1. INTRODUCTION:

In contemporary food system the use of packaging materials in food products is considered as most critical component that acts as a protective covering^1,2of the product from physical impact and microbial contamination which improves the shelf life.^3,4 Synthetic petroleum-based packaging is the most widely used packaging type at the present time⁵. However, its environmental impacts have suffered from its low degradation into non-degradable litter⁶. It has been developed initially as a friendlier to the environment packaging, edible film-based packaging materials⁷. However, bio-composite materials have also developed ⁸ and are also being increased with pectin. They could also ultimately be an edible film⁹ further delivering all of the ingredient’s bonding¹⁰. Initially with edible films, the most important quality of the film is that it must be resistant to food destruction by microorganisms^11,12–14 and also for its mucoadhesive properties¹⁵.

Chitosan, derived from chitin, discovered in 1859 by Professor C. Rouget has antimicrobial properties useful in various fields¹⁶. SA, from brown seaweed, first isolated by British chemist E.C.C. Stanford in 1881 is used for gelling, stabilizing, and antimicrobial properties in various industries¹⁷. The antibacterial activities of chitosan are achieved through the polymer being positively charged as well as the bonded amine group. However, chitosan has a drawback of leaching under the conditions of high-water absorption and generalized acidity¹. This defect can be either removed with the making of a PEC¹⁸ or a PEC film.^19,20,21.

PECs are developed mainly from polycationic polymers, like chitosan, and polyanionic polymers, including alginate, κ-carrageenan²², or sodium carboxymethyl cellulose (CMC-Na)²³. Synthesis occurs due to the electrostatic interactions that take place at acceptable pH levels, and the parameters that affect PEC yield and properties include ratios of each polymer, pH, and the stirring speed. The characterization of PECs has been done using several techniques (FTIR, SEM, XRD, EDS) ²⁴ and confirmed PEC formation and some of its structural properties^22,23. PECs are often produced as films with uneven, fibrous surface structures, or as aerogels that have high specific surface areas (380-400 m2/g) and porous structures^22,25. One remarkable property of these PEC materials is their improved antibacterial activity against gram-positive and gram-negative bacteria, like E. coli, S. aureus, Bacilus Subtilis ^23,25. It is thought that this property is a function of the amine groups that become protonated (NH3+) in the formulation, which leads to a positive charge, allowing the PEC to interact directly with bacterial cell membranes, increasing permeability, and cell death^22,23. Both studies reported the benefits of using natural, non-toxic polymers, in combination with the simple method to prepare PEC, suggesting large-scale use of PEC materials could occur across several industries.^{22,23,25,26,27}

In this work we focus on worked on a novel PEC. using a method where the chitosan was used as polycationic and formed together as a film with SA and carboxylated-sodium alginate which acts as a polyanionic. The chitosan is acting as a polycationic and sodium alginate and carboxylated-sodium alginate are acting as polyanionic, inner-face materials that formed electrostatic bonds with no physical crosslinking (NH3+) into the films, lining them with are polymer electrolyte complex films for primary use to block oxidation²⁸. The physical appearance supports the idea that if there are any changes in structure. FTIR support there are formation of complex study. The antimicrobial study will determine some of the effectivity on complex activity towards antimicrobial the gram (+) and gram (-) means broad spectrum or narrow spectrum. Therefore, the proposed ultimate goal of this study is a development of how to obtain an effective environmentally friendly materials with good antimicrobial properties and their related supportive test for food preservation and packaging^29–31.

2. MATERIALS AND METHODS:

2.1. Materials:

CS of medium Molecular weight 150-500m. Pas, obtained from SRL chem laboratory, SA mol weight was obtained from LOBA Chemie of 30 CPS viscosity (1% Solution) Laboratory, glacial acetic acid, NaOH and deionized water are obtained from SRL chem laboratory and Solid agar media obtained from Hi Media Laboratories Limited and Escherichia coli having stain number ATCC 25922 and Bacillus subtilis having stain number ATCC 6633. Both strains were cultured and maintained under standard laboratory conditions to ensure viability and reproducibility in antimicrobial testing.

2.2 Chitosan-Sodium Alginate Complex Preparation:

CS-SA Complex was prepared by mixing 1:1 molar ratio. 1% of CS was dissolved in 20ml of glacial acetic acid while 4% of SA was dissolved in 20ml of deionized water under magnetic stirrer (REMI 2MLH) at 600rpm for 4hours at room temperature. Then previously dissolved CS solution was added to SA solution to obtained a white coloured complex which then centrifuged, homogenized and poured on a Petri plate and then confirm of uniform thickness and dried at 55^oC at Hot air Oven until a constant weight of that complex film was obtained. Then it was preserved in a hermitically sealed container till further use²².

2.3 Preparation of Carboxylated-Sodium Alginate:

SA was derivatized to carboxylated-sodium alginate having substitution in following method. In brief, the process of producing carboxylated-sodium alginate which comprises suspending required quantity of SA in an ice-cold deionized aqueous solution of not more than 60% w/v sodium hydroxide. Then the dispersion was kept at 5-8℃ for 1h. After that the resulting solution was mixed with 1.5gm of monochloroacetic acid solution and the temperature was raised slowly to 15-18℃, after 30 mins, temperature was increased to 75℃ and maintained for additional 30mins. Wetted mass was collected and washed with 20ml 80% methanol three times and maintained pH at neutral by glacial acetic acid and dried at 50-60℃, dried product was milled, washed and redried³².

R-OH (Alginate) + ClCH₂COONa → R-OCH₂COONa (Carboxymethyl Alginate) + NaCl

Where R represents the alginate polymer backbone.

2.4 Chitosan-Carboxylated Sodium alginate film preparation:

CS-CMA film was prepared with chitosan: CMA of 1:1 ratio. CS was dissolved in glacial acetic acid in a ratio following the same procedure as stated before of 1% and carboxylated-Sodium Alginate was dissolved in deionized water in a ratio of 4% with the help of magnetic stirrer at 600rpm (REMI 2MLH) for 4 hours at room temperature. Subsequently, centrifuged, homogenized and poured on a Petri plate and then confirm of uniform thickness and dried at 55^oC at Hot air Oven until a constant weight of that complex film was obtained. Then it was preserved in a hermitically sealed container till further use²².

2.5 Physical Characteristic test:

2.5.1 Loss on Drying: Weigh 1g of each film sample PEC-CS&SA, PEC-CS&CMA) accurately. Now, Place samples in a hot air oven at 60°C for 3hours. After that, put the samples in a desiccator and reweigh. Calculate the loss on drying using the formula (Ferreira et al. 2022)

Loss on Drying (%) = [(Initial weight - Final weight) / Initial weight] × 100

2.5.2 Optical Microscopy: Prepare thin sections of each film sample. Observe under an optical microscope at 400x magnification. Now, capture images and describe observed features³³.

2.5.3 Solubility: Cut 2cm × 2cm squares of each film and weigh. Immerse samples in 50mL of distilled water at room temperature for 24 hours. Remove undissolved film, gently blot dry, and weigh. Calculate solubility using the formula³⁴:

Solubility (%) = [(Initial weight - Final weight) / Initial weight] × 100

2.6 FTIR:

IR spectra were recorded on a Bruker, FTIR spectrophotometer using KBr optics. Each sample was mixed with KBr and converted into disc at 100kg pressure using a hydraulic press. The spectra were recorded within 4000-400 cm^-1 wave numbers.

2.7 NMR Study of CMA:

To confirm the Carboxylation of Sodium alginate 13C NMR spectroscopy analysis was conducted on a VARIAN 400 MHz NMR instrument using deuterium oxide (D2O) as the solvent. The data comprises several important chemical shifts in parts per million (ppm), spanning from the aliphatic to the carboxylic regions, indicative of the structural components of the sample.

2.8 Antibacterial activity test:

The antimicrobial properties of PEC films are evaluated via Agar plate method in which the Solid agar media is used to check the bacterial sensitivity which contains Bacillus subtills as gram (+) and Escherichia coli (gram (-)) as a colony forming unit at a concentration of approximately 10⁶ CFU/ml. Sterilized paper discs were carefully placed on the inoculated agar and dosed with 20 microliters of the hydrosol film solution. As a positive control, 10milligrams of Amoxycillin were applied to another disc per manufacturer's instructions while deionized water served as the negative control. Following incubation at 37°C for 24hours, the diameter of any observable inhibition zones surrounding each disc was measured. All experiments were conducted thrice to ensure reliability and reproducibility of the findings²².

3. RESULT:

3.1. Formation of PEC films:

The polymer complexation process yielded PEC-CS&SA with a drying to constant weight efficiency of 88.59%, and PEC-CS&CMA with a drying to constant weight efficiency of 90%. From a physical examination, the films were observed to have a light-yellow colour, were rough in texture, and initially had a slightly pungent odour, which dissipated. The films were prepared as dry films once dimensioned to size 9×9 cm before cutting of the films to 2×2cm pieces for experimental assessments.

3.2FTIR Result:

The FTIR spectra (Figure 2) of the separate polymers and their complexes showed discernible spectral features. CS exhibited distinct peaks at 3390-3263 cm⁻¹ (O-H and N-H stretching), 2989-2823 cm⁻¹ (C-H stretching), 1649 cm⁻¹ (amide I), and 1595 cm⁻¹ (amide II). SA exhibited distinct peaks at 3600-3200 cm⁻¹ (O-H stretching), 2900 cm⁻¹ (C-H stretching), 1649 cm⁻¹, and 1436 cm⁻¹ (carboxylate stretching).

PEC-CS&SA represented multiple shifted peaks which include a broad band at 3390-3263 cm⁻¹ (O-H and N-H stretching), 2989-2823 cm⁻¹ (C-H stretching), 1694 cm⁻¹ (shifted amide I), 1562 cm⁻¹ (amide II), a new peak at 1515 cm⁻¹ (ionic interaction), and 1029 cm⁻¹ (C-O-C stretching).

PEC-CS&CMA exhibited peaks at 3428.94 cm⁻¹ (O-H and N-H stretching),1694.55 cm⁻¹ (C=O stretching), 1640.20 cm⁻¹ (N-H bending), and 1594.83 cm⁻¹ (asymmetric COO⁻ stretching). The changes in spectra seen in the complexes in comparison to the individual polymers confirm that the polyelectrolyte complex formed due to ionic interactions.

3.7 Antimicrobial Activity test: Antimicrobial testing (Figure 5, Table 2), demonstrated variability in the efficacy of the PECs against the test microorganisms. PEC-CS&SA showed zones of inhibition (ZOI) measuring 14.3mm against Escherichia coli (Gram-negative), and 10.4mm against Bacilus subtilis (Gram-positive), demonstrating broad-spectrum antibacterial efficacy.

In contrast, PEC-CS&CMA was demonstrated to have selective antimicrobial action with a 14.0mm ZOI against Escherichia coli, but only 1.5mm against Bacilus subtilis, demonstrating preferential activity against Gram-negative bacteria. Collectively, these results suggest that both Polylactide-pea protein complexes contain greater antimicrobial activity compared to their individual polymer components, with PEC-CS&SA demonstrating activities against both Gram-negative and Gram-positive bacteria.

4. DISCUSSION:

4.1 Formation of PEC films:

The high yields for PEC-CS&SA and PEC-CS&CMA (88.59% and 90%, respectively) are indicative of efficient complex formation through electrostatic interactions of oppositely-charged polysaccharides. Our results are in agreement with Ferreira et al. (2022) who found similar yields (85-92%) in their study of preliminary studies on polyelectrolyte complexes between chitosan and carboxymethylcellulose made under similar preparation conditions³³. Evidence of successful complex formation can be attributed to the strong electrostatic attractions between the positive amine groups of chitosan and the negatively charged carboxyl groups of the alginate derivatives³⁵.

The physical characteristics of the films, including pale yellow colour and uneven surface texture, is consistent with a study by Ismillayli et al. (2020) describing chitosan-based polyelectrolyte complex films²². We believe that the phantom-like surface roughness observed at about the film surface is caused by a heterogeneous distribution of the polymer chains through the complex formation; as recently reported by Yang et al.(2023) in the study of chitosan-carboxymethyl cellulose complexes. The dimensionally stable size of the films (9×9cm) exhibits good film forming capability; Barik et al. (2024) included film formability as an important characteristic for forthcoming food packaging applications³⁶. The increasingly less pungent smell after three weeks also suggests that the remaining acetic acid molecule vapourised or evaporated after storage, and agrees with Zhang et al. (2021) who reported similar sensory behaviour post- processing in hydrogel composites³⁷.

4.2 FTIR:

FTIR spectra analysis confirms effective polyelectrolyte complex formation as indicated by significant peak shifts typical of ionic interactions between the polymers. The shifts of the amide I band from 1649 cm⁻¹ in pure chitosan to 1694 cm⁻¹ in the PEC-CS&SA and 1694.55 cm⁻¹ in the PEC-CS&CMA are as reported by Brugnerotto et al. (2001), who recorded similar spectral shifts in complexes of chitosan formed as a result of electrostatic interaction³⁷.

The appearance of an additional peak at 1515 cm⁻¹ in PEC-CS&SA is particularly significant because it indicates the occurrence of ionic interaction between NH₃⁺ groups present in chitosan and COO⁻ groups present in alginate. The observation concurs with the findings of a study by Xu et al. (2007) that exhibited similar peaks in crosslinked blends of chitosan and alginate (38). Zhao et al. (2021) also presented additional evidence that the spectral changes are coupled with improved mechanical properties along with stability of the resulting complexes²⁶.

The wide bands between 3600-3200 cm⁻¹ corresponding to N-H and O-H stretching are less intense in the PECs than in pure polymers. This intensity weakening is equivalent to hydrogen bonding between the polymers, as earlier elucidated by Zhang et al. (2018) for porous polyelectrolytes²⁸. Recent work by Gorshkova et al. (2024) revealed that these hydrogen bond interactions are largely accountable for stability and functionality of chitosan-alginate complexes²⁵.

For PEC-CS&CMA, stronger asymmetric COO⁻ stretching peak at 1594.83 cm⁻¹ reflects higher negative charge density due to carboxymethylation, which Seidi et al. (2021) theorized increases electrostatic interaction with positively charged chitosan²⁰.

4.3 Loss on Drying: The identical water content values found for PEC-CS&CMA (9.5%) and PEC-CS&SA (9.7%) suggest that both complexes have equivalent hygroscopic characteristics. According to Pirsa and Mohammadi (2021), these values are within the ideal range (8–12%) for polysaccharide-based films. They showed that this moisture content offers appropriate flexibility without sacrificing stability³⁴. Since too much moisture can cause premature degradation or decreased mechanical strength, and too little moisture can result in brittleness, as Rajabi et al. (2021)²¹ have observed, intermediate water retention is advantageous for preserving film integrity throughout storage and application.

4.4 Optical Microscopy Discussion:

Microscopic examination of the samples showed results identical to the findings of Yang et al. (2023) who studied chitosan-cellulose polyelectrolyte complexes²³. The irregular surface with visible fibers in PEC-CS&SA and more pronounced fibrous structures in PEC-CS&CMA result from different electrostatic interaction patterns during complex formation. Rodríguez Sánchez et al. (2022) showed that particle fibrous shape enhances efficacy because its large surface contact facilitates microbial cell interaction³⁹.

4.5 Solubility:

The PEC-CS&SA and PEC-CS&CMA solution levels (62.4% and 60.8% respectively) achieve equilibrium by incorporating alginate derivative water solubility with the restricted pH neutral solubility of chitosan⁴⁰. The PECs exhibit suitable water solubility according to Ardean et al. (2021) that enables sustained antimicrobial effects because intermediate dissolution rates are beneficial for controlled drug delivery systems (19). The slightly reduced solubility of PEC-CS&CMA is attributed to strong electrostatic effects stemming from increased carboxyl content in agreement with Dangi et al. (2023) who studied gum-based films¹¹.

4.6 NMR:

¹³C NMR spectral data confirming that carboxymethylation of sodium alginate is successful was due to the appearance of characteristic carbon resonances in the 173 – 181ppm range corresponding to carboxyl groups. This supports previous research by Yue et al. (2021) who identified similar chemical shifts for modified alginate derivatives⁴¹. Maintaining such peaks at 63.321ppm (C2 carbon atom in alginate backbone), and 61.135ppm (C3 carbon atom in alginate backbone) is observed and is in alignment with Furevi et al., (2022) (42) for observing that the primary polysaccharide structure is retained during modification.

The power of the carboxylate region peaks indicates hydroxyl groups are successfully substituted by carboxymethyl moieties. McNeely and O'Connell's foundation³² work establishes that this subsitution pattern primarily affects the guluronic acid residues of alginate, and thus CMA has a markedly higher negative charge density relative to native alginate. In recent studies, Rodríguez Sánchez et al. (2022) have proven that this specific pattern of selective modification improved functional properties while leaving the polysaccharide structure intact³⁹.

4.7 Antimicrobial Activity test:

The presence of complex formation enhances the antimicrobial activity of the polyelectrolyte complexes versus their individual polymer components in a synergistic effect. Despite broad spectrum activity against both Gram positive B subtilis (10.4mm ZOI) and Gram-negative Escherichia coli (14.3mm ZOI), PEC-CS&SA also demonstrated selective activity against Escherichia coli (14.0mm ZOI) with little effect against B subtilis (1.5mm ZOI), PEC-CS&CMA.

Such antimicrobial effects are attributable to a number of underlying mechanisms. According to Ismillayli et al. (2020), these NH₃⁺ in the PEC structure can interact with the anionic parts of the bacterial cell membranes, which increases permeability and eventually results in cell death²². We demonstrate in our study that PEC-CS&CMA is particularly effective against Gram negative bacteria since the bigger zones of inhibition seen for Escherichia coli against the two complexes, which is in good accordance with Mai-Prochnow et al. (2016) who found that cell wall structure strongly correlated with antimicrobial susceptibility²³. The difference in the depth of the peptidoglycan layer in Gram-positive bacteria (20-80nm) from Gram-negative bacteria (<10nm) is hypothesized to hinder the interaction between the complex and cell components, thereby decreasing the efficacy against B. subtilis.

Gorshkova et al. (2024) recently showed that alginate brought about similar selective antimicrobial activity with a higher activity against Gram negative bacteria due to stronger interaction with the outer membrane lipopolysaccharides²⁵. Barik et al. (2024) also noted that modification of polysaccharides with carboxymethylation may affect the antimicrobial profile by changing the charge distribution and polymer conformation³⁶.

Our complexes have better comparative antimicrobial efficacy compared to the systems of Yang et al. (2023) for similar polyelectrolyte systems²³, indicating that the use of specific polyelectrolyte ratio and preparation methods in our study were more effective in achieving antimicrobial properties. In addition, PEC-CS&SA has dual action capability against both bacterial types, which is more versatile than selective agents—similar to Zhao et al. (2021) study of polysaccharide polyelectrolyte complexes’ versatility¹⁹.

4.6 Potential Applications and Future Directions:

Because of the demonstrated antimicrobial properties of the PEC films along with the fact that the films are biodegradable, they are promising candidates for food packaging applications⁴³. Infact, Hegab et al., also stated that there is a general need for active packaging products to provide both protective and antimicrobial functions in order to prolong shelf life of food (Ghoshal, 2018) (3). With respect to this trend, our PEC films fall well aligned, requiring no preservatives or packaging in synthetic forms.

Seidi et al. (2021) suggests that the controlled solubility of the complexes is a good opportunity for targeted drug delivery applications referring to the use of chitosan-based blends in biomedical field ²⁰. In particular, the variable antimicrobial profiles of PEC-CS&SA and PEC-CS&CMA could be utilized in the different therapeutical needs⁴⁴: general antimicrobial purposes by broad spectrum profiles and selective antimicrobial activity with respect to Gram negative pathogens in devoted indications⁴⁵.

The optimization of particular properties is also possible in future research along with additional bioactive compounds for multifunctional capabilities and study of scaling parameters for industrial production⁴⁶. The commercialization potential of biopolymer complexes⁴⁷ depends on reproducible performance in many environmental conditions⁴⁸ as the ability of the water-soluble biopolymers (complexing agent) to complex metal ions is influenced by temperature, pH, and humidity (49).

5. CONCLUSION:

The study demonstrates the successful synthesis of polyelectrolyte complexes (PECs) through ionic interaction between CS and SA or carboxylated-sodium alginate, which was confirmed by physical observation and further supported by FTIR analysis. The PECs exhibit enhanced antibacterial activity compared to parent polymers, particularly against Bacilus subtilis and Escherichia coli. The antimicrobial activity of PEC-CS &SA against Gram (+) bacteria was ZOI of 10.4 mm and Gram (-) bacteria it was 10.3 mm. But PEC-CS&CMA exhibited very high Gram (-) activity with ZOI of 14 mm whereas it exhibits limited activity towards Gram (-) bacteria with ZOI of 1.5 mm.

The biodegradable, non-toxic, and edible nature of these complexes, along with increased antimicrobial activity, makes such complexes valuable candidates for application as edible films or coatings in food preservation. The electrostatic interactions between the polymers, as confirmed by FTIR, and the fibrous and rough surface texture as determined by optical microscopy, are critical determinants of the unique properties of these PECs. In summary, these findings suggest that PEC films have tremendous potential to prevent bacterial contamination and growth in food systems and are therefore a safe and effective method to extend shelf life and ensure food safety.

7. ACKNOWLEDGEMENTS:

The authors are extremely thankful to Instrumentation Laboratories in Department of Pharmaceutical Technology in Jadavpur University.

8. AUTHOR CONTRIBUTIONS:

Arindam Sarkar and Sanchita Mandal planned the experiment. Arindam Sarkar performed the experiment, carried out the calculations, interpreted results and discussion, and wrote the manuscript. Shila Barman contributed to the interpretation of the result. Sanchita Mandal contributed to the overall supervision and guided on manuscript writing, editing and reviewing.

15. REFERENCE:

1. Li J, Van Ewijk G, Van Dijken DJ, Van Der Gucht J, De Vos WM. Single-Step Application of Polyelectrolyte Complex Films as Oxygen Barrier Coatings. ACS Appl Mater Interfaces. 2021 May 12;13(18):21844–53.

2. Abbas HA, Serry FM, EL-Masry EM. Combating Pseudomonas aeruginosa Biofilms by Potential Biofilm Inhibitors. 2(2).

3. Ghoshal G. Chapter 10 - Recent Trends in Active, Smart, and Intelligent Packaging for Food Products. In: Grumezescu AM, Holban AM, editors. Food Packaging and Preservation [Internet]. Academic Press; 2018 [cited 2024 Nov 22]. p. 343–74. (Handbook of Food Bioengineering). Available from: https://www.sciencedirect.com/science/article/pii/B9780128115169000105

4. Pathak KK, Pateria MA, Deshmukh K. Comparative Study of Optical and Electrical Properties of CdSe: Sm and CdSe: Nd Nanocrystalline Thin Film. Rese Jour of Engin and Technol. 2018;9(1):67.

5. Patil A, Basawaraj R, Ali K, Joshi A, Nanjwade B. Design and Evaluation of Gatifloxacin Ocular Films for Sustained Release.

6. Pitrubhakta JR, Kere TA, Shinde SS, Soni SA, Jadhav VR. Synthesis, Characterization, and Gas Sensing performance of Nanometer TiO2 thick film by Hydrothermal method. Asian Journal of Research in Chemistry. 2020;13(5):360–4.

7. Sadique S, Ramya SS. Preparation and Evaluation of Fast Dissolving Oral Film of Losartan Potassium. Rese Jour Pharmaceut Dosag Form and Technol. 2020;12(1):13.

8. Roy S, Priyadarshi R, Rhim JW. Gelatin/agar-based multifunctional film integrated with copper-doped zinc oxide nanoparticles and clove essential oil Pickering emulsion for enhancing the shelf life of pork meat. Food Research International. 2022 Oct; 160:111690.

9. Savale PA. Comparative Study of Various Chemical Deposition Methods for Synthesis of Thin Films: A Review. Asia Jour Rese Chem. 2018;11(1):195.

10. Shrivastava K, Sahu S, Mishra SK, De K. In vitro Antimicrobial Activity and Phytochemical Screening of Syzygium aromaticum. 4(1).

11. Dangi N, Attri S, Tomar S, Guleria P. Characterization of Novel Films and Coatings Based on Gums. In: Biopolymer-Based Films and Coatings. CRC Press; 2023.

12. Thakur M, Sharma I, Moda M, Sharma N. Fast Dissolving Films: A Review. AJPS. 2023 Aug 26;211–7.

13. Tiwari P. Antimicrobial Activity of Ashwagandharishta Prepared by Traditional and Modern Methods. 4(3).

14. Varsha A, Yadav KS, Bindaiya S. Formulation Development and Evaluation of Fast Dissolving Films of Oloptadine HCl. AJPS. 2021 May 10;11(2):103–8.

15. Arindam Sarkar SB. Sodium Alginate in Mucoadhesive Drug Delivery Systems: A Comprehensive Review of Properties, Dosage Forms, And Characterization Methods. 2025 Jan 13 [cited 2025 Apr 21]; Available from: https://zenodo.org/doi/10.5281/zenodo.14636331

16. Masuelli M, Renard D. Advances in Physicochemical Properties of Biopolymers (Part 2). Bentham Science Publishers; 2017. 497 p.

17. Volod’ko AV, Davydova VN, Petrova VA, Romanov DP, Pimenova EA, Yermak IM. Comparative Analysis of the Functional Properties of Films Based on Carrageenans, Chitosan, and Their Polyelectrolyte Complexes. Marine Drugs. 2021 Dec 12;19(12):704.

18. Zhang S, Lu Y, Ding Q, Yu Y, Huo P, Shi W, et al. MOF derived NiO thin film formed p-n heterojunction with BiVO4 photoelectrode for enhancement of PEC performance. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2022 Dec; 655:130282.

19. Ardean C, Davidescu CM, Nemeş NS, Negrea A, Ciopec M, Duteanu N, et al. Factors Influencing the Antibacterial Activity of Chitosan and Chitosan Modified by Functionalization. International Journal of Molecular Sciences. 2021 Jan;22(14):7449.

20. Seidi F, Khodadadi Yazdi M, Jouyandeh M, Dominic M, Naeim H, Nezhad MN, et al. Chitosan-based blends for biomedical applications. International Journal of Biological Macromolecules. 2021 Jul 31; 183:1818–50.

21. Rajabi M, McConnell M, Cabral J, Ali MA. Chitosan hydrogels in 3D printing for biomedical applications. Carbohydrate Polymers. 2021 May 15; 260:117768.

22. Ismillayli N, Andayani IGAS, Honiar R, Mariana B, Sanjaya RK, Hermanto D. Polyelectrolyte Complex (PEC) film based on chitosan as potential edible films and their antibacterial activity test. IOP Conf Ser: Mater Sci Eng. 2020 Oct 1;959(1):012009.

23. Yang D, Gong L, Li Q, Fan B, Ma C, He YC. Preparation of a biobased polyelectrolyte complex from chitosan and sodium carboxymethyl cellulose and its antibacterial characteristics. International Journal of Biological Macromolecules. 2023 Feb; 227:524–34.

24. Sahoo P, Sharma A, Padhan S, Thangavel R. Cu doped NiO thin film photocathodes for enhanced PEC performance. Superlattices and Microstructures. 2021 Nov; 159:107050.

25. Gorshkova NA, Brovko OS, Palamarchuk IA, Ivahnov AD, Bogdanovich NI, Vorob’eva TYa. Preparation of an Antibacterial Composite Aerogel for Biomedical Purposes Based on an Alginate–Chitosan Complex and Calcium Carbonate. Appl Biochem Microbiol. 2024 Apr;60(2):194–200.

26. Zhao T, Li X, Gong Y, Guo Y, Quan F, Shi Q. Study on polysaccharide polyelectrolyte complex and fabrication of alginate/chitosan derivative composite fibers. International Journal of Biological Macromolecules. 2021 Aug; 184:181–7.

27. Bala A, Mandal S. Spectrophotometric and HPLC analysis of amoxicillin trihydrate in presence of acetaminophen in different pH media. Futur J Pharm Sci. 2024 Dec 3;10(1):174.

28. Zhang W, Zhao Q, Yuan J. Porous Polyelectrolytes: The Interplay of Charge and Pores for New Functionalities. Angewandte Chemie International Edition. 2018;57(23):6754–73.

29. ‪History, Factors, Mechanism, Formulation, Evaluation, Application, And Advancement Of Gastro Retentive Drug Delivery System [Internet]. [cited 2025 May 10]. Available from: https://scholar.google.com/citations?view_op=view_citation&hl=en&user=m8Esiy4AAAAJ&sortby=pubdate&citation_for_view=m8Esiy4AAAAJ:eO3_k5sD8BwC

30. ‪A Detailed Discussion on Mucoadhesive Drug Delivery System [Internet]. [cited 2025 May 10]. Available from: https://scholar.google.com/citations?view_op=view_citation&hl=en&user=m8Esiy4AAAAJ&sortby=pubdate&citation_for_view=m8Esiy4AAAAJ:rbm3iO8VlycC

31. ‪Spectrophotometric Simultaneous Quantitative Analytical Analysis Method For Pcm-Met Developed And Validated [Internet]. [cited 2025 May 10]. Available from: https://scholar.google.com/citations?view_op=view_citation&hl=en&user=m8Esiy4AAAAJ&sortby=pubdate&citation_for_view=m8Esiy4AAAAJ:zGdJYJv2LkUC

32. Mcneely WH, O’connell JJ. Carboxymethyl alginate product and methods of preparing carboxymethyl alginate [Internet]. US2902479A, 1959 [cited 2024 Nov 28]. Available from: https://patents.google.com/patent/US2902479A/en

33. Ferreira DCM, Ferreira SO, de Alvarenga ES, Soares N de FF, Coimbra JS dos R, de Oliveira EB. Polyelectrolyte complexes (PECs) obtained from chitosan and carboxymethylcellulose: A physicochemical and microstructural study. Carbohydrate Polymer Technologies and Applications. 2022 Jun 1; 3:100197.

34. Pirsa S, Mohammadi B. Conducting/biodegradable chitosan-polyaniline film; Antioxidant, color, solubility and water vapor permeability properties. Main Group Chemistry. 2021 Jan 1;20(2):133–47.

35. Brugnerotto J, Lizardi J, Goycoolea FM, Argüelles-Monal W, Desbrières J, Rinaudo M. An infrared investigation in relation with chitin and chitosan characterization. Polymer. 2001 Apr 1;42(8):3569–80.

36. Barik M, BhagyaRaj GVS, Dash KK, Shams R. A thorough evaluation of chitosan-based packaging film and coating for food product shelf-life extension. Journal of Agriculture and Food Research. 2024 Jun 1; 16:101164.

37. Zhang M, Wang G, Zhang X, Zheng Y, Lee S, Wang D, et al. Polyvinyl Alcohol/Chitosan and Polyvinyl Alcohol/Ag@MOF Bilayer Hydrogel for Tissue Engineering Applications. Polymers (Basel). 2021 Sep 17;13(18):3151.

38. Xu Y, Zhan C, Fan L, Wang L, Zheng H. Preparation of dual crosslinked alginate-chitosan blend gel beads and in vitro controlled release in oral site-specific drug delivery system. Int J Pharm. 2007 May 24;336(2):329–37.

39. Rodríguez Sánchez RA, Matulewicz MC, Ciancia M. NMR spectroscopy for structural elucidation of sulfated polysaccharides from red seaweeds. International Journal of Biological Macromolecules. 2022 Feb; 199:386–400.

40. Phonrachom O, Charoensuk P, Kiti K, Saichana N, Kakumyan P, Suwantong O. Potential use of propolis-loaded quaternized chitosan/pectin hydrogel films as wound dressings: Preparation, characterization, antibacterial evaluation, and in vitro healing assay. International Journal of Biological Macromolecules. 2023 Jun;241:124633.

41. Yue W, Zhang HH, Yang ZN, Xie Y. Preparation of low-molecular-weight sodium alginate by ozonation. Carbohydrate Polymers. 2021 Jan;251:117104.

42. Furevi A, Ruda A, Angles d’Ortoli T, Mobarak H, Ståhle J, Hamark C, et al. Complete 1H and 13C NMR chemical shift assignments of mono-to tetrasaccharides as basis for NMR chemical shift predictions of oligo- and polysaccharides using the computer program CASPER. Carbohydrate Research. 2022 Mar;513:108528.

43. Dos Santos VS, Lorevice MV, Baccarin GS, Da Costa FM, Da Silva Fernandes R, Aouada FA, et al. Combining Chitosan Nanoparticles and Garlic Essential Oil as Additive Fillers to Produce Pectin-Based Nanocomposite Edible Films. Polymers. 2023 May 9;15(10):2244.

44. Kodan N, Ahmad M, Mehta BR. Charge carrier separation and enhanced PEC properties of BiVO4 based heterojunctions having ultrathin overlayers. International Journal of Hydrogen Energy. 2021 Jan 1;46(1):189–96.

45. Zhang M, Zhang M, Zhao Z, Zhu J, Wan X, Lv Y, et al. Preparation and characterization of intelligent and active bi-layer film based on carrageenan/pectin for monitoring freshness of salmon. International Journal of Biological Macromolecules. 2024 Sep 1;276:133769.

46. He M, Pan J, Hong M, Shen Y, Zhang H, Jiang Y, et al. Fabrication of antimicrobial packaging based on polyaminopropyl biguanide incorporated pectin/polyvinyl alcohol films for fruit preservation. Food Chemistry. 2024 Nov 1;457:140106.

47. Sreedhar A, Ta QTH, Noh JS. Rational engineering of morphology modulated Ti-ZnO thin films coupled monolayer Ti3C2 MXene for efficient visible light PEC water splitting activity. Journal of Electroanalytical Chemistry. 2022 Sep 15;921:116703.

48. Santana JV, Marangoni Júnior L, Cassol GZ, Sato HH, Vieira RP. Advanced pectin-based films: Enhancing antioxidant, antibacterial, UV barrier, and physicochemical properties upon oligomeric limonene derivative incorporation. Food Hydrocolloids. 2024 Apr 1;149:109558.

49. Xu F, Yan W, Pang D, Ren B, Tang Q, Wang X, et al. High-performance photoelectrochemical (PEC) type self-powered ultraviolet photodetectors (PDs) based on three-dimensional ZnO film/carbon fiber paper. Optical Materials. 2024 Apr; 150:115185.

Received on 20.03.2025 Revised on 23.08.2025

Accepted on 04.12.2025 Published on 02.07.2026

Available online from July 15, 2026

Asian J. Res. Pharm. Sci. 2026; 16(3):207-216.

DOI: 10.52711/2231-5659.2026.00032

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Polymer(complex)	*Gram (-) (Escherichia coli)* ZOI (mm)**			Average± SD	*Gram (+) (Bacillus subtilis) ZOI (mm)*			Average± SD
PEC-CS&SA	14.1	14.5	14.3	14.3 ± 0.20	10.2	10.5	10.6	10.4 ± 0.21
PEC-CS&CMA	13.9	14.0	14.1	14.0 ± 0.10	1.4	1.5	1.6	1.5 ± 0.10