobial finishing of textiles. Some of these include silver,
titanium dioxide (TiO2) [46, 47], zinc [48-50], copper [51, 52] and cobalt [53]. Among
the antimicrobial metal ions, silver is widely used in textiles due to its high efficiency and
20
low toxicity to humans. The studies showed that metal nanoparticles are found to be more
effective compared to the bulk material. The increased antimicrobial efficacy is attributed
to small particle size which provides large specific surface area that leads to greater
interaction with micro-organisms [54]. The silver nanoparticles have been applied on
cotton[55, 56], cotton/ polyester (PET) blends [57], PET, polypropylene (PP) [58],
polyethylene (PE) [59] based textile materials; in most cases the finish was applied by a
padding technique. Most of the synthetic fibers can also be modified and made
antimicrobial by doping the polymer with silver particles before extrusion [60]. The
finished material loses antimicrobial efficacy slowly due to gradual release of the finish.
Polyacrylonitrile (PAN) fibers were finished with TiO2 solution by a dip-coating method
at low temperature. This showed UV-protection properties and, according to the report,
the finished material can be a potential antimicrobial candidate [61]. Antimicrobial rayon
fibers were prepared by adding TiO-SiO complexes of two different sizes (30 nm and
90nm) to a spinning solution of rayon. It was reported that rayon fibers modified with the
30nm particle size of TiO-SiO complex showed better performance than the 90 nm
particle size [62, 63].
In order to improve the durability of antimicrobial finishes to washing and to
prolong the release of metal nanoparticles, several techniques have been reported. These
include treatment of cellulosics with polyvinyl pyridine [64] or succinic acid anhydride
[65] which immobilized Ag ,Cu or Zn ions on the fabric. The other notable technique
reported is use of a sol-gel technique to entrap Ag+ ions in silica matrix [66-68]. In the
case of protein fibers [69-71], it is reported that apart from free carboxylic groups present
in the fibers for metal binding, the number of sites were further increased by treatment
21
with tannic acid or ethylenediaminetetraacetic (EDTA) dianhydride which helped to
chelate metal ions and thus immobilize metal ions on the fiber. The major limitation of
metal finishes is environmental problems and recently concerns were raised regarding the
use of silver in the finishing due to development of resistant strains of microbes [72].
Quaternary compounds
Quaternary ammonium compounds (QACs) are well known important biocides
which have been used for many years. In fact, the first industrial production of
antimicrobial textiles using QACs in the late 1930s for German and US army uniforms to
reduce odor and infections [73]. QACs are effective against a wide variety of microbes
such as Gram-positive and Gram-negative bacteria, fungi and certain classes of viruses
[37, 74-76]. QACs generally contain four organic substituents covalently attached to a
nitrogen atom which can be similar or dissimilar in properties [77]. Bioactivity of these
agents depend upon the type of substituent, the number of quaternary nitrogen atoms and
counterions. The exact mechanism of the antimicrobial action of QACs is still under
debate but it is widely believed that the QACs damage the cell membrane of microbes
[78, 79]. According to the generally accepted hypothesis, the long hydrophobic
substituent chain in a QAC intercalates with the hydrophobic component of the cell
membrane while the positively charged QAC interacts with the negatively charged cell
membrane. This electrostatic interaction disrupts the ionic integrity of the membrane,
leading to cell death [7, 38, 63, 80, 81].
QACs can be applied on anionic fiber surfaces by the exhaustion method. The
binding action between fiber and QACs takes place predominantly due to ionic
22
interaction [82]. QACs show ease of application and have an excellent antimicrobial
properties; however QACs generally show poor wash durability because they tend to
leach out from the fiber.
Wool fabrics have been modified by the application of cationic agents such as
cetylpyridinium chloride (CPC), benzyldimethyl-hexadecylammonium chloride
(BDHAC), (Figure 1.19) and cetyltrimethylammonium bromide (CTAB). These agents
were reported to bind with anionic sites in wool through ionic interactions at appropriate
pHs and thereby modifying wool fibers giving antimicrobial properties [83, 84]. The
percent exhaustion of CPC and BDHAC on cotton fabric was increased by creating
anionic sites on fiber. The cotton fabric was treated with 4-aminobenzenesulfonic acid–
chloro–triazine adduct which generated anionic sulfonate groups on fiber. The modified
cotton showed antimicrobial efficacy [85]. It has also been shown that CPC can be
exhausted at boiling conditions on synthetic fibers such as Acrilan® or Orlon® acrylic
fibers which contain anionic carboxylate or sulfonate groups [86-88].
a) b)
Figure 1.19: Structures of a) cetylpyridinium chloride (CPC) and b)
benzyldimethyl-hexadecylammonium chloride (BDHAC)
23
The exhaustion of QACs on synthetic polyamide fibers such as nylons dyed with
anionic dyes was achieved. The process was developed based on the hypothesis that
anionic dyes can act as bridging links between synthetic fibers and cationic functional
finishes. The work showed that acid dyeing of nylon fabrics increased the number of
available binding sites for QACs to interact with, and thus improved the durability of the
finish [82, 89, 90]. The polyamide fibers were also modified by a graft copolymerization
technique to provide antimicrobial properties to fibers. Two different monomers, namely
methacryloyloxyethyl trimethylammonium chloride and methacryloyloxyethyl
dimethyldodecylammonium bromide, with quaternary amine groups were grafted on
knitted fabric using sodium persulfate initiator [91]. The grafting technique was also
utilized in developing an antimicrobial finish on polypropylene (PP) fabrics. The PP
fabric was irradiated with an electron-beam accelerator and subsequently grafted with 2-
N-morpholino ethyl methacrylate (MEMA) (Figure 1.20). The amino groups from the
grafted moieties were finally quaternized with different alkylating agents [92].
Figure 1.20: Modification of PP with cationic derivative of 2-N-morpholino ethyl
methacrylate (MEMA)
To improve the durability of antimicrobial finishes on wool, attempts have been made to
covalently attach the finishing agent on to the fiber. The synthesized new agent, N-
dodecyl-aminobetaine-2-mercaptoethylamine hydrochloride (DABM) (Figure 1.21), is
reported to react with wool through its thiol group. The binding is proposed to take place
between the thiol group and cysteine-S-sulphonate residues (Bunte salts) of wool treated
24
with sodium bisulphite, or with the disulfide groups from the cystine component of wool
[63, 93].
Figure 1.21: Structure of N-dodecyl-aminobetaine-2-mercaptoethylamine
hydrochloride (DABM)
Silane chemistry is also used to covalently attach quaternary compounds on
textiles. The alkoxysilane (-SiOR) moiety can hydrolyze to form silanol (-SiOH) in the
presence of a catalyst. The hydrolyzed silanol can further react with hydroxyl groups in
the fiber or react with each other to form crosslinks. The commercially available product,
3-trimethoxysilylpropyl dimethyloctadecyl ammonium chloride (AEM 5700, formerly
known as Dow Corning 5700) (Figure 1.22), has been shown to bind irreversibly to
textile fibers such as cotton, polyester and nylon. The aqueous solution of this
antimicrobial agent can be applied by padding, spraying or foam finishing [94, 95].
Figure 1.22: Structure of 3-trimethoxysilylpropyldimethyloctadecyl ammonium
chloride
Among the cationic agents, polycationic polysiloxanes have also shown effective
antimicrobial properties. The synthesized copolymers of polydimethylsiloxane,
polymethylsiloxane and the quaternary ammonium salt or imidazolium salt-based
25
polysiloxane were found to be effective against a broad spectrum of bacteria (Figure
1.23). The copolymer of imidazolium salt-based polysiloxane had an added advantage of
better thermal stability compared to the quaternary ammonium salt-based polysiloxane
copolymers [63, 96].
Figure 1.23: Structure of polycationic imidazolium-modified polysiloxane
Chitosan
Chitin is a polymer of N-acetylglucosamine, a derivative of glucose and the
second most ubiquitously found natural polysaccharide on earth after cellulose. It is the
main component of the exoskeletons of crabs, lobsters and shrimp, insects and other
animals[97]. The chemical structure of chitin is similar to cellulose, a polysaccharide.
When chitin is deacetylated above ~ 60% it is called chitosan, which is a β-(1, 4)-linked
polysaccharide of D-glucosamine. Chitosan is a nontoxic, antimicrobial and
biodegradable polymer (Figure 1.24). The primary amine group at C2 position from
glucosamine has a pKa of ~6.5, which can be easily protonated in acidic pH (below 6.5)
making it water soluble. Chitosan’s polycationic nature in acidic pH makes it antifungal
and antimicrobial by its action in which chitosan binds to the anionic sites of the microbe
protein, similar to QACs. It is also believed that oligomeric chitosan can penetrate the
cell of microbe and inhibit ribonucleic acid (RNA) transcription, leading to the
prevention of microbial growth [63, 98].
26
Figure 1.24: Deacetylation of chitin
The antimicrobial activity of chitosan depends on the molecular weight, degree of
deacetyalation and pH. It is believed that a molecular weight of at least 10,000 is required
for better antimicrobial efficacy [99]. The increase in degree of deacetylation creates
more amino groups, which improves solubility in acidic pH. The increase in the charge
density caused by protonation of chitosan leads to better antimicrobial potency [100].
Chitosan has been mainly used for modification of cellulosics and their blends,
but it has low durability to washing as it releases gradually from fabric. This occurs due
to its weak binding action on fabric [101]. Chitosan also has the drawback of
antimicrobial activity over a limited pH range. To circumvent this problem, derivatives of
chitosan and crosslinking agents are used. Some of the synthesized derivatives of
chitosan include N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride (Figure
1.25) [102, 103], methylated N -(4-N,N -dimethylaminobenzyl) chitosan chloride
(MDMBzCh) (Figure 1.26), methylated N -(4-pyridylmethyl) chitosan chloride
(MPyMeCh) (Figure 1.27), and N-p-(N-methylpyridinio) methylated chitosan chloride
[104]. These contain a stable quaternary amine that leads to improved antimicrobial
efficacy and a wider pH application range[63].
27
Figure 1.25: Structure of N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride
Figure 1.26: Methylated N - (4-N, N -dimethylaminobenzyl) chitosan chloride
(MDMBzCh)
Figure 1.27: Methylated N -(4-pyridylmethyl) chitosan chloride (MPyMeCh)
A fiber reactive derivative of chitosan was developed, which contains a
quaternary amine and a fiber reactive acrylamidomethyl group. The modified version of
chitosan, O-acrylamidomethyl-N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan
chloride (NMA-HTCC) (Figure 1.28), showed improved durability compared to chitosan
[63, 105].
28
Figure 1.28: Structure of NMA-HTCC
In another strategy, crosslinking agents are used to form covalent linkages
between hydroxyl groups of chitosan and cellulose [103]. Some of the crosslinking
agents employed so far include 1,2,3,4- butanetetracarboxylic acid (BTCA) [106-108],
dimethylol dihydroxyl ethylene urea (DMDHEU) [109], citric acid(CA) [110, 111],
glutaraldehyde [112], and glyoxal [113]. In another approach, the use of core-shell
assemblies of chitosan as shell materials and poly (n-butyl acrylate) or poly (N-
isopropylamide) polymers as core material to produce nanoparticles has been reported.
Cotton fabric treated with synthesized nanoparticles showed excellent antimicrobial
activity and improved durability without hampering the physical properties of the fabric
[114]. The application of chitosan oligomers on synthetic fibers such as polypropylene
has been reported to create antimicrobial properties, but the finish had an adverse effect
on hand and tensile properties of the material [63, 115].
Poly (hexamethylenebiguanide) (PHMB)
Polybiguanides are polycationic polymers with quaternary amine based biguanide
repeating units which are separated by hydrocarbon chains of the same or varying lengths
[100]. Among the polybiguanides, poly (hexamethylenebiguanide) (PHMB) (Figure 1.29)
has been proved to be a very effective antimicrobial agent and contains an average of 11
29
biguanide units. Due to its low toxicity and fairly broad spectrum antimicrobial efficacy,
it finds applications in the food industry, swimming pool water treatments[116], wound
dressings, mouthwashes and textiles. The antimicrobial activity of PHMB is attributed to
its ability to impair microbial cell membrane integrity, such as is observed in other
quaternary compounds. PHMB has been extensively studied for imparting antimicrobial
activity to cellulosic materials[63].
Figure 1.29: Structure of PHMB
It is reported that PHMB, being cationic in nature, can bind to the anionic sites on
cotton which are generated through preparatory processes such as bleaching or
mercerization. PHMB can be easily applied on cellulosic materials by the exhaust method
at neutral pH and room temperature, or by a pad-dry-cure process. The binding of PHMB
with cellulose is a combination of electrostatic interaction and hydrogen bonding. At
lower concentrations of PHMB, the binding action is dominated by electrostatic
interactions between PHMB and cellulose; however, with an increase in PHMB
concentration hydrogen bonding between cellulose and PHMB dominates the binding
[117]. It is observed that the adsorption of PHMB on cellulosic fibers increases if the
fibers are previously dyed with anionic reactive dyes. Anionic groups such as the sulfonic
acid functionality of dyes provide additional binding sites for PHMB, but strong
interactions between dye and PHMB have an adverse effect on antimicrobial activity [73,
118]. Recently, wool fabric treated with PHMB along with peroxymonosulfate and
30
sodium sulfate showed good antimicrobial efficacy but with adverse effects on the
physical properties of the fabric [119, 120].
Another promising biguanide is chlorhexidine (Figure 1.30), which is an
antiseptic used in many household products such as antimicrobial soap, mouthwash,
contact lens solutions and medical applications. The research reported was conducted to
study application of chlorhexidine on cotton [63, 121].
Figure 1.30: Structure of chlorhexidine
Regenerable antimicrobial finishes
The regeneration principle was first proposed by Gagliardi in 1962 for
antimicrobial textiles, but some important significant advances in the area were achieved
almost 30 years later .[122] Regenerable antimicrobial agents can be repeatedly made
active by an external step, usually laundering in bleach after use. There are two kinds of
chemistries reported thus far; one is N-halamine based chemistry, which is been widely
studied, and the other is peroxyacid based chemistry[63].
N-halamine is defined as a compound with one or more covalent linkages between
nitrogen and the halogen (chlorine or bromine). Carefully designed N-halamine
compounds, unlike the inorganic halogens, are more stable, less corrosive, are broad
spectrum disinfectants and have been used in water treatment.[123, 124] The active
halogen in the N-halamine structure, usually chlorine, is responsible for its antimicrobial
31
activity. The chlorine in the N-Cl bond is replaced with H in an electrophilic substitution
reaction which can take place in the presence of water. The resultant positive halogen
(Cl+) ion from N-halamine penetrates through the charged microbial cell membrane/wall
and binds with appropriate receptor sites in the microbe, disrupting important enzymatic
and metabolic intracellular processes and leading to cell death. The generated N-H
containing product does not have an antimicrobial efficacy, but the antimicrobial
properties of the agent can be regenerated by exposing the agent to bleach solution (dilute
sodium hypochlorite), replacing the H in N-H with active chlorine (Figure 1.31).
Figure 1.31: Regenerable antimicrobial activity of N-halamine
A variety of derivatives of N-halamine agents have been developed and
covalently attached to cellulosic fibers [17, 125-128], nylon [129, 130], polyester [131,
132], acrylic, other synthetic fibers, and protein fibers. The strategy of using N- halamine
based derivatives has been extended to create N-halamine based monomers with vinyl
reactive group (Figure 1.32). These monomers can be polymerized on cellulose [133]
fiber surfaces under suitable reaction conditions to form antimicrobial coatings with
excellent effectiveness and washing durability[63].
32
a) b)
Figure 1.32: Polymerizable N-halamine derivatives, a) 3-(4'-vinylbenzyl)-5,5-
dimethylhydantoin, b) N-chloro-2,2,6,6-tetramethyl- 4-piperidinyl methacrylate
N-halamine based regenerable finishes are found to be durable and very effective
against broad range of microbes. However, the treated fabric tends to adsorb additional
active chlorine other than that covalently attached after each bleach wash. This adsorbed
residual chlorine causes unpleasant odors, discoloration of colored fabrics and can be a
skin irritant for users. The research showed that treatment with a reducing agent (sodium
sulfite) has been successfully used to remove the unbonded residual chlorine without
lowering the performance of the finish. [63, 134]
Among the peroxyacids, peroxyacetic acid is a well-known strong oxidizing agent
which is used in waste water plants, cooling towers, hospitals, food-processing and the
beverage industry. [135] The antimicrobial activity of peroxyacids is related to the
generation of reactive oxygen species which can damage DNA and lipids, denature
proteins and enzymes, and can also disrupt cell membranes. [136, 137] Peroxyacids are
transformed to carboxylic acid during the deactivation process. The active peroxyacids
33
can be regenerated by treatment with suitable oxidants (peroxide bleach) (Figure 1.33)
[63, 138].
Figure 1.33: Regenerable antimicrobial activity of peroxyacids
The research study also demonstrated that cotton treated with BTCA and CA
provides necessary carboxylic acid groups which can be converted to peroxyacids with
the use of oxygen bleach or sodium perborate. The finished cotton fabric showed good
antimicrobial properties but the efficacy was reduced after several washing and
recharging cycles [63, 138-140].
Triclosan
Triclosan (Figure 1.34) is a broad spectrum antimicrobial agent and has been
utilized in a wide range of products such as mouthwash, toothpastes, soaps, body wash,
deodorants, shaving creams, plastics and textiles [141]. It is believed that at lower
concentrations it acts as bacteriostatic. The mode of action of triclosan involves blocking
of lipid biosynthesis by binding with enoyl-acyl carrier protein reductase enzyme (ENR),
which prevents fatty acid synthesis required for lipid production in the microbe [142,
143].
34
Figure 1.34: Structure of triclosan (2, 4, 4’-trichloro-2’-hydroxydiphenyl ether)
Triclosan can be used on polyester and nylon fibers by exhaustion before dyeing,
simultaneously with dyeing or after dyeing step, which is possibly due to its relatively
small molecular size and similarities to disperse dyes. [95] Synthetic polymers can also
be modified by adding triclosan directly into melt-spinning. [144] Triclosan has a
disadvantage of low durability and is released in normal wash and wear. The durability of
triclosan finished cotton fabric was improved by using crosslinking agents such as BTCA
and citric acid (CA). [145] The other approach followed was incorporation of triclosan in
β-cyclodextrins [146] or cationic derivatives of β-cyclodextrins [147, 148] which formed
a host-guest inclusion complex which can be then incorporated in the fiber or polymer
film. Encapsulation has also been used to encapsulate triclosan molecules in
biodegradable polylactide based microspheres which were then applied on rayon
nonwoven textiles. [63, 149, 150]
Abundant use of triclosan as a biocide has led to resistance by many microbes,
which is a major concern. One example is Pseudomonas aeruginosa bacteria which can
pump out triclosan effectively from its bio-system [151]. The other issue is its low
stability to sunlight. It undergoes phototransformation in aqueous solutions to form 2,8-
dichlorodibenzo-p-dioxin, which contributes dioxin toxicity in the environment [152].
35
Dyes
Some of the dyes used in coloration of textiles have shown antimicrobial
properties based on their molecular structures; this mainly includes metal based dyes. The
use of chromium (Cr) and copper (Cu) based dyes in coloration of silk showed effective
antimicrobial efficacy. The observed functionality was caused by a slow release of metal
ions from dyed fabric [153]. The dye-mordant chemistry of CI Direct Blue 168 and
copper sulfate was effectively utilized and antimicrobial properties were endowed to
acrylic fabric [154]. Some synthetic dyes were designed to show antimicrobial activity.
For example, novel cationic dyes were synthesized by covalently linking quaternary
ammonium group on an aminoanthraquinioid chromophore (Figure 1.35). This showed
antimicrobial efficacy but had low washing durability when applied on acrylic fabric
[155, 156]. In another strategy reactive cationic dyes were developed based on
aminoanthraquinone-cyanuric chloride derivative (Figure 1.36) which formed covalent
bonds with cellulosic fiber [157]. A series of monoazo based cationic dyes were
developed by following a diazotization –coupling reaction between two aromatic amino
compounds in which one contained quaternary ammonium salts of varying alkyl chain
length and a second contained N,N-dimethyl-benzeneamine or 1-phenyl-3-methyl-5-
pyrazolone as a coupling group (Figure 1.37). The synthesized dyes showed
antimicrobial performance through minimum inhibitory concentration (MIC) results [63,
158].
36
Figure 1.35: Structure of synthetic cationic aminoanthraquinone dye
Figure 1.36: Structure of cationic reactive dye
Figure 1.37: Structure of cationic monoazo dye
Research in natural dyes has shown that some dyes have useful antimicrobial
properties [159]. Some of the examples include a natural cationic colorant, berberine
chloride (Figure 1.38) [160], which was applied on cotton fabric, and curcumin (1,7-bis
(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione), (Figure 1.39) an active
component of turmeric, was applied on wool. [63, 161]
37
Figure 1.38: Structure of Berberine chloride
Figure 1.39: Structure of curcumin
One study showed that use of natural dyes in conjunction with metal oxide based
mordants improved antimicrobial as well as UV-protection properties of treated cotton
fabric [63, 162].
In textiles, many of the current available antimicrobial agents are leachable type
of agents and possess low durability. The finishes become ineffective after few wash and
wear cycles. Recently, the leachable antimicrobial agents raised environmental concerns
as there are reported cases of evolution of resistant bacterial strains against these agents.
In our work, we tried to tune the chemistry of well-known antimicrobial quaternary PEI
polymers for textile fibers so that they can be easily incorporated in to the current
production processes, and the fiber reactive groups from polymers were expected to form
covalent linkages with the fiber which could improve durability of finish.
38
Research objectives
Project 1. Photocrosslinkable antimicrobial copolymer
The carefully designed polymeric biocidal agents have the advantages of being
stable, non-volatile, durable, non-permeable through the skin, non-leachable, efficient
and selective.[15] The immobilization of biocidal polymers on the surface avoids its
release in the environment, which is a distinct advantage. However, the covalent
attachment of biocidal polymers on common and inert plastic surfaces such as
polyethylene, polypropylene, and polystyrene is challenging due to lack of functional
groups in the polymer. Generally, oxidative treatments used to render surface
functionality for immobilization of additives include flame, corona discharge, gamma
rays, ion or electron beams, and plasma and laser treatments [163, 164], and are
expensive treatments. In this work, an attempt is made to conveniently attach ultrathin
biocidal polymer coatings covalently on the surfaces that have inert functionality. The
antimicrobial copolymer was designed with pendant benzophenone groups that act as a
photo-crosslinkers for the covalent attachment of the antimicrobial polymer to any
substrate containing a C-H bond upon irradiation with UV light.
The primary goal of the study was to develop new biocidal polymer which can be
easily attached covalently to the surfaces of inert plastic and textile materials. Some of
the specific objectives of the study were as follows:
1. Synthesis and characterization of a photocrosslinkable antimicrobial copolymer
2. Characterization of the coatings (kinetics of attachment, thickness, smoothness of
coating)
3. Evaluation of antimicrobial activity
39
4. Optimization of application conditions for the synthesized copolymer
5. Surface modification of a variety of commodity plastics and textiles with the
synthesized copolymer
Project 2. Reactive antimicrobial copolymers for textile fibers
The use of polymeric antimicrobial agents for textile materials holds much
promise, and polymeric antimicrobial agents can be designed to endow desired functional
properties to the finish. There are number of different antimicrobial polymers and co-
polymers reported but there is limited research available on the application of these
polymers to textile materials.
Many of the reported textile modification methods with polycations involve
multistep modification of procedures, use of harmful solvents, and lengthy process
times.[8, 22, 75] These factors are constraints for their industrial scale-up or
incorporation into current production processes. Almost all the chemical preparatory or
finishing steps in textiles are carried out using water as media as it is economical and
safe. To this end, an attempt was made to develop a non-leachable polymeric
antimicrobial finish which can be applied through current textile processing procedures.
The primary objective of this work is to design and optimize novel reactive
biocidal copolymers for application to cellulosic textile materials using an existing simple
application method such as the exhaust method. The covalent attachment of these
polymers was expected to enhance the durability of the finish. In this research, the goal
was to incorporate fiber reactive groups on the backbone of (PEIs) polymers. Vinyl
sulfone and chlorotriazine groups, which are well known reactive groups in reactive dyes,
40
were grafted on to PEI polymers to create new copolymers. Some of the specific
objectives of the study were as follows:
1. Synthesis of new cationic PEI polymers with reactive vinyl sulfone and
chlorotriazine pendant groups
2. Characterization of new copolymers
3. Study of the antimicrobial efficacy of new the copolymers
4. Evaluation of the durability of new copolymers to accelerated laundering
41
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144. Kalyon, B.D. and U. Olgun, Antibacterial efficacy of triclosan-incorporated
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146. Lu, J., et al., Formation of antibiotic, biodegradable polymers by processing with
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62
CHAPTER 2
PHOTOCROSSLINKABLE ANTIMICROBIAL COPOLYMER
Accepted to the ACS Journal of Applied Materials and Interfaces, June 2011
Dhende, V. P.; Samanta, S.; Jones, D. M.; Hardin, I. R.; Locklin, J., One-Step
Photochemical Synthesis of Permanent, Nonleaching, Ultrathin Antimicrobial Coatings
for Textiles and Plastics. ACS Applied Materials & Interfaces 2011
63
Abstract
Recently, antimicrobial agents have gained much attention in both academic and
industrial circles due to their ability to provide protection to a wide range of materials
against microbial attacks. Commercial applications of antimicrobial agents include
textiles, food packaging and storage, the shoe industry, water purification, medical
devices, and dental surgery equipment. There are ranges of antimicrobial agents with
differing chemistries available commercially today and many more are being studied in
academic laboratories. Quaternary polymeric compounds have proven to be very
effective antimicrobial agents because of their unique structural properties. The scientific
literature is replete with reports of surface modifications of different substrates by
covalent attachment of these biocidal polymers. However, covalent surface attachment of
biocidal polymers on common inert plastic materials such as polypropylene, polyethylene
and polyvinyl chloride is very challenging with very few examples in the literature. In
our study, we have successfully synthesized quaternary polyethylenimine based
copolymers with a photoactive benzophenone pendant group that allows covalent
attachment of copolymer on any surface with C-H bonds upon irradiation with mild UV
light. The coating showed impressive antimicrobial activity against both Gram positive
and Gram negative bacteria. A simple spray application technique was used to coat the
substrates uniformly with copolymer to create permanent ultrathin biocidal coating.
Keywords: Polyethylenimine, photocrosslinker, antibacterial, antimicrobial, antifouling
64
In the last two decades, there is significant amount of work done in the area of
antimicrobial polymers at an academic and industrial level. There are number of research
articles published on different synthetic strategies, polymer architectures and
immobilization of these biocidal polymers on variety of surfaces such as glass,[34-36]
polymer,[21, 165-170] paper,[37] and metal.[171]
Some cationic polymers, like
quaternary polyetheleneimines (QPEIs), have proven effective at killing bacteria because
of their unique structural and hydrophobic properties.[172-177] The generally accepted
hypothesis for antimicrobial activity of polycations with hydrophobic side chains is that
the pendant hydrophobic groups can intercalate into the hydrophobic portion of a cell
membrane, while the electrostatic interaction of the positively charged backbone and the
negatively charged bacterial cell membrane/wall disrupts the ionic integrity of the
membrane, causing cell death. But surface immobilization of biocidal polymers on inert
plastic materials is still very challenging and there are not many articles published on this
area till date. The other important aspect is irreversible or covalent attachment of these
biocidal polymers on the surface to avoid its release in the environment. Recently, Hsu
and Klibanov [43] reported a system in which an aryl azide based biocidal PEI copolymer
was used to modify cotton fabrics. In this case, the nitrophenylazide based crosslinker
reacts preferentially with the hydroxy functionality on the cellulose surface. While this
methodology is achievable with surfaces that contain reactive functional groups
(examples include hydroxy, amine, carboxylic acid, and chloro), the covalent attachment
of biocidal polymers on common and inert plastic surfaces such as polyethylene,
polypropylene, and polystyrene is more challenging with very few examples in the
literature.[33, 178-180]
65
The ability of benzophenone (BP) to act as a cross-linking agent and abstract
hydrogen from a suitable hydrogen donor has been well studied and utilized in various
chemical systems for many years.[181-187] BP is an ideal choice for crosslinking organic
thin films, because it can be activated using mild UV light (345 – 365 nm), avoiding
oxidative damage of the polymer and substrate that can occur upon exposure to higher
energy UV. The benzophenone moiety is more chemically robust than other organic
cross-linkers and reacts preferentially with C-H bonds in a wide range of different
chemical environments. Triggered by UV light, benzophenone undergoes an n-π*
transition, resulting in the formation of a biradical triplet excited state that can abstract a
hydrogen atom from a neighboring aliphatic C-H group to form a new C-C bond.[188]
Figure 2.1: Photoreaction of benzophenone (BP)
66
This photoreaction has recently been used to attach thin polymer layers to metal
and oxide surfaces,[189-194] along with applications in microfluidics,[195] organic
semiconductors,[196] redox polymers,[197, 198] and biosensors.[199]
Material and Methods
Materials
Silicon wafers (Universitywafer.com) with native oxide and glass slides (VWR)
(cut into 2.5 × 2.5 cm pieces) were used as substrates. The other textile and commodity
plastic substrates include, 100% cotton print cloth with specifications of weave 78 × 76,
weight 102 g/m2 (Testfabric, Inc.), polypropylene nonwoven geotextile (provided by
TenCate Geosynthetics and Industrial Fabrics), polyethylene transparent sheets (Great
Value storage bag, Wal Mart, Inc.) and polyvinyl chloride transparent sheets (Wal Mart,
Inc.) were purchased. Poly(2-ethyl-2-oxazoline) (Mw = 50,000 g/mol) (Aldrich), tert-
amylalcohol (Aldrich), 1-bromododecane (Alfa Aesar), iodomethane (Alfa Aesar), 4-
hydroxybenzophenone (Alfa Aesar), 1, 6-dibromohexane (Alfa Aesar), trypticase soy
broth (TSB) (Difco), trypticase soy agar (TSA) (Difco), were used as received.
Instrumental Methods
Atomic force microscopy (AFM) experiments for quaternized PEI based polymer
films were performed using a Multimode Nanoscope IIIa (Digital Instruments/Veeco
Metrology Group). All measurements were performed using tapping mode. Null
ellipsometry was performed on a Multiskop (Optrel GbR) with a 632.8 nm He-Ne laser
beam as the light source. Both and values were measured and thickenss was
calculated by integrated specialized software. At least three measurements were taken for
67
every layer, and the average thickness was calculated. UV-vis spectroscopy was
performed on a Cary 50 spectrophotometer (Varian). Infrared spectroscopy studies of
polymer coated films were done using a Thermo-Nicolet Model 6700 spectrometer
equipped with a variable angle grazing angle attenuated total reflection (GATR-ATR)
accessory (Harrick Scientific). The UV light source was an OmniCure, Series 1000 with
365 nm bandpass filter, equipped with a liquid filled fiber optic waveguide. The
substrates were held 2 cm from the source and irradiated with a power of 180 mW/cm2.
The synthesized compounds were analyzed using proton (1H) and carbon (
13C) Nuclear
Magnetic Resonance (NMR) spectroscopy and spectra were recorded using a Varian
Mercury 300 NMR spectrometer working at 300 MHz. An internal standard of
tetramethylsilane is used to report relative chemical shifts.
Antimicrobial Test Method
The antimicrobial efficacy was determined by using a modified version of test
method published by Haldar et al.[200] The antimicrobial test method followed in this
work mimics the practical scenario of airborne bacteria coming in contact with substrates
which is simulated by spraying the bacterial aerosol. The common way of infection
spreading includes respiratory droplets produced by sneezing, coughing, laugh, or
breathing.
Trypticase soy broth (TSB) (10 mL) was inoculated with one loopful of bacteria
Staphylococcus aureus (ATCC 6538) culture or Escherichia coli (ATCC 25922) and
incubated overnight in a water shaker bath at 37°C with 45 linear strokes per minute. The
new TSB (10 mL) was again inoculated with 100 μl of an overnight bacterial culture and
incubated for 4 hours in the above-mentioned conditions in the shaker bath. One milliliter
68
of this culture was transferred to a 1.5 mL centrifuge tube and was centrifuged at 5000
rpm for 1 minute at 21°C to precipitate bacteria and form a bacterial pellet. (Centrifuge =
accuSpin Micro 17R, Fisher Scientific, Tubes = Micro Centrifuge Tube, VWR
International). The supernatant solution was discarded and 1 mL of sterile water was
added to the microbial pellet in the tube. The microbes were re-suspended in the solution
by using a vortex mixer (Vortex Genie 2) and was transferred to 9 mL of sterile water to
make a bacterial concentration of ~ 3×106 cfu (colony forming units) and subsequently
transferred to thin layer chromatography (TLC) sprayer bottle which was connected to
pneumatic dispense regulator (EFD 1500XL). The polymer coated substrates were
uniformly sprayed on one side in a controlled fashion from the TLC sprayer for 1 second
at 30-40 psi pressure. The distance between the sprayer and glass slide was
approximately 1-1 ½ feet. The sprayed sample was air dried for approximately 1 minute
and the sample was carefully mounted on a Difco™ Trypticase soy agar (TSA) plate.
TSA plates were incubated for 24 hours at 37 °C. Finally, the number of colonies grown
on the slide was counted.
Syntheses:
Linear Polyethylenimine (PEI): The deacylation reaction was performed according to
literature procedures.[201] 3 g of poly (2-ethyl-2-oxazoline, Mw, 50 kDa) (POEZ) was
added to 120 mL of 24 % (wt/vol) HCl, followed by refluxing for 96 h. The POEZ
dissolved completely in 1 h, but after overnight reflux a white precipitate appeared. The
precipitate was filtered and then air-dried. The resultant protonated, linear PEI was
dissolved in water and neutralized with aqueous KOH to precipitate the polymer. The
white powder was isolated by filtration, washed with distilled water until the pH became
69
neutral, and dried under vacuum. Yield: 1.15 g (88 %). 1H NMR (CDCl3): , 2.72 (s, 4H,
NCH2CH2N), 1.71 (1H, NH).
4-[(6-Bromohexyl) oxy] benzophenone: 4-Hydroxy benzophenone (5.94 g, 30 mmol),
1,6 dibromohexane (8.05 g, 33 mmol), potassium carbonate (5.95 g, 45 mmol) and DMF
(60 mL) were stirred at room temperature for 16 h under inert atmosphere. The reaction
mixture was poured into ice water (300 mL) and extracted with ether (100 mL). The
organic layer was collected and the solvent was removed with a rotary evaporator. The
crude product was purified on a silica gel column by using 10:1 hexane:ethyl acetate
mixture (Scheme 2.1). Yield: 8.2 g (76 %). 1H NMR (CDCl3): , 7.81 (d, 2H, J = 8.4 Hz),
7.75 (d, 2H, J = 7.8 Hz), 7.54 (t, 1H, 7.5 Hz), 7.47 (t, 2H, J = 6.9 Hz), 6.93 (d, 2H, J = 9.0
Hz), 4.06 (t, 2H, J = 6.3 Hz), 3.43 (t, 2H, 6.6 Hz), 1.86 (m, 4H), 1.50 (m, 4H). 13
C NMR
(CDCl3): , 25.47, 28.10, 29.11, 32.86, 33.95, 68.2, 114.2, 128.37, 129.92, 129.94,
132.06, 132.78, 138.55, 162.9, 195.7.
Scheme 2.1: Synthesis of 4-[(6-Bromohexyl) oxy] benzophenone
Linear Copolymer of N,N-dodecyl methyl and N,N-[(6-hexyl) oxy] benzophenone
methyl PEI: 0.5 g (12 mmol of the monomer unit) of the PEI was dissolved in 6 mL of
tert-amyl alcohol, followed by the addition of 2.1 g (15 mmol) of K2CO3, 1.99 g (8
mmol) of 1-bromododecane, and 1.44 g (4 mmol) of 4-[(6-bromohexyl) oxy]
benzophenone and the reaction mixture was stirred at 95 C for 96 h. After removing the
solids by filtration under reduced pressure, 1.5 mL of iodomethane was added, followed
70
by stirring at 60 C for 24 h in a sealed, heavy walled pressure vessel. After reaction, the
solution was dried using a rotary evaporator. The yellow solid was dissolved in a
minimum volume of dichloromethane and then the solution was added to excess hexane
to precipitate the polymer. The light yellow solid was filtered and dried at room
temperature under vacuum for 12 hours (Scheme 2.2). Yield: 2.3 g (46 %). 1H NMR
(CDCl3): , 7.77 (bs, 4H); 7.56 (bs, 1H), 7.45 (bs, 2H); 6.96 (bs, 2H); 4.19 – 3.26 (m,
21H); 1.83 (bs, 6H); 1.65 (bs, 16H); 1.23 (bs, 34H), 0.87 (bs, 6H). 13
C NMR (CDCl3): ,
195.73, 162.88, 138.24, 132.56, 131.72, 129.71, 128.25, 114.32, 67.95, bs 53.45, 31.90,
29.65, 29.59, 29.53, 29.47, 29.36, 22.67, 14.11.
Scheme 2.2: Synthesis of linear copolymer N, N-dodecyl methyl and N, N-[(6-hexyl)
oxy] benzophenone methyl PEI
Preparation of self-assembled monolayers on glass substrates: Glass slides were cut
into rectangles. The substrates were sonicated with Fisherbrand sonicating soap, 18.2 MΩ
deionized water, isopropanol, and acetone for 10 min each and finally dried in an oven
for 1 h. After cleaning, a self-assembled monolayer (SAM) of octyltrichlorosilane was
formed from the vapor phase by suspending the substrates in a vacuum desiccator and
placing two drops of silane on a glass substrate at the bottom. The substrates were kept in
71
a vacuum flux (constant pressure of 100 millitorr) for 20 min. After venting with
nitrogen, the substrates were sonicated with acetone and dried under air.
Surface bound PEI Polymer (2): 15 mg of quaternized polymer (2) was dissolved in 1
mL of acetone solvent. The solution was filtered through 0.25 m filter. The polymer
film was developed on functionalized glass substrate by spin coating with 0.5 mL of
solution at 1000 rpm. The glass substrate was irradiated with UV light (365 nm, 180
mW/cm2) for 15 mins to covalently bind the polymer on the glass surface through the
pendant benzophenone moiety. The substrate was sonicated with acetone for one minute
and dried under air.
72
Results and Discussion
Copolymer 2, which contains both hydrophobic and benzophenone side chains,
was prepared by reacting linear PEI with 4-[(6-Bromohexyloxy)] benzophenone and 1-
bromododecane (Scheme 2.2) along with subsequent quaternization using iodomethane.
The copolymer composition was checked by NMR spectroscopy, which revealed that the
polymer composition matched the pendant group feed ratio. Based on the NMR
integration values, the benzophenone side-chain constitutes 33% of total polymer pendant
groups with the dodecane constituting the other 66%. We were unable to characterize the
copolymer using gel permeation chromatography but using the initial molecular weight
of the poly (2-ethyl-2-oxazoline) before hydrolysis and functionalization (Mw = 50,000
g/mol), the approximate molecular weight of the quaternized copolymer was ~194kDa.
Copolymer 2 is soluble in halogenated solvents, acetone, and slightly soluble in alcohols.
As described above, the benzophenone component of 2 can act as a cross-linker between
the hydrophobic PEI polymer and any organic substrate through C-H activation. Initially,
we have used glass and silicon wafers functionalized with alkyl SAMs to analyze the
polymer film thickness before and after crosslinking, kinetics of functionalization, and to
observe any surface morphology changes through atomic force microscopy. Flat
substrates also simplify the antimicrobial activity assays because of the ease of analytical
quantification.
73
Scheme 2.3: Surface attachment of benzophenone-PEI copolymer
The cross-linking and structure of the covalently bound polymer surfaces is
shown in Scheme 2.3. Initially, the oxide surfaces were functionalized with
octyltrichlorosilane (OTS) to generate C-H alkyl groups on the surface. To this modified
surface a thin layer of copolymer 2 was deposited using spin coating (15 mg/mL in
acetone, 1000 rpm). Covalent attachment was generated by exposure to UV irradiation
(365 nm, 180 mW/cm2) for 15 minutes. The crosslinked films were then washed with
acetone and sonicated in acetone for one minute to remove any residual, unbound
materials. The polymer film thickness was measured before and after sonication and was
observed to be 93 and 77 nm respectively, indicating that approximately 80% of the
coating remained after cross-linking. The thickness of the cross-linked coating did not
change upon prolonged sonication in any organic solvent.
74
Figure 2.2: Change in UV spectra of benzophenone in polymer 2 with UV exposure
with time (365 nm).
The kinetics of surface attachment of copolymer 2 was investigated by UV-vis
spectroscopy on OTS functionalized quartz substrates. Time dependent changes in the
absorption spectra of the film under UV light irradiation are shown in Figure 2.2.
Photon absorption at 365 nm results in the promotion of one electron from a nonbonding
n-orbital to an antibonding *-orbital of the carbonyl group on the benzophenone moitey.
The n- * transition yields a biradicaloid triplet state where the electron-deficient oxygen
n-orbital is electrophilic and therefore interacts with weak C-H -bonds, resulting in
hydrogen abstraction to complete the half-filled n-orbital.[202, 203] The two resulting
75
radical species can then combine to form a new C-C bond. The reaction progress can be
monitored indirectly by following the decrease in the -* transition of benzophenone at
290 nm. As expected, this peak decreases with increasing irradiation time. After ~30
minutes, the reaction is complete as observed, with no further changes in the spectrum
with prolonged irradiation.
Figure 2.3: FTIR spectra of a thin film of copolymer 2, before (A) and after (B) UV
exposure
The photochemical attachment of copolymer 2 was also confirmed using grazing
incidence attenuated total internal reflection fourier transform infrared spectroscopy
(GATR-FTIR). Copolymer 2 was spincast onto a silicon wafer that was modified with a
SAM of OTS. Figure 2.3 shows the GATR-IR spectrum of a silicon wafer modified with
copolymer 2 (A) before and (B) after UV irradiation. In Figure 2.3A, the peaks at 2920
76
and 2849 cm-1
are due to C-H stretching of the aliphatic backbone and pendant groups.
The C=O of the benzophenone pendant group is observed at 1648 cm-1
. The C-C ring
vibrations are assigned at 1600 cm-1
along with the C-N+ stretch at 1468 cm
-1. Peaks at
1253 and 1020 cm-1
are assigned to the C-O-C asymmetric and symmetric stretches
respectively. Figure 2.3B shows the polymer film after irradiation. A significant
reduction in the C=O strecth at 1648 cm-1
is readily apparent, which indicates photo-
decomposition of the carbonyl group along with the covalent attachment of 2 onto the
OTS functionalized SiO2 surface. The overall decrease in all peak intensities correlates
with the decrease in film thickness after crosslinking and subsequent sonication.
Figure 2.4: Tapping mode AFM image for the film of copolymer 2 (A) as cast before
sonication (thickness 93 nm, RMS roughness 0.48 nm) and (B) after sonication (thickness
77 nm, RMS roughness 0.83 nm).
AFM was used to characterize the surface morphology of copolymer (2) film
before and after sonication to remove any non-covalently bound polymer from the
surface. Before and after sonication, the irradiated film of 2 was very smooth. A
77
representative morphology for both is shown in Figure 2.4. The thickness of the film is
93 nm (measured with ellipsometry) with an RMS roughness 0.48 nm by AFM. Figure
2.4B shows the morphology of the film after sonication. The overall film thickness
decreased to 77 nm after sonication, with an increase in surface roughness to 0.83 nm due
to removal of non-covalently attached polymer from the surface.
Table 2.1: Antimicrobial test with S. aureus along with percent bacterial reduction.
There were four sets of samples tested: (1) Control glass substrate with OTS coated
SAM, (2) spin coated glass substrate with 5 mg/mL polymer concentration, (3) spin
coated glass substrate with 10 mg/mL polymer, and (4) spin coated glass substrate with
15 mg/mL concentration.
Control
(CFU)
5 mg/ml polymer
conc.
10 mg/ml polymer
conc.
15 mg/ml polymer
conc.
Uncoated
glass
slides
SUV*
Film
Thickness
35nm
SUVS*
Film
Thickness
31nm
SUV
Film
Thickness
55nm
SUVS
Film
Thickness
53nm
SUV
Film
Thickness
93nm
SUVS
Film
Thickness
77nm
1 258 1 15 0 3 0 4
2 247 4 16 0 4 0 2
3 158 0 10 0 3 3 2
Average 221 1.66 13.66 0 3.33 1 2.66
%
Reduction - 99.24 93.81 100 98.49 99.54 98.79
*SUV= Spin-coated UV radiated unsonicated glass slides
*SUVS= Spin-coated UV radiated sonicated glass slides
78
Figure 2.5: Digital pictures of the glass substrates sprayed with S. aureus and incubated
for 24 hours at 37 °C (A) control glass slide and (B) copolymer (15 mg/mL) coated glass
slide
The glass substrates coated with three different coating thicknesses of copolymer
2 showed excellent activity against S. aureus before sonication and demonstrated more
than 99 % bacterial reduction. However, after sonication the performance was lowered to
93 % bacterial reduction in the case of slides with 31 nm coating thickness, and slides
with 53 and 77 nm coating thicknesses showed reduction of more than 98 % (Figure 2.5)
(Table 2.1). A similar trend was observed against E. coli and slides after sonication
showed bacterial reduction of 80 % with 31 nm film thickness (Table 2.2). The slides
after sonication with film thicknesses of 77 nm performed well with close to 100 %
reduction (Figure 2.6). The exact mechanism of dependence of film thickness on bacterial
reduction is currently unknown for our polymer system, and further experimental analysis
is required in the direction.
79
Table 2.2: Antimicrobial test with E. coli along with percent bacterial reduction. There
were four sets of samples tested: (1) Control glass substrate with OTS coated SAM, (2)
spin coated glass substrate with 5 mg/mL polymer concentration, (3) spin coated glass
substrate with 10 mg/mL polymer, and (4) spin coated glass substrate with 15 mg/mL
concentration.
Control
(CFU)
5 mg/ml polymer
conc.
10 mg/ml polymer
conc.
15 mg/ml polymer
conc.
Uncoated
glass
slides
SUV*
Film
Thickness
35nm
SUVS*
Film
Thickness
31nm
SUV
Film
Thickness
55nm
SUVS
Film
Thickness
53nm
SUV
Film
Thickness
93nm
SUVS
Film
Thickness
77nm
1 91 0 11 1 0 0 1
2 81 2 24 0 11 0 0
3 136 2 26 0 6 0 1
Average 102.66 1.33 20.33 0.33 5.66 0 0.66
%
Reduction - 98.70 80.19 99.67 94.48 100 99.35
*SUV= Spin-coated UV radiated unsonicated glass slides
*SUVS= Spin-coated UV radiated sonicated glass slides
80
Figure 2.6: Digital pictures of the glass substrates sprayed with E.coli and incubated for
24 hours at 37 °C (A) control glass slide and (B) polymer (15 mg/mL) coated glass slide
81
Figure 2.7: Digital pictures of the textiles and plastic substrates sprayed with S. aureus.
(A) untreated cotton, (B) cotton sprayed coated with 15 mg/ml polymer 2, (C) untreated
polypropylene (nonwoven geotextile fabric), (D) polypropylene spray-coated with 15
mg/ml polymer 2, (E) untreated poly(vinyl chloride) substrate, (F) poly(vinyl chloride)
substrate spray coated with 15 mg/ml polymer 2, (G) untreated polyethylene substrate,
and (H) polyethylene substrate spray coated with 15 mg/mL polymer 2.
In order to investigate the versatility of these copolymers on commodity plastics
and textile fabrics, variety of substrates such as cotton, polypropylene, polyethylene and
poly (vinyl chloride) were photochemically modified with copolymer 2 using simple
spray coating technique. The copolymer, dissolved in acetone, was uniformly sprayed
coated with a laboratory TLC sprayer. The substrates were air dried and irradiated (365
nm, 180 mW/cm2) to covalently attach the polymer to the plastic surface. After UV
(E) (F)
(G) (H)
82
curing, the substrates were thoroughly washed in acetone to remove any non-covalently
attached copolymer. For all substrates, there were no major changes observed to either
the hand or physical properties. On the cotton pieces, the coated samples showed mild
yellowing after UV irradiation. The copolymer treated and untreated substrates were
challenged against S. aureus with the antibacterial test method described earlier. Figure
2.7 shows bacterial proliferation on the untreated substrates and excellent antibacterial
activity on the treated substrates. The results demonstrate covalent immobilization of
copolymer 2 on all substrates, including those with reactive functional groups such as
cotton as well as on inert plastic surfaces such as polypropylene, poly (vinyl chloride)
and polyethylene.
Testing in aquatic environments:
The effectiveness of the polymer coating on polyvinylchloride (PVC) substrates
was tested by submerging 1 ft2 of the substrates shown in (Figure 2.8), in the Pacific
Ocean off the coast of Chile and the Atlantic Ocean off the coast of Canada (Figure 2.9).
The substrates were examined after 50 and 40 days of testing in the ocean water off the
coast of Chile and Canada, respectively. The substrates that were coated with copolymer
2 were effective at preventing fouling of the grids. The uncoated samples were
completely covered with bacteria, algae, barnicles, and other sea creatures, while the
substrates coated with copolymer 2 were free of fouling, except for a residual thin film.
This thin film of fouling was easily wiped away, while the fouled, uncoated substrates
were very difficult to clean by hand, and required excessive pressure washing with a
stream of high pressure water.
83
Figure 2.8: Biofouling testing in the ocean water off the coast of Chile for 50 days
Figure 2.9: Biofouling testing in the ocean water off the coast of Canada for 40 days
84
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90
CHAPTER 3
REACTIVE ANTIMICROBIAL COPOLYMERS FOR TEXTILE FIBERS
To be submitted to the Journal of AATCC review
Dhende, V., Samanta, S., Hardin, I., and Locklin, J.
91
Abstract
In recent years, the uses of antimicrobial agents in textile finishing has gained
interest due to increased consumer awareness of hygiene and the need to bestow
protection to natural textile fibers against microbial attacks. The main aim of the work
was to design and optimize novel reactive copolymers, which can be applied on cellulosic
textile materials using a simple application method such as the exhaustion. In this
research, the goal was to incorporate hydroxy reactive groups on the backbone of
quaternary polyethyleneimine (PEI) polymers. The covalent attachment of antimicrobial
agent with fiber would improve its durability and avoid its release in the environment. To
this end, three new copolymers were synthesized namely sulfated quaternary based PEI
(SQ-PEI), monochlorotriazine based quaternary PEI (MCT-PEI) and dichlorotriazine
based (DCT-PEI) which contain both fiber reactive vinyl sulfone, monochloro and
dichlorotriazine side chains, respectively, and hydrophobic side chains (dodecane, C12).
The polymeric antimicrobial agents were chosen because they have the advantages of
being non-permeable through the skin, non-volatile, stable, efficient and selective. These
polymers were tested for their antimicrobial properties and durability to accelerated
laundering.
Keywords: Polyethylenimine, triazine, vinyl sulfone, antimicrobial, durability
92
In recent years, the use of antimicrobial agents in textile finishing has gained
increased interest due to the heightened consumer concern with hygiene and textiles. In
2000, worldwide production of antimicrobial textiles was 100,000 tons and 30,000 tons
in Western Europe. Production increased more than 15% a year from 2001 to 2005 in
Western Europe. [95]
Most of the natural fibers used in textiles are prone to microbial attacks. These
microbes break down the natural polymers to simple sugars, which then are used as food
sources by the microbes. Finishing textile materials with antimicrobial agents protects the
user of the textiles against microbes related to aesthetic, hygienic or medical problems,
and protects the textile material itself against biodeterioration from mold, mildew and rot-
producing fungi. There are three different means by which these finishing agents work,
namely 1) a controlled release mechanism, 2) the regeneration principle, and 3) the
barrier or blocking action. In the first mechanism, the textile material is finished with a
leachable type of antimicrobial agent which is consumed over a period of time. This type
of finishing agents loses effectiveness after few laundry washes. Another problem
associated with this type of finishing agent is microbes developing resistant strains
against the finish. The widespread use of triclosan, which falls under this category, has
led to the development of triclosan-resistant bacteria such as Pseudomonas aeruginosa
which can effectively pump out the triclosan from the cell [151]. In the second
mechanism, the finish must be reactivated by some additional step after use. For
antimicrobial halamine finished fabrics, the reactivation is done by using chlorine bleach.
In the third mechanism, the fabric can be finished with an inert physical barrier coating
material or surface coatings which can kill microbes on contact. [204]
93
Material and Methods
Materials
The following chemicals were used as received in the synthesis and antibacterial
testing:
Poly (2-ethyl-2-oxazoline) (Aldrich), tert-amylalcohol (Aldrich), dimethylsulfoxide
(DMSO) (Aldrich), 4-hydroxythiophenol (TCI america), 2-bromoethanol (Alfa Aesar),
Oxone™ (2KHSO5.KHSO4
.K2SO4) (Alfa Aesar), 1-bromododecane (Alfa Aesar),
Iodomethane (Alfa Aesar), 1, 6 dibromohexane (Alfa Aesar), cyanuric chloride (TCI
america), sulfanilic acid (Alfa Aesar), nutrient agar (NA) (Difco™), nutrient broth (NB)
(Difco™), dialysis tubing (Fischer Scientific, 12mm diameter, cutoff at 12000 Daltons),
freeze dryer (Labconco). The desized and bleached 100% cotton print cloth was
purchased from Testfabric Inc, West Pittston, PA, with specification of (weave 78×76,
weight 102 g/m2) as a test fabric. The fabric was further cleaned by treatment with
boiling water for 30 mins and oven dried. Gram positive and Gram negative bacteria,
namely S. aureus (ATCC 6538) and E. coli (obtained from UGA dept of microbiology),
were used in antibacterial testing.
Instrumental Analysis
The synthesized compounds were analyzed using proton (1H) and carbon (
13C)
nuclear magnetic resonance (NMR) spectroscopy. Spectra were recorded using a Varian
Mercury 300 NMR spectrometer working at 300 MHz. An internal standard of
tetramethylsilane was used to report relative chemical shifts. Fourier transform infrared
(FTIR) measurements were taken with a Nicolet model 6700 instrument at 128 scans
with 4 cm-1 resolution for analysis of compounds. The compound was thoroughly mixed
94
and crushed with dry potassium bromide (KBr). A transparent pellet of mixture was made
by using Beckman pelletizer to take FTIR spectra.
Finishing of Fabric
The copolymers were applied by the exhaust method to bleached cotton fabrics.
The copolymers were added to water and stirred to create a dispersion. The bleached
cotton fabrics were treated with finishing solutions for 20-30 min at 80°C in the case of
sulfated quaternary PEI (SQ-PEI) and monochlorotriazine based PEI (MCT-PEI)
copolymers and at room temperature for DCT-PEI copolymer in conical flasks with
magnetic stirring. To these finishing solutions, 0.2% (Wt/Vol) Na2CO3 was added and
treatments were continued for 30 minutes at their respective temperatures. The fabric were
rinsed thoroughly with water after the application process and dried in the air. The fabrics
were treated with a 5% finish on the weight of fabric (owf) with a material to liquor ratio
of 1: 40.
Accelerated Laundering (AATCC 61 2003)
The finished fabrics were tested by AATCC Test Method 61: Colorfastness to
Laundering, Home and Commercial: Accelerated, which is a useful test to evaluate the
durability of finishing agent against laundering and detergents. Among the five different
test conditions available in the test method, AATCC 61 2A was selected. One cycle of
2A test conditions is equivalent to five home or commercial machine launderings done at
38±3°C. The test was performed with 150 mL water, 0.15% owf detergent (1993 AATCC
standard reference detergent), 50 steel balls (0.6cm diameter) in a closed stainless steel
canisters. The closed canisters were loaded on Launder-O-meter and rotated at 40±2 rpm
95
for 45 minutes at 49°C. The washed samples were washed with distilled water after the
cycle and air dried.
Antimicrobial Test (AATCC 100-2004)
The treated fabrics were tested by ‘AATCC Test Method 100-2003: Antibacterial
Finishes on Textile Materials’, which is a quantitative procedure for the evaluation of
antibacterial activity. The test was carried out using S. aureus and E. coli, representing
Gram positive and Gram negative bacteria, respectively. Bacteria are classified into Gram
positive or Gram negative categories based on the reaction of bacteria to the Gram stain
test. Gram stain results depend on the bacterial cell wall structure. The Gram positive
bacterial cell wall consists of plasma membrane, periplasmic space and thick layer of
peptidoglycan. The Gram negative bacterial cell wall is more complex and is made up of
plasma membrane, periplasmic space, and a thin layer of peptidoglycan. The outer layer
consists of lipopolysaccharides and proteins. Because of the different cell wall structures
the bacteria have different defense mechanisms and therefore it is important to assess the
efficacy of antibacterial agent against both types of bacteria to confirm broad range
activity.
Three replications were done for each treatment. The bacteria were incubated in a
nutrient broth for 24 hours at 37°C. The finished fabrics and control fabrics were cut into
circular shape 4.8±0.1cm in diameter. The sample was kept in a 250 mL wide-mouth
glass jar with screw cap inoculated with 1 mL of inoculum overnight. The inoculum was
nutrient broth culture containing ~1×108/mL colony forming units (CFU). The inoculated
samples were then incubated at 37°C for 18 to 24 hours. After incubation, 100 mL of
96
sterilized water was added to the jar. The jar was closed immediately and shaken
vigorously for one minute. The supernatant was diluted to 101 and 10
2 in series. One
hundred microliters was placed on the nutrient agar plate and spread evenly on the agar
surface using a spreader. The nutrient agar plates were incubated for 24 hours at 37°C in
an incubator before taking pictures.
Syntheses
Linear Polyethylenimine (PEI): The deacylation reaction was performed according to a
literature procedure [8]. Three grams of the poly (2-ethyl-2-oxazoline, Mw, 50 000 Da)
(POEZ) was added to 120 mL of 24 % (wt/vol) HCl, followed by refluxing for 96 hours.
The POEZ crystal dissolved completely in 1 hour, but a white precipitate appeared after 3
hours of refluxing. The precipitate was filtered and then air-dried. The protonated
polymer was dissolved in water and neutralized with KOH solution and isolated by
filtration. The white powder was isolated by filtration, washed with distilled water until
the pH became neutral, and dried under vacuum. The yield of the reaction was 1.15 g (88
%). The product was confirmed by proton NMR spectroscopy and the peak values were
1H NMR (CDCl3): , 2.72 (s, 4H, NCH2CH2N), 1.71 (1H, NH).
Scheme 3.1: Synthesis of linear PEI
4-(2-hydroxyethylsulfanyl) Phenol: The phenolic intermediate of sulfur (a) (4-(2-
hydroxyethylsulfanyl) phenol) was synthesized by stirring 4-hydroxythiophenol
97
(mercaptophenol) (6.00 g, 47.61 mmole) in dimethylformamide (DMF, 50 mL) at -5°C
with, 2-bromoethanol (5.90 g, 47.6 mmole) in the presence of K2CO3 (6.6 g, 47.48
mmole) for 30 minutes. The reaction mixture was then stirred for 12 hours at room
temperature. The reaction mixture was poured in ice water (300 mL) and extracted with
dichloromethane (DCM) (200 mL). The organic layer was removed using a rotary
evaporator. The crude product was purified on silica gel column by using a
chloroform:methanol (94:6) solvent mixture. Solid white product was obtained after
removal of solvent mixture. The yield of the reaction was 72.31%. The product was
confirmed by proton NMR and the peak values are 1H NMR (CDCl3): , 8.01 (s, OH,
1H), 7.33 (d, ArH, 2H, J= 8.7 Hz), 6.78 (d, ArH, 2H, J = 8.7 Hz), 4.52 (s, OH, 1H), 3.67
(t, 2H, J = 6Hz), 2.99 (t, 2H, J = 5.7).
4-(2-hydroxyethansulfonyl) phenol: In the next step, the reaction was carried out
according to a literature procedure [9] in which 4-(2-hydroxyethylsulfanyl) phenol (5.85
g, 34.41 mmole) in methanol was stirred with Oxone™ (2KHSO5.KHSO4
.K2SO4) (30.24
g) at 10°C for 20 minutes and then at room temperature for 12 hours to create 4-(2-
hydroxyethansulfonyl) phenol (b). The reaction mixture was filtered, 1 mL of 38-40%
aqueous NaHSO3 was added, and the pH adjusted to 7 using aqueous NaOH (28%). The
mixture was again filtered and the solvent removed by rotary evaporator. The crude
product was purified on a silica gel column using DCM:methanol (91:9) solvent mixture.
Solvent was removed to yield a solid white product. The yield of the reaction was
75.10%. The product was confirmed by proton NMR spectroscopy and the peak values
were 1H NMR (DMSO): , 10.56 (s, OH, 1H), 7.67 (d, ArH, 2H, J = 7.8), 6.9 (d, ArH,
2H, J = 7.5), 3.62 (t, 2H, J = 6.9), 3.31 (t, 2H, J = 6.6).
98
Scheme 3.2: Synthesis of 4-(2-hydroxyethansulfonyl) phenol
2-(4-(6-bromohexyloxy) phenylsulfonyl) ethanol: The intermediate (b) (5.22 g, 30.70
mmole) was then stirred with dibromohexane (31.52 g, 130.24 mmole) to create the
intermediate (c). The reaction was carried out at room temperature for 16 hours under
nitrogen atmosphere in DMF (70 mL) solvent in the presence of K2CO3 (4.3 g). The
reaction mixture was poured in ice water (300 mL) and extracted with DCM (200 mL).
The organic layer was removed by rotary evaporator. The crude product was purified on
silica gel column using a DCM:methanol (95:5) solvent mixture. The yield of the reaction
was 54.25%. The product was confirmed by proton and carbon NMR spectroscopy and
the peak values are 1H NMR (CDCl3): , 7.84 (d, ArH, 2H, J = 9Hz), 7.06 (d, ArH, 2H, J
= 9Hz), 4.04 (t, 2H, J = 6Hz), 3.98 (t, 2H, J = 6.9Hz), 3.43 (t, 2H, J = 6.9Hz), 3.32 (t, 2H,
J = 3.6Hz), 1.9-1.7 (m, 4H), 1.6-1.4 (m, 4H). 13
C NMR (CDCl3): , 163.76, 130.39,
115.26, 68.56, 58.69, 56.72, 33.89, 32.77, 31.13, 29.00, 28.02.
99
Scheme 3.3: Synthesis of 2-(4-(6-bromohexyloxy) phenylsulfonyl) ethanol
Quaternary PEI Copolymer: The intermediate (c) and 1-bromododecane were stirred
with deacylated PEI (0.6 g, 13.95 mmole) intermediate at 95°C for 96 hours in DMSO
solvent. The reaction mixture was filtered and CH3I (2.94 g, 20.92 mmole) was added to
the filtrate. The mixture was stirred at 60°C for 24 hours to obtain quaternized PEI
copolymer (d). Upon cooling, the reaction mixture was dialyzed in distilled water
overnight and freeze dried to obtain pure product. The yield of the reaction was 48%. The
product was confirmed by proton NMR spectroscopy and the peak values are 1H NMR
(DMSO): , 7.8 (bs, 2H), 7.13 (bs, 2H), 3.65-3.32 (m, 22H), 1.8-0.7 (m, 31H).
Scheme 3.4: Synthesis of quaternary PEI copolymer
100
Sulfated Quaternary PEI Copolymer (SQ-PEI): The copolymer (638 mg, 0.89 mmole)
in DMF was sulfated with pyridine sulfur-trioxide complex (427 mg, 2.68 mmole). The
mixture was stirred for 2 hours at 80°C. The reaction mixture was dialyzed in distilled
water overnight and freeze dried to yield final product. The yield of the reaction was
51%. The product was confirmed by FTIR spectroscopy.
Scheme 3.5: Sulfation of quaternary PEI
6-bromohexan-1-ol: A mixture of hexane-1, 6-diol (23.8 g, 0.2 mol) and HBr (48%
aqueous solution, 25 mL) in 50 mL benzene was refluxed for 72 hours. Upon cooling, the
reaction mixture was decanted and the aqueous phase extracted with ether and
chloroform. The combined organic layers were concentrated and the residue was
dissolved in ether and washed with saturated NaHCO3, and then with water until neutral
pH. The organic phase was dried with magnesium sulfate (MgSO4), filtered and
concentrated. The crude product was purified by high vacuum distillation to yield 21.5g
of 6-bromohexane-1-ol as colorless oil. 1H NMR (CDCl3): , 3.65 (t, 2H, J= 6.4Hz), 3.41
(t, 2H, J = 6.4Hz), 1.9-1.8 (m, 4H), 1.4-1.3 (m, 4H).
101
Scheme 3.6: Synthesis of 6-bromohexan-1-ol
4-(4,6-dichloro-1,3,5-triazin-2-ylamino)benzenesulfonic acid: The reaction was done
according to that reported in the literature with some modifications in the procedure.[205]
Cyanuric chloride (4.66 g, 2.52 mmole) was dissolved in acetone (50 mL). The ice-water
mixture (125 mL) was then added to this solution to generate a white precipitate.
Sulfanilic acid (4.32 g, 2.5 mmole) was added to 25 mL of water. The compound was
dissolved and the pH was adjusted to 4.8 by adding 2M NaOH solution. The solution of
sulfanilic acid was added in a drop-wise fashion. The reaction was carried at -5 to 0 ºC
under inert atmosphere for 1 hour. The reaction mixture was filtered to separate a white
solid powder. The powder was first washed with an excess amount of acetone and later
with diethyl ether. The compound was finally dried under vacuum. The product was
confirmed by proton NMR and the peak values were 1H NMR (DMSO): , 11.14 (s, 1H),
7.5 (q, ArH, 4H, J= 10.9 Hz).
Scheme 3.7: Synthesis of 4-(4, 6-dichloro-1,3,5-triazin-2-ylamino)
benzenesulfonic acid
Hydroxy based Quaternary PEI Copolymer: The linear deacylated PEI (500 mg,
11.62 mmole) was stirred with 1-bromododecane (1.44 g, 5.81 mmole) and 6-
102
bromohexane-1-ol (1.05 g, 5.81 mmole) at 95 °C for 96 hours in the presence of tert-
amyl alcohol solvent and potassium carbonate (1.9 g, 13.74 mmole). The reaction
mixture was filtered and immediately treated with CH3I (1.36 mL, 23.24 mmole). The
reaction mixture was stirred at 60 °C for 24 hours and, after cooling the reaction mixture,
the copolymer was precipitated by adding excess diethyl ether. The precipitated product
was filtered and dried under vacuum to obtain hydroxyl based quaternized PEI copolymer
(f). The yield of the reaction was 45%. The product was confirmed by proton NMR
spectroscopy and the peak values were 1H NMR (DMSO): , 4.3 (bs, 2H), 3.6-3.39 (m,
22H), 1.8-0.7 (m, 31H).
Scheme 3.8: Synthesis of hydroxyl based quaternary PEI copolymer
Monochlorotriazine Based Quaternary PEI Copolymer (MCT-PEI): The reaction
was done by following a similar reaction reported in the literature.[206] The intermediate
(e) (251.3 mg, 0.78 mmole) and triethyl amine (52.09 mg, 0.51 mmole) was dissolved in
dimethyl sulfoxide (DMSO). The temperature of the reaction mixture was raised to 40ºC.
The solution of copolymer (c) (500 mg, 0.78 mmole) dissolved in DMSO was added
drop-wise to the reaction mixture. The reaction mixture was stirred at 60 ºC for 1 hour.
The product was precipitated by adding excess ethyl acetate. The product was filtered and
103
dried under vacuum to yield final copolymer (d). 1H NMR (DMSO): , 7.5 (bs, 4H), 4.42
(bs, 2H), 3.70-3.39 (m, 22H), 1.8-0.7 (m, 31H).
Scheme 3.9: Synthesis of monochlorotriazine based quaternary PEI copolymer
Dichlorotriazine Based Quaternary PEI Copolymer (DCT-PEI): Quaternary PEI
(3.25 g, 5.10 mmole) was dissolved in DMF solvent (35 mL) and added in a dropwise
fashion to the solution of cyanuric chloride (940 mg, 5.10 mmole) and K2CO3 (774 mg,
5.61 mmole) at 0 to 5 °C for one hour. The reaction mixture was filtered and precipitated
by adding excess ethyl acetate solvent at lower temperature. The mixture was filtered and
dried under vacuum to yield final solid yellow product. 1H NMR (DMSO): , 4.49 (bs,
2H), 3.7-3.39 (m, 22H), 1.8-0.7 (m, 31H).
Scheme 3.10: Synthesis of dichlorotriazine based quaternary PEI copolymer
104
Results and Discussion
Synthesized copolymers SQ-PEI, MCT-PEI and DCT-PEI contain both fiber
reactive vinyl sulfone, monochloro and dichlorotriazine side chains, respectively, and
hydrophobic side chains (dodecane, C12). The composition of copolymers quaternary PEI
(d), MCT-PEI and DCT-PEI was checked by NMR spectroscopy, which revealed that the
polymer composition matched with the pendant group feed ratio. Based on the NMR
integration values, the fiber reactive side-chain constitutes 50% of total polymer pendant
groups with the dodecane constituting the other 50%. The synthesized new copolymers
are soluble in DMF, DMSO and sparingly soluble in alcohols.
The FTIR spectra of quaternized PEI and sulfated quaternary PEI exactly
matched peak by peak except there were additional peaks around ~1000-1090 cm-1
for S-
O-C stretching vibrations after introducing the sulfate group to the polymer (Figure 3.1).
The bands around 1400 and 1200 cm-1
were attributed to asymmetric and symmetric
stretching vibrations of sulfone groups (-SO2-) in the polymer. The other major peaks in
the figure 2.22 were peaks at 2920 and 2849 cm-1
due to C-H stretching of the aliphatic
backbone and pendant groups, C-C ring vibrations at 1600 cm-1
and C-N+ stretch at 1468
cm-1
.
105
Figure 3.1: FTIR spectra (a) Quaternary PEI (b) Sulfated quaternary PEI
(SQ-PEI)
The cotton fabrics were finished with method typically used in reactive dyeing of
cellulosics. The reactive dye classes containing monochlorotriazine and vinyl sulfone
groups are applied around 80 to 95 °C and often called hot brand dyes. On the other hand,
the dichlorotriazine containing reactive dye class, being very reactive, is applied at room
temperature and is often called cold brand dyes. These dyes are covalently attached on
cellulosic textiles by adding suitable alkali and this step is called fixation step.[207] In
the case of MCT-PEI and SQ-PEI copolymers, a finishing temperature of 80 °C was used
is, similar to monochloro and vinyl sulfone based reactive dyes.
In the case of SQ-PEI, the copolymer forms a dispersion in water at a neutral pH
and dissolves completely in water at alkaline pH due to salt formation at the sulfated
3500 3000 2500 2000 1500 1000
0.00
0.01
0.02
Abs
orba
nce
Wavenumber (cm-1
)
After sulfation
Before sulfation
S=O
1223 cm-1
C-O-S 1090 cm-1
(a)
(b)
106
group. The polymer is expected to undergo Michael addition reaction to form a covalent
bond with the substrate under alkaline conditions at 80 °C. The vinyl group generated
from β-sulfatoethyl sulfate under alkaline conditions can react with the nucleophile such
as hydroxyl groups in cellulose to form a covalent bond. The general reaction schematic
of polymer with substrate is shown in figure 3.2.
Figure 3.2: Finishing of SQ-PEI with cotton fabric under alkaline conditions
The MCT-PEI and DCT-PEI copolymers have reactive triazine linkers on the
polymer backbone which can undergo nucleophilic substitution reaction (Figure 3.3) with
hydroxyl groups in cellulose to form a covalent bond. The copolymer DCT-PEI is
expected to be more reactive than the MCT-PEI as it has two chlorine atoms on the
triazine ring; therefore finishing was done at room temperature and the MCT-PEI
copolymer was treated at 80°C.
Figure 3.3: Finishing of MCT-PEI with cotton fabric under alkaline conditions
107
Microbiological Testing:
The antibacterial efficacy of the copolymers was evaluated by the AATCC 100
test. The fabrics finished with the copolymers (SQ-PEI, MCT-PEI and DCT-PEI) showed
excellent performance against S. aureus before washing, with ~100% reduction (Figure
3.5a, 3.7a, and 3.9a). However, the antibacterial performance was drastically reduced
after one accelerated laundering cycle (Figures 3.5b, 3.7b, and 3.9b). A similar trend was
observed in the case of E. coli testing too, with significant reduction in bacterial count
before washing and a dramatic reduction in antibacterial performance after one
accelerated laundering cycle (Figures 3.6, 3.8, and 3.10).
To investigate the poor performance of the copolymers, after washing, the
nitrogen content of the fabrics was analyzed through elemental analysis on the finished
fabrics and after washing. The elemental analysis was performed by The Chemical
Analysis Laboratory at The University of Georgia. The nitrogen content on the fabric was
an indication of the presence of the copolymer on the fabric. In the case of DCT-PEI,
after the wash cycle there was no nitrogen present on the fabric suggesting the polymer
did not covalently attach to the fabric and was washed away. Similarly, in the case of SQ-
PEI more than 80 percent of the finishing agent was removed after the wash cycle.
Interestingly, MCT-PEI showed an increase in the nitrogen content after the wash cycle;
this may be caused by the uneven distribution of copolymer on the fabric surface (Table
3.1). The large variation in the nitrogen content readings of SQ-PEI before washing may
also be caused by uneven distribution of copolymer on the fabric.
108
Table 3.1 Percentage nitrogen content on the treated and untreated fabrics before wash
and after wash by elemental analysis
SQ-PEI MCT-PEI DCT-PEI Control
%N on
fabric
before
wash
cycle
%N on
fabric
after
wash
cycle
%N on
fabric
before
wash
cycle
%N on
fabric
after
wash
cycle
%N on
fabric
before
wash
cycle
%N on
fabric
after
wash
cycle
%N on
untreated
cotton
fabric
1 0.068 0.027 0.094 0.209 0.146 0.0 0
2 0.22 0.0 0.075 0.081 0.116 0.0 0
Avg. 0.144 0.027 0.084 0.145 0.131 0.0 0
Although all the new copolymers showed good antibacterial properties before
washing, the optimized application conditions must be developed to improve the
durability results. Corrective measures should be taken to suspend the copolymers in
water homogeneously. This could be achieved through creation of an emulsion with a
suitable surfactant. This would allow uniform distribution of the copolymer on the fabric
surface during the application stage. The other parameters which might improve the
reaction between copolymers and fabric are use of stronger alkali, and increasing
treatment temperature and time. An alternative application technique could be a typical
pad-dry-steam method used in reactive dyeing of textiles in which the finishing solution
is forced on the fabric during padding followed by a drying step. The fabric is then
passed through alkali bath by padding process and subsequently wet steam is used to fix
the finishing agent.
109
(a) (b)
Figure 3.4: Untreated cotton fabrics (a) control S. aureus and (b) control E. coli
(a) (b)
Figure 3.5: Evaluation against S. aureus, (a) treatment with SQ-PEI before washing (b)
treatment with SQ-PEI after washing
110
(a) (b)
Figure 3.6: Evaluation against E. coli, (a) treatment with SQ-PEI before washing (b)
treatment with SQ-PEI after washing
(a) (b)
Figure 3.7: Evaluation against S. aureus, (a) treatment with MCT-PEI before washing
(b) treatment with MCT-PEI after washing
111
(a) (b)
Figure 3.8: Evaluation against E. coli, (a) treatment with MCT-PEI before washing (b)
treatment with MCT-PEI after washing
(a) (b)
Figure 3.9: Evaluation against S. aureus, (a) treatment with DCT-PEI before washing (b)
treatment with DCT-PEI after washing
112
Figure 3.10: Evaluation against E. coli, (a) treatment with DCT-PEI before washing (b)
treatment with DCT-PEI after washing
113
References
95. Gao, Y. and R. Cranston, Recent advances in antimicrobial treatments of textiles.
Textile Research Journal, 2008. 78(1): p. 60-72.
151. Willey, J.M., L.M. Sherwood, and C.J. Woolverton, Prescott's Principles of
Microbiology. 2009: McGraw-Hill Higher Education. 960.
204. Bajaj, P., Finishing of textile materials. Journal of Applied Polymer Science,
2002. 83(3): p. 631-659.
205. Renfrew, A.H.M., D.A.S. Phillips, and I. Bates, 4-Arylamino-6-chloro-1,3,5-
triazin-2(1H)-ones: nucleophilic substitution of a model compound in acid
medium to produce novel fibre reactive triazinyl derivatives. Dyes and Pigments,
2003. 59(1): p. 99-106.
206. Zhao, T., G. Sun, and X. Song, An antimicrobial cationic reactive dye: Synthesis
and applications on cellulosic fibers. Journal of Applied Polymer Science, 2008.
108(3): p. 1917-1923.
207. Shore, J., ed. Cellulosics dyeing / edited by John Shore. 1995, Society of Dyers
and Colourists: Bradford, West Yorkshire, England. ix, 408 p.
114
CHAPTER 4
SUMMARY
In project one, we have demonstrated a novel and efficient approach to covalently
attach antimicrobial polymer on any substrate with a C-H bond. A hydrophobic PEI
copolymer substituted with benzophenone side chain was spin-casted or spray-coated on
a wide range of surfaces from cotton to inert plastics and photo-crosslinked by UV
irradiation. After the covalent attachment of polymer on the surface, the biocidal activity
was investigated against both Gram-positive (S. aureus) and Gram-negative (E. coli)
bacteria. The surface grafted with a high density of polymers exhibited relatively high
biocidal activity. When the thickness of the polymer layer was greater than 50 nm,
essentially almost all the bacteria were killed. The PVC grids coated with the copolymer
showed excellent performance against bio-fouling. The grids put in different ocean
environments (coast of Chile and Canada) for more than 40 days had a very little biofilm
formation. Based on the initial studies, the polymer coating can be a good candidate for
anti-fouling applications. Overall, this one step photochemical attachment process of an
ultrathin antimicrobial coating is both simple and scalable for industrial applications.
A detailed study of cytotoxicity and biocompatibility of BP-PEI is needed in the
future. The favorable results will open up its applications in medical implants and other
in-vivo uses. Moreover, it is very much necessary to continue efforts in the better
understanding of antimicrobial mechanism of surface immobilized antimicrobial
polymers along with the polymer structural parameters which influence biosafety.
115
Finally, the demonstrated strategy of photochemical attachment of biocidal polymers
presents numerous opportunities to create new more effective antimicrobial polymers. In
project two, three new copolymers SQ-PEI, MCT-PEI and DCT-PEI were synthesized
containing the reactive pendant groups vinyl sulfone, monochlorotriazine and
dichlorotriazine, respectively, and a cationic PEI backbone. All the copolymers showed
good antibacterial properties against both Gram positive (S. aureus) and Gram negative
(E. coli) before laundering, but the performance decreased substantially after laundering.
The elemental analysis results indicate copolymers were washed away in the laundering
process.
There is need to develop optimized application conditions to improve the
reactions between the copolymers and cotton fabric. Some of the possible parameters
could be use of stronger alkali, and increasing the treatment temperature and time to
improve copolymer fixation. The other area of focus should be achieving uniform
distribution of copolymer on the fabric surface which by formulating the finishing agent
with suitable surfactant. An alternative application technique could be a typical pad-dry-
steam method which may improve fixation of copolymers on the fabric surface.