Synthesis and characterization of starch-g-polyacrylamide-co-polylactic acid hydrogel for the potential wound dressing application

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Ibrahim H. Mondal, H. Jahan Kadri, Firoz Ahmed, Md. Hasinur Rahman This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4946153/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Jun, 2025 Read the published version in Polymer Bulletin → Version 1 posted 12 You are reading this latest preprint version Abstract A starch-based hydrogel was synthesised by direct grafting of polylactic acid (PLA) and acrylamide onto starch. Potassium persulphate (K 2 S 2 O 8 ) was used as an initiator and citric acid (CA) was used as an eco-friendly cross-linker. The purpose of the graft was to test an alternative anti-microbial wound dressing material. The FTIR, XRD, SEM and EDX data confirmed that the copolymerisation and cross-linking of the hydrogel was successful. Tests, with chemical reactions; yielded TGA data, which confirmed the enhanced thermal and mechanical properties of the augmented hydrogel. The hydrogel swelled up to 481% at pH 7.20 and exhibited a water vapour transmission rate of 148 g/m 2 per day. The hydrogel also showed anti-microbial activity against both gram-positive ( S. aureus) and gram-negative ( E. coli) bacteria. Its zone of inhibition was 21 mm and 19 mm with a mild anti-oxidant property. This synthesised hydrogel is completely non-toxic and bio-degradable, making it especially suitable as a wound dressing material. Starch Hydrogel Anti-bacterial Biodegradable Anti-oxidant Wound dressing cell viability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Hydrogels, based on biopolymers, have been getting greater attention since the discovery of a ‘super absorbent’ polymer in the 1950s. Over the past seven decades, researchers from academic institutions and businesses have focused on the industrial uses of these hydrogels [ 1 ]. A variety of potential applications, including pharmaceuticals, sanitary napkins, biomedicine, tissue engineering, agriculture, veterinary medicine and waste water treatment, was quickly explored [ 2 – 4 ]. The need for hydrogels which have three-dimensional (3-D) structures, physically or chemically, with partly cross-linked polymeric networks, is apparent. Such hydrogels have a good capacity for absorption of water, as well as other biological fluids and saline solutions. Using a range of copolymers and cross-linkers, the physical and chemical properties of the hydrogels can be easily customised. The cross-linked structure of the hydrogel network protects it from dissolution. Munim and Raja [ 5 ] reported that the presence of amine, hydroxyl, amide and sulfate groups of polysaccharides increases the water absorption tendency of hydrogels. Biopolymer-based hydrogels are appealing because they are readily available naturally, inexpensive, biodegradable, non-toxic and have desired physico-chemical properties. These features set them apart from petroleum-based synthetic counterparts, which pollute the environment [ 6 – 10 ]. Hydrogel dressings in the form of a gel or film can absorb exudates from a wound surface and maintain a moist environment. Translucent and flexible hydrogel dressings are easy to remove from wounds. Exudate removal from the wound surface, moisture control, gas transport, infection prevention, biocompatible, biodegradable, cost-effective, non-toxic, pain relief, wound necrosis reduction, low skin adhesion and mechanical stability are all features of an ideal wound dressing material [ 11 ]. Starch is one of the most abundant and cheap polysaccharides. It features inherent biodegradability and renewability, it is available in nature and it is an energy source obtained from plants that contain cellulose. Starch in cereal grains, such as corn, wheat, rice, barley, rye, oats, buckwheat, roots (potatoes, sweet potatoes and cassava), sago, stems and seeds/ legumes (lentils, beans and peas), are valuable for human nutrition. Starch consists of two types of anhydro-glucose units: amylose (α-1, 4-linkage), which is linear, and amylopectin (α-1, 6-linkage), which is branched. These are joined by an α-D-glycosidic bond. Amylopectin and amylose, the major components of starch both include glucose. Therefore, starch is classified as a homo-polymer of glucose units but it is a hetero-polymer of amylose and amylopectin. Starch is a naturally semi-crystalline biopolymer. Amylose usually constitutes around 20–30% of a granule of starch and amylopectin constitutes around 70–80% of it. The molecular formula of starch is (C 6 H 10 O 5 ) n . Different starches contain different percentages of amylose and amylopectin. Soluble starch contains 30% amylose and 70% amylopectin [ 12 – 19 ]. Polylactic acid (PLA) is a naturally occurring polymer PLA has proved notable for its biodegradability, biocompatibility, lack of toxicity and ability to be thermally-processed. A thermoplastic aliphatic polyester, made from agricultural waste, PLA is environmentally-friendly. PLA degrades quickly and safely on human skin. So, PLA is a prime candidate for applications such as drug delivery systems, sutures and clips for biomedical applications. PLA has also proved suitable for pharmaceuticals use in food packaging [ 20 – 22 ]. Torres et. al., [ 23 ] synthesised a novel hydrogel of poly (acrylamide) (PAAm) and starch at different ratios. They found that their hydrogel was a potential platform for controlled release of amoxicillin. The hydrogel, loaded with amoxicillin, was tested by disc diffusion against Escherichia coli ATCC-25922, Staphylococcus aureus ATCC-25923 and a carbapenemase producer, Pseudomonas aeruginosa . These hydrogels proved to be powerful bacteria growth inhibitors when loaded with amoxicillin in this fashion. Thus, they were found suitable for biomedical applications. However, the hydrogels were not antimicrobial without antibiotics. They also exhibited an improved rate of water uptake with increasing starch content but not at a super-absorbent level [ 23 ]. Pal et. al., [ 24 ], prepared corn-starch-based hydrogel membranes by cross-linking them with polyvinyl alcohol. The membranes had sufficient mechanical strength and water-retention capacity but no anti-microbial capacity. At the same time, they also developed another novel hydrogel by crosslinking polyvinyl alcohol with starch suspension, using glutaraldehyde as a crosslinking agent. The membrane had sufficient strength to be used as artificial skin but water absorption and retention properties were not reported by the authors [ 24 , 25 ]. Pattra et.al. prepared starch-based super water absorbent (SWA) hydrogel and validated the practical benefits of SWA for agricultural applications. SWA was successfully prepared in an up-scaling production process by radiation-induced graft polymerisation of acrylic acid onto cassava starch. Although SWA showed high swelling ability and was able to increase the survival rate of young rubber trees planted in an arid area by up to 40%, it did not show any anti-microbial activity [ 26 ]. The design and preparation of novel biodegradable hydrogels developed by the free radical polymerisation of acrylamide acrylic acid was reported. Some formulations with bis-acrylamide, in the presence of a corn starch/ethylene-co-vinyl alcohol copolymer blend (SEVA-C), were also reported. Although the hydrogel was highly bio-degradable and was appropriate for drug delivery, it showed low absorption capacity [ 27 ]. The graft co-polymerisation of polyacrylic acid and/or acrylamide on potato starch was reported by Al-Aidy and Amdeha [ 28 ]. This hydrogel was able to adsorb malachite green (MG) dye from the environment but the applicability of this hydrogel to medical application, as a super-absorbent, was not reported by the author, nor was its anti-microbial activity investigated. Sethi et. al., [ 17 ] synthesised a hydrogel, using a hybrid backbone of karaya gum starch and grafted with polyacrylic acid, which was tested as an oral drug delivery carrier. Paracetamol and aspirin were the test drugs. Due to its low tensile strength, this hydrogel was found not to be suitable for use as wound-dressing material, nor was it effective as an anti-microbial. Demitri et al., [ 29 ]. developed a novel, superabsorbent, cellulose-based hydrogel, cross-linked with citric acid. Although the hydrogel was bio-degradable, its anti-microbial activity and tensile strength were not reported. A hydrogel based on polyacrylamide, grafted to starch/clay nano-composite, was used for enhanced oil recovery, but its bio-medical applications were not investigated properly [ 30 ]. The hydrogel, made of cassava starch grafted to poly [Acrylamide-co-(Maleic Acid)] was found to be superabsorbent of γ-irradiation and used for packaging material [ 31 ]. Hydrogel prepared from the graft copolymerization of starch with acrylamide and 2-acrylamido-2-methylpropanesulfonic acid (AMPS) was responsive but its anti-microbial activity was not investigated [ 32 ]. Another hydrogel, prepared by graft co-polymerisation of ethyl acrylate/acrylamide on to corn starch, using potassium permanganate–citric acid initiation, was optimised but its characterisation was not reported properly [ 33 ]. The cellulose-based hydrogels were prepared by free radical graft copolymerisation reaction of cotton with acrylic acid (AA) and acrylamide (AM), using N,N-methylne-bis-acrylamide (MBA) as a crosslinker in the presence of potassium persulphate (K 2 S 2 O 8 ) as an initiator. These hydrogels’ optimisation was studied but their applications were not examined properly [ 34 ]. All these above-mentioned hydrogels have swelling, absorption, mechanical, anti-microbial, and diffusivity properties but the study of their applications was unidimensional. For this reason, it is necessary to study hydrogels now specifically to learn how they will function in wound healing. Therefore, here, potato starch-based hydrogel was developed through graft copolymerisation. Acrylamide and polylactic acid were grafted on to starch, using citric acid (CA) as a cross-linker, in the presence of potassium persulphate as a redox initiator. The goal was to develop a highly-porous, absorbent hydrogel, with high mechanical strength, which was also active against micro-organisms. The synthesised hydrogel of starch-g- (poly acrylamide-co-PLA) was characterised via various physico-chemical and instrumental analyses. The hydrogel was found to be useful complicated wound-healing management, as an advanced wound dressing. Experimental Materials Fresh potatoes ( Solanum tuberosum ) were collected from the local market in Rajshahi, Bangladesh. Polylactic acid (Uni-Chem, China), acrylamide (BDH, England), citric acid (Merck, India), 1,4- dioxane, (BDH, England), DPPH (2,2-diphenyl-1-picrylhydrazyl) (Sigma Aldrich), methanol (Merck, Germany), potassium persulphate (Merck, Germany), etc. of analytical grade chemicals were purchased and used in the experiments reported here. Extraction of potato starch and synthesis of hydrogel Starch was extracted from fresh potatoes. For this purpose, the peeled potatoes were blended and then the residual mass was removed by means of a cloth filter. The remains were again filtered using Whatman filter paper. The filtrate was kept in a beaker for about 12 hours, to allow it to settle down. Then the extracted starch was separated, using a centrifuge. Next, the resulting extracted starch was sun-dried and then crushed into a fine powder. This starch powder was then levelled and preserved in an air-tight container for further use [ 35 ]. The whole extraction process is represented in Fig. 1 . To summarise the process used, 1.0 g (2% w/v) of potato starch was gelatinised at 60 o C for one hour, with continuous stirring. Then the gelatinized starch was cooled to room temperature and poured into a three-neck round-bottom flask, equipped with a thermometer, a mechanical stirrer and a nitrogen line (Fig. 1 (b )). After that, acrylamide (300% w/w based on potato starch weight), potassium persulphate 0.2% and 1%, 10 ml of PLA solution, were added to it. The ratio of starch and acrylamide was 1: 3. PLA was dissolved in 1, 4-dioxane by heating at 50 o C for 30 minutes and cooled down to room temperature. Then dissolved crosslinking agent 2%, 10 ml CA was added to the solution and stirred. The reaction flask was then purged with nitrogen gas for 30 minutes, to remove any dissolved oxygen which might remain. After constant stirring for 30 minutes, the reaction continued for 4 hours at 50 o C, using a water bath to complete the polymerisation reaction. A transparent, sticky solution was then obtained. After completing the reaction, the prepared hydrogel was washed several times with distilled water, poured into a petri dish, cut into small pieces and dried in a drier for 3 days at 60 o C. The dried gel was ground and screened through a 200-mesh sieve. The resulting white granular product was then preserved in an air-tight container. Characterisation Swelling studies The swelling behaviour of the synthesised hydrogel was determined by treating it with distilled water and immersing it in solutions of different pH (pH = 1.2, 4.4, 7.4, 8.1, 10.02). The test samples were also immersed in organic solvent (ethanol and acetone). A known initial weight W i of the sample was immersed in 100 ml of solvent at room temperature for 48 hours. The samples were filtered and the excess solvent was removed with the help of filter paper. Then the final weight, W f, was determined. The percentage of swelling was calculated as per following Eq. (1) [ 16 ]. $$\:Swelling\:Percentage\:\left(\%\right)=\frac{{W}_{f}-{W}_{i}}{{W}_{i}}\times\:100\dots\:\dots\:\dots\:\dots\:\dots\:\dots\:\dots\:\left(1\right)$$ Where W i is the initial weight and W f is the final weight of the sample after swelling. Gel content The pre-weighted hydrogel sample was dried to a constant weight and then immersed in distilled water for 24 hours to remove the solid fraction. The swelled sample was taken out from the distilled water and dried to a constant weight in an oven. The gel content was calculated as Eq. (2) [ 36 , 37 ] $$\:Gel\:Content\:\left(\text{%}\right)=\frac{{\text{W}}_{\text{d}}}{{\text{W}}_{\text{i}}}\times\:100\dots\:\dots\:\dots\:\dots\:\dots\:\dots\:\dots\:\left(2\right)$$ Where W d is the weight of dry gel after extraction from water and W i is the initial weight of dry gel. Water vapour transmission rate test A small bottle was filled with 10 ml distilled water to assess the Water Vapour Transmission Rate (WVTR). The mouth of the bottle was covered with a hydrogel membrane and Teflon tape was used to keep it airtight. The bottle was weighed and kept at 40 o C in an oven. After 24 hours, the bottle was taken out from the oven and weighed again. The WVTR (g/m 2 per day) was determined using the Eq. (3) [ 38 ]. $$\:Water\:vapour\:transmission\:rate=({\text{W}}_{\text{i}}-{\text{W}}_{\text{t}})/\left(\text{A}\times\:24\right)\times\:{10}^{6}\dots\:\dots\:\dots\:\dots\:..\:(3)$$ where W i and W t are the masses of the bottle before and after heating, respectively, and A is the inner area of the round mouth of the bottle. The experiment was conducted at 40 o C, as the hydrogel would be used as a wound dressing on human skin, which has a temperature of 37.2 o C. Moisture retention capability The moisture content of the synthesised hydrogel was determined by a moisture analyzer (RADWAG, 26–600 Radom, Poland). The moisture retention capability was calculated according to the following Eq. (4) [ 39 ]. $$\:Moisture\:Retention\:Capability\:\left(\text{%}\right)=\:\frac{{\text{W}}_{1}-{\text{W}}_{2}}{{\text{W}}_{1}}\times\:100\dots\:\dots\:\dots\:\dots\:..\left(4\right)$$ where W 1 is the initial weight of the sample and W 2 is the weight of the dry sample after being heated at 40 o C for 8 hours. Antioxidant property The capability of synthesised hydrogel to scavenge free radical cations was assessed, using 1,1-diphenyl-2-picrylhydrazyl (DPPH). A stock solution of 0.004% (w/v) DPPH was prepared. using 50 ml methanol. and was kept in the dark. 1 ml of prepared test samples of different concentrations (5, 10, 15, 20, and 25 µg/mL) were taken, in test tubes and 3 ml of DPPH, in methanol solution, was added to each test tube. Then the test tubes were incubated at room temperature for 30 minutes, in a dark place, to allow the reaction to be completed. Then the absorbance of the solution was measured at 517 nm, using a spectrophotometer (T60 UV-Visible spectrophotometer, UK), against blank. The antiradical activity was measured as % scavenging of free radicals, by measuring the decrease in absorbance percentage compared to the control solution. The percent inhibition or scavenging activity was calculated [ 40 ] using the following Eq. (5). $$\:\text{S}\text{c}\text{a}\text{v}\text{e}\text{n}\text{g}\text{i}\text{n}\text{g}\:\text{A}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y},\:\text{I}\text{%}]=\frac{{\text{A}}_{0}-{\text{A}}_{1}}{{\text{A}}_{0}}\times\:100\dots\:\dots\:\dots\:\dots\:\dots\:\dots\:\dots\:(5)$$ where A 0 is the absorbance of the control and A 1 is the absorbance in the presence of hydrogel. Antibacterial property Antibacterial activity was measured by the agar well disc diffusion method reported by [ 39 , 41 ]. The antibacterial activity of the prepared hydrogel was tested against gram-positive bacteria Staphylococcus aureus and gram-negative bacteria Escherichia coli . The prepared nutrient agar (Hi media, India) medium was sterilised in an autoclave at 121ºC for 15 minutes. This medium was then transferred to sterilised Petri dishes in a laminar airflow chamber. The media solidified in 20 minutes and then the cultures of S. aureus and E. coli were spread on the media. Then the solid hydrogel sample was embedded in the agar well. Starch, polylactic acid, citric acid, and solvent 1,4-dioxane, and prepared hydrogel were loaded (10µl) on to a sterilised disc and the plates were incubated at 37º C, for 24 hours. After incubation, the antimicrobial activity was assessed in terms of the zone of inhibition. Cell viability test Cell viability of the control sample (blank culture media) was 91.87% ± 2.51. The experimental sample was approximately 1.5 x 0.9 (1 x w,cm) and approximately 50 mg. The experimental sample was placed in 10 mL of culture media. The result was obtained after 3 days of observation. Biodegradability test Starch has a tendency to degrade when buried in the soil, as it contains glycosidic linkages. To study in-soil degradation of the prepared hydrogel, grafted samples were weighed individually and buried in soil for 56 days. After that period, samples were withdrawn, carefully washed with distilled water and dried at 105ºC for 20 minutes. Then the samples were kept at room temperature for 24 hours before weighing. Finally, the weight loss of the degraded hydrogel was determined, using the following Eq. (6) [ 42 , 43 ]. $$\:Weight\:Loss,\:\left(\%\right)=\frac{{W}_{i}-{W}_{d}}{{W}_{i}}\times\:100\dots\:\dots\:\dots\:\dots\:\dots\:\dots\:\dots\:.\left(6\right)\:$$ where W i is the initial weight of hydrogel and W d is the weight of the degraded hydrogel after a period of burial. FTIR analysis The raw potato starch, acrylamide, citric acid, synthesized hydrogel, PLA and KBr (potassium bromide) were dried in an oven at 105 0 C, to remove all moisture. Then each starch, the synthesised hydrogel and PLA was mixed with dried KBr to make a powder, using a mortar pestle and pellet. Then the samples were analysed in an FTIR spectrotmeter (Model: FTIR- 8900, Shimadzu, Japan) They were found to be within 400-4000 cm -1 wavenumber. Scanning electron microscopy and Energy-dispersive X-ray analysis The surface morphology of extracted starch, PLA and the prepared hydrogel was scanned Electron Microscope (SEM) (JEOL, Model- JSM7600 F, Japan). The micrographs were taken at a magnification of 300 times and the scans were performed at 5.0 kV accelerating voltage. At the same time, Energy Dispersive X-Ray Analyses (EDX) were performed with the same instrument. The samples were subjected to an electron beam of 15 kV of energy-accelerating voltage and the counting rate was 305 cps. X-Ray diffraction analysis The crystallinities of extracted starch, PLA and prepared hydrogel were investigated by a PAN Analytical X Pert PRO X-ray diffractometer (Model: Empyrean, PAN Analytical, Netherland). Cu-Kα radiation of wavelength 1.5406 Å was used as the X-ray source. The scanning rate was 2 minutes with a scan angle from 5˚ to 50˚. Thermal analysis Thermal analyses of all samples were carried out with a Perkin-Elmer Simultaneous Thermal Analyzer (STA 8000, Germany). The tests were carried out in an inert nitrogen atmosphere.at a temperature of 30º C - 600º C. The heating rate and the airflow rate were 20 °C/min and 200 ml/minute, respectively. Results and Discussion Gel content The gel content of a hydrogel is important because it indicates the degree to which the hydrogel network is cross-linked. The hydrogel was submerged in distilled water for 24 hours. The polymer part, which was not cross-linked, was dissolved in water. The remaining part of the hydrogel is called the gel content. A higher gel content means a highly cross-linked as well as mechanically-strong hydrogel. The amount of gel in the prepared Starch/PLA hydrogel was 75% [ 38 ]. Swelling behavior In Fig. 2 , the dependence of swelling on the pH of the aqueous solution in which the hydrogel is placed is shown. The percentage of swelling of the prepared hydrogel was very low in acidic medium and increased with increased pH. At pH 7.4 (neutral) the DS increased sharply to 481%. Further increase of pH to alkaline caused swelling increase negligibly and level off. In acidic medium, the prepared hydrogel was unable to donate protons (protonation) due to common ion effects of hydrogen ions. Both intra- and inter-molecular hydrogen bonds were formed in the hydrogel network in acidic solution. The prepared hydrogels were therefore more compact such acidic solutions (pH 1.2–3.5), which inhibited their swelling. De-protonation began at pH 3.1 like the carboxyl group of citric acid [ 44 ]. This de-protonation allowed more water to enter into the hydrogel structure, which is the meaning of “swelling”. It is also clear that the swelling is both capillary- and diffusion-controlled (45). Water vapour transmission rate The hydrogel wound dressing should have the ability to minimise the fluid loss from the wound by maintaining a moist environment under the wound. Lower WVT is therefore a good characteristic for hydrogel to be used as wound dressing. [ 38 ] reported that the loss of water from second- and third-degree wounded skin is 178.55 ± 4.5 and 143.2 ± 4.5 g/m 2 per day, respectively. The prepared hydrogel had a water vapour transmission rate ( WVTR) of 148 g/m 2 h, which is within the range of water loss from wounded skin. Therefore, this prepared hydrogel will tend to keep wounds dry. Yet the moisture retention capability of the prepared hydrogel was 84.84% at 40⁰C. So, while keeping the wound from oozing fluid, the hydrogel will still maintain the moist environment which maximises healing of wounds, which is the ideal combination for a wound dressing. FTIR analysis The FTIR spectra of starch, acrylamide, PLA, citric acid and prepared hydrogel are presented in Fig. 3 . In Fig. 3 (a) , the characteristic FTIR spectrum of starch is represented. The broad peak at 3168 cm − 1 , in the region of 3100–3600 cm − 1 , was assigned to the stretching vibrations of –OH groups. The peak at 2931 cm − 1 corresponds to asymmetric and symmetric stretching vibrations of methylene groups. Triplets peak at 1160, 1080 and 989 cm − 1 were found, with the band at ~ 989 cm − 1 assigned to C–O stretching vibration in the C–O–C glucose ring. Intra-molecular hydrogen bonding of OH groups at C-6 is attributed to the amorphous structure in starch. Peaks at 1080 cm − 1 and 1160 cm –1 are associated with C–O and C–OH bending vibration and determine the crystalline phase [ 46 ]. The characteristics of starch samples observed in the FTIR is also similar to that found by Sethi et al., [ 17 ] and Waghmare et al., [ 47 ]. In Fig. 3 (b) , the peaks at 3355 cm − 1 and 3182 cm − 1 were assigned to the NH– stretching vibration of acrylamide. The peak at 1673 cm − 1 was assigned to C = O stretching vibration of carboxyamide. A peak at 1612 cm − 1 was assigned to C = C vibrations. A C-N stretching band appears at 1431 cm − 1 which was assigned to primary amides. The peak at 980 cm − 1 was assigned to the vibration of H in C = C-H [ 48 ]. The peaks at 1140 cm − 1 and at 980 cm − 1 also represented the crystal and amorphous characteristics, respectively, of acrylamide. In Fig. 3 (c) , the peak at 1760 cm − 1 was assigned to the strong C = O stretching of PLA. The peak at 1184 cm − 1 was assigned to the C-O stretching of the –CH (CH 3 )-OH end group of PLA. [19,20] The peak at 1184 cm − 1 for PLA also showed its high crystallinity. XRD analysis also showed the evidence of the high crystallinity of PLA. In Fig. 3 (d) , citric acid showed a peak at 3332 cm − 1 , assigned to O-H stretching (R-C(O)-OH. The peak at 1721 cm − 1 was assigned to C = O stretching (C (O)-OH). The peak at 1207 cm − 1 was assigned to C-O stretching vibration Pichitchai et al. [ 49 ]. The peak of citric acid at 1080 cm − 1 represented the high crystallinity of citric acid and also the same peak at 1080 cm − 1 , in the FTIR spectrum of the prepared hydrogel proved the crosslinking of two starch-PLA-acrylamide polymer chains by citric acid. From Fig. 3 (e) , it can be seen that the peak at 1643 cm − 1 shifted to a strong peak at 1667 cm − 1 of the prepared hydrogel, which is attributed to C = O stretching vibration. That vibration was proof of the superposition of C = O in amide and C = O in COOH (carboxylic group) of PLA due to grafting. The small peak at 1760 cm − 1 shown in the FTIR of the prepared hydrogel was assigned to the strong C = O stretching of PLA. This is also proof of the successful co-polymerisation of PLA with starch. On the other hand, the shifting of the peak at 1673 cm − 1 to 1667 cm − 1 and the absence of the characteristic peak of C = C at 1612 cm − 1 for acrylamide proved the grafting and co-polymerisation of acrylamide with PLA and starch. Citric acid showed a characteristic peak for C = O stretching (C (O)-OH) at 1721 cm − 1 . But in the prepared hydrogel, this peak had shifted to 1667 cm − 1 , proving the cross-linking of citric acid with the starch-PLA-acrylamide copolymer. The FTIR spectra of the prepared hydrogel (Fig. 3 e) has a comparatively sharp peak at 1106 cm − 1 , which was assigned to the C-O-C in glycosidic linkage in the hydrogel. This was also assigned to the co-polymerisation of starch with PLA. All this FTIR data of starch, PLA, acrylamide, citric acid and prepared hydrogel suggested the following chemical reactions and structure of the prepared hydrogel [ 50 , 51 ]. 3.5 Thermal gravimetric analysis The thermograms of PLA, starch and the synthesized hydrogels are presented in Fig. 4 . Three steps of weight loss, as well as thermal degradation in the TGA curves, are shown. The first step can be seen up to 125 ºC, which is related to the loss of water from the starch and the prepared hydrogel. In the second step, the weight loss is due to the thermal decomposition of the polymeric chain. Starch decomposes at about 320 ºC [ 43 ]. In this step, the thermal decomposition of amide and carboxylate side-groups of the polyacrylamide also occur, as reported by Singh and Mahto [ 30 ]. The third step of weight loss represents carbon burning, at about 400–450 ºC. It is also observed that the decomposition temperature of starch is lower than that of PLA. The thermal stability of newly-prepared hydrogel was different than that of potato starch and PLA. From this thermogram, it is clear that the prepared hydrogel is amorphous in nature. Its greater elasticity than its components suggests that prepared hydrogel could be suitable as wound dressing. X-Ray diffraction analysis Figure 5 (a-c) represents the XRD patterns of PLA, starch and prepared hydrogel. In Fig. 5 (b) , the X-Ray diffraction analysis ( XRD) of starch shows four broad peaks, at 2θ value of starch, at 16̊, 17̊, 22̊ and 27̊, respectively [27]. In Fig. 5 (a) , pure PLA has two sharp peaks, at about 17̊-16̊ and a small peak at 19.5 ̊ indicating its crystallinity [19, 20]. From Fig. 5 (c) , the XRD pattern of the prepared hydrogel showed new peaks, which are, comparatively, less sharp and in different positions than its monomers. This suggests that, after the incorporation of PLA and acrylamide with starch and cross-linking with citric acid, the XRD of prepared hydrogel was quite different to that of starch. These changes were the result of the formation of the hydrogel. Basically, the sharpness of the XRD peaks indicates the degree of crystallinity. The fact that the XRD peaks of the hydrogel were very low indicates that the prepared hydrogel was less crystalline than the monomers and highly amorphous. This amorphous portion increases its elasticity and thus makes it more suitable for wound dressing materials. On the other hand, the amorphous nature of this hydrogel will make it more bio-degradable and thus better for the environment. 3.8 Energy-dispersive X-ray analysis The Energy dispersive X-ray analysis (EDX) and the elemental compositions of starch, PLA and synthesised hydrogel, in both mass and atomic percentages, are listed in Table 1 . The number of elements present in the synthesised hydrogel was quite different from that of its reactant. The elements in the hydrogel come from acrylamide and PLA, composed of Carbon, Oxygen and Nitrogen with different mass and atomic percentages. The presence of a Nitrogen atom indicates the hydrophilic nature of the hydrogel, which ensures the successful grafting and co-polymerisation of acrylamide with the starch and PLA. Table 1 EDX analyses of starch, PLA, acrylamide and the synthesised hydrogel. Types of Samples Elements keV Mass % Atomic % Starch C K 0.277 58.78 65.51 O K 0.525 41.22 34.49 PLA C K 0.277 64.01 70.32 O K 0.525 35.99 29.68 Acrylamide C K 0.277 67.93 72.07 N K 0.392 11.33 10.43 O K 0.525 20.74 16.70 Synthesised Hydrogel C K 0.277 45.89 51.78 N K 0.392 19.74 19.1 O K 0.525 34.37 29.12 Surface morphology analyses The surface morphologies of starch, PLA and the prepared hydrogel are shown in Fig. 7 (a-c) . The SEM micrograph represents the granular and oval-shaped structure of starch. The surface of the PLA was rough. The surface of the hydrogel was quite different and more uniform than that of the starch or PLA. The changes in morphology after the grafting with polyacrylamide and poly-lactic acid onto the starch confirmed the formation of the hydrogel. Starch-g-PLA-polyacrylamide cross-linked with citric acid has a fibrillar structure and it is amorphous in nature. Antimicrobial Activity From the structure of hydrogel, it is clear that the middle carboxyl group, of citric acid, is free in the hydrogel structure. It is able to donate a hydrogen ion in wet conditions, as free citric acid was able to donate three hydrogen ions in such conditions. Basically, this hydrogen ion is responsible for the anti-microbial activity of the hydrogel. Yoon et. al. [ 52 ], established that citric acid is anti-microbial. The hydrogen ions released from the prepared hydrogel enter the microbial cells and decrease.their pH, rendering them acidic. ATP then hydrolyses, becoming ADP plus di-hydrogen phosphate. Consequently, the concentration of ATP in the cells decreases, which causes the multiplication, and cell-wall formation, of the micro-organism to stop. Table 2 Antibacterial activity (Measured by size of the zone of inhibition). Sample Inhibition zone (mm) against S. aureus bacteria (a) Inhibition zone (mm) against E. coli bacteria (b) Ampicillin (10µL) 42 40 Starch - - PLA (1%) 16 15 Citric acid (2%) 25 23 Hydrogel 21 19 The antimicrobial impacts of the Ampicillin (positive control), starch, PLA, CA and the prepared hydrogel were determined by testing them against the gram-negative bacteria, Escherichia coli and the gram-positive bacteria, Staphylococcus aureus . In Table 2 , the sizes of the zones of inhibition (of the microorganisms’ growth) in the samples were measured, in millimeters, at the end of the incubation period, and tabulated. The results showed that the hydrogel was more effective against S. aureus bacteria than against Escherichia coli . In the tabulated data, starch showed no antibacterial activity but the synthesised hydrogel showed a significant, clear zone of inhibition, of about 21 mm for S . aureus and 19 mm for E. coli bacteria. This result ensures that his hydrogel was effective against micro-organisms. Anti-oxidant test The synthesised hydrogel showed exceptional antioxidant activity as per the DPPH assay. About 37% inhibition of free radicals was noted at the highest concentration of 25µg/ml (Fig. 7 a). This is assigned to the prepared hydrogel combining more effectively with the free radicals formed by DPPH in the working solution at the higher concentration. As a result, increased scavenging was observed. Biodegradability In Fig. 7 (b) , the bio-degradability study of the prepared hydrogel is represented. Biodegradability is one of the expected properties of hydrogels for a safe and sustainable environment. From this point of view, agro-based, as well as polysaccharide-based hydrogels are the best choice, as they contain glycosidic linkages between glucose units and finally degrade to CO 2 , H 2 O and other non-toxic material. But the bio-degradation can be delayed when an antimicrobial agent is added into hydrogel preparation. In such a case, citric acid acts as an antimicrobial agent, as it is well known that citric acid provides multiple functional groups and is also able to create active binding sites. It is well-known that citric acid has antibacterial properties. In this experiment, the prepared potato starch-based hydrogel degraded more slowly, due to the introduction of the antimicrobial agent. The synthesised hydrogels proved their biodegradability by 72% degradation after 8 weeks’ burial. The graphical representation and the trend line show a very clear, linear relationship between biodegradability and time up to 42 days. After that, degradation seems to stop. From this experiment, we can also say that the prepared hydrogel was bio-degradable in nature and no adverse effect on the environment like conventional wound dressing materials have. Cell viability test Figure 9 shows that the cell viability for control (using vero cells in blank culture medium) was 91.87% ± 2.51%. The sample, using prepared hydrogel of approximately 1.5 x 0.9 cm 2 dimensions and with a weight of approximately 50 mg, was placed in 10 mL of culture medium. After 3 days in the medium, cell viability was 86.97% ± 2.11%. From this result, it can be concluded that the cell viability was not changed significantly [p value, ns (using two-tailed unpaired t test)]. Conclusion The newly-developed potato starch-based hydrogel was synthesised and characterised successfully. The FTIR data proved the successful copolymerisation of PLA and acrylamide with starch and also the cross-linking of copolymer chains by citric acid molecules. The presence of nitrogen atoms in the prepared hydrogel network, found by EDX analysis, also proved the successful grafting of acrylamide on to PLA and starch. The XRD data, along with FTIR data, confirmed the lower crystallinity and higher elastic properties of the prepared hydrogel compared to its raw components. The swelling and water vapour transmission rates (WVTR) was 481 (at pH 7.2) and 148 g/m 2 h, respectively, which suggest that this prepared hydrogel will be a good absorbent, and wound dressing material. The anti-microbial test of the prepared hydrogel also showed good activity against both gram-positive ( S. aureus) and gram-negative ( E. coli) bacteria, having a zone of inhibition 21mm and 19mm, respectively. This good result is assigned to hydrogen ion donation from the carboxyl group of citric acid (as an anti-microbial) in the hydrogel network. The prepared hydrogel showed a good cell viability. It was found to be non-toxic and harmless to human skin. Therefore, dermal use is indicated. The synthesised hydrogel was found to have a mild antioxidant property (scavenging of 37.2% for 25µg/ml). It also showed high bio-degradability (72% within 8 weeks). All these properties of the newly-developed hydrogel lead to the conclusion that the hydrogel has potential application as a wound dressing material. The high swelling rate of the prepared hydrogel ensured its high porosity and prevalence of interstitial spaces. Further investigation of the suitability of this hydrogel as a drug-carrier for specific drugs is recommended. Declarations Author Contributions Md. Ibrahim H. Mondal : Conceptualization, Resources, Supervision, Funding acquisition, formal analysis, Writing – Reviewing and editing. H. Jahan Kadri: Methodology, Investigation, Formal analysis, Visualization, Writing – Original draft. Firoz Ahmed: Methodology, Investigation, Formal analysis, Visualization, Writing – Original draft, Writing – Reviewing and editing. Md. Hasinur Rahman: Formal analysis, Visualization, Writing – Reviewing and editing. Data availability Data are contained within the article. 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Supplementary Files Graphicalabstract.jpg Graphical abstract Cite Share Download PDF Status: Published Journal Publication published 11 Jun, 2025 Read the published version in Polymer Bulletin → Version 1 posted Editorial decision: Revision requested 03 Feb, 2025 Reviews received at journal 02 Feb, 2025 Reviews received at journal 26 Jan, 2025 Reviewers agreed at journal 23 Jan, 2025 Reviewers agreed at journal 16 Jan, 2025 Reviewers agreed at journal 15 Jan, 2025 Reviewers agreed at journal 14 Jan, 2025 Reviewers agreed at journal 14 Jan, 2025 Reviewers invited by journal 14 Jan, 2025 Editor assigned by journal 22 Aug, 2024 Submission checks completed at journal 21 Aug, 2024 First submitted to journal 20 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4946153","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":347198734,"identity":"b16fbae1-7eb6-491c-a9e6-5df9b73b2312","order_by":0,"name":"Md. 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Hydrogel.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4946153/v1/36c48add7906b568146f6e1f.jpg"},{"id":64940690,"identity":"2e363e95-48f5-496b-abc5-fa2785d46751","added_by":"auto","created_at":"2024-09-20 15:35:07","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":36450,"visible":true,"origin":"","legend":"\u003cp\u003eThe XRD pattern of (a) PLA, (b) Starch and (c) Hydrogel.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4946153/v1/a4f62fee7228adbe7ab966ac.jpg"},{"id":64940142,"identity":"792c7867-df90-4cc3-b471-01a4ace44afe","added_by":"auto","created_at":"2024-09-20 15:27:07","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":102163,"visible":true,"origin":"","legend":"\u003cp\u003eSEM of (A) Raw Starch (B) PLA and (C) Synthesised Hydrogel.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4946153/v1/8af52c8be0efb10ff89ff309.jpg"},{"id":64940691,"identity":"249725e6-b374-44d9-befa-cb53d6b747ba","added_by":"auto","created_at":"2024-09-20 15:35:07","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":73332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 7 \u0026nbsp;\u003c/strong\u003eReaction mechanism behind antibacterial activity of hydrogel [52].\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4946153/v1/25233bffbc52e7d71464e36e.jpg"},{"id":64940145,"identity":"174480a9-4fe8-4d57-81a3-6efa1eb6c00c","added_by":"auto","created_at":"2024-09-20 15:27:07","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":40366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 8.\u003c/strong\u003e (a) Anti-oxidant property (b) Bio-degradability of prepared hydrogel.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4946153/v1/f3a6938e639f118ed52f43b3.jpg"},{"id":64940146,"identity":"f4ed59fc-7794-4516-bce6-37da5c7c6a10","added_by":"auto","created_at":"2024-09-20 15:27:08","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":98691,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 9 \u003c/strong\u003e\u0026nbsp;Image of cell viability on vero cell (a), (b) and (c) for control and (d), (e) and (f) for hydrogel samples on the day 1,2 and 3, respectively.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4946153/v1/f7018e6ab64a74d919bb9b3a.jpg"},{"id":84726532,"identity":"725010e3-f086-486f-a934-6fa1d1724aba","added_by":"auto","created_at":"2025-06-16 16:06:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1680797,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4946153/v1/24350c46-bbd9-42c6-8d62-3fbf4e250fa7.pdf"},{"id":64940139,"identity":"105f06be-8788-44e6-a47f-2e5c645f2c8f","added_by":"auto","created_at":"2024-09-20 15:27:07","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":70577,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4946153/v1/e36224e6692b2f1574edacf9.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis and characterization of starch-g-polyacrylamide-co-polylactic acid hydrogel for the potential wound dressing application","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHydrogels, based on biopolymers, have been getting greater attention since the discovery of a \u0026lsquo;super absorbent\u0026rsquo; polymer in the 1950s. Over the past seven decades, researchers from academic institutions and businesses have focused on the industrial uses of these hydrogels [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. A variety of potential applications, including pharmaceuticals, sanitary napkins, biomedicine, tissue engineering, agriculture, veterinary medicine and waste water treatment, was quickly explored [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe need for hydrogels which have three-dimensional (3-D) structures, physically or chemically, with partly cross-linked polymeric networks, is apparent. Such hydrogels have a good capacity for absorption of water, as well as other biological fluids and saline solutions. Using a range of copolymers and cross-linkers, the physical and chemical properties of the hydrogels can be easily customised. The cross-linked structure of the hydrogel network protects it from dissolution.\u003c/p\u003e \u003cp\u003eMunim and Raja [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] reported that the presence of amine, hydroxyl, amide and sulfate groups of polysaccharides increases the water absorption tendency of hydrogels. Biopolymer-based hydrogels are appealing because they are readily available naturally, inexpensive, biodegradable, non-toxic and have desired physico-chemical properties. These features set them apart from petroleum-based synthetic counterparts, which pollute the environment [\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHydrogel dressings in the form of a gel or film can absorb exudates from a wound surface and maintain a moist environment. Translucent and flexible hydrogel dressings are easy to remove from wounds. Exudate removal from the wound surface, moisture control, gas transport, infection prevention, biocompatible, biodegradable, cost-effective, non-toxic, pain relief, wound necrosis reduction, low skin adhesion and mechanical stability are all features of an ideal wound dressing material [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStarch is one of the most abundant and cheap polysaccharides. It features inherent biodegradability and renewability, it is available in nature and it is an energy source obtained from plants that contain cellulose. Starch in cereal grains, such as corn, wheat, rice, barley, rye, oats, buckwheat, roots (potatoes, sweet potatoes and cassava), sago, stems and seeds/ legumes (lentils, beans and peas), are valuable for human nutrition.\u003c/p\u003e \u003cp\u003eStarch consists of two types of anhydro-glucose units: amylose (α-1, 4-linkage), which is linear, and amylopectin (α-1, 6-linkage), which is branched. These are joined by an α-D-glycosidic bond. Amylopectin and amylose, the major components of starch both include glucose. Therefore, starch is classified as a homo-polymer of glucose units but it is a hetero-polymer of amylose and amylopectin. Starch is a naturally semi-crystalline biopolymer. Amylose usually constitutes around 20\u0026ndash;30% of a granule of starch and amylopectin constitutes around 70\u0026ndash;80% of it. The molecular formula of starch is (C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e)\u003csub\u003en\u003c/sub\u003e. Different starches contain different percentages of amylose and amylopectin. Soluble starch contains 30% amylose and 70% amylopectin [\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePolylactic acid (PLA) is a naturally occurring polymer PLA has proved notable for its biodegradability, biocompatibility, lack of toxicity and ability to be thermally-processed. A thermoplastic aliphatic polyester, made from agricultural waste, PLA is environmentally-friendly. PLA degrades quickly and safely on human skin. So, PLA is a prime candidate for applications such as drug delivery systems, sutures and clips for biomedical applications. PLA has also proved suitable for pharmaceuticals use in food packaging [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTorres et. al., [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] synthesised a novel hydrogel of poly (acrylamide) (PAAm) and starch at different ratios. They found that their hydrogel was a potential platform for controlled release of amoxicillin. The hydrogel, loaded with amoxicillin, was tested by disc diffusion against \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC-25922, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC-25923 and a carbapenemase producer, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. These hydrogels proved to be powerful bacteria growth inhibitors when loaded with amoxicillin in this fashion. Thus, they were found suitable for biomedical applications. However, the hydrogels were not antimicrobial without antibiotics. They also exhibited an improved rate of water uptake with increasing starch content but not at a super-absorbent level [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePal et. al., [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], prepared corn-starch-based hydrogel membranes by cross-linking them with polyvinyl alcohol. The membranes had sufficient mechanical strength and water-retention capacity but no anti-microbial capacity. At the same time, they also developed another novel hydrogel by crosslinking polyvinyl alcohol with starch suspension, using glutaraldehyde as a crosslinking agent. The membrane had sufficient strength to be used as artificial skin but water absorption and retention properties were not reported by the authors [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Pattra \u003cem\u003eet.al.\u003c/em\u003e prepared starch-based super water absorbent (SWA) hydrogel and validated the practical benefits of SWA for agricultural applications. SWA was successfully prepared in an up-scaling production process by radiation-induced graft polymerisation of acrylic acid onto cassava starch. Although SWA showed high swelling ability and was able to increase the survival rate of young rubber trees planted in an arid area by up to 40%, it did not show any anti-microbial activity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe design and preparation of novel biodegradable hydrogels developed by the free radical polymerisation of acrylamide acrylic acid was reported. Some formulations with bis-acrylamide, in the presence of a corn starch/ethylene-co-vinyl alcohol copolymer blend (SEVA-C), were also reported. Although the hydrogel was highly bio-degradable and was appropriate for drug delivery, it showed low absorption capacity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The graft co-polymerisation of polyacrylic acid and/or acrylamide on potato starch was reported by Al-Aidy and Amdeha [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This hydrogel was able to adsorb malachite green (MG) dye from the environment but the applicability of this hydrogel to medical application, as a super-absorbent, was not reported by the author, nor was its anti-microbial activity investigated.\u003c/p\u003e \u003cp\u003eSethi et. al., [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] synthesised a hydrogel, using a hybrid backbone of karaya gum starch and grafted with polyacrylic acid, which was tested as an oral drug delivery carrier. Paracetamol and aspirin were the test drugs. Due to its low tensile strength, this hydrogel was found not to be suitable for use as wound-dressing material, nor was it effective as an anti-microbial. Demitri et al., [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. developed a novel, superabsorbent, cellulose-based hydrogel, cross-linked with citric acid. Although the hydrogel was bio-degradable, its anti-microbial activity and tensile strength were not reported. A hydrogel based on polyacrylamide, grafted to starch/clay nano-composite, was used for enhanced oil recovery, but its bio-medical applications were not investigated properly [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The hydrogel, made of cassava starch grafted to poly [Acrylamide-co-(Maleic Acid)] was found to be superabsorbent of γ-irradiation and used for packaging material [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Hydrogel prepared from the graft copolymerization of starch with acrylamide and 2-acrylamido-2-methylpropanesulfonic acid (AMPS) was responsive but its anti-microbial activity was not investigated [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Another hydrogel, prepared by graft co-polymerisation of ethyl acrylate/acrylamide on to corn starch, using potassium permanganate\u0026ndash;citric acid initiation, was optimised but its characterisation was not reported properly [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The cellulose-based hydrogels were prepared by free radical graft copolymerisation reaction of cotton with acrylic acid (AA) and acrylamide (AM), using N,N-methylne-bis-acrylamide (MBA) as a crosslinker in the presence of potassium persulphate (K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e) as an initiator. These hydrogels\u0026rsquo; optimisation was studied but their applications were not examined properly [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAll these above-mentioned hydrogels have swelling, absorption, mechanical, anti-microbial, and diffusivity properties but the study of their applications was unidimensional. For this reason, it is necessary to study hydrogels now specifically to learn how they will function in wound healing.\u003c/p\u003e \u003cp\u003eTherefore, here, potato starch-based hydrogel was developed through graft copolymerisation. Acrylamide and polylactic acid were grafted on to starch, using citric acid (CA) as a cross-linker, in the presence of potassium persulphate as a redox initiator. The goal was to develop a highly-porous, absorbent hydrogel, with high mechanical strength, which was also active against micro-organisms. The synthesised hydrogel of starch-g- (poly acrylamide-co-PLA) was characterised via various physico-chemical and instrumental analyses. The hydrogel was found to be useful complicated wound-healing management, as an advanced wound dressing.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eFresh potatoes (\u003cem\u003eSolanum tuberosum\u003c/em\u003e) were collected from the local market in Rajshahi, Bangladesh. Polylactic acid (Uni-Chem, China), acrylamide (BDH, England), citric acid (Merck, India), 1,4- dioxane, (BDH, England), DPPH (2,2-diphenyl-1-picrylhydrazyl) (Sigma Aldrich), methanol (Merck, Germany), potassium persulphate (Merck, Germany), etc. of analytical grade chemicals were purchased and used in the experiments reported here.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExtraction of potato starch and synthesis of hydrogel\u003c/h2\u003e \u003cp\u003eStarch was extracted from fresh potatoes. For this purpose, the peeled potatoes were blended and then the residual mass was removed by means of a cloth filter. The remains were again filtered using Whatman filter paper. The filtrate was kept in a beaker for about 12 hours, to allow it to settle down. Then the extracted starch was separated, using a centrifuge. Next, the resulting extracted starch was sun-dried and then crushed into a fine powder. This starch powder was then levelled and preserved in an air-tight container for further use [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The whole extraction process is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eTo summarise the process used, 1.0 g (2% w/v) of potato starch was gelatinised at 60\u003csup\u003eo\u003c/sup\u003eC for one hour, with continuous stirring. Then the gelatinized starch was cooled to room temperature and poured into a three-neck round-bottom flask, equipped with a thermometer, a mechanical stirrer and a nitrogen line (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e(b\u003c/b\u003e)). After that, acrylamide (300% w/w based on potato starch weight), potassium persulphate 0.2% and 1%, 10 ml of PLA solution, were added to it. The ratio of starch and acrylamide was 1: 3. PLA was dissolved in 1, 4-dioxane by heating at 50\u003csup\u003eo\u003c/sup\u003eC for 30 minutes and cooled down to room temperature. Then dissolved crosslinking agent 2%, 10 ml CA was added to the solution and stirred.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe reaction flask was then purged with nitrogen gas for 30 minutes, to remove any dissolved oxygen which might remain. After constant stirring for 30 minutes, the reaction continued for 4 hours at 50\u003csup\u003eo\u003c/sup\u003eC, using a water bath to complete the polymerisation reaction. A transparent, sticky solution was then obtained. After completing the reaction, the prepared hydrogel was washed several times with distilled water, poured into a petri dish, cut into small pieces and dried in a drier for 3 days at 60\u003csup\u003eo\u003c/sup\u003eC. The dried gel was ground and screened through a 200-mesh sieve. The resulting white granular product was then preserved in an air-tight container.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCharacterisation\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eSwelling studies\u003c/h2\u003e \u003cp\u003eThe swelling behaviour of the synthesised hydrogel was determined by treating it with distilled water and immersing it in solutions of different pH (pH\u0026thinsp;=\u0026thinsp;1.2, 4.4, 7.4, 8.1, 10.02). The test samples were also immersed in organic solvent (ethanol and acetone). A known initial weight W\u003csub\u003ei\u003c/sub\u003e of the sample was immersed in 100 ml of solvent at room temperature for 48 hours. The samples were filtered and the excess solvent was removed with the help of filter paper. Then the final weight, W\u003csub\u003ef,\u003c/sub\u003e was determined. The percentage of swelling was calculated as per following \u003cb\u003eEq.\u0026nbsp;(1)\u003c/b\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Swelling\\:Percentage\\:\\left(\\%\\right)=\\frac{{W}_{f}-{W}_{i}}{{W}_{i}}\\times\\:100\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\left(1\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the initial weight and \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e is the final weight of the sample after swelling.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGel content\u003c/h2\u003e \u003cp\u003eThe pre-weighted hydrogel sample was dried to a constant weight and then immersed in distilled water for 24 hours to remove the solid fraction. The swelled sample was taken out from the distilled water and dried to a constant weight in an oven. The gel content was calculated as \u003cb\u003eEq.\u0026nbsp;(2)\u003c/b\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:Gel\\:Content\\:\\left(\\text{%}\\right)=\\frac{{\\text{W}}_{\\text{d}}}{{\\text{W}}_{\\text{i}}}\\times\\:100\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\left(2\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e is the weight of dry gel after extraction from water and \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the initial weight of dry gel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWater vapour transmission rate test\u003c/h2\u003e \u003cp\u003eA small bottle was filled with 10 ml distilled water to assess the Water Vapour Transmission Rate (WVTR). The mouth of the bottle was covered with a hydrogel membrane and Teflon tape was used to keep it airtight. The bottle was weighed and kept at 40\u003csup\u003eo\u003c/sup\u003eC in an oven. After 24 hours, the bottle was taken out from the oven and weighed again. The WVTR (g/m\u003csup\u003e2\u003c/sup\u003e per day) was determined using the \u003cb\u003eEq.\u0026nbsp;(3)\u003c/b\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:Water\\:vapour\\:transmission\\:rate=({\\text{W}}_{\\text{i}}-{\\text{W}}_{\\text{t}})/\\left(\\text{A}\\times\\:24\\right)\\times\\:{10}^{6}\\dots\\:\\dots\\:\\dots\\:\\dots\\:..\\:(3)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere W\u003csub\u003ei\u003c/sub\u003e and W\u003csub\u003et\u003c/sub\u003e are the masses of the bottle before and after heating, respectively, and A is the inner area of the round mouth of the bottle. The experiment was conducted at 40\u003csup\u003eo\u003c/sup\u003eC, as the hydrogel would be used as a wound dressing on human skin, which has a temperature of 37.2 \u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eMoisture retention capability\u003c/h2\u003e \u003cp\u003eThe moisture content of the synthesised hydrogel was determined by a moisture analyzer (RADWAG, 26\u0026ndash;600 Radom, Poland). The moisture retention capability was calculated according to the following \u003cb\u003eEq.\u0026nbsp;(4)\u003c/b\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:Moisture\\:Retention\\:Capability\\:\\left(\\text{%}\\right)=\\:\\frac{{\\text{W}}_{1}-{\\text{W}}_{2}}{{\\text{W}}_{1}}\\times\\:100\\dots\\:\\dots\\:\\dots\\:\\dots\\:..\\left(4\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e is the initial weight of the sample and \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e is the weight of the dry sample after being heated at 40\u003csup\u003eo\u003c/sup\u003eC for 8 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eAntioxidant property\u003c/h2\u003e \u003cp\u003eThe capability of synthesised hydrogel to scavenge free radical cations was assessed, using 1,1-diphenyl-2-picrylhydrazyl (DPPH). A stock solution of 0.004% (w/v) DPPH was prepared. using 50 ml methanol. and was kept in the dark. 1 ml of prepared test samples of different concentrations (5, 10, 15, 20, and 25 \u0026micro;g/mL) were taken, in test tubes and 3 ml of DPPH, in methanol solution, was added to each test tube. Then the test tubes were incubated at room temperature for 30 minutes, in a dark place, to allow the reaction to be completed. Then the absorbance of the solution was measured at 517 nm, using a spectrophotometer (T60 UV-Visible spectrophotometer, UK), against blank. The antiradical activity was measured as % scavenging of free radicals, by measuring the decrease in absorbance percentage compared to the control solution. The percent inhibition or scavenging activity was calculated [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] using the following \u003cb\u003eEq.\u0026nbsp;(5).\u003c/b\u003e\u003cdiv id=\"Eque\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Eque\" name=\"EquationSource\"\u003e\n$$\\:\\text{S}\\text{c}\\text{a}\\text{v}\\text{e}\\text{n}\\text{g}\\text{i}\\text{n}\\text{g}\\:\\text{A}\\text{c}\\text{t}\\text{i}\\text{v}\\text{i}\\text{t}\\text{y},\\:\\text{I}\\text{%}]=\\frac{{\\text{A}}_{0}-{\\text{A}}_{1}}{{\\text{A}}_{0}}\\times\\:100\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\dots\\:(5)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere A\u003csub\u003e0\u003c/sub\u003e is the absorbance of the control and A\u003csub\u003e1\u003c/sub\u003e is the absorbance in the presence of hydrogel.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial property\u003c/h2\u003e \u003cp\u003eAntibacterial activity was measured by the agar well disc diffusion method reported by [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The antibacterial activity of the prepared hydrogel was tested against gram-positive bacteria \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and gram-negative bacteria \u003cem\u003eEscherichia coli\u003c/em\u003e. The prepared nutrient agar (Hi media, India) medium was sterilised in an autoclave at 121\u0026ordm;C for 15 minutes. This medium was then transferred to sterilised Petri dishes in a laminar airflow chamber. The media solidified in 20 minutes and then the cultures of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e were spread on the media. Then the solid hydrogel sample was embedded in the agar well. Starch, polylactic acid, citric acid, and solvent 1,4-dioxane, and prepared hydrogel were loaded (10\u0026micro;l) on to a sterilised disc and the plates were incubated at 37\u0026ordm; C, for 24 hours. After incubation, the antimicrobial activity was assessed in terms of the zone of inhibition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell viability test\u003c/h2\u003e \u003cp\u003eCell viability of the control sample (blank culture media) was 91.87% \u0026plusmn; 2.51. The experimental sample was approximately 1.5 x 0.9 (1 x w,cm) and approximately 50 mg. The experimental sample was placed in 10 mL of culture media. The result was obtained after 3 days of observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBiodegradability test\u003c/h2\u003e \u003cp\u003eStarch has a tendency to degrade when buried in the soil, as it contains glycosidic linkages. To study in-soil degradation of the prepared hydrogel, grafted samples were weighed individually and buried in soil for 56 days. After that period, samples were withdrawn, carefully washed with distilled water and dried at 105\u0026ordm;C for 20 minutes. Then the samples were kept at room temperature for 24 hours before weighing. Finally, the weight loss of the degraded hydrogel was determined, using the following \u003cb\u003eEq.\u0026nbsp;(6)\u003c/b\u003e [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003cdiv id=\"Equf\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equf\" name=\"EquationSource\"\u003e\n$$\\:Weight\\:Loss,\\:\\left(\\%\\right)=\\frac{{W}_{i}-{W}_{d}}{{W}_{i}}\\times\\:100\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\dots\\:\\dots\\:.\\left(6\\right)\\:$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere W\u003csub\u003ei\u003c/sub\u003e is the initial weight of hydrogel and W\u003csub\u003ed\u003c/sub\u003e is the weight of the degraded hydrogel after a period of burial.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFTIR analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw potato starch, acrylamide, citric acid, synthesized hydrogel, PLA and KBr (potassium bromide) were dried in an oven at 105 \u003csup\u003e0\u003c/sup\u003eC, to remove all moisture. Then each starch, the synthesised hydrogel and PLA was mixed with dried KBr to make a powder, using a mortar pestle and pellet. Then the samples were analysed in an FTIR spectrotmeter (Model: FTIR- 8900, Shimadzu, Japan) They were found to be within 400-4000 cm\u003csup\u003e-1\u003c/sup\u003e wavenumber.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eScanning electron microscopy and Energy-dispersive X-ray analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe surface morphology of extracted starch, PLA and the prepared hydrogel was scanned Electron Microscope (SEM) (JEOL, Model- JSM7600 F, Japan). The micrographs were taken at a magnification of 300 times and the scans were performed at 5.0 kV accelerating voltage. At the same time, Energy Dispersive X-Ray Analyses (EDX) were performed with the same instrument. The samples were subjected to an electron beam of 15 kV of energy-accelerating voltage and the counting rate was 305 cps.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eX-Ray diffraction analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe crystallinities of extracted starch, PLA and prepared hydrogel were investigated by a PAN Analytical X Pert PRO X-ray diffractometer (Model: Empyrean, PAN Analytical, Netherland). Cu-K\u0026alpha; radiation of wavelength 1.5406 Å was used as the X-ray source. The scanning rate was 2 minutes with a scan angle from 5˚ to 50˚.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThermal analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThermal analyses of all samples were carried out with a Perkin-Elmer Simultaneous Thermal Analyzer (STA 8000, Germany). The tests were carried out in an inert nitrogen atmosphere.at a temperature of 30\u0026ordm; C - 600\u0026ordm; C. The heating rate and the airflow rate were 20 \u0026deg;C/min and 200 ml/minute, respectively.\u0026nbsp;\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eGel content\u003c/h2\u003e \u003cp\u003eThe gel content of a hydrogel is important because it indicates the degree to which the hydrogel network is cross-linked. The hydrogel was submerged in distilled water for 24 hours. The polymer part, which was not cross-linked, was dissolved in water. The remaining part of the hydrogel is called the gel content. A higher gel content means a highly cross-linked as well as mechanically-strong hydrogel. The amount of gel in the prepared Starch/PLA hydrogel was 75% [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eSwelling behavior\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the dependence of swelling on the pH of the aqueous solution in which the hydrogel is placed is shown. The percentage of swelling of the prepared hydrogel was very low in acidic medium and increased with increased pH. At pH 7.4 (neutral) the DS increased sharply to 481%. Further increase of pH to alkaline caused swelling increase negligibly and level off. In acidic medium, the prepared hydrogel was unable to donate protons (protonation) due to common ion effects of hydrogen ions. Both intra- and inter-molecular hydrogen bonds were formed in the hydrogel network in acidic solution. The prepared hydrogels were therefore more compact such acidic solutions (pH 1.2\u0026ndash;3.5), which inhibited their swelling. De-protonation began at pH 3.1 like the carboxyl group of citric acid [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This de-protonation allowed more water to enter into the hydrogel structure, which is the meaning of \u0026ldquo;swelling\u0026rdquo;. It is also clear that the swelling is both capillary- and diffusion-controlled (45).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eWater vapour transmission rate\u003c/h2\u003e \u003cp\u003eThe hydrogel wound dressing should have the ability to minimise the fluid loss from the wound by maintaining a moist environment under the wound. Lower WVT is therefore a good characteristic for hydrogel to be used as wound dressing. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] reported that the loss of water from second- and third-degree wounded skin is 178.55\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 and 143.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 g/m \u003csup\u003e2\u003c/sup\u003e per day, respectively. The prepared hydrogel had a water vapour transmission rate \u003cb\u003e(\u003c/b\u003eWVTR) of 148 g/m\u003csup\u003e2\u003c/sup\u003eh, which is within the range of water loss from wounded skin. Therefore, this prepared hydrogel will tend to keep wounds dry. Yet the moisture retention capability of the prepared hydrogel was 84.84% at 40⁰C. So, while keeping the wound from oozing fluid, the hydrogel will still maintain the moist environment which maximises healing of wounds, which is the ideal combination for a wound dressing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eFTIR analysis\u003c/h2\u003e \u003cp\u003eThe FTIR \u003cem\u003espectra\u003c/em\u003e of starch, acrylamide, PLA, citric acid and prepared hydrogel are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e(a)\u003c/b\u003e, the characteristic FTIR spectrum of starch is represented. The broad peak at 3168 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, in the region of 3100\u0026ndash;3600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, was assigned to the stretching vibrations of \u0026ndash;OH groups. The peak at 2931 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to asymmetric and symmetric stretching vibrations of methylene groups. Triplets peak at 1160, 1080 and 989 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were found, with the band at ~\u0026thinsp;989 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e assigned to C\u0026ndash;O stretching vibration in the C\u0026ndash;O\u0026ndash;C glucose ring. Intra-molecular hydrogen bonding of OH groups at C-6 is attributed to the amorphous structure in starch. Peaks at 1080 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1160 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e are associated with C\u0026ndash;O and C\u0026ndash;OH bending vibration and determine the crystalline phase [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The characteristics of starch samples observed in the FTIR is also similar to that found by Sethi et al., [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and Waghmare et al., [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e(b)\u003c/b\u003e, the peaks at 3355 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3182 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were assigned to the NH\u0026ndash; stretching vibration of acrylamide. The peak at 1673 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was assigned to C\u0026thinsp;=\u0026thinsp;O stretching vibration of carboxyamide. A peak at 1612 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was assigned to C\u0026thinsp;=\u0026thinsp;C vibrations. A C-N stretching band appears at 1431 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which was assigned to primary amides. The peak at 980 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was assigned to the vibration of H in C\u0026thinsp;=\u0026thinsp;C-H [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The peaks at 1140 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and at 980 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e also represented the crystal and amorphous characteristics, respectively, of acrylamide.\u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e(c)\u003c/b\u003e, the peak at 1760 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was assigned to the strong C\u0026thinsp;=\u0026thinsp;O stretching of PLA. The peak at 1184 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was assigned to the C-O stretching of the \u0026ndash;CH (CH\u003csub\u003e3\u003c/sub\u003e)-OH end group of PLA. \u003csup\u003e[19,20]\u003c/sup\u003e The peak at 1184 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for PLA also showed its high crystallinity. XRD analysis also showed the evidence of the high crystallinity of PLA.\u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e(d)\u003c/b\u003e, citric acid showed \u003cb\u003ea\u003c/b\u003e peak at 3332 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, assigned to O-H stretching (R-C(O)-OH. The peak at 1721 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was assigned to C\u0026thinsp;=\u0026thinsp;O stretching (C (O)-OH). The peak at 1207 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was assigned to C-O stretching vibration Pichitchai et al. [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The peak of citric acid at 1080 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represented the high crystallinity of citric acid and also the same peak at 1080 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, in the FTIR spectrum of the prepared hydrogel proved the crosslinking of two starch-PLA-acrylamide polymer chains by citric acid.\u003c/p\u003e \u003cp\u003eFrom Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e(e)\u003c/b\u003e, it can be seen that the peak at 1643 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e shifted to a strong peak at 1667 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of the prepared hydrogel, which is attributed to C\u0026thinsp;=\u0026thinsp;O stretching vibration. That vibration was proof of the superposition of C\u0026thinsp;=\u0026thinsp;O in amide and C\u0026thinsp;=\u0026thinsp;O in COOH (carboxylic group) of PLA due to grafting. The small peak at 1760 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e shown in the FTIR of the prepared hydrogel was assigned to the strong C\u0026thinsp;=\u0026thinsp;O stretching of PLA. This is also proof of the successful co-polymerisation of PLA with starch. On the other hand, the shifting of the peak at 1673 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1667 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the absence of the characteristic peak of C\u0026thinsp;=\u0026thinsp;C at 1612 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for acrylamide proved the grafting and co-polymerisation of acrylamide with PLA and starch. Citric acid showed a characteristic peak for C\u0026thinsp;=\u0026thinsp;O stretching (C (O)-OH) at 1721 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. But in the prepared hydrogel, this peak had shifted to 1667 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, proving the cross-linking of citric acid with the starch-PLA-acrylamide copolymer. The FTIR \u003cem\u003espectra\u003c/em\u003e of the prepared hydrogel (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee) has a comparatively sharp peak at 1106 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was assigned to the C-O-C in glycosidic linkage in the hydrogel. This was also assigned to the co-polymerisation of starch with PLA.\u003c/p\u003e \u003cp\u003eAll this FTIR data of starch, PLA, acrylamide, citric acid and prepared hydrogel suggested the following chemical reactions and structure of the prepared hydrogel [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e3.5 Thermal gravimetric analysis\u003c/h3\u003e\n\u003cp\u003eThe thermograms of PLA, starch and the synthesized hydrogels are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Three steps of weight loss, as well as thermal degradation in the TGA curves, are shown. The first step can be seen up to 125 \u0026ordm;C, which is related to the loss of water from the starch and the prepared hydrogel. In the second step, the weight loss is due to the thermal decomposition of the polymeric chain. Starch decomposes at about 320 \u0026ordm;C [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In this step, the thermal decomposition of amide and carboxylate side-groups of the polyacrylamide also occur, as reported by Singh and Mahto [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe third step of weight loss represents carbon burning, at about 400\u0026ndash;450 \u0026ordm;C. It is also observed that the decomposition temperature of starch is lower than that of PLA. The thermal stability of newly-prepared hydrogel was different than that of potato starch and PLA. From this thermogram, it is clear that the prepared hydrogel is amorphous in nature. Its greater elasticity than its components suggests that prepared hydrogel could be suitable as wound dressing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eX-Ray diffraction analysis\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e(a-c)\u003c/b\u003e represents the XRD patterns of PLA, starch and prepared hydrogel. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e(b)\u003c/b\u003e, the X-Ray diffraction analysis \u003cb\u003e(\u003c/b\u003eXRD) of starch shows four broad peaks, at 2θ value of starch, at 16̊, 17̊, 22̊ and 27̊, respectively [27]. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e(a)\u003c/b\u003e, pure PLA has two sharp peaks, at about 17̊-16̊ and a small peak at 19.5 ̊ indicating its crystallinity [19, 20].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e(c)\u003c/b\u003e, the XRD pattern of the prepared hydrogel showed new peaks, which are, comparatively, less sharp and in different positions than its monomers. This suggests that, after the incorporation of PLA and acrylamide with starch and cross-linking with citric acid, the XRD of prepared hydrogel was quite different to that of starch. These changes were the result of the formation of the hydrogel. Basically, the sharpness of the XRD peaks indicates the degree of crystallinity. The fact that the XRD peaks of the hydrogel were very low indicates that the prepared hydrogel was less crystalline than the monomers and highly amorphous. This amorphous portion increases its elasticity and thus makes it more suitable for wound dressing materials. On the other hand, the amorphous nature of this hydrogel will make it more bio-degradable and thus better for the environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Energy-dispersive X-ray analysis\u003c/h2\u003e \u003cp\u003eThe Energy dispersive X-ray analysis (EDX) and the elemental compositions of starch, PLA and synthesised hydrogel, in both mass and atomic percentages, are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The number of elements present in the synthesised hydrogel was quite different from that of its reactant. The elements in the hydrogel come from acrylamide and PLA, composed of Carbon, Oxygen and Nitrogen with different mass and atomic percentages. The presence of a Nitrogen atom indicates the hydrophilic nature of the hydrogel, which ensures the successful grafting and co-polymerisation of acrylamide with the starch and PLA.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEDX analyses of starch, PLA, acrylamide and the synthesised hydrogel.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTypes of Samples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElements\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ekeV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMass %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAtomic %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStarch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e58.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e65.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eO K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.525\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e64.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e70.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eO K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.525\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAcrylamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e67.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e72.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.392\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eO K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.525\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eSynthesised Hydrogel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e51.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.392\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eO K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.525\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eSurface morphology analyses\u003c/h2\u003e \u003cp\u003eThe surface morphologies of starch, PLA and the prepared hydrogel are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e(a-c)\u003c/b\u003e. The SEM micrograph represents the granular and oval-shaped structure of starch.\u003c/p\u003e \u003cp\u003eThe surface of the PLA was rough. The surface of the hydrogel was quite different and more uniform than that of the starch or PLA. The changes in morphology after the grafting with polyacrylamide and poly-lactic acid onto the starch confirmed the formation of the hydrogel. Starch-g-PLA-polyacrylamide cross-linked with citric acid has a fibrillar structure and it is amorphous in nature.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eAntimicrobial Activity\u003c/h2\u003e \u003cp\u003eFrom the structure of hydrogel, it is clear that the middle carboxyl group, of citric acid, is free in the hydrogel structure. It is able to donate a hydrogen ion in wet conditions, as free citric acid was able to donate three hydrogen ions in such conditions. Basically, this hydrogen ion is responsible for the anti-microbial activity of the hydrogel. Yoon et. al. [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], established that citric acid is anti-microbial. The hydrogen ions released from the prepared hydrogel enter the microbial cells and decrease.their pH, rendering them acidic. ATP then hydrolyses, becoming ADP plus di-hydrogen phosphate. Consequently, the concentration of ATP in the cells decreases, which causes the multiplication, and cell-wall formation, of the micro-organism to stop.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibacterial activity (Measured by size of the zone of inhibition).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInhibition zone (mm) against\u003c/p\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e bacteria (a)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInhibition zone (mm) against\u003c/p\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e bacteria (b)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmpicillin (10\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStarch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePLA (1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCitric acid (2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHydrogel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe antimicrobial impacts of the Ampicillin (positive control), starch, PLA, CA and the prepared hydrogel were determined by testing them against the gram-negative bacteria, \u003cem\u003eEscherichia coli\u003c/em\u003e and the gram-positive bacteria, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. In Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the sizes of the zones of inhibition (of the microorganisms\u0026rsquo; growth) in the samples were measured, in millimeters, at the end of the incubation period, and tabulated.\u003c/p\u003e \u003cp\u003eThe results showed that the hydrogel was more effective against \u003cem\u003eS. aureus\u003c/em\u003e bacteria than against \u003cem\u003eEscherichia coli\u003c/em\u003e. In the tabulated data, starch showed no antibacterial activity but the synthesised hydrogel showed a significant, clear zone of inhibition, of about 21 mm for \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eaureus\u003c/em\u003e and 19 mm for \u003cem\u003eE. coli\u003c/em\u003e bacteria. This result ensures that his hydrogel was effective against micro-organisms.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eAnti-oxidant test\u003c/h3\u003e\n\u003cp\u003eThe synthesised hydrogel showed exceptional antioxidant activity as per the DPPH assay. About 37% inhibition of free radicals was noted at the highest concentration of 25\u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). This is assigned to the prepared hydrogel combining more effectively with the free radicals formed by DPPH in the working solution at the higher concentration. As a result, increased scavenging was observed.\u003c/p\u003e \n\u003ch3\u003eBiodegradability\u003c/h3\u003e\n\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e(b)\u003c/b\u003e, the bio-degradability study of the prepared hydrogel is represented. Biodegradability is one of the expected properties of hydrogels for a safe and sustainable environment. From this point of view, agro-based, as well as polysaccharide-based hydrogels are the best choice, as they contain glycosidic linkages between glucose units and finally degrade to CO\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eO and other non-toxic material. But the bio-degradation can be delayed when an antimicrobial agent is added into hydrogel preparation. In such a case, citric acid acts as an antimicrobial agent, as it is well known that citric acid provides multiple functional groups and is also able to create active binding sites. It is well-known that citric acid has antibacterial properties.\u003c/p\u003e \u003cp\u003eIn this experiment, the prepared potato starch-based hydrogel degraded more slowly, due to the introduction of the antimicrobial agent. The synthesised hydrogels proved their biodegradability by 72% degradation after 8 weeks\u0026rsquo; burial. The graphical representation and the trend line show a very clear, linear relationship between biodegradability and time up to 42 days. After that, degradation seems to stop. From this experiment, we can also say that the prepared hydrogel was bio-degradable in nature and no adverse effect on the environment like conventional wound dressing materials have.\u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003eCell viability test\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e shows that the cell viability for control (using \u003cem\u003evero\u003c/em\u003e cells in blank culture medium) was 91.87% \u0026plusmn; 2.51%. The sample, using prepared hydrogel of approximately 1.5 x 0.9 cm\u003csup\u003e2\u003c/sup\u003e dimensions and with a weight of approximately 50 mg, was placed in 10 mL of culture medium. After 3 days in the medium, cell viability was 86.97% \u0026plusmn; 2.11%. From this result, it can be concluded that the cell viability was not changed significantly [p value, ns (using two-tailed unpaired t test)].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe newly-developed potato starch-based hydrogel was synthesised and characterised successfully. The FTIR data proved the successful copolymerisation of PLA and acrylamide with starch and also the cross-linking of copolymer chains by citric acid molecules. The presence of nitrogen atoms in the prepared hydrogel network, found by EDX analysis, also proved the successful grafting of acrylamide on to PLA and starch. The XRD data, along with FTIR data, confirmed the lower crystallinity and higher elastic properties of the prepared hydrogel compared to its raw components. The swelling and water vapour transmission rates (WVTR) was 481 (at pH 7.2) and 148 g/m\u003csup\u003e2\u003c/sup\u003eh, respectively, which suggest that this prepared hydrogel will be a good absorbent, and wound dressing material. The anti-microbial test of the prepared hydrogel also showed good activity against both gram-positive (\u003cem\u003eS. aureus)\u003c/em\u003e and gram-negative (\u003cem\u003eE. coli)\u003c/em\u003e bacteria, having a zone of inhibition 21mm and 19mm, respectively. This good result is assigned to hydrogen ion donation from the carboxyl group of citric acid (as an anti-microbial) in the hydrogel network. The prepared hydrogel showed a good cell viability. It was found to be non-toxic and harmless to human skin. Therefore, dermal use is indicated. The synthesised hydrogel was found to have a mild antioxidant property (scavenging of 37.2% for 25\u0026micro;g/ml). It also showed high bio-degradability (72% within 8 weeks).\u003c/p\u003e \u003cp\u003eAll these properties of the newly-developed hydrogel lead to the conclusion that the hydrogel has potential application as a wound dressing material. The high swelling rate of the prepared hydrogel ensured its high porosity and prevalence of interstitial spaces. Further investigation of the suitability of this hydrogel as a drug-carrier for specific drugs is recommended.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMd. Ibrahim H. Mondal\u003c/strong\u003e:\u0026nbsp;Conceptualization, Resources, Supervision, Funding acquisition, formal analysis, Writing\u0026nbsp;–\u0026nbsp;Reviewing and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH. Jahan Kadri:\u0026nbsp;\u003c/strong\u003eMethodology,\u0026nbsp;Investigation, Formal analysis, Visualization,\u0026nbsp;Writing\u0026nbsp;–\u0026nbsp;Original draft.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFiroz Ahmed:\u003c/strong\u003e Methodology,\u0026nbsp;Investigation, Formal analysis, Visualization,\u0026nbsp;Writing\u0026nbsp;–\u0026nbsp;Original draft, Writing\u0026nbsp;–\u0026nbsp;Reviewing and editing.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eMd. Hasinur Rahman:\u003c/strong\u003e Formal analysis, Visualization,\u0026nbsp;Writing\u0026nbsp;–\u0026nbsp;Reviewing and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are contained within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest or competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMondal MIH, Haque MO, Islam MA, Ahmed F (2017) Preparation and Characterization of Carboxymethyl Cellulose-g-Acrylamide Hydrogel. 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Int J Molecul Sci 19(4):1114. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms19041114\u003c/span\u003e\u003cspan address=\"10.3390/ijms19041114\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"polymer-bulletin","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pobu","sideBox":"Learn more about [Polymer Bulletin](http://link.springer.com/journal/289)","snPcode":"289","submissionUrl":"https://submission.nature.com/new-submission/289/3","title":"Polymer Bulletin","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Starch, Hydrogel, Anti-bacterial, Biodegradable, Anti-oxidant, Wound dressing, cell viability","lastPublishedDoi":"10.21203/rs.3.rs-4946153/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4946153/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA starch-based hydrogel was synthesised by direct grafting of polylactic acid (PLA) and acrylamide onto starch. Potassium persulphate (K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e) was used as an initiator and citric acid (CA) was used as an eco-friendly cross-linker. The purpose of the graft was to test an alternative anti-microbial wound dressing material. The FTIR, XRD, SEM and EDX data confirmed that the copolymerisation and cross-linking of the hydrogel was successful. Tests, with chemical reactions; yielded TGA data, which confirmed the enhanced thermal and mechanical properties of the augmented hydrogel. The hydrogel swelled up to 481% at pH 7.20 and exhibited a water vapour transmission rate of 148 g/m\u003csup\u003e2\u003c/sup\u003e per day. The hydrogel also showed anti-microbial activity against both gram-positive (\u003cem\u003eS. aureus)\u003c/em\u003e and gram-negative (\u003cem\u003eE. coli)\u003c/em\u003e bacteria. Its zone of inhibition was 21 mm and 19 mm with a mild anti-oxidant property. This synthesised hydrogel is completely non-toxic and bio-degradable, making it especially suitable as a wound dressing material.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Synthesis and characterization of starch-g-polyacrylamide-co-polylactic acid hydrogel for the potential wound dressing application","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-20 15:27:02","doi":"10.21203/rs.3.rs-4946153/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-03T08:22:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-02T15:31:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-26T07:58:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"82406963448679640440027547215770802263","date":"2025-01-23T06:12:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"198920231103251602052303812994478486649","date":"2025-01-16T16:08:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"304359923381441375909504562650390169175","date":"2025-01-15T06:59:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"264043048583484957078651257390770267390","date":"2025-01-14T23:18:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"238027967389958310927124079981341254265","date":"2025-01-14T16:00:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-14T15:53:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-22T07:49:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-21T07:42:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Polymer Bulletin","date":"2024-08-20T15:27:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"polymer-bulletin","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pobu","sideBox":"Learn more about [Polymer Bulletin](http://link.springer.com/journal/289)","snPcode":"289","submissionUrl":"https://submission.nature.com/new-submission/289/3","title":"Polymer Bulletin","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b369e78f-535e-4661-98f0-80e43e957271","owner":[],"postedDate":"September 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-04T09:08:56+00:00","versionOfRecord":{"articleIdentity":"rs-4946153","link":"https://doi.org/10.1007/s00289-025-05840-1","journal":{"identity":"polymer-bulletin","isVorOnly":false,"title":"Polymer Bulletin"},"publishedOn":"2025-06-11 15:56:54","publishedOnDateReadable":"June 11th, 2025"},"versionCreatedAt":"2024-09-20 15:27:02","video":"","vorDoi":"10.1007/s00289-025-05840-1","vorDoiUrl":"https://doi.org/10.1007/s00289-025-05840-1","workflowStages":[]},"version":"v1","identity":"rs-4946153","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4946153","identity":"rs-4946153","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}