Instituto Tecnológico y de Estudios Superiores de Monterrey

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Instituto Tecnológico y de Estudios Superiores de Monterrey

Campus Monterrey

School of Engineering and Sciences

Optimization of Nanocomposite Hydrogels for the Treatment of Diabetic Foot Ulcers

A thesis presented by

Angel Manuel Villalba Rodríguez

Submitted to the

School of Engineering and Sciences

in partial fulfillment of the requirements for the degree of Master of Science

In

Nanotechnology

Monterrey Nuevo León, November 24^th, 2020

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Dedication

First, I would like to thank God, for he is the beginning and the end of everything. He who makes the impossible possible and gave me a new opportunity in life.

I dedicate this to my beautiful wife Monica Guerrero, who has been the greatest support, friend, and partner in this new episode of our lives. To my parents, brother, and sister, for they have always believed in me and supported me in all my decisions. To my all- time best friend Angel Fonseca, and all my beloved ones, because in one way or another, they have always been there for me.

I deeply thank my mentor, advisor, friend and brother-like figure, Dr. Hafiz Iqbal for giving me a chance since the day we met and offered to teach me all he knew about research, which he has done exceedingly well up to this point. For his patience, trust, and time. Also Dr. Roberto Parra for taking me in and allowing me to join his research group, and always having a way to cheer me up.

Finally, with all my love I dedicate this to Laura Hoesterey Baker. Teacher, counselor, friend, and mother-like figure. Had she not been used by God in a critical part of my life I would not be able to write this. I thank her for always believing in me and making me believe I could be a better version of myself. I know you are now in God’s presence, rest in peace. I love you and will always try my best to make you proud.

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iv Acknowledgements

I would like to express my deepest and most sincere gratitude to all the people who supported me throughout this challenging and exciting journey of my master’s degree. To my family and friends for always being there for me. To Dr. Hafiz Iqbal and Dr. Roberto Parra since they first received and accepted me in their amazing research group and the group members because they all have taught me something and supported me along the way. I would like to thank Osamu Katagiri for teaching me Python language and Dr. Eduardo Sosa for supporting me with the experimental design, as well as Centro del Agua para América Latina y el Caribe and the staff members at Centro de Innovación y Diseño Estratégico de Productos (CIDEP) for allowing me to use their laboratories.

I would like to also give special acknowledgements to Instituto Tecnológico de Estudios Superiores de Monterrey for opening its doors to me and the support on tuition, and to CONACYT for the economical support #795810.

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Optimization of Nanocomposite Hydrogels for the Treatment of Diabetic Foot Ulcers

By

Angel Manuel Villalba Rodríguez

Abstract

In the present, diabetic foot ulcers are a very common pathology all around the world, and most products in the market offer to solve a specific need for the patient such as battling infection or allowing wound debridement. In such regard biomaterial-based nano cues with multi-functional characteristics have been engineered with high interests around the globe.

The ease in fine tunability with maintained compliance makes an array of nanocomposite biomaterials outstanding candidates for the biomedical sector of the modern world. In this context, the present work intends to tackle the necessity of alternatives for the treatment of diabetic foot ulcers through the formulation of nanoclay/polymer-based nanocomposite hydrogels. Laponite RD, a synthetic 2-Dimensional nanoclay that becomes inert when in physiological environment, while mixed with water, becomes a clear gel with interesting shear-thinning properties. Optimization of the hydrogel formulation is approached by preparing the samples at several concentration ratios (Nanoclay-Chitosan and Nanoclay- Gelatin; 35-95% of nanoclay in steps of 15%), where the mechanical properties such as viscosity, shear-thinning and injectability are observed to change. Zero shear viscosity of the samples was calculated by the Cross Model Equation, and was observed to increase for the chitosan samples as the nanoclay ratio increased (3.41x10³ - 6.44x10⁴ Pa⋅s at 35-65% of nanoclay, respectively) and then decreasing after that concentration of nanoclay. Gelatin had the opposite behavior, while both formulations having a zero shear viscosity of ~3.5x10⁴

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vi Pa⋅s at 95% of nanoclay. Injection force values of the samples in 3 mL plastic syringes with 20G needles, ranged between 1 and 4 N in average, approximately.

Adding Laponite RD to chitosan or gelatin, allows for the modification of mechanical properties of such materials. The setup explored in this research allows for a promising polymeric matrix that can potentially be loaded with active compounds for antibacterial support in foot ulcers, as well as enzymes for wound debridement.

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List of Figures

Figure 1. Schematic representation of the formulation of an optimized nanocomposite material by

encapsulating enzymes such as collagenase, gelatinase or trypsin within a polymeric matrix for treating the

main setbacks in the wound healing process of diabetic foot ulcers. ... 10

Figure 2. MMPs expressed by fibroblasts and inflammatory cells such as neutrophils and macrophages regulate the wound healing process (Modified from Kurahashi et al., 2015; Martins et al., 2012; Nguyen, et al., 2016). ... 12

Figure 3. Rheological measurements from NCxCHIy (red) and NCxGELy (blue) (35:65) of viscosity (η) and shear stress (τ) vs shear rate (𝛾). ... 28

Figure 4. Cross model fitting (blue) of sample NC35CHI65 for the calculation of the zero shear viscosity. .. 29

Figure 5. Cross model fitting (blue) of sample NC35GEL65 for the calculation of the zero shear viscosity. . 29

Figure 7. Cross model fitting (blue) of sample NC50CHI50 for the calculation of the zero shear viscosity. .. 30

Figure 8. Cross model fitting (blue) of sample NC50GEL50 for the calculation of the zero shear viscosity. . 31

Figure 10. Cross model fitting (blue) of sample NC65CHI35 for the calculation of the zero shear viscosity. 32 Figure 11. Cross model fitting (blue) of sample NC65GEL35 for the calculation of the zero shear viscosity. 32 Figure 12. Rheological measurements from NCxCHIy (red) and NCxGELy (blue) (80:20) of viscosity (η) and shear stress (τ) vs shear rate (𝛾). ... 33

Figure 13. Cross model fitting (blue) of sample NC80CHI20 for the calculation of the zero shear viscosity. 33 Figure 14. Cross model fitting (blue) of sample NC80GEL20 for the calculation of the zero shear viscosity. 34 Figure 15. Rheological measurements from NCxCHIy (red) and NCxGELy (blue) (95:05) of viscosity (η) and shear stress (τ) vs shear rate (𝛾). ... 34

Figure 16. Cross model fitting (blue) of sample NC95CHI05 for the calculation of the zero shear viscosity. 35 Figure 17. Cross model fitting (blue) of sample NC95GEL05 for the calculation of the zero shear viscosity. 35 Figure 18. Zero shear viscosity values with respect to increase in NC concentration. ... 36

Figure 20. Compilation of FTIR measurements of NC-CHI samples. ... 37

Figure 21. Compilation of FTIR measurements of NCxGELy samples. ... 38

Figure 22. Schematic representation of the universal testing machine setup. ... 39

Figure 23. Injection force vs time of sample CHI100 at a) 2 mL/hr and b) 8 mL/hr (no needle). ... 40

Figure 24. Injection force vs time of sample NC35CHI65 at a) 2 mL/hr and b) 8 mL/hr. ... 41

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Figure 29. Injection force vs time of sample NC100 at a) 2 mL/hr and b) 8 mL/hr. ... 46

Figure 30. Injection force vs time of sample NC100 at a) 2 mL/hr and b) 8 mL/hr (no needle). ... 47

Figure 31. Injection force vs time of sample NC35GEL65 at a) 2 mL/hr and b) 8 mL/hr. ... 48

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List of Tables

Table 1. Relation of concentrations of Nanoclay-Chitosan hydrogels. ... 23 Table 2. Relation of concentrations of Nanoclay-Gelatin hydrogels. ... 23 Table 3. Injection force for each of the samples in 60 seconds of extension by compression uniaxial testing. ... 52

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x

Chapter 1

1. Introduction

Nanotechnology, first introduced by Richard Feynman in 1959 as a new concept in science (Feynman, 1960), was formally and experimentally introduced to the world in 1981 when IBM scientists Gerd Binnig & Heinrich Rohrer developed the first scanning tunneling microscope (STM). Such technology allowed them to see single atoms for the first time by scanning a tiny probe over the surface of a silicon crystal (Binning & Rohrer, 1981; Vithun et al., 2020).

By studying the differences in surface area to volume ratio, physicochemical stability and drug delivery capabilities at molecular to atomic levels, compared to the micro- or even the more, with the macroscale, it has been found that there are vast variations in the properties of matter in terms of mechanical strength, thermal and/or electric conductivity, surface roughness, to mention some examples. This is due to techniques such as “bottom-up”

nanofabrication which allow tunability of materials at the molecular scale (Gleiter, 2000).

Materials properties such as shape, size, crystal structure and surface roughness can be taken advantage of, and are currently being applied to practically any area of the biomedical field such as wound healing and drug delivery around the globe with exceedingly successful results as reported by Lata et al., 2017.

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3 Materials science is a field in which nanotechnology is being greatly explored, due to how much the bulk and surface properties previously mentioned such as structural tunability, functionalization, and physicochemical stability, etc. are observed to change with diverse synthetization protocols in order to form customized nanostructured materials (Bhushan, 2017; van Assenbergh et al., 2018).

The use of nanostructured materials in the form of nanoparticles, nanofibers and any shape given at the nanoscale (1-100 nm), applied towards biotechnological and/or biomedical applications such as wound healing, treatment of emerging pollutants and drug delivery has been exponentially growing over the past few decades (Pelegrino & Seabra, 2017; Paumo et al., 2020; Jacob et al., 2018).

Researchers, engineers and renowned companies have become very interested in the future of applied nano-systems and are currently striving for achieving more advanced technological options by using the outstanding, and often surprising, properties of nanomaterials in order to overcome emerging challenges and solve current problems of the modern era (Aithal, 2016).

The scope of nanostructured materials used for biotechnological and biomedical applications is very broad due to the usage of not only metallic nanoparticles, nanostructured biopolymers and ceramics in the form of nanoclays (Peña-Parás et al., 2018). Each of these classifications have their own derivations of new subclasses of materials such as nanocomposite hydrogels and carbon-based nanostructures which have gained an important role on the field of nanotechnology as to even be considered and named as stand-alone materials (Dhama et al., 2017).

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4 By decreasing the size of a system to nanometric scale, its contact surface increases exponentially, while decreasing its volume, leading to particular physico-chemical properties that are viable for numerous biomedical applications such as wound healing (Hamdan et al., 2017). The size and shape of nanostructured systems are key factors that determine their potential in wound healing processes, by influencing drug/growth factor delivery efficiency, diffusion through cell membranes, and cellular/tissue reactions (Negut et al., 2018).

Proteolytic enzymes promote cleansing of wound exudates, decrease the concentration of pathogenic microflora on wound surfaces, prevent secondary infections, and normalize the circulation of blood to the inflamed area (Sinclair & Ryan, 1994).

Immobilization of enzymes on different carriers for the development of wound care products, allows the localization of enzyme action and increases their stability during various processes in wounds, which significantly accelerates the entire healing process (Vernikovskii, 2012).

In the present work, a revision of research related to biomaterials used in the wound healing process for diabetic patients is made. The focused enzyme family are proteases to target the issues of wound healing.

Furthermore, a discussion is held towards the gaps found and how nanotechnological approaches can solve other biomedical problems.

1.1 Motivation

To satisfy existing and emerging needs of the bio-sector through the use of new techniques, technological development, innovation and research for such area through the application of the scientific method and the properties of materials as well as the combination and modification thereof. Likewise, to contribute with scientific articles based on the data

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5 obtained during the project, as well as a product that can satisfy a demanded need for the biomedical field and human healthcare.

1.2 Problem Statement and Context

In the present world, there are not many commercially available products for the treatment of diabetic foot ulcers, and the great majority of them generally only focus on treating one of the possible attend the infection of the wound or another specific process such as debridement or oxygenation.

1.3 Hypothesis

Due to the nature of synthetic nanoclays, they can be used to tailor the mechanical properties of polymers such as chitosan and gelatin to reach desirable injectability due to the shear- thinning behavior of the resulting hydrogel.

1.4 Solution overview

The synthetization of a biocompatible hydrogel improved with drug-loadable nanoparticles as a topical wound healing dressing for the promotion of the wound healing process in patients in which debridement and coagulation is difficult. The main focus is to formulate and analyze two different biopolymers combined with synthetic nanoclay in order to optimize their properties for the aforementioned application.

1.5 Key objectives

1.5.1 To develop nanoclay/polymer-based nanocomposite hydrogels with mechanically injectable characteristics.

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6 1.5.2 To optimize the preparation and processing of the developed hydrogel.

1.5.3 To characterize the mechanical properties of the developed nanoclay/polymer-based nanocomposite hydrogels.

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Chapter 2

2. Background

2.1 Chitosan-based composite materials

Due to its physicochemical properties, chitosan is a biopolymer that has drawn much attention for biomedical applications, such as drug delivery and tissue engineering. However, it is possible to improve the properties of chitosan by mixing it with other materials to obtain chitosan-based composites. Such composite materials have tunable properties that make them more efficient than using chitosan alone. One of the main disadvantages of chitosan is its poor solubility at basic pH levels (Elgadir et al., 2015). Recent studies have been carried out to modify the properties of chitosan-based composite materials for a wide range of applications such as wound healing and drug delivery (Azuma et al., 2015; Rubentheren et al. 2015). Some examples of chitosan-based composites for wound healing include systems such as lyophilized, water-absorbent hydrogels with antibacterial effects and no toxicity for its application as wound dressing (Hu et al., 2018). Also, the addition of nanoparticles, such as silver nanoparticles, to the polymeric matrix of chitosan has shown to improve antibacterial properties and stability of the material, proving to be an effective alternative to treat dermal pathogens and allow for wounds to regenerate properly (Ye, Cheng & Yu, 2019)

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8 2.2 Nanoclay-based materials

Nanoclays (NC) are ultra-fine (≈1 nm thick, 30 nm wide) polar nanomaterials that consist of nanoparticles that contain minerals found in bones such as silicates, calcium, sodium, aluminum, zinc, iron and magnesium. Such nanoclays can successfully interact and disperse within the networks of polymeric hydrogels by virtue of their charge. It should be noted that nanoclays can be considered as promising synthetic materials as an alternative to silica nanoparticles and other ceramic materials that are used in the bio-sector, due to their diverse mineral composition and their ability to combine. Among many different types of nanoclay, Laponite RD (Na0.7 (Mg5.5Li0.3) Si8O20 (OH)4) is a brand name for a synthetic nanoclay made of platelet-shaped silicates that have strong cationic interactions which have shown great potential in the field of tissue engineering (Mahdavinia & Shokri, 2017).

Laponite consists of high aspect ratio nanoplatelets. Their negatively charged surface (due to the presence of OH groups) makes them easy to disperse in water at low concentrations. The cell regulatory properties of laponite have been studied extensively within gelatin (GEL), chitosan (CHI) and polyethylene glycol (PEG), and although PEG alone does not affect cell attachment, incorporation of laponite into PEG promotes

cytoskeletal alignment of F- proteins, actin and cell binding (Sheikhi et al., 2018; Yang et al., 2011; Maeda et al., 2019). Subsequent studies with nanoclay-gelatin methacryloyl compounds have revealed their profound impact on the proliferation and osteogenic differentiation of preosteoblast cells. The compounds were exceptionally strong since the Young's modulus of compression increased almost four times compared to pure GelMa, in addition, the observation that laponite could induce bone mineralization without the aid of osteoinductive growth factors was of particular interest. Therefore, hydrogels incorporated

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9 with laponite are currently being investigated for use as promising solutions without growth factors for tissue engineering (Cidonio et al., 2019). Synthetic silicate nanoplatelets, such as Laponite RD, are highly charged nanoparticles that have been demonstrated to induce blood coagulation (Baker et al., 2007).

2.3 Complications in wound healing

Certain complications may arise during the wound healing process which include dehiscence, herniation, wound infection, delayed healing, and excessive scar formation (Franz et al., 2007). A combination of overlapping factors, such as local tissue ischemia, repeated trauma and ischemia/reperfusion injury, tissue necrosis, compromised cellular and systemic stress response, and essential bacterial infection, can result in non-healing or chronic wounds (Stojadinovic et al., 2008). Additional to the local factors, systemic ones include: age, gender, hormones, stress, ischemia, diseases (diabetes, keloids, fibrosis, hereditary healing disorder, jaundice uremia), obesity, medications (glucocorticoid steroids, non-steroidal anti-inflammatory drugs, chemotherapy), alcoholism and smoking, immunocompromised conditions (cancer, radiation therapy), nutrition, etc. (Guo et al., 2010).

Chronic wounds can generally be classified into three most common categories:

diabetic foot ulcers (DFUs), pressure ulcers (PUs) and venous leg ulcers (VLUs). Given variations in molecular etiology, these wounds share similar elements: i) modified expression of proteases, ii) dysregulation of pro-inflammatory cytokines, iii) presence of senescent cells, iv) high oxidative stress and low oxygen levels, and v) formation of biofilms (Chin et al., 2019).

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10 Moreover, a plethora of causes feed the unfavorable microenvironment that impedes cutaneous repair such as hyperglycemia, persistent inflammation, and growth factor and cytokine deficiencies lead to impaired stem cell recruitment for sufficient angiogenesis (Sorg et al., 2016).

Therefore, an increasing interest in understanding the mechanisms that contribute to non-healing wounds such as reduced bioavailability of growth factors and receptors, irregular production/modification of matrix proteins, decreased proliferative capacity of resident cells, and inadequate or impaired wound perfusion (Demidova-Rice, et al., 2012). The previously mentioned elements shared between chronic wounds involve, in different levels, the activity of enzymes that impact negatively or positively on the wound healing process.

2.4 Role of enzymes in topical wound healing

Injured tissues increase the formation of reactive oxygen products and reduce various enzymatic and non-enzymatic free- radical scavengers and the presence of reactive oxygen radicals adversely affects the wound healing process. Additionally, excess of reactive oxygen species (ROS) causes inflammation, death of cells, tissue damage and reduction of the healing process (Kurahashi et al., 2015).

Figure 1. Schematic representation of the formulation of an optimized nanocomposite material by encapsulating enzymes such as collagenase, gelatinase or trypsin within a polymeric matrix for treating the

main setbacks in the wound healing process of diabetic foot ulcers.

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11 To protect themselves from oxidative stress, cells have developed several systems to detoxify ROS. The enzymatic strategy consists of ROS-detoxifying enzymes, including superoxide dismutases (SODs), the seleno-enzyme GSH peroxidase (SeGPx), and catalase.

SODs catalyze the dismutation of superoxide anions to molecular oxygen and hydrogen peroxide. The latter can be further detoxified by catalase or by GSH peroxidases (GPx), which include, for example the SeGPx and members of the peroxiredoxin (Prx) family (Auf dem Keller, 2006).

Another type of enzymes involved in the complex process of tissue remodeling and repairing of wounds are proteases. It is thought that there is a burst of protease activity at the start of acute wound healing, and that in normally-healing wounds, the activity peaks in the first few days and then declines to very low levels after one week, as healing progresses (Singh, et al., 2017 ). However, in non-healing wounds, it is thought that high protease activity may arise through two main paths: relating to both the host cells (human) and to bacteria in the wound. It is thought that the two types of protease activity have a synergistic mechanism (Westby et al., 2018).

During inflammatory response, the synthesis of dermal enzymes is accelerated leading to degradation of extracellular matrix (ECM). It has been suggested that the expression of dermal enzymes and the downregulation of fiber synthesis play a major role in the process of skin wound. Hyaluronic acid depolymerized by hyaluronidase, elastase hydrolyzes fibrin and elastin fibers, and matrix metalloproteinases-1 particularly break the type I collagen (Boran et al., 2018; Mukherjee et al., 2011).

The degradation and remodeling of the ECM by proteases, particularly the matrix metalloproteinases (MMPs), is a key element of tissue repair and plays a role in the influx of

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12 leukocytes, angiogenesis and re-epithelialization (McCarty & Percival, 2013). ECM degradation clears the path for platelets, cell growth, neutrophils and macrophages to remove pathogens (Velnar et al., 2009; Murray & Wynn, 2011).

Neutrophils produce high levels of reactive oxygen species (ROS), proteases and pro- inflammatory cytokines to sanitize the wound. When this process is complete, apoptosis occurs on neutrophils and become phagocytosed by the newly arrived macrophages (Frykberg and Banks, 2015).

Macrophages participate in phagocytosis and the process of killing bacteria or removing debris, by secreting matrix metalloproteinases, for example, collagenase, or elastase (Eming et al., 2009). On the other hand, they are the main source of cytokines and growth factors stimulating the proliferation of fibroblasts and collagen biosynthesis.

Releasing the plasminogen activator, they cause the removal of fibrin clot (Olczyk et al., 2014).

Figure 2. MMPs expressed by fibroblasts and inflammatory cells such as neutrophils and macrophages regulate the wound healing process (Modified from Kurahashi et al., 2015; Martins et al., 2012; Nguyen, et al., 2016).

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13 2.5 Role of proteases in the wound healing process

Proteases and their inhibitors are key factors during the wound healing process.

Proteolytic enzymes are present in different proportions during acute and chronic injuries.

Proteolytic enzymes (proteases, proteinases and peptides) are a family of proteins that serve to degrade necrotic debris derived from cell breakdown. They are produced endogenously often as precursor proteins whose activation is precisely regulated. These activated enzymes serve many functions in normal as well as pathological situations as mentioned before.

Additionally, they are involved particularly in the regulation of cell maturation and multiplication; collagen synthesis and turnover. The development and removal of the perivascular fibrin cuffs found in venous insufficiency and leg ulceration as well as the removal of dead tissues following inflammation. As a limited number of enzymes perform all these functions, it is difficult to predict the effects of applying synthetic proteolytic enzymes to a wound (Sinclair And, R. D., & Ryan, T. J., 1994).

Proteases play a pivotal role in wound management. They are present in acute and chronic wounds in different proportions. Balance between protease and their inhibitors are crucial for healing of wounds because irregularity can lead to excessive extracellular matrix (ECM) degradation and deposition, thus leading to impaired healing. Recent advancements in wound care established several means to control the level of proteases, such as matrix metalloproteinases (MMP) modulators including protease-modulating dressing, signaling molecules, peptides, and micro-RNA (Singh, N., & Bhattacharyya, D., 2017).

The presence of high concentration levels of protease within chronic wounds are responsible for extracellular matrix degradation and inhibition of cell proliferation and migration within the wounds. Furthermore, bacterial toxins are responsible for excessive

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14 inflammatory responses and tissue damage that can lead to osteomyelitis, abscess, cellulitis, or limb loss (e.g. in diabetic patients). Proteases can break down AMPs into nonfunctional products and limit their therapeutic efficacy (Thapa, R. K., et al, 2019). The following list enumerates the participation of proteolytic enzymes in wound healing processes.

● They play a pivotal role in wound management and the balance between them prevents irregularities such as excessive extracellular matrix (ECM) degradation and deposition leading to impaired healing. No balanced process usually leads to abnormal scarring (Xue and Jackson, 2015). Furthermore, when conjugated to other complications such as diabetes leads to continuous inflammation and non-healing (Pastar et al., 2018).

● Enzymes that catalyze protein hydrolysis into smaller fragments, i.e. peptides.

They can be classified based on their protein substrate, pH optima, their cut specificity (the amino acid peptide bond hydrolyzing), their catalytic site configuration.

● Proteases are divided into two main categories, exopeptidases and endopeptidases.

● Exopeptidases target the C- and N- terminal peptide linkages, whereas endopeptidases cleave peptide bonds distant from the termini of the protein substrate.

● Based on the active site configuration, proteases are classified as serine, aspartic, cysteine and metalloproteases.

● Other proteases that do not fall into the conventional classifications are the ATP-dependant proteases.

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● One of their major functions is to regulate the balance between tissue synthesis and tissue degradation.

● Essential role in migration and activation of fibroblasts, remodeling of extracellular matrix and activation of growth factors.

● During inflammatory stages they have a role in debridement and removal of the necrotic tissues, foreign bodies, and bacterial load at the wound site.

● In the proliferative phase, they are expressed quickly at the budding tip of blood vessels to facilitate vascular invasion during angiogenesis.

● In the last phase of maturation and remodeling, they digest the extracellular matrix and assist in tissue remodeling. It was estimated that more than 100 enzymes are involved in this phase.

● The major proteases involved in wound healing are Metalloproteases of the metzincin family (MMPs and ADAM (TS)s), serine proteases, tolloids, meprins, and pappalysins (Chakraborti S. et al. 2017).

2.6 Classification of proteases involved in the wound healing process

2.6.1 Matrix metalloproteinases (MMPs)

MMPs are a group of calcium-dependent, zinc-containing enzymes. In different tissues, they have been identified 24 different MMPs, varying substrate specifications, and multiple functions (Puente X. S. et al. 2003 & Fanjul M. et al. 2010). Besides, during microbial infection, the MMPs play an essential role by degrading the extracellular matrix products from different organs that exhibit antimicrobial activity against wound pathogens (Levi E. et al. 1996). Based on MMPs domain organization and substrate preference, these

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16 can be classified into the four most relevant groups: 1) collagenases, 2) gelatinases, 3) stromelysins and 4) matrilysins.

2.6.2 Collagenase

This group comprises three enzymes, such as MMP-1, MMP-8, and MMP-13, whose are the only enzymes in the mammals with the capacity to cleave the triple helix of fibrillar collagen (Armstrong et al., 2002). These MMPs can also degrade several other ECM and non-ECM molecules. Interstitial collagenase (MMP-1) cleaves type II collagen and appears to have preferential activity against type III. Polymorphonuclear collagenase (MMP-8) has the most significant activity against type I. MMP-13 has a unique intensive ability to cleave type I, type II, and type III collagen (Hatz et al., 1994).

2.6.3 Gelatinases

MMP-9 (gelatinase B) and MMP-2 (gelatinase A) are the principal enzymes that are upregulated in chronic wounds (Gearing et al., 1995). Fibroblasts secrete MMP-2, and the molecularly larger MMP-9 is produced predominantly by leukocytes and perhaps also by keratinocytes. These enzymes play an essential role in the remodeling because they have additional fibronectin located inside the catalytic domain. One of the main functions of gelatinase A is to accelerate migration, while gelatinase B promotes cell migration and re- epithelialization (Gomiz et al., 2009).

2.6.4 Stromelysins

This group is composed of three members: Stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and stromelysin-3 (MMP-11), which play a varied role in the degradation of the

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17 extracellular matrix (Rechardt et al., 2000). Stromelysins are expressed by epithelial, and fibroblast cells and are secreted to the extracellular space where they play essential roles in biological processes such as mammary gland development, immunity, and wound healing (Page-McCaw et al., 2009).

2.6.5 Matrilysins

During the process of tissue remodeling, MMP-7, also known as matrilysins-1 is believed to degrade components of the extracellular matrix (ECM) like laminin, entactin and type IV collagen (Overall et al., 2002). Additionally, in humans, MMP7 expression is observed in IPF lung tissue but not healthy control samples. It is also detectable in BAL fluid, where levels are increased in patients with IPF and inversely correlated with FVC (McGuire et al., 2003).

2.6.6 Serine proteases

Serine proteases are proteins with abundant sources distributed among all living cells and are important enzymes because some of them hydrolyze peptide bonds (Toth et al., 2007). These proteins contain serine residues in their active catalytic center, which has a molecular mechanism similar to esterase. The presence of nucleophilic serine residue in the active site that attacks the carbonyl moiety of substrate derives its name as a serine protease (Puente et al., 2005).

The enzymatic activities of serine proteases are tightly regulated within translation transcription, zymogen activation, autolysis, and interaction with natural inhibitors.

Thrombin, is one of the most noticeable members of the serine protease family, is a 36‐kDa

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18 protein comprised of two chains, A and B, linked by a disulfide bond (Di Cera, 2008).

Grouping peptidases classify all known proteolytic enzymes based on sequence similarity and structure into clans and families based on catalytic mechanism, PA Clan proteases and the E*form, and homology (Rawlings et al., 2007).

● Catalytic mechanism: Enzymes exhibiting proteolytic activity are classified as cysteine, glutamic, serine, threonine, aspartic, asparagine, or metalloproteases.

Activation of many trypsin-like serine proteases requires proteolytic processing of an inactive zymogen precursor. Nearly all PA Clan proteases utilize the canonical catalytic triad and hydrolyze the peptide bond via two tetrahedral intermediates (Duffy et al., 2008).

● PA Clan Proteases: The largest family of serine proteases are the clan PA proteases that are bearing by the trypsin fold and perhaps the best-studied group of enzymes (Harper et al., 1972). Most clan PA proteases have trypsin-like substrate specificity and prefer Arg or Lys side chains at the P1 position of the substrate. Furthermore, trypsin and chymotrypsin are known to be digestive enzymes cleave polypeptide chains at positively charged (Arg/Lys) or large hydrophobic (Phe/Trp/Tyr) residues, respectively. PA Clan proteases have relied upon several crucial biological processes such as blood coagulation and the immune response, which involve cascades of sequential zymogen activation (Barrett et al., 1995).

● E*form: The critical serine protease in recent kinetic studies on thrombin showed that the blood coagulation cascade, vouch for unexpected plasticity of the trypsin fold (Cera et al., 2006). Thrombin exists in three forms at equilibrium, such as Na+-free form E, Na+-bound form E, and E* (Bah et al., 2006). Where Na+ are the low and

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19 high activity conformations of the enzyme; Na+-bound being responsible for the procoagulant, prothrombotic, and signaling functions. A third form, E*, is in equilibrium with E and is inactive toward the substrate and unable to bind Na+ (Cera et al., 2009).

Devitalized tissues present in a wound bed serve as a reservoir for bacterial growth and contain elevated levels of inflammatory mediators that promote chronic inflammation and impair cellular migration necessary for wound repair. Effective wound cleansing and debridement are essential for granulation and re-epithelization. Among various debridement methods, enzymatic debridement is a highly selective method that uses naturally occurring proteolytic enzymes. Papain combined with urea has been widely used to remove necrotic/devitalized tissues (Shoba et al., 2014).

2.7 Trypsin delivery via nanostructured cues

Trypsin is well known to hydrolyze the arginine and lysine residues of proteins.

However, in practical applications, free trypsin suffers several problems such as a high expense, instability, and difficulties with its recovery from the reaction system. (Sun et al., 2017). Besides, because of the self-digestion of the trypsin, the digestion of proteins in solution is usually slow and inefficient. (Silva et al., 2016). To address these problems, the immobilization of the enzyme, leading to improved performance, is to attach the enzyme to reliable support, such as membranes, nanoparticles, or porous materials. The notable advantages of immobilization include improved enzyme stability, ease of isolation from the protein digestion solutions, and the possibility of repeated use. (Gorodia et al., 2005).

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20 2.7.1 Nanoparticle-immobilized trypsin

Immobilization method, in which trypsin is covalently bound to the nanoparticles, can increase its stability and reusability by preventing the leakage of the trypsin (Allcok H.

R et al., 1986 & Migneault I. et al, 2004). Previous work by Sun et al. reported a covalent immobilization on Fe3O4 magnetic nanoparticles modified by carboxymethyl chitosan (CM- CTS) via EDC and glutaraldehyde (GA) cross-linking (Sun J. et al., 2017). They concluded that immobilized trypsin exhibited greater pH stability, which may be attributed to the conformational stabilization of the immobilized trypsin resulting from multipoint covalent cross-linking. Besides, the immobilized trypsin showed greater temperature stability than free trypsin. Another example of trypsin immobilization is given by Atacan et al. they covalently immobilized trypsin onto modified magnetic nanoparticles where the surface modification method improved the dispersibility, stability, biocompatibility of the magnetic nanoparticles for specific purposes (Atacan, K. et al. 2017).

2.7.2 Nanofiber-immobilized trypsin

Recent works have reported different trypsin immobilization strategies such as onto electrospun nanofibers, also by direct covalent attachments dispersed nanofibers of PS/PSMA, NNMs made from a mixture of polystyrene and poly[styrene-co-(maleic anhydride)] copolymer (PS/PSMA) (Jungbae, K. et al. 2009). Silva et al. reported the immobilization of trypsin onto poly(ethylene terephthalate)/poly(lactic acid) nonwoven nanofiber mats; where they assessed, three different immobilization strategies (Silva, T. et al. 2015), such as: a) Employing a carbodiimide, direct covalent attachment immobilization was carried out (Kulik, E. et al. 1993); b) immobilization by adsorption and crosslinking with glutaraldehyde and, c) immobilization by covalent attachment of crosslinked trypsin

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21 aggregates to amine-derivatized PET/PLA mats mobilization by covalent attachment of crosslinked trypsin aggregates to amine-derivatized PET/PLA mats. It was concluded that the lowest immobilized enzyme activity was performed onto the PET/PLA mats by direct covalent attachment to the carboxylic groups.

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22

Chapter 3

3. Materials and Methods

3.1 Materials

Low molecular weight chitosan from shrimp exoskeleton with a deacetylation degree of ≥75 %, Type A gelatin from porcine skin and Acetic Acid 99.7%, were purchased from Sigma-Aldrich. Laponite RD Nanoclay was a sample supplied by the company High Chem Specialties México, S.A. de C.V.

3.2 Methods

For the elaboration of the Nanoclay-Chitosan and Nanoclay-Gelatin hydrogels, chitosan, nanoclay and gelatin solutions were made into aqueous solutions. The solutions of the three materials were prepared separately, at different temperatures of the aqueous medium, and under the following preparation protocol.

The gelatin solution was prepared at 18% (m/v) in ultrapure water preheated in an incubator at 37 °C. The gelatin was weighed at 1.8 grams in plastic trays and introduced into the water, later incubated for 4 hours at the same temperature (37 °C) in order for the gelatin to dissolve completely, since gelatin at room temperature tends to solidify and becomes very viscous, making it difficult to handle.

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23 Laponite RD, on the other hand, requires that the aqueous medium be at low temperatures when introducing the nanoclay into it so that the nanoclay can overcome the surface tension of the water and lower its gelation rate, thus being able to dissolve properly.

The nanoclay was weighed at 4.5 g and then added to 5 mL of ultrapure water cooled for approximately 1 hour in a fridge (5 °C) in a 15 mL tube. Right after introducing the nanoclay into the water, it was immediately mixed by vortex for 5 minutes to obtain a homogeneous hydrogel. Approximately 8 to 10 samples were prepared and kept in the fridge until used.

The chitosan was prepared at a concentration of 4.5% (m/v) at 50 °C. The polymer was introduced into the aqueous medium with 1% of acetic acid and subjected to magnetic stirring for 24 hours slowly adding the chitosan powder as it dissolved with time.

Once the nanoclay hydrogel and the two polymer solutions had been prepared, they were weighed and mixed as shown in the following tables:

Table 1. Relation of concentrations of Nanoclay-Chitosan hydrogels.

Table 2. Relation of concentrations of Nanoclay-Gelatin hydrogels.

Nanoclay Gelatin Water Total mass

35% - 1.54 g 65% - 1.43 g 4.23 g 7.2 g

50% - 2.20 g 50% - 1.10 g 3.90 g 7.2 g

65% - 2.86 g 35% - 0.77 g 3.57 g 7.2 g

80% - 3.52 g 20% - 0.44 g 3.24 g 7.2 g

95% - 4.18 g 5% - 0.11 g 2.91 g 7.2 g

Nanoclay Chitosan Water Total mass

35% - 1.54 g 65% - 5.72 g 0.00 g 7.2 g

50% - 2.20 g 50% - 4.40 g 0.60 g 7.2 g

65% - 2.86 g 35% - 3.08 g 1.26 g 7.2 g

80% - 3.52 g 20% - 1.76 g 1.92 g 7.2 g

95% - 4.18 g 5% - 0.44 g 2.58 g 7.2 g

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24 Each sample was prepared separately ensuring that the polymer was still warm and the nanoclay was cold while weighing, then they were immediately mixed with a vortex for 10-15 minutes until a homogeneous hydrogel was obtained.

The calculations in each table were made in order to have a total of 5.5% of solids within the hydrogel, taking into consideration the amount of water from dissolving both the polymer and the nanoclay, before mixing.

After obtaining the nanocomposite hydrogel samples, they were stored in sample tubes at 5°C before being taken for characterization.

3.3 Characterization

3.3.1 Rheological evaluation

The rheological tests for the measurement of hydrogel viscosity and shear stress were done on an Anton-Paar Physica MCR 301 rotational rheometer with a cone and plate geometry, setting a physiological temperature of 37°C, and a shear rate from 0.1 to 100 s^-1. It is important to note that each test was done by triplicate and the results presented are the average from each set of samples. Rotational-type test was chosen because it simulates applications such as a fluid moving through a tube (in this case a syringe) where there is a dependance on volume flow rate and flow velocity.

3.3.2 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy was carried out using a Perkin Elmer Frontier FT-IR Spectrometer with Perkin Elmer Universal ATR Sampling Accessory. The samples were placed in the ATR accessory and then were run a total of 30 scans each in a wavelength range from 4500 cm^-1 to 450 cm^-1, in transmittance mode at room temperature. After each sample was

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25 characterized, the ATR accessory was carefully cleaned with acetone and filled with a new, different sample.

3.3.3 Injectability

Aside from viscosity measurements being relevant for the evaluation of the mechanical properties of polymers, it is important to also assess whether a material is applicable for proper topical, or subcutaneous administration. This can be achieved by measuring the force required to inject such material through a system (syringe and needle) and obtain such register such values by means of a force sensor or other approaches such as converting the variations in voltage from the injection system (such as syringe pumps) into force values. In this study, injection forces of the different sample formulations were measured using a universal testing machine (UTM) Instron 3365.

Approximately 3 mL of each sample was previously loaded into its respective syringe (BD Plastik 3 mL) through the back while the plunger was removed. Then the plunger was put back into the syringe and pushed just so that it reached the 3 mL mark. All the samples were left face up without needle in a vacuum chamber for 24 hours to remove air bubbles and, in some cases, leave no empty spaces in the syringe. Afterwards, the plunger was pushed in each syringe in order for the samples to reach the 2.1 mL mark and parafilm was applied to the tip of each syringe in order to safely transport them for characterization without losing any additional material.

Once in the UTM, each sample was placed as shown in Figure 22, with a 20G needle (inner diameter = 0.603 mm) and a collector below it to receive the injected hydrogel. The load cell (piston) was placed just touching the plunger and pushed about 0.1 mL to ensure there was proper contact between the plunger and the load cell.

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26 The UTM was configured to a flow rate of 0.53 mm/min (2 mL/hr) and 2.13 mm/min (8 mL/hr) for 60 seconds in each sample. Some of the formulations marked in the figures as

“NN” were ran without needle due to clogging of this.

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27

Chapter 4

4. Results

4.1 Rheological tests

Figures 3, 6, 9, 12 and 15 show the shear-thinning behavior of NCxCHIy samples (red) and NCxGELy samples (blue), where the dotted lines are the viscosity curves, and the lines with triangles are the shear stress curves for each respective sample.

With the data obtained from the rheometer, the zero-shear viscosity for each sample was calculated on Python by using the Cross Model Equation

𝜂 = 𝜂_∞+ 𝜂₀ − 𝜂_∞ 1 + (𝐶𝛾̇)^𝑚

Where 𝜂 is the measured viscosity, 𝜂_∞ is the infinite shear viscosity (how the material tends to behave at very high shear rates), 𝜂₀ is the zero shear viscosity (viscosity at the lower Newtonian plateau). C is the Cross time constant, and finally, m is the dimensionless Cross rate constant which is a measurement of the degree of dependance of the viscosity with respect to shear rate in the shear-thinning region.

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28 The zero shear viscosity values obtained from the mathematical modelling (Figures 4, 5, 7, 8, 10, 11, 13 and 14) were graphed with respect to the nanoclay concentration increases for NCxCHIy and NCxGELy samples, shown in figure 18. It is observed that as the nanoclay concentrations increase for the chitosan samples, the zero shear viscosity values tend to increase, reaching a maximum viscosity value, and then decreasing. This behavior can be attributed to the polymeric chain arrangement within the hydrogel along with the nanoplatelets. In the case of the gelatin samples, the behavior is the opposite, first it decreases, and then it increases, reaching a very similar value as the NC95CHI05 sample.

Figure 3. Rheological measurements from NCxCHIy (red) and NCxGELy (blue) (35:65) of viscosity (η) and shear stress (τ) vs shear rate (𝜸̇).

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29

Figure 4.Cross model fitting (blue) of sample NC35CHI65 for the calculation of the zero shear viscosity.

Figure 5. Cross model fitting (blue) of sample NC35GEL65 for the calculation of the zero shear viscosity.

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30

Figure 6. Rheological measurements from NCxCHIy (red) and NCxGELy (blue) (50:50) of viscosity (η) and shear stress (τ) vs shear rate (𝜸̇).

Figure 7.Cross model fitting (blue) of sample NC50CHI50 for the calculation of the zero shear viscosity.

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31

Figure 9. Rheological measurements from NCxCHIy (red) and NCxGELy (blue) (65:35) of viscosity (η) and shear stress (τ) vs shear rate (𝜸̇).

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32

Figure 10. Cross model fitting (blue) of sample NC65CHI35 for the calculation of the zero shear viscosity.

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33

Figure 12. Rheological measurements from NCxCHIy (red) and NCxGELy (blue) (80:20) of viscosity (η) and shear stress (τ) vs shear rate (𝜸̇).

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34

Figure 15. Rheological measurements from NCxCHIy (red) and NCxGELy (blue) (95:05) of viscosity (η) and shear stress (τ) vs shear rate (𝜸̇).

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35

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36

Figure 18. Zero shear viscosity values with respect to increase in NC concentration.

4.2 Fourier Transform Infrared (FTIR) Spectroscopy

Fourier transform infrared spectroscopy (FTIR) is a characterization technique commonly used to obtain the infrared absorption spectrum of a material. The wavelength maximum absorption peaks are determined by the energy transition, which corresponds to the wavelength vibrating in a bond or group of molecules. The intensity of absorption peaks is a direct measurement of the amount of material present. Through the characteristic absorption of infrared radiation, the types of chemical bonds (functional groups) and possible elements can be identified. The application of FTIR allows a simultaneous collection of spectral data in a wide spectral range in just a few seconds with improved sensitivity.

In this specific research work, FTIR was used to study the potential interactions between individual counterparts, such as chitosan (CHI), gelatin (GEL) and nanoclay (NC), in the developed hydrogel composites. Figure 20 shows the typical FTIR spectrum of NC-

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37 CHI-based hydrogels prepared using different NC-CHI ratios. The FTIR spectra show a similar pattern in the absorption peaks in the regions of approximately 3,300 cm^-1 which indicates the presence of water, and CO bonds extend in the region between 1,150 cm^-1 and 1,000 cm^-1. In particular, the FTIR spectra show that the predominant absorption bands at 1650 cm^-1 and 990 cm^-1 correspond to amide I caused by C=O stretching vibrations, and Si- O from nanoclay, respectively. The peaks from 1550 cm^-1 to 1,590 cm^-1 and the smaller peaks at around 1,400 cm^-1 correspond to the extension of amides (-NH2) and carboxyl groups, respectively. The distinctive absorption band located at around 990 cm^-1-1000 cm^-1 is due to the Si–O stretching vibration from the nanoclay.

Finally, the peaks tending to form at approximately 600 cm^-1 confirms the presence of nanoclay in the samples. (Fan et al., 2007; Shi et al., 2008; Tao et al., 2017)

Figure 19. Compilation of FTIR measurements of NC-CHI samples.

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38

Figure 20. Compilation of FTIR measurements of NCxGELy samples.

4.3 Injection Force Tests

As it is observed in the figures 23-35, the formulations tested at 2 mL/hr had a tendency of requiring more force to inject the material after one minute of testing, with the exception of samples NC35GEL65 and NC50GEL50 (31-a and 32-a, respectively), which reached a plateau and maintained a constant injection force.

In the case of the formulations tested with a flow rate of 8 mL/hr, all formulations showed a peak in the first 10 seconds of injection, a minor decrease and then a plateau with near-to-constant injection force, except for samples NC80CHI20, GEL100, NC35GEL65, NC65GEL35 and NC80GEL20 (figures 27-b, 30-b, 31-b, 33-b and 34-b, respectively), which showed inconsistent behavior in the form of oscillatory force values (repetitive peaks), except for Figure 34-b. The samples without needle were CHI100 and GEL100 at 8 mL/hr which clogged the needle and were used as control samples.

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39 It is important to note that none of the formulations were above 20 N, except for NC80CHI20 and NC80GEL20 (>12 N with tendency to keep increasing), which is a recommended force value limit for subcutaneous injection (Vo et al., 2016; Chen et al., 2017). All the injection force value ranges were summarized and shown in Table 3.

Figure 21. Schematic representation of the universal testing machine setup.

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40

Figure 22. Injection force vs time of sample CHI100 at a) 2 mL/hr and b) 8 mL/hr (no needle).

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41

Figure 23. Injection force vs time of sample NC35CHI65 at a) 2 mL/hr and b) 8 mL/hr.

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42

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43

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44

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45

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46

Figure 28. Injection force vs time of sample NC100 at a) 2 mL/hr and b) 8 mL/hr.

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Figure 29. Injection force vs time of sample NC100 at a) 2 mL/hr and b) 8 mL/hr (no needle).

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48

Figure 30. Injection force vs time of sample NC35GEL65 at a) 2 mL/hr and b) 8 mL/hr.

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49

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50

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51

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52

Table 3. Injection force for each of the samples in 60 seconds of extension by compression uniaxial testing.

CHI100 NC35CHI65 NC50CHI50 NC65CHI35 NC80CHI20 NC95CHI05

Chitosan samples

2 mL/hr; 8 mL/hr >4 N; 1.5-2 N >2 N; 1.5-2.5 N >3.5 N; 2-2.5 N >2.5 N; 2.3-2.6 N >7 N; 10-30 N >2 N: 0.5-1.5 N

Gelatin samples

2 mL/hr; 8 mL/hr >4 N; ~12 N 1-1.7 N; 1.6 N 2.5 N; 1-3 N >2 N; 0.5-2 N >3 N; >12 N >1.2 N; ~1.5

GEL100 NC35GEL65 NC50GEL50 NC65GEL35 NC80GEL20 NC95GEL05

>: Force keeps increasing after the read value; ~: Force stays approximately at the read value.

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53

Chapter 5 5.1 Conclusions

The present study presents several injectable, shear-thinning hydrogel formulations for a biomaterial that can potentially be further improved by drug-loading and enzymatic encapsulation to act as a drug delivery system/ wound dressing for diabetic foot ulcers, to mention an example. As it was observed in the rheology tests, the samples had a shear- thinning behavior which was affected by the ratio between the nanoclay and the corresponding polymer matrix (chitosan or gelatin). This property is favorable for polymers that are aimed at being used as injectable systems, since it is needed for the material to flow while under shear stress (at the walls of the syringe). Also, the addition of nanoclay to each polymer presented significant changes to the zero shear viscosity values modelled with the 50:50 concentration ratio marking a critical point for such behavior in both polymers, where the zero shear viscosity values would tend to revert to their initial values. This is a demonstration of how increasing the nanoclay concentration beyond 50% decreases the viscosity and the polymeric entanglements in the case of gelatin, making the flow of the material less continuous. This means that the 50:50 ratio was also the most unique in terms of viscosity at very low shear rates, while the 95:05 (NC:Polymer) ratio had almost the same value in both cases.

The injectability tests are also a reflection of the nanoclay effect on the hydrogel formulations which complements the data obtained in the rheological tests. While most of the samples tended to keep increasing, the injectability tests were done for only 60 seconds and the required forces (with the exceptions of the control samples) were low (1-4 N). It is

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54 important to notice that the flow rates were very low in order to be able to observe the steadiness of the injection forces required. The higher flow rate (8 mL/hr) showed that as the flow rate increases, so does the initial required force to inject the material, but also the force reaches a plateau that maintains the injection force nearly constant. Although a 20G needle was used for this experiment, enzymatic wound debridement agents for diabetic foot ulcers and other cases of necrotic wounds are commonly applied with a syringe without needle or directly from a tube. In which case the injection forces required reach a plateau as shown in the figures presented. In this study, the formulation with ratio of NC50CHI50 and NC50CHI50 showed a more stable behavior during injection, while also taking into consideration that enough amount of nanoclay solids are needed if drug-loading is to be studied in further research, as well as enzymatic encapsulation.

5.2 Future considerations

The current experimental section of this work focuses on the formulation and characterization of a nanocomposite hydrogel. It is also of great interest to explore the potentiality of the hydrogel to be drug-loaded, as well as used for the encapsulation of enzymes (specifically Collagenase) in further research.

Drug loading is planned to be done by measuring the loading efficiency of amikacin, and analyze release kinetics from samples NC50CHI50 and NC50GEL50, as well as its impact on diabetic topical wounds from rat models as published by El-Naggar et al., 2016.

Furthermore, a similar model would be tested by adding collagenase into the polymeric matrix, once the proper drug dosage is selected.

It is important to mention that due to the current COVID-19 pandemic throughout the world, there was very limited access for use of laboratories and school services, allowing only some students to use certain equipment and materials a few hours a day. Such situation made the follow-up of the aforementioned experiments impossible and the ones done, extremely difficult to achieve. Therefore, such tests, especially the animal models, would take a very long time with the current restrictions, but hopefully this work can continue in the future.

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Instituto Tecnológico y de Estudios Superiores de Monterrey