Comparative efficacy of citric acid/tartaric acid/malic acid additive-based polyvinyl alcohol-starch composite films

Abstract

To ascertain upon the ideal configuration of physico-mechanical qualities, efficient processing techniques, and network stability of the prepared bio-composite films in real-world applications, the polymeric materials shall be subjected to a careful manipulation. Such bio-composite films have outstanding combinations of biocompatibility and toxicity-associated safety qualities. Such research interventions will be beneficial for the packaging, pharmaceutical, and biomedical industries that wish to target and adopt them for commercial applications. In this article, three alternate organic acids, i.e., citric acid (CA), tartaric acid (TA), and malic acid (MA), are blended separately into polyvinyl alcohol (PVA)-starch (St)-glycerol (Gl) composite films and for the targeted purpose of enhanced crosslinking, plasticizing, and antibacterial capability of the polymer network. The organic acid-based bio-composite polymeric films were assessed in terms of swelling index (SI), in vitro degradation, tensile strength (TS), percentage elongation (%E), antibacterial activity, and cytotoxicity attributes. Among these, the MA-based PVA composite films outperformed the CA-based PVA composite film in terms of absorbency (SI 739.29%), mechanical strength (TS 4.88 MPa), and elasticity (%E 103.68%). Furthermore, following a 24-h incubation period, the MA-based films exhibited the highest proliferative effect of 215.59% for the HEK cells. In conclusion, the MA has been inferred to be the most relevant organic acid for the desired optimality of film composition, physical and biological properties, and cost.

Introduction

Low-cost, biodegradable wound dressing materials necessitate upon the amalgamation of both synthetic and natural polymers. Accordingly, they facilitate the unavoidable utilization of synthetic polymers and overcome the higher cost of the natural polymers. Among several natural polymers, starch (St) is the most inexpensive, easily available, processable, and renewable natural polymer (Torres et al. 2013; Mogoşanu and Grumezescu 2014). It can be easily blended with various synthetic polymers for subsequent enhancement in the biodegradability. Further, the non-toxic nature of the St has been promising for the relevant application as a scaffold and tissue engineering material (Shen et al. 2019). Among various synthetic polymers, polyvinyl alcohol (PVA) has been significantly investigated in conjunction with the St. This is due to the biodegradability, non-toxicity, and transparent film-forming capability of the PVA-St composite (Ramaraj 2007; Popescu et al. 2018). However, along with the mentioned promising perspectives, the PVA-St composite has been characterized with a poorer combination of mechanical strength and higher water solubility (Raj and Somashekar 2004). To overcome this aspect, the crosslinking with suitable agents can improvise upon the compatibility attributes of the PVA-St composite films. Also, due to being non-toxic and antibacterial agents, the organic crosslinkers can promote the shape stability and strength characteristics of the composite film (Sekhavat Pour et al. 2015). Among several alternative additives, the citric acid (CA) has been a unique example due to the fact that few investigations suggested its efficacy in customizing the PVA-St hybrid hydrogel sheets for wound dressing applications (Shi et al. 2008; Wu et al. 2017). Devoting towards the optimality of various additives such as CA and Gl, Do et al. (2006b) opted for a constant constitution of PVA-St blend and assessed upon the influence of CA and Gl concentrations on various characterization parameters such as degree of swelling (DS), tensile strength (TS), percentage elongation (%E), and solubility (S) of the PVA-St composite films. The authors inferred that except for the solubility, the CA and Gl have been instrumental to enhance all other parameters. This is attributed to the multiple carboxyl groups (–COOH) that exist in their structures (Do et al. 2006a). In this regard, it shall be noted that while the Gl structure has only three hydroxyl groups (–OH), the CA has three –COOH groups and one hydroxyl group (–OH group) per monomer, and the compositional optimality reflects upon the optimal contribution of these groups to the mentioned parameters. The comprehensive research investigations reported by Shi et al. (2008) did convey that in the PVA-St-Gl-CA composite films, the %E always increased to reach greater values with CA concentration. However, the corresponding influence on the DS and TS values has been primarily detrimental. In other words, the composite’s residual free CA enhanced the % E and exacerbated the plasticization effect. Due to its chemical structure that contained multiple –COOH groups, the CA has been opined to participate in the esterification reaction and thereby enable the creation of hydrogen bonding in conjunction with the –OH groups of the PVA-St. As a result, the required properties of the composite film, namely TS, elasticity, water resistance, and swelling capability, have improved considerably (Shi et al. 2008). Thus, based on such structural analysis, other polycarboxylic organic acids such as tartaric acid (TA, with 2 OH and 2 COOH groups) and malic acid (MA, with 1 OH and 2 COOH groups) shall be also able to ascertain a comparable influence in terms of the enhanced grafting and crosslinking between the polymer chains. Thereby, positive influence upon the mentioned parameters shall be possible for both TA and the MA.

Recently, Chang et al. 2022 developed citric acid (CA) crosslinked WL hydrogel films for potential wound dressing applications. The conducted studies also elucidated upon the significance of using citric acid in terms of the esterification crosslinkage and its subsequent influence on the hydrogel’s structural integrity, swelling capacity, porosity, and mechanical properties (Chang et al. 2022). The reported findings are in very good agreement with those inferred from the earlier work of Shi et al. (2008). The CA crosslinked hydrogels exhibited pH-responsive water absorption, sustained drug release, superior biocompatibility, and long-lasting antibacterial activity. All these ascertained the prepared films to be highly promising for wound care application. The research investigation reported by Aizaz et al. (202e4) on the hydrogel films prepared through the crosslinked polyethylene oxide (PEO) and guar gum (GG) and with rosemary (RM) and citric acid (CA) optimal loading is also corroborative to the above-mentioned trends. Such porous hydrogel structure demonstrated efficacy in terms of moisture retention, cell attachment, and proliferation. Among the mentioned ingredients, the CA has been instrumental to promote neovascularization which is confirmed by the chorioallantoic membrane assay. Accordingly, enhancing tissue regeneration has been facilitated in the wound dressing film which is a highly desired property in the healing of a wound. Also, the reported hydrogel films exhibited antibacterial efficacy and controlled drug release. Such efforts render them to be a suitable candidate for the advanced burn care needs (Aizaz et al. 2024).

Further, few investigations targeted the assessment of the possible role of other organic acids for the improved characteristics of the PVA-St composite films. However, the investigators did not detail upon the optimality of their concentrations and as well did not delineate upon the application of PVA-St-organic additive-based mixed films for any industrial or commercial application. Do et al. (2006b) assessed the sensitive influence of various organic acids in the PVA-St composite films. For a constant composition of the PVA-St mix (5% each), the authors altered the MA and TA concentrations and in the range of 0 to 54.28 mmol. Accordingly, it was inferred that while %E and DS increased, the TS reduced (Do et al. 2006b). Similarly, Yun et al. (2006) deliberated upon the effects of CA, MA, and TA concentrations on the properties of the PVA-St blend-based composite films. The authors inferred that the TA produced the second-best results and after the CA. Comparatively, the MA performed poorly (Yun et al. 2006). Olivato et al. (2012) fabricated St-polyester-blown synthetic packaging films and with alternative organic acid additives. The authors considered the chemical alteration as follows. For a total of 90 wt% of polymer contents, 10 wt% of additives (Gl: organic acids (CA/MA/TA)) were added, and the CA or TA or MA concentrations were varied between 0.375 and 1.5 wt%. It was assessed that the composite films being prepared with higher organic acid constitution had higher combinations of water vapor permeability (WVP) and WL values. The second highest WL value was achieved for a 1.5 wt% composition of the TA additive. This is followed by the CA additive. For the MA case, the highest WVP values were achieved for a concentration of 1.5 wt%, and these were higher in comparison to those achieved with the TA or CA. Nevertheless, the WVP reduced for enhanced TA and CA concentrations beyond 0.75 wt%. Additionally, improved TS was noted for a concurrent increase in the TA and CA concentration. Conversely, the TS improved up to the enhanced MA concentration of 0.75 wt%. Beyond such concentration, the TS was detrimentally influenced. An increase in MA concentration improved %E in the composite film. For the CA case, the %E values increased upto 0.75 wt% of the organic acid additive and thereafter declined abruptly. In summary, the TA increment detrimentally influenced %E (Olivato et al. 2012). Thus, the relevant prior art precisely did not address upon the sensitive influence of various organic acids (MA and TA) on the plasticization capability, antimicrobial efficacy, and in vitro biocompatibility (Do et al. 2006b; Yun et al. 2006; Olivato et al. 2012). However, such studies have been targeted and reported for the CA-based PVA-St composite film and for various applications (Shi et al. 2008; Wu et al. 2017). While Olivato et al. (2012) focused on a qualitative analytical method and discussed upon the role of alternate organic acid additives in altering the physical characteristics of the films, the authors did not utilize the PVA as a base synthetic polymeric for the packaging application (Olivato et al. 2012). Such studies utilizing the PVA composite films will be as well promising for appropriate customization and design of synthetic hydrogel films for wound dressing applications.

Considering the above summarized research gaps, the central objective of this article is to examine the influence of the TA, MA, and CA as alternate organic acid additives in PVA-St composite films. Thereby, the desired optimal combinations of swelling index (SI), solubility, gel fraction (GF), in vitro degradation or weight loss (WL), mechanical properties (TS, %E), antibacterial efficacy, and relative growth rate (RGR) of PVA-St-Gl films are to be assessed. For this purpose, the four best inferred compositions of the CA-based PVA-St composite films were chosen from the findings of our investigation (Das et al. 2020a). Accordingly, the CA has been substituted with either TA or MA. Finally, based on both physical characterizations and qualitative analysis of wound dressing application, a comparative study has been conducted to assess upon the efficacy of the mentioned organic acids. Thus, the best organic acids among CA, MA, and TA would be evaluated to serve as cost-effective additives for wound dressing compatible films. Thereby, associated deeper introspections have been anticipated to provide useful insights into the exact role of alternate organic acids in altering and improvising composite film characteristics.

Experiments

Materials

The base PVA polymer was acquired from Merck Co. in Germany and has an average molecular weight of 72,000 g/mol with 98% hydrolyzation. Merck Limited, Mumbai, is the source of the following products: natural polymer starch (soluble for analysis (ACS)), citric acid (monohydrate for analysis (ACS)) (molar mass (M) = 210.14 g/mol), L( +)-tartaric acid (for analysis (ACS)) (molar mass (M) = 150.08 g/mol), DL-malic acid (M = 134.08 g/mol), and anhydrous glycerol (M = 92.09 g/mol). De-ionized water (DI), which was obtained from the analytical laboratory of the Department of Chemical Engineering at the Indian Institute of Technology in Guwahati, Assam, India, was used for the experimental investigation.

Film fabrication methodology

The overall PVA/St/organic acid/Gl casting solution blend fabrication, solution casting methodology, and the subsequent drying process of the casted film were similar to those being reported in our recent work (Das et al. 2019). In the former published articles, the adopted optimal drying parameters and formulations have been summarized by our research group (Das et al. 2020a, 2020b). In those research articles, the targeted novelty was to achieve an optimal composition of the CA-based PVA-St-Gl composite film. This was targeted through the variation of all four constituent concentrations. Thereby, the four best compositions being achieved for a combinatorial increment in the physical properties of the films were identified. Hence, flawless composite films were prepared and were subjected to additional characterization and analyses and through the sole replacement of the CA with other organic acids (TA or MA) in the manufactured films. Thus, while maintaining a similar film constitution of those inferred from our previous findings, only MA/TA have been replaced and relevant investigations were targeted. Table 1 summarizes the targeted compositions of the organic acid additive-based PVA-St-Gl composite films.

Table 1 A summary of deployed solution compositions for the fabrication of alternate organic acid-based PVA composite films

Characterization

ATR-FTIR spectra

The ATR spectra of the films were measured with a two FTIR spectrometer (Perkin Elmer (Singapore) Spectrum). The details were outlined in a preceding work (Das et al. 2019).

Swelling index

Representing the absorption capacity of the fabricated PVA composite films, the SI was evaluated with the procedure summarized by (Zou et al. 2007). The same has been detailed in our earlier research work that referred to film sample pieces (1 × 1 cm²) being subjected to appropriate treatment procedures (Das et al. 2019). Accordingly, the following expression was used to evaluate the SI:

$$SI=\frac{{W}_{s}}{{W}_{0}}\times 100\%$$

(1)

where ${W}_{0}$ and ${W}_{s}$ refer to the weights taken for the samples before experimentation and after 24 h of soaking in the phosphate buffer saline (PBS) solution, respectively.

In vitro degradation or hydrolytic degradation

The in vitro degradation (WL) was characterized for the PVA/St/organic acid/Gl composite film and with a hydrolytic media for a time duration of 27 days. Procedures elaborated in our earlier work were followed (Das et al. 2019, 2020b). Accordingly, the response was determined using respective terms and as follows:

$$WL=\frac{\left({W}_{0}-{W}_{f}\right)}{{W}_{0}}\times 100\%$$

(2)

where ${W}_{0}$ indicates the original sample’s dry weight, and ${W}_{f}$ represents the sample weight being dried at 37 °C following soaking in the PBS solution for 27 days.

Mechanical properties

For the fabricated PVA composite films based on organic acids, the TS and %E characteristics were determined with a 5-kN electromechanical universal testing machine (UTM) (Zwick Roell: Z005TN equipped with 100 N load cell). Relevant adopted procedures for sample preparation, UTM system parametric choice, etc., have been summarized in our earlier work (Das et al. 2020a, 2020b). Prior to the determination of the TS and %E, the sample thickness was evaluated using a digimatic micrometer (APB-2D, Mitutoyo Corporation, Japan; accuracy level 0.001).

Antibacterial efficacy

Gram-negative bacteria Escherichia coli (ATCC 25922) and Gram-positive bacteria Listeria monocytogenes (ATCC 19115) were used to evaluate the antimicrobial properties of PVA-St organic acid Gl films. In the tests, one of these bacteria was aseptically inoculated in 10 mL of nutritional broth, and the system was then incubated at 37 °C with constant stirring (160 rpm) in a shaking incubator. A UV–Vis spectrophotometer (Model No.: GENESYS 10S UV–Vis, Make: Thermo Scientific) was used to measure the absorbance at 600 nm every 2 h to assess bacterial growth. In a sterile setting, the broth was spread out on a perti-plate over nutritional agar media once the microbial growth had attained an optical density of 0.6. Thereafter, the films (19.63 mm²) were put on top of the plate and incubated at 37 °C for 12 h. Subsequently, the agar plate’s zone of inhibition was assessed by visual inspection.

Cell growth evaluation

Cell line and cell culture conditions

Human embryonic kidney-293 cells were procured from the National Centre for Cell Science, Pune, cell repository. The cells underwent cultivation by following the conditions summarized in our previous article (Das et al. 2020a).

Biocompatibility

The in vitro biocompatibility of the HEK cells has been presented in terms of RGR and was verified using the procedure described in our previous work (Das et al. 2020a). The subsequent expression has been deployed to evaluate the RGR:

$$R\text{GR}=\frac{\text{Absorbance of the treated cells}}{\text{Absorbance of the control or untreated cells}}\times 100\%$$

(3)

The outcomes of all the PVA-St composite films that have been assessed for their biocompatibility were determined as per the GB/T16175-1996 standard (Shi et al. 2008).

Statistical analysis

Statistical analysis was administered through the Tukey test and with the one-way ANOVA.

Results and discussion

ATR analysis

The spectral analysis in the wavelength range of 3700–2700 and 900–1800 cm⁻¹ for different situations related to the alternative organic acid composition in PVA-St composite films is shown in Fig. 1. The figure shows that in the 3200–3400 cm⁻¹ spectral area, there is a broad and prominent peak for all formulations. This supports the stretching vibration of the composite structure’s –OH groups. Next to this band, another band exists (2910–2950 cm⁻¹), which is indicative of the C–H bond being stretched. Another sharp band may be seen in the 995–1050 cm^–1 area, which is consistent with the C–O groups’ deformation vibration. Adjacently, two little peaks (1060–1110 and 1130–1160 cm⁻¹) are visible to this sharp spectrum, and they are consistent with the stretching vibrations of the C–C and C–O groups as well as the symmetric vibration of the C–C groups that exist in a symmetric manner to represent the zigzag chain’s trans-configuration that occurs repeatedly and regularly in the crystalline area. Subsequently, at around 1205–1208 and 1400–1420 cm^–1, there are minor, consecutive peaks that are consistent with the symmetric bending mode of CH₂ groups and corroborate as well with the wagging vibration of C–H groups, respectively. Additionally, a coalescence peak in the 1700–1750 cm^–1 area is present and corroborates with the ester bonds and carboxyl C = O groups that exist in the organic acids (Das et al. 2019).

Fig. 1

Influence of CA, TA, and MA on the ATR spectra of alternate organic acid-based PVA-St composite films a EXP-%E, b EXP + %E, c RSM-%E, and d RSM + %E, in the spectral region of 1800–900 and 3700–2700 cm.⁻¹

In the 3200–3400 cm⁻¹ spectral range (Fig. 1), the peak intensity is highest for TA in comparison with MA or CA for most formulations. In comparison to the CA, TA, and MA have a higher number of –OH groups to participate in the crosslinking reaction. Moreover, among TA and MA, MA has a total lesser number of participating groups. Hence, TA has a higher constitution of free –OH groups and henceforth corroborates with the higher peak intensity in the 3200–3400 cm⁻¹ spectral region. Adjacent to this, in the 2910–2950 cm⁻¹ spectral range, for the case where the formulation contains an equal amount of PVA and St (EXP + %E and RSM + %E), the peak intensity representing the C–H bond stretching of the polymer composite is the comparatively lowest for the CA additive-based case. However, for the combinations including more St, the application of CA as an additive fostered a comparative increment in the peak intensity. This may occur due to the better intermolecular interaction between St and CA in comparison to the interaction between PVA and CA.

All formulations confirmed peaks related to ester bonds in the 1700–1750 cm⁻¹ spectral range. However, the TA-based formulations exhibited a shift towards higher wavenumbers. This depicted a reduced bond length and corroborated to the TA inhibiting intermolecular hydrogen bonding with the PVA or St. The FTIR spectrum analysis of this region and the 3200–3400 cm⁻¹ spectral range also corroborates with the extent of crosslinking (kindly refer to the relevant table in the supporting material). In summary, it has been observed that the formulation based on CA possesses the highest degree of crosslinking. In the 995–1050 cm⁻¹ spectral region, the TA-based formulations indicate that the increasing peak intensity corroborates with the intermolecular interactions between TA and PVA molecules in the polymer chains. Especially for the formulation (EXP-%E) with lower additives concentration, the peak position shifted, and the absorbance of this band reduced significantly. This is attributed to the ether band (Bozdoğan et al. 2020).

Swelling index characteristics

Figure 2 depicts the altered PVA-St composite film SI properties with respect to the concentrations of CA, TA, and MA. The ideal formulations labeled as EXP-%E, EXP + %E, RSM-%E, and RSM + %E are those that correspond to those mentioned in our earlier work. Consequently, with corresponding modifications in CA, TA, and MA, the SI values increased from 292.63 to 338.37, 322.68 to 355.45, and 347.56 to 739.29%, respectively. Thus, the MA-based films exhibited the highest SI features in a hierarchical comparison with both CA and TA-based films (level p ≤ 0.001, for EXP + %E, RSM-%E, and RSM + %E formulations). This could be due to the greater amount of –COOH groups (CA > TA and MA) and henceforth comparatively improved, stronger, and effective crosslinking ability (Shi et al. 2008). The MA has the comparatively fewest appropriate functional groups for their involvement during the esterification process (Yun et al. 2006). However, it can be observed that the higher swelling capacity of the films based on MA in comparison with the other organic acids is less prominent for the formulation with a higher amount of St and is more prominent for the formulation with an almost equal amount of PVA and St. Such an effect is especially relevant for the case of RSM + %E formulations. Such an effect could occur due to the enhanced participation of St than the PVA in the esterification reaction (Shi et al. 2008). The MA-based formulation with a higher amount of St in comparison to the PVA easily creates hydrogen bonds with the MA and henceforth exhibited better crosslinking ability. However, the MA-based formulation with equal amounts of St and PVA fostered lesser crosslinking but with a higher swelling index.

Fig. 2

Influence of CA, TA, and MA on the swelling index characteristics of organic acid-based PVA-St composite films

Wound dressing materials are usually designed to have a much larger swelling capacity to enable efficient absorption of wound exudates. From the standpoint of SI analysis, films prepared with three alternative organic acid-based formulations achieved an SI value larger than 260%. This is confirmed that they are superabsorbent and are suitable for their utility as wound dressing materials (Ahmed et al. 2018). Additionally, it can be analyzed that the SI variation has been minimal for the EXP-%E formulation-based film. For the case, minimum concentration of the additive has been ascertained. Also in comparison to the CA, the TA-based film had a higher SI and at a significant level of p ≤ 0.05. Henceforth, at the 15 wt% additive concentration, it is expected that any of the three mentioned alternative organic acids had a comparable degree of crosslinking (and swelling properties).

In vitro degradation characteristics

The in vitro degradation or WL data patterns for the PVA-St composite films based on alternative organic acids have been depicted in Fig. 3. The WL fluctuated for a time period of 27 days of incubation for various formulations and the EXP-%E data set. The parameter altered from 25.56 to 53.27, 31.05 to 76.75, and 32.54 to 61.73% for CA, TA, and MA, respectively. For the PVA-St composite film and for the EXP + %E formulation case, the corresponding values altered in the range of 40.62 to 56.38, 51.65 to 65.9, and 37.57 to 57.44%, respectively, Conversely, the in vitro degradation characteristics varied as follows for the alternative organic acid-based PVA-St composite films that were prepared with RSM-%E formulation: 54.51 to 76.78, 37.42 to 73.96, and 43.57 to 73.99% for CA, TA, and MA, respectively. Comparably, the variable trends for the composite films being prepared with the RSM + %E formulation respectively altered from 1.93 to 64.79, 35.32 to 93.47, and 27.96 to 66.56%. The time-dependent features confirmed a large initial value (day 1) which eventually escalated progressively (from 3rd to 7th day), and eventually reached a steady WL trend for the majority formulations. This may have occurred due to the loss of the free available –OH and –COOH groups from the composite and their non-participation in the crosslinking reaction. The free available –OH and –COOH groups from the composite film reduced gradually with respect to the time and thereby affirmed a steady weight loss after 7 days. A similar release profile has been confirmed by Aizaz et al. (2024) for the polyethylene oxide (PEO)/guar gum (GG)/rosemary (RM)/CA hydrogel films (Aizaz et al. 2024). Till 14 days, except for the films being prepared with the EXP + %E formulation, the TA-based PVA composite films demonstrated inadequate general deterioration properties. However, after 14 days, all formulations containing TA as the organic acid component exhibited a strong escalation (with significance levels of p ≤ 0.001 for the RSM + %E and RSM-%E formulations and p ≤ 0.01 for the EXP-%E formulation).

Fig. 3

Influence of CA, TA, and MA on the in vitro degradation characteristics of alternate organic acid-based PVA-St composite films a EXP-%E, b EXP + %E, c RSM-%E, and d RSM + %E constitutions

Furthermore, an analysis of the films being prepared with the EXP + %E formulation revealed that the films prepared with the TA were comparatively soluble (kindly refer to Fig. S1 in the supporting material). It follows that in comparison to the formulations being achieved with the CA and MA, the identical formulation will have the highest time-altered WL characteristics. The 27th day confirmed identical degradation characteristics for the CA and MA-based films, but for the films being manufactured with an EXP-%E optimized formulation. On the other hand, the MA-based film’s daily WL rate was somewhat noticeable. On the first day, the MA-based film exhibited a WL lower trend with respect to that of the CA-based film (p ≤ 0.001 and ≤ 0.01, respectively, for the RSM + %E and RSM-%E formulations). After 27 days, nevertheless, the WL steadily improved to match the degradation value reported by the film prepared with CA. A wound healing product should ideally enable a consistent but gradual increase in the medications’ or antibiotics’ release from the hydrogel film matrix. Such a release shall be high during the initial period, relatively better later onwards, and should involve a gradual reduction in the desired release of the materials. The hydrogel film's shortened shelf life is discussed in the later scenario. The MA-based films demonstrated desirable in vitro degrading characteristics in an ideal situation. This was followed by the CA-based films. The desired drug release characteristics in composite films have been opined to be far more effective in the MA in comparison to the CA in the PVA composite films and for the wound dressing applications.

Mechanical characteristics

The mechanical property trends for the PVA composite films being prepared with variant CA, TA, and MA concentrations have been depicted in Fig. 4a and b, respectively. For all cases of the optimized PVA-St composite films (EXP-%E, EXP + %E, RSM-%E, and RSM + %E) and for corresponding constituent concentration variations, the TS and %E varied as 3.36 to 7.65, 2.98 to 7.75, 1.91 to 5.83 MPa and 9.13 to 147.86, 16.76 to 49.58, 18.4 to 103.68%, for CA, TA, and MA, respectively. The TA-based films exhibited weaker characteristics for both mechanical properties in comparison to the individuals equipped with the CA and MA (increased TS for the CA-based EXP + %E formulation with p-value ≤ 0.01, higher %E for the MA-based RSM-%E formulation with p-value ≤ 0.001). Also, it shall be noted that the TS value range of normal human skin is about 2.5 to 35 MPa. Along with this value range, about 70 to 78% elongation is to be set as the minimum criterion for the composite film to qualify as a wound healing film for compatible application to withstand external stress and maintain its integrity at the time of application (Khaliq et al. 2023). Considering this clause, it can be analyzed that while all the organic acid-based composite films demonstrated adequate TS value, only CA and MA-based films exhibited a reasonable %E.

Fig. 4

Influence of CA, TA, and MA on the a tensile strength and b percentage elongation characteristics of alternate organic acid-based PVA-St composite films

It can be inferred that the TS and %E values of the films prepared with CA as an additive were significantly higher (with a significance threshold of p ≤ 0.01 for the EXP + %E formulation and p ≤ 0.001 for the RSM + %E formulation) in comparison to those prepared with the MA and TA. This is due to the reason that the superior crosslinking features of the higher concentrations of CA ascertained upon its comparatively better performance for the assessed parameter (TS) and with respect to the MA and TA (Shi et al. 2008). Similar trends have been found by Shi et al. (2008). The authors hypothesized that with the higher constitution of –OH and –COOH groups, the CA has the best crosslinking capability. Furthermore, Shi et al. (2008) already elucidated upon the plasticizing capability of the CA. This is due to the freely available –COOH groups. For this reason, greater concentrations ascertained more availability of –COOH groups and henceforth larger %E (Shi et al. 2008). However, this has not been the case for the MA and TA-based PVA composite films. In terms of the improvisation of the mechanical properties of the PVA-St hybrid sheets, the MA is the second-best choice among all three considered organic acids. Furthermore, the MA-based films showed better mechanical features (increased TS for RSM-%E formulation with a significant level of p ≤ 0.001) than the CA-based films. The higher constitution of St can be analyzed to propel the pertinent crosslinking characteristics. This is due to the greater coherence between St and MA, a subject being detailed in the “Swelling index characteristics” section of the article.

Antibacterial efficacy

Using E. coli and L. monocytogenes, PVA/St/organic acid/Gl film’s antibacterial efficacy concerning TA and MA was studied. As a positive control, 10 µg/mL of the antibiotic kanamycin was used. Photographs confirming the antibacterial efficacy of films based on TA (a) and MA (b) for gram-positive (Fig. S3) and gram-negative (Fig. S2) have been presented in the supplementary materials. For most cases, a distinct inhibition zone exists and in the surrounding area of the film surface. Both TA and MA-based PVA-St composite films exhibited antibacterial efficacy against L. monocytogenes and E. coli. However, in comparison to the MA-containing films, the films prepared with TA confirmed a more pronounced effect. This is due to the reason that the TA is a powerful organic acid. The lower pKa values for organic acids (pKa in the TA < CA < MA order) were associated with more effective and powerful antibacterial action (Francesco et al. n.d.). Conversely, the organic acids’ pKa level created an acidic environment that encourages a breakage in the bacterial cell’s protein and cell membrane manufacturing pathway (Wu et al. 2017). Furthermore, it should be mentioned that, in comparison to the E. coli, the PVA composite films based on CA for every four alternate situations (EXP-%E, EXP + %E, RSM-%E, and RSM + %E) demonstrated greater antibacterial efficacy against L. monocytogenes (Das et al. 2020a). On the other hand, the PVA composite films based on TA and MA, which had comparable film constitutions, had significant antibacterial efficacy against both L. monocytogenes and E. coli.

The antibacterial efficacy of MA-based PVA-St composite films was found to be lower in the EXP + %E and EXP-%E cases. This is in contrast to the RSM + %E and RSM-%E cases. This is for the reason that the organic acid concentration of the film matrix in both the MA-based PVA composite films and for the EXP + %E and EXP-%E cases had lower percentages than those achieved for the RSM + %E case. Furthermore, within the composite matrix, the PVA constitution has been significantly reduced for the RSM-%E instance. As a result, the matrix aided the leaching of extra organic acids that were present in the composite’s matrix and created an artificial acidic surrounding environment. The analyses of the CA-based PVA composite affirmed a similar pattern (Das et al. 2020a). However, this is not applicable for the TA-based composite films. Also, it should be mentioned that the TA-based PVA composite films showed stronger weight loss characteristics (described in the “Swelling index characteristics” section) for every PVA composite film that was prepared with a film composition based on the EXP case and especially for the EXP + %E scenario. Thus, it is evident that in this instance, the amount of TA that leaches from the films is far more than that of films being prepared for any other formulation. Consequently, such a trend could be due to the increased antibacterial activity.

In vitro biocompatibility

After 48 h of treatment with the HEK cells, the PVA-St composite films based on CA, MA, TA, and their in vitro biocompatibility were examined for their RGR%-based cell growth capacity. The RGR% values with respect to the extract (treatment solution) prepared with different organic acid-based PVA-St composite film formulations have been shown in Figs. 5 and 6, respectively, and after 24 and 48 h of incubation. After soaking in the FBS-supplemented DMEM media for 24 and 48 h, each of the four alternative PVA-St film compositions based on organic acids was assessed as per the details presented in our earlier work (Das et al. 2020a).

Fig. 5

Influence of CA, TA, and MA on the relative growth rate characteristics of 24h extracted sample with HEK cells and alternate organic acid-based PVA-St composite films a EXP-%E, b EXP + %E, c RSM-%E, and d RSM + %E cases

Fig. 6

Influence of CA, TA, and MA on the relative growth rate characteristics of 48h extracted sample with HEK cells and alternate organic acid-based PVA-St composite films a EXP-%E, b EXP + %E, c RSM-%E, and d RSM + %E cases

The ISO recommendations (GB/T16175-1996 standard) state that the test sample with less than 75% RGR, as compared to the control sample, has a higher risk for toxicity and should not be used as a material for wound dressings. Following a 48-h extraction interval, the experimental studies confirmed that the PVA composite film based on TA demonstrated such hazardous potential. As HEK cells were treated with 100 µL of extract sample or treatment for experimentally optimized formulations, their RGR decreased significantly (p ≤ 0.01 for EXP-%E and p ≤ 0.001 for EXP + %E) and in comparison to the control sample. According to a prior analysis, these two formulations with different TA values had stronger deterioration characteristics (“In vitro degradation characteristics” section). Consequently, increased acidity and toxicity were enabled by the weight loss-driven TA leaching from both formulations. Therefore, compared to CA and MA, TA-based experimentally improved formulations have been found to have poorer in vitro biocompatibility but higher antibacterial efficiency (“Antibacterial efficacy” section).

The produced treatment solution after the 24-h extraction process of all the optimized MA-based PVA-St composite films exhibited maximum RGR value in comparison with the films prepared with the TA and CA. The p-values were less than 0.05 for TA-based films and were less than 0.01 for CA at the significance level. For the formulations of EXP-%E, EXP + %E, and RSM + %E, there was a significant rise in the RGR of the HEK cells being treated with 100 µL of the treatment sample (p-values were less than 0.05 for EXP-%E and ≤ 0.001 for all other cases). On the other hand, a notable decrease in RGR was obtained for the PVA composite films containing MA after 48 h of extract treatment. Furthermore, following 48 h of incubation, the PVA composite films containing CA had consistently enhanced cell development characteristics along with incremental in vitro degradation that was driven with increased leaching of the organic acid.

With respect to this effect, it should be remembered from the “In vitro degradation characteristics” section that during the first and second days of the degradation-related incubation period, the WL rate of PVA composite films containing MA was noticeably greater than those being prepared by CA. Conversely, it has been predicted that the leached MA into the media during a 48-h extraction process may adversely affect cell proliferation. This led to a reduced prolific activity for the scenario in which the extraction sample was treated for 48 h. However, the PVA composite films containing CA exhibited consistent prolificity. This was consistent with the WL’s gradual increase. For the 24-h extracted sample being used as the treatment case, the PVA composite films prepared with MA and RSM + %E film composition affirmed the greatest RGR of 215.59% (relative to the data from the control sample) and among all the films being tested for RGR features (Fig. 5). In contrast, the films prepared with CA showed much reduced values (149.55%). Subsequently, co-culturing techniques confirmed that, for the same case, the biocompatibility of the PVA composite films containing MA profoundly decreased for the treatment solution extracted for 48 h (104.78% in comparison to the control). On the other hand, the biocompatibility of the PVA composite films containing CA has been significantly higher (155.67%). This is for the reason that the MA leaching has a more significant negative impact during the extraction process (which was not seen with the film prepared with CA). In conclusion, PVA-St-Gl composite films based on MA and CA both reacted favorably and sensitively to cell development and accordingly confirmed their potential uses as materials suitable for tailored wound dressings.

Conclusions

This work leads to several useful deductions. The overall deducation is that excepting the TA for its higher acidic properties that induced toxicity towards cell growth, the other two alternate acids namely CA and MA have been the interesting options for the generation of PVA composite films that are devoid of defects and for wound dressing applications.

Other notable deductions are as follows. Firstly, the composite film prepared with MA and RSM + %E formulation had the greatest swelling values (739.29 ± 5.24%). However, the case did not refer to comparable solubility levels and is endowed with the weakest crosslinking features. The solubility of the MA-based film was 61.59 ± 1.21%, which is not appreciably greater than the solubility of the CA-based (75.88 ± 4.34%) and TA (61.81 ± 1.63%)-based PVA composite films. Henceforth, despite ascertaining a comparatively weaker crosslinking capability with respect to the CA and TA, the MA can prevent the loss of the polymer network in a hydrolytic environment and along with its capability to promote the absorbing capacity of the polymeric composite.

Secondly, during the initial period, the PVA composite film containing MA and RSM + %E formulation exhibited comparatively lower WL (27.96 ± 1.81%) with respect to the CA-based film (51.93 ± 2.71%). On the other hand, for the 27-day duration, the weight loss of the films containing MA significantly rose to 66.56 ± 3.51%, a value which is comparable to that of the CA-based PVA composite films (64.79 ± 3.65%). The corresponding gradual degradation trait of the MA-based films is promising for the delayed drug release scenario, a feature being sought as the most ideal for the wound dressing materials. Henceforth, the MA-based films can be assessed for further investigations that target the effective drug release kinetics.

Thirdly, despite being inferred with a relatively poorer crosslinking strength, the MA instigated good mechanical qualities in the PVA composite films and with the desired promising combinations of the SI and in vitro degradation features. The PVA films containing MA exhibited comparatively advanced and adequate mechanical properties for a film to be considered to be applicable for wound dressing application (4.88 ± 1.47 MPa tensile strength and 103.68 ± 6.87% elasticity) and especially with respect to the TA-based films and the CA-based PVA films (RSM + %E formulation being the best with 5.14 ± 0.63 MPa tensile strength and 147.86 ± 7% elasticity). Additionally, the characterization targeting wound dressing applicability of the PVA-St composite films containing different organic acids confirmed the favorable antibacterial efficiency and HEK cell proliferation for the MA-based films. When the MA film sample was soaked and extracted for 24 h, the ideal composite film for PVA and St containing MA affirmed the greatest RGR value of 215.59 ± 7.36%. This result is much larger than the equivalent value attained for the PVA composite films containing CA (149.55 ± 1.92%). The lengthy testing did, however, confirm that there is still a need to adjust the MA concentration. This is due to its leaching that occurs during the therapy.

In summary, among all considered alternate organic acid blended composite films, the PVA composite films containing MA affirmed encouraging qualities. However, they still require further refinement in the film composition for further advancements towards the affirming of promising combination of the quantitative and qualitatative properties. Thereby, the MA-based composite films can open newer avenues for enriched applications as affordable hydrogel materials for the advanced wound dressing scenarios.

Nomenclature

–OH Hydroxyl

–COOH Carboxyl

PVA Polyvinyl alcohol

St Starch

Gl Glycerol

CA Citric acid

MA Malic acid

TA Tartaric acid

DI water De-ionized water

DMEM Dulbecco’s modified eagle’s medium

FBS Fetal bovine serum

PBS Phosphate buffer saline

FTIR Fourier transform infrared spectroscopy

HEK Human embryonic kidney

E. coli Escherichia coli

L. monocytogenes Listeria monocytogenes

ISO International organization for standardization

ANOVA Analysis of variance

RSM Response surface methodology

EXP-%E Experimental investigation-based optimal formulation that did not consider %E as an additional response variable

EXP + %E Experimental investigation-based optimal formulation that considers %E as an additional response variable

RSM-%E Design expert software-based optimal formulation that did not consider %E as an additional response variable

RSM + %E Design expert software-based optimal formulation that considers %E as an additional response variable

TS Tensile strength, MPa

%E Percentage elongation or elongation at break, %

WL Weight loss, %

S Solubility, %

DS Degree of swelling, %

SI Swelling index, %

GF Gel fraction, %

RGR Relative growth rate, %