Fabrication of Lipid Combination for Bioavailability Enhancement of Isoniazid

 

Shaveta Ahalwat*, D. C. Bhatt, Surbhi Rohilla

Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology,

Hisar -1250­01, India.

 

ABSTRACT:

The objective of this work was to precisely design novel lipid composition and investigate for improved characteristics to enhance the bioavailability of isoniazid (INH) loaded in nanostructured lipid carrier (NLC). Response surface methodology (RSM)-Central composite rotatable design (CCRD) was used to evaluate the effect of formulation variables, the ratio of two different solid lipids, solid lipid: liquid lipid, and the drug concentration on response variables. The encapsulation efficiency (EE) of optimized formulation was found to be 72.82±0.49%, drug loading (DL) was 15.15±0.10%, mean particle size (PS) was 285.1±4.71nm and in vitro drug release (DR) was 75.30±1.95% in 24 h. The optimized formulation was investigated via Differential Scanning Calorimetry (DSC), X-Ray Diffraction Pattern (XRD) analysis, in vitro release kinetics and Transmission electron microscopy (TEM). The polydispersity index (PDI) and zeta potential (ZP) were determined as 0.484±0.026 and +21±1.44mV respectively. Formulations were found to be most stable up to 25°C for 6 months. The present study successfully optimized the lipid combination at different concentrations and introduced the best composition of lipids with improved characteristics of INH-NLC formulation and expected to increase the bioavailability of isoniazid to replace the conventional drug delivery system for tuberculosis.

 

 

1. INTRODUCTION:

Isoniazid (INH) is an extensively used, safest first-line oral antitubercular drug used for the management of clinical symptoms of both active and latent tuberculosis (TB)^1,2. It works by inhibiting the synthesis of mycolic acid, the basic unit of the bacterial cell wall^3,4. It belongs to the BCS class III drug (high solubility and low permeability)^5,6. Isoniazid is exemplified by a short biological half-life (1-3 hours) therefore quickly excretes out from the body^7-9. The present therapeutics of TB are effective but the short half-life and long treatment resulted in emergence of resistance¹³. So, there is a need for the discovery of new drugs or redevelopment of present formulations to achieve a better treatment profile for tuberculosis^11-12.

 

 

The incorporation of hydrophilic moieties into a hydrophobic lipid matrix is a challenge as they tend to migrate more towards the aqueous phase throughout the manufacturing process^13,14. Preparation of NLCs is one of the strategies to overcome this problem¹⁵. These are formed by mixing incompatible liquid lipid into solid lipid, which increases the solubility of a drug into the lipid matrix¹⁶. Also, responsible for formations of imperfect crystal structures, which provides more space for drug payloads¹⁷. This resulted in the reduction of drug dose, dosing frequency, and thereupon reducing the drug resistance¹⁸. NLCs has the potential to avoid drug explosion due to the lipid crystallization phenomenon during the manufacturing process and storage periods and showed good sustained drug release profiles^19-20.

 

This work aimed to optimize and formulate isoniazid loaded NLCs employing the RSM-CCRD model. The lipid-excipients compatibility was investigated by using Fourier transform infrared spectroscopy (FTIR) analysis. The optimized batch with optimum formulation variables was further prepared and analyzed using DSC, PXRD techniques. Homogeneity of the INH-NLC formulation was determined by PDI and ZP analysis. Morphologic evaluation and internal drug loading of optimized formulation was carried out via photomicrographs of TEM. Drug release kinetic modeling and stability studies were also conducted to obtain drug release behavior and stability of the formulations.

 

2. MATERIALS AND METHODS:

2.1. Materials:

Isoniazid was purchased from Merck (St. Louis, MO, USA). Other excipients used for lipid nanocarrier formulations were the following: Glyceryl monostearate (Hi-Media Laboratories, Mumbai), Stearyl amine, Linoleic acid, Pluronic F-127, and Tween 80 (Merck, St. Louis, MO, USA). Analytical reagent (AR) grade chemicals along with double distilled water were employed throughout the study.

 

2.2. Method of preparation of NLC:

Hot homogenization and ultrasonication method was used with some modification to produce INH-NLC²¹. Briefly, solid lipids and liquid lipid together with tween 80 were heated up to 80°C and blended with continuous stirring. Aqueous emulsifier solution of pluronic F-127 was prepared in concentration of 1 gm/100ml in doubled distilled water with heating to an identical temperature. Isoniazid was dissolved in 1 ml of this aqueous solution and incorporated into the oily phase under homogenization at 18,000rpm (PRO 25D, Pro Scientific Inc, Oxford, CT, USA). A hot aqueous emulsifier solution was dispersed dropwise into the melted lipids beneath identical conditions. The acquired product was sonicated for 10 min using a probe sonicator (Q55, SONICA, New Town, CT. USA) with an amplitude of 75%. The INH-NLC dispersion was cool down to room temperature and centrifuged (CPR 24 PLUS, REMI, India) with a speed range up to 18000 rpm, 45 minutes, 4°C temperature to get NLC pellet. The pellet was washed thrice with double-distilled water, vacuum dried and freshly reconstituted into normal saline to make nano suspension before use¹². The blank NLC was formulated by the identical procedure without adding INH.

 

2.3. Design of Experiments:

The RSM-CCRD was used to evaluate the relationship between formulation variables such as GMS: SA (A), GMS+SA: LA (B), drug concentration (C), and on response variables namely, EE (%), DL (%), DR (%), and mean PS (nm) of INH-NLC as they were the key determinants impacting bioavailability of INH-NLC formulation. The experimental value ranges and criteria for independent and dependent variables were based on the literature and preclinical experiment results for preparing NLC (Table 1). The extreme points were +1.682 and -1.682 for each factor (high and low), which provide two-way interaction by setting each of the factors at five levels, thus providing precise results.

 

Table 1. Description of RSM-CCRD for independent and dependent variables

Independent Variables (Formulation variables)	Levels
	-1.682	-1	0	+1	+1.682
(A) GMS: SA (w/w)	33.1821	40	50	60	66.8179
(B) GMS+SA: LA (w/w)	53.1821	60	70	80	86.8179
(C) Drug concentration (INH) (mg)	115.91	150	200	250	284.09
Dependent variables (Response variables)	Constraints
Encapsulation Efficiency (%)	Maximum
Drug Loading (%)	Maximum
Drug release (%)	Maximum
Mean Particle Size (nm)	Minimum

 

2.4. Optimized conditions for formulation and model validation:

The final selection of optimized formulation parameters depends upon the desirability function. The desirability value of unity represents the highest desirable response value (acceptable value), whereas 0 represents the lowest desirable response value (unacceptable value).

 

2.5. Characterization of NLC:

2.5.1. Fourier Transform Infrared Spectroscopy:

FTIR spectrophotometer (Spectrum BX, Perkin Almer, US) was utilized to perform the FTIR analysis for functional group determination and compatibility study of isoniazid, all proposed lipids, physical mixture (PM), and an optimized INH-NLC formulation. The analysis was carried out using the KBr pellet technique having a sample: KBr ratio of 1:10 and a frequency range of 4500 cm^-1 to 350 cm^-1 was used with 4 cm^-1 resolution^22,23 _.

 

2.5.2. Differential Scanning Calorimetry Analysis:

DSC thermogram was investigated by DSC 25 (DSC 25, TA Instruments, England). All proposed lipids, free drug, physical mixture, and optimized INH-NLC formulation were put individually in aluminum pans and heating was carried out in a temperature range from 25 to 300°C with a heating rate of 5°C/min under nitrogen atmosphere having a flux of 50 cm³ /min. Thermograms were recorded and compared with raw materials²⁴.

 

2.5.3. Powder X-ray Diffraction Pattern Analysis:

The crystalline behavior of the drug and other excipients together with the physical mixture and optimized INH-NLC formulation was analyzed employing X-ray Diffractometer (Rigaku Miniflex-II, USA) and copper (Cu) was used as a radiation source. Scanning was done in a range of 0-80° at the diffraction angle 2θ²⁵.

 

2.5.4. Determination of Mean Particle Size, Particle Size Distribution and Zeta Potential Measurements:

Zetasizer (Microtec S-3500, USA was used to determine the mean PS and PDI for all INH-NLC formulations at a temperature of 25°C and backscattered at 90°²⁶. PDI reflects the homogeneity of the lipid nanoparticles in INH-NLC formulation and its value varies from 0 to 1 and a value near 0 shows highly homogeneous nanoparticles in INH-NLC formulation²⁷. ZP displays the particle surface charge of INH-NLC formulation^28,29.

 

2.5.5. Determination of Encapsulation Efficiency and Drug Loading:

A UV spectrophotometer (UV-1800, Shimadzu Corporation, Japan’) was used to evaluate the EE and DL of INH-NLC at λ_max 262nm. After separation of INH-NLC pellet, aqueous phase containing non-encapsulated drug was separated and diluted with distilled water and filtered using 45µm filter paper and assessed for drug content³⁰.

                                                   W_i - W_f

Encapsulation Efficiency (%) = ------------ x 100 ……(1)

                                                     W_i

                                    W_i - W_f

Drug Loading (%) = ---------------- x100      …………(2)

                                 W_i - W_f  + W_l

Here, W_i denotes the initial weight of INH used for the preparation of NLCs, W_f denotes the final weight of unentrapped drug found in supernatant, W_l denotes the total weight of lipids used in the formulation³¹.

 

2.5.6. In vitro Drug Release Analysis and Release Kinetics:

The dialysis bag approach was utilized to study the DR of isoniazid from pure drug solution and INH-NLC formulation using a dialysis tube (molecular weight cut off 12,400 Da, Hi-Media Laboratories, Mumbai). Both corresponding to 5mg of INH were taken in 2ml of phosphate buffer saline (PBS) pH 7.4 and placed into a dialysis tube and dispersed in 200ml of same in a beaker. The temperature and rotation were maintained at 37°C±0.5°C and 100 rpm respectively. The beakers with dialysis bag were covered with parafilm and samples (5 ml) were withdrawn at different time points up to 24 h. The same volume was replaced with fresh solvent to maintain the sink condition. The samples were analyzed spectrophotometrically for the amount of INH released³². The obtained release data was fitted in these release kinetic models i.e. zero-order, first-order, Higuchi square root, and Korsmeyer-Peppas to identify the release behaviour from matrix. The highest correlation value was used to identify the best fit (R²)³³.

 

2.5.7. Transmission Electron Microscopy:

The surface morphology and internal drug loading of INH-NLC were examined by employing a TEM instrument (Tecnaii, china) operated at 200 kV. To prepare the sample, carbon-coated copper grid was used (Boston Industries, Inc, Walpole, MA) and the images were seen with the help of TEM Imaging Analysis Software (TIA)³⁴.

 

2.5.8. Stability Studies:

The influence of temperature and relative humidity on the stability of the optimized INH-NLC was investigated at different temperatures under refrigerator condition (4 ± 2°C), room condition (25 ± 3°C/60 ± 5RH) and accelerated condition (40 ± 3°C/75 ± 5RH) as per ICH guidelines in climate humidity chamber (Binder GmbH^® Ltd, Munchen, Germany) over 6 months and were assessed at different time intervals for standard parameters i.e. PS (nm), ZP (mV), PDI, and residual drug content (%) at 262 nm³⁵.

 

2.5.9. Data Analysis:

Design Expert^® v 11 software was used to correlate the formulation variable with response variables of all experiments. Prism 8.4.0^® software was used to estimate the experimental data and their statistical prevision. All experimental data were demonstrated as the mean and standard deviation (mean ± SD) and the differences were deemed statistically significant if p ≤ 0.05.

 

3. RESULTS AND DISCUSSION:

3.1. Central Composite Rotatable Design-Response Surface Methodology:

3.1.1. Model Fitting and Statistical Analysis:

A set of experiments with their experimental results were used to estimate the coefficients of the polynomial equation. Various polynomial models were used to fit the data and found best fitted into a quadratic model (p < 0.05) with no transformations of data. The relationship between independent variables and dependent variables could be represented by quadratic polynomial equations (3 to 6). The positive and negative signs indicated the synergistic and antagonistic effect of representative variables in polynomial equation.

 

EE (%)= +73.80+1.80A+0.9971B+3.78C-6.04AB +1.82AC-0.6469BC-3.71A²+0.7640B²-4.48C²   ……. (3)

 

DL=+14.76+0.4139A+0.1805B+3.96C-1.24AB+ 0.4519 AC-0.0804BC-0.7202A²+0.1737B²-0.7437C²  …….. (4 )   

DR (%) = +74.64+1.51A-0.8381B-0.3544C+2.93AB-0.3461AC+0.7807BC+0.5961A²-3.88B²-2.31C²     ….(5)

 

PS (nm)= +297.93-50.10A+10.82B-83.70C-0.4875AB +28.86AC-0.8875BC+19.15A²-16.14B²-25.45C²   …..(6)

 

3.1.2. Response Surface Analysis:

Model equations were used to prepare three-dimensional (3D) response surface and two-dimensional (2D) contour plots with the help of Design Expert software. Figures 1 exemplify the consequences of two variables at a time, while the third one is kept at zero in coded unit level. 3.1.1.

 

The minimum and maximum EE were estimated to be 55.66 and 81.73% respectively. Response surface and contour plots, Figure 1[i(a,b)], showed the quadratic effect of the ratio of two different solid lipids (GMS: SA), Figure 1[i(a,c)] exhibited the linear effect of solid lipids: liquid lipid (GMS+SA: LA) and Figure 1 [i(b,c)] showed the quadratic effect of drug concentration (INH) on EE of INH-NLC formulation. Optimized maximum encapsulation efficiency was found to be 74.44% at optimum values of all formulation variables.

 

 

The DL was found in a range of 6.46 to 20.43%.

Response surface plots, Figure 1[ii(a,b)] and in Figure 1[ii(a,c)] showed the quadratic effect of two different solid lipids (GMS: SA) and solid lipids: liquid lipid (GMS+SA: LA) and Figure 1[ii(b,c)] showed a linear increment in drug loading in INH-NLC formulation. The maximum drug loading was found to be 15.38% at optimum values of independent variables for optimized INH-NLC formulation.

 

The DR was found to be in a range of 62.14 to 79.32%. Response surface plot, Figure 1[iii(a,b)] illustrated the linear effect of the ratio of two different solid lipids (GMS: SA) on in vitro drug release. On the contrary, Figure 1[iii(a,c)] and Figure 1[iii(b,c)] showed the quadratic effect of the ratio of solid lipid: liquid lipid (GMS+SA: LA) and drug concentration (INH) on DR of INH-NLC formulation. The optimized maximum drug loading was observed 74.61% at optimum values of all formulation variables.

 

The minimum and maximum values for mean PS were found to be 94.63nm and 476.4nm respectively. Response surface plots, Figure 1[iv(a,b)] and Figure 1[iv(b,c)] represented the linear antagonistic effect of the ratio of two different solid lipids (GMS: SA) and drug concentration (INH) on mean PS of INH-NLC formulation. On the contrary, quadratic effect was seen for the ratio of solid lipid: liquid lipid (GMS+SA: LA) on mean PS of formulation. The optimized maximum mean PS was found to be 281.8nm at optimum values of all formulation variables.

 

Figure 1: Consequences of interactions (a) AB, (b) AC, (c) BC, on (i) EE (ii) DL (iii) DR (iv) mean PS of INH-NLC formulation

 

3.2. Optimized Conditions for Formulation and Model Validation:

The optimum values of formulation variables were finalized by numerical optimization with maximum desirability of 0.636 (Table 2). A minimum percentage bias was found between the experimental values and the predicted values. This proved that the model was reliable and reasonable. PDI and ZP values for the optimized batch were estimated as 0.484±0.026 and +21.36 ± 1.44 respectively.

 

Table 2. Optimized independent variables for most desired formulation and estimation of the percent bias by comparing the observed and predicted values in the optimized INH-NLC

S. No.	(A) GMS: SA (w/w)	(B) GMS+SA: LA (w/w)	(C) Drug Concentration (mg)	Desirability
NLC	50.82	70.71	207.51	0.636
S.no.	Response Variables	Predicted	Actual	Bias (%)*
1.	Encapsulation efficiency (%)	74.44	72.82 ± 0.49	2.18
2.	Drug Loading (%)	15.38	15.15 ± 0.10	1.49
3.	In vitro drug release (%)	74.61	75.30 ± 1.95	0.92
4.	Mean particle size (nm)	281.83	285.1 ± 4.71	1.16

*Bias was calculated as (Predicted value-observed value) / predicted value*100

3.3. Characterization of Optimized Nanostructured Lipid Carriers:

3.3.1. Fourier Transform Infrared Spectroscopy:

Figure 2(i) represented that all characteristic peak of drug and excipients were present at their place resulted in the structural, and functional conformation of compounds^36-39. There was absence of any new absorption band in the FTIR spectra of both the physical mixture and optimized formulation, resulting in the compatibility between drug and excipients. All the characteristic bands of isoniazid and other excipients were present with minor shifting resulted in the increased solubility of the drug in the lipid nanocarriers. The magnitude of these shifts in stretching frequencies is proportional to the extent of increased drug solubility in the nanocarriers resulted in better EE in the formulation as compared to the physical mixture³⁰.

 

3.3.2. Differential Scanning Calorimetry Analysis:

Figure 2(ii) shows the sharp endothermic peak of isoniazid at 173.01°C and enthalpy (ΔH) of 258.26 J/g suggesting the crystalline structure of the drug⁴⁰. The decrease in this endothermic peak and enthalpy (ΔH) to 164.25°C and 75.34 J/g respectively in the physical mixture indicated that the solubility of the drug was increased in lipids. This endothermic peak was absent in the optimized formulation associated with complete solubilization of the drug in the lipids. This resulted in the decreased crystalline behavior or increased amorphous nature of the drug as well as the formulation as described in an earlier study⁴¹. Glyceryl monostearate and stearyl amine showed isotherm at 46.48°C, 43.55°C respectively, associated with the melting of lipids^41,43.

 

 

 

Figure 2: (i) FTIR spectra (ii) DSC thermogram (iii) X-ray diffraction pattern of isoniazid, proposed lipids, physical mixture, and optimized batch (iv) in vitro drug release profile of plain drug solution and optimized INH-NLC formulation

 

3.3.3. Powder X-ray Diffraction Pattern Analysis:

Isoniazid and other excipients showed sharp and intense diffraction peaks as shown in Figure 2(iii))^40,41. In optimized formulation, these sharp peaks were diminished associated with an increase in amorphous nature of drug  due to drug lipid solubilization. The comparable research was found, whereby isoniazid decreased its crystalline behavior when loaded in SLNs³⁰. The reason behind that heating of lipids during the process causes polymorphic crystalline transformation of lipids⁴².

 

3.3.4. In vitro Cumulative Drug Release and Release Kinetics:

The fast drug release from INH-NLC formulation was observed in the initial hours thereafter a sustained release effect for 24 hours (Figure 2(iv)). This corresponding to the deposition of some liquid lipid-containing INH on the outer surface of the formulation, which takes place due to melting point differences in lipids. Higher melting point solid lipid crystallizes first form a little/no liquid lipid core and outer shell consist of the majority of the liquid lipid resulted in the fast release of the drug at the initial stage^43,9. In contrast, the drug was fast released from the pure drug solution and was close to 99 % in just 4 hours whereas about 75% of the drug was released in 24 hours representing the sustained release behavior of INH-NLC formulation¹². The R² values of different models were found to be 0.8945 for both zero-order and first-order, 0.9785 for Higuchi’s square root, and 0.9605 for Korsmeyer–Peppas models (Figure 3). The highest R² value was found for Higuchi’s square root model indicated that the drug release from the lipid nanocarriers was regulated by a diffusion-controlled mechanism^44,45.

 

Figure 3: In vitro drug release kinetic models together with their values

 

3.3.6. Transmission Electron Microscopy:

Photomicrographs were captured using a transmission electron microscope. Figure 4 (A) is a close view of the nanoparticles, clearly indicated the formation of imperfections within the NLC nanoparticles formed by lipids blend during the preparation process and confirmed the drug loading in it. The NLC particles were of spherical morphology and around 120 nm in size. Whereas, diameter determined by the particle size analyzer is 285.1 nm. The reason behind the difference in particle size is that the two different instruments working on different principles and sample preparation techniques¹².

 

Figure 4: Transmission electron microphotographs of drug-loaded INH-NLC

 

3.3.7. Stability Studies:

The storage stability of INH-NLC was analyzed for 6 months (Table 3). The physical appearance of INH-NLC samples stored at 4 ± 3°C and 25 ± 3°C/60± 5% RH were preserved throughout the study period, while aggregation of particles took place in samples stored at 40 ± 3°C/75± 5%RH after 3 months and stability parameters were not in measurable condition. The average PS of INH-NLC was increased significantly (p < 0.05) to a small extent with respect to time due to accumulation of small-sized particles, which in turn depends on several factors like size, density, and charge of nanoparticles within the INH-NLC formulation^46,47. ZP and PDI were changed insignificantly (p > 0.05 at 4 ± 3°C and 25 ± 3°C/60± 5% RH temperature throughout the study period. Furthermore, residual drug content was slightly decreased throughout the storage period with a significant p value of less than 0.05. The reason behind that is diffusion of drug towards the surface⁴⁸. Slight differences were found in storage parameters for INH-NLC during the entire storage period. Hence, INH-NLC were found to be chemically and physically stable and retain their pharmaceutical properties below 25°C over 6 months.

 

Table 3. The stability study data of optimized INH-NLC formulation stored at 4 ± 3°C, 25 ± 3°C/60 ± 5%RH and 40 ± 3°C/75± 5%RH for 6 months (n = 3)

Stability at 4 ± 3°C
Parameters	0 month	1 month	2 months	3 months	6 months
PS (nm)	285.1 ± 4.71	287.17 ± 5.50	290.6 ± 5.62	294.03 ± 4.08	305.47 ± 5.72
PdI	0.48 ± 0.03	0.53 ± 0.05	0.50 ± 0.03	0.47 ± 0.03	0.52 ± 0.19
ZP (mV)	21.36 ± 1.44	20.92 ± 1.25	20.21 ± 0.78	19.78 ± 1.16	21.01 ± 0.76
Drug Content (%)	100	99.64 ± 0.12	99.34 ± 0.22	99.05 ± 0.13	98.17 ±0.13
Stability at 25 ± 3°C/60± 5%RH
Parameters	0 month	1 month	2 months	3 months	6 months
PS (nm)	285.1 ± 4.71	289 ± 4.60	292.83 ± 4.88	296.77 ± 4.66	309.47 ± 6.33
PdI	0.48 ± 0.03	0.40 ± 0.08	0.33 ± 0.06	0.41 ± 0.02	0.53 ± 0.18
ZP (mV)	21.36 ± 1.44	20.59 ± 0.39	20.54 ± 0.75	19.78 ± 1.71	20.02 ± 1.44
Drug Content (%)	100	99.57 ± 0.01	99.13 ± 0.02	98.54 ± 0.24	97.23 ± 0.65
Stability at 40 ± 3°C/75± 5%RH
Parameters	0 month	1 month	2 months	3 months	6 months
PS (nm)	285.1 ± 4.71	429.43 ± 4.83	576.93 ± 11.47	720.23 ± 12.46	NM
PdI	0.48 ± 0.03	0.53 ±0.05	0.57 ± 0.03	0.61 ±0.03	NM
ZP (mV)	21.36 ± 1.44	22.32 ± 1.52	23.84 ± 1.80	25.41 ± 1.82	NM
Drug Content (%)	100	97.36 ± 1.53	93.71 ± 1.26	89.02 ± 0.82	NM

 

 

4. CONCLUSION:

The isoniazid was successfully incorporated in lipid nanocarriers by using hot homogenization and ultrasonication technique. The polymer combination and amount of drug were optimized at different concentrations using RSM-CCRD model. Increased amorphous behavior of the formulation was observed from DSC and PXRD study analysis. Spherical-shaped nanoparticles loaded with drug micro-crystals were seen in TE micrographs. The optimized formulation showed a fast drug release in the initial hours thereafter a slow release over 24 hours. The INH-NLC formulation showed good physical and chemical stability up to 25°C during 6 months. This research work successfully optimized the lipid combination and introduced the best composition of lipids with improved characteristics of INH-NLC formulation and expected to increase the bioavailability of isoniazid to replace the conventional drug delivery system for tuberculosis.

 

5. ACKNOWLEDGMENTS:

Authors thanks to Mrs. Chanda, Department of Electron Microscopy, AIIMS, New Delhi for the TEM analysis and Dr. Ashish Aggarwal, Department of Physics, Guru Jambheshwar University of Science and Technology, Hisar for XRD analysis.

 

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Accepted on 30.12.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5589-5596.