Chitosan-Linseed mucilage polyelectrolyte complex nanoparticles of Methotrexate: In vitro cytotoxic efficacy and toxicological studies

2.5.1 Compatibility study by fourier transform infrared (FTIR) spectroscopy

FTIR spectra was acquired between 4000 and 500 cm⁻¹ wavenumbers using the instrument IR Prestige-21 spectrophotometer (Shimadzu, Kyoto, Japan) (Sindhu et al., 2015). For identification of the interaction between the drug and the polymers, spectral analysis of natural polymer used (LSM), synthetic polymer used (CS), pure drug (MTX), blank LSMCSNPs and MTX-LSMCSNPs were carried out by making a KBr disc.

2.5.2 Differential scanning calorimetry (DSC)

A thermal analyzer (Q600 SDT - TA, USA) was used to check the samples for thermal stability. Powdered samples were precisely weighed and placed on aluminum plates, temperature range of 50–400 °C was applied at 10 °C/min under a dynamic N₂ atmosphere. For the DSC thermograms, an empty pan was utilized as a reference (Ali et al., 2011).

2.6.1 Encapsulation efficiency of MTX-LSMCSNPs

MTX-LSMCSNPs were centrifuged at 4 °C for 45 min at 12000 rpm to separate the non-encapsulated drug to assess the encapsulation efficiency (EE). Measurement of absorbance at the wavelength of 306 nm was done using UV-spectrophotometer (Schimadzu, Japan UV-1650). To determine the amount of unentrapped drug following equation was used (Ong et al., 2016, Bashir et al., 2021). $$\mathrm{E}\mathrm{n}\mathrm{c}\mathrm{a}\mathrm{p}\mathrm{s}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{E}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y}=\frac{\left(\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}\mathrm{a}\mathrm{d}\mathrm{d}\mathrm{e}\mathrm{d}\right)-\left(\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}\right)}{(\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}\mathrm{a}\mathrm{d}\mathrm{d}\mathrm{e}\mathrm{d})}\mathrm{X}100$$

2.6.2 Drug content and yield

MTX-LSMCSNPs (5 mg) were suspended in 0.5 mL of water, followed by the addition of 9.5 mL DMSO. Suspensions were centrifuged at 12000 rpm for 45 min at 4 °C under the chilling conditions to determine % yield and drug content. The nanoparticles pellet was dried after removing the supernatant. The % drug content and yield of MTX were calculated by UV-Spectrophotometer using the following equations (Gooneh-Farahani et al., 2020, Bashir et al., 2021). $$\%\mathrm{D}\mathrm{r}\mathrm{u}\mathrm{g}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}=\frac{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{n}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e}\mathrm{s}}{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{n}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e}\mathrm{s}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{d}}\mathrm{X}100$$ $$\%\mathrm{Y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d}=\frac{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{n}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e}\mathrm{s}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{d}}{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{d}(\mathrm{p}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{m}\mathrm{e}\mathrm{r}\mathrm{s}+\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g})}X100$$

2.6.3 Physical appearance

All the formulations prepared (F1, F2, and F3), of MTX-LSMCSNPs were checked visually for turbidity.

2.6.4 Zeta size and Zeta potential analysis

The particle size, polydispersity index (PDI), and zeta potential of the formulations F1, F2, and F3), of MTX-LSMCSNPs were evaluated using Malvern Instruments Nano ZS 90 Zeta-sizer (Malvern, UK). To eliminate repetitive scattering, a He-Ne laser and water as a dispersion were used (Refractive index: 1.33). Zeta potential and polydispersity index were also determined. Concentrated solution may lead to false results of particle size analysis due to multiple scattering. Therefore, 1 mg/mL of MTX-LSMCSNPs diluted solution was used for analysis. Data was analyzed by cumulant method of analysis in Malvern software. This software considered each particle as a sphere and considered that in bulk distribution. To avoid particle aggregation tendency, samples were added by using a 0.2 µm syringe filter. Collected results were averaged of thrice analysis of each formulation. Data obtained was also used for polydispersity index (PDI) analysis (Bhattacharjee 2016). PDI was calculated by dividing the square of standard deviation by the average particle diameter as mentioned in following equation. $${\mathit{PDI}=(\frac{\mathit{\sigma }}{2a})}^2$$

2.6.5 Scanning electron microscopy (SEM) analysis

For surface and morphological analysis of MTX-LSMCSNPs, scanning electron microscopy (SEM) analysis was performed. Using a scanning electron microscope (SEM) (JEOL, IT100LA) with a 20- kV accelerating voltage, the form and surface morphology of contained Methotrexate loaded PEC stabilized NPs were studied. The small drop of NP suspension was dried on room temperature for 24 h as placed on SEM stub using double-sided adhesive tape and digital images of the samples were taken (Sahu and Das 2014).

2.6.6 Transmission electron microscopy (TEM) analysis

MTX-LSMCSNPs were also analyzed by Transmission electron microscopy. TEM analysis provides much higher resolution of nanoparticles image than other light-based techniques to analyze the quality, shape and size of nanoparticles. The picture was captured using a Transmission Electron Microscope (TEM) by placing the single drop of NP suspension to dry on room temperature on carbon-coated copper grids (CF400-Cu) (Hashad et al., 2016). The sample was examined using a TEM (Philips Technai-20) apparatus with a beam current of 104.1A and a voltage of 100 kV.

2.6.7 Swelling ratio of MTX-LSMCSNPs

To measure the swelling ratio weighed dried (Wd) samples of MTX-LSMCSNPs were immersed in phosphate buffer (37 °C) at different pH values for 24 h to attain equilibrium state. Samples were removed after 24 h, dried using filter paper and weighed (Wg) again. Swelling ratio was calculated by using following formula. $$\mathrm{S}\mathrm{w}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}=\frac{\mathit{Wg}-Wd}{\mathit{Wd}}$$

2.6.8 In vitro drug release

The drug release from prepared MTX-LSMCSNPs formulations was checked by applying the dialysis bag approach using a dialysis membrane i.e., regenerated cellulose membrane (cut off 10,000kD). Dissolution rate apparatus, USP type-II (Pharma-Test, Hainburg, Germany) was used for this analysis. Formulations (containing 2.5 mg of MTX) were placed in a dialysis bag and then this bag was put in 250 mL dissolution medium (0.1 N HCL) and the study was carried out for 2 h. After this, the dissolution medium was replaced by 250 mL of another dissolving media (Phosphate Buffer pH 5.5 and pH 7.4), where they were studied for another 24 h at 37 °C with continuous stirring at a speed of 100 rpm. Studies were carried out at pH 5.5 to confirm the release rate in tumor microenvironment (Chickpetty and Raga 2013, Weng et al., 2020). At regular intervals of every 30 min, the 5 mL of sample was extracted and replaced with an equivalent volume of fresh dissolving media, then examined by UV-spectrophotometry at the wavelength of 306 nm. The concentration was obtained by the calibration curve of MTX at a given time (t) and release was determined by using the following equation (Kou et al., 2020). $$\%\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}\mathrm{r}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}=\frac{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{M}\mathrm{T}\mathrm{X}\mathrm{r}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{d}}{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{l}\mathrm{o}\mathrm{a}\mathrm{d}\mathrm{e}\mathrm{d}\mathrm{M}\mathrm{T}\mathrm{X}}\mathrm{X}100$$

2.6.9 Kinetics of drug release

Different model-dependent approaches were employed to analyze the drug release kinetics from the controlled release formulation, including zero order, first order, Hixson–Crowell, Higuchi, and Korsmeyer–Peppas. The dissolution profiling of MTX-LSMCSNPs was carried out by applying these models via DDsolver software to ensure that the drug dissolution from the prepared formulations was occurring in an appropriate manner (Mircioiu et al., 2019).

Zero Order Kinetics: This model highlights that MTX-LSMCSNPs were at a concentration-independent release rate. The zero order kinetics is represented in following equation. $$Q_t=Q_o+K_o\mathrm{t}$$

Where Q_t signifies the amount of drug dissolved in time t, Q₀ the initial drug concentration in the solution, and K₀ the zero-order release constant (Libo and Reza 1996, Freitas and Marchetti 2005).

First-order kinetics: The model was used to explore the hydrophilic drugs release from a porous matrix and compared the drug content inside the drug carrier as represented as below. $$\mathrm{L}\mathrm{o}\mathrm{g}\mathrm{Q}\mathrm{t}=\mathrm{L}\mathrm{o}\mathrm{g}\mathrm{Q}0-\frac{\mathrm{K}1}{2.303}t$$

Where, Q_t = amount of drug dissolved at time t; Q₀ = Initial drug concentration in the solution. The first-order rate constant is denoted by K (Bravo et al., 2002, Ramteke et al., 2014).

Higuchi model: This model is mainly used to find out the cumulative percent release of hydrophilic drugs from polymeric matrixes of hydrophobic nature in relation to the square root of time as represented in the following equation. $$Q_t=K_H·t^{1/2}$$

Where, Q_t = amount of drug dissolved at time ‘t; K_H = Higuchi model constant (Higuchi 1963, Grassi and Grassi 2005).

Hixson–Crowell model: The model explained the drug release changes by changes in surface area and diameter of the particles. Here drug release is linked to dissolution changes, not by diffusion and was determined using the following equation. $$W_o^{1/3}-W_t^{1/3}=Kt$$

W₀ represents the initial drug quantity in the pharmaceutical dosage form, and W_t represents the drug quantity remaining in the pharmaceutical dosage form at time t (Chen et al., 2007).

Korsmeyer–Peppas Model: This model was used to explain the cumulative drug release from the polymeric matrix. The release rate constant for the Korsmeyer–Peppas model was calculated using the following equation. $${\frac{\mathit{Mt}}{M\infty }=Kt}^n$$

Where Mt/M $\infty$ represents a drug-fraction released at time t, k represents the release rate constant, n represents the release exponent, and (kappa) represents a constant that integrates the surface volume relationship.

3.2.1 Fourier transform infrared (FTIR) spectroscopy

The FTIR spectra of MTX, CS, LSM, blank LSMCSNPs and MTX-LSMCSNPs was shown in Fig. 3.The IR spectra of the MTX (Fig. 3a) showed absorption bands at 3338 cm⁻¹ (O—H stretching), 3080 cm⁻¹ (N—H stretching), 1670–1600 cm⁻¹ (C⚌O stretching), 1550–1500 cm⁻¹ (N—H bending), 1400–1200 cm⁻¹ (C—O stretching), 930 cm⁻¹ (O—H bending), and 820 cm⁻¹ (C—H bonding) (Boni et al., 2018, Nosrati et al., 2018, Gao et al., 2021). The spectra of CS (Fig. 3b) showed the peak of the OH group at 3356–3284 cm⁻¹, the peak of —CH₂ stretch at 2921 cm⁻¹, the peak of C⚌O stretching at 1651 cm⁻¹, the peak of —CH₂ bend at 1430–1384 cm⁻¹, the peak of N—H at 1592 cm⁻¹, the peak of C—O—C glycoside linkage at 1154 cm⁻¹, and the peak of Carbonyl, C—O at 1070 cm⁻¹ (Ahyat et al., 2017, Priya et al., 2020). The IR spectrum of LSM (Fig. 3c) revealed a larger band for —OH group stretching, ranging from 3674.39 cm⁻¹ to 3332.92 cm⁻¹; —CH₃ was being stretched at 2856.23 cm⁻¹ (Kurra et al., 2019). It moreover exhibited significant bands for —CH group stretch at 1685.06 cm⁻¹ (Ghumman et al., 2020). Other notable features include a sharp peak at 1440.83 cm⁻¹ owing to —CH stretching, a characteristic peak at 1112.95 cm⁻¹ due to glyosidic bond (C—O—C) of polysaccharide, and a characteristic peak at 1037.20 cm⁻¹ due to —CO bending (Rocha et al., 2021). The spectra of blank LSMCSNPs (Fig. 3d) showed the peak of the OH group at 3274 cm⁻¹, the peak of —CH₂ stretch at 2126 cm-¹, the peak of C⚌O stretching at 1634 cm⁻¹, the peak of —CH₂ bend at 1390 cm⁻¹, C—O—C glycoside linkage represents a peak at 1282 cm⁻¹, and the peak of Carbonyl, C—O at 1070 cm⁻¹. The IR spectra of MTX-LSMCSNPs (Fig. 3e), showed absorption bands at 3290 cm⁻¹ that was linked to O—H stretching, the peak of —CH₂ stretch at 2126 cm-¹, 1640–1600 cm⁻¹ represented C⚌O stretching, 1550–1500 cm⁻¹ showed N—H bending, 1400–1100 cm⁻¹ was linked to C—O stretching. Furthermore, IR spectra exhibited a shifting in the COO— peak shift to 1647 cm⁻¹, suggesting the formation of electrostatic bonds between two LSM and CS. The results revealed that the functions of the physical mixture of MTX, LSM, and CS do not interact.

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Fig. 3

Drug polymer compatibility FTIR studies (a) MTX (b) CS (c) LSM (d) Blank LSMCSNPs (e) MTX-LSMCSNPs.

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3.2.2 Differential scanning calorimetry (DSC)

LSM bears three endothermic and one shoulder peak (Fig. 4a); a high denaturation temperature of 114.7 °C was observed. Melting point and oxidation temperature were shown at 55–56 °C and 146–153 °C respectively (Athukorala et al., 2009). The DSC thermogram of CS (Fig. 4b); revealed a large exothermic peak at 297 °C; the degradation of chitosan amine units was linked to this exothermic peak. Chitosan has shown an endothermic peak at 80 °C This endothermic peak was related to the hydrophilic groups of chitosan associated with the loss of water (Dey et al., 2016). The DSC thermogram of MTX showed two endothermic peaks 129 °C due to release of water and (Fig. 4c); and broad endothermic peak in the range of 195–210 °C has showed the melting point of pure MTX (Taheri et al., 2011, Ray et al., 2015, Toomari and Namazi 2016). In MTX-loaded nanoparticles thermogram (Fig. 4e), it was observed that the melting point of MTX was shifted towards a lower temperature. It could be because MTX was not in crystalline form, but was in an amorphous state (Fini et al., 2010).

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Fig. 4

Differential scanning calorimetry (DSC) Thermogram (a) LSM (b) CS (c) MTX (d) Blank LSMCSNPs (e) MTX-LSMCSNPs.

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3.3.1 Encapsulation efficiency, drug content and yield

Methotrexate-loaded PEC stabilized nanoparticles based on LSM-CS were found to have drug entrapment efficiency (%) ranging from 49.97 ± 0.91 to 72.25 ± 0.66 %. The % drug content was determined to be between 82.52 ± 0.95 and 92.83 ± 0.72 %, while the % yield was found to be between 72.33 ± 1.01 % and 88.08 ± 0.88 % as shown in Table 3. With increasing LSM concentrations, the entrapment efficiency, drug content, and yield all improved. Because the quantity of LSM in formulations F1 and F2 (1:1) and (1:2) was equal, there was little variation in the outcomes. LSM-CS content was increased in formulation F3 (2:1) which might be due to adequate interaction between the viscous LSM solution and chitosan does not allow the drug to leach out of loosely aggregated polyelectrolyte particles.

3.3.2 Physical appearance

All MTX-LSMCSNPs formulations were colorless and transparent. No apparent particulates or turbidity was seen. The formed nano-suspensions were found to be clear with no signs of precipitation.

3.3.3 Particle size distribution and morphology of nanoparticles

The size distribution and ζ-potential values of MTX-LSMCSNPs are shown in Table 4. The particle size was determined to be between 192.1 nm and 246.6 nm, with a PDI of 0.326 to 0.502. The smallest particle size (192.1 nm) was found in formulation F3, which might be associated with the lowest CS content and highest LSM concentration. Formulation F3 with zeta potential −16.6 mv, which might be related to the lowest CS content and greatest LSM concentration, as the chitosan is cationic and LSM is anionic in nature. Formulation F2 with zeta potential of + 9.8 mv, which might be due to the high CS content that, is cationic in nature. The polydispersity index (PDI) tells us the heterogeneity in the detected particle size. PDI is a dimensionless parameter and if the value of PDI is less than 0.1, then particles are considered as “Monodisperse” and particle size is considered “Polydisperse” with PDI value, greater than 0.7 (Souza et al., 2016, Danaei et al., 2018). As evidenced by our samples; PDI was from 0.3 to 0.5; all samples showed suitable particle size distributions (values below 0.5), they also show a comparatively less susceptibility of agglomeration (Dantas et al., 2018). Dynamic light scattering (DLS) analysis was done to calculate the PDI results. Particle size was smallest when the LSM-CS ratio was 2:1. Changes in the mass ratio of LSM and CS resulted in a shift of particle size and showed that the LSM-CS ratio affects the particle size (Bhattacharya 2020). Secondly, ζ-potential was reduced upon increasing the ratio of LSM to Chitosan (Fig. 5A & B). SEM analysis and chemical composition of LSM was also checked (Fig. 5C & 5G). Further, SEM and TEM analysis for the formulations (F1-F3) along with their respective histogram are shown in (Fig. 5D-5F), representing the size distribution of nanoparticles. In Fig. 5F, particle size was measured by SEM image using ImageJ software for F1, F2, F3 formulations. SEM and TEM analysis showed that all MTX-LSMCSNPs had solid, smooth, and nearly spherical shapes (Noreen et al., 2022). The mean size distribution of NPs was in accordance to DLS analysis which was confirmed by applying the Gaussian fit of counts on distribution frequency (Souza et al., 2016). DLS analysis revealed the particle size distribution but SEM and TEM analysis was done to see the exact shape of MTX-LSMCSNPs and more reduced size had been seen at a ratio of 2:1 (Fig. 5D-F).

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Fig. 5

(A) Zeta -average d n.m of F3 (LSM-CS in ratio (2:1)) (B) Zeta-potential (mV) of F3, F2, F1 of LSM:CS ratio (2:1, 1:2 and 1:1). (C) Chemical composition of LSM (D) SEM images of LSM (E) SEM images of F3, F2, F1 LSM:CS ratio (2:1, 1:2 and 1:1) (F) TEM images of F3, F2, F1 of LSM:CS ratio (2:1, 1:2 and 1:1). G) Size distribution histograms of F3, F2, F1 of LSM:CS ratio (2:1, 1:2 and 1:1).

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3.3.4 pH-dependent swelling behavior of MTX-LSMCSNPs

pH of the medium is the most important factor that affect the swelling of behavior of nanoparticles, so swelling behavior was checked at various pH values and results have been plotted in Fig. 6. At acidic pH the amine groups with in polymeric layer of the LSM-CS having positively charged and thus protonated so an increase in swelling ratio results from the ensuing repelling force. Swelling ratio diminished when the pH was raised to a neutral level. It was anticipated to be have minimal surface charges because of this reduced swelling ratio values. Nevertheless, the trend towards higher pH levels (i.e., basic condition), swelling ratio grew once again, that specified most likely as a result of the medium's increased ion concentration (Najafipour et al., 2020).

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Fig. 6

Swelling ratio of MTX-LSMCSNPs at various pH values.

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3.3.5 Drug release studies

For the proper treatment of cancer disease, tumor- targeted drug delivery is required with minimum or no release of drug before the targeted site. Otherwise, the first pass effect can reduce the efficacy of the drug and leads to failure of treatment. Therefore, sustained and targeted delivery of hydrophobic drugs is required with maximum efficacy and minimum toxicity. The diffusion bag technique was used to achieve in-vitro drug release, and data were collected for all the prepared MTX-LSMCSNPs formulations. The release studies were carried out both at normal simulated conditions (pH 1.2 and pH 7.4) and pH linked to the tumor microenvironment (pH 5.5). At pH 1.2, all formulations showed drug release within the range of 8 to 14 % and results were insignificant between all formulation (F1, F2 and F3). At pH 5.5, cumulative % release was 58–69 %, while at pH 7.4, it was recorded around 47–58 % up to 24 h. The drug released quickly at pH 5.5, due to protonation of carboxyl and amino groups present on LSM-CS surface enhanced the swelling capacity of both polymers. Faster release at pH 5.5 was also due to repulsion happened between similar charges. Whereas at pH 7.4 release was slow as the swelling index was low due to deficient surface charges (Najafipour et al., 2020). The in vitro release data indicated that the MTX-LSMCSNPs formulation showed sustained drug release with a steady rise as shown in Fig. 7. Overall, the F3 formulation had superior and sustained drug release than F1 and F2 due to a higher concentration of LSM than chitosan. On the other hand, in the F2 formulation, the percentage of drug release was reduced due to higher concentration of chitosan than LSM. Whereas, F1 formulations exhibited a percentage drug release between the F2 and F3 formulations due to equal concentrations of LSM and chitosan.

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Fig. 7

In vitro cumulative drug release percentage, (CDR %) of MTX-LSMCSNPs (A) pH 5.5, (B) 7.4 at 37 °C. (n = 3/ Mean ± S.D.).

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While constructed like a nanoparticles systems, MTX-LSMCSNPs formulations (F3) with smaller particle size showed higher in vitro therapeutic characteristics in the dissolution experiment. The impact of therapeutic substance-loaded dosage forms with decreased particle size on dissolution rate has subsequently become the subject of extensive research (Takano et al., 2008). That enormous surface region enabled through fabricating the hydrophobic constituents using nanoparticle systems resulted for the in vitro disintegration behavior being consistent with certain other biological molecules, which facilitated drug solubility through achieving fast immersion throughout the container (Hintz and Johnson 1989). The hydrophobic substance was reduced to such nanometer size because of a rise in molecule curving, which also resulted in a rise in dissolving pressures. Greater permeability due to the reduced size permitted for increased drug absorption concentrations. That results in enhanced physical and chemical qualities and therefore should resolve the in vivo permeation issue with the poorly soluble MTX as BCS class II drugs (Mauludin et al., 2009, Kou et al., 2020).

3.3.6 In-vitro drug release kinetics

Five different model-dependent kinetic approaches were employed to analyze the drug release kinetics from the of MTX-LSMCSNPs. DDsolver software was used to study that the drug release kinetic behavior from the prepared formulations (Mircioiu et al., 2019). The findings are summarized in Table 5. All of the formulations of MTX- LSMCSNPs (F1, F2, and F3) followed the Higuchi model at both pH 5.5 and pH 7.4, with the highest R² values as compared to all other models. R² values were near to one that represents these MTX-LSMCSNPs formulations following the Higuchi model approach. Higuchi model approach is chiefly used for studying the drug release kinetic from the nanoparticles formulations. The Higuchi model is generally linked to three types of hypothesis. Firstly, drug concentration is higher initially than drug solubility. Secondly, the drug particles are encapsulated inside the nanoparticles having no drug on the surface of the nanoparticles, because the particles are much smaller than the thickness of the prepared system. Thirdly, this Higuchi model follows the porous system because hydrophilic polymer solubilizes easily to create pores, drug diffusivity remains constant and drug release occurs through pores created in the matrix (Mircioiu et al., 2019). Further, we checked the data by Korsmeyer-Peppas model that showed the second highest R² values as compared to other models after the Higuchi model and N value of F1 and F2 formulations was between 0.45 and 0.89, representing the non -fickian transport while F3 formulation, has N value less than 0.45, representing the fickian transport at pH 5.5 and N value for all formulations (F1, F2, and F3) was less than 0.45 at pH 7.4, representing fickian transport behavior for drug release.