Formulation, Optimization and Pharmacodynamic Studies of Pioglitazone HCl Solid Lipid Nanoparticles

Pioglitazone HCl is an oral anti-diabetic agent used for the treatment of diabetes mellitus type II. The aim of the present work is to evaluate the pharmacodynamic activity of solid lipid nanoparticles of pioglitazone HCL prepared by using solvent injection technique and to compare with the control and test group. Among all the formulations, F5 was found to possess highest in-vitro drug release within 24 hrs i.e., 95.02±1.26%. The in vivo studies were performed using male albino rats of wistar strain (150-200g). Rats were divided in to five groups (n=6), group-I normal, group-II diabetes control, group-III placebo control, group-IV reference, group-V test group. Diabetes was induced by streptazocin (60 mg/kg) by intraperitonial route. The reference group was treated with marketed tablet of pioglitazone HCL, test groups were treated with SLNs suspended in 0.1% Tween 80 and given to animals through oral gavages. Blood samples were collected by retro-orbital puncture before treatment, and after treatment at time intervals 0, 2, 4, 6, 8, 10, 12 and 24h in anticoagulated vials. Parameters like glucose, tri glycerides (TG), total cholesterol (TC) and HDL-C were estimated by calorimetric method. Diabetes induced rats showed elevated levels of glucose, TG, TC and reduced HDL. The oral administration of drug loaded SLNs in 0.1% Tween 80 solution Original Research Article Muzib et al.; JPRI, 33(46A): 329-341, 2021; Article no.JPRI.75554 330 showed reduced levels of glucose, TG and elevated levels of HDL-C and slightly reduced levels of TG in 24 h where as the marketed tablet showed reduced levels of glucose, TG and TC up-to 12 h and in 24 th h the glucose levels get elevated. Thus the optimized SLNs showed prolonged activity.

showed reduced levels of glucose, TG and elevated levels of HDL-C and slightly reduced levels of TG in 24 h where as the marketed tablet showed reduced levels of glucose, TG and TC up-to 12 h and in 24

INTRODUCTION
Carrier based delivery of a drug molecule to specific organ sites is one of the major thrust area in pharmaceutical sciences. So, a high potential for drug delivery has been attributed to particulate drug carriers, especially small particles such as microparticles and colloidal system in nanometer range [1]. Nanotechnology, as defined by the National Nanotechnology Initiative (NNI), is the study and use of structures roughly in the size range of 1 to 100nm. The goal of nanotechnology is the same as that of medicine, to diagnose as accurately and early as possible and to treat as effectively as possible without any side effects using controlled and targeted drug delivery approach [2]. The efficient drug delivery depends on their ability to penetrate through several anatomical barriers, sustained release of their contents and their stability in the nanometer size. However, the scarcity of biocompatible polymers with regulatory approval and their high cost have limited the wide spread application of nanoparticles to clinical medicine [3].To overcome these limitations of polymeric nanoparticles, lipids have been put forward as an alternative carrier, particularly for lipophilic pharmaceuticals. These lipid nanoparticles are known as Solid Lipid Nanoparticles(SLNs) [4]. Pioglitazone HCl is an oral anti-diabetic agent used for the treatment of diabetes mellitus type II [5]. It is the drug of choice for the preparation of SLNs since it is highly lipophilic in nature. It is formulated in the form of SLNs to increase the aqueous solubility and for sustained release over a prolonged period of time to maintain the glucose levels by preventing the hyperglycemia [6].
The present work aimed to prepare SLNs using ethyl cellulose as polymer, cholesterol as solid lipid, tween80 and span 60 as surfactants. The formulated SLNs were subjected to evaluation tests like; entrapment efficiency and in vitro diffusion studies and characterized by using Scanning electron microscopy (SEM), particle size by zeta seizer and polymer compatibility studies were done by using FT-IR and DSC. The optimized formulation was subjected to in vivo pharmacodynamics activity.

Materials
Pioglitazone hydrochloride was gift sample received from Hetero drugs Pvt.Ltd, Cholesterol and Ethyl Cellulose were purchased from SD Fine-chem. Laboratories. Tween 80 and Span 60 were purchased from Merck, Mumbai, India. All the reagents used were of analytical grade.

Preparation of Pioglitazone hydrochloride loaded Solid lipid nanoparticles
Pioglitazone HCl Solid lipid nanoparticles were prepared by solvent injection method [7]. An accurately weighed amount of Pioglitazone hydrochloride (30 mg), cholesterol (solid lipid), ethyl cellulose (polymer) and span 60 (surfactant) were dissolved in methanol and heated upto 60±2°C (Table 1). The aqueous phase was prepared by dissolving Tween 80 in distilled by stirring which is maintained at same temperature. The organic phase containing lipid mixture was added drop wise with stirring to aqueous phase with the help of hypodermic needle. The mixture was then sonicated for 15 min to get nanoemulsion and it is lyophilized for 48h to obtain the freeze dried SLNs. a) FT-IR spectroscopy: This study was carried to find out the compatibility between drug (Pioglitazone HCl), polymer (Ethyl cellulose). Sample and KBR were taken in the ratio of 1:100 in a mortar and triturated. A small amount of triturate was taken into a pellet maker and was compressed at 10 kg/cm 2 to form a transparent pellet using a hydraulic press. The pellet was kept in a sample holder and scanned from 4000 cm -1 to 400 cm -1 in FT-IR spectrophotometer [9]. The possible interaction between Pioglitazone HCl and Ethyl cellulose was accessed by comparing FTIR spectra of pure drug, polymer and nanoparticle formulation. b) Differential scanning calorimeter (DSC): DSC studies were performed to understand the behavior of ethyl cellulose on application of thermal energy. DSC was performed on a DSC (METTLER STAR˚ SW 8.10) at a heating rate of 10°C/min in the temperature range of 0-250°C using empty aluminum pan as reference standard [10].

Determination of particle surface morphology by SEM
The morphological characteristics of nanoparticles were determined by scanning electronic microscopic (SEM, Zeiss Ultra-60 (FE SEM)). Specified quantity of nanoparticles were taken and mounted directly on the SEM stub, using double sided, sticking tape and scanned in a high vacuum chamber with a focused electron beam.

Particle size determination:
The average particle sizes, polydispersity index and zeta potential of the lipid particulate dispersions were determined using a Zetasizer (Horiba SZ-100). The sample of dispersion was diluted to 1:9 v/v with double distilled water to ensure that the light scattering intensity was within the instrument's sensitivity range. Double distilled water was filtered through 0.45μm membrane filters prior to particle size determination.

In-vitro drug release from SLNs
The in vitro drug release studies were performed using the USP paddle method. The dissolution study was carried out in two dissolution media; 0.1N HCl pH 1.2, and phosphate buffer pH 6.8 and the entire system was kept at 37°±0.5° with continuous agitation speed of 50rpm [11,12]. At appropriate time intervals 5ml of release medium was removed and 5ml fresh medium was added into the system to maintain sink condition. The amount of pioglitazone in the release medium was evaluated by U.V Spectrophotometer at 269nm. The concentration of pioglitazone HCl in test samples was corrected and calculated by using the regression equation of the calibration curve.

Kinetic Modeling of Drug Release:
Data obtained from in-vitro studies were fitted to various kinetic models such as zero order, first order, Higuchi model and korsmeyer-peppas model [13].

In vivo-Pharmacodynamic Studies
The drug loaded Solid lipid nanoparticles were optimized based on the performance of in vitro tests. The optimized drug loaded formulation was used to study in vivo glucose and cholesterol reducing activity on male albino rats according to the protocol (Table 1). Wistar strain of male albino rats weighing 150-200 g were used in the study (n=6). The animals were housed in standard polypropylene cages and maintained under controlled room temperature and humidity with 12h light/dark cycle [14]. All the rats were fed with commercially available rat normal pellet diet (NPD) and water and libitum, prior to the dietary manipulation. The experimental protocol was designed and approved by the Institutional Animal Ethical Committee (CPCSEA/1677/PO/Re/S /IAEC-04/2016) Diabetes was induced by inducing Streptozocin (STZ) (60 mg/kg) by intra peritoneal route except for normal group [14]. To confirm the induction of hyperglycemia, after 24h blood samples were collected from the retro-orbital puncture. The glucose and cholesterol concentration of the blood samples was then determined using a standard enzymatic assay kit. The rats were then divided into 5 groups of 6 animals [15,16].
The blank, reference, test groups were treated with SLNs suspended in 0.1% Tween 80 and given to animals through oral gavages. Blood samples were collected by retro-orbital puncture at predetermined time intervals, viz., before treatment, and after 0, 2, 4, 6, 8, 10, 12 and 24h in anticoagulant filled vials. Plasma was separated by centrifugation at 1000rpm for 15min. The serum was quantitatively analyzed for blood glucose, triglycerides, cholesterol and HDL using calorimeter.

Determination of Blood Glucose:
Blood glucose levels was determined by using glucose estimation kit GOD-POD method [17].

Calculation:
Triglycerides (mg/dl) = × 200 Total Cholesterol and HDL-C: The cholesterol levels were calculated by using the kit CHOD-POD method.

Statistical Analysis
The data obtained from the entrapment efficiency and in-vitro drug release of Pioglitazone HCl SLNs were statistically analyzed with one way ANOVA (p<0.05) using Graph pad prism 5.0.

RESULTS AND DISCUSSION
Pioglitazone loaded SLNs were subjected to know the effect of lipid, polymer and surfactant of the formulation on entrapment efficiency and in-vitro drug release to optimize the best formulation. The optimized formulation was characterized by SEM, particle size, drugexcipient compatibility studies.

Entrapment Efficiency
Among all the prepared SLNs, entrapment efficiency (EE) of Pioglitazone HCl -SLNs sample F5 was found to be 95.39% (Table 3).High incorporation was possible because lipid (cholesterol) increased the solubility of Pioglitazone HCl and span 60 as a lipid surfactant helps to solubilise the drug in to lipid which further increased entrapment of drug. The EE mainly depended on the varied concentrations of polymer (ethyl cellulose) and Tween 80 which is used as an aqueous surfactant. On increasing the amount of ethyl cellulose up to 120 mg, the EE was increased and on further increase the EE get decreased, the probable cause of this behaviour was drug expulsion from the surface of SLNs.

Particle Size and Zeta Potential
The mean particle size and zeta potential of optimized formulation was found to be 34.5±7.7 nm and -16.9 mV respectively and was shown in (Figs. 2 and 3). Particle size was found to be nanometric size with PDI lesser than 1 and zeta potential of -16.9mV indicating the stability of the formulation.

Drug-polymer Interaction Study
The interaction study between the drug (Pioglitazone HCl) and drug loaded Solid Lipid Nanoparticles were evaluated using ATR-FT-IR and DSC analysis.
Attenuated total reflectance -Fourier transform infrared (ATR-FTIR) The possible interactions between the drug and the excipients were studied by IR spectroscopy for pure drug pioglitazone HCl in (Fig. 4). The FT-IR spectra of polymer and their physical mixtures with drug were shown in (Fig. 5). For optimized formulation (F5) of SLNs all peaks which have been obtained as of the pure drug in the IR spectrum shown in (Fig. 6). From the physical mixtures of drug, lipid, and polymer there were no major shifting as well as no loss of any functional peaks between the spectra of drug and physical mixtures as shown in (Tables 4-6).
Hence it was confirmed that there was no interaction between the drug and polymer. This finding was further supported by DSC studies.

Differential Scanning Calorimeter
The differential scanning calorimeter was carried out for the pioglitazone HCl SLNs, to study the compatibility or any interaction of drug and polymer after the formation of SLNs and is shown in (Figs. 7 and 8). DSC thermogram of pure drug exhibited a sharp endothermic peak at 190.84°C corresponding to its melting point and the peak obtained for the pioglitazone HCl SLNs is at 186.9°C for the drug. Thus there was no significant change in the position of peak of the drug in the Pioglitazone HCl SLNs, but there is change in the relative intensities of the peak. It may be due to reduced drug crystallinity and also indicates that the drug is only physically entrapped in the polymer matrix and there is no interaction between drug and polymers.

In-vitro Dissolution of Formulated Solid Lipid Nanoparticles
The in vitro release results of Pioglitazone HCl loaded SLNs formulations are shown in (Fig.  9).In vitro drug release study of Pioglitazone HCl loaded SLNs showed the sustained release behaviour. Almost all formulation dissolution profiles showed biphasic behaviour consisting of initial burst release followed by a slow release phase.Among all the formulations, F5 was found to possess highest in-vitro % drug release within 24h i.e., 95.02%. The release profiles of these SLN resemble the drug enriched core model. The initial burst release can be attributed to the presence of free drug in the external phase and drug adsorbed on the surface of particles while the slow release was due to drug encapsulated within lipid matrix and also an outer phase of increased viscosity. The increase in polymer concentration had significant effect on the drug release which prolonged the release from SLNs [17,18]. The increase in amount of polymer concentration appeared to substantially decrease the release in all formulations. This may be attributed to the slower rate of drug diffusion from these SLNs into the dissolution mediums due to increased viscosity of the polymeric outer membrane. Thus, with the use of the Pioglitazone HCl loaded SLNs, it is possible to achieve the loading dose of the drug due to initial burst release and followed by maintenance dose due to the sustained release. Therefore, F5 formulation was considered as the optimized formulation based on entrapment efficiency and in-vitro drug release.

Kinetic Modeling of Drug Release
The data was analyzed by zero order, first order, Higuchi and Peppas equation models, their plots and 'R 2' values were shown in (Table 7). When the release data were analyzed as per zero and first order models, the 'r' values were relatively higher in first order model with all the formulations of SLNs indicating that the drug release from all these formulations followed first order kinetics. Pioglitazone HCl release data also obeyed Higuchi and peppas equation models with 'R 2 ' values greater than 0.943. When percent release was plotted against √time, linear

Time (hrs)
regressions with 'R 2 ' > 0.943 were observed with all the SLNs formulations prepared indicating that the drug release from all these formulations was diffusion controlledfrom the pores on the particle surface. As per peppas equation, the release exponent 'n' was found in the range 0.4693 to 0.8801 indicating non-fickian diffusion as the release mechanism from all the SLNs formulations.

In-vivo -Pharmacodynamic Studies
The Pharmacodyanamic studies were carried in male wistar albino rats with F5 formulation by comparing placebo,reference and test formulations. The Glucose, TC, TG and HDL-C were estimated in plasma samples of different groups as given in (Figs. 10-13). Hypoglycemic activity of pioglitazone HCl causes reduction in plasma glucose levels and this action is due to increase in both insulinstimulated glucose uptake in peripheral tissues and insulin sensitivity in hepatic and adipose tissues, thereby lowering plasma glucose [19]. At the same time, pioglitazone HCl SLNs also shows beneficial effects on lipid metabolism by significant reduction in TG levels and elevation of the HDL-C levels and also slight decrease in TC levels.
In the present Study, diabetes induced rats showed elevated levels of glucose, TG, TC and reduced HDL. The oral administration of drug loaded SLNs in 0.1% Tween 80 solution showed reduced levels of glucose, TG and elevated levels of HDL-C and slightly reduced levels of TG in 24 h where as the marketed tablet showed reduced levels of glucose, TG and TC up-to 12 h and in 24 th hr the glucose levels get elevated. Thus the optimized SLNs showed prolonged activity.
On the other hand, the diabetic rats treated with blank SLNs showed equivalent levels as in diabetic group i.e., no reduction in glucose, TG and TC levels. So, the results proved that the SLNs without drug didn't show any possible effects.
The statistically significant (P<0.05) decrease in glucose, TG, TC and elevated levels of HDL-C levels were found in reference and test groups indicating that the Pioglitazone HCL was released from conventional marketed tablet and prepared SLNs and showed hypoglycemic activity.
Though there was no significant difference in Pharmacodyanamic effects with reference and test formulation upto 12 hrs, but on prolonged use possess more advantages with extended release drug delivery system compared to the conventional release drug formulation in respect to dose, side effects etc.
These results indicated that the optimized formulation (F5) could function as a prolonged release hypoglycemic drug delivery system to prevent hyperglycemia by lowering the plasma glucose levels for extended period of time without maintenance dose.

CONCLUSION
Pioglitazone HCl SLNs successfully optimized to achieve increased solubility of Pioglitazone as well as the higher and sustained release. The obtained SLNs showed nanosized homogeneous particles with high entrapment efficiency and low recrystallization properties. The in-vivo studies showed the significant decrease in glucose levels on oral administration of Pioglitazone HCl SLNs for 24 hrs. This work facilitates the use of SLNs as a novel delivery system to enhance the solubility of pioglitazone HCl and could be effective in sustaining drug release for a prolonged period. The SLNs acts as efficient carrier for pioglitazone HCl for an antidiabetic activity that can increase the therapeutic efficacy of pioglitazone HCl in the oral treatment of type II diabetes. In-vivo experimentation was conducted for the optimized formulation and the levels of glucose and lipid profile (triglycerides, total cholesterol, HDL) were estimated. From the results it was found that there was decrease in the levels of glucose which proved that nanoparticles through oral route released the drug up to 24 h and showed the hypoglycemic activity. This could be due to slow release of drug from lipid particles and maintained peak plasma concentration over a prolonged period.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
As per university standard written ethical approval has been collected and preserved by the author(s).