Gustavo Hernández Vargas Development of bio-composites with requisite characteristics for biomedical settings School of Engineering and Sciences Instituto Tecnológico y de Estudios Superiores de Monterrey

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(1)Instituto Tecnológico y de Estudios Superiores de Monterrey Campus Monterrey. School of Engineering and Sciences. Development of bio-composites with requisite characteristics for biomedical settings A dissertation presented by. Gustavo Hernández Vargas Submitted to the School of Engineering and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Engineering Science Major in Biotechnology. Monterrey Nuevo León, November 23th, 2020.

(2) Here’s to the crazy ones, the misfits, the rebels, the troublemakers, the round pegs in the square holes. The ones who see things differently — they’re not fond of rules. You can quote them, disagree with them, glorify or vilify them, but the only thing you can’t do is ignore them because they change things. They push the human race forward, and while some may see them as the crazy ones, we see genius, because the ones who are crazy enough to think that they can change the world, are the ones who do. Steve Jobs, 1997 V.

(3) Acknowledgements The most difficult section to write in all works are the acknowledgements, as you have the risk to forget someone important or don't express yourself properly. In this case, I would try to be brief. First of all, I would say thank you to my whole family, without your unconditional support all my achievements would be only a brief time frame without meaning. Mom and Dad, thank you to for your love. Your patience and advices have made me the person who I am. Secondly, I would like to acknowledge the wise man who directed this work, my great advisor Prof. Hafiz MN Iqbal. Once you told me the meaning of Hafiz, since then I see you with great respect, not only in academia but also in life. You are a very good advisor. I truly belief that the greatest thing that happens along my PhD was to be your student. Thank you for all your support and words of support. This work belongs to you. I hope someday to be as loyal, clever and patience as you are. Thanks also to all the friends and collogues that I met along this road. You know that I will always have time for you. To my committee, thank you to be present in this important moment. I know that if you are here today is because you had an important role in my formation. Thank you for your time and comments. Is an honor for me to have you here. Thank you Tecnólogico de Monterrey and CONACyT for the financial support to fulfill this goal. And to all of you who read this section waiting to see your name, thank you. After all this time the most important lesson that I will keep forever and share with you is that it doesn't matter when, how or where, you will find that important thing that will make you happy… then the rest will no longer matter. Neither to read your name in a thesis work. Finally, I would say thank you Sara. You are my greatest adventure and the most amazing serendipity. I love you.. VII.

(4) Development of bio-composites with requisite characteristics for biomedical settings By Gustavo Hernández Vargas Abstract There is a dire need to engineer biologically robust constructs to meet the growing needs of 21st-century medical sector. The increasing (re)-emergence of human-health related pathogenic microbes has caused a havoc and serious challenge to health care services. In this context, herein, we report the development and characterization of various polymeric bio-composites with unique structural and functional attributes. For a said purpose, chitosan and graphene were used to engineer bio-composites, which were then functionalized by loading silver and platinum nanoparticles. A microwave-assisted approach was adopted to construct silver and platinum nanoparticles loaded graphenebased bio-composites. While, “one-pot” synthesis approach was used to engineer silver and platinum nanoparticles loaded chitosan-based bio-composites. As developed biocomposites were designated as GO-Ag-S1 to GO-Ag-S5 (silver nanoparticles loaded graphene-based bio-composites), GO-Pt-P1 to GO-Pt-P5 (platinum nanoparticles loaded graphene-based bio-composites), CHI-Ag-S1 to CHI-Ag-S5 (silver nanoparticles loaded chitosan-based bio-composites), and CHI-Pt-P1 to CHI-Pt-P5 (platinum nanoparticles loaded chitosan-based bio-composites). Finally, the nanoparticles loaded bio-composites of graphene and chitosan were subjected to characterization via UV-Visible spectrophotometric analysis, percent loading efficiency (%LE) analysis, Fourier-transform infrared (FTIR) spectroscopy, mechanical measurements, and antibacterial attributes. The UV-Visible spectrophotometric analysis revealed characteristic peaks appeared at the λmax 420 nm and 266 nm which belongs to the silver and platinum nanoparticles, respectively. The graphene-based bio-composites, i.e., GO-Ag-S3, GO-Ag-S4, and GOPt-P3 showed optimal %LE of 88, 92, and 89%, respectively. Whereas, CHI-Ag-S4, CHIPt-P3, and CHI-Pt-P4 bio-composites showed optimal %LE of 94, 86, and 94%, respectively. Two regions, i.e., (1) between 3600-3100 cm-1, and (2) between 1,800 and VIII.

(5) 1,000 cm-1 in the FTIR spectra were found of particular interest. The FTIR profile exposed the available functional moieties at the surface of respective bio-composites. Variable mechanical attributes of silver and platinum nanoparticles loaded bio-composites were recorded from the stress-strain curves. All developed bio-composites showed bactericidal activities up to certain extent against both test strains. As compared to the initial bacterial cell count (control value, i.e., 1.5 × 108 CFU/mL), the bio-composites with higher %LE showed almost complete inhibition, with a log reduction from 5 to 0, and bactericidal activities up to certain extent against both test strains, i.e., Bacillus subtilis (B. subtilis), and Escherichia coli (E. coli). In conclusion, the notable structural, functional, mechanical and antimicrobial attributes suggest the biomedical potentialities of newly in-house engineered silver and platinum nanoparticles loaded graphene and chitosan-based biocomposites.. IX.

(6) List of Figures Figure 1. General principles for antimicrobial surfaces development. A) Use of repelling surfaces to avoid biofilm formation. B) Use of contact active microbial materials to kill microorganisms.. 28. Figure 2. Scheme of an electrochemical biosensor. Biological sensing elements are coupled to electrodes. These traduce the signal to deliver a readable output.. 30. Figure 3. A generalized schematic representation of heart tissue engineering. (A) affected patient, (B) specific cells: (i) fibroblast and other cell lines, (ii) iPS, and (iii) cardiomyocytes and other necessary cells, (C) three-dimensional porous biomaterial-based heart scaffold of different nature and architecture, and cultured under dynamic conditions in (D) bioreactors systems (i) perfusion bioreactor and (ii) spinner tank bioreactor, which nurture the development of heart tissue by supporting efficient nutrition of cultured cells and applying mechanical stimuli that are critical for functional regeneration and (E) engineered heart as a potential alternative.. 33. Figure 4. UV-Vis spectral analysis of in-house engineered silver nanoparticles at numerous intervals within 0 to 24 h range. The λmax values were noted from 0 h to each hour until 6 h and then after every 6 h until 24 h by taking an aliquot from the same mother liquor.. 44. Figure 5. UV-Vis spectral analysis of in-house engineered platinum nanoparticles at numerous intervals within 0 to 24 h range. The λmax values were noted from 0 h to each hour until 6 h and then after every 6 h until 24 h by taking an aliquot from the same mother liquor.. 45. Figure 6. Percent loading efficiency of newly in-house engineered silver and platinum nanoparticles into/onto graphene-based bio-composites.. 46. Figure 7. Percent loading efficiency of newly in-house engineered silver and platinum nanoparticles into/onto chitosan-based bio-composites.. 47. Figure 8. FTIR profiles of respective polymeric materials and nanoparticles.. 48. Figure 9. Typical FTIR profiles of silver nanoparticles loaded graphene-bio-composites. 48 Figure 10. Typical FTIR profiles of platinum nanoparticles loaded graphene-biocomposites.. 49. Figure 11. Typical FTIR profiles of silver nanoparticles loaded chitosan-bio-composites.49 Figure 12. Typical FTIR profiles of platinum nanoparticles loaded chitosan-biocomposites.. 50. Figure 13. Evaluation of the antibacterial activities of silver and platinum nanoparticles loaded graphene-bio-composites. (A) and (B) are representing the antibacterial attributes of silver nanoparticles loaded bio-composites against B. subtilis, and E. coli, respectively. (C) and. X.

(7) (D) are representing the antibacterial attributes of platinum nanoparticles loaded bio-composites against B. subtilis, and E. coli, respectively. The reduction in the initial bacterial count i.e. 1.5 × 108 CFU/mL showed bacteriostatic and bactericidal activities of the respective samples. A 2-log reduction was considered to claim an antibacterial activity.. 53. Figure 14. Evaluation of the antibacterial activities of silver and platinum nanoparticles loaded chitosan-bio-composites. (A) and (B) are representing the antibacterial attributes of silver nanoparticles loaded bio-composites against B. subtilis, and E. coli, respectively. (C) and (D) are representing the antibacterial attributes of platinum nanoparticles loaded bio-composites against B. subtilis, and E. coli, respectively. The reduction in the initial bacterial count i.e. 1.5 × 108 CFU/mL showed bacteriostatic and bactericidal activities of the respective samples. A 2-log reduction was considered to claim an antibacterial activity.. XI. 54.

(8) List of Tables Table 1. Summary of different applications of chitin in tissue engineering and regenerative medicine. The information showed in the table are support material; novel features in mechanical properties, water uptake, and cell proliferation; cell culture; proposed application; and reference.. 21. Table 2. Summary of different applications of alginate in tissue engineering and regenerative medicine. The information showed in the table are support material; novel features in mechanical properties, water uptake, and cell proliferation; cell culture; proposed application; and reference.. 23. Table 3. Sample IDs of newly in-house engineered silver and platinum nanoparticles loaded graphene-based bio-composites.. 40. Table 4. Sample IDs of newly in-house engineered silver and platinum nanoparticles loaded chitosan-based bio-composites.. 42. Table 5. DMA-mechanical properties of silver and platinum nanoparticles loaded graphene-based bio-composites.. 51. Table 6. DMA-mechanical properties of silver and platinum nanoparticles loaded chitosan-based bio-composites.. XII.

(9) Table of contents 1.. INTRODUCTION .................................................................................................................................... 13 1.1. MICROBIAL COLONIZATION MECHANISM ................................................................................................. 15 1.2. THESIS STRUCTURE .................................................................................................................................. 16 1.3. MOTIVATION .............................................................................................................................................. 17 1.4. HYPOTHESIS ............................................................................................................................................. 17 1.5. OVERALL AIM AND OBJECTIVES ............................................................................................................... 18 1.5.1. General objective ............................................................................................................................ 18 1.5.2. Specific objectives........................................................................................................................... 18. 2.. LITERATURE BACKGROUND – STATE-OF-THE-ART AND OPPORTUNITIES ........................... 19 2.1. POLYSACCHARIDES CANDIDATES ............................................................................................................ 20 2.1.1. Chitosan............................................................................................................................................ 21 2.1.2. Alginate ............................................................................................................................................. 23 2.2. NANOPARTICLES OF INTERESTS .............................................................................................................. 25 2.2.1. Silver ................................................................................................................................................. 25 2.2.2. Platinum ............................................................................................................................................ 26 2.3. BIOMEDICAL APPLICATION ....................................................................................................................... 26 2.3.1. Antimicrobial surfaces .................................................................................................................... 26 2.3.2. Biosensors........................................................................................................................................ 28 2.3.3. Tissue Engineering ......................................................................................................................... 30 2.4. FUTURE TRENDS AND CONCLUSIONS ....................................................................................................... 33. 3. DEVELOPMENT AND CHARACTERIZATION OF NANOPARTICLES-LOADED BIOCOMPOSITES FOR BIOMEDICAL SETTINGS ............................................................................................ 34 3.1. INTRODUCTION .......................................................................................................................................... 36 3.2. MATERIALS AND METHODS ....................................................................................................................... 37 3.2.1. Reagents/consumables .................................................................................................................. 37 3.2.2. Synthesis of silver and platinum nanoparticles ........................................................................... 38 3.2.3. Synthesis of graphene oxide ......................................................................................................... 38 3.2.4. Nanoparticles-loaded graphene-based bio-composites ............................................................ 39 3.2.5. Nanoparticles-loaded chitosan-based bio-composites .............................................................. 40 3.2.6. Instrumental analysis of pristine and engineered bio-composites ........................................... 41 3.2.7. Evaluation of antibacterial attributes ............................................................................................ 42 3.3. RESULTS AND DISCUSSION....................................................................................................................... 43 3.3.1. Percent loading efficiency (%LE) .................................................................................................. 44 3.3.2. Fourier-transform infrared spectroscopy (FTIR) ......................................................................... 46 3.3.3. Dynamic Mechanical evaluation ................................................................................................... 49 3.3.4. Antibacterial attributes of engineered bio-composites ............................................................... 51 3.4. CONCLUSIONS .......................................................................................................................................... 55 3.5. ACKNOWLEDGEMENT ............................................................................................................................... 55 3.6. CONFLICT OF INTERESTS .......................................................................................................................... 55 4.. CONCLUSIONS AND FUTURE WORK .............................................................................................. 56. 5.. REFERENCES ....................................................................................................................................... 59. CURRICULUM VITAE ..................................................................................................................................... 77 APPENDIX ....................................................................................................................................................... 78. XIII.

(10) 1. Introduction The Biomaterials field, with the application in the medical sector, has evolved along the 21st century due to health and environmental crises. Special attention has been focused on the increasing emergence of microbial resistance and the development of green methods for biomaterials synthesis. Antimicrobial resistance arose as a result of the excessive use of antibiotics. The main concerns with antibiotics resistance development are microbial infections post-implant device interventions, as this can result in implant rejections or wound healing delay [1–3]. Microbial infection post-implant results from a complex interaction between the bacteria, the implant and the host immune system response to both. Without the presence of a foreign body, bacterial tissue infections can be controlled by the host immune system. However, when a medical device is implanted, the device’s materials can trigger local tissue response, which can lead to acute and chronic inflammation. This can then evolve to fibrosis and finally to a niche of immune depression, which allows microbial infections [4]. To address this concern, there is a great emphasis on the development of novel biomaterials that incorporate antimicrobial agents such as antibiotics, antimicrobial peptides or metal nanoparticles. Metal nanoparticles of noble metals such as gold, silver, and platinum represent a potential improvement for current biomaterials due to their different outstanding characteristics, including their antimicrobial activity and high surface-to-volume ratio. Metal nanoparticles-based therapies have a broad range of applications in the biomedical field, including wound healing care. There are two main strategies to apply metal nanoparticles in health care, either as a reinforcement material or as a carrier molecule [5,6]. As a reinforcement material, metal nanoparticles have helped to improve the morphology and physicochemical characteristics of engineered materials. This is important as these properties play a key role in the correct development and maintenance of tissular structures and functions [7,8]. Also, these have helped to avoid microbial infections [2,9]. Although metal nanoparticles have shown their importance, the harsh reaction conditions for their synthesis (use of numerous solvents and surfactants) have limited their application. To overcome this challenge, the use of natural polymers to engineer nanoparticles-loaded bio-composites is proposed. 13.

(11) Natural polymers are naturally occurring materials with outstanding functional properties. These also present good biocompatibility and biodegradability. Among the different natural polymers, polysaccharides have excelled due to their unique structure, which eases chemical and physical modification. This property is important as modifications allow to include specific cues that can trigger specific cell behaviors. Another important characteristic is their close structural relation with proteoglycans (PGs) [10,11]. Despite these biological advantages, polysaccharides lack of mechanical strength has limited their full application in tissue engineering. The incorporation of nanoparticles such as graphene, silver, and platinum has supported the improvement of these materials. Nanoparticles selection are considered upon the characteristic to improve. It has been proved that graphene/chitosan-based bio-composites supplemented with other functional entities offer high stability and strong mechanical behaviors as well as self-healing properties. Electrical conductivity is also improved. This is important for hearth tissue engineering applications, as graphene-chitosan composites have shown a faster spontaneous beating rate compared with pristine composite [12]. Graphenechitosan composites have been also suggested as material for application into artificial cartilage [13] and bone tissue engineering [14,15]. Addition of silver and platinum are important for application where composites are used as antimicrobial wound dressings [16,17]. Given the changing dynamics of 21st-century bio-composites with medical potencies, incorporating biologically active and therapeutic cues/agents into/onto the pristine materials to engineer bio-composite matrices with significant bioactivities, such as antibacterial potentialities, has received limited attention so far. Thus, keeping in mind the points mentioned above and critics, the main aim of this dissertation work is to present the novel formation of bio-composites with medical attributes. For a said purpose, in the first step, silver and platinum nanoparticles were synthesized by reducing AgNO3 into Ag+ and H2PtCl6 into Pt0, respectively. Secondly, a microwave-assisted approach was adopted to construct silver and platinum nanoparticles loaded graphene-based biocomposites. In the third step, the “one-pot” synthesis approach was used to engineer silver and platinum nanoparticles loaded chitosan-based bio-composites. Fourthly, as developed bio-composites were designated as GO-Ag-S1 to GO-Ag-S5 (silver 14.

(12) nanoparticles loaded graphene-based bio-composites), GO-Pt-P1 to GO-Pt-P5 (platinum nanoparticles loaded graphene-based bio-composites), CHI-Ag-S1 to CHI-Ag-S5 (silver nanoparticles loaded chitosan-based bio-composites), and CHI-Pt-P1 to CHI-Pt-P5 (platinum nanoparticles loaded chitosan-based bio-composites). Finally, the analytical characterization of nanoparticles loaded bio-composites of graphene and chitosan was performed via UV-Visible spectrophotometric analysis, percent loading efficiency (%LE) analysis, Fourier-transform infrared (FTIR) spectroscopy, mechanical measurements, and antibacterial attributes.. 1.1.. Microbial Colonization Mechanism. Over their evolution, bacteria have developed mechanisms that allow them to adapt and resist extreme conditions where life cannot normally exist. These include complex mechanisms against toxic substances, rapid division cycles and the ability to transfer genetic information between species. Within the human body, bacteria can be found either as a free microorganism floating in solution (i.e., blood) or as a biofilm adhered to a surface. A biofilm is a dense surface area where bacteria have adhered and embedded in a polysaccharide matrix [18,19]. Biofilm development is the most common bacterial infection of implantable devices. This follows with a defined order where the first step is the adsorption of host proteins within the surface of the medical device. This adsorption can be led by the surface hydrophobicity, roughness, porosity or its chemical composition. Once the surface is cover with these proteins, microorganisms adhesion start allowing them to attach, growth and proliferate modulating their gene expression and metabolism [20]. Adhesion can also be regulated through unspecific reversible adhesion. In this scenario, the bacterial cell wall recognizes adhesive matrix molecules, which allow them an ionic association. To overcome this situation, it is necessary to acknowledge each case to propose the best strategy. Actually, there are two proposed strategies to avoid bacterial infection. The first one requires the incorporation of antimicrobial systems into the devices. Although effective, this strategy can lead to antimicrobial resistance development as, in some cases, exposure to antimicrobial systems is not necessary. The. 15.

(13) second strategy is to coat the device surface with polycations, which in contact with the negatively charged bacteria cell wall can cause its death.. 1.2.. Thesis structure. The present work aims to prove and describe the development and characterization of novel nanoparticles-loaded bio-composites with unique structural and functional attributes, especially antimicrobial activity. To achieve this goal, this dissertation work has been divided into four different chapters. This first chapter, used as an introduction, gives a general overview of the subject addressed in this work. Particular motivations, its importance, the hypothesis and objectives of this thesis are also presented in this chapter. The second chapter presents the background and the state of the art that motivates this work. In this chapter, the reader would find important information about the different naturally occurring biomaterials subjected to this work, as well as valuable information about the trends on noble metal nanoparticle design. This chapter also aims to show the importance to develop novel composites for biomedical applications. Chapter third presents a full-research article already accepted and published in a Scopus indexed scientific journal. This work is entitled “Development and characterization of nanoparticles-loaded bio-composites for biomedical settings” and presents the results obtained to obtain the degree of Doctor of Philosophy in Engineering Science, with a major in Biotechnology. It is important to mention that since this chapter has already gone through a peer to peer review, it has its own introduction, methods, and acknowledgment section. To ease the reading, the references for this chapter are included in the reference chapter. Figure numbers and formats were edited accordingly for the purposes of this document. Finally, a fourth chapter with the general conclusions and further work recommendations is presented. In this chapter, the result from international residences and abroad experiences are also presented. All the titles and abstract of the scientific work obtained in this dissertation are presented at the end of this document.. 16.

(14) 1.3.. Motivation. Antimicrobial resistance is one of the most significant health care challenges of 21st Century. Nowadays, it has been estimated a global socioeconomic impact of $100 trillion per annum with a potential loss of 10 million lives annually by 2050. Although this risk has been acknowledged since 2001, significant advances have been developed recently [21]. At the same time, the number and kind of implantable devices, such as temporary urinary catheters or tissue engineering grafts has increased. Because the chemical and physical composition of these devices, combined with the antimicrobial resistance crisis, the risk of developing a device-related infection has increased. This makes an apparent necessity for the development of novel bio-composite materials to engineer antibacterial matrices. In this context, nanoparticles-loaded/decorated constructs have received significant attention due to their potential application in the biomedical sector as a reinforcement material.. 1.4.. Hypothesis. The 21st-century medical sector has required to engineer biologically robust biocomposites to meet specific biochemical and physicochemical characteristics of organs and tissues. Although great advances have been achieved, there is still a necessity to propose biomaterials able not only to provide the cues to control cell behavior or tissue repair but also to avoid health care challenges as microbial resistance. In this regard, the present dissertation work is developed around the following hypothesis: •. Control over silver- and platinum-nanoparticles load into pristine chitosanor graphene-based composite will improve its mechanical and antimicrobial properties. As a result, we will obtain a pair of novel nanoparticles-loaded bio-composites with unique structural and functional properties.. 17.

(15) 1.5.. Overall aim and objectives. 1.5.1. General objective To develop chitosan- and graphene-composites loaded with silver- and platinumnanoparticles to improve their mechanical performance (e.g., Young’s modulus, tensile strength and elongation) and their antibacterial properties against B. subtilis and E. coli.. 1.5.2. Specific objectives 1.5.2.1.. To obtain the silver- and platinum-nanoparticles through a reduction. synthesis. 1.5.2.2.. To synthesize graphene oxide from graphite powder.. 1.5.2.3.. To load different silver- and platinum-nanoparticles concentration into the. chitosan- and graphene-based bio-composites. 1.5.2.4.. To measure the mechanical performance of the pristine and engineered. bio-composites evaluating their Young’s modulus, tensile strength and elongation at break. 1.5.2.5.. To evaluate the antibacterial properties of the pristine and engineered bio-. composites against B. subtilis and E. coli.. 18.

(16) 2. Literature background – State-of-the-art and opportunities Biomaterial development holds a key role on biomedical engineering due to the challenge of mimic the complexity of the extracellular matrix (ECM). Nowadays, it is well known that the ECM not only provides adhesions sites for cells, but also has an important role in cell behavior (e.g. growth, migration and proliferation) [22]. To meet ECM functions, biomaterials research has been focused on study the effect of specific physical cues on the nanoscale to obtain specific biological responses that affect cellular function [23]. Naturally, the ECM is composed of collagen, elastin and proteoglycans which provide specific cues to nascent cells. Direct use of these materials on biomedical application has been limited due to their high production costs or poor mechanical properties. In this scenario, natural polymers such as gelatin, chitosan, alginate and cellulose hold important attention due to their flexibility in surface modification, mechanical performance, biocompatibility, and non-toxic, and non-immunogenic properties. Natural polymers are polymeric networks based on natural occurring products such as proteins and sugars. These have used in different biomedical application (e.g. drug delivery systems, tissue engineering scaffolds) due to their functional properties [24]. Among the different natural polymers, polysaccharides are promising materials which hold. outstanding. biocompatibility. and. biodegradability. properties.. Structurally,. polysaccharides also resemble the proteoglycans (PGs) present in the ECM. PGs are core proteins covalently attached to one or more glycosaminoglycans (GAGs) chains. PGs have key functional and structural roles in the ECM and also participate in cell functions such as migration, proliferation and adhesion [25,26]. Polysaccharides are long sugar monomers chains join together by glycosidic bonds. These are usually found or extracted from plants and animals. Natural polymers can be classified using different criteria; most relevant for biomedical application are: i) their morphology: linear or branched; ii) their electrical charge: neutral, cationic or anionic; and iii) their degradability. Besides their biocompatibility, their close structural relation with PGs make these to show good hemocompatibility and good interaction with living cells. Polysaccharides are also a low-cost option for biomedical application [10,11]. Polysaccharides structure make them also an ideal biomaterial as this enable chemical and physical backbone modifications. Modifications are implemented to generate 19.

(17) biological cues that can induce specific cell behavior. An example of this is the conjugation with the conserved RGD motif which allows cell adhesion [27,28]. Despite their functional properties, polysaccharides lack of mechanical strength has limited their application in complex tissue engineering application such as heart tissue engineering or skin tissue engineering. Regardless this limitation, there is a growing interest to use these natural polymers to engineer nanoparticles-loaded bio-composites with biomedical potential. Nanoparticles, especially those made of noble metals such as gold, silver and platinum have received extensive attention due to their multi-functional attributes for potential applications in the bio- and non-bio sectors of the modern world. For biomedical application, metal nanoparticles have a broadly range of functions such as the treatment of diseases [29], antimicrobial agents [16,17], biosensor [30], drug delivery and as a reinforcement of pristine materials. Nevertheless, the presence and utilization of numerous solvents or surfactants, along with other harsh reaction conditions all confines the synthesis and development of nanoparticles or nanoparticles-loaded bio-composites. To overcome these challenges, the principal proposed strategy is the development of biologically active bio-composites loaded with metal nanoparticles. Special attention has been given to the development of elastic and electroconductive materials as well as the development of antibacterial matrices.. 2.1.. Polysaccharides Candidates. Bio-based materials possess several complementary functionalities e.g. unique chemical. structure,. bioactivity,. non-toxicity,. biocompatibility,. biodegradability,. recyclability, etc. that position them well in the modern world’s materials sector. In this context,. the. utilization. of. biomaterials. provides. extensive. opportunities. for. experimentation in the field of interdisciplinary and multidisciplinary scientific research. With an aim to address the global dependence on petroleum-based polymers, researchers have been redirecting their interests to the engineering of biological materials for targeted applications in different industries including cosmetics, pharmaceuticals, and other biotechnological or biomedical applications [31].. 20.

(18) 2.1.1. Chitosan Chitosan is a linear polysaccharide obtained through chitin deacetylation. Deacetylation process take place when the acetyl group from the molecular chain of chitin is removed and replaced by an amino group (-NH2). Deacetylation of chitin involves chemical hydrolysis under alkaline conditions (e.g. sodium hydroxide solution) or by enzymatic hydrolysis in the presence of chitin deacetylase. For general applications, commercial chitosan has an average deacetylation degree from 70 to 90%, but for biological applications it may have a deacetylation degree higher than 95%, since biocompatibility increases with deacetylation degree [32,33]. Chitosan has been proven to act as an excellent biomaterial showing biocompatibility, biodegradability, hydrophilicity and having antimicrobial [34], analgesic properties [35]. Besides these characteristics, injection, ingestion, implantation and topical application of chitosan have not shown anti-inflammatory or allergic response in humans. Structurally, chitosan is similar to GAGs; this, combined with it highly hydrophilic surface, allows cells adherence and proliferation. This property also gives chitosan-based biomaterials the ability to influence on the modulation of cytokines and growth factors [36]. Despite their outstanding characteristics, chitosan lack of mechanical strength and instability prior implantation have limited its application on biomedical field. To overcome these drawbacks, cross-linking of this biomaterial with other natural polymers or nanocomposites have been proposed.. 21.

(19) Table 1. Summary of different applications of chitin in tissue engineering and regenerative medicine. The information showed in the table are support material; novel features in mechanical properties, water uptake, and cell proliferation; cell culture; proposed application; and reference. Reprinted with permission from Ref. [31]. Source Chitosan Chitosan Chitosan. Chitosan Chitosan. Support material Poly(vinyl alcohol)/genipin Xylan hemicellulose Glycol and nanohydroxyapatite. Gelatin and aloe vera Poly(vinyl alcohol). Mechanical Yes Yes Yes. Yes Yes. Yes No. No Yes. Poly(L-lactic acid). Chitosan. B-1,3-glucan and hydroxyapatite. No. No. Yes. Chitosan Chitosan. Enzymatically modified with ferulic acid B-glycerol phosphate and gelatin. No Yes. Yes Yes. Yes No. Carboxymethyl chitosan Chitosan. Graphene oxide. Yes. No. Yes. Collagen and bioactive glass nanoparticle. Yes. Yes. Yes. Poly(lactide-co-glycolide). No. Chitosan Chitosan. Genipin Xanthan. Yes Yes. Yes. CP No Yes Yes. Chitosan nanoparticles. Chitosan. Yes. Novel features WU Yes Yes No. No Yes Yes. 22. Yes. Yes No Yes. Cell culture None Lymphocytes Human bone marrow mesenchymal stem cells and embryonic cell line (HEK293T) None Umbilical cord bloodderived mesenchymal stem cells Glioblastoma cell line (U87 MG) and Neuroblastoma cell line [Be(2)] Human osteoblast Mesenchymal stem cells Adipose-derived mesenchymal stem cells Bone marrow mesenchymal stem cells Human bone marrowderived mesenchymal stem cell Schwann-like cells and human adipose-derived stem cells SAOS-2 cells Mesenchymal stromal cells. Proposed application Tissue engineering Tissue engineering Tissue engineering. Reference [37] [38] [39]. Tissue engineering Tissue engineering and regenerative medicine Tissue engineering. [40] [41]. [42] Regenerative medicine Tissue engineering Tissue engineering Tissue engineering. [43] [44] [45] [46]. Tissue engineering [47] Regenerative medicine Tissue engineering Tissue engineering. [48] [49] [50].

(20) 2.1.2. Alginate Alginate is a linear copolymer of β (1-4) linked D-mannuronic (M) acid and α (1-4) linked L-guluronic acid (G). This is extracted from native brown seaweed, where its main function is to provide the strength and flexibility necessary to resist the conditions where the seaweed grows [51]. Alginate is recognized to form a physical gel either at low pH or under the presence of divalent cations such as Ca2+ or Ba2+. Physical properties of alginate gels can be widely controlled varying M and G residue ratio or its sequential order, the polymer molecular weight and the cation concentration at the time of gelation [52]. This make alginate a promising material for cell encapsulation or 3D printing applications. Some properties of alginate scaffolds derivate in its solubility, hydrophobicity, affinity for specific proteins, low toxicity, and biocompatibility. Although it can be a highly flexible material, alginate has natural poor cell adhesion and poor in vivo degradation performance. Support systems have been developed with the incorporation of different materials such as collagen, poly(lactic-co-glycolic acid) (PLGA), Poly-L-lysine, polycaprolactone (PCL), polyethers, and chitosan [36]. Other works have developed such support systems by covalently cross-linking alginate with adipic hydrazide and polyethylene glycol (PEG). Also, cross-linking density and thus mechanical strength properties can be increased by varying the ratio of beta-D-mannuronic acid and alpha-Lglucuronic acid blocks and molecular weight of the polymeric chain [53]. Currently, there are several medical-related applications of alginate. They vary from drug delivery systems to tissue engineering scaffolds. Some alginate-based scaffolds attempt to mimic bone and cartilage, nerve, and connective tissues [36]. Also, alginate scaffolds have been widely explored for their application on the generation of liver and pancreas tissues and as a wound dressing for the treatment of acute or chronic wounds [54,55].. 23.

(21) Table 2. Summary of different applications of alginate in tissue engineering and regenerative medicine. The information showed in the table are support material; novel features in mechanical properties, water uptake, and cell proliferation; cell culture; proposed application; and reference. Reprinted with permission from Ref. [31]. Source Alginate Alginate Alginate. Alginate. Support material Gelatin, Gellan gum, carboxymethylcellulose, and lignin Hyaluronic acid and collagen Hyaluronic acid. Novel features Mechanical WU CP Yes Yes No Yes No. No No. Yes Yes. Cell culture L929 cell line Rat chondrocytes Bone marrow- and adipose tissuemesenchymal stem cells Embryonic stem cells. Poly(N-iso-propylacrylamide)poly(ethylene glycol) Carboxymethyl-chitosan and agarose Polycaprolactone Nanocellulose. Yes. No. Yes. Yes. No. Yes. Yes Yes. No No. Yes No. Poly(ethylene oxide) and trifluoroacetic acid. No. Yes. Yes. Glycine-arginine-aspartateRGD-peptide. Yes. Alginate. Chitosan. No. No. Yes. Cardiomyocyte Cells. Alginate Alginate. Poly(L-glutamic acid) Silk. Yes Yes. Yes No. No Yes. Chondrocytes D3 mouse embryonic stem cells. Alginate Alginate Alginate Alginate Alginate. Yes. Yes. Alginate. None. No. No. Yes. Alginate. Gelatin. No. No. Yes. Alginate. Hydroxyapatite. Yes. Yes. No 24. Cortical human neural stem cells Chondrocytes Human nasoseptal chondrocytes Fibroblast cells (NIH/3T3-cells) C2C12 mouse skeletal myoblast. Neuron-like PC12 cells Human mesenchymal stem cells Rat osteoblast cells. Proposed application Tissue engineering and regenerative medicine Tissue engineering Regenerative medicine Tissue engineering Regenerative medicine Tissue engineering Tissue engineering Tissue engineering and regenerative medicine Tissue engineering and regenerative medicine Regenerative medicine Tissue engineering Tissue engineering and regenerative medicine Regenerative medicine Tissue engineering Tissue engineering. Reference [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70].

(22) 2.2.. Nanoparticles of Interests. Nanoparticles such as silver and platinum have been extensively used on polysaccharides modification due to their capacity to improve their mechanical, electrical and antimicrobial properties. Furthermore, nanoparticles also provide large surface areas which have an important effect on their biological properties. These characteristics allows nanoparticles to display a larger number of functional groups as a ligand and also affect their absorption and release behavior. 2.2.1. Silver Silver nanoparticles (AgNPs) possess special physicochemical properties such as size, shape and composition which make them a candidate material for biomedical, environmental and industrial applications. In the biomedical field, AgNPs have highlighted due to their antimicrobial [2,9] and anticancer [71–73] effect. Nowadays, regarding worldwide spreading of microbial resistance, much attention has been focused on AgNPs antimicrobial properties. Microbial infections are the main cause of healing delay of closed wounds. Excessive use of antibiotics has led to microbial resistance, which has resulted in a health crisis. Such problems have opened the opportunity for AgNPs based antiseptics. Ag ions and Ag compounds are widely acknowledged as highly toxic compounds to microorganism with a broad spectrum activity and a far lower propensity to induce microbial resistance than antibiotics [2]. Today, even different medical tools used in hospital are coated with AgNPs to avoid bacterial contamination. In the biomaterial design field, the use of natural polymers as safe reductants have support the development of bio-composite with improved antimicrobial properties. Today there are different hypothesis on AgNPs antimicrobial mechanism. The main hypothesis suggest that larger AgNPs surface allow a prolonged release of Ag ions, which are uptake by bacteria disrupting ATP production and DNA replication. Another hypothesis also proposes a direct damage to bacteria cell wall. This has been proved in a study where AgNPs effect. 25.

(23) on E. coli and S. aureus where compared being the concentration of peptidoglycans the parameter which affect its sensitivity. This also explains why AgNPs affect bacteria but not mammalian cells [74]. In clinical trial, chitosan-AgNPs wound healing efficiency were compared with 1% silver sulfadiazine showing a higher healing rate after 13 days and a shorter healing period of 13.51 ± 4.56 days compared with 17.45 ± 6.23 [75]. ChitosanAgNPs composite have also prove to be safe for human cells [76]. Other modification includes an AgNPs-alginate/gelatin hydrogel with an improved antibacterial activity against P. aeruginosa and S. aureus and non-cytotoxic effects against fibroblast cells. This also shown the capacity to reduce a wound in an in vivo study [77]. 2.2.2. Platinum Platinum (Pt) has been used for a long time in biomedical application due to its biocompatibility, electrical conductivity, radiopacity, and durability. Despite to be a high cost material it is included in medical devices such as stents, catheters and pacemaker. Currently, there is an interest to explore platinum nanoparticles (PtNPs) application on biomedical field, especially for the development of biosensors, nanocarriers or treatments for diseases related to oxidative stress [78]. PtNPs possess unique characteristics such as surface functionalities, shape, porosity and agglomeration which make them an interesting material for biomedical application. In this field, this have proved to be useful as antimicrobial and anticancer agent [79,80], a useful drug delivery system [81], and a tool for the development of more sensitive bioimaging and biosensing tools.. 2.3.. Biomedical Application. 2.3.1. Antimicrobial surfaces Development of contact active antimicrobial surfaces has received great attention in the last years due to the increase challenge of microbial resistance. Microbial growth. 26.

(24) and colonization of medical implantable devices, food processing equipment or water treatments systems leads to human diseases developments, environment contamination and food spoilage. Microbial colonization of different surfaces can follow different mechanisms; however, the biggest challenge arises when the bacteria builds biofilms on the material’s surface. A biofilm is a dense surface area where bacteria have adhered and embedded in a polysaccharide matrix [18,19]. Once embedded into this matrix, bacteria are up to 1000 times more resistant compared with single free bacteria cells. In recent years, two different approaches have been suggested to overcome biofilms developments and also the use of antimicrobial agents (Figure 1) [82].. Repelling. Killing. A). B). Ag. Ag. Ag. Ag. Ag. Figure 1. General principles for antimicrobial surfaces development. A) Use of repelling surfaces to avoid biofilm formation. B) Use of contact active microbial materials to kill microorganisms.. First approach is the use of is superhydrophobic surfaces which reduces bacteria adhesion, and therefore avoids the biofilm formation. Superhydrophobic surfaces were inspired by the lotus leaf and its use on biomedical applications is relatively new. The mechanism behind this approach is the use of different contact angles. When contacts angles are between 0º and 90º the resulting surfaces is hydrophilic. Superhydrophilic surfaces are those with a contact angle less than 5º. On the contrary, hydrophobic surfaces are those with contact angles between 90º and 180º and superhydrophobic surfaces are obtained when the contact angle is greater than 150º [83]. Hydrophobicity surfaces helps to repel water, which enables dirt particles to be carried away while the 27.

(25) liquid droplet rolls off. This also weak bacteria attachment and avoids biofilm formation. In addition, it has been suggested that the nanostructures needed to achieve this effect may induce the mechanical disruption of bacteria cell membrane [84–86]. Second approach is the use of contact active antimicrobial materials. This approach proposes the attachment of antimicrobial moieties to material surface. This approach, unlike the use of antibiotics, avoids the release of pollutants to the environments and also reduce the development of microbial resistance. Also, as the antimicrobial is directly attached to the surfaces, it can extend its life cycle. Although there are different moieties for this purpose, the use of nanoparticles such as silver and platinum have great interest. In the past, the antibacterial mechanisms of these particle have shown to be effective to kill bacteria with multidrug-resistance. Until now, there is not a define mechanism for which metal nanoparticles may have an effect of bacterial. One hypothesis support that small metal nanoparticles are responsible of the greatly increase production of reactive oxygen species (ROS) which damages and inactivates essential biomolecules such as nucleic acids, proteins, and lipids [87–89]. Other hypothesis also takes account of the difference between the cell membrane of the gramnegative and gram-positive bacteria. This suggest that the presence of an outer membrane in gram-negative bacteria make them more resistance to the direct killing mechanism. This hypothesis assumes that the direct contact of metal nanoparticles with the cell membrane changes its structure and increase its permeability. This results in an a uncontrolled movement through membrane creating disorder and toxic effects on the cell structure [90–92]. 2.3.2. Biosensors One of the applications of nanotechnology in the biomedical field has been the development of biosensors. These are sophisticated devices which use biological elements such as enzymes or proteins to sense signals on different samples [93]. For biomedical applications, biosensors can overcome common bottleneck of traditional methods (e.g., chromatography) such as low sample concentration and the lack of selectivity and sensitivity. Moreover, conventional methods require long and specialized 28.

(26) sample pre-treatment, which may potentially translate to time-consuming processes. In this context, electrochemical biosensors have proven to be useful tools to detect small sample volumes, low concentrations of biological components, and sometimes miniaturized analytical devices [94,95]. Electrochemical-based techniques for sensing can be categorized as potentiometric, amperometric or coulometric, voltammetric (incorporating preconcentration and stripping steps), and conductometric. The ability to design highly specific recognition sites makes biosensors a suitable alternative to traditional chromatography-based methods [96]. Among existing biosensors, electrochemical biosensors have various advantages such as real-time monitoring, miniaturization, and the enhancement of selectivity and sensitivity. Also, electrochemical reactions deliver electronic signals, thus, it is not necessary to use complicated signaling elements. This facilitates the development of portable systems for clinical testing and on-site environmental monitoring [94]. Electrodes used in biosensors allow the conversion of biological signals into a readable output signal. The selectivity and sensitivity of these signals can be achieved via modification with specific biological elements such as DNA, enzymes, or cells (Figure 2). Based on the nature of the biological modification, electrochemical biosensors can be classified as either biocatalytic or affinity sensors. Electrochemical biocatalytic sensors are modified with biological elements able to recognize a target and induce a response of an electroactive molecule (e.g., enzymes). Meanwhile, electrochemical affinity sensors have a binding recognition element that releases a signal when it is coupled to the target (e.g., antibodies) [97]. On biosensor field, metal nanoparticles and natural polymers have been used to improve its sensitivity. This improvement is obtained due to a high degree of modification, bulky surface, high refractive index and more reactivity.. 29.

(27) Figure 2. Scheme of an electrochemical biosensor. Biological sensing elements are coupled to electrodes. These traduce the signal to deliver a readable output. Reprinted with permission from Ref. [98].. 2.3.3. Tissue Engineering Tissue engineering (TE) field results from the application of principles from engineering and life science into the development of biological substitutes. Its main goal is to provide solutions for the replacement, repair, and regeneration of tissues and organs [7,8]. Over the last decade, advancements in this field have contributed to understand the significance of specific physical cues in the proper development and maintenance of tissular structure and function. TE strategies can be classified either as scaffold-based or scaffold-free, being the scaffold-based strategy the most widely used [99]. In the scaffoldbased strategy, cells are seeded or encapsulated within a scaffold. Scaffold’s materials are commonly biodegradable polymers such as the presented before. This strategy has the advantage to protect cell from an immune rejection while allows proper interchange of nutrients and waste products. Although application of this strategy has shown promising results, some specific application such as heart, skin and bone tissue engineering still require the development of material with specific properties. 2.3.3.1.. Heart tissue engineering. Heart tissue engineering (HTE) (Figure 3) arise as a strategy to ameliorate the lesion produced after a myocardial infarction (MI). Many researches have already 30.

(28) stablished, that, in order to use any biomaterial for this application, it must at least possess the following essential properties [100,101]: •. Mechanical strength. •. Architectural anisotropy. •. Allows vascularization. •. Electrophysiological stability. •. Contractility at physiologically relevant rates. •. Excitation-contraction coupling (conversion of electrical stimulation to mechanical responses). Other important criterion includes easy harvest of cells, high proliferation, nonimmunogenicity, resistance to ischemia, and ability to differentiate into mature, functional cardiomyocytes. Whereas, from the materials perspective, scaffold cues must be nontoxic, non-immunogenic and should trigger functional cardiogenic differentiation [102,103]. The four main approaches being used to create complex cardiac constructs, i.e. (I) biodegradable scaffolds seeded with cardiac cell precursors, (II) molded polymercell mixtures, (III) cell sheets, and (IV) in situ TE. Figure 2 illustrates a generalized schematic representation of heart tissue engineering. Seeded biodegradable scaffolds and polymer-cell moldings techniques provide a unique platform to construct in-vitro templates that can get remodel and become functional upon implantation. In the first approach, natural or synthetic polymers are used to create a matrix onto which progenitor cardiomyocytes are seeded. Upon implantation, the matrix degrades and is substituted by extracellular matrix (ECM) produced by the transplanted cells [104]. The second approach works in a very similar fashion with the main difference being that the cells and the polymer form a heterogeneous mixture that is gelled in a mold before implantation [105]. Zimmermann and colleagues (2006)[106] successfully developed an “engineered heart tissue” (EHT) using a myocardial infarcted rat model. The EHT showed mature cardiomyocyte differentiation, formed patent vasculature that anastomosed to the recipient’s vessels, had strong electrical coupling to host tissue with conduction velocities comparable to the healthy myocardium, was non31.

(29) arrythmogenic, and improved the systolic and diastolic function of the left ventricle. Other applications of these two approaches have been thoroughly reviewed, elsewhere [107]. Sekine and colleagues (2012) [105] described the cell sheet-based TE. Further studies were conducted to address vascularization in which endothelial cells (EC) were cocultured on the same cell sheet as SMs or MSCs. After implantation, these constructs showed the formation of capillary networks congruent with the amount of ECs seeded [105,108]. Mayor challenge related with the design of biomaterials for the development of heart substitutes is the improvement of their electromechanical properties. In the past, the improvement of pristine chitosan scaffolds with CNT have shown excellent in vitro results supporting cell electrical signaling in cardiac function.. Figure 3. A generalized schematic representation of heart tissue engineering. (A) affected 32.

(30) patient, (B) specific cells: (i) fibroblast and other cell lines, (ii) iPS, and (iii) cardiomyocytes and other necessary cells, (C) three-dimensional porous biomaterial-based heart scaffold of different nature and architecture, and cultured under dynamic conditions in (D) bioreactors systems (i) perfusion bioreactor and (ii) spinner tank bioreactor, which nurture the development of heart tissue by supporting efficient nutrition of cultured cells and applying mechanical stimuli that are critical for functional regeneration and (E) engineered heart as a potential alternative. Reprinted with permission from Ref. [100].. 2.4.. Future trends and conclusions. Tissue engineering and regenerative medicine have an enormous potential in medical applications such as organ regeneration or cartilage repreparation. However, there is still some work that needs to be done. Some prospects that have improved the results in the last years are the combination of bio-based materials with synthetic materials. Some of the most used materials are chitosan, cellulose, graphene, gold nanoparticles, silver nanoparticles, and many others. These materials usually give the strength necessary for scaffolds to be considered as potential candidates for tissue engineering. Furthermore, the results of the combination of novel synthetic materials with biomaterials can be improved with the implementation of novel techniques and scaffold geometries. Some of the techniques that have been used are electrospinning, wet spinning, and microfluidics which allow scientists to find more opportunities in construction design. Another field closely related to tissue engineering and regenerative medicine is drug delivery, in the sense that the cells must be carried in a system such as nanoparticles, nanofibers, microspheres, and others. This is done, in order to, be able to reach a specific region of the body, and after that adhere and proliferate to start the process of tissue regeneration, thus making drug delivery a field with high impact in the biomedical line of investigation. In conclusion, novel biological (bio)-materials are improving the works being done in tissue engineering and regenerative medicine. When speaking of materials, in general, it is important to consider which properties and methods for their characterization will be used to have a better idea of the data needed for specific applications.. 33.

(31) 3. Development and characterization of nanoparticles-loaded bio-composites for biomedical settings Abstract There is a dire need to engineer biologically robust constructs to meet the growing needs of 21st-century medical sector. The increasing (re)-emergence of human-health related pathogenic microbes has caused a havoc and serious challenge to health care services. In this context, herein, we report the development and characterization of various polymeric bio-composites with unique structural and functional attributes. For a said purpose, chitosan and graphene were used to engineer bio-composites, which were then functionalized by loading silver and platinum nanoparticles. A microwave-assisted approach was adopted to construct silver and platinum nanoparticles loaded graphenebased bio-composites. While, “one-pot” synthesis approach was used to engineer silver and platinum nanoparticles loaded chitosan-based bio-composites. As developed biocomposites were designated as GO-Ag-S1 to GO-Ag-S5 (silver nanoparticles loaded graphene-based bio-composites), GO-Pt-P1 to GO-Pt-P5 (platinum nanoparticles loaded graphene-based bio-composites), CHI-Ag-S1 to CHI-Ag-S5 (silver nanoparticles loaded chitosan-based bio-composites), and CHI-Pt-P1 to CHI-Pt-P5 (platinum nanoparticles loaded chitosan-based bio-composites). Finally, the nanoparticles loaded bio-composites of graphene and chitosan were subjected to characterization via UV-Visible spectrophotometric analysis, percent loading efficiency (%LE) analysis, Fourier-transform infrared (FTIR) spectroscopy, mechanical measurements, and antibacterial attributes. The UV-Visible spectrophotometric analysis revealed characteristic peaks appeared at the λmax 420 nm and 266 nm which belongs to the silver and platinum nanoparticles, respectively. The graphene-based bio-composites, i.e., GO-Ag-S3, GO-Ag-S4, and GOPt-P3 showed optimal %LE of 88, 92, and 89%, respectively. Whereas, CHI-Ag-S4, CHIPt-P3, and CHI-Pt-P4 bio-composites showed optimal %LE of 94, 86, and 94%, respectively. Two regions, i.e., (1) between 3600-3100 cm-1, and (2) between 1,800 and 1,000 cm-1 in the FTIR spectra were found of particular interest. The FTIR profile exposed the available functional moieties at the surface of respective bio-composites. Variable mechanical attributes of silver and platinum nanoparticles loaded bio-composites were 34.

(32) recorded from the stress-strain curves. All developed bio-composites showed bactericidal activities up to certain extent against both test strains. As compared to the initial bacterial cell count (control value, i.e., 1.5 × 108 CFU/mL), the bio-composites with higher %LE showed almost complete inhibition, with a log reduction from 5 to 0, and bactericidal activities up to certain extent against both test strains, i.e., Bacillus subtilis (B. subtilis), and Escherichia coli (E. coli). In conclusion, the notable structural, functional, mechanical and antimicrobial attributes suggest the biomedical potentialities of newly in-house engineered silver and platinum nanoparticles loaded graphene and chitosan-based biocomposites. Keywords: Nanoparticles; Bio-composites; Functional attributes; Characterization; Loading efficiency; Mechanical properties; Bactericidal; Biomedical. ACCEPTED AS: Hernandez-Vargas G, Parra-Saldivar R, Iqbal HMN. Development and Characterization of Nanoparticles-Loaded Biocomposites for Biomedical Settings. J Pure Appl Microbiol. 2020;14(4): Article Number: 6721. Available at the link: https://microbiologyjournal.org/development-and-characterization-of-nanoparticlesloaded-bio-composites-for-biomedical-settings/. 35.

(33) 3.1.. Introduction. An array of multi-functional biomaterials is evolving with enormous curiosity for researchers due to their range of applications. Research is underway around the globe to engineer pristine or hybrid polymer-based bio-composites that plays a substantial role in a field of catalysis, enzymology, environmental, pharmaceutical, and biomedical applications [109–113]. Bio-composites can be engineered using naturally occurring biopolymers either in pristine form or the combination of multi-materials. Multi-functional materials-based bio-composites and/or nano-cues/nano-constructs have now become a high requisite for new applications [114]. Likewise, synthetic polymer-based biocomposites, nano-composites (based on pristine or hybrid biopolymers) also exhibit inherited. or. improved. structural. and. multi-functional. attributes,. for. example,. biocompatibility, biodegradability, (re)-generatability, renewability, recyclability, high and efficient functionality against various substrates, induced turn-over, and overall costeffectiveness are of high interest for numerous applications. Individually or collectively, all those properties of bio-composites open new and interesting perspectives with notable incidences in the environmental, biomedical, and biotechnological sector of the modernday world [113,115–117]. Nowadays, the great emphasis has been given to exploit novel bio-composites to improve the existing characteristics or impart new properties of applied interests. To effectively address this concern, numerous nanoparticles, such as silver nanoparticles, platinum nanoparticles, and so on, are novel candidates that can be effectively used to engineer functionalized or nanoparticle-decorated bio-composites. Incorporation of some nanoparticles furnishes new or improve the existing properties of the sample, such as antimicrobial, water vapor permeability, UV protection, facilitating the bio-composite suitability for food packaging, biomedical, and therapeutic applications [87,118]. For instance, the introduction of blended silica nanostructured particles in polymer matrix composites improved the creep resistance owing to homogeneous nanoparticle distribution [119]. A great deal of research has been given to the nanoparticles-loaded/decorated constructs with multi-functional attributes for potential applications in the bio- and non-bio 36.

(34) sectors of the modern world. Aiming to strengthen further the surface functionality critics of pristine or engineered bio-composites, numerous measures have been developed and exploited, such as re-functionalization of engineered constructs via a surface coating, dipping and decorating with other bioactive entities, e.g., nanoparticles. Nevertheless, the presence and utilization of numerous solvents or surfactants, along with other harsh reaction conditions all confines the synthesis and development of nanoparticles or nanoparticles-loaded bio-composites [87,118]. Regardless of the limitations mentioned above, the idea of using biopolymers, such as chitosan, cellulose, or graphene to engineer nanoparticles-loaded bio-composites has provoked significant research interests [120]. Though a massive spectrum of biopolymers is available, however, chitosan, and graphene are very promising and have been broadly employed in the biotechnology sector at large and biomedical, in particular [121,122]. In view of the changing dynamics of 21st-century bio-composite materials with medical potencies, the incorporation of biologically active and therapeutic cues/agents into/onto the pristine bio-composite materials to engineer antibacterial matrices has received limited attention so far. Thus, keeping in mind the points mentioned above and critics, herein, we report the development and characterization of nanoparticles-loaded bio-composites with unique structural and functional attributes. For a said purpose, chitosan and graphene were used to engineer bio-composites, which were then functionalized by loading silver and platinum nanoparticles on the surface. Finally, the nanoparticles-loaded bio-composites were then fully characterize using various instrumental techniques.. 3.2.. Materials and methods. 3.2.1. Reagents/consumables All consumables or reagents/chemicals used in this study were of analytical laboratory-grade with purity >99% and utilized deprived of any purification unless otherwise stated. Chitosan (MW 100-300 kDa with 82% degree of deacetylation), graphite powder (with a purity >99.99%), hydrogen peroxide (H2O2), sodium borohydride (NaBH4),. 37.

(35) glutaraldehyde, silver nitrate (AgNO3), chloroplatinic acid (H2PtCl6), sodium nitrate (NaNO3), sulfuric acid (H2SO4), potassium permanganate (KMnO4), and ethylene glycol were obtained from the local suppliers and distributors of Sigma-Aldrich. 3.2.2. Synthesis of silver and platinum nanoparticles The silver nanoparticles were synthesized using 50 mM silver nitrate solutions. The AgNO3 reduction into Ag+ ions was followed by mixing silver nitrate solutions. The reduction reaction was performed under continuous stirring for 10 min on a magnetic stirrer, and the solution was incubated at 28±2 °C for 2 h. A significant change in color from light yellow to blackish-brown was recorded that further indicates the reaction termination. While, platinum nanoparticles were developed by the reduction of H2PtCl6 in solution with a stabilizing or capping agent, i.e., ethylene glycol/NaBH4 to form colloidal nanoparticles. As H2PtCl6 reduced to neutral platinum metal (Pt0), the reaction mixture becomes supersaturated, the Pt0 begins to precipitate in the form of nanoscale particles. Both silver and platinum nanoparticles containing reaction mixtures were subjected to the centrifugation at 4,000 g for 5 min, to purify the silver and platinum nanoparticles. The obtained supernatants were oven-dried at 50 °C and subjected to UV-Visible spectrophotometric analysis. The λmax values were noted from 0 h to each hour until 6 h and then after every 6 h until 24 h by taking an aliquot from the same mother liquor. The resultant final nanoparticles were stored and used for further characterization and loading onto graphene and chitosan-based bio-composites. 3.2.3. Synthesis of graphene oxide Prior to the development of bio-composites, as received graphite powder was used to synthesize graphene oxide. For a said purpose, a mixture of graphite powder (4 g) and NaNO3 (2 g) was prepared using the 2:1 ratio, respectively, in the presence of concentrated H2SO4 (200 mL). The above mixture was placed in an ice bath to maintain the temperature and cooled the mixture down to 0±1 oC. After attaining the requisite temperature (0±1 oC), the mixture was supplemented with KMnO4 (10 g) consecutively with small doses and considered as a reaction solution. The reaction solution was heated up to 35±3 oC and continuously stirred for 2 h. This was followed by adding sterilized 38.

(36) water (300 mL) to finalize the oxidation process. The unreacted KMnO4 from the reaction mixture was eliminated by adding H2O2 (30 mL). After 30 min continuous stirring, the reaction mixture was centrifuged at 1500 g for 30 min. The obtained supernatant was poured off, and the solid material was placed in water and recentrifuged under the same conditions. Finally, brown-yellow graphene oxide was recovered, quantified (5 mg/mL), and stored in a sterilized plastic tube. 3.2.4. Nanoparticles-loaded graphene-based bio-composites Herein, silver and platinum nanoparticles loaded graphene-based bio-composites were prepared using a microwave-assisted approach. Briefly, graphene oxide powder (10 mg) was poured into the ethylene glycol (100 mL) and treated by ultrasonication at a supersonic power of 500 W for 5 min. This was followed by the addition of AgNO3 into the graphene oxide - ethylene glycol solution, the presence of NaBH4 as an additional reducing agent, and heated to 80±5 oC. This new mixture was then subjected to the irradiation in a microwave oven at 700 W for 2 min. The resultant mixture was centrifuged at 1500 g for 20 min, and solids were washed and dried in a hot air oven at 80±5 oC for 12 h to obtain silver nanoparticles loaded graphene-based bio-composites. Likewise, for the development of platinum loaded graphene-based bio-composites, AgNO3 was replaced with H2PtCl6 as a platinum precursor. Sample IDs of newly in-house engineered silver and platinum nanoparticles loaded graphene-based bio-composites are summarized in Table 3.. 39.

(37) Table 3. Sample IDs of newly in-house engineered silver and platinum nanoparticles loaded graphene-based bio-composites. Sample IDs. Nanoparticle. NPs (%). NPs precursor. Bio-composite IDs. S1. Silver. 1. AgNO3. GO-Ag-S1. S2. Silver. 2. AgNO3. GO-Ag-S2. S3. Silver. 3. AgNO3. GO-Ag-S3. S4. Silver. 4. AgNO3. GO-Ag-S4. S5. Silver. 5. AgNO3. GO-Ag-S5. P1. Platinum. 1. H2PtCl6. GO-Pt-P1. P2. Platinum. 2. H2PtCl6. GO-Pt-P2. P3. Platinum. 3. H2PtCl6. GO-Pt-P3. P4. Platinum. 4. H2PtCl6. GO-Pt-P4. P5. Platinum. 5. H2PtCl6. GO-Pt-P5. 3.2.5. Nanoparticles-loaded chitosan-based bio-composites Herein, silver and platinum nanoparticles loaded chitosan-based bio-composites were prepared using the “one-pot” synthesis approach. For a said purpose, chitosan solution (0.5%, w/v) was sequentially added dropwise in 5.0% (w/v) acetic acid solution under continuous stirring conditions at 28±2 °C for 1 h. Aiming to cast the chitosan-based bio-composite membrane, the above reaction mixture was poured into the pre-labeled and sterilized Petri dishes, followed by incubation in a hot air oven at 60±5 °C for 24 h. At the end of the stipulated incubation period, the casted chitosan-based bio-composite membranes were recovered and washed twice with sterilized water to remove the excessive acetic acid from the surface. The loading of silver and platinum nanoparticles onto the newly developed chitosan-based bio-composite membranes was performed via surface dipping and incorporation technique. For a said purpose, a pre-weight (Wi) chitosan-based bio-composite was dipped in a freshly synthesized silver and platinum nanoparticles solution, each separately. The reaction was activated using 0.5% (w/v) glutaraldehyde solution (newly prepared within a 50 mM Na‒malonate buffer of pH 4.5). A pre-optimized reaction period (60 min) was used to incubate the surface dipped 40.

(38) chitosan-based bio-composite in a hot air oven at 50±5 °C until thoroughly dried, and final dry weight (Wf) was recorded. Both Wi and Wf were used to calculate the nanoparticles loading efficiency. Sample IDs of newly in-house engineered silver and platinum nanoparticles loaded chitosan-based bio-composites are summarized in Table 4. Equation (1) was used to calculate the percent loading efficiency (%LE): !"#$%&' )**%+%)&+, (%) =. !"#!$ !$. × 100,. (1). Where Wf = final dry weight and Wi = initial weight (chitosan without NPs). Table 4. Sample IDs of newly in-house engineered silver and platinum nanoparticles loaded chitosan-based bio-composites. Sample IDs. Nanoparticle. NPs (%). NPs precursor. Bio-composite IDs. S1. Silver. 1. AgNO3. CHI-Ag-S1. S2. Silver. 2. AgNO3. CHI-Ag-S2. S3. Silver. 3. AgNO3. CHI-Ag-S3. S4. Silver. 4. AgNO3. CHI-Ag-S4. S5. Silver. 5. AgNO3. CHI-Ag-S5. P1. Platinum. 1. H2PtCl6. CHI-Pt-P1. P2. Platinum. 2. H2PtCl6. CHI-Pt-P2. P3. Platinum. 3. H2PtCl6. CHI-Pt-P3. P4. Platinum. 4. H2PtCl6. CHI-Pt-P4. P5. Platinum. 5. H2PtCl6. CHI-Pt-P5. 3.2.6. Instrumental analysis of pristine and engineered biocomposites A Shimadzu UV-Visible spectrophotometer was used to record the absorbance of freshly synthesized silver and platinum nanoparticles. A quartz cell (1.0 cm path length) was used to record the absorbance of 300 µL of silver and platinum nanoparticles, each separately, at the wavelengths from 200 to 600 nm. The λmax values were noted from 0 h to each hour until 6 h and then after every 6 h until 24 h by taking an aliquot from the same mother liquor.. 41.

(39) Fourier-transform infrared spectroscopy (FTIR) was used to investigate the available functional groups on the bio-composite surface. FTIR spectra were recorded at a wavelength range of 4000‒500 cm-1 with 64 scans at a resolution of 4.0 cm-1 using a Perkin-Elmer System 2000 FTIR spectrophotometer. The possible peak numbers were assigned accordingly. Mechanical measurements, i.e., Young's modulus, tensile strength, and elongation at break attributes of pristine and nanoparticles-loaded bio-composites were performed using a Perkin-Elmer Dynamic Mechanical Analyzer (DMA) Q800. For all analysis, the load was set within a range of 1-6000 mN with a crosshead speed of 200 mN min−1 at a constant tensile rate.. 3.2.7. Evaluation of antibacterial attributes The antibacterial attributes of newly engineered bio-composites were trialed using a conventional spread-plate method. For a said purpose, two bacterial strains, i.e., Bacillus subtilis (B. subtilis), and Escherichia coli (E. coli). Before culturing the bacterial strains, each test specimen was UV-sterilized followed by controlled inoculation of freshly overnight grown bacterial suspensions (containing 105 CFU/mL) on the surfaces of the test bio-composites. The bacterially inoculated bio-composites were incubated at 35 oC for 24 h. At the end of the incubation, the overnight grown bacterial cells from the control and test bio-composites were wash away twice using 50 mL phosphate buffer (pH, 7.0). The bacterial cell count in terms of CFU/mL was determined by conventional spread-plate method. For each test sample, the CFU/mL values were used to calculate the antibacterial efficacy of the test composites using Equation 2. !"'4)$5+6%"& = !"' 789 7"&64": − !"' 789 <4)#6)$. (2). Due to the intrinsic variability of the antibacterial test results, at least a 2-log reduction was considered necessary to claim an antibacterial activity, as reported by Elegir et al. [123].. 42.