THE UNIVERSITY OF CRAIOVA

Doctoral School of Sciences

Physics

PHD THESIS

- SUMMARY -

LASER PROCESSING OF BIO-POLYMERS:

APPLICATIONS IN BIOLOGY AND MEDICINE

PhD Supervisor:

Prof. C.S. I Dr. Maria Dinescu

PhD Student: Simona Brajnicov

Craiova

2019

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Acknowledgments

I would like to start by thanking the members of the evaluation committee of this doctoral thesis: CS I Dr.

Mariana Braic, CS II Dr. Alexandra Palla-Papavlu, Prof. Univ. Dr. Cristian Focșa, who have dedicated their

time and energy to read and evaluate this thesis.

First and foremost, I would like to thank Prof. Dr. Maria Dinescu for her guidance and trust she has offered

me since the first steps in the group "Photonic Processing of Advanced Materials" at the National Institute for

Laser, Plasma and Radiation Physics. I would like to particularly thank her for the opportunities and support

she offered me to reach a position of professional maturity and not only.

Furthermore, I would like to thank all my colleagues in the "Photonic Processing of Advanced Materials"

group for the help and advice provided whenever I needed it.

In particular, I would like to thank Valentina Dinca, Andreea Matei, Alexandra Palla-Papavlu, Antoniu

Moldovan, and Mihaela Filipescu for their unconditional help, patience, and understanding with which they

have permanently surrounded me. I am truly grateful for their support, encouragement, and advice that have

been of great use to me throughout my PhD thesis and will be useful to me in the future.

I would like to thank Dr. Anișoara Cîmpean and Dr. Patricia Neacșu from the Faculty of Biology of the

University of Bucharest, and also to Dr. Alina Vasilescu from the International Centre of Biodynamics for the

contribution made to this PhD thesis.

In addition, I am also grateful to my colleagues from the "Plasma Processes for Functional Materials and

Surfaces" group for their support and availability.

I would also like to thank my friends for all the love, constructive discussions and sacrifices they have made

for me.

Last, but not least, I thank my family and Ionuț for the love and support they have always offered me.

All these thanks are addressed from the heart and with utmost gratitude!

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LASER PROCESSING OF BIO-POLYMERS: APPLICATIONS IN BIOLOGY

AND MEDICINE

Table of Contents

1. Introduction ................................................................................................................................................... 4

2. The structure of the thesis and a short description of the contents of each chapter ....................................... 5

3. Conclusions of the research carried out within the present PhD thesis ....................................................... 18

3.1. Articles in ISI journals for the period 2016-2019 ................................................................................. 18

3.2. Presentations at international conferences ............................................................................................ 19

3.3. Participation in summer schools and scientific sessions ...................................................................... 20

4. Bibliography ................................................................................................................................................ 21

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1. Introduction

Laser deposition techniques are increasingly used in the field of organic and inorganic materials. A significant

technological progress is the development of thin films fabrication techniques, due to the need for using them

in a wide range of applications in fields such as electronics, sensors, optics, pharmacology, biology, or

medicine. Polymers, proteins, and bacteria play an important role for thin films used in applications targeting

biology and medicine. Depending on the type and field of application, polymers can be used in the form of

microemulsions, microspheres, hydrogels or thin layers.

In particular, due to their specific properties, such as stability in biological environment, mechanical strength,

protection against corrosion, biocompatibility, polymers, proteins and bacteria can offer a wide range of

functionalities, being used in various applications in the biomedical and pharmaceutical fields such as

orthopaedic materials, antibacterial surfaces, drug-controlled release coatings, tissue engineering and

biosensors materials [1].

This PhD thesis entitled "LASER PROCESSING OF BIO-POLYMERS: APPLICATIONS IN BIOLOGY

AND MEDICINE" is the result of the work carried out within the “Photonic Processing of Advanced

Materials” group from the Laser Department of the National Institute for Laser, Plasma and Radiation Physics

– INFLPR, in collaboration with the Faculty of Biology of the University of Bucharest and with the

International Centre for Biodynamics. Part of the results presented in this thesis were obtained in the

framework of three projects, i.e. PN-III-P2-2.1-PED-2016-1715 - HERMESH (results presented in chapter II),

PN-II-RU-TE-2014-4 -2434 - BIOSINTEL (results presented in chapter III) and PN-III-P2-2.1-PED-2016-

0221 - IPOD (results presented in chapter V) through CNCS-UEFISCDI-funded research programs.

The main objective of this thesis is three-fold, i.e. to obtain biopolymeric and antibacterial coatings, as well as

coatings based on hybrid composite materials using laser-based techniques, aiming at the development of

biomedical applications and the fabrication of biosensors.

Among the laser-based techniques available, i.e. pulsed laser deposition (PLD), matrix assisted pulsed laser

evaporation (MAPLE), and laser-induced forward transfer (LIFT), in this thesis I have focused on using

MAPLE in order to obtain thin films with potential applications in biology and medicine.

The present PhD thesis follows three directions to highlight the versatility of the MAPLE technique in creating

functional bio-interfaces and thin films:

1. Development of functional bio-interfaces and thin films with potential in applications related to

the functionalization of commercial polypropylene and polyester meshes used to repair parietal

defects, as well as the functionalization of bone implants, namely obtaining multifunctional hybrid

coatings with controllable chemical and physical characteristics which allow the integration of the

foreign prosthetic material in the body with a minimal risk of infections.

2. Obtaining functionalized cell-based surfaces of Micrococcus lysodeikticus (ML), so that they can

be used as cell optical biosensors.

3. Obtaining shellac coatings with possible applications as enteric coatings for oral medicines.

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The schematic representation of the directions followed in this thesis, as well as the chapters in which the

corresponding results are described, are shown in figure 1.

Figure 1. Schematic representation of the structure of this thesis.

2. The structure of the thesis and a short description of the contents of each chapter

The thesis is divided in 6 chapters.

The first chapter, "Thin film deposition methods and characterization techniques", is dedicated to a short

classification of the deposition methods used to obtain thin films, where the focus is on laser-based techniques

with emphasis on MAPLE. This chapter describes i) the MAPLE experimental setup used for processing thin

films ii) the investigation techniques used to provide information on the properties of the coatings obtained by

MAPLE: scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier-transform infrared

spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS),

Raman spectroscopy, spectro-ellipsometry (SE); iii) the determination of the films’ wetting properties and in

vitro biocompatibility assessment.

The technique chosen for carrying out the experiments presented in this thesis with the final goal of obtaining

coatings with biomedical applications is MAPLE. The schematic representation of the technique is shown in

figure 2. In recent years, remarkable results have been reported in literature regarding MAPLE deposition of a

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significant number of polymers, bioactive compounds, proteins, ceramic materials in the form of thin films

[2,3,4,5,6,7,8,9]. MAPLE has been developed in the late 1990s at the US Naval Research Laboratory [3Error!

Bookmark not defined.], for the transfer of organic and inorganic materials on solid substrates, proving to be

a flexible method for controlling deposited organic compounds, polymers, proteins or biological materials [4].

There are various applications in the field of medicine and biology, for which MAPLE has proven its versatility

in the deposition of natural and synthetic polymers [5,6], nanoparticles [7] or incorporating various drugs and

proteins into a polymeric matrix [8,9].

Even if it is considered a relatively expensive technique (as it involves the use of pulsed laser sources and

vacuum systems), MAPLE is unique by providing all of the following advantages simultaneously: (1) no

significant degradation of the polymer or biomaterial; (2) the ability to monitor and control in real time the

deposition rate; (3) the ability to deposit selectively onto different areas by using proper masks, thus avoiding

the need for subsequent patterning, which might alter the chemistry of the deposited material; (4) offers the

possibility to deposit coatings with a wide range of thicknesses, from a few nanometers to a few microns with

accurate thickness control; (5) the ability to control the surface morphology (roughness, granulation, etc.) for

specific requirements; (6) the ability to deposit multilayer structures, thus solving the problem of solvent

orthogonality, and the possibility to obtain composite films by irradiating a target made of different materials

dissolved in the same solvent or in different solvents [10,11, ,12,13,14,15,16,17,18,19,20,21,22]. In this thesis,

MAPLE has been optimized for obtaining thin films with possible applications in biology and medicine.

Figure 2. Schematic representation of the MAPLE setup.

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The second chapter, "Functionalization of flexible substrates for medical applications 1", presents an

application of MAPLE, i.e. obtaining hybrid coatings for commercial polypropylene and polyester meshes

used for parietal defect repairs.

At the moment, meshes are the most common implant materials used in general surgery, over 20 million

implants of this type are used every year in the world [23]. The use of meshes to repair parietal defects is

accepted as a general standard, both in minimally-invasive (laparoscopic) procedures and in conventional

surgical procedures. Over 70 types of mesh implants are used in this field (general surgery), and among the

most used are those based on polypropylene (PP) and polyester (PE), due to their mechanical properties,

optimum biological tolerance, chemically inert character, and also their flexibility and biocompatibility [24].

In addition to the chemical and physical properties, another important aspect is bacteria adherence to the mesh

implants and the formation of biofilms, which leads to the increase of antibiotics use and, ultimately, to the

removal of the mesh from the body [25]. Although the use of mesh implants in surgery is more and more

frequent, their optimum integration in the body is still a complex subject, and the risk associated with infections

continues to be one of the main unresolved problems [26].

In this thesis, we propose an innovative solution that allows limiting risks from infections. The solution

proposed is covering the commercial polypropylene and polyester meshes (figure 3) with thin films of

polyethylene oxide (PEO), and also a mixture of polyethylene oxide and carbon nanotubes (CNT), by the laser-

based deposition method MAPLE. Several groups of researchers have already obtained polymeric and hybrid

thin layers using the MAPLE technique. Therefore, the successful use of MAPLE for thin film deposition has

already been proven in several published articles. [27,28,29,30,31]

Figure 3. Optical images of polypropylene (PP) and polyester (PE) meshes used in MAPLE experiments

The novelty of this approach is the use of MAPLE technique to obtain hybrid thin films (polyethylene oxide

and polyethylene oxide with different concentrations of carbon nanotubes) to be used as layers with

1 This section of the thesis was published in Applied Physics A ((2019) 125:424), the article title

„Tuning the physico-chemical properties of hernia repair meshes by matrix-assisted pulsed laser evaporation”,

authors: Alin, CD; Grama, F; Papagheorghe, R; Brajnicov, S; Ion, V; Vizireanu, S; Palla-Papavlu, A; Dinescu,

M.

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antimicrobial properties for hernia repair meshes. Also, the morphological and chemical properties of the

deposited thin layers indicate that MAPLE is an appropriate method for the fabrication of thin layers which

could be used for controlled drug release.

The coatings were obtained by depositing (in different experiments) polymers of polyethylene oxide, carbon

nanotubes, and the mixture of polyethylene oxide and carbon nanotubes (PEO:CNT mixture) on hernia repair

meshes. SEM images on different areas of the interfaces of polypropylene (images 4.a and 4.c) and polyester

(4.b) hernia repair meshes , and meshes coated with a thin layer of PEO polymer (4.d), carbon nanotubes (4.e),

or PEO:CNT mixture (4.f and 4.g) are shown in figure 4.

Figure 4. SEM images of the macroporous monofilament commercial hernia repair meshes, before and after

deposition by MAPLE: low magnification (the first two columns) and a single wire (the third column). On the

first line are the SEM images obtained on different areas of polypropylene (PP) (a) and polyester (PE)

meshes (b) and the enlarged image of a single wire in a polyester mesh (c), before deposition; on the second

line are images of the meshes covered with a thin layer of PEO polymer (d); on the third line are images for

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meshes covered with carbon nanotubes (arrows in the figure indicate clusters of CNTs) (e); on the fourth line

are images for meshes covered with PEO80:CNT20 mixture (f); and on the last line images for the meshes

covered with PEO98:CNT2 mixture (g)

The PEO, CNT, and PEO:CNT coatings were deposited by MAPLE directly on the surface of commercial

meshes, without any previous functionalization. FTIR, XPS, SE and SEM investigations showed no chemical

modification of the deposited materials.

The optical properties of the PEO:CNT mixture were determined by spectro-ellipsometry, highlighting the

presence of carbon nanotubes (by comparing the absorption spectrum of the deposited material with that of the

initial product) [32,33]. In addition, the wetting properties of the hernia repair mesh surfaces are significantly

modified by coating with a thin layer of PEO, CNT or PEO:CNT mixture, from a hydrophobic character

(contact angle 131° ̶ 96°), to a hydrophilic character (contact angle 37° ̶ 11°). A larger number of

nanotubes on the surface of hybrid thin films leads to a more hydrophilic surface, confirmed by the results of

contact angle measurements. The presence of nanotubes changes both the topography and the chemistry of the

surfaces covered with the protective layers.

In recent years, the antimicrobial properties of CNTs have received special attention, so in the future we intend

to investigate the antimicrobial effect of coatings of polymer mixtures with carbon nanotubes deposited by

MAPLE. In addition, it will be interesting to determine the minimum amount of carbon nanotubes in PEO:

CNT mixtures leading to the greatest antimicrobial effect against different bacteria. This study provides a

solution for potential applications for hernia repair.

In chapter three, ’’Obtaining new hybrid nanocomposite coatings2’’, the results on obtaining

multifunctional coatings with controlled chemical and physical characteristics (functionality, morphology and

roughness) with possible applications in the functionalization of bone implants are presented.

In recent decades there has been an increasing interest in the use of different types of synthetic biodegradable

polymers in the field of biomedicine, as versatile materials that lead to a low immune response and have well-

defined physical-chemical, biological, biomechanical, and degradability characteristics

[34,35,36,37,38,39,40,41,42,43,1]. The main challenge is to optimize the functionality of the material

according to the application requirements. Therefore, different types of degradable polymers have been and

are being investigated, in order to be correlated with the needs of the desired applications.

Biocompatible and biodegradable coatings with controlled chemical and physical characteristics (e.g.

morphology and roughness) are of great interest in applications related to the functionalization of orthopaedic

implants. Metal implants used in orthopaedics are usually made of Ti or its alloys, and have an inert surface

upon contact with the tissue, requiring some functionalization to facilitate the implant's integration into the

body and to minimize the inflammatory response.

2 The first part of this section of the thesis was published in Applied Physics A ((2017) 123:707), the

article title "Tailored biodegradable triblock copolymer coatings obtained by MAPLE: a parametric study",

authors: Brajnicov, S; Neacsu, P; Moldovan, A; Marascu, V; Bonciu, A; Ion, R; Dinca, V; Cimpean, A;

Dinescu, M.

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In the context of this research direction, this chapter presents the results of two systematic studies on:

(i) deposition parameters that influence a series of novel biodegradable coatings based on tribloc poly

(lactide-co-caprolactone) -block-poly (ethylene-glycol) -bloc-poly (lactide-co-caprolactone)

copolymers (PLCL-PEG-PLCL) and,

(ii) obtaining new hybrid nanocomposite coatings for an improved osteoblast response by

incorporating tricalcium β-phosphate nanoparticles (TCP) and HidroMatrix into the co-polymer

(PLCL-PEG-PLCL) tested in the first study (i), obtained by MAPLE, for use in biomedical

research applications.

The morphological characteristics and their roughness were modified by the variation of the composition of

the target material and the laser fluency. Coatings were used for preliminary in vitro testing with MC3T3-E1

pre-osteoblast cells.

In the first part of this study, new biodegradable coatings based on triblock PLCL-PEG-PLCL copolymers

were deposited by MAPLE. The morphological characteristics and the roughness were modified by the

variation of the laser fluence, with significant changes of the morphology, from the droplet accumulation of

materials for the surfaces of the PLCL-PEG-PLCL copolymer coatings obtained using the laser fluence values

between 0.228 J/cm2 and 0.377 J/cm2 (F1 – F4), to wrinkles or carpet-like structures for the surfaces of the

copolymer coatings obtained at larger values, between 0.437 J/cm2 and 1.300 J/cm2 (F5 – F8).

(A) (B)

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Figure 5. AFM images (~ 40 μm × 40 μm) (A) and SEM images (B) on PLCL– PEG–PLCL layers deposited

by MAPLE at different fluences: F1=0,228 J/cm2; F2=0,261 J/cm2; F3=0,333 J/cm2; F4=0,377 J/cm2;

F5=0,437 J/cm2; F6=0,640 J/cm2; F7=0,900 J/cm2; F8=1.300 J/cm2 ; scale bar is 10 μm for AFM images

and 50 μm for SEM images; all vertical AFM scales are represented in nm.

Due to the interaction between the laser beam and the target, at increasing fluence, the polymer molecules are

evacuated into larger solvent clusters, and the evaporation of the solvent during transfer and after deposition

on the substrate is responsible for the formation of the surface polymer characteristics. These characteristics

are observed in the AFM images (figure 5.A) and SEM images (figure 5.B) of the films deposited by MAPLE

[44,45].

The FTIR analysis correlated with the surface morphology led to the selection of the coatings from the triblock

copolymer that had optimal properties; these were used in preliminary in vitro experiments on cell adhesion

and morphology of MC3T3-E1 pre-osteoblasts, in order to evaluate the relative biocompatibility of the tested

substrates.

MC3T3-E1 cells showed differences in their adhesion capacity and morphological characteristics in response

to the variations of the analysed polymeric surfaces (figure 6). Thus, on PLCL-PEG-PLCL copolymer coatings

obtained by MAPLE at different values of laser fluence between 0.228 J/cm2 and 0.333 J/cm2 (F1 –F3) pre-

osteoblasts adhered appropriately and spread well after 24 hours of culture, suggesting that these surfaces

provide favourable growth conditions. On contrary, the adhesion and morphology of MC3T3-E1 cells induced

by the PLCL-PEG-PLCL copolymer coatings obtained at larger values of laser fluence values between 0.377

J/cm2 and 0.437 J/cm2 (F4 – F5) were adversely affected by the physico-chemical features of their surfaces.

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Figure 6. Fluorescence microscopy images showing the adhesion capacity of MC3T3-E1 cells after 2 hours

of culture (A) and morphological features at 24 h post-seeding (B) induced by different coatings of PLCL–

PEG–PLCL (F1=0,228 J/cm2; F2=0,261 J/cm2; F3=0,333 J/cm2; F4=0,377 J/cm2; F5=0,437 J/cm2). Cells

were stained with AlexaFluor 488-Phalloidin to detect actin (green) and with DAPI to detect the nuclei

(blue). Scale bar: 20 μm.

In the second part of this study, TCP particles and HydroMatrix were distributed in a controlled manner in the

biodegradable synthetic copolymer PLCL-PEG-PLCL in order to obtain new biodegradable composite

biointerfaces that follow the improved response of the osteoblast MC3T3-E1 cells.

SEM images confirmed that TCP nanoparticles and HydroMatrix were successfully incorporated into PLCL-

PEG-PLCL copolymer by MAPLE method. The coatings obtained with the MAPLE method have a relatively

uniform distribution of the material throughout the surface, as opposed to those obtained by drop-cast, in which

the material forms agglomerations. SEM images obtained on different areas of the composite coatings

deposited by the drop-cast method (7.A) and MAPLE (7.B) are shown in figure 7.

(A) (B)

Figure 7. SEM images obtained on different areas of the reference composite coatings obtained by drop-cast

(A) and of the composite coatings obtained by MAPLE (B).

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The in vitro biological evaluation of all the biomaterials analysed confirmed their functionality was confirmed

by bio-tests with osteoblast cells (figure 8).

The results presented in this chapter highlight the versatility of the MAPLE technique in the creation of bio-

functional interfaces as coating layers for studies related to the interaction of mammalian cells at the cell-

material interface.

Figure 8. Morphological characteristics of MC3T3-E1 osteoblasts cultured on materials 24 hours after

seeding (40x); Fluorescence marking: cytoskeletal actin - green; vinculin (adhesion protein) - red; the

nucleus - blue

The fourth chapter, "Fabrication of functional bacterial layers of Micrococcus lysodeikticus (ML)

deposited by MAPLE on glass substrates, for optical detection of serum lysozyme3", highlights another

applicability of the MAPLE method: obtaining functionalized surfaces based on Micrococcus lysodeikticus

(ML) cells so that they can be used as cellular optical biosensors.

Laser-based deposition techniques - relatively new in the fields of biointerfaces and biosensitization, especially

in cell transfer - offer promising prospects for obtaining coatings with controlled properties, from natural or

synthetic compounds. These techniques eliminate most of the shortcomings usually associated with "classic"

surface functionalization techniques (drop-cast, immersion coating or centrifugal coating): surface unevenness,

contamination, use of solvents, etc.

Cellular biosensors for detection by optical [46,47] or electrochemical [48,49,50] methods, achieved by

immobilizing whole and functional cells (algae, bacterial cells, mammalian cells) on different substrates, have

3 This section of the thesis was published in Colloids and Surfaces B: Biointerfaces ((2018) 162: 98-

107), the article title "Functional Micrococcus lysodeikticus layers deposited by laser technique for the optical

sensing of lysozyme", authors: Dinca, V; Zaharie-Butucel, D; Stanica, L; Brajnicov, S; Marascu, V; Bonciu,

A; Cristocea, A; Gaman, L; Gheorghiu, M; Astilean, S; Vasilescu, A.

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applications in various fields, from ecological testing and ecotoxicity to biopharmaceutical production [51] or

medical diagnosis [52].

In this study we aimed to obtain functionalized cell-based surfaces of Micrococcus lysodeikticus, which can

be used as biosensors for optical detection of lysozyme in serum.

Lysosyme is an enzyme present in biological fluids of the body (tears, urine, serum or saliva), whose

concentration in human serum is a specific biological marker for conditions such as AIDS, pulmonary

tuberculosis, sarcoidosis, inflammatory bowel disease (IBD) [53], nephrosis, acute bacterial infection,

ulcerative colitis [54] etc.

ML, a gram-positive bacterium belonging to the Micrococcacea family, is a typical substrate for this enzyme

and is used to determine the enzymatic activity of lysozyme by turbidimetry [55].

In this study, the optimal conditions for obtaining the bacterial interfaces functionalized by the MAPLE method

were determined by the morphological and structural characterization of the interfaces as well as by testing

them with different concentrations of human lysozyme, so that they can be used as cellular optical biosensors.

To validate the results of this study, reference interfaces were used for the optical detection of lysozyme in

serum - obtained by the layer-by-layer method of deposition (LBL).

As a first step in the functionalization of bacterial interfaces, the glass slides were coated with Poly

Diallyldimethylammonium Chloride (PDDA) ̶ a high density cationic polymer ̶ used as a positively charged

adsorbent on the glass slides surface to facilitate the adhesion of negatively charged ML bacteria.

AFM and SEM images and FTIR measurements on bacterial films confirmed that ML bacteria were

successfully deposited as functional cells on the PDDA layer by MAPLE. The layers deposited by MAPLE

were less dense, having a different appearance from those deposited by LBL.

Prior to serum testing, ML/PDDA layers, obtained by MAPLE or LBL, were further coated with graphene

oxide (GO). This step was considered necessary to avoid the desorption of bacteria in the serum in the absence

of lysozyme, similar to the observations in literature [56].

GO/ML/PDDA interfaces were investigated for their usefulness in detecting lysozyme, by applying a volume

of 15 µl of foetal bovine serum (FBS) with different concentrations of human lysozyme (0-10 µg/ml) on a

sensor surface area. The changes in the intensity of the analysed areas after exposure to the serum, correlated

with the images of phase contrast microscopy, were evaluated by two alternative methods: 1) by UV-VIS

spectrophotometry, measuring the absorption of the functional ML slides in the range 350-700 nm and 2) by

scanning the slides and analysing the optical image through ImageJ.

The optimal value for the incubation period of the biosensors with serum samples was 10 min. After incubation

with the serum samples, the absorption spectra of the ML films change significantly. The lysozyme destroys

the bacteria and induces their desorption from the PDDA interface depending on its concentration in the serum.

In particular, the absorption at 700 nm is a reliable analytical parameter, allowing the construction of

calibration curves that indicate the proportional dependence of the absorption on the lysozyme concentration,

for concentrations between 1 and 10 µg/µl (figure 9) with the calculated detection limit of 0.5 µg/µl. These

data confirmed the feasibility of using ML functional interfaces for quantitative detection of lysozyme in

serum.

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Figure 9. Absorbance spectra in the range 350-700 nm of PDDA/ML/GO slides following incubation with

bovine serum spiked with different concentrations of lysozyme from 0 to 1, 3, 5, 7 and 10 µg/mL (A).

Corresponding calibration curve for lysozyme obtained by taking as analytical parameter the variation in

absorbance at 700 nm following incubation with spiked serum (B).

The functionalized bacterial interfaces obtained by MAPLE were also tested to analyse human serum samples

from IBD patients. Two samples with human serum and two samples with FBS were analysed with the

functionalized slides obtained by MAPLE and LBL. As shown in Table 1, there is a good agreement between

the results obtained on the ML interfaces deposited by MAPLE and the reference ones deposited by LBL.

Moreover, the calculated recovery values of the concentration of lysozyme in bovine serum, detected with ML

slides and compared with the theoretical value of lysozyme concentration, indicate a good accuracy of

detecting lysozyme with both ML interfaces.

Table 1 Analysis of serum samples with ML-functionalised interfaces

Sample Lysozyme (µg/mL )

ML-functionalized

interfaces by MAPLE

Reference ML-functionalized

interfaces via LBL

Human serum 1 5.14±0.06 5.13±0.87

Human serum 2 6.10±0.24 6.72±0.91

Bovine serum ND ND

Bovine serum with

7 µg/mL lysozyme

6.75±0.35

7.23±2.14

In conclusion, the bacteria deposited by MAPLE retained their sensitivity to lysozyme and led to similar results

for the detection of lysozyme in real serum samples with the "reference" interfaces obtained by LBL.

Compared to LBL, where the steps of the slide functionalization were executed manually, MAPLE offers a

good control of the deposition. Given the importance of the architecture of detection interfaces, MAPLE is a

promising technique for the controlled transfer of other compounds, to increase the stability and sensitivity of

serum lysozyme detection interfaces.

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In chapter five, "Shellac coatings tested for enteric coatings4", the results of a systematic study on obtaining

shellac coatings by MAPLE are presented, with possible applications as enteric coatings for orally

administered drugs.

Enteric coatings are polymeric coatings applied to drugs that are administered orally, with the purpose of

preventing their disintegration or dissolution in the stomach.

The transport of drugs with direct absorption into the colon, or the delayed release of the active substances are

particularly important processes in the field of pharmacotherapy, for the more effective treatment of certain

conditions. These include irritable bowel syndrome [57], Crohn's syndrome [58], nocturnal asthma, angina or

arthritis [59].

Shellac is a biomaterial, a resin secreted by the female insect Laccifer (Tachardia) Iacca Kerr (Fam. Coccidae)

on several tree species. It consists of esters and polyesters of polyhydroxy acids. The main components of the

shellac are aleuritic acid and shellolic acid [60]. Other components may be butyl acid and jalaric acid [61]. The

colour can vary between light yellow and dark red, depending on the area of collection of the raw material

from which the shellac results. Due to remarkable properties such as UV radiation resistance, waterproofing,

relatively high electrical resistivity [62], shellac is used in various fields: for encapsulation and

microencapsulation as [63], as an enteric coating [64,65,66] (in the pharmaceutical field), as a protective

coating for fruit [67], can also be added to the composition of packaging materials [68] (in the food industry).

It can also be used in the preparation of various protective lacquer [69], to the protection of art objects [70],

can be used as a substrate for organic field effect transistors as well as as a dielectric material for the gate [60],

etc.

The objectives of this study were achieved in three stages: obtaining the layers of shellac using the MAPLE

technique; morphological and structural analysis of the layers; testing of layers in simulated gastric fluid to

evaluate the ability to be used as enteric coatings.

Following the MAPLE experiments, it has proven to be an efficient deposition method for obtaining adherent

and smooth shellac coatings. After the optimization of the deposition parameters, films with a thickness of

2000 nm were obtained, with the roughness value less than 1% of the thickness. The obtained films do not

show drops. It has been observed that laser fluence is an important factor in maintaining the chemical structure

of the shellac. For small fluences, all the characteristic absorption bands were kept and the spectra were in

good agreement with the initial shellac spectra.

The best thin films in terms of chemical structures and surface roughness, obtained at the wavelength of 266

nm, the fluence of 0.6 J / cm2, from targets with a concentration of 2% shellac were used for SGF tests.

The films were immersed in a simulated gastric fluid solution for 15, 30, 60, 120 and 240 minutes. The time

intervals chosen for this study were selected according to Ref. [71], the 240-minute period corresponding to a

typical passage of an element through the digestive system.

4 This section of the thesis was published in Coatings ((2018) 8:275), the article title „Shellac Thin

Films Obtained by Matrix-Assisted Pulsed Laser Evaporation (MAPLE)”, authors: Brajnicov, S; Bercea, A;

Marascu, V; Matei, A; Mitu, B

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After immersion in the simulated gastric fluid, the film thickness did not change after 240 minutes of

immersion. The surface changed, through the appearance of the pores, even for an immersion time of up to 15

minutes, as can be seen in figure 10, and the roughness increased. The density and size of the pores increased

with the increase of the SGF exposure period, but the largest pore measured after 240 minutes in the SGF had

a depth of only 85 nm (figure 11).

IR spectrometry measurements showed no significant change in the shellac films immersed in the GHS, and

the analysed absorption maxima and bands remained relatively constant even after an exposure of 240 min.

Figure 10. AFM images (5 μm x 5 μm) on a reference film and on the layers exposed to SGF.

Figure 11 Variation of roughness and number of pores on a surface of 5 μm × 5 μm of films, depending on

the exposure time to SGF. MAPLE films have a thickness of 2000 nm.

18

The analysis presented in this chapter confirms that shellac films deposited by MAPLE can be used as enteric

coatings for pharmaceutical applications.

In the last chapter, chapter six "Conclusions", the conclusions of the research and the personal contributions

of the thesis are presented.

3. Conclusions of the research carried out within the present PhD thesis

In the chapter of general conclusions, in addition to the conclusions presented at the end of each chapter, the

personal contributions from the thesis are included.

Dissemination of results:

3.1. Articles in ISI journals for the period 2016-2019

1. Ionita, I; Bercea, A; Brajnicov, S; Matei, A; Ion, V; Marascu, V; Mitu, B; Constantinescu, C; "Second

harmonic generation (SHG) in pentacene thin films grown by matrix assisted pulsed laser evaporation

(MAPLE)"; APPLIED SURFACE SCIENCE 480, 212-218 (2019);

2. Alin, CD; Grama, F; Papagheorghe, R; Brajnicov, S; Ion, V; Vizireanu, S; Palla-Papavlu, A; Dinescu, M;

"Tuning the physicochemical properties of hernia repair meshes by matrix-assisted pulsed laser

evaporation"; APPLIED PHYSICS A-MATERIALS SCIENCE & PROCESSING 125 (6) 424 (2019);

3. Icriverzi, M; Rusen, L; Sima, LE; Moldovan, A; Brajnicov, S; Bonciu, A; Mihailescu, N; Dinescu, M;

Cimpean, A; Roseanu, A; Dinca, V; "In vitro behavior of human mesenchymal stem cells on poly(N-

isopropylacrylamide) based biointerfaces obtained by matrix assisted pulsed laser evaporation"; APPLIED

SURFACE SCIENCE 440, 712-724 (2018);

4. Dinca, V; Viespe, C; Brajnicov, S; Constantinoiu, I; Moldovan, A; Bonciu, A; Toader, CN; Ginghina,

RE; Grigoriu, N; Dinescu, M; Scarisoreanu, ND; "MAPLE Assembled Acetylcholinesterase-

Polyethylenimine Hybrid and Multilayered Interfaces for Toxic Gases Detection"; SENSORS 18 (12)

4265 (2018);

5. Brajnicov, S; Bercea, A; Marascu, V; Matei, A; Mitu, B; "Shellac Thin Films Obtained by Matrix-

Assisted Pulsed Laser Evaporation (MAPLE)"; COATINGS 8 (8) 275 (2018);

6. Dinca, V; Zaharie-Butucel, D; Stanica, L; Brajnicov, S; Marascu, V; Bonciu, A; Cristocea, A; Gaman, L;

Gheorghiu, M; Astilean, S; Vasilescu, A; "Functional Micrococcus lysodeikticus layers deposited by laser

technique for the optical sensing of lysozyme"; COLLOIDS AND SURFACES B-BIOINTERFACES 162,

98-107 (2018);

7. Mitran, V; Dinca, V; Ion, R; Cojocaru, VD; Neacsu, P; Dinu, CZ; Rusen, L; Brajnicov, S; Bonciu, A;

Dinescu, M; Raducanu, D; Dan, I; Cimpean, A; "Graphene nanoplatelets-sericin surface-modified Gum

alloy for improved biological response"; RSC ADVANCES 8 (33) 18492-18501 (2018);

19

8. Brajnicov, S; Neacsu, P; Moldovan, A; Marascu, V; Bonciu, A; Ion, R; Dinca, V; Cimpean, A; Dinescu,

M; "Tailored biodegradable triblock copolymer coatings obtained by MAPLE: a parametric study";

APPLIED PHYSICS A-MATERIALS SCIENCE & PROCESSING 123 (11) 707 (2017);

9. Rusen, L; Brajnicov, S; Neacsu, P; Marascu, V; Bonciu, A; Dinescu, M; Dinca, V; Cimpean, A; "Novel

degradable biointerfacing nanocomposite coatings for modulating the osteoblast response"; SURFACE &

COATINGS TECHNOLOGY 325, 397-409 (2017);

10. Rusen, L; Neacsu, P; Cimpean, A; Valentin, I; Brajnicov, S; Dumitrescu, LN; Banita, J; Dinca, V;

Dinescu, M; "In vitro evaluation of poly(ethylene glycol)-block-poly(epsilon-caprolactone) methyl ether

copolymer coating effects on cells adhesion and proliferation"; APPLIED SURFACE SCIENCE 374, 23-

30 (2016).

3.2. Presentations at international conferences

1. Brajnicov, S; Marascu, V; Rusen, L; Moldovan, A; Dinca, V; Dinescu, M; "Copolymer PLCL-PEG-PLCL

functional bio-coating obtained by Matrix Assisted Pulsed Laser Evaporation: a deposition parametric

study";10th International Conference On Photoexcited Processes And Applications (ICPEPA-10), August

29 – September 2, 2016, Brasov, Romania; P77 – poster presentation;

2. Brajnicov, S; Neacsu, P; Dinca, V; Marascu, V; Bonciu, A; Cimpean, A; Dinescu, M; "Polylactide-co-

caprolactone based coatings deposited by Matrix Assisted Pulsed Laser Evaporation: an optimization

study", 17th International Conference On Plasma Physics And Applications, June 15 – 20 Magurele 2017,

Romania, Topic 8: Laser plasmas and their applications, P8-02 – poster presentation;

3. Brajnicov, S; Neacsu, P; Dinca, V; Marascu, V; Bonciu, A; Cimpean, A; Dinescu, M; "Tunability of the

surface morphology of PLCL-PEG-PLCL co-polymer coatings deposited by Matrix Assisted Pulsed Laser

Evaporation", IONS Balvanyos 2017 (International OSA Network of Students), July 25-28, Balvanyos,

Transylvania, Romania, 2017 – poster presentation – III prize for best poster;

4. Brajnicov, S; Dinca, V; Marascu, V; Dinescu, M; "Thin films of PLCL-PEG-PLCL co-polymer coatings

deposited by Matrix Assisted Pulsed Laser Evaporation", "The fifth edition of the International

Colloquium 'Physics of Materials' – PM-5", November 10-11, Bucuresti, Romania, 2016, O.2.6 – oral

presentation;

5. Brajnicov, S; Ion, V; Marascu, V; Rusen, L; Dinca, V; Dinescu, M; "Characterization and degradation

behavior of hybrid coatings obtained by Matrix Assisted Pulsed Laser Evaporation" ; 16th International

Balkan Workshop on Applied Physics (IBWAP 2016), July 7-9, 2016, Constanta, Romania, S2 P4 – poster

presentation;

6. Brajnicov, S; Marascu, V; Bonciu, A; Moldovan, A; Vlad, A; Dinca, V; Dinescu; M; "Tailored

biodegradable triblock copolymer coatings obtained by MAPLE for bioresponsive interfaces",

EMRS2017, Section X: New frontiers in laser interaction:from hard coatings to smart materials; May 22-

26, Strasbourg, France - EMRS2017, X P_1.32 – poster presentation;

20

7. Brajnicov, S; Bonciu, A; Marascu, V; Moldovan, A; Dinca V; Dinescu, M; "Biofunctional PLCL based-

coating obtained by Matrix Assisted Pulsed Laser Evaporation", 17th International Balkan Workshop on

Applied Physics and Materials Science – IBWAP 2017, Constanta, Romania,11-14, July, 2017, Laser

Plasma and Radiation Physics and Application, S2 P1 – poster presentation;

3.3. Participation in summer schools and scientific sessions

1. Dinca, V; Zaharie-Butucel, D; Stanica, L; Brajnicov, S; Marascu, V; Bonciu, A; Cristocea, A; Gamang,

L; Gheorghiu, M; Astilean, S; Vasilescuc, A; Dinescu, M; "Functional Micrococcus lysodeikticus layers

deposited by laser technique for the optical sensing of lysozyme"; Sixth Intl. School on Lasers in Materials

Science - SLIMS, July 8-14, S. Servolo Island, Venice, Italy, 2018 – oral presentation and poster

presentation;

2. Brajnicov, S; "Functional Polymeric Coatings Obtained by Matrix-Assisted Pulsed Laser Evaporation"

LASER IGNITION SUMMER SCHOOL, July 19-22, Brasov, Romania, 2017, P12_37 – poster

presentation;

3. Brajnicov, S; Dinca, V; Marascu, V; Rusen, L; Neacsu, P; Cimpean, A; Dinescu, M; "Multifunctional

coatings obtained by Matrix Assisted Pulsed Laser Evaporation" 15th IUVSTA School - Lasers for the

Nano-Engineering of Surfaces Intl. School on Lasers in Materials Science – SLIMS, July 10-17, S.

Servolo Island, Veneția, Italia, 2016 – poster presentation;

4. Brajnicov, S; Marascu, V; Dinca, V; Dinescu, M; "Tunability of the surface morphology of PLCL-PEG-

PLCL co-polymer coatings deposited by matrix assisted pulsed laser evaporation", Sesiunea Știintifică a

Facultații de Fizică, June 17, București, Romania, 2016, 10:00-10:15 – oral presentation.

21

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