1. Introduction
Nanoparticles have gained huge attention as drug carriers. In particular, the polymer-based on nanoparticles [
1,
2], lipid-based [
3,
4], magnetic nanoparticles [
5,
6], and liposomes [
7] are broadly studied under nanoformulations. Amongst them, the polymeric nanoparticles seemed to be particularly explored on account of their distinct physicochemical attributes [
8]. There are natural and synthetic polymers that are flexible and are employed for many uses, inclusive of the pharma industry [
9]. The polymers of natural origin are preferred more than synthetic, because of their eco-friendly cost-effective nature, biological effects, and biocompatibility.
The natural hydrogel polymers such as alginate, gelatin, and collagen have the potential to hold abundant water, keeping the structure intact, and are thus employed to carry hydrophilic drugs. The majority of the alginate produced annually is utilized for pharma and biomedical purpose, and the rest in the food industry [
10,
11]. Following the post-development of alginate in the 1980s, their application was expanded as microparticles for encapsulation and several studies were conducted to formulate alginate nanoparticles [
12,
13]. The nanomaterials of alginate denote a rapid growth field, especially in the food and pharma industry, and also in the academe [
14].
Chitosan is a hydrophilic linear cationic polysaccharide found profusely in nature, consisting of glucosamine and N-acetyl-glucosamine associated with glycosidic bonds [
15]. It is acquired by the removal of amino acetyl groups from chitin, a major constituent in the fungi cell wall, crustacean shells, and the cuticle of insects. Chitosan is insoluble at neutral and high pH regions due to its molecular structure and pKa (6.2–7.0). This means that chitosan can be protonated at low pH in aqueous solutions. Apart from biological degradation and biocompatibility, chitosan has a distinct bioadhesive feature that enables the interactivity of positive amino ions with a negatively charged mucous membrane. Owing to these characteristics, it is employed as a preferential matrix in the pharma industry [
16].
Among the available natural polysaccharides, two polyelectrolyte polymers, chitosan and alginate having opposite charges are chosen. The advantage of the quick gelling nature of chitosan and alginate is utilized to develop polycations and polyanions of composite polyelectrolyte [
17]. Employing polyelectrolytic complexation, the chitosan amine (-NH2) group and alginate carboxylic (-COOR) group interact and result in the formation of polyionic chitosan-alginate complex [
18]. As this interactivity lessens the complex porosity, it can protect the encapsulant and effectually control the release than the individual chitosan or alginate [
19]. At minimal pH, the high solubility of chitosan is decreased by the poor solubility of the alginate system, although at greater pH alginate is stabilized by chitosan that has low solubility.
Simvastatin (SIM) has less bioavailability (<5%) and greatly encounters metabolism by microsomal enzymes. SIM is classified as a Biopharmaceutics Classification System (BCS) Class-II compound with poor aqueous solubility and an acceptable permeability through biomembranes. The cytochrome enzyme CYP3A4 majorly targets the lactone framework of SIM and notably decreases the uptake by the intestine. The aquaphobic nature averts the drug dissolution entirely in the intestinal medium and accounts for low bioavailability. In general, statins decrease blood cholesterol by impeding HMG-CoA (3-hydroxy-3-methyl glutaryl coenzyme A) and are efficacious in maintaining cholesterol levels [
20]. SIM was also recognized for its prospects in the management of various cancers by impeding the cell cycle, preventing metastasis, and promoting apoptosis [
21]. Inhibition of HMG-CoA reductase results in alteration of the prenylation of small G proteins such as Ras, which regulate cell growth and survival via the downstream signaling pathways. Accordingly, inhibition of HMG-CoA reductase by statins was found to trigger apoptosis in several cancer cells. Masashi et al., recently showed that statins decreased the activation of the Ras/extracellular regulated kinase 1/2 (ERK1/2) pathway and Ras/phosphoinositol-3 kinase/Akt pathway. In malignant glioma cells, statins induce apoptosis by the activation of c-Jun N-terminal kinase 1/2 (JNK1/2) or by increasing the expression of Bim [
22].
To accomplish quality by design (QbD), the strategy of Design of Experiments (DoE) is broadly employed in research and industrial setup. The conventional one factor at a time (OFAT) method for screening, development and analysis of drugs has largely been displaced by the QbD process. This novel approach renders fine results with fewer experiment trails and encompasses screening and design standardization; also it depicts the effects of various input elements and their interference in a cost-efficient mode [
23]. SIM has a short half-life (about 2 h) and undergoes extensive first-pass metabolism in the intestinal gut wall and liver, thus minimizing its therapeutic efficacy. On the other side, alginate chitosan delivery systems have the potential power to improve drug stability, increase the duration of the therapeutic effect and permit administration through enteral or parenteral administration, which may prevent or minimize the drug degradation and metabolism as well as cellular efflux [
24]. On basis of the above statements, the present study aims at developing alginate-chitosan nanocarriers for SIM encapsulation. Many investigators have explored the behavior of chitosan and alginate nanoparticles individually in delivering various drugs. However, SIM in chitosan nanoparticles was studied in a different manner [
25], using quercetin as a doping agent. The study was not focused on cytoskeleton images and, as yet, no literature is evident on the direct use of chitosan-alginate nanoparticles as a carrier for SIM.
3. Discussion
IR Spectrum of the pure SIM shows the characteristic peaks at 3550, 2931, 1465, and 1072 cm−1. From the IR spectral data of SIM formulation, it is evident that there were no interactions of the drug as it exhibited similar peaks with a slight change in the intensity. This confirms the undisturbed structure of the drug in the formulation. Thus, this proves the fact that there is no potential incompatibility of the drug with the chosen polymers for formulation.
The root effect interrelationship among selected variables and the independent responses could be demonstrated by the recommended quadratic polynomials and the corresponding statistical significance, determined by ANOVA. The Predicted R² for both the responses of 0.891, 0.9223 is in accordance with the Adjusted R² of 0.9209 and 0.9463, respectively, as the variation falls below 0.2. In addition to this fit, summary data were applied to ensure the effectiveness and fitness of the chosen model. The model repeatability can be assured with the value of the coefficient of variation (CV). CV of the selected quadratic model should be <10%, to confirm the reproducibility. Relatively low CV values (7.35-PS & 5.98-EE) were noted in the study which ensures model accuracy and reliability. Adequate Precision quantifies S/N (signal to noise) proportion. In general, a fraction greater than four is preferable. PS and EE show a ratio of 13.1592 and 20.6875, denoting an appropriate signal, thus confirming the efficiency of the model to run the design space. Lack of fit can result in an ineffective model to represent the complete data. Therefore, lack of fit is a prerequisite to determining that the equations developed by the model are coherent in forecasting the responses. The lack of fit
p values of PS, EE, and SI were observed as insignificant and so the model chosen was appropriate [
27]. The Model F-value of both the responses were found to be 28.92 and 43.33, inferring the applicability of the model. Only a probability of 0.01% exists that a large F-value may arise because of the noise and as required, the model
p-value was observed to be significant with
p values of 0.0002 and <0.0001.
Further, the effect of test orders on the adapted model was illustrated by the residuals versus test order [
28]. In the present work, linear distribution of the external studentized residuals with a slight variation was noted, denoting that the selected model was admissible statistically [
29].
Figure 1b, depicts experimental operations set against the residuals, indeed a working method to recognize the slinking variables which may alter the study results. An arbitrary distribution pattern is noted in the chart that denotes time-dependent variables lurking in the framework.
ANOVA results outranged the statistical significance developed by the quadratic equation; furthermore, the p-value was <0.0500, representing the significance of model terms. The test method stipulated that PS was greatly influenced by (a) adversary effect of A with p-value 0.0121 and (b) synergic effects of B, and polynomial terms of A and B with a p-value of 0.0002, 0.0004, and 0.0002, correspondingly. Response 2 was greatly impacted by (i) adversary effect of polynomial term of A with p-value of 0.0067 and (ii) synergism effect of B with a p-value of <0.0001, and amongst the crucial parameters, term B affected the EE with high enormity.
The contour plot which gives the association of chosen responses with the variables ensures the variable effects. RSM was employed to estimate and interpret the response of independent parameters against the obtained discrete responses. Three-dimensional surface graphs are crucial to illustrate the interactivity and main effect. The obtained responses are forecasted by contour plots [
30]. As seen in contour plots, PS was found to be less as the chitosan concentration increased; in contrast to this, a high concentration of sodium alginate will be responsible for higher particle sizes. The interaction of these variables (blue color region) at specified concentration can yield the nanoparticles with minimum PS. Maximum EE was observed (orange color zone) with a high concentration of sodium alginate and varied concentration of chitosan. All these results will comply with ANOVA and regression analysis.
To standardize the model’s order obtained from statistical analysis, the function of global desirability (D) was employed. Every response was laid a limit (PS-Minimum and EE-Maximum) to draft an inlay plot to enhance the independent variables. All the feasible individual parameters were included in the method for standardization. The optimized concentrations of chitosan and sodium alginate were found to be 0.258 g and 0.353 g with desirability of 0.880. An optimized formulation (O-SIM-CAN) was prepared and evaluated for PS and EE to validate the study design. As required, the relative error was observed below 5%, which confirms the design accuracy (
Table 6). The same formulation was used to evaluate the remaining parameters.
Initially, in the preparation of SIM-CA-NP chitosan droplets were formed while stirring with tween 80. Subsequently, solidification was observed because of ionic crosslinking with alginate solution. Chitosan acquired a positive group, owing to the presence of amine groups at the aqueous solution of pH 4 to 5.5, while alginate dissolved in a neutral pH solution where the carboxylic groups were charged negatively. Hydrogel is formed due to the interactivity of amino groups of chitosan and carboxylic groups of alginates in the aqueous solutions of nearly 5.2 pH. At the same time, SIM—which is positively—charged was complexed with negatively charged alginate to attain a greater drug loading to the nanoparticle. The preparation rendered an opalescent suspension with a positive value of zeta potential. Chitosan acquired a positive group, owing to the presence of amine groups at the aqueous solution of pH 4 to 5.5, while alginate dissolved in a neutral pH solution where the carboxylic groups were charged negatively. Hydrogel is formed due to the interactivity of amino groups of chitosan and carboxylic groups of alginates in the aqueous solutions of nearly 5.2 pH. At the same time, SIM—which is positively charged—was complexed with negatively charged alginate to attain a greater drug loading to the nanoparticle. The preparation rendered an opalescent suspension with a positive value of zeta potential. Final NPs were collected after freeze-drying. The high positive surface charge of the formulation is an additional benefit while employing NPs in drug delivery as they can be easily transported by the negative channels in the plasma membrane. The variation in the zeta potential is attributed to the neutralization of the chitosan charge by the powerful negative charge of STTP versus alginate at the working pH. The standardized PDI validates the monodispersity of the formulation. Various structures of smooth to rough structures were noticed, and these may likely be developed by the formation of the hydrogen bond between eNH2, eOH, and eNHCOCH2 groups of chitosan backbone. Further SEM images clearly showed the well-separated and disperse nanoparticles. In addition to this, the SEM image gave a rough estimation regarding the particle size, which was about 20–40 nm in contrast to particle size determination by the DLS method. This can be explained since DLS measures the hydrodynamic diameter and also the swelling capability of polymeric hydrogel in the solution, while SEM depicts the pictures of dried particles.
The cumulative SIM release from plain SIM and the optimized formulation was studied as a function of time using PBS solution of pH 7.4. In the beginning, a quick release of SIM from both plain SIM and O-SIM-CAN is seen up to 24 h. It contributes around 40–45% of SIM from the total encapsulated quantity. This initial rapid release of SIM from NPs was mainly attributed to the occurrence of SIM at the NP’s surface, allowing a great extent of water diffusion through the liquid matrix, and thus accounts for rapid drug release. Further, a sustained phase with consistent drug release is seen for the next 72 h. The two profiles had a similar pattern of release, yet variation exists in the quantity released. The total amount of drugs released from the optimized formulation was around 86.25%. This was because of the gelling action of chitosan and alginate, which were responsible for controlling the drug release. On the contrary, the amount of total drug released from plain SIM was nearly 35%. It certainly denotes that the release of SIM was decelerated due to the encapsulation of NPs.
Two treatments (plain SIM, optimized formulation) at distinct concentrations (10–50 µg/mL) and the control group were tested for cell viability. This test affirms that all the treatments decreased cell viability with the given dose (in a dose-dependent manner). The observed percentage cell viability of O-SIM-CAN was less than the plain SIM, denoting that the effect resulted in high cytotoxicity on HSC-3 cells. The cytoskeleton images indicate that O-SIM-CAN showed uniform distribution and extreme cellular spreading. This nature was observed the same for 24 h and 72 h of cell culture in contrast to other samples. This can be due to, the surface nature of the chitosan alginate nanoparticles, which further promotes the strong affinity to the HSC-3 cells on a porous surface. Cell migration and vascularization were further noticed. That leads to differentiation and proliferation of the cells for the new tissue growth. In the case of pure SIM, this nature of differentiation was observed to be poor, owing to its inability towards the HSC-3 cells. The slightly augmented property was observed after 72 h of cell treatment. Hence, the chitosan alginate carrier system assisted in improved cell proliferation for SIM [
31]. Literature suggests that chitosan and its derivatives can selectively pervade through the cancer cells and exhibit antineoplastic effects employing cellular enzymes, apoptosis, antiangiogenic, enhanced immunity, and antioxidant defense mechanisms. On the other hand, alginate-based carriers modified with several drugs are presumed to accumulate in the liver and have a high level of targeting efficiency to hepatocytes. Hence, both these ingredients were majorly responsible for the enhancement of the anti-proliferative activity of SIM. The caspase-3 enzyme assay and cell viability studies ensure that the occurrence of apoptosis with optimized formulation increased significantly compared to other treatments, which can be credited to the use of chitosan and alginate in designing the current formulation.