Tasks and contents: Tasks: Extraction and encapsulation of betel leaf phytochemicals into chitosan nanoparticles and synthesis of thermosensitive chitosan hydrogel composite for deliver
LITERATURE REVIEW
Wound healing process and the impacts of betel leaf extracts on wound
1.1.1 Wound and the physiological processes in wound healing
The term “wound” describes damaged living tissues as a result of accidents, surgical operations or diseases A wound typically disrupt the continuity of the skin, rendering it unable to protect underlying cells and tissues [1] Effective first aid for wounds is critical to promote accelerated healing, which can make a significant difference in the healing process Therefore, understanding the physiology of wound healing is critical for effective wound management Generally, depending on the injured site and the severity of the wound, there are four common types of tissues that might be damaged: epithelial (skin), muscles, connective tissues (bone, blood and lymphatic vessel), and nervous tissues Among these, damage to epithelial tissue is the most common as the occurrence of an open wound results in the breakdown in epithelial tissues and skin integrity at the wound site [1, 2] Without protection from the skin, underlying tissues are exposed to foreign bodies, which can cause infection Thus, immediately after injury, the human body issues a cascade of biological events in respond to promote the healing process [1]
The wound healing process consists of four or sometimes three different interconnected and overlapping phases: hemostasis, inflammation, proliferation, and remodeling[1-5] The role of the first stage is to prevent excessive cell loss caused by bleeding During this stage, fibrin clots form as preliminary matrices, promoting hemostasis, and allowing for cell migration in subsequent phases [1, 3] After the formation fibrin clot, activated platelets send out signaling factors (cytokines) to attract neutrophils to the wound site [2-6] This event marks the beginning of the inflammatory phase, in which, the permeability of blood vessels is increased to allow the entry of different cells, including white blood cells Additionally, neutrophils are activated and led to the damaged site to clear out pathogens and debris such as dead cell via phagocytosis After a period of 48-72 h,
3 monocytes are drawn to the area and transform into macrophages that digest the remnants of apoptotic cells, bacteria, and debris [1, 3, 4] The inflammatory response commonly last around few hours or up to days depending on the severity of the wound [1-5, 7] In the subsequent proliferative phase, keratinocytes, fibroblasts, endothelial cells and leukocytes accumulate in the wound area In this phase, the synthesis of extracellular matrix components is promoted, as well as enhanced angiogenesis, granulation tissue formation, collagen deposition and re- epithelialization [1-3, 5] The last and final phase of wound healing is remodeling, in which involves remodeling of extracellular matrix, substitution of collagen III by collagen I, coupled with increased activity of matrix metalloproteinases (MMPs) Apoptosis of temporary endothelial cells, fibroblasts and myofibroblasts also takes place in this phase [1-4, 7]
Figure 1.1 Primary phases of wound healing [1]
As previously mentioned, the healing phases are interconnected and overlapping with one another Thus, the healing process does not follow a linear
4 order, yet progressing forward or backward between stages [8] Delays in any of these stages can lead to persistent wounds One particular case that prolongs the wound healing is due to exacerbated inflammatory response, which causes cellular damage, and interferes with other healing stages [2, 7] The concept about inflammation will be clarified further in the next section
1.1.2 Inflammatory response phase and its role in wound healing
Inflammation or inflammatory response is a defense mechanism of the human body, in which multiple cellular processes occur to restore the tissue to its preinjury state [7].The function of inflammation is to remove dead cells, pathogens, and debris in the wound area This “clean up” process involves white blood cells and various other type of signaling cells and growth factors The immediate inflammatory phase that occurs right after an injury is described as acute inflammation, while the one that persists through this time window is called chronic inflammation Usually, the outcomes of acute inflammation include either the reversal or resolution of the inflammatory phase, or progression to chronic inflammation stage [7] Ideally, resolution is the preferred outcome as it contributes to the regeneration cells and tissues that return the organ to its normal function [7,
9, 10] However, in some cases, improper resolution can lead to the development of chronic inflammation or other adverse health effects [9], consequently damaging other cells and tissues [2, 7, 11]
Figure 1.2 Outcomes of acute inflammatory response [9]
Acute inflammation can be described as a cascade of initial responses that happen in the first few hours or days after the body has sustained an injury [3, 7] The mechanism of inflammatory responses features different vascular and tissue responses Vascular responses involve various changes in the vascular compartment with at least six intravascular events First of all, is the activation of endothelial cells as these cells produce adhesion sites for leukocytes to adhere to the endothelium Activated endothelial cells also make and release proinflammatory cytokines and chemokines that attract and activate neutrophils (polymorphonuclear leukocytes, PMNs) [3, 7].Meanwhile, the attracted neutrophils also undergo alterations in surface molecules and adhere to the endothelium to prevent their transmigration beyond the vascular compartment The neutrophils then release proteolytic enzymes and oxygen-derived free radicals to phagocyte any foreign matters and pathogen [3,
5, 7] A second change in the vascular compartment is the reversible opening of endothelial cells tight junctions, which allows the leakage of body fluids to the extravascular compartment This prevents extensive edema-excessive build-up of fluid in a closed compartment that can cause organ dysfunction Platelet aggregation
6 is also prompted as they adhere together and to the endothelial cells to create intravascular thrombosis Finally, hemorrhage may also be induced by acute inflammation due to vascular structural damage However, all of these events are reversible and can be resolved as the acute inflammatory response reaches the resolution stage [7]
Figure 1.3 Biological events in acute inflammatory response [7]
Although chronic inflammation usually progresses in a longer time frame, the definition of it does not relate to the persistence of the inflammatory response but is rather defined by the presence of specific cells Cells such as lymphocytes, macrophages, and plasma cells are predominantly present at the injured site in contrast to the acute inflammatory response, during which the population of
7 neutrophils present at the injured site is most dominant [7] In addition, chronic wound beds also exhibit features such as: low concentration of growth factors and disorganized extracellular matrix [12].Chronic inflammation can extends for long periods of time, usually months or even years The cause for this persistent inflammation is due to the constant engagement of the innate and acquired immune responses Ongoing inflammatory state induce redundant secretion of proteolysis enzymes in response to bacteria and infection Unfortunately, excessive proteolysis also hinder the development of new blood vessels, which can starve cells off oxygen and nutrients needed for granulation and tissue deposition [5, 12] As a result, cell proliferation is impaired and the healing process is also delayed Chronic inflammation can also cause extensive scarring of the affected tissues, with large collagenous scars and the development of fibrosis [7] The mechanism of chronic inflammation includes vascular responses, in which, the interaction of lymphocytes and monocytes to the adhesion molecules on the vascular (endothelial) cells occur, which ultimately results in their extravascular transmigration Once the monocytes have reached the extravascular compartment, they will undergo differentiation into macrophages Along with lymphocytes, macrophages secrete factors such as the transforming growth factor β (TGF-β) These factors are responsible for the activation of fibroblast, which leads to the production of cross-linked collagen, sometimes resulting in extensive collagenous scars [7, 12] Persistent chronic inflammation can subsequently result in an intense fibrotic response in vital organs or in some serious cases, might cause organ dysfunction or even destruction of the affected site [7, 13]
Figure 1.4 Biological events in chronic inflammation [7]
1.1.3 Termination of inflammatory responses to promote wound healing
Although the inflammation process is a crucial part of the human body’s immune system, ongoing inflammatory responses can also cause adverse effects, causing extensive tissue or organ injury [7, 8, 12] Therefore, in the body also exist naturally occurring anti-inflammatory factors These anti-inflammatory factors include cytokines (IL10, TGF-β), prostaglandin E2, indoleamine 2,3-dioxygenase, and M2 phenotype macrophages [14, 15] These cells can interfere with the inflammatory response by inhibiting the production of proinflammatory mediators and reducing the secretion of proteolytic enzymes and reactive oxygen species from PMNs [7, 8] Consequently, regulate the inflammatory responses before they cause serious damage to tissues However, in the case of chronic inflammation, there is an ongoing conflict between pro- and anti-inflammatory cytokines, along with excessive oxygen free radicals and proteases, as a result, prolonging the inflammation state [16] Therefore, requiring the use of drugs or other medical products to aid wound healing
1.1.4 Over-the-counter anti-inflammatory drugs and their effects on wound healing
In some cases, perpetuation of inflammatory response is undesirable as it can cause serious damage to organs or excessive scarring of the afflicted site, and relying on the body’s anti-inflammatory factors is not enough Thus, demand the use of anti- inflammatory medications to suppress the inflammatory responses Commonly, nonsteroidal anti-inflammatory drugs (NSAIDs) are clinically used to counteract with the inflammation process [9] Available over-the-counter NSAIDs such as aspirin, ibuprofen, and naproxen are widely distributed and can be found in many local drugstores These drugs can help alleviate the symptoms of inflammation by inhibiting pro-inflammatory mediators such as prostaglandins and thromboxane Specifically, NSAIDs inhibit the activity of Cyclooxygenase (COX) enzymes, which is involved in the generation of pro-inflammatory mediators such as thromboxane and prostaglandins There are two types of isozymes of COX, COX-1 and COX-2 [7] While, COX-1 aids the secretion of natural mucus lining that act as a protective barrier for the inner stomach, COX-2 is generally unexpressed in most cells in normal conditions [20] However, during inflammation, the levels of COX-
2 are elevated in cells where prostaglandin is upregulated Unselective inhibition of both COX-1, and COX2 may result in reduced levels of protective prostaglandins in the stomach, leaving it vulnerable to being irritated by acidic molecules in gastric fluids This leads to common gastrointestinal problems that are commonly encountered when using NSAIDs such as nausea, vomiting, indigestion, gastric ulcers, stomach bleeding, and diarrhea Generally, NSAIDs is divided into two categories: unselective and COX-2 selective to avoid causing adverse effect to gastric compartment However, most of the available NSAIDs are non-selective and actively inhibit both COX-1 and COX-2 that aside from inhibiting inflammation, also inhibit platelet aggregation and increase risks of developing stomach ulcers and stomach bleeding [21]
1.1.5 Natural extracts and their anti-inflammatory properties
As already mentioned, the use of NSAIDs only result in symptomatic relief, yet also bring about side effects, and health risks Moreover, NSAIDs are chemical composition also contain complex organic compounds that are costly to produce As a result, recent trends in anti-inflammatory medications have now shifted attention to natural extracts since phytochemicals from plants also possess anti-inflammatory properties [17, 18] In the last few years, many studies have been conducted to investigate the anti-inflammatory effect of various types of plant extract from many species of vegetation and have shown decent results in inhibiting the inflammatory responses However, these herbal extracts are often vulnerable to external stimuli such as heat, moisture, pH of the environment, and can readily be consumed by enzymes in the human body [19] For that reason, a delivery system is needed for the administration of bioactive substances to avoid the degradation of the active compounds in the extracts
1.1.6 Betel leaf extract as a therapeutic agent to alleviate inflammation in wounds
The betel plant is a type of climbing vine belonging to the family Piperaceae, which also includes plant species such as pepper and kava Piper betle L is native to South Asian and Southeast Asian regions Betel has glossy heart-shaped leaves that are widely used in traditional remedies The cultivation and use of betel plants play an important role in various cultural, social, and religious occasions in many Asian countries [20]
Carrier system and their relevance in delivering natural extracts
1.2.1.1 Concept and properties of nanoparticles
In the scope of drug delivery, the problem of targeted and controlled delivery of therapeutic agent is a major obstacle in treatment of diseases The conventional use of drug is often limited by safety dosage, poor bioavailability, and poor selectivity [34] Therefore, to address these problems, the research community has extensively studied the use drug delivery system, including nanoparticles Nanoparticles are materials that have dimension in the range of 1 to 100nm, which grant them unique characteristics compared to their bulk counterparts [35, 36] As a drug delivery system, nanoparticles possess many unique physicochemical and
15 biological properties such as the ability to deliver a wide variety of therapeutic agents, rapid cellular uptake, ability to traverse biological barriers, tunable degradation rate and release rate of encapsulated content [36] Moreover, surface modifications of nanoparticle allow targeted drug delivery and controlled release in response to environmental stimuli (temperature, pH, osmolality, and enzymatic activity) [34] Nonetheless, nanoparticles intended for medical applications must also meet several requirements such as biocompatibility, non-cytotoxicity
1.2.1.2 Preparation-methods and application in delivering natural extracts
There are various methods for the synthesis of nanoparticles as shown in
Figure 1.6 For the specific aim of delivering phytochemicals in natural extract, liposomes, micelles, and polymeric nanoparticles have been synthesized as nanocarrier system The encapsulation of natural extract into nanocarriers offer many advantages, such as protection against external stimuli and enzymatic activity, enhanced bioavailability, improved endocytosis, and controlled release of active compounds [37] The primary methods for fabrication of polymeric nanocarriers consist of: ionic gelation (coacervation), nanoprecipitation (solvent displacement), solvent evaporation, and spray drying
Figure 1.6 Methods for synthesis of nanoparticles [37]
Ionic gelation is the simplest method for fabrication of nanoparticles The principle of this method involves the formation of coacervate phase from a mix of polyelectrolytes, encapsulating the extract molecules within the newly-formed matrices This synthesis method can be influenced by different factors such as polymer concentration, polymer ratio, ionic strength, and pH [37] Generally, the size of particles synthesized by this method is between 100 to 600 nm
Nanoprecipitation of solvent displacement involves the precipitation of polymers from an organic solvent and the distribution of this solvent into an aqueous phase with or without a stabilizer In this method, the ingredients including polymers, active compounds, and surfactant are firstly dissolved in a semi-polar solvent that is miscible with water such as ethanol or acetone The mixture is then introduced to an aqueous solution with surfactant under constant stirring The formation of nanoparticles take place simultaneously due to rapid solvent diffusion
Subsequently, the solvent is evaporated under low pressure and the nanoparticles can be collected In this method, the addition rate of organic phase into the aqueous phase as well as polymer concentration can greatly impact the particle size and encapsulation efficiency [38, 39] This method is most relevant for delivering poorly soluble compounds
Solvent evaporation involves the preparation of an oil-in-water (o/w) emulsion to yield nanospheres Initially, organic polymers are dissolved a polar organic solvent, then mixed with the active compounds either by dispersion or dissolution In addition, an aqueous phase containing surfactant is also prepared, the organic solution is then emulsified into this aqueous phase Then ultrasonication or homogenization is employed to ensure uniform dispersion of the nanodroplets Following the evaporation of solvent, a nanoparticle suspension is obtained [37]
The spray drying method is widely applied in the pharmaceutical sector for converting liquid drug formulation into powder form This method employs pressure nozzles and rotary atomizers, which yield fine droplet via vibrating mesh technology For fabrication of extract loading nanoparticles, an emulsion between aqueous polymer solution and the polyphenol solution is produced with the help of an emulsifier such as Tween 80 The emulsion is then passed through a 0.45 μm syringe to prevent nozzle blockage during the spray drying process The resulting mixture is then sprayed within a temperature range of 30 to 80°C, under specific flowrates and pressures The formation of uniform droplets is facilitated by either air pressure or the vibration of the mesh These droplets are subsequently dried using hot air and collected as a fine powder via the use of an electrostatic particle collector [40]
1.2.2 Hydrogel as a macro-delivery system
1.2.2.1 Concept and properties of hydrogel
There are currently several challenges associated with biomedical applications, including tissue engineering, wound healing, and controlled drug delivery Addressing these challenges is essential to creating the best possible therapeutic conditions for human health One potential solution being proposed is the use of hydrogels for these applications
Hydrogels are polymer chains that are cross-linked to form a 3D network, and they can absorb large amounts of water because they contain hydrophilic groups such as -NH2, -COOH, -OH, -CONH2, -CONH, and -SO3H [17, 41-43] Hydrogels can be produced in a variety of physical forms, including coatings, microparticles, nanoparticles, slabs, and films [43] Hydrogels have biomimetic properties, such as softness, flexibility, biocompatibility, biodegradability, remarkable absorption capacity, and tunable mechanical properties, which distinguish them from other nanomaterials (e.g., nanoparticles, nanofibers, thin films, etc.) and make them particularly suitable for certain biomedical applications [44] However, there are some limitations to their use, such as low tensile strength, which can lead to early drug release before the medication reaches the target location The hydrophilic polymeric core may not be the best place to retain incompatible hydrophobic medicines, and this can be a challenge for drug delivery [43] Physical properties of hydrogels can be tailored to produce a solid, semi-solid, or liquid substance by adjusting their physicochemical properties and crosslinking reaction Therefore, hydrogels have solid-like properties, such as infinite viscosity, a defined shape, and modulus, as well as liquid-like properties, allowing solutes to diffuse freely, as long as the size of the solute is not larger than the average mesh size (the distance between crosslinks) [45]
By modifying the hydrophilic and hydrophobic ratios, initiator or polymer amounts, reaction circumstances, and hydrogel properties (such as swelling- deswelling rate, stiffness, mesh size, and degradability), it is possible to alter these
19 properties (such as time, temperature, container, etc.) Swelling behavior, in particular, is a feature of hydrogels that makes them appropriate for drug delivery Hydrogels, when came into contact with water can absorb it in great amount Thus, results in an expanded and softened structure, becoming supple and loose and displaying no interfacial tension with either water or bodily fluids The swelling degree of hydrogels is controlled directly by the penetration of water into the hydrogel network through diffusion This process includes three stages: The first stage is called as primary bonded water, in which water molecules connect to hydrophilic groups present in the polymer network In the following stage, intermediate water – during this stage, water molecules interact with existing hydrophobic groups Lastly, free water state – after the diffusion reached equilibrium, the voids in the crosslinked network are filled with water Degree of crosslinking and polymer concentration can influent the swelling rate of hydrogels, as water penetrating the polymer network, causing the hydrogel to swell, while the crosslinks help the material to hold its shape Eventually, these opposing forces will reach an equilibrium state, at which the structure will not increase further in size Hydrogels with higher crosslinking degree tend to swell up less and also show an increased brittleness [46]
The categorization of hydrogels is dependent on various characteristics, as depicted in Figure 1.5, these includes several features such as their source, composition, environmental stimuli, crosslinking, property, configuration, and ionic charge [42, 46]:
Figure 1.7 Classification of hydrogels based on several features [46]
This classification is based on origin of ingredients used for hydrogel synthesis For instance, hydrogel based on cellulose, chitosan, gelatin, agarose, and collagen are classified as natural hydrogel While synthetic hydrogels that have artificial polymer constituents like polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene oxide (PEO), and polyacrylamide (PAAM) The last category is semi-synthetic hydrogels, which are made up of both biopolymer and synthetic polymer
Based on the composition of the polymer
Hydrogels can be classified into four groups: homopolymers, copolymers, semi-interpenetrating networks (semi-IPN), and interpenetrating networks (IPN), as shown in Figure 1.6 Homopolymer hydrogels are made from a single type of monomer, while copolymer hydrogels are made from two or more types of monomers Depending on the order of monomer composition, copolymers can be categorized as block, alternative, or random copolymers The key difference between homopolymer and copolymer hydrogels is that homopolymer hydrogels only contain one type of polymer chain, while copolymer hydrogels contain multiple
Research status on chitosan-based hydrogel composite delivery system 30
Due to the frailty of CS/GP hydrogel system, many studies have been carried out with the purpose of enhancing the mechanical performance of this system The hydrogel composite system containing chitosan and gelatin have been widely studied in the research community in recent years In 2018, Cheng et al reported a thermosensitive chitosan-gelatin-based hydrogel system containing curcumin- loaded nanoparticle and latanoprost as topic eye drop formulation The results show that the hydrogel composite is capable of achieving gelation at 34°C in around 81.196 ± 3.581 seconds Additionally, the cumulative release of curcumin loaded nanoparticles and latanoprost after 7 days are 7.13 ± 1.12 %, and 23.63 ± 2.41 %, respectively[69] In another research published in 2020, an injectable thermosensitive chitosan/gelatin-based hydrogel carrying erythropoietin (EPO) was reported The gelation time in the chitosan/gelatin was reported to be 110 ± 15s at pH 7.0 and the cumulative release of EPO reached 94% after 15 days [70]
The concept of employing chitosan-based nanoparticles and chitosan hydrogel as a carrying system of natural extracts has been extensively studied in the research community However, the combination of both materials is rarely reported, with most articles report about the use of chitosan hydrogel to deliver inorganic nanoparticles such as silver or metal oxides A summary of existing research about employing hydrogel as a delivery system of extract chitosan nanoparticles loading drug or natural extracts is shown in Table 1.3
In summary, with the lack research about thermosensitive CS/GP/GEL hydrogel composite carrying BLE-loaded chitosan nanoparticles, and with the recent trend in utilizing natural extract in hydrogel The synthesis of a novel thermosensitive hydrogel composite incorporating Piper Betle L extract-loaded nanoparticles represents a captivating and innovative area of research The content for this study includes studying the extraction of betel leaf, quantification of bioactive content, and synthesis of thermosensitive chitosan-gelatin hydrogel composite, as well as in situ loading of betel extract-loaded nanoparticles Followed by studying the effect of gelatin and co-gelling agent-NaHCO3 on physicochemical properties of the hydrogel and cumulative release of betel leaf extract In addition, characterizations of the material such as morphology and chemical constituents, mechanical performance are also included
Table 1.3 Summary of research combining hydrogel with nanoparticles
Loading type Key points Application Ref
Solanum nigrum L leaf extract Chitosan Alginate
15 nm -Zeta potential: -31.5 ± 2.4 mV -EE: 91.6 % -Cumulative release of extract: 12% after 6 days
-Alginate Chitosan In situ loading
-Particles size: 252 nm -Zeta potential: 36.3 ±
28 days -Complete release of berberine after 264 hours
Carbopol Chitosan In situ loading
-Particles size: 676 ± 2.76 nm -PDI: 0.465 -Hydrogel swelling ratio: 51 ± 3.7%
-Hydrogel degradation after 3-4 weeks -Cumulative release of extract: 44% after 17 hours
Dexamethasone sodium phosphate Silk fibroin Chitosan In situ loading
-Particles size: 488.05 ± 38.69 nm -PDI: 0.15 ± 0.07 -Zeta potential: 32.12 ± 2.42 mV -EE: 67.6 ± 6.7%; LC:
100-150 nm -Zeta potential: 20 mV -Complete release of encapsulated drug after 24 hours
EXPERIMENT
Research objective and contents
Synthesis of thermosensitive hydrogel composites from chitosan and gelatin delivering chitosan nanoparticles loaded with betel leaf extract
Content 1: Extraction and quantification of bioactive compounds in ethanolic betel leaf extract
Content 2: Investigating the influence of initial extract loading concentration on the properties of betel extract loaded chitosan nanoparticles
Content 3: Fabrication and characterization of thermosensitive hydrogel composites
Content 4: Investigating the influence of synthesis conditions on the properties of the chitosan/gelatin hydrogel composites encapsulating the as- fabricated nanoparticles to deliver betel leaf extract
Content 5: Evaluating in vitro release kinetics and biodegradability of the synthesized chitosan/gelatin hydrogel composites
Materials, equipment, and instrumentations
High molecular weight chitosan (DA>80%, 324 kDa ,1000-1500 cP) was supplied by Vietnam Food β-Glycerophosphate disodium salt, Folin-Ciocalteu reagent, gallic acid, disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, hydrated aluminum trichloride, and sodium nitrite were purchased from Sigma-Aldrich (USA) Gelatin, acetic acid, lactic acid, dimethyl sulfoxide (DMSO), methanol, sodium carbonate, and sodium bicarbonate were purchased from suppliers in China Absolute ethanol (99.5%) was purchase from
36 local supplier Rutin was purchased from Institute of Drug Quality Control Ho Chi Minh City
Fresh betel leaves cultivated in Ba Diem commune, Hoc Mon province and were harvested in March 2024 No pretreatment steps were carried out except for rinsing with water to remove dirt and contaminants.
Extraction and determination of bioactive content in betel leaf ethanolic
The extraction of betel leaf phytochemicals was carried out using ultrasound- assisted extraction method Briefly, an appropriate amount of dried betel leaf powder was dispersed in ethanol 80% (v/v) with a solid-liquid ratio of 1:10 The mixture was then sonicated using a probe ultrasonicator (UP400St, Hielscher) The solid-liquid mixture was placed in an ice bath for temperature control The extracting temperature was kept around 40-45℃ After a predetermined period of time, the extract solution was then separated from solid material via vacuum filtration The filtration was performed two times to remove solid residue The obtained extract solution was concentrated using a rotary evaporator (Yamato RE-301-CW) The dried extract moisture content was analyzed using a moisture analyzer (Yoke DSH-10A) Dried betel leaf extract was stored in a freezer prior to quantification of active ingredients
2.3.2 Determination of total flavonoid and total polyphenolic content in BLE
2.3.2.1 Determination of total flavonoid content (TFC)
Total flavonoid content was measured by aluminum chloride colorimetric assay developed by Zhisen, Mencheng, and Jiangming (1999) [76] with some modifications [77, 78]
The biological sample was prepared by dissolving dry extract in methanol and further dilute to appropriate concentrations using methanol Flavonoid colorimetric assay was performed by mixing 200àl of sample with 800àl RO water, followed by 60àl of 5% NaNO2 and 60àl AlCl3 10% The mixture was then left to react in dark conditions After 5 minutes, 400àl of NaOH 1M was added, and the total volume of the solution is made up to 3ml using RO water The experiment was repeated three times to ensure an error < 5%
Rutin was used as a reference standard, from which a calibration curve was constructed accordingly TFC of betel leaf extract is calculated as follows:
TFC (mg RE/g extract): Total flavonoid content
RE (àg RE/ml sample): Rutin equivalent
V sample (ml): Total volume of biological sample m sample (àg): Mass of dissolved extract
Figure 2.2 Aluminum chloride colorimetric assay
2.1.1.1 Determination of total polyphenolic content (TPC)
Determination of the total phenolic content is carried out according to the Folin-Ciocalteu (FC) method proposed by Singleton (1999) [79] The principle of
FC analysis is based on electron transfer, when the heteropolyphosphotungstates- molybdates complexes are exposed to oxidant species, reduction of phosphomolybdate occurs and result in products with blue color The presence of several phenolic species can lead to very broad absorption peaks and since other components in biological sample do not absorb at 760nm Therefore, this particular wavelength is generally chosen for FC method
The FC method for determination of TPC in ethanolic extract of betel leaf is briefly described as follows: a biological sample was prepared by dissolving a small amount of betel extract in DMSO, the solution is then diluted to appropriate concentrations solution using distilled water A small volume of biological sample (40 àl) was added into a glass vial via the use of a micropipette, and mixed with 200àl of FC reagent and put in an ultrasonic bath sonicator for 5 minutes After sonication, 600àl of 20% sodium carbonate solution is added into the solution, followed by another sonication for 30 minutes Finally, 3160àl of distilled water is added to the sample Absorbance measurement was carried out using a UV-vis spectrophotometer at 760nm The reaction is kept in dark and repeated three times to ensure an error of 7), due to the basic nature of NaHCO3 and the formation of CO2 that contribute to increasing pH of the solution [64, 65]
However, raising the concentration of gelatin leads to the contradictory effect compared to that of sodium bicarbonate The reason for this phenomenon is that COO - groups from gelatin can react with NH3+ groups in chitosan [95], competing with other gelling agents and possibly acting as a barrier preventing CS chains from aggregation Furthermore, since gelatin solution was prepared using lactic acid, the acidic nature of the solution evident by decreasing pH, therefore increase the solubility of chitosan, hindering gel formation Likewise, higher polymer concentration also results in a higher density of intermolecular hydrogen bonding between CS and GEL [94], requiring more energy to break up these bonds and trigger gelation Consequently, a longer gelation time and higher sol-gel transition temperature are observed in hydrogel samples with higher gelatin concentration
The SEM images display uneven pore size and random interconnected porous network In Figure 3.6, hydrogel composites display increase brittleness with increasing NaHCO3 concentration as evident in the crosslink density of the polymer network However, excessive addition of this gelling agent also results in collapsed network due to high brittleness as displayed in Figure 3.6C On the other hand, Figure 3.7 demonstrates that increasing gelatin content does not show any significant alteration in the morphology of hydrogel composites The average pore size of all hydrogel composites was determined using ImageJ software The results are shown in Table 3.4, it is evident that at a gelatin concentration of 0.25% gelatin
67 and sodium bicarbonate content of 0.075M, the hydrogel have greatest average pore size among all samples
Figure 3.6 SEM images of hydrogel composites with various NaHCO 3 concentration: A) 0.05M, B) 0.075M, C) 0.1M
Figure 3.7 SEM images of hydrogel composites with various gelatin concentration: A) 0.2%, B) 0.25%, C) 0.3%, D) 0.35%, E) 0.4%
Table 3.2 Average pore size of different hydrogel composites
The swelling ratio is an important parameter in hydrogel fabrication as its not only influent the structure integrity of the material, but also affect the release of encapsulated substances from the matrix Swelling degree also imply the crosslinking density and microporous nature of hydrogel
As illustrated in Figure 3.8, an increased NaHCO3 concentration contribute to a more swellable hydrogel As mentioned above, CO2 is a byproduct generated in the process of hydrogel synthesis, forming tiny bubbles inside the hydrogel solution that will eventually burst after gelation Therefore, leaving vacant spaces (micropores) inside the polymer structure The more crosslinker added into the hydrogel solution, the more CO2 bubbles were generated, thus leaving more pore sites in the gel structure [64] When immersed in PBS, the solvent can easily fill up the pores and causing polymer chains to swell up
Figure 3.8 Effect bicarbonate on swelling ratio of hydrogel composites of: A) gelatin and B) sodium bicarbonate
Gelatin is a hydrophilic polymer that can absorb water and swell [96] Consequently, the addition of gelatin into hydrogels can increase swelling ratio As shown in Figure 3.6, when gelatin concentration is increased from 0.2 to 0.3%, the hydrogels swell up more, from 192.92% to 258.51% at 37°C and from 268.77% to 324.33% at 39°C Nevertheless, as the gelatin concentration reached 0.35 and
70 higher, swelling ratio starts to decline, with swelling ratio of 0.4% gelatin sample only reached 218.41% and 249.82% at 37° and 39°C, respectively This is due to more crosslinked polymer network between gelatin and chitosan Additionally, amino groups present in both chitosan and gelatin structure can easily be protonated in acidic condition [97] In this case, the hydrogel composites were immersed in PBS pH 6.5, which causes protonation of amino groups, resulting in an increased hydrogen bonds density As a result, higher gelatin content brings about more hydrogen bonds in the polymer network that reduces swelling
At higher temperature, all samples swelled up even more due to the breaking of hydrogen bonds between polymeric components in the hydrogel structure The swelling degree at 39°C also shows the same trend compared to the results at physiological temperature
The addition of NaHCO3 as a co-gelling agent along with GP also helps enhancing the mechanical performance of CS/GP/GEL hydrogel Sodium bicarbonate not only actively neutralizes NH3+ groups to NH2 groups, reducing repulsion force between CS molecules, but also increase the crystallinity of the system, resulting in a stiffer gel Figure 3.9 shows that a moderate addition of sodium bicarbonate at a concentration of 0.075M is most appropriate for reinforcing the hydrogel composite While, other samples show inferior mechanical performance, and suffered plastic deformation to their structure before reaching the deformation target at 70% strain The hydrogel with 0.075M NaHCO3 reached the 70% deformation target without compromising its structural integrity with peak load at the target strain was around 30kPa
Figure 3.9 Effect of sodium bicarbonate concentration on mechanical performance of hydrogel composites
In this research, gelatin was used as a reinforcing agent to resolve the frailty of thermosensitive CS/GP hydrogel As mentioned in section 3.3.1, higher gelatin content results in higher crosslinking density in the hydrogel structure Thus, increasing the gelatin concentration means the polymer network becomes more brittle due to more crosslinking As demonstrated in Figure 3.8, a gelatin concentration of 0.25% brings about the most compression-resistant hydrogel among all samples At the concentration of 0.2%, the content of gelatin was still too low to reinforce the hydrogel structure However, higher gelatin content (>0.25%) also results in stiffer and more brittle gel that suffered from plastic deformation, breaking the hydrogel structure Additionally, Young’s modulus values of different hydrogel composites are shown in Table 3.3, ranging from 3.18 to as high as 12.72 kPa This result indicates that the hydrogel composite with 0.075M NaHCO3 and 0.25% gelatin demonstrates appropriate flexibility and durability, ideal for wound dressing applications
Figure 3.10 Effect of gelatin concentration on mechanical performance of hydrogel composites Table 3.3 Young’s modulus of different hydrogel composites
3.3.2 Effect of NCS-BLE loading on hydrogel properties
3.3.2.1 Sol-gel behavior of hydrogel solution
Inclusion of NCS-BLE into the hydrogel composites also affects the sol-gel behavior as shown in Figure 3.11 When the initial BLE concentration in NCS is at 2000ppm, the hydrogel composites transform into gel at 38°C However, as the BLE concentration increases, gelation temperature decreases With an increase in BLE concentration, CS and STPP concentration also increase as mentioned in Table 2.1 Thus, introducing more polymer constituent to the hydrogel system, and possibly unreacted ions from STPP Consequently, these molecules can participate in reinforcing the hydrogel network via hydrogen bonding or electrostatic crosslinking [72, 73] Additionally, charged amino groups in the structure of chitosan can also hinder gelatin by keeping the chitosan molecules soluble [48], requiring more time to achieve gelation [98] Therefore, as the BLE concentration loaded into the nanocarriers increase, the gelation temperature decreases This result also corresponds with the explanations about increased positive charges neutralization in section 2.3.1
Figure 3.11 Effect of chitosan nanoparticles loaded with different BLE concentrations on gelation temperature
The effect of extract-loaded nanoparticles on the swelling ratio of the hydrogel composites is illustrated in Figure 3.12 As mentioned earlier, the addition of chitosan nanoparticles into hydrogel leads to an increase in the number of functional groups, especially amino groups In acidic medium such as PBS pH 6.5, these amino groups can become charged groups, thereby increasing intermolecular hydrogen bonding within the system For that reason, the swelling ratio of hydrogel composites loading NCS-BLE is lower than those without the nanoparticles On the other hand, nanoparticles have high surface to volume ratio that make them more absorbent, due to more rapid water penetration [99] Nevertheless, this effect is only observable at a BLE concentration lower than 3500ppm, as higher BLE content leads to reduction in swelling ratio
At 39°C, the hydrogel composites exhibit higher swelling ratio from 169.75 to 297.10%, compared to 118.13 to 192.38% at physiological temperature The reason behind higher swelling ratio at higher temperature also attribute to the same explanation stated in section 3.3.1.3 This result is comparable to that of chitosan- alginate hydrogel composite encapsulating berberine-loaded chitosan nanoparticles, which also reported a swelling ratio of around 225% [72]
Figure 3.12 Effect of BLE-loaded chitosan nanoparticles on swelling ratio of hydrogel composites
Cumulative release of BLE from hydrogel/nanoparticle matrix
The cumulative release profile of flavonoid from chitosan-gelatin hydrogel composites was obtained PBS pH 6.5 containing lysozyme at 39°C The release profile of hydrogel composites carrying NCS-BLE with different BLE concentration is illustrated in Figure 3.17 Initially, a burst release phenomenon occurs in all hydrogel composites within the first 15 minutes This is due to high crosslinking density in the hydrogel network, shrinking the polymer network and expel water along with some nanoparticles and free extract molecules Therefore, as the chitosan concentration increases, the hydrogel structure can shrink even more and expel more water and flavonoid compounds [94] After the initial burst, the release profile of flavonoid followed a gradual release from the hydrogel composites as the matrix is slowly eroded by lysozyme After 3h, the release rate of most
CGNBE1CGNBE2CGNBE3CGNBE4CGNBE5
80 hydrogel samples has reached equilibrium, excluding the hydrogel composite with 800ppm BLE The cumulative flavonoid release percentage of hydrogel composites with 400 to 800ppm BLE after 3h is 28.37%, 34.73%, 42.43%, and 57.71%, 60.54%, respectively
Figure 3.17 Cumulative flavonoid release profile of hydrogel composites carrying chitosan nanoparticles loaded with different BLE concentration
3.5 Release kinetics and release mechanism of BLE from hydrogel/nanoparticle matrix
The release data of hydrogel composites was fitted to different drug release models in order to study the release kinetics of betel leaf extract from the hydrogel matrix Table 3.4 shows the drug release models used in this study and their parameters, as well as adjusted coefficient of determination (R 2 adj)
CGNBE1CGNBE2CGNBE3CGNBE4CGNBE5
Table 3.4 Parameters and adjusted R 2 value of drug release models
Model Parameter CGNBE1 CGNBE2 CGNBE3 CGNBE4 CGNBE5
From the adjusted R 2 values demonstrated in the table above, the most fitting release models are the Korsmeyer-Peppas and Peppas-Sahlin Comparison between actual release data and predicted data from the Korsmeyer-Peppas model is shown in Figure 3.18
Figure 3.18 Cumulative release data fitted to Korsmeyer-Peppas release model
The release mechanisms of BLE can be determined via the release exponent n in the Korsmeyer-Peppas model For cylindrical matrix, the release mechanisms can be identified as Fickian diffusion when n = 0.45, non-Fickian diffusion when 0.45 < n< 1, and super case II transport when n > 1 [102] As shown in Table 3.4, the values of release exponent n of all hydrogel composites are smaller than 0.45, implying that the release mechanism is Fickian [103] or pseudo-Fickian diffusion [104] Comparing to other research on drug release kinetics from chitosan composite scaffold, Hadjianfar et al studied the release of 5-fluorouracil from Polycaprolactone/chitosan matrix and reported n values ranging from 0.136-0.370 [103] Another research involves the release of tetracycline hydrochloride from poly(ω-pentadecalactone-co-ε-caprolactone)/gelatin/chitosan ternary nanofibers recorded small n values (0.245-0.268) Additionally, the author also concluded that the release mechanism is pseudo-Fickian diffuision [104] Pseudo-Fickian diffusion refers to deviation from ideal Fickian diffusion and account for other release mechanisms such as relaxation or erosion of the polymer network [105, 106] Since the release medium used in the release experiment includes PBS and lysozyme, it is
CGNBE1CGNBE2CGNBE3CGNBE4CGNBE5
83 likely that relaxation and erosion mechanisms have significant effect on the release profile of BLE Further study on the effect of other release mechanisms on the release profile of BLE can be done using the Peppas-Sahlin release model [107]
In the Peppas-Sahlin model, the release kinetics is described according to the following equation:
The first term of the right side of the equation represents the Fickian diffusional contribution, denoted as F, whereas the second term, denoted as R is attributed to Case II (non-Fickian) relaxational contribution [107] The exponent m in this model is the diffusional exponent, similar to exponent n in the Korsmeyer- Peppas model In an ideal scenario, in which the relaxational mechanism is negligible and Fickian diffusion is the main driving force of the release process, then m should be equal to n = 0.45 [108] However, all of the m values shown in Table
3.4 are < 0.45, which suggest that the release mechanism of BLE also depend on relaxational contribution In order to determine the main driving force of the release process, a ratio between R and F can be used to discover which is the more dominant between the two contributions The ratio of both contributions is represented as follows:
If the R/F ratio is > 1, then relaxation is the dominant release mechanism Inversely, when R/F