Application of Box-Behnken Experimental Design in Process Parameter Optimization for Production of Berberine HCl loaded Chitosan Coated Sodium Alginate Nanoparticles

 

Vinod Kumar¹*, Prashant Kumar², Saurabh Sharma¹

¹Research Scholar, School of Research, Pharmaceutical Sciences, CT University,

Ferozepur Road, Sidhwan Khurd, Punjab, India - 142024.

¹School of Pharmaceutical Sciences, CT University, Ferozepur Road, Sidhwan Khurd, Punjab, India - 142024.

²College of Pharmacy, Teerthanker Mahaveer University, Moradabad, Uttar Pradesh India - 244001.

 

ABSTRACT:

Psoriasis is the most common chronic autoimmune disease. The pathophysiology, genetics, comorbidities, and biologic therapies of plaque psoriasis have seen the most rapid advances. Only a tiny percentage of the supplied dose reaches the target site in most situations (traditional dosage forms), while the balance is distributed throughout the body according to its physicochemical and biochemical properties. The current worker used nanotechnology to carry out study on the formulation and evaluation of nanoparticulate Berberine HCl loaded Chitosan coated sodium alginate nanoparticles. The Berberine HCl Nanoparticles were optimised using the Box-Behnken design. Particle size (68.82-275.78nm), zeta potential ((10.90)– (47.1 mv), percentage yield (80.75 percent - 96.21percent), percentage drug entrapment (50.95 -77.28percent), and percentage release in pH 7.4 phosphate buffer (60.848 - 95.869percent) were all found to be positive with Berberine HCl nanoparticles. Nanoparticles were found to be spherical in shape with rough surfaces, according to surface morphology (SEM). In a pH 7.4 phosphate buffer, an in-vitro drug release investigation on an optimised batch of Berberine HCl nanoparticles (BE-OPT) revealed 96.242percent (12 h) release. The release kinetic investigation with the optimised batch of Berberine HCl nanoparticles (BE-OPT) demonstrated that Higuchi's release kinetic model was followed. The chosen (optimised) nanoparticles were sealed in amber-coloured bottles with cotton plugs and caps. All were kept for six months at 40±2°C/75±5% RH and examined for their physical appearance and drug content at certain intervals.

 

 

 

INTRODUCTION: 

Psoriasis is a chronic naturally resistant skin disease that affects roughly 3% of the US population and an anticipated 125 million people across the globe. The most prevalent type of psoriasis is plaque psoriasis, which accounts for even more than 80% of all occurrences. Men and women are equally afflicted by psoriasis, albeit adults are more impacted than children. Plaque psoriasis has witnessed the most rapid advancements in pathogenesis, genetics, comorbidities, and biologic treatments^1-2.

 

The medical attributes of skin disease vary based on the type of psoriasis. Plaque scabies, guttate psoriasis, erythrodermic psoriasis, and pustular psoriasis are all types of psoriasis³.

 

Herbal medicines have been used all over the world from last many years. Especially in India, there is wide market for herbals. Herbal medicines have less adverse effects as compared with modern medicines. Iriventi P. et al., (2021) formulated herbal cream that consists of Azadirachta indica (Neem) extract used in treating Psoriasis⁴. The delivery of plant/herbal therapeutic molecules as drugs is problematic due to poor solubility, poor permeability, low bioavailability, instability in biological milieu and extensive first pass metabolism. These limitations of herbal drugs can be overcome by attaching or encapsulating them with suitable nanomaterials⁵. It has been substantially proposed to combine natural medicine with nanotechnology, due to the fact nanostructured structures might to potentiate the motion of plant extracts, lowering the required dose and aspect effects, and enhancing activity⁶. Herbal resources now play a major role in the treatment of skin and inflammatory illnesses. According to several research, psoriasis symptoms can be alleviated by altering one's diet and lifestyle. Psoriasis symptoms have been relieved by a fasting period, a low-energy diet, and a vegetarian diet. Because omega-3 fatty acids and Vitamin E are included in fish oil, it has a favourable effect in several treatments⁷.

 

Berberine (BBR) is a natural plant alkaloid isolated from the Chinese herb Rhizoma coptidis and is commonly used for the treatment of diarrhoea⁸. Verma D. et al., (2020) evaluated the analgesic effect of Berberine and Asiatic acid alone, and in combination for their synergistic effect in animal models⁹. Ramachandran Ramachandran V. et al., (2020) investigated the effect of berberine an anti-oxidant and anti-inflammatory molecule, on cognitive functions, oxidative-nitrosative stress and inflammation in streptozotocin (STZ)-induced diabetic rats¹⁰. The low bioavailability and non-definitive mechanism of berberine HCl action limit its clinical applications¹¹. Berberine is an individual of the original berberine alkaloid family. It mainly exists in the roots, trunks and barks of various trees and has anti-psoriatic activity. In any case, according to the berberine organization, limited bioavailability and low assimilation rate are the two main obstacles, because only 0.5% of the ingested berberine is consumed in the small digestive tract, and this rate is when it enters the intestines. It is further reduced to 0.35%¹².

 

Among several novel drug delivery payloads, such as in realm of dermal/transdermal administration for the treatment of dermatological disorders such as psoriasis, the nano-sized carrier has established a distinct position. called nanocarriers are only employed in dermal treatments¹³. They have a number of advantages over other transdermal/topical drug delivery systems. Thus, the present worker formulated the nanoparticles of berberine HCl utilizing Chitosan coated sodium alginate nanoparticles.

 

MATERIALS AND METHODS:

Preparation of Berberine HCl loaded Chitosan coated Sodium Alginate Nanoparticles:

The berberine HCl (BE) loaded chitosan coated sodium alginate nanoparticles were prepared by method given Elsayed M.M.A. et al with some modifications. Sodium alginate nanoparticles were made by dissolving 0.25g in 30ml hot water and then stirring the solution with a magnetic stirrer for 10 minutes to distribute the drug. After homogenization, a 5 percent (w/v) suspension of CaCO3 was added to the Alg-drug solution, which was then disseminated in paraffin oil (30 percent internal phase ratio, v/v) containing varied concentrations of span 80 percent as an emulsifying agent and emulsified by stirring at various speeds. 20mL paraffin oil with 0.2 mL glacial acetic acid (acid/Ca molar ratio of 3.5) was added to the w/o emulsion after homogenization for 15 minutes. To allow calcium carbonate solubilization and the creation of calcium alginate microspheres, mixing was continued for another 20 minutes. After 10 minutes, the gelled microspheres were segmented and recouped with a CaCl₂ (0.05 M) solution containing 1% Tween 80 to remove the leftover oil. Microspheres were recovered from the oily phase using an acetate buffer at pH 4.5 (USP XXVII) and rinsed with this buffer until no more oil was visible under the optical microscope. As a polymer, chitosan was employed at concentrations of 0.5, 1, and 1.5 percent. The microspheres were immersed in chitosan solution (0.5, 1, 1.5 percent w/v) containing 0.05 M CaCl2 (pH 5.5) while being magnetically stirred. Microspheres were cleaned as indicated earlier after 30 minutes¹⁴.

 

Optimization of Berberine HCl loaded Chitosan Sodium alginate Nanoparticles:

With minor modifications to Yetilmezsoy et al., method, a Box-Behnken experimental design was used to examine and optimize the formulation parameters of BE nanoparticles production for highest percent EE and regulated drug distribution after two and 12 hours¹⁵. In scenarios with more than two dependent variables, this design is utilised because it requires less treatment combinations than other designs¹⁶. Controlled release nanoparticles of BE were created using a three-factor, three-level architecture. D:P ratio (X1), revolution per minute (X2), and span 80 percent were the three independent formulation factors investigated during the study (X3). In the above design, three levels of speed were used: 200, 400, and 600 rpm, which corresponded to the numbers -1, 0 and +1, respectively. The drug:polymer ratio was changed to (1:1), (1:2), and (1:3), which corresponded to -1, 0 and +1, respectively. Finally, span 80 percent was decided to be 1 percent, 1.5 percent, and 2 percent, respectively, denoting -1, 0 and +1 value. The entrapment efficiency (Y1), particle size (Y2), and release at the end of 12 hours (Y3) were chosen as the dependent variables to be examined for the manufactured BE microspheres.

 

Evaluation of Berberine HCl loaded Chitosan coated Sodium Alginate Nanoparticles:

Nanoparticles of Berberine HCl capable of giving an improved release profile were synthesised in this study. The preparation process was found to be easy and repeatable. Percentage yield, Percent Entrapment Efficiency, Particle Size, Zeta Potential, SEM analysis, Percentage drug release, Release Kinetics, and Stability tests were all performed on the produced      nanoparticles^17-23.

 

RESULTS:

Preparation and Optimization of Berberine HCl loaded Chitosan coated Sodium Alginate Nanoparticles:

Berberine HCl loaded Chitosan coated Sodium Alginate Nanoparticles and for designing an optimized formulation, Box-Behnken experimental design was employed. In total, 15 batches were created and tested for a variety of criteria.

 

Evaluation and optimization of Berberine HCl loaded Chitosan coated Sodium Alginate Nanoparticles:

Berberine HCl nanoparticles were examined for various post formulation parameters viz., percentage yield, entrapment efficiency, particle size and percent drug release. Nanoparticle optimization was carried out with the help of design expert software. Using Design-Expert version 13 software, a 3-factor, 3-level Box-Behnken design was used to optimise grafting. The results of the evaluation of the prepared batches were shown in the table 1. The statistical validity of the polynomials was determined using the Design expert Software's ANOVA feature. Design Expert software was used to create three-dimensional (3D) response surface plots and two-dimensional (2D) contour plots based on the model polynomial functions.

 

Box-Behnken statistical analysis:

A number of studies were carried out using the experimental runs obtained at various factor level combinations. The experimental matrix for the independent variables and observed responses from the randomised runs is shown in table 2.

 

Table 1: Evaluation of Formulation design

 

For responses R1, R2 and R3 (percent Entrapment Efficiency, Particle Size and Drug release), the best-fitting model was quadratic, according to Design Expert software. To build a full model (FM) polynomial equation, all answers were fitted to the model. The percent Entrapment Efficiency (R1), ranged between 50.95–75.18 percent, Particle Size (R2) was in between 68.82–275.78 micrometer while the percent drug release was in the range of 60.85-95.85 %. All responses were fitted to a second quadratic model and the adequacy of this model was verified by ANOVA, lack of fit and coefficient of determination (R²) tests. The results of lack of fit tests and ANOVA of the quadratic models for all responses are revealed in table 3. Here, in the ANOVA test, the p values of F-statistic of the model for responses Y1, Y2 and Y3 were <0.0500, indicate model terms are significant. As a consequence, from the p values for this model it can be accomplished that all of the responses (Y1, Y2 and Y3) fitted the quadratic model well (p <0.10 or smaller) lacks prediction efficiency, so a non-significant lack of fit value in the model is highly desirable.

 

Table 2: Box-Behnken design of experiment and Evaluation of Optimized Design

 

 

All of the responses fitted in the quadratic model showed a non-significant lack of fit (p> 0.1), proving the adequacy of the model fit. Table 4 shows a summary of the multiple regression analysis of the responses for the second-order quadratic model. Signal-to-noise ratio, a measure of the range of a predicted response relative to its associated error, is called “adequate precision”. For navigating the design space, a ratio greater than 4 is desirable. The ratios of “adequate precision” for Y1, Y2 and Y3 were 8.994, 42.174 and 8.104, respectively, indicating an adequate signal.

 

The predicted results were compared with the obtained results and standard deviation was determined. Saraf A. et al., (2019) applied box-behnken experimental design (BBD) for optimization of polymeric nanoparticles for site specific delivery of curcumin in colon cancer. The results of the study suggested that hydrophobic herbal molecule like curcumin, with evident anti-cancer activity in colon cancer, can be successfully encapsulated in nanoparticulate system for improving therapeutic efficiency and site-specific delivery to colon²⁴. Sharma A. et al., (2021) optimized Aceclofenac-loaded microsponges using Box-Behnken design (BBD) and desirability function. It was concluded that the BBD was a valuable tool for the development of optimized microsponges with desired properties²⁵.

 

Coefficient estimation and equations for responses:

The responses obtained at various levels of independent variables were subjected to multiple regression to give quadratic polynomial Eq. (1) for Y1 in terms of coded factors where intercept is 62.11, coefficient value for A:X1 is 0.5125, B:X2 is 6.92 and C:X3 is 3.31; quadratic polynomial Eq. (2) for Y2 in terms of coded factors where intercept is 238.31, coefficient value for A:X1 is 88.84, B:X2 is 27.40 and C:X3 is 22.34 and quadratic polynomial Eq. (3) for Y3 in terms of coded factors where intercept is 79.69, coefficient value for A:X1 is 0.577, B:X2 is 7.70 and C:X3 is 4.80.

 

Y1 (% Entrapment Efficiency):

62.11-0.5125*A-6.92*B+3.31*C-1.94*AB-2.43*AC+2.29*BC-2.10*A²-1.27*B²+6.41*C²+7.90*A²B+4.61*A²C-6.31*AB² …(1)

 

Y2 (Particle Size):

238.31+88.84*A-27.40*B-22.34*C+2.83*AB+0.525*AC-30.20*BC-53.85* A²-53.85-31.08*B²-11.64*C²+27.65*A²B+7.70 A²C-43.73*AB² …………………………………………..(2)

 

Y3 (Drug release):

79.69+0.577*A-7.70*B+4.80*C-2.39*AB-3.11*AC+4.24*BC-2.95*A²-2.65*B²+8.50*C²+8.08*A²B+3.28*A²C-11.81*AB² (3)

 

Response surface, contour plot analysis and Optimization of the formulation:

To further elucidate the relationship between the independent and dependent variables, two-dimensional (2D) contour plots and three-dimensional (3D) response surface plots of the responses across the selected factors were constructed as shown in figure 1. These types of plots are very useful for studying the interaction effects between two factors and for understanding how the effect of one factor will be influenced by the change in the level of another factor. Here, one independent variable must always be fixed as these types of plots can only express two independent variables at a time against the response²⁶. The independent variables were simultaneously optimized for all three responses by using the desirability function after studying the effects of the dependent and independent variables on the responses. Responses Y1, Y2 and Y3 were transformed into individual desirability scales. Constraints were set against all of the responses. Equal weight and importance were given to all of the responses. Finally, by combining the individual desirability function as the geometric mean by abroad grid search and practicability search over the domain by the Design-Expert software (StatEase Inc.), the global desirability value was calculated. The optimized formulation was achieved at A=0.25:0.25, B=1.5% and C=600 RPM with the corresponding desirability (D) value of 1.000. This factor level combination predicted the responses Y1 = 77.280 (%), Y2 = 68.82 nm and Y3 = 95.85%.

 

Figure 1: Contour plots of EE (A), Particle size (B), Drug release (C) and 3D plot of EE (D), Particle size (E), Drug release (C)

Table 3: Characterization of Optimized design Formulation

 

Table 4: Evaluation parameters of Optimized batch BE-OPT

 

Figure 2: Particle size (A) Zeta potential (B) of Optimized formulation BE-OPT

 

Finally, three batches of the optimized formulations were prepared to confirm the validity of the optimal parameters and predicted responses calculated. All of the responses were evaluated for each optimized formulation. The comparisons of predicted and experimental results were shown in table 3. It can be seen that the experimental values were in very close agreement with the predicted values, indicating the triumph of the BBD pooled with a desirability function for the assessment and optimization of berberine HCl formulations.

 

Characterization of Optimized Design Formulation:

The experimental values of these responses by the optimised grafting solutions (Table 4) were found to be in agreement with the predicted values of Entrapment Efficiency, Particle size, and Drug release generated by design expert software, implying that the optimised formulation was rational and reliable.

 

Particle size and Zeta potential:

The mean particle size of the optimised formulation BE-OPT was 68.50nm and the poly dispersity index (PDI) was 25.0percent. The zeta potential of aqueous dispersion of nanoparticles, which was determined to be 12.7mV, indicating that the formulation was stable (Figure 2).

 

Figure 3: SEM of Optimized formulation BE-OPT

 

Scanning Electron Microscopy (SEM):

The optimized formulation BE-OPT had a spherical and homogeneous dispersion in scanning electron microscopy pictures. The SEM picture revealed an unevenly smooth and rough surface. The increased size of SA–CH nanoparticles after coating with CH was confirmed.

 

FTIR Analysis:

Peaks were observed at 3362.11cm-1 (C-H stretching), 1642.96cm-1 (C=C and C=N stretching), 1381.94cm-1 (deformation in C-H), and 1041.80 cm-1 in the spectrum of the optimized formulation BE-OPT, as shown in figure 4. (C-O stretching). There was a small change in the drug's distinctive peaks, indicating that the drug and excipient had a meaningful interaction.

 

 

Figure 4: FTIR Spectrum of Optimized Formulation BE-OPT

 

Figure 5: DSC Thermogram (A) and XRD analysis (B) of Optimized Formulation BE-OPT

 

 

Figure 6: Percent (%) drug release profile of optimized batch (BE-OPT) of Berberine HCl Nanoparticles

 

 

Figure 7: Zero order (A), Higuchi graph (B), First order (C) and Korsmeyer peppas (D) graphs of Optimized batch of Berberine HCl nanoparticles (BE-OPT) in pH 7.4 phosphate buffer

DSC Thermogram and x-ray diffraction analysis:

The thermogram showed a shift in one of the endothermic peaks in the formulation thermal curves. Tonset = 314.96°C; Tpeak = 314.96°C) displayed a large endothermic peak on the DSC curve. The x-ray diffraction analysis revealed that in the formulation with chitosan and sodium alginate, drug crystallinity peaks were still observable (Figure 5).

 

In-vitro dissolution study:

The in-vitro release characteristics of the improved batch were also tested and presented in figure 6. The optimised batch (BE-OPT) had a percent (percent) drug release of 96.242% in 12 hours.

 

Release kinetic study:

The release kinetic studies performed with optimized batch (BE-OPT) of Berberine HCl nanoparticles, revealed that drug followed Higuchi as release kinetic model Figure 7).

 

Accelerated Stability Study:

The stability test was carried out under accelerated settings on the developed formulations in accordance with ICH recommendations. It was found that the formulations were stable at 40±2°C and 75±5% RH relative humidity.

 

DISCUSSION:

Psoriasis is an immune-mediated chronic skin condition that causes about 3% of the US population and an estimated 125 million individuals globally. Psoriasis affects both men and women equally, although adults are more affected than youngsters. The pathophysiology, genetics, comorbidities, and biologic therapies of plaque psoriasis have seen the most rapid advances. When used in traditional dose forms, the majority of dosagic concentrations are wasted. The ability of many medications to reach the site of therapeutic action limits their efficacy. Only a tiny percentage of the supplied dose reaches the target site in most situations (traditional dosage forms), while the balance is distributed throughout the body according to its physicochemical and biochemical properties. The current worker used nanotechnology to carry out study on the formulation and evaluation of nanoparticulate Berberine HCl loaded Chitosan coated sodium alginate nanoparticles. The main advantages of the nanoparticles are their stability and long-term storage. The particle size and surface characteristics of nanoparticles can be easily modified for controlled and targeted drug delivery²⁷. Polymeric nanoparticles are made from bio­degradable and biocompatible polymers, represent an option for controlled drug delivery and are promising formulation used for drug delivery systems, because they can be targeted²⁸. Experimental design methodology is a strategy that allows to study different variables simultaneously, the relationship between them and their influence on different experimental responses, by running a small number of experiments. Furthermore, through mathematical models it may determine the optimum level of the variables required for a given response. This technique can be successfully used to optimize nanoparticle preparation conditions²⁹. Fadhila F., et al., (2019) analysed of nanoparticle extract of Clove (Syzygium aromaticum L.) by Differential Scanning Colorimetry and their activity toward HeLa cell line. The result of synthesis showed that nanoparticle extract with yield 75% yield. The percentage yield of Berberine HCl nanoparticles was in close proximity of the result stated above³⁰. The observed response values were comparable to the predicted ones, with low percentage bias (±5%), thus suggesting the optimized formulation is trustworthy and that the model’s prediction ability is quite good.

 

CONCLUSION:

Nanotechnology has a big impact on the drug delivery industry, and nanoparticles are on the cutting edge with a lot of potential in clinical treatment and research. Nanotechnology's application in different fields, particularly health care, is critical, and it is leading to the progressive replacement of traditional treatments. Developing a medication delivery system that promotes a drug's pharmacological efficacy while decreasing toxic/side effects in vivo is a difficult undertaking. The use of colloidal drug carriers, which can provide site-specific or targeted drug delivery with optimal drug release patterns, is one strategy. Liposomes and micro/nanoparticles have been studied the most among these carriers. The current worker aimed to create the above-mentioned effective Berberine HCl formulation with a controlled release mechanism that would lower dosagic concentration and frequency of application while also improving patient compliance with the lowest potential harmful parameters. This study could provide a vision for different approaches and could be used as a platform in design and optimization of different nanoparticle formulations. In conclusion, a Box-Behnken experimental design was successfully used in order to obtain QU-nanoparticles with optimized characteristics.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

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Accepted on 20.08.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1139-1146.