Kinetic study of in vitro release of curcumin from chitosan biopolymer and the evaluation of biological efficacy

2.1 Extraction of curcumin from turmeric rhizome

Curcuma longa L (turmeric) rhizomes were collected, washed, and peeled to remove any impurities on the outer surface. They were cut into thin pieces and oven-dried at $45$  °C overnight. Then, they were ground using a mechanical grinder to obtain the turmeric powder. Turmeric powder (10 g) was subjected to double organic extraction using 100 mL of 80 % ethanol as the solvent following the heated sonication ( $45$  °C for 3 h) to obtain the curcumin extract. Soner 203H Heated Ultrasonic Cleaner, 35 KHz, was used for the extraction process. The crude extract of curcumin was obtained after successful evaporation of ethanol.

2.2 Synthesis of chitosan from waste shrimp shells

Waste shrimp shells of shrimp varieties Penaeus monodon and Penaeus vannamei were collected and cleaned by removing the pleopods and pereiopods. The resulting shrimp shells were washed several times with tap water and distilled water, followed by washing with 70 % isopropyl alcohol to remove impurities adhered to the shells. Cleaned shells were then oven-dried at $80$  °C for 3 h and ground using a grinder until a fine powder resulted. Demineralization, which is the removal of CaCO₃ and Ca₃(PO₄)_2, was carried out using 10 % (v/v) HCl solution for 24 h under constant stirring. The samples were washed with distilled water to obtain a neutral pH. The resulting powder was then suspended in a 3 % (w/v) NaOH solution for 5 h under constant stirring for the deproteination step. Deacetylation was conducted by reflux condensation (2 h at $80$  °C) using a 50 % (w/v) solution of NaOH. Through this step, chitin present in the shrimp shell was converted to higher molecular weight chitosan, and the resulting powder was washed using distilled water until a neutral pH was obtained. Chitosan was oven-dried overnight at $80$  °C and stored in a reagent bottle for future usage and analysis (Mendis et al., 2023).

2.3 Synthesis of the curcumin-chitosan composite material

Chitosan (1 g) powder was dispersed in the ethanolic extract of curcumin (0.1 g/ml) and allowed for casting, absorbing, and grafting of curcumin to the chitosan biopolymer under slow constant stirring at 100 rpm for three days at room temperature. Then, the resulting composite material (chitosan: curcumin, 1:10) was oven-dried at $40$  °C to remove any moisture retained and stored in an airtight container for future use and analysis. The total synthesis procedure of the curcumin chitosan composite is illustrated in Scheme 1.

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Scheme 1

Schematic representation – the synthesis of chitosan curcumin composite from the raw materials.

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2.4 Characterization of synthesized materials

The Rigaku Ultima IV system, which uses Cu Kα (λ = 0.154 nm) radiation varying the 2θ from 10 ${}^{°}$ to 80 ${}^{°}$ at a scan speed of 2 ${}^{°}$ /min, was utilized to obtain crystallographic structure patterns of turmeric powder, chitosan, and the synthesized composite material. The surface morphology of the synthesized chitosan and composite was evaluated using ZEISS EVO 18 RESEARCH field-emission SEM.

2.5 Anti-inflammatory activity – Egg albumin denaturation assay

The in vitro anti-inflammatory activity of the curcumin extract and drug composite was assessed according to Yesmin et al.(Yesmin et al., 2020) Banerjee et al.(Banerjee et al., 2014) with slight modifications. The reaction mixture (5 mL) consisted of 0.2 ml of 1 % (w/v) egg albumin with 2.8 mL phosphate buffer saline (PBS, pH 6.4) and 2 mL of changing concentrations (100 μg/mL − 1600 μg/mL) of curcumin extract and composite material in methanol. A mixture of 0.2 mL of 1 % egg albumin, 2 mL methanol (without the plant extract), and 2.8 mL of PBS was used as the control substance. Samples were incubated at 37 °C for 30 min and heated in a water bath for 15 min at 70 °C. After that, absorbance was measured at 660 nm, and the experiment was triplicated. The following formula (1) was used for calculating the percentage inhibition of protein denaturation: (Banerjee et al., 2014)

2.6 Antioxidant activity − diphenyl picrylhydrazyl (DPPH) assay

According to the protocol reported by Ak & Gülçin (Ak & Gülçin, 2008), utilizing a stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution, the overall antioxidant potential of curcumin and the composite material was assessed. The 1 mg/mL stock solution of the samples was diluted with methanol to produce 5 mL of reaction mixture with the final concentrations of $5,10,15,20and25\mu g/mL$ for curcumin extract and $10,20,30,40,50,60and70\mu g/mL$ for the composite material. For each reaction mixture, 0.5 mL from the one mM stock DPPH solution was added. The DPPH absorbance was measured against a blank (methanol 5 mL and 0.5 mL of DPPH) after 30 min of incubation in the dark. The radical scavenging ability (RSA%) of the extract is proportional to the decline of the absorbance after adding the stable DPPH radical to the extract. This analysis was performed in triplicate (n = 3), and the results were expressed as mean % inhibition of the DPPH radical/ RSA% ± SE, which was calculated as follows (2): (Ak & Gülçin, 2008)

2.7 In vitro drug release studies

In vitro drug release of the synthesized composite material was assessed over different pH conditions varying from pH 1, 2.5, 4, 5.5, 7, 7.4, 8.5, and 10. Further, the ionic strength of the release medium was changed by varying the NaCl concentration from 0.1 M – to 0.5 M. For the analysis, 5 mg of the composite material was added to the release medium in a cuvette, and the release data was collected at 15-minute intervals for 10 h using a visible range spectrophotometer. From the absorbance values, the pH media and the NaCl concentration, which showed the best release, were selected. To determine dose-dependent release, a media with a NaCl concentration of 0.3 M and pH of 7.4 was prepared. Drug release was measured in the prepared media by changing the composite weight to 2.5 mg, 5 mg, 7.5 mg, and 10 mg. Drug release data was converted to the cumulative drug release % (%CDR) using the Eq. (3):

2.8 Antibacterial activity determination

2.9 Statistical analysis

Data from each experiment were statistically analyzed using SPSS Statistics (Version 27, SPSS Inc., Chicago, Illinois, USA), and results were expressed as mean ± standard error (SE). The confidence level of 95 % was set as the Statistical significance. One-way analysis of variance (ANOVA) was used to determine the differences among the treatment means.

3.1 Characterization

3.1.1

3.1.1 XRD analysis

The XRD patterns were collected to study the crystallographic orientation of turmeric powder, synthesized chitosan, and the synthesized composite material (Fig. 1). The XRD pattern of the chitosan shows a significant peak at 19.47° which is attributed to the (1 1 0) crystalline plane pattern and the shoulder peak at 20.91°, which represents the (1 2 0) crystalline plane, and the small broad peaks at 23.46°and 26.36° correspond to the (1 0 1) and (1 3 0) crystalline planes of chitosan, respectively (Mendis et al., 2023). The d spacing values calculated by Bragg’s law using Eq. (11) of the respective crystalline planes are 0.4555, 0.4220, 0.3807, and 0.3355 nm, respectively.

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Fig. 1

XRD patterns of the materials.

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The XRD pattern of curcumin shows peaks at 14.66°, 17. 20°, 18.36°, 19.9°, 22.1° 23.48° and 25.66° matching with the CCDC No. 82–8842 and JCPDS 9–816 (Van Nong et al., 2016). The inter-layer distances corresponding to the respective atomic planes are 0.5999, 0.5151, 0.4026, 0.4784, 0.4448, 0.3710, and 0.3430 nm, respectively.

The peak at 19.55° of the XRD pattern of the curcumin-chitosan composite, which corresponds to the (1 1 0) crystalline plane of chitosan, indicates the presence of chitosan in the composite and the presence of peaks corresponding to curcumin appear at 23.35°, 26.55°, 37.12°, 17.20° and 14.66° in the XRD pattern of the composite confirms the presence of curcumin crystallized on chitosan in the composite. The calculated d spacings values of the respective crystalline planes are 0.4537, 0.38070, 0.3355, 0.242, 0.5151, and 0.5999 nm, respectively.

The crystallite size was calculated by the Scherrer equation given in Eq. (12), using the highest intense peak of curcumin, chitosan, and the composite located at 2θ values of 17.20°, 19.47° and 19.55°.

(11)

$$\lambda =2d\sin \theta$$

(12)

$$L=\frac{K\lambda }{\beta \cos \theta }$$ Where λ represents the wavelength of the X-ray source; θ: diffraction angle; L: crystallite size; β: half maximum of the peak in radians; K: Scherer’s constant (0.89).

Table 1 summarizes the parameters related to crystalline structures. Curcumin was crystallized on the surface of chitosan during the preparation of the composite, resulting in the growth of the crystallite size, as evidenced by the increase in the crystallite size and the number of planes of the composite material in XRD results.

Table 1 Crystallographic parameters were calculated from XRD data.

Sample	2θ°	L (nm)	d (nm)	L/d
Curcumin	17.20	28.47	0.5151	55.27
Chitosan	19.47	55.01	0.4555	120.77
Composite	19.55	66.11	0.4537	145.71

3.1.2

3.1.2 Scanning electron microscopy

SEM images were collected to study the 3D geometry of the surface of the prepared materials. A well-established oval-shaped macropore structure was established on chitosan, with the removal of protein present in the chitin upon treatment with 3 % NaOH (Fig. 2 (a)). In the composite, the macropore structure is disturbed by the crystallization of curcumin on the porous structure of chitosan, as shown in the SEM image in Fig. 2 (b).

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Fig. 2

SEM images of (a)Chitosan (b)Chitosan-curcumin composite.

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3.2 Egg albumin denaturation assay

Curcumin extract has a substantial anti-inflammatory property, as shown in Fig. 3 (a). Curcumin extract has acted in a dose depending on manner in the range of 100 μg/ml to 1600 μg/ml. The composite, which consists of curcumin extract and chitosan, has been shown to possess strong anti-inflammatory capabilities due to the grafting of curcumin onto the surface of the delivery system. The IC₅₀ values of curcumin and composite materials are 0.285 mg/ml and 1.083 mg/ml, respectively. Lower IC₅₀ values indicate higher activity against the inflammatory reactions (Elisha et al., 2016). Further, it can be concluded that the ethanolic extract of the turmeric powder consisting of phytochemicals such as polyphenols, terpenoids, and flavonoids (curcumin)(Gonfa et al., 2023) has contributed to the substantial anti-inflammatory potential and has been expressed in the developed novel delivery system.

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Fig. 3

Anti-inflammatory activity of (a) curcumin extract (b) curcumin chitosan composite.

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Steroid and nonsteroidal anti-inflammatory drug therapy is the conventional method of treating inflammatory diseases. However, these medications can have serious side effects like myocardial infarction (Truong et al., 2019). Therefore, with minimal probability of causing adverse drug reactions, curcumin-loaded chitosan composites can act as a potential plant-based drug to replace synthetic drugs due to their substantial anti-inflammatory activity.

3.3 DPPH radical scavenging ability

When endogenous reactive oxygen species, or ROS, generation exceeds the body's antioxidant capability, oxidative damage typically occurs (Shaaban et al., 2021). As a well-known potent antioxidant, curcumin has shown a dose-dependent increment in the RSA% with the concentration. According to Fig. 4, the radical scavenging ability has reached a saturated level (80.62 ± 2.45 %) when the concentration is above 25 $\mu g/ml$ , where it no longer represents a linear behavior. As the linear range of the RSA% was not prominent for higher concentrations (>25 $\mu g/ml)$ , curcumin’s antioxidant potential was determined over a concentration range of 5–––25 $\mu g/ml$ . For the composite, a concentration series of 10–––70 $\mu g/ml$ was used where the RSA% reached the maximum value of 88.78 ± 0.32 % at 70 $\mu g/ml$ in the concentration series tested. From 10 − 70 μg/ml, the RSA% varied from 26.57 ± 1.94 to 88.78 ± 0.32, acting in a dose-dependent manner. The IC₅₀ values of curcumin and composite materials were determined to be 13.47 μg/ml and 36.76 μg/ml, respectively. Ascorbic acid was used as the positive control, and the values obtained were significantly higher than those of the composite samples. At 50 μg/ml, the composite showed 78.93 %, whereas the positive control showed 91.66 % DPPH radical scavenging activity.

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Fig. 4

DPPH antioxidant assay (a) curcumin (b) drug composite.

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The IC₅₀ of the novel composite is mainly attributed to the high DPPH radical scavenging ability of the bioactive curcumin, which has been grafted upon chitosan biopolymer. Sánchez-Machado et al.(Sánchez-Machado et al., 2018) and Tomida et al.(Tomida et al., 2009) have reported that chitosan also possesses antioxidant activity.

Anraku et al.(Anraku et al., 2008) indicate that chitosan exhibits antioxidant properties at varying molecular weights. Human serum albumin is protected from oxidation by low molecular weight chitosan by inhibiting the production of carbonyl and hydroperoxide groups (Anraku et al., 2008). Curcumin is a lipid-soluble substance with antioxidant activity that can bind to the cell membrane and gets transformed into phenoxyl radical by seizing the action of lipid radicals. Thereafter, penoxyl radicals can be restored by water-soluble substances such as ascorbic acid after moving to the cell membrane surface. This transformation is facilitated by the higher polarity nature of the phenoxyl radicals than that of curcumin (Ak & Gülçin, 2008; Jovanovic et al., 1999). The study conducted by Priyadarsini et al.(Priyadarsini et al., 2003) has provided experimental and theoretical outcomes that collectively affirm curcumin's superior antioxidant effectiveness, primarily resulting from its phenolic OH group, with a minor role played by the CH₂ site. Further, the phenolic OH group in curcumin hinders lipid peroxidation induced by free radicals and exhibits antioxidant qualities (Priyadarsini et al., 2003).

Therefore, both the curcumin and chitosan’s antioxidant ability have resulted in a higher antioxidant potential in the composite material. Both the samples showed an intense colour change over the concentration, where the purple colour at low concentrations converted to light yellow colour at higher concentrations of the sample.

3.4 Drug release and statistical analysis

A one-way ANOVA test was conducted to determine the differences in the percentage of cumulative drug release (%CDR) with varying pH levels and ionic strengths of the release media. The results are summarized in Table 2 (A) and Table 2. (B). Table 3..

According to the results, the pH ranges from pH 1 – 4 have shown a significant difference in the % CDR profile with that of the media with pH 7.4 (p-value < 0.05). This can be further confirmed by the significant F values reported in the pH range of 1–4. However, in the pH range from 5.5 to 8.5, the drug release profile does not show a significant difference with that of pH 7.4 (p-value > 0.05). When the pH is increased to 10 drug release behaviour was changed significantly concerning that in pH 7.4 (p-value < 0.05, significant F value). These behaviours correspond to the structural differences and breakdown of the curcumin with the change of the pH in the release medium. Further, it can be seen from the % CDR values that were recorded for 10 h that the drug release increased with increasing alkalinity of the release medium, in which the best release profile over time was obtained at pH 10. Since the mode of administration of the drug delivery systems is either the oral or the intravenous (IV) routes, pH values higher than 7.4 were not taken into consideration when analyzing the results with kinetic models, even though the %CDR was higher. Furthermore, at pH > 7 conditions, curcumin breaks down in 30 min to produce vanillin, 4-dioxo-5-hexanal, ferulic acid, feruloylmethane, and Trans-6-(40-hydroxy-30-methoxyphenyl)-2 (W. Liu et al., 2016). Curcumin breaks down much more slowly in acidic environments; after one hour, less than 20 % of the total curcumin had broken down, as depicted by Cas & Ghidoni (Cas & Ghidoni, 2019) and W. Liu et al.(W. Liu et al., 2016). Moreover, the blood pH is known to be 7.4 in general conditions. Therefore, pH 7.4 media was taken as the media with a higher % CDR for further analysis. Sodium chloride was used to change the ionic strength of the medium, and the %CDR of the media with molarity ranging from 0.1 M−0.5 M was compared with that of 0.3 M. All the release profiles were shown to be significantly different at the 95 % confidence interval when compared to that resulted with 0.3 M NaCl. A better release profile was obtained for the 0.3 M NaCl concentration, and %CDR was found to decrease when the NaCl concentration was increased further. According to Jafari et al. (Jafari et al., 2023), the concentration range of 0.1 M – 1 M NaCl has affected the stability of the curcumin nanoemulsion. Nanoemulsion aggregates in media with higher ionic strength because the electrostatic repulsion between the particles tends to be superseded by the more prominent attractive forces such as van der Waals and hydrophobic interactions. They further indicate that the zeta potential of the nanoemulsions was decreased drastically (near 0) at the higher NaCl concentrations, which in return facilitated the destabilization of nanoemulsion by overcoming the electrostatic repulsion. Conversely, at lower NaCl concentrations, electrostatic repulsion dominated the weaker van der Waals forces and hydrophobic interactions of the nanoemulsions. Peng et al. 2018) indicated that nanoparticles are stabilized against aggregation at NaCl concentrations lesser than 500 mM, but at NaCl concentrations of 500 mM − 1000 mM, nanoparticles tend to aggregate because of the strong, attractive forces compared to the electrostatic repulsions. In the current study, ionic strength varied in a range below 500 mM, and the media with the best-released profile was used for further analysis. Supporting these facts, a media consisting of a pH of 7.4 and 0.3 M NaCl concentration was prepared to assess the drug release profile of the drug with changing the drug dosage (2.5 mg – 10 mg).

Even though curcumin exhibits anti-oxidant, anti-inflammatory, particularly anti-neuroinflammatory, anti-tumor, antithrombotic, immunomodulatory, antidiabetic, analgesic, and microglia-activation inhibitory effects (Bagheri et al., 2020), curcumin cannot be used as a direct medication orally or intravenously because of its’ poor pharmacokinetic properties. The primary objective of developing a delivery system for curcumin is to enhance pharmacokinetic properties compared to free curcumin (Fig. 5). Because of the positively charged nature of the chitosan biopolymer, it can open the epithelial cell tight junctions of the mucous membrane (Choukaife et al., 2022) to deliver the curcumin to the target sites effectively when administrated orally, as shown in Fig. 5 (b). Due to its pleiotropic therapeutic properties on the neurological system, curcumin has been considered a possible therapeutic factor for a wide range of central nervous system-associated diseases. Curcumin can also pass the blood–brain barrier (BBB) (Mirzaei et al., 2017; Yavarpour-Bali et al., 2019). Numerous experimental and clinical investigations have demonstrated the beneficial effects of curcumin in neuroinflammatory illnesses, such as Alzheimer's disease (Hamaguchi et al., 2010b), Multiple Sclerosis (Xie et al., 2011), and Parkinson's disease (B. et al. Bharath, 2012). Therefore, the suggested novel drug delivery system possesses the ability to deliver curcumin to the brain target tissues as well (Fig. 5 (c)). Upon uptake of the drug intravenously or orally, the cellular lifespan of the drug composite is shown in Fig. 6. Following the endocytosis of the curcumin-loaded chitosan drug composite by cells, endosome complexes are formed, entrapping the drug molecules. An endosome/lysosome hybrid is formed with the fusion of lysosome to this complex, which, in return, is responsible for the release of the drugs to the cytoplasm by digestion and rupturing the hybrid complex. Subsequently, the release of curcumin to the extracellular matrix is driven by higher Ca²⁺ levels in the extracellular space (Liang et al., 2021). Considering these therapeutic potentials, the release of the drug from the delivery system can be assessed through an in vitro study to confirm whether the incorporation of a chitosan biopolymer-based delivery system has improved the pharmacokinetic properties of curcumin.

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Fig. 5

Drug composite’s pharmacokinetic properties (a) Enhanced bioavailability of chitosan-curcumin drug composite over time in the gastrointestinal tract (b) Schematic representation of drug composite structure and interaction with the mucus layer (c) Curcumin penetrating the epithelial cell tight junctions in the blood–brain barrier.

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Fig. 6

Cellular lifespan of the drug composite.

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All the experimental drug release data are fitted into kinetic models considering only the first 60 % of the drug release. Supplementary Table 1A, Table 1B, and Table 1C summarize the model parameters of each of the six models used in the study. Considering the R² values (R² > 0.99) of the model fits, KP and PS models were selected to explain the release mechanism of the curcumin-coated chitosan composite material. Linear models like first order, zero order, and HC models were not taken into consideration when interpreting the results because of the lower R² values obtained for the fitted % CDR data (R² < 0.95).

Supplementary Table 1A, Table 1B, and Table 1C show the model parameters of the KP model, which are attributed to the n: diffusional exponent and k_p: release constant about the curcumin release from the chitosan polymeric matrix. According to the parameter “n,” the model offers information about the release profile that can be either a Fickian distribution (Case I) or a non-Fickian distribution (Case II, Anomalous Case, and Super Case II). (Ritger & Peppas, 1987) (Ritger & Peppas, 1987)state that the Fickian diffusion controls the drug release mechanism when n = 0.43, anomalous (non-Fickian) transport occurs when 0.43 < n < 0.85, and case II transport occurs when n = 0.85 (Marcos et al., 2015; Unagolla & Jayasuriya, 2018). These n values explained by the KP model apply to samples of monodispersed polymers; however, Ritger & Peppas (Ritger & Peppas, 1987) and Unagolla & Jayasuriya (Unagolla & Jayasuriya, 2018)reported that n values of approximately 0.3 ± 0.1 in polydisperse spherical samples are also feasible for Fickian diffusion. The n values obtained in this study for all the varied parameters are approximately 0.30 ± 0.01, which is less than 0.43 and hence propose the occurrence of the Fickian diffusion mechanism in polydispersed polymer matrices. Fig. 7 indicates the Ritger-Peppas model's nonlinear curve fitting to the obtained experimental data. The physical and structural characteristics of the drug and polymer matrix both affect the release constant (k_p), which is directly proportional to the diffusion constant (Unagolla & Jayasuriya, 2018). As shown in Supplementary Table 1A, Table 1B, and Table 1C, the highest kp value was obtained in media consisting of a pH value of 10, 0.3 M NaCl concentration, and a drug dosage of 2.5 mg. While pH ten has shown a higher release rate, pH 7.4 has also exhibited a better release rate over time, as discussed under 3.5.

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Fig. 7

Fitted %CDR data for Korsmeyer–Peppas model of the drug composite under varying media conditions; (a-h) pH,(i-m) ionic strength,(n-q) drug concentrations.

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Fig. 7

Fitted %CDR data for Korsmeyer–Peppas model of the drug composite under varying media conditions; (a-h) pH,(i-m) ionic strength,(n-q) drug concentrations.

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The PS model considers both the Fickian contribution and the case II relaxation contribution (second term of Eq. (7)) (Marcos et al., 2015). The purely Fickian diffusion exponent is related to the exponential coefficient m. The values of n and m in Eqs. (6) and (7) should be equal if the relaxational mechanism is insignificant (Abdul Hameed et al., 2020; Marcos et al., 2015; Unagolla & Jayasuriya, 2018). Therefore, the difference between the n and m exponents indicates that case II relaxation, as well as Fickian diffusion, are important mechanisms in the curcumin release process from chitosan. The relaxational over Fickian contribution ratio can be computed using Eq. (8). Supplementary Fig. 1 represents the Fitted %CDR data, and supplementary Fig. 2 shows the variation of R/F values of the Peppas-Sahlin model. Supplementary Figs. 3, 4, 5, and 6 show the variation of Fitted %CDR data of Higuchi, Zero order, First order, and Hixson–Crowell models.

Fig. 2 displays the variation of the R/F ratio against time in different media conditions. The extremely low R/F values initially show that the Fickian diffusion governs the drug release during 0–30 min at some media compositions. It is observed that the relaxation mechanism contributes to the release of curcumin from chitosan biopolymer over time, as depicted by the increase in the R/F values. When R/F is equal to 1, erosion and diffusion equally contribute to the release of curcumin, where diffusion predominates when R/F < 1, and relaxation (erosion) predominates when R/F > 1 (Jadidi et al., 2020).

Peppas-Sahlin model indicates higher rates of curcumin release (k_D and k_R) in media with pH 10, 0.3 M NaCl, and 2.5 mg of curcumin-chitosan composite, separately, being similar to the KP and Higuchi models, suggesting significantly higher release of curcumin under those conditions. Curcumin release at pH 1 deviated where polymer relaxation did not play a major part in drug release (m = n), where polymer relaxation had proven to play a predominant role in the release of the curcumin from the drug when the pH was increased. Low R/F values at the beginning of the release (less than 0) gradually increased over time until pH 7.4, suggesting that diffusion predominated in the first phase of the drug release (0–30 min) and with time relaxation became more prominent. In the media with the highest ionic strength (0.5 M), polymer relaxation has not been a mechanism in releasing the curcumin (R/F < 0), which can be further proven by the m = n value in Supplementary Table 1(B). Therefore, it is suggested that the increase in the NaCl concentration aggregation of the drug composite might have hindered the relaxational mechanism. At pH 1, curcumin might not have been released effectively due to its’ weaker degradation at an acidic medium (R/F < 0). Jafari et al.(Jafari et al., 2023) indicated that the particles tend to aggregate as the surface electrical charge of the particles reduces the electrostatic repulsive forces at lower pH values, which might have contributed to the lower release of curcumin at low pH. However, it should be emphasized that the Fickian diffusion was actively responsible for the release of the curcumin in all the cases.

The Higuchi model is not theoretically applicable to the analysis of swellable drug delivery systems because the model discusses only the effect of time on the release. As this model completely neglects the swelling or dissolution effect of the polymeric carrier system in its’ basic assumptions, using this model alone for the analysis can lead to erroneous conclusions on the drug delivery mechanism (Peppas & Narasimhan, 2014). Nonetheless, the model has been frequently applied in swellable polymer systems to provide a general understanding of the drug transport mechanism because of its incredibly simple nature. In a diffusion-controlled drug delivery system, proportionality between the CDR and the squared of time is seen to be a reliable metric for studying drug release. Consequently, if the Higuchi model is applied, more mathematical analysis must be required to conclude the drug release mechanism (Peppas & Narasimhan, 2014; Siepmann & Siepmann, 2008).

HC model discusses drug release when it dominates by dissolving velocity instead of diffusion. In the current drug delivery system, diffusion has proven to be the major factor in the release of drugs according to the higher R² values reported for the KP model, which is further proven by the lower R² values obtained for the HC model and therefore, can be concluded that curcumin is not released from the chitosan matrix by dissolution solely (Rostami et al., 2014).

The reservoir diffusion-type drug delivery system is reflected in both the first-order and zero-order models (Wójcik-Pastuszka et al., 2019). Case II transport is assumed in the zero-order model (Marcos et al., 2015). Furthermore, the first order is theoretically equal to the KP model in the cases where n = 1, indicating that the case II transport mechanism is the responsible factor for the drug release (Unagolla & Jayasuriya, 2018). Supplementary Table 1 (A), Table 1 (B), and Table 1 (C) exhibit lower R² values for zero and first order, indicating inadequate fitting of the experimental data. Thus, case II transport does not entirely cause curcumin release from chitosan.

In summary, both the Fickian diffusion and polymer relaxation (case II) transport play govern the release of curcumin from chitosan over time according to non-linear curve fitting data reported in the in vitro study.

3.5 Antibacterial activity

According to the results shown in Fig. 8 (a) and (b), both curcumin and composite material have shown antibacterial activity against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus. Out of all three samples, curcumin extract, chitosan, and composite, chitosan was effective against only Pseudomonas aeruginosa, Klebsiella pneumonia, and Escherichia coli bacterial strains. Among these three bacterial strains, chitosan was highly effective against Escherichia coli, resulting in zones of inhibition of 11.75 ± 0.61 mm, 13.17 ± 1.77 mm, and 12.17 ± 0.60 mm for 5 mg/mL, 10 mg/mL, and 20 mg/mL concentrations respectively. The zones of inhibition against Pseudomonas aeruginosa were reported to be 11 ± 0.58 mm, 11.67 ± 1.20 mm, and 13 ± 1.32 mm, while Klebsiella pneumonia showed 10.00 ± 1.22 mm, 11.67 ± 0.17 mm and 11.00 ± 0.58 mm for the three concentrations tested. However, chitosan did not show any antibacterial effect against the Gram-positive bacterial strain, Staphylococcus aureus.Fig. 9.

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Fig. 8

Antibacterial activity of (a) curcumin and (b) chitosan (c) drug composite against Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli. *Footnote: The obtained zones of inhibition of curcumin, chitosan, and drug composite against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus.

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Fig. 9

Antibacterial activity of Curcumin extract (CE), Chitosan (Chi), and composite material against (a) Staphylococcus aureus, (b) Klebsiella pneumoniae, and (c) Pseudomonas aeruginosa.

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Among the four pathogenic microorganisms tested, Staphylococcus aureus was found to be the most susceptible to the inhibitory activity of curcumin extract and the newly synthesized curcumin grafted composite. In terms of varied concentrations of the curcumin extract and composite tested, 10 mg/mL was proven to have the highest antibacterial effects than 5 mg/mL and 20 mg/mL against Pseudomonas aeruginosa, Klebsiella pneumonia, and Staphylococcus aureus. The reported zones of inhibition of curcumin extract and composite material against S.aureus at 10 mg/mL were 15.67 ± 0.67 mm and 16.34 ± 0.60 mm, whereas the zones of inhibition of curcumin extract and composite material at 5 mg/mL were 12.34 ± 0.34 mm, 13.34 ± 0.34 mm and at 20 mg/mL the values were, 12.84 ± 0.60 mm and 13.34 ± 0.73 mm. Considering the given results, it was apparent that when the concentrations of the samples increased from 10 mg/mL to 20 mg/mL, zones of inhibition decreased, indicating that higher molecular weight and bulkiness of the chitosan polymer could have contributed to lowered diffusion of the extract in Mueller–Hinton agar medium and composite in the MHA growth media (Kong et al., 2010; Verlee et al., 2017).

The positive control Amoxicillin showed higher zones of inhibition than Curcumin, Chitosan, and Composite against all test microorganisms at 5 mg/ mL, 10 mg/ mL, and 20 mg/ mL. The highest zone of inhibition for Amoxicillin was recorded against Staphylococcus aureus with a zone of 40.5 ± 0.73 mm at 5 mg/ mL, whereas the same in Curcumin, Chitosan and Composite were 12.34 ± 0.34, 0 and 13.34 ± 0.34.

Moreover, the composite material has shown higher inhibitory activity against the most susceptible S. aureus than the curcumin extract itself. This indicates that the grafting of curcumin to chitosan has significantly improved (p < 0.05) the bioavailability of the newly developed composite material, contributing to higher antibacterial activity. Since chitosan is a positively charged molecule, it often interacts well with negatively charged cell membranes (around − 70 mV) as a result of ionic exchanges mediated by the Na⁺/ K⁺ pump between the intracellular and extracellular medium. Because of the positive charge, the cells tend to absorb chitosan nanoparticles more frequently, increasing the biocompatibility of the drug (Rodrigues et al., 2012).

Between K. pneumoniae and P. aeruginosa, K. pneumoniae was more susceptible to both curcumin extract and composite. The zones of inhibition that were reported for curcumin extract against K. pneumoniae were 11.50 ± 1.04 mm, 11.84 ± 1.01 mm, and 11.67 ± 0.34, while P. aeruginosa showed zones of inhibition of 10.67 ± 0.67 mm, 10.84 ± 0.44 mm, and 9.67 ± 0.34 mm at 5 mg/ mL, 10 mg mL, and 20 mg/mL concentrations, respectively. Enhanced antibacterial activity of composite material was observed at 5 mg/mL concentration against K. pneumoniae and P. aeruginosa, which is depicted by the zones of inhibition of 12 ± 0.76 mm and 11.67 ± 0.88 mm, respectively. When the concentration increased (>5 mg/mL), the zones of inhibition for the composite material against K. pneumoniae were 10.67 ± 0.67 mm and 11.00 ± 0.58 mm for the concentrations of 10 mg/mL and 20 mg/mL, respectively. Whereas for P. aeruginosa, it was reported to be 10.84 ± 0.60 mm and 10.84 ± 0.44 mm, respectively.

No zones of inhibition were reported for curcumin and composite material against the E. coli in the tested concertation range. The structure of the outer membrane of E. coli consists of outer membrane proteins (OMPs) that can act as selective channels to regulate the molecule uptake into the cells. Y. F. Liu et al.(Y. et al. et al., 2012) and Azucena et al.(Azucena et al., 2019) have indicated that curcumin does not show antibacterial activity against E. coli due to this complex cellular structure of Gram-negative bacteria. However, chitosan showed antibacterial activity against E. coli at the tested 5 mg/mL, 10 mg/mL, and 20 mg/mL concentrations due to the higher affinity and absorption of positively charged chitosan to the negatively charged cellular membrane of the gram-negative E.coli. Conversely, when curcumin is grafted onto the surface of the chitosan carrier, the net positive charge has been decreased. In comparison, fewer positively charged amino groups are usually available when chitosan is prepared as a carrier and combined with a drug. Its ability to interact with cell membranes and its surroundings is subsequently impacted by its reduced charge, which may limit its absorption and, ultimately, its potential toxicity (Rodrigues et al., 2012). The proposed antibacterial mechanism of the drug composite is shown in Fig. 10.

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Fig. 10

Antibacterial mechanism of drug composite.

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MIC and MBC results are reported in Table 4, where all samples except for curcumin against K. pneumoniae and chitosan against S. aureus showed bacteriostatic effects with MBC/MIC $\le 4$ . The MIC and MBC assays offer essential information about the mode of action of an antibacterial agent. The lowest concentration of bactericidal activity of an antibacterial agent is known as the minimum bactericidal concentration or MBC. MIC is defined as the lowest concentration of a material that can inhibit the visible growth of an organism. The MBC/MIC ratios were determined to evaluate the mode of antibacterial activity. The MBC/MIC ratio ≤ 4 indicates the bactericidal effect, whereas MBC/MIC > 4 indicates the bacteriostatic effect (Jayanetti et al., 2024).

The effectiveness of curcumin as a bacteriostatic agent varies significantly among gram-positive and gram-negative bacteria due to the differences in the components of their cell walls and membranes. Curcumin may interfere with the protofilament polymerization process linked to the FtsZ protein, which is crucial in cell division in microorganisms (Yun & Lee, 2016).

Curcumin has had higher antibacterial activity against Gram-positive bacteria in comparison to Gram-negative bacteria (Adamczak et al., 2020; Azucena et al., 2019). In the current study, curcumin and the composite showed the highest antibacterial effects on S. aureus. Furthermore, curcumin damages the bacterial membrane to prevent S. aureus from growing in size. It is presumed that the nature of the cell wall explains curcumin's greater efficacy against Gram-positive (+) bacteria as opposed to Gram-negative (−) bacteria. Peptidoglycan and hydrophobic compounds make up the majority of gram-positive bacteria's cell walls, which facilitate easier penetration. On the other hand, the complicated composition of the outer membrane of Gram-negative bacteria acts as a barrier, preventing the entry of most hydrophobic compounds and antimicrobial drugs such as colistin, quinolones, and β-lactams (Górski et al., 2022). Being a hydrophobic molecule, gram-negative bacterial cell walls do not allow the penetration of the active curcumin inside the microbial cell.

Chitosan showed antibacterial activity against E. coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, as shown in Fig. 8 (b). At lower concentrations, it binds to the negatively charged surface of cells, particularly Gram-negative bacteria, disrupting cell membrane permeability and causing cell death by externalizing intracellular components. Conversely, higher concentrations of positively charged chitosan, attributed to amino groups, can coat the cell surface, trapping intracellular components inside the cell of microorganisms (Hosseinnejad & Jafari, 2016; Lim & Hudson, 2004). Moreover, in the case of Gram-positive bacteria, the positive charges on both bacterial cells and chitosan create a repelling effect, preventing their close interaction (Hosseinnejad & Jafari, 2016). Gram-negative bacteria are thought to respond to chitosan more than Gram-positive bacteria because of their hydrophilic nature. Chung et al. (Chung et al., 2004); Kong et al. (Kong et al., 2010), and Pavinatto et al. (Pavinatto et al., 2014) indicate a stronger bactericidal effect on Gram-negative bacteria, attributed to the higher affinity between chitosan's amino groups and anionic radicals in the cell wall. Conversely, Hassan et al.(Hassan et al., 2018) and Helander et al.(Helander et al., 2001) suggest that Gram-positive bacteria may be more susceptible due to the presence of the gram-negative outer membrane barrier. The antimicrobial action involves chitosan being absorbed onto bacterial cell surfaces, causing increased lipid membrane permeability and the release of essential compounds, ultimately leading to cell death (Hosseinnejad & Jafari, 2016). Table 5 shows a summary of the relevant studies compared to the current study.