ABSTRACT

Objectives

Tuberculosis (TB) remains a major worldwide health challenge causing morbidity and mortality, necessitating novel approaches for its effective therapy. Pyrazinamide (PYZ), a nicotinamide analogue, is a key frontline drug significantly involved in the treatment of TB. However, its dose-dependent hepatotoxicity is a major concern that needs to be addressed. The aim of the current research was to develop PYZ-loaded solid lipid nanoparticles (PYZ-SLNs) as a potential therapeutic intervention for treating TB.

Materials and Methods

The PYZ-SLNs were formulated by a high-pressure homogenization technique and optimized using a 23-factorial design. The drug concentration, emulsifier concentration, and homogenization cycles were considered critical formulation and processing parameters to study their effects on essential attributes of quality of PYZ-SLNs, i.e., entrapment efficiency (EE%), drug loading (DL%), and particle size.

Results

The optimized PYZ-SLNs showed a particle size of 401±08 nm, EE% of 86.24±1.15, DL% of 14.38±0.85. The in vitro lipolysis studies revealed that PYZ-SLNs exhibited an anti-lipolytic effect due to stabilization by poloxamer 188. Moreover, the in vitro gastrointestinal (GI) stability results demonstrated that the PYZ-SLNs were stable in GI tract media (at pH 1.2, pH 4.5, pH 6.8, and pH 7.4). The in vitro drug release studies showed the best fit with the Hixon-Crowell model. The accelerated stability studies revealed no significant changes in PYZ-SLNs for 6 months.

Conclusion

PYZ-SLNs could be a promising carrier for the treatment of TB via the oral intestinal lymphatic pathway, circumventing its hepatic first-pass metabolism and thereby preventing hepatic adverse effects.

Keywords:

Full factorial design, lipolysis model, long-chain lipids, lymphatic delivery, drug release kinetics

INTRODUCTION

Tuberculosis (TB) is a major worldwide health issue causing morbidity and mortality. It is an ailment that strikes in conditions of malnutrition, poverty, and limited healthcare access. Approximately 10.6 million people developed TB, and 1.3 million people died from TB in 2022, as per the World Health Organization Global TB Report. Primarily, developing countries such as India, Bangladesh, Pakistan, Philippines, Nigeria, and Indonesia bear the brunt of the worldwide TB burden.¹The currently available TB treatment lasts from 6 months to 12 months, depending on its type.^2-6 The drug treatment for TB is complex due to its adverse effects, multiple drug regimens, and longer duration. Thus, effective drug formulations are necessary that can provide sustained release over a ,more extended period and thereby increase medication adherence in patients.⁷

A nicotinamide analogue, pyrazinamide (PYZ), is an essential frontline drug utilized for TB treatment. PYZ has a noteworthy role in shortening the TB treatment schedule from a duration of 9 to 12 months to a schedule of 6 months.⁸The capability of PYZ to shorten the schedule is due to its activity against a population of persistent tubercle bacilli dwelling in acidic pH environments that are not destroyed by other TB drugs. The acidic pH facilitates the intracellular accumulation of pyrazinoic acid, the active derivative of PYZ formed by the conversion of PYZ by pyrazinamidase. Due to high dosing frequency and high drug payload, the regimen for PYZ bactericidal activity is limited to two months. A PYZ dose of 15-30 mg/kg, or 1.5 to 2 g (depending on the patient’s weight) is generally given. However, dose-dependent hepatotoxicity is a severe adverse effect of PYZ. Furthermore, major adverse effects of PYZ also include gouty arthritis, hyperuricemia, and, in rare cases, nephritis.^{2, 3, 6, 9, 10}

Several research studies have been conducted on formulating and developing nano-carriers to deliver PYZ, namely, liposomes,⁹ colloidosomes,¹¹ polymeric nanoparticles,^{6, 12}etc. Liposomes are potential carriers offering unique advantages; however, issues such as rapid leakage of water-soluble drugs, low encapsulation efficiency, instability in plasma, and poor storage stability persist.¹² Moreover, polymeric nanocarriers often require organic solvents in their fabrication, leading to toxicity and environmental risk.¹³ Additionally, researchers explore inhalation therapy to deliver PYZ. However, challenges in using specialized delivery devices and implementing them on a large scale are some of the drawbacks that need to be addressed.¹⁴

The oral route is the most convenient for drug delivery. The key contributing factors affecting the oral bioavailability of drugs are gastric mean residence time, pH of the gastrointestinal (GI) tract, and drug solubility and permeability. Moreover, the pre-systemic metabolism also affects the bioavailability of drugs. The lymphatic pathway is a preferable alternative for avoiding first-pass metabolism. Several colloidal nano-carriers such as nano-micelles, self-emulsifying delivery systems, polymeric nanoparticles, liposomes, microemulsions, and solid lipid nanoparticles (SLNs) have been explored for achieving oral delivery via intestinal lymphatic transport.^15-17

SLNs have emerged as promising nanoparticles as they amalgamate the benefits of all colloidal carriers mentioned and thereby improve the effectiveness of encapsulated drugs. They enhance the drug’s lymphatic uptake. They are taken up via lymphatic circulation and enter blood vessels via jugular and subclavian veins, thus preventing the first-pass metabolism of the drug, and thereby reducing hepatic adverse effects.

Incorporating PYZ in SLNs would also promote drug efficacy, lessen the drug dose, and provide a sustained release.^18-20 Furthermore, it has been reported that the drug must have log P≥4.7 to be suitable for lymphatic uptake. PYZ exhibits a log P of -1.884; hence, it cannot enter the lymphatics and thereby undergoes hepatic first-pass metabolism.²¹ Thus, based on the facts above, there is a dire need to develop therapeutic strategies that reduce PYZ hepatic toxicity.

To surmount the limitations associated with PYZ therapy, we propose an encapsulation of PYZ in SLNs to reduce hepatic adverse effects. The fabricated PYZ-SLNs contain long-chain lipids that facilitate absorption through the lymphatic system compared to medium or short-chain lipids. Moreover, the SLNs were developed utilizing poloxamer 188 (surfactant), which develops a steric crown on the SLNs surface, thus decreasing their contact with pancreatic and gastric lipase. Thus, poloxamer 188 plays a significant role in maintaining the integrity of PYZ-SLNs during its migration via the GI tract into the lymphatic system (Figure 1).²²

MATERIALS AND METHODS

Materials

PYZ (99% pure) was obtained as a gift sample from S. Kant Healthcare Ltd., Mumbai, India. Stearylamine and stearic acid were acquired from Himedia Laboratories in Mumbai, India. Compritol 888 ATO (Glyceryl di- and tri-behenate) was acquired as a kind gift from Gattefosse India Pvt. Ltd., Mumbai, India. Glyceryl monostearate was donated by Hallstar, USA. Softisan 154 and Dynasan (114, 116, and 118) were donated by CremerOleo, Germany. Crodamol cetyl palmitate was acquired as a gratis sample from Croda, Mumbai, India. Tween 80, tween 20, sorbitan monooleate (span 80), and sorbitan monolaurate (span 20) were acquired from Central Drug House (CDH) Pvt. Ltd., New Delhi, India. Cremophor EL, cremophor RH 40, poloxamer 407 (P407), poloxamer 188 (P188), and solutol HS 15 samples were acquired as samples from BASF India Ltd., Mumbai, India. Soy Lecithin (Source: Soy), lipoid S 75 phospholipon 90 H, and phospholipon 90 G were acquired as samples from Lipoid GmbH, Germany. Sephadex G-50 (grade: coarse) was procured from MP Biomedicals, USA. Mannitol was acquired from CDH, New Delhi, India. The solvents used were of high performance liquid chromatography (HPLC) grade, and all additional chemicals were of analytical grade.

Surfactants and solid lipids screening

The lipids were heated to 10 °C higher than their melting temperature in a glass vial. PYZ (in increments of 2 mg) was incorporated into the melted lipid, which was stirred at 100 rpm for 24 h by employing a water bath incubator shaker (EIE Instruments Pvt. Ltd., Ahmedabad). To confirm whether insoluble drug crystals were present or absent, the lipid melts were examined against a black-and-white background. The solubility of the drug was also assessed in several surfactant solutions (1% w/v) that were kept at a temperature of 25±2 °C. All the above-mentioned studies were carried out in triplicate sets.^23-26

Preparation of PYZ-SLNs

The high-pressure homogenization method was used for the fabrication of PYZ-SLNs.^27-29 In brief, soy lecithin (0.3% w/v), stearyl amine (0.1% w/v), and stearic acid (1% w/v) were heated (10 °C above the melting point of the lipids) in a beaker using a water bath to obtain a uniform lipid phase. In the lipid melt phase, PYZ (30% w/w) was added and dissolved with constant stirring. Poloxamer 188 (3% w/v) was melted at a temperature similar to the lipid phase to obtain an aqueous surfactant solution. A high-speed stirrer (12000 rpm) (RQ 122/D Remi mechanical stirrer, Remi Mumbai, India) was used to mix aqueous surfactant solution and melted lipid phase, to form a pre-emulsion. A high-pressure homogenizer (GEA Lab Homogenizer PandaPLUS 200, Niro Soavi, Italy) was employed for the nanonization of pre-emulsion. It was subjected to 12 homogenization cycles at 1200 bar pressure in the homogenizer. The formulated SLNs were then lyophilized after the addition of cryoprotectant (Mannitol: 5% w/v) employing a bench-top freeze-dryer (FD-10-MR, Labfreez Instruments, Beijing, China) and were stored in a cool place.²⁶^,^30-35

Statistical analysis

Statistical optimization of PYZ-SLNs

The PYZ-SLNs were optimized for various processing and formulation variables by applying the design of experiments using Design Expert software (Stat-Ease, Inc., Minneapolis, MN, USA). The preliminary batches (data not shown) were fabricated and characterized to investigate the formulation and processing variables influencing the PYZ-SLNs. A 2³full factorial design (two levels and three factors) was applied to optimize PYZ-SLNs. A 2³ full factorial design estimates the impact of three factors (screened via preliminary trials) at two levels (high and low) on the dependent variables.

PYZ-SLNs characterization studies

Determination of particle size, polydispersity index (PDI), and zeta potential (ZP)

The particle size analysis was conducted by employing Zetasizer Nano ZS90 (Malvern Panalytical Ltd., Malvern, UK), which functions based on the principle of dynamic light scattering. The sample was diluted, for particle size analysis, to the required concentration using water for injection. Particle size distribution results were acquired, indicating the PDI of formulated PYZ-loaded SLNs. Each sample had a run time of 120 seconds. The ZP calculates the particle’s electrophoretic mobility in an electric field using about 12 to 15 runs for every measurement. All the above-mentioned studies (D90, PDI, and ZP) were carried out in triplicate sets.²⁴^,²⁵^,^33-35

Evaluation of DL% and EE%

The DL% and EE% of the PYZ-SLNs were estimated based on the size exclusion chromatography principle by utilizing the Sephadex G-50 minicolumn and centrifugation technique (as discussed elsewhere).³⁴^,³⁶

The drug-loaded SLNs, after centrifugation, collected from Sephadex G-50 mini column were diluted (20 times) using a 2:1 ratio of dichloromethane, methanol to determine the entrapped drug (W_entrapped). A validated HPLC method was utilized to analyze the EE% of the aforementioned sample. All the above-mentioned procedures were carried out in triplicate. The following formula was used to calculate the EE%:

Where W_entrapped is the amount of entrapped PYZ in the SLNs (Separated via Sephadex G-50 column), W_total is the whole PYZ quantity that was added into the formulation.

The following formula was used to calculate the DL%:³⁷

Differential scanning calorimetry (DSC) analysis

A Shimadzu DSC 60 A (Kyoto, Japan) linked to a TDA trend line program was utilized for calorimetric analysis. Samples were weighed (5 mg) in an aluminum pan, while an empty aluminum pan served as the reference. Thermograms were scanned at 10 °C per minute from 40 °C to 300 °C. Thereafter, the samples were cooled down to 40 °C using liquid nitrogen.²⁶^,32-34,38

Transmission electron microscopy (TEM) analysis

The PYZ-SLN’s morphology was examined by using a CM-200 TEM (Philips, the Netherlands). On Formvar-coated TEM grids (Ted Pella, Redding, CA), SLNs (diluted 50X) were mounted and then negatively stained with phosphotungstic acid. The PYZ-SLNs sample was dried for 5 min at 25 °C and then observed using a TEM with a resolution of 0.23 nm at 200 kV.^{19,24-26,33,34,39,40}

Powder X-ray diffraction (PXRD) studies

To investigate the crystallinity of PYZ and the SLNs’ structure after preparation, an X-ray diffractometer (Bruker D8 Discover, Germany) was used. The diffraction patterns of pure PYZ, stearic acid (solid lipid), blank SLNs, and optimized PYZ-SLNs (batch 11) were analyzed. The samples (500 mg) were kept in the sample compartment and exposed to Ni-filtered Cu Kα- radiation at a wavelength of 1.5406 Å at 30 kV, 10 mA. Each diffractogram was recorded from 10° to 40° two theta angles, and plots (intensity vs. 2θ) were generated utilizing software OriginPro 2017 (OriginLab Corporation, USA).²⁴^,²⁵^,³³^,³⁴^,⁴⁰

In vitro characterization studies of PYZ-SLNs

In vitro release study

Using the dialysis membrane method, the in vitro release from PYZ-SLNs was conducted in various solutions: 0.1 N HCl (pH 1.2) to mimic gastric pH phosphate-citrate buffer (pH 4.5) to mimic endosomal alveolar macrophage and phosphate buffer (pH 6.8 and 7.4) to mimic intestinal pH.^41-45The volume of various release media for conducting the in vitro release study was 100 mL. The freeze-dried SLNs (comprising the 10 mg equivalent of entrapped PYZ) were added to the dialysis tube (molecular weight cut-off 12-14 KDa, HiMedia Laboratories Pvt. Limited, Mumbai) for the investigation. The beaker containing dialysis tubing suspended in the media was maintained at 37.0±0.5 °C with magnetic stirring (5-MLH Remi, Remi labworld, India) at a speed of 100 rpm. The 5 mL aliquots were drawn at predetermined time points (0, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 96, and 120 hours) and replenished with an equal amount of fresh release media to sustain the sink conditions. A validated HPLC technique was used to analyze the samples and determine the quantity of drug released.²⁵^,²⁶^,³³^,³⁴

Drug release kinetics

The drug release data were further subjected to drug dissolution (DD) solver to determine the drug release kinetics.⁴⁶Various mathematical models, namely first order, zero order, Higuchi, Weibull, the Hixon-Crowell, and the Korsemeyer-Peppas were evaluated to determine the drug release mechanism from SLNs. The release data were fitted to all the above-mentioned models in the DD solver program and the scatter plots were generated with the fitted curve. The model with the highest R-square value and highest f-value was the best fit for dissolution profiling. The formula for the models to which the release data were fitted is mentioned below:

Where, in all models, F is the fraction (%) of drug released in time t, k₁is first order release constant, k₀is zero order release constant, is the Higuchi release constant, is the maximum drug released at infinite time, α (scale parameter) defines the time scale of the process, β (shape parameter) characterizes a curve as exponential, sigmoid, or parabolic; k_HCis release constant in the Hixson-Crowell model, is the korsemeyer-peppas release constant incorporating the geometric and structural characteristics of the formulation, and n is the diffusional exponent defining the release mechanism.⁴⁷

In vitro lipolysis test

The in vitro lipolysis test of PYZ-SLNs was conducted as per the previously described procedure using simulated empty-state intestinal conditions.⁴⁸ A lipolytic medium was prepared and reacted with the formulated PYZ-SLNs. It contained calcium chloride (5 mM), sodium taurodeoxycholate (5 mM), sodium chloride (150 mM), soy lecithin (1.25 mM), pancreatic lipase (0.525 g/300 IU/mL), and sodium dodecyl sulphate (0.5% w/v). The lipolytic medium was buffered with tris-maleate (pH 6.8). Herein, simulated digestive media (14 mL) was mixed with PYZ-SLNs (6 mL). The above blend was stored at 37±0.5 °C in a thermostatic water bath. The entire lipolysis process was maintained at pH 6.8 by using an Auto Titrator (Titra+, LabIndia Analytical Instruments Pvt. Ltd., Thane, India). The autotitrator used 200 mM sodium hydroxide to counterbalance the fatty acid generated by the lipid digestion. When the pH change at 15 minute intervals was less than 0.05 units, the digestive process was considered to be complete.

Where V is the titrant volume utilized amidst the digestion at 6.8 pH, C is the titrant concentration, M.W denotes the lipids molecular weight (g/mol) utilized for the formulation of SLNs, 3 describes that one triglyceride molecule can release a maximum of 3 fatty acids, ρ indicates the lipid density (g/mL), ν is the lipids volume in SLNs within the lipolysis medium.^24-26,33,34

In vitro investigation of GI stability of PYZ-SLNs

GI stability of PYZ-SLNs was assessed by determining their EE%, PDI, and particle size across different pH ranges, viz. pH 1.2 (0.1 N HCl), pH 4.5 (sodium acetate buffer), pH 6.8 (PBS), and pH 7.4 (PBS), simulating the GI physiology of humans. To assess the stability of the formulated PYZ-SLNs, they were kept at pH 1.2 and 4.5 for 2 h, and incubated at pH 6.8 and 7.4 for 6 h.¹⁹^,⁴⁹^,⁵⁰

Stability analysis of PYZ-SLNs

The stability studies of the optimized lyophilized PYZ-SLNs were conducted according to the previously reported literature.¹⁹^,⁵¹The lyophilized PYZ-SLNs were stored in amber-colored glass vials, sealed, and placed in an upright position in a stability chamber (Nova Instruments Pvt. Ltd. in Ahmedabad). Thereafter, the SLNs were stored at 8 °C, 30±2 °C/65±5% RH (intermediate conditions), and 40±2 °C/75±5% RH (accelerated conditions) for 6 months to assess their stability. At predetermined intervals, namely, initial, 1 month, 3 months, and 6 months, samples were investigated for ease of redispersibility, any alterations in physical appearance, EE%, PDI, and particle size.^24-26,32,33

RESULTS

Lipid excipients and surfactants screening

Solid lipids screening

The solid lipid was selected based on the criterion that the drug has maximum solubility in that lipid. PYZ has a log p of -1.884 as it is lipophilic in nature. The results showed that stearic acid had the highest solubility of PYZ (Figure 2A). The partitioning of PYZ in various solids was evaluated.²³ Stearic acid was selected for developing PYZ-SLNs because PYZ partitioning was highest in stearic acid.

Screening of surfactants

The surfactant was selected based on the least solubility of PYZ in it. As shown in Figure 2B, the investigation was carried out using different surfactant solutions (1% w/v). Drug moieties are incorporated in the lipid matrix and are strongly associated with the solid lipid core due to surfactants with low drug solubility. It was necessary to select surfactants with the lowest solubility of PYZ. Drug moieties are materialized in the lipid matrix and are strongly associated with the solid lipid core by surfactants having a low drug solubility.³³^,³⁴ It was necessary to select surfactants having the least solubility for PYZ. The PYZ solubility was found to be highest in polysorbate 80 (20 mg/mL), followed by cremophor RH40 (18 mg/mL), polysorbate 40 (16 mg/mL), polysorbate 20 (16 mg/mL), and cremophor EL (16 mg/mL). PYZ was found to be moderately soluble in poloxamer 407 (14 mg/mL) and solutol HS15 (14 mg/mL). The solubility of PYZ in P188 was the lowest (12 mg/mL). Thus, P188 was selected for formulating PYZ-SLNs.

It has been reported that P188 provides an anti-lipolytic effect in the GI tract. A steric crown is created on SLNs surface, which reduces the interaction of SLNs with pancreatic and gastric lipase.^50-52Batches CSP2 (1% w/v P188) and CSP3 (3% w/v P188) were subjected to an in vitro lipolysis study. Batch CSP2 degraded by 21.23±1.86%, while batch CSP3 degraded by 10.12±0.69% (Figure 2C). Thus, 3% w/v P188 was selected for the optimization of PYZ-SLNs as it resulted in minimum lipolysis.

PYZ-SLNs optimization by response surface methods

Optimization of PYZ-SLNs utilizing 2³ factorial design

Based on the preliminary trial results (data not shown), it was observed that among various formulation and process variables, the parameters viz. homogenization cycles, emulsifier concentration, and drug concentration demonstrated significant impact on particle size, DL%, and EE% of PYZ-SLNs. The emulsifier concentration influenced particle size and EE%, while the drug concentration primarily impacted DL% and EE%. The homogenization cycles impacted the EE% and particle size of PYZ-SLNs. To comprehensively optimize the PYZ-SLNs, these primary observations were taken into account, and a 2³ factorial design (two levels and three factors) was applied, as shown in Table 1 and Table 2. The following mathematical model was used to derive equations showing the link between independent and dependent variables:

Where X₁, X₂, and X₃ are the factors (chosen from primary studies), Y_iis the dependent variable, B₁ to B₃₃ is the regression coefficients range and B₀ is the intercept.

3D response surface plots were generated, and data were investigated by employing Design-Expert 10 software (version 10.0.6) (Stat-Ease, Inc., Minneapolis, MN, USA). To identify a design space with higher desirability, restraints were applied to responses.

The responses [particle size (D90), EE%, and DL%] achieved at various levels of the independent variables (X₁, X₂, and X₃) were put through multiple linear regression to obtain second-order polynomial Equations (11), (12), and (13). The equations are mentioned below:

The equations signify the influence of X₁, X₂, and X₃ (independent variables) on Y₁, Y₂, and Y₃ (dependent responses). The correlation coefficient R² values were established to be 0.9484, 0.7094, and 0.9317 for dependent responses Y₁, Y₂, and Y₃, respectively, signifying a good match.

The impact of factors and independent variables on particle size is shown in Figure 3A-C. Equation (11) indicates that the X₂ and X₃ variables have a notable inverse effect on the PYZ-SLNs particle size. This suggests that the particle size (D90) of PYZ-SLNs is reduced with increasing homogenization cycles (X₃). The possible cause of the decrease in particle size might be the cavitation forces in the high-pressure homogenizer leading to the reduction of PYZ-SLNs to nano-scale size.⁵³^,⁵⁴Moreover, the particle size of PYZ-SLNs reduces with an increase in emulsifier (X₂) concentration. This might be owing to increased stabilization of pre-emulsion by an emulsifier, leading to a diminution in particle size as well as its aggregation.⁵³

The impact of the factors on EE% is represented in Figure 3D-F. It is evident from Equation (12) that factors X₁ and X₂ have a positive impact, whereas factor X₃ has a negative impact on EE% of PYZ-SLNs. The direct correlation between the drug concentration and EE% is described by the Equation. The EE% increases with an increase in drug concentration, which might be due to the increased drug availability for accommodation in a lipid matrix. The EE% rises as the emulsifier (X₂) concentration increases. The high drug solubilization and incorporation into the lipid core could be responsible for an increase in EE%.⁵³^,⁵⁵ The EE% was negatively impacted by homogenization cycles (X₃). The EE% decreased as the number of homogenization cycles increased. This might be owing to the increased surface area of particles caused by the cavitational forces generated with increased cycles of homogenization, resulting in drug leaching during SLN formation.⁵³^,⁵⁴ The impact of factors on response DL% is represented in Figure 3G-I. It is clear from Equation (13) that the variables X₁ and X₂have a positive impact on DL%. The increase in drug concentration was attributed to more drug able to be incorporated into the lipid matrix. Furthermore, the drug is more solubilized in the lipid matrix with the addition of an emulsifier. Thus, an increase in emulsifier concentration increases the DL%. It was found that as the homogenization cycles increased, the DL% was reduced. This could be the result of greater homogenization cycles, which further cause drug leaching during the preparation of SLNs.

Optimization and validation

The 3D RSP results were based on polynomial models, which depicted the impact of significant independent factors on responses. Two additional checkpoint batches, P9 and P10, with the predicted values, were prepared to verify the validity of the optimization process. The checkpoint batches were prepared, and the variance between the observed and predicted values was examined using the Student’s t-test (Table 2). The outcomes showed a high degree of closeness between observed and predicted values, and the difference between them was not significant (p>0.05). As a result, the obtained mathematical Equation was found to be accurate in predicting the responses Y₁, Y₂, and Y₃ (Table 3).

The software Design-Expert 10 (version 10.0.6) was used to create optimum conditions for PYZ-SLNs, with a desirability value of 0.878. The optimized batch P11 was selected by setting the criteria of particle size <500 nm, and DL% and EE% as being the highest. For the optimization batch, the software projected the following process parameters: drug concentration (PYZ) =30% w/w, emulsifier concentration (Soy lecithin) =0.3% w/v, and homogenization cycles =12. Furthermore, the Design-Expert 10 software predicted that the optimized batch P11 would have a particle size of 404 nm (Figure 4), with 85.74% (EE%) and 14.33% (DL%). Table 3 shows that the predicted values produced by the software and the practical values (P11-optimized PYZ-SLNs) were in good agreement.

In vitro drug release analysis of PYZ-SLNs

The lyophilized PYZ-SLNs (batch P11) in vitro drug release study was conducted at different pH values: 1.2 (0.1 N HCl), 6.8, and 7.4 (PBS), 4.5 (citrate-phosphate buffer) (Figure 5A and B). At a pH of 1.2, the PYZ-SLNs (Batch P11) exhibited ~10% drug release in 2 h. However, at pH 4.5, 6.8, and 7.4, the PYZ-SLNs (Batch P11) were observed to have a biphasic drug release pattern, releasing about 90% of the drug in 5 days (120 h) at different pH ranges. This pattern consisted of an initial burst release (~30% in 12 h) and thereafter, a sustained release (~90% in 120 h). The drug release pattern was found to be the same in the entire pH range. Therefore, it was concluded that the in vitro release of PYZ-SLNs was pH-independent.

Drug release kinetics

Furthermore, the optimized PYZ-SLNs, (Batch P11), in vitro drug release profile was fitted to first-order, zero-order, Baker-Lonsdale, Hixson-Crowell, Korsmeyer-Peppas, Higuchi, and Weibull models (Table 4). To further describe the drug release mechanism, the best model with the lowest f-value and the highest R-square value was selected. The Hixson-Crowell model (Figure 5C) best fits the drug release profile of the PYZ-SLNs (Batch P11), with the lowest f-value and the highest R-squared when compared to other models (Table 4). The Hixson-Crowell model explains drug release through a dissolution mechanism, which depends on the surface area of the drug particles in contact with the aqueous phase. Hence, the larger the surface area, the faster the particle dissolution.⁵⁶^,⁵⁷

DSC analysis

DSC analysis demonstrates the alterations in thermal behavior occurring because of the interactions amongst lipid components and drug during fabrication of PYZ-SLNs.^58-60 The pure PYZ transition temperature (Figure 6A) was determined to be 191.78 °C (endothermic peak) by DSC analysis.⁶¹^,⁶² The stearic acid (lipid excipient) transition temperature (Figure 6B) was determined to be 60.44 °C. Due to the addition of excipients such as soy lecithin and stearylamine (emulsifier), the blank SLNs peak (Figure 6C) was altered to 69.38 °C. Furthermore, in optimized PYZ-SLNs batch P11 (Figure 6D), the peak for the pure drug was not obtained. This might be attributed to the encapsulation of PYZ in a distributed form inside the SLN lipid core.

Morphological examination by TEM analysis

The TEM analysis of the PYZ-SLNs (batch P11) showed that the particles had a smooth surface and even size and shape (Figure 7A). There was no discernible particle aggregation, and the mean diameter was <500 nm. These analyses validate the outcomes of the DLS method.

PXRD analysis

The OriginPro 2017 software (OriginLab Corporation, USA) was used to analyze the diffraction patterns of the PYZ-SLNs (Batch P11) and PYZ, as shown in Figure 7B. The p-XRD patterns of PYZ showed its crystalline character, as clear sharp peaks were observed in the 2θ scale. In the PYZ-SLNs (Batch P11), the data showed a decline in the relative integrated peak intensity, and there was no discrete PYZ peak, signifying that PYZ was encapsulated in the lipid core of the SLNs in an amorphous form. Therefore, it can be concluded that the PYZ, which was incorporated into the lipid core of SLNs, was completely solubilized and changed into an amorphous state.

GI stability studies (in vitro)

PYZ-SLNs were uniformly distributed without any drug precipitation, according to the outcomes of stability studies (Table 5). Furthermore, after being incubated with different United States Pharmacopeia buffers, with a pH range of 1.2 to 7.4, statistically non-significant (p>0.05) variations in PDI values, EE%, and particle size were observed.

Stability studies for PYZ-SLNs

The stability of SLNs was determined at freezer (8 °C), intermediate conditions (30±2 °C/RH 65±5%), and accelerated conditions (40±2 °C/RH 75±5%) for 6 months. At predetermined intervals (initial, 1 month, 3 months, and 6 months), samples were analyzed for any alterations in physical appearance, ease of redispersibility, EE%, PDI, and particle size (D90). Table 6 represents the stability results for the lyophilized formulations (PYZ-SLNs). The lyophilized powder had a fluffy appearance, and upon reconstitution with water, it was found to be easily re-dispersed. Furthermore, there were no noteworthy changes in the parameters, suggesting that the prepared nanoparticles were stable and had a long shelf-life.

DISCUSSION

The PYZ-SLNs were fabricated using a high-pressure homogenization technique. In the preliminary trials, it was observed that three parameters, namely, homogenization cycles, drug concentration, and emulsifier concentration, had a noteworthy impact on DL%, EE%, and particle size of PYZ-SLNs. A 2³ factorial design was applied to optimize PYZ-SLNs. By employing Design-Expert software, the 3D response surface plots were generated and investigated. The independent variables emulsifier concentration (X₂) and homogenization cycles (X₃) had a significant reverse impact on particle size of PYZ-SLNs. With the increase in homogenization cycles, the decrease in particle size might be owing to the cavitation forces in the high-pressure homogenizer, leading to the reduction of PYZ-SLNs to nano-scale.^{53, 54} Moreover, an increase in emulsifier concentration reduced the particle size of PYZ-SLNs. This might be owing to increased stabilization of pre-emulsion by an emulsifier, leading to a diminution in particle size as well as its aggregation.⁵³ Furthermore, the drug concentration and emulsifier concentration had a positive impact on the EE% of PYZ-SLNs. The high drug solubilization and incorporation into the lipid core could be responsible for an increase in EE%.^{53, 55} The EE% decreased with an increase in homogenization cycles (X₃). This might be owing to the increased surface area of particles owing to the cavitational forces generated with increased cycles of homogenization resulting in drug leaching during SLN formation.^{53, 54} Thereafter, the impact of independent variables on DL% showed that an increase in drug concentration and emulsifier concentration led to an increase in DL%. The increase in drug concentration was attributed to more drug accessible to get incorporated into the lipid matrix. Furthermore, the drug gets solubilized more in the lipid matrix with the addition of an emulsifier as it acts as a solubilizer. Thus, an increase in emulsifier concentration increases the DL%. However, an increase in homogenization cycles led to a decrease in DL%. This could be the result of greater homogenization cycles which further cause drug leaching during the preparation of SLNs.^{53, 54}

To validate the optimization process, checkpoint batches (P9 and P10) with predicted values were prepared. The predicted and observed values represented a high degree of closeness as the difference among them was not significant (p>0.05). Thus, the obtained mathematical Equation was accurate in predicting the responses Y₁, Y₂, and Y₃. The optimized batch (P11) was fabricated by setting the optimum conditions (particle size: <500 nm and DL% and EE% of highest). The predicted and observed values of the P11 batch were in close agreement.

The in vitro drug release from the optimized batch P11 (lyophilized), evaluated in different pH media, represented an initial burst release followed by a sustained release. Further, the Hixson-Crowell model was found to provide the best fit for the PYZ-SLNs drug release profile as it had the highest R-square and lowest f-value. The Hixson-Crowell model explains the drug release by a dissolution mechanism, which depends on the outer surface of the aqueous phase. Hence, the larger the surface area, the faster the particle dissolution.^{56, 57} DSC analysis demonstrates the alterations in thermal behavior occurring because of the interactions among lipid components and drug during fabrication of PYZ-SLNs.^58-60 Herein, the DSC results of optimized PYZ-SLNs (P11 batch) did not show the peak of the pure drug. This might be attributed to the encapsulation of PYZ in a distributed form inside the SLNs lipid core.^{61, 62} Moreover, the TEM analysis results did not depict any particle aggregation. The TEM results validated the outcomes of the DLS method. The PXRD indicated a decrease in peak intensity and did not detect any PYZ peaks. Thus it was concluded that the PYZ was incorporated into the lipidic core of SLNs solubilized, and changed into an amorphous state. Furthermore, the PYZ-SLNs were determined to be stable under accelerated stability conditions for 6 months, as there were no changes in EE%, D90, PDI, or their physical appearance.

CONCLUSION

The current research work emphasizes the prospects of SLNs as an efficient carrier for oral delivery of the anti-tubercular drug PYZ. For optimizing the formulated PYZ-SLNs systematically, three factors and a two-level fractional factorial design were applied. The in vitro drug release study represented a biphasic release of PYZ-SLNs, consisting of an initial burst release followed by sustained release, fitting best with the Hixson-Crowell model, indicative of a release via a diffusion mechanism. The in vitro lipolysis analysis showed that PYZ-SLNs demonstrated an anti-lipolytic action in the fasted-state simulated intestinal (lipase-containing) fluid. PYZ-SLNs demonstrated good resistance in vitro and easily withstood varied GI tract-resembling media. The accelerated stability assessment confirmed that the PYZ-SLNs were stable even after 6 months, indicating their long shelf-life. Hence, from the aforementioned outcomes, it may be inferred that the optimized and formulated PYZ-SLNs can avert the PYZ degradation in varied GI media, and can be employed as a promising nano-based treatment for TB.

Ethics

Ethics Committee Approval: Not required.

Informed Consent: Not required.

Acknowledgment

Authors are thankful to the Institute of Pharmacy, Nirma University, Ahmedabad, Gujarat, India for providing the necessary facilities to generate the manuscript that is a part of Doctor of Philosophy (Ph.D.) research work of Mr. Nimitt Chokshi, submitted to Nirma University, Ahmedabad, India.

Authorship Contributions

Concept: N.C., M.P., Design: N.C., M.P., Data Collection or Processing: N.C., P.V., S.C., M.P., Analysis or Interpretation: N.C., P.V., M.P., Literature Search: N.C., S.C., Writing: N.C., P.V., S.C., M.P.

Conflict of Interest: The authors declare no conflicts of interest.

Financial Disclosure: The authors are highly grateful to Department of Science and Technology, Fund for Improvement of S&T Infrastructure (grant number: SR/FST/LSI-607/2014), Government of India for providing the necessary funding to establish equipment facility in laboratory.

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Formulation and Optimization of Pyrazinamide-Loaded Solid Lipid Nanoparticles by Employing a Design of Experiments Approach