Design, Fabrication, and Characterization a Sprayable

Methacrylic anhydride-modified gelatin hydrogel combined

with Graphene Oxide-Silver Nanoparticle and Quercetin

Flavonoids for wound healing

Submitted by: Adrielle Jamila S. Picar

Submitted to: Ma’am Maria Cecilia Santiago

I
 CHAPTER I

INTRODUCTION
BACKGROUND OF THE STUDY

Chronic wounds are defined as wounds that fail to proceed through the normal

phases of wound healing in an orderly and timely manner. Often, chronic wounds stall in

the inflammation phase of healing. Skin damage may lead to the intrusion of

microorganisms or other exogenous harmful substances. In recent years, researchers

have developed biological therapy methods for wound treatment, but they do not

adequately resolve skin ulcers. Chronic wounds are a significant medical and financial

burden on the health care system. Wounds represent 1% of all skin disease diagnoses

in the United States, and account for 6.07% of all cutaneous-related deaths annually..

Patients with chronic wounds report major negative impacts on their quality of life, with

statistically significant increases in anxiety and depression and decreased perceived

social support. Beyond the morbidity and mortality, wounds are the most expensive skin

ailment, with more than $14 billion spent annually on the management of venous leg

ulcers alone.

¹Frykberg, Robert G., and Jaminelli Banks. 2015. “Challenges in the Treatment of Chronic Wounds.”
Advances in Wound Care 4 (9): 560–82.
https://doi.org/10.1089/wound.2015.0635.
²Darwin, Evan, and Marjana Tomic-Canic. 2018. “Healing Chronic Wounds: Current Challenges and
Potential Solutions.” Current Dermatology Reports 7 (4): 296–302. https://doi.org/10.1007/s13671-018-
0239-4
 The nomenclature is far from agreed upon, and these wounds are sometimes

referred to as hard-to-heal or difficult to heal wounds/ulcers, and the time span required

for chronicity has been defined in the range from 4 weeks up to more than 3 months.

Based on the causative aetiologies, the Wound Healing Society classifies chronic

wounds into four categories: pressure ulcers, diabetic ulcers, venous ulcers and arterial

insufficiency ulcers. Older people are the highest risk group for chronic wounds given

that wound repair slows down as the body ages and incidences of cardiovascular

disease and diabetes (which increase the incidence of chronic wounds) also increase

with age. According to various research reports and data published by Mission Regional

Medical Center in 2020, it was estimated that around 6.7 million people in the world

were suffering from chronic wounds. The global chronic wound care market is projected

to grow from USD 12.36 billion in 2022 to USD 19.52 billion by 2029, at a CAGR of

6.7% in the forecast period. On a slightly different note, treatment for wounds that cause

scarring is as crucial as chronic wounds. This is due to the immense numbers of

patients, which is estimated to be around 100 million patients per year in the developed

world, that were affected by scars from 55 million elective surgeries and 25 million

trauma-related surgeries.

________________________

³Järbrink, Krister, Gao Ni, Henrik Sönnergren, Artur Schmidtchen, Caroline Pang, Ram Bajpai, and Josip
Car. 2017. “The Humanistic and Economic Burden of Chronic Wounds: A Protocol for a Systematic
Review.” Systematic Reviews 6 (1).
https://doi.org/10.1186/s13643-016-0400-8.  
⁴Zulkefli, Nabilah, Che Nur Mazadillina Che Zahari, Nor Hafiza Sayuti, Ammar Akram Kamarudin,
Norazalina Saad, Hamizah Shahirah Hamezah, Hamidun Bunawan, et al. 2023. “Flavonoids as Potential
Wound-Healing Molecules: Emphasis on Pathways Perspective.” International Journal of Molecular
Sciences 24 (5): 4607.
https://doi.org/10.3390/ijms24054607
 In preliminary studies, Hao Cheng et. al presents a sprayable hydrogel-based

wound dressing loaded with cerium oxide nanoparticles (CeONs) and an antimicrobial

peptide (AMP), for combined reactive oxygen species (ROS)-scavenging and

antibacterial properties. They adopted a mussel-inspired strategy to chemically

conjugate gelatin with dopamine motifs and prepared a hydrogel dressing with improved

binding affinity to wet skin surfaces. The broad-spectrum antibacterial ability, the ROS

scavenging capacity, and the adhesive ability of the fabricated hydrogel dressing were

systematically verified in vitro and in vivo. Another study from Zulkefli Nabilah in which

they compiled existing evidence on the manipulation of flavonoids towards achieving

skin wound healing, together with current limitations and future perspectives in support

of these polyphenolic compounds as safe wound-healing agents.

It has been proven that the conductivity of graphene-based materials resembles

that of the skin; thus, it could stimulate normal wound healing. Furthermore, being a

good antibacterial, graphene could inhibit microbial infection, thus allowing more proper

and rapid wound healing. Graphene-platforms could be functionalized by different

antibacterial agents such as metal-nanoparticles, natural compounds, and antibiotics.

The Graphene structure can absorb near-infrared wavelengths, allowing it to be used as

antimicrobial photodynamic therapy. Therefore, Graphene-based material could be

used to inhibit pathogens that cause serious skin infections and destroy their biofilm

community

.________________________
Liu, Changfeng, Huajing Zeng, Ziyan Chen, Zhenlin Ge, Bei Wang, Bin Liu, and Zengjie Fan. 2022.
“Sprayable Methacrylic Anhydride-Modified Gelatin Hydrogel Combined with Bionic Neutrophils
Nanoparticles for Scar-Free Wound Healing of Diabetes Mellitus.” International Journal of Biological
Macromolecules 202 (January): 418–30. https://doi.org/10.1016/j.ijbiomac.2022.01.083.
 Quercetin is a kind of polyhydroxy flavonoids, which is frequently found in

flowers, leaves, and fruits of various plants. It possesses wide pharmacological activities

such as anti-cancer, anti-oxidation, anti-fibrosis, and anti-inflammation effects, and

therefore shows high medicinal value. Importantly, quercetin can improve common

wound healing by increasing fibroblast proliferation, while decreasing fibrosis and scar

formation. Moreover, it can reduce obesity-induced hepatic inflammation through

macrophage phenotype switching, and induce shift of macrophage phenotype from M1

to M2 after spinal cord injury in rats. However, it remains unclear whether quercetin

promotes diabetic wound healing via macrophage polarization from M1 to M2

phenotype. Quercetin possesses powerful antioxidant and anti-inflammatory effects,

which support its potential use in wound healing. Additionally, quercetin can reduce both

acute and chronic inflammatory stages.

Therefore, this study aims to Design, Fabricate, and Characterize a Sprayable

Methacrylic anhydride-modified gelatin hydrogel combined with Graphene Oxide-Silver

Nanoparticle and Quercetin Flavonoids for wound healing with antioxidative and anti-

inflammatory activities, to come up with a biodegradable, adhesive, air permeability, and

elastic hydrogel wound dressing, to measure the antioxidative ability and anti-

inflammatory of Graphene Oxide-Silver Nanoparticle and Quercetin Flavonoids and to

reduce the number of colony-forming units in biopsies extracted from the wound.

________________________
Zulkefli, Nabilah, Che Nur Mazadillina Che Zahari, Nor Hafiza Sayuti, Ammar Akram Kamarudin,
Norazalina Saad, Hamizah Shahirah Hamezah, Hamidun Bunawan, et al. 2023. “Flavonoids as Potential
Wound-Healing Molecules: Emphasis on Pathways Perspective.” International Journal of Molecular
Sciences 24 (5):. https://doi.org/10.3390/ijms24054607.
STATEMENT OF THE PROBLEM

This study aims to ascertain the biodegradability, antimicrobial, sprayability, anti-

inflammatory and antioxidative activities of the sprayable hydrogel wound dressing

embedded with Graphene Oxide-Silver Nanoparticle and Quercetin Flavonoids.

Therefore, this study wants to address the following:

1. Does combining Graphene Oxide-Silver Nanoparticle and Quercetin Flavonoids

enhance the following characteristics of the sprayable hydrogel wound dressing in terms

of:

a. Biodegradable

b. Antimicrobial

c. Anti-Inflammatory

d. Sprayability

e. Antioxidative

4. Does sodium alginate that contains sodium ion and group of alginate helps the
antibacterial and antioxidant transport in the skin and to fasten the changes in a wound

_______________________
________________________
Parasuraman, Subramani, AlexanderVictor Anand David, and Radhakrishnan Arulmoli. 2016. “Overviews
of Biological Importance of Quercetin: A Bioactive Flavonoid.” Pharmacognosy
Reviews.https://doi.org/10.4103/0973-7847.194044.
Li, Yu-Jia, Shih-Chun Wei, Han-Wei Chu, Hong-Jyuan Jian, Anisha Anand, Amit Nain, Yu-
Fen Huang, Huan-Tsung Chang, Chih-Ching Huang, and Jui-Yang Lai. 2022. “Poly-Quercetin-Based
NanoVelcro as a Multifunctional Wound Dressing for Effective Treatment of Chronic Wound Infections.”
Chemical Engineering Journal 437 (June): 135315. https://doi.org/10.1016/j.cej.2022.135315

STATEMENT OF THE OBJECTIVES

The researchers’ objects to Design, fabricate and characterize a Sprayable Methacrylic

anhydride-modified gelatin hydrogel combined with Graphene Oxide-Silver Nanoparticle

and Quercetin Flavonoids for wound healing.

Specific Objectives:

•To Fabricate a Sprayable Methacrylic anhydride-modified gelatin hydrogel combined

with Graphene Oxide-Silver Nanoparticle and Quercetin Flavonoids for wound healing

•To extract quercetin flavonoid using Conventional solvent extraction (CSE) and

Ultrasound-assisted extraction (UAE)

•To extract Polysaccharide from fresh purple sweet potato using hot water centrifugal

filtration vacuum concentration

•To prepare deionized solution for synthesizing C. retusa with Polysaccharide, sodium

alginate, and petroleum ether

STATEMENT OF THE HYPOTHESIS

This study is designed to assess the hypothesis of the wound dressing performance

and possible outcomes in Designing, Fabricating, and Characterizing of a Sprayable

Methacrylic anhydride-modified gelatin hydrogel combined with Graphene Oxide-Silver

Nanoparticle and a Quercetin Flavonoids for wound healing. Thus, this study

hypothesizes the following:

1. If Graphene Oxide Silver nanoparticles and Quercetin Flavonoids are related to

antioxidative and anti-inflammatory activities, then increasing the amount of the

substances enhance the efficiency and properties of the wound dressing and helps the

antioxidative and anti-inflammatory transport in the skin faster.

2. If Methacrylic anhydride-modified gelatin hydrogel combined with Graphene Oxide-

Silver Nanoparticle and Quercetin Flavonoids has an increase in amount, then the

biocompatibility, antioxidative and anti-inflammatory activities of the wound dressing will

enhance and be improved.

3 The biocompatibility, antioxidative and anti-inflammatory activities of the wound

dressing will enhance and be improved when Methacrylic anhydride-modified gelatin

hydrogel combined with Graphene Oxide-Silver Nanoparticle and Quercetin Flavonoids

has an increase in amount.

SIGNIFICANCE OF THE STUDY

Every research study has implications for people, society, countries, and the

world. The generalization of this present would be a significant contribution to the vast

knowledge in relation to individuals with chronic wounds and skin infections and those

individuals whose suffering from it. Skin injuries present a special challenge since

wound healing is a difficult and complicated process. Designing, Fabricating, and

Characterizing a sprayable hydrogel aims to provide benefits from people who have

those who suffered from chronic wounds and prevent the surgical wound infection

caused by Gram positive bacteria that is typically present on human skin and to harm

live tissue caused by a cut, blow, or other force; the skin is frequently fractured or cut.
Hydrogel is a great dressing candidate that overcomes the drawbacks of standard

dressings due to its 3D structure, superior permeability, outstanding biocompatibility,

and capacity to offer a moist environment for wound repair . Both Graphene Oxide-Silver

Nanoparticle and Quercetin Flavonoids have shown anti-inflammatory and antioxidative

activities against chronic wounds. Hence, it can be used as a source of anti-

inflammatory and antioxidative agent cross linked with graphene oxide-silver

nanoparticle that has been proven in previous study has antibiotic and shows strong

bactericidal activity against Gram-positive bacteria.

SCOPE AND LIMITATIONS

The scope of this study is limited to Designing, Fabricating, and Characterizing a

Sprayable Methacrylic anhydride-modified gelatin hydrogel combined with Graphene

Oxide-Silver Nanoparticle and Quercetin Flavonoids for wound healing. The methods

and procedures will be conducted in selected laboratories such as; Research Center for

the Health Sciences, ADMATEL (Advanced Device and Materials Testing Laboratory),

Standards and Testing Division, Research Institute for Tropical Medicine, Research and

Analytical Services Laboratory (RASL) since these laboratories offers: Hydrogen Bond,

Ionic Interaction, Conjugation Reaction, Crystallization, Enzymatic Reaction, Biological

synthetic methods, Electron irradiation, Gamma irradiation, Laser abiation and Vitro

screening.

This study will be divided into two: experimental groups and control group. The

experimental group will have 2 sets: Set A will have Methacrylic anhydride with

graphene oxide-silver nanoparticle. Set B will have Methacrylic anhydride with quercetin
flavonoids. While the control group will be commercially produced sprayable hydrogel

wound dressing.

CHAPTER II

REVIEW OF RELATED LITERATURE & STUDIES RELATED LITERATURE

Methacrylic Anhydride
The copolymerization of methacrylic anhydride at 36.6oC produces its polymer. It

can be prepared directly from the corresponding acids through the use of dehydrating

agents and indirectly from the reaction of sodium salts of the acids with corresponding

acid chlorides. Gelatins prepared from methayacrylamide hydrogel have high storage

modulus. Methacrylic anhydride can be used as a precursor to synthesize bio-

renewable resins for composite applications. For example, it can be used to prepare

soybean oil and a eugenol-based resin system. This resin possesses high thermal

stability, and good mechanical properties, which makes it a suitable matrix for the

pultrusion process. It can be used as a methacrylating agent to prepare high-

performance lignin-based thermosets. It can also be used as a starting material to

synthesize Methacrylated chondroitin sulfate pH-sensitive hydrogels by

copolymerization reaction. These hydrogels can be used in drug delivery systems.

Intramolecularly cross-linked macromolecules (ICMs) with higher intramolecular cross-

link density via iniferter-mediated polymerization

¹Sigma Aldrich. 2021. “Blue-White Screening & Protocols for Colony Selection.” Merck.
https://www.sigmaaldrich.com/MX/en/technical-documents/technical-article/genomics/cloning-and
expression/blue-white-screening
‘

Hydrogel Wound Dressing

Hydrogel dressings are an emerging area for wound care, as they increase the

speed and success of wound healing. The success of hydrogel dressings is thought to

be due to their ability to maintain an optimum wound healing environment, which is

warm and moist, rather than dry whilst keeping out infective agents. They are capable of

superseding conventional dressings such as natural or synthetic cotton, lint, and gauze

bandages. Hydrogel dressings are composed of about 90% water suspended in a gel of

insoluble hydrophilic polymers that swell up on contact with water. They are typically

made from polymers of synthetic molecules, such as polymethacrylate and

polyvinylpyrrolidine, and some are combined with alginate dressings. They control the

exchange of fluid at the wound-bandage interface, with sodium and other molecules in

the wound discharge being exchanged for hydrogel compounds.The hydrogel provides

moisture, enabling painless debridement of necrotic and infected tissue, promoting

granulation and complete healing. Since they have a high water content, they are not

completely absorbent, which makes them appropriate for wounds with light to moderate

exudation. In other situations, the accumulation of water can result in skin maceration

and multiplication of microbes, leading to a foul-smelling infected wound.

_________________________

¹AZoNetwork. 2022. Review of Hydrogel Dressings. News Medical Life Sciences. Dr. Liji Thomas.
October 12, 2022. https://www.news-medical.net/health/Hydrogel-Dressings.aspx.
Hydrogels may also cool the wound which is helpful in alleviating pain. The gel flattens

out the wound surface contours to prevent dead space from becoming infected, besides

providing support for surface healing.

Chronic Wounds

Chronic wounds are those that do not progress through a normal, orderly, and

timely sequence of repair. They are common and are often incorrectly treated. The

morbidity and associated costs of chronic wounds highlight the need to implement

wound prevention and treatment guidelines. Common lower extremity wounds include

arterial, diabetic, pressure, and venous ulcers. Physical examination alone can often

guide the diagnosis. All patients with a nonhealing lower extremity ulcer should have a

vascular assessment, including documentation of wound location, size, depth, drainage,

and tissue type; palpation of pedal pulses; and measurement of the ankle-brachial

index. Atypical nonhealing wounds should be biopsied. The mainstay of treatment is the

TIME principle: tissue debridement, infection control, moisture balance, and edges of

the wound. After these general measures have been addressed, treatment is specific to

the ulcer type. Patients with arterial ulcers should be immediately referred to a vascular

surgeon for appropriate intervention. Treatment of venous ulcers involves compression

and elevation of the lower extremities, plus exercise if tolerated

_________________________

¹Bowers, Steven, and Eginia Franco. 2020. “Chronic Wounds: Evaluation and Management.” American
Family Physician 101 (3): 159–66.
https://www.aafp.org/pubs/afp/issues/2020/0201/p159.html
Diabetic foot ulcers are managed by offloading the foot and, if necessary, treating the

underlying peripheral arterial disease. Pressure ulcers are managed by offloading the

affected area.

A chronic wound is one that fails to progress through a normal, orderly, and

timely sequence of repair, or in which the repair process fails to restore anatomic and

functional integrity after three months.1 In 2014, wound care for Medicare beneficiaries

cost an estimated $28 billion to $96.8 billion. 2 A 2012 German study found a 1% to 2%

prevalence of chronic nonhealing wounds in the general population. 3 Chronic wounds,

typically diabetic ulcers, preceded 85% of amputations. 4 Some chronic wounds can take

decades to heal, thus contributing to secondary conditions such as depression, and can

ultimately lead to isolation and family distress. The five-year mortality rate after

developing a diabetic ulcer is approximately 40% 5.

Graphene Oxide-Silver Nanoparticle

Ag-GO nanoplatform could be considered for use in biomedical applications.

However, additional studies are needed to evaluate Ag-GO nanoplatform for specific

applications, including wound dressings. The nanoplatform coated with the silver

nanoparticles and graphene oxide composite showed strongest antibacterial

properties.11

Wierzbicki, M., Jaworski, S., Sawosz, E. et al. Graphene Oxide in a Composite with Silver Nanoparticles
Reduces the Fibroblast and Endothelial Cell Cytotoxicity of an Antibacterial Nanoplatform. Nanoscale Res
Lett 14, 320 (2019).https://doi.org/10.1186/s11671-019-3166-9
 Quercetin Flavonoid

Quercetin is a flavonoid with antioxidant properties. The ability of quercetin is

claimed to exert many beneficial effects on health, including protection against various

diseases such as osteoporosis, lung cancer, and cardiovascular disease. Quercetin is

said to be one of the most widely used bioflavonoids for the treatment of metabolic and

inflammatory disorders. It is one of the most abundant dietary flavonoids found in fruits

(mainly citrus), green leafy vegetables as well as many seeds, buckwheat, nuts, flowers,

barks, broccoli, olive oil, apples, onions, green tea, red grapes, red wine, dark cherries,

and berries such as blueberries and cranberries. The highest concentrations of

flavanols were found in vegetables such as onions and broccoli, fruits such as apples,

cherries, and berries, and drinks such as tea and red wine.

RELATED STUDIES

Sprayable hydrogel dressing accelerates wound healing with combined

reactive oxygen species-scavenging and antibacterial abilities

Wound management poses a considerable economic burden on the global

healthcare system, considering the impacts of wound infection, delayed healing and

scar formation.

¹Parasuraman, Subramani, AlexanderVictor Anand David, and Radhakrishnan Arulmoli. 2016. “Overviews
of Biological Importance of Quercetin: A Bioactive Flavonoid.” Pharmacognosy Reviews 10 (20): 84.
https://doi.org/10.4103/0973-7847.194044.
²Cheng, Hao, Zhe Shi, Kan Yue, Xusheng Huang, Yichuan Xu, Chenghao Gao, Zhongqi Yao, Yu Shrike
Zhang, and Jian Wang. 2021. “Sprayable Hydrogel Dressing Accelerates Wound Healing with Combined
Reactive Oxygen Species-Scavenging and Antibacterial Abilities.” Acta Biomaterialia 124 (April): 219–32.
https://doi.org/10.1016/j.actbio.2021.02.002.
 To this end, multifunctional dressings based on hydrogels have been developed

to stimulate skin healing. Herein, we describe the design, fabrication, and

characterization of a sprayable hydrogel-based wound dressing loaded with cerium

oxide nanoparticles (CeONs) and an antimicrobial peptide (AMP), for combined reactive

oxygen species (ROS)-scavenging and antibacterial properties. We adopted a mussel-

inspired strategy to chemically conjugate gelatin with dopamine motifs and prepared a

hydrogel dressing with improved binding affinity to wet skin surfaces. Additionally, the

release of AMP from the hydrogel demonstrated rapid release ablation and contact

ablation against four representative bacterial strains, confirming the desired

antimicrobial activities. Moreover, the CeONs-loaded hydrogel dressing exhibited

favorable ROS-scavenging abilities. The biocompatibility of the multifunctional hydrogel

dressing was further proven in vitro by culturing with HaCaT cells. Overall, the benefits

of the developed hydrogel wound dressing, including sprayability, adhesiveness,

antimicrobial activity, as well as ROS-scavenging and skin-remodeling ability, highlight

its promissing translational potentials in wound management. STATEMENT OF

SIGNIFICANCE: Various hydrogel-based wound-dressing materials have been

developed to stimulate wound healing. However, from the clinical perspective, few of

the current wound dressings meet all the intended multifunctional requirements of

preventing infection, promoting rapid wound closure, and minimizing scar formation,

while simultaneously offering the convenience of application. In the current study, we

adopted a mussel-inspired strategy to functionalize the GelMA hydrogels with DOPA to

fabricate GelMA-DOPA hydrogel which exhibited an enhanced binding affinity for wound
surfaces, AMP HHC-36 and CeONs are further encapsulated into the GelMA-DOPA

hydrogel to confer the hydrogel wound dressing with antimicrobial and ROS-scavenging

abilities. The GelMA-DOPA-AMP-CeONs dressing offered the benefits of sprayability,

adhesiveness, antimicrobial activity, as well as ROS-scavenging and skin-remodeling

ability, which might address the therapeutic and economic burdens associated with

chronic wound treatment and management.

Flavonoids as Potential Wound-Healing Molecules: Emphasis on Pathways

Perspective

Wounds are considered to be a serious problem that affects the healthcare

sector in many countries, primarily due to diabetes and obesity. Wounds become worse

because of unhealthy lifestyles and habits. Wound healing is a complicated

physiological process that is essential for restoring the epithelial barrier after an injury.

Numerous studies have reported that flavonoids possess wound-healing properties due

to their well-acclaimed anti-inflammatory, angiogenesis, re-epithelialization, and

antioxidant effects. They have been shown to be able to act on the wound-healing

process via expression of biomarkers respective to the pathways that mainly include

Wnt/β-catenin, Hippo, Transforming Growth Factor-beta (TGF-β), Hedgehog, c-Jun N-

Terminal Kinase (JNK), NF-E2-related factor 2/antioxidant responsive element

(Nrf2/ARE), Nuclear Factor Kappa B (NF-κB), MAPK/ERK, Ras/Raf/MEK/ERK

¹Zulkefli, Nabilah, Che Nur Mazadillina Che Zahari, Nor Hafiza Sayuti, Ammar Akram Kamarudin,
Norazalina Saad, Hamizah Shahirah Hamezah, Hamidun Bunawan, et al. 2023. “Flavonoids as Potential
Wound-Healing Molecules: Emphasis on Pathways Perspective.” International Journal of Molecular
Sciences 24 (5): 4607. https://doi.org/10.3390/ijms24054607
phosphatidylinositol 3-kinase (PI3K)/Akt, Nitric oxide (NO) pathways, etc. Hence, we

have compiled existing evidence on the manipulation of flavonoids towards achieving

skin wound healing, together with current limitations and future perspectives in support

of these polyphenolic compounds as safe wound-healing agents, in this review.

Graphene oxide-based injectable conductive hydrogel dressing with

immunomodulatory for chronic infected diabetic wounds

The main challenges of chronic wounds represented by diabetic wounds are that

the infection is not easily controlled and the healing is difficult, mainly due to the high-

glucose microenvironment, M1 macrophage aggregation, and persistent inflammation.

Previous studies inspired by endogenous electric fields have demonstrated that

conductive materials can promote cell proliferation, migration, etc., to promote wound

healing. However, no study has been conducted on the effect of conductive materials

on local immunity. To this end, this study designed a graphene oxide-based conductive

hydrogel to repair infected diabetic wounds. Self-healing injectable conductive

hydrogels were synthesized by dynamic Schiff-base reaction and electrostatic

interactions between oxidized hyaluronic acid, N-carboxyethyl chitosan, graphene

oxide, and polymyxin B. The study found that the hydrogel has good biocompatibility

and antibacterial properties. In addition, in vivo experiments,confirmed that hydrogel.

_________________________

¹Ou, Xiaolan, Lin Guan, Wenlai Guo, Xi Zhang, Siyu Wu, Deming Guo, Ruiyan Li, Andrei V. Zvyagin,
Quan Lin, and Wenrui Qu. 2022. “Graphene Oxide-Based Injectable Conductive Hydrogel Dressing with
Immunomodulatory for Chronic Infected Diabetic Wounds.” Materials & Design 224 (December): 111284.
https://doi.org/10.1016/j.matdes.2022.111284.
could modulate macrophage polarization to improve the local inflammatory

microenvironment, promote neovascularization, and significantly accelerate the healing

of diabetic wounds. Most notably, graphene oxide-based conductive hydrogels can

modulate local immunity in diabetic wounds to promote healing. This study provides

new guidelines for expanding research on conductive materials and new directions for

promoting healing in infected diabetic wounds.

Silver nanoparticles-immobilized-radiation grafted polypropylene fabric as

breathable, antibacterial wound dressing

In this work, a simplistic approach for developing an antibacterial and hydrophilic non-

woven polypropylene (NWPP) fabric with excellent breathability has been presented via

a two-step process. In the first step, acrylic acid was introduced onto NWPP fabric using

simultaneous irradiation graft polymerization and pre-irradiation graft polymerization

methods. Different grafting parameters, e.g., absorbed dose, monomer concentration,

homopolymer inhibitor, reaction time, etc., were studied to optimize the grafting process.

Subsequently, the acrylic acid grafted NWPP (NWPP-g-AA) fabric was immobilized with

silver nanoparticles (Ag NPs), prepared from silver nitrate using trisodium citrate as

reducing agent. After optimizing the grafting parameters, the NWPP-g-AA and Ag NPs-

(NWPP-g-AA) samples were thoroughly characterized by grafting yield, FTIR, SEM,

TEM, water contact angle, water vapor transmission rate (WVTR) and mechanical

_________________________

¹Mehta, Kanchan, Virendra Kumar, Bhuvaneshwar Rai, Meenu Talwar, and Gulshan Kumar. 2023. “Silver
Nanoparticles-Immobilized-Radiation Grafted Polypropylene Fabric as Breathable, Antibacterial Wound
Dressing.” Radiation Physics and Chemistry 204 (March): 110683.
https://doi.org/10.1016/j.radphyschem.2022.110683.
properties. The Ag NP-(NWPP-g-AA) fabric samples exhibited desired hydrophilicity,

breathability and mechanical strength. Ag NP-(NWPP-g-AA) fabric samples were tested

for antibacterial activity using plate count method after serial dilution, and exhibited

excellent antibacterial efficacy against S.aureus (gram positive) and E.coli (gram

negative) bacteria. This strategy demonstrated that a hydrophilic NWPP fabric having

long-lasting antimicrobial activity was developed by gamma radiation-induced graft

polymerization, which offers an ideal material for antibacterial wound dressing

applications.

An adhesive gelatin-coated small intestinal submucosa composite hydrogel

dressing aids wound healing

It is a challenging clinical task to determine how to repair large-area skin defects

better. Traditional wound dressings (e.g., cotton and gauze) can only be used as a

dressing; consequently, there is an increasing demand for wound dressings with

additional properties (i.e., antibacterial and pro-repair) in clinical practice. In this study, a

composite hydrogel with o-nitrobenzene-modified gelatin-coated decellularized small

intestinal submucosa (GelNB@SIS) was designed for the repair of skin injuries. SIS is a

natural extracellular matrix with a 3D microporous structure and also contains high

levels of growth factors and collagen. GelNB provides this material photo-triggering

tissue adhesive property. The structure, tissue adhesion, cytotoxicity, and bioactivity to

_________________________

¹Wang, Lu, Fengling Liu, Xinrang Zhai, Wei Dong, Wei Wei, and Zhenhua Hu. 2023. “An Adhesive
Gelatin-Coated Small Intestinal Submucosa Composite Hydrogel Dressing Aids Wound Healing.”
International Journal of Biological Macromolecules 241 (June): 124622.
https://doi.org/10.1016/j.ijbiomac.2023.124622.
cells were investigated. Based on in vivo study and histological analysis, we found the

combination of GelNB and SIS improved the healing process by promoting vascular

renewal, dermal remodeling, and epidermal regeneration. Based on our findings,

GelNB@SIS is a promising candidate for tissue repair applications.

Quercetin functionalized hybrid electrospun nanofibers for wound dressing

application

Quercetin is a potential phytochemical with high anti-inflammatory and antioxidant

activities known to treat numerous ailments. In this study, quercetin loaded

polycaprolactone (PCL)/gelatin (GLN) electrospun nanofiber was synthesized and

their wound healing efficiency was evaluated. The results showed that the quercetin

loaded nanofibers had a continuous, smooth and bead-free morphology with

improved surface hydrophilicity. The quercetin incorporated nanofibers exhibited

greater mechanical strength with bactericidal activity. The biocompatibility of the

synthesized nanofibers on 3T3 fibroblast cells was examined by MTT assay and

AO/EtBr staining. The in vivo study revealed a significant contraction of the wounds

within 18 days with increased angiogenesis, reepithelization and collagen synthesis

for quercetin nanofibers treated wounds. Overall, as synthesized nanofibers provided

desired integrity and hydrophilicity to develop the therapeutic system for wound

dressing applications.

¹Karuppannan, Sathish Kumar, Mohammed Junaid Hussain Dowlath, Raghavendra Ramalingam,

Shazia Anjum Musthafa, Munuswamy Ramanujam Ganesh, V. Chithra, Balasubramani Ravindran,
anDKantha Deivi Arunachalam. 2022. “Quercetin Functionalized Hybrid Electrospun Nanofibers for
Wound Dressing Application.” Materials Science and Engineering: B 285 (November)
https://doi.org/10.1016/j.mseb.2022.115933

CHAPTER III

METHODOLOGY

1. Collection of materials

In the collection of materials, In the collection of materials, methacrylic anhydride

will be purchased from Mt.Natib, Barrio San Jose, Caloocan. the graphene oxide will

be purchased from Artdeshine Philippines, Biñan, Laguna. The onion that will be the

source of quercetin flavonoids will be purchased at Puregold Paco.

2. Extraction of Quercetin Flavonoids

2.1 Conventional solvent extraction (CSE) and Ultrasound-assisted extraction

(UAE)

The ratio of raw materials (Onion flesh/skin) to solvent is 1:10 (w/v). The dried material

(15 g) will be used for each treatment. There are two factors affect the CSE and UAE

efficacy: the concentration of ethanol solution (from 50 to 80%) and the time of

extraction (from 15 to 60 mins). The process of extraction began by mixing 15 g of dried

Onion flesh/skins in 150 mL of ethanol solution. The temperature of the sample will be

maintained in the water bath or ultrasonic bath at 60oC for different extraction time as

mentioned above. Thereafter, centrifugation will be applied using a centrifuge with

12,000 rpm for 30 mins at room temperature (25oC). Subsequently, the centrifuged
clear liquid will be obtained and kept in dark condition at low temperature (2-3 oC) until

further analysis of quercetin. Each treatment will be performed three times.

2.2 Quercetin analysis

2.2.1 Sample preparation

Onion extract (0.2 mL) will be centrifuged (3 mins, 15,000 rpm) to remove the solid

substance. Then, the sample will be purified by protein precipitation and solidphase

extraction. Briefly, the cartridge will be activated by methanol and conditioned by

distilled water. Then sample will be loaded sample through the cartridge and followed by

de-salting with distilled water. Finally, the analytes were eluted with 60% methanol.

3. Synthesis of silver nanoparticles embedded in graphene oxide

GO will be dissolved in deionized water (1.0 g to 100 mL) and heated to 60 °C.

Subsequently, silver precursor, AgNO3 (0.118 mmol), will be added to the solution and

vigorously stirred for 24 h. Then, the solution will be dialyzed through SnakeSkin™ 3500

Da dialysis tubing (Thermo Scientific, USA) for two days to remove the unreacted

components. The solution will be further lyophilized and kept at -20 °C until use.

4. Preparation of Materials

4.1 Preparation of Sprayable Methacrylic Anhydride modified gelatin hydrogel

The prepared GelMA hydrogel has good sprayability and film-formation ability under

blue light illumination (wavelength = 435–480 nm). Nanoparticles mimicking neutrophils

belong to a double enzyme system that are encapsulated in ZIF-8 nanoparticles, which

can consume glucose to produce HClO, ensuring a decrease in the glucose

concentration of the wound and growth inhibition in bacteria.

5. Characterization of the sprayable wound dressing

5.1 Antioxidative

DPPH Assay is a stable free radical that changes color when it reacts with antioxidants.

The change in color is measured spectrophotometrically, and the degree of color

change indicates the antioxidative capacity of the wound dressing.

5.2 Anti-inflammatory

Employ techniques like Western blotting or quantitative polymerase chain reaction

(qPCR) to analyze specific molecular markers associated with inflammation. This can

include assessing the expression levels of inflammatory mediators, such as

cyclooxygenase-2 (COX-2) or inducible nitric oxide synthase (iNOS), in tissue samples

treated with the wound dressing.

5.3 Antimicrobial

Zone of Inhibition Test helps determine the antimicrobial activity of the wound dressing

against specific microorganisms. It involves inoculating a standardized concentration of

the microorganism onto an agar plate and then applying the sprayable wound dressing

onto a paper disc placed on the agar surface. The antimicrobial activity of the dressing

is indicated by a clear zone of inhibition around the disc, where the growth of the

microorganism is inhibited.

5.4 Biodegradability
The biodegradability of the hydrogel will be also measured by immersing them in PBS

containing 20 Units/mL hyaluronidase and observing the number of days they took to

degrade.

5.5 Sprayability

Hydrogel solution at different concentrations [3 − 10% (w/v)] mixed with a red food dye

will be transferred into a commercial 100- mL plastic spray bottle with plastic sprayer,

the sprayability of the hydrogel will be tested at room temperature.