INTRODUCTION:

Lovastatin ([(1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxooxan-2-yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8 a hexa hydro naphthalen-1-yl] (2S)-2-methyl butanoate) (Figure 1), is a lactone metabolite isolated from the fungus Aspergillus terreus as a prodrug with cholesterol-lowering activity¹. It is known to be a highly effective and generally, well-tolerated treatment for patients with moderate hypercholesterolemia² as a cholesterol-lowering agent³ and is a highly lipophilic drug (log P=4.3). It also having bone formation and chemopreventive activity^4,5. The poor oral bioavailability of <5% due to rapid metabolism in the gut and liver⁶, and short half-lives (1–2 h) of its active metabolite beta- hydroxy acid, makes lovastatin a potential candidate for sustained release carriers as it takes 2-3 days to achieve steady-state plasma concentrations (Cp_ss)⁷. Hence a formulation with sustained release action and an alternative route would be desirable for lovastatin.

Figure 1: Molecular structure of Lovastatin

Transdermal delivery provides a leading-edge over injectable and oral routes due to the reduction of dosing frequencies, avoiding the first-pass metabolism, and is also suitable for patients who are unconscious or vomiting, or those who rely on self-administration⁸ and hence increasing patient compliance⁹. This delivery route allows both convenient and painless drug delivery as well as sustained release profile with reduced side effects.so transdermal delivery can provide a non-invasive alternative to parenteral routes, thus circumventing issues such as needle phobia. Furthermore, the pharmacokinetic profiles of drugs are more uniform with fewer peaks, thus minimizing the risk of toxic side effects¹⁰. However, physiological barriers in the skin undermine the delivery efficiency of conventional patches, limiting drug candidates to small-molecules and lipophilic drugs.Drug delivery through the skin has a very high potential in therapy since it presents a large surface area of skin and ease of access allows many placement options on the skin for transdermal absorption¹¹. Lipid nanoparticles like solid lipid nanoparticles (SLNs) have been explored during the last few decades for the delivery of drugs. SLNs are mainly composed of Physiological lipids which are solid at room temperature and classified as GRAS (generally recognized as safe)¹² like glyceryl monostearate, glyceryl behenate, palmitic acid, various fatty acid and many more.The unique ability of SLNs to encapsulate both hydrophilic and lipophilic drugs¹³, to improve bioavailability¹⁴, to achieve sustained release of actives ¹⁵,increase the chemical stability of molecules against oxidation, hydrolysis and light^16,17,18. SLNs when applied to the skin form an adhesive occlusive film layer and show an occlusive effect on the skin, which results in reduced water loss from the skin and consequently an increase in skin hydration. The skin penetration of active substances can also be enhanced as a consequence of the occlusive properties of SLNs¹⁹.

The present work aimed to develop Solid lipid nanoparticles loaded with lovastatin for transdermal delivery which provide a sustained release profile and may overcome the bioavailability issues of lovastatin.

MATERIALS AND METHODS:

Materials:

Lovastatin was obtained as a gift sample from Shreeji Pharma International, Vadodara Gujarat, India. Tween 80, glyceryl monostearate, stearic acid, chloroform, acetone, isopropyl alcohol, sodium chloride, and potassium bromide were purchased from from Central Drug House, New Delhi. Methanol and ethanol were purchased from Qualigens Fine Chemicals (A Division of Glaxo India Limited), Mumbai.

Screening of Lipids:

The lipid was screened based on solubility of the drug in various lipids like glyceryl monostearate, glyceryl behenate andstearic acid. Solubility of drug in different lipid was determined visually by taking 10mg of lovastatin in a wide mouth screw-capped bottle. The solid lipid was separately heated above its melting point and was gradually added in portions to the bottle with continuous stirring using a vortex mixer. The maximum amount of lipid required to solubilize the drug required to form a clear, pale yellow solution of molten lipid was noted visually²⁰.

Preparation of Lovastatin loaded Solid Lipid Nanoparticles:

The solvent emulsification diffusion technique²¹ was used for the preparation of lovastatin-loaded solid lipid nanoparticles (LSLNs). Nine formulations with different compositions were prepared (Table 1Glyceryl monostearate lipid in various ranges from 20 to 80mg were weighed and dissolved in 2ml solution of ethanol and chloroform (in ratio of 1:1) as internal oil phase. Twenty milligrams of lovastatinwas dispersed in the above solution 0.8ml of aqueous surfactant solution (1.0- 2.0% w/v) was taken in a homogenizer tube and the above drug dispersion was added drop by drop and homogenized at 4000rpm for 30 minutes to prepare o/w emulsion. The organic solvent was completely removed by evaporating on a rotary evaporator (Buchi type) at 400 mbar, 45°C, 30 min.Ice cold water was added to this emulsion to make up the volume up to 50ml. withcontinuous stirring (3000rpm) for about 3 hours after that diffusion of organic solvent into external aqueous phase taken place. Again, dispersion was centrifuged for 30 minutes at 15000rpm (Remi, India) for the separation of solid lipid materials along with drugfollowed by redispersing in 1.5% w/v aqueous surfactant (Tween 80) solution and sonicated for 5 minutes.

Table 1: Composition of different batches of lovastatin loaded solid lipid nanoparticles.

Formulation code	Drug to lipid ratio	Surfactant Conc.(w/v)
LSLN1	1:1	1
LSLN2	1:1	1.5
LSLN3	1:1	2.0
LSLN4	1:2	1
LSLN5	1:2	1.5
LSLN6	1:2	2.0
LSLN7	1:3	1
LSLN8	1:3	1.5
LSLN9	1:3	2.0

Evaluation of Lovastatin loaded Solid lipid nanoparticles:

Determination of Particle Size, Polydispersity Index and Zeta Potential:

Particle size was calculated as mean particle size by Photon Correlation Spectroscopy (Nano ZS, Malvern, UK) at room temperature (298°K). For study 1ml of LSLNs suspension was diluted ten times (10ml) with distilled water and average particle size, polydispersity index, and zeta potential²²were measured.

Determination of Shape and Surface Morphology:

The shape and surface morphology of the solid lipid nanoparticles were visualized by Transmission electron microscopy (TEM).

Determination of Percent Drug Loading:

Ten milliliters of SLNs suspension was taken with a pipette (10ml, Borosil), and transferred into a centrifuge tube and centrifuged at 5000rpm for 50 min at room temperature (Centrifuge, REMI), the lipid portion was isolated, and the absorbance of the drug in the supernatant was determined spectroscopically using UV-VIS Spectrophotometer (Shimadzu, Japan) at 238nm. The concentration of drug was calculated from the calibration curve. The drug loading and entrapment of solid lipid nanoparticle was calculated by the following equation²¹

Percentage Drug Loading = (W_T-W_S/W_T-W_S+W_L) × 100

Where,

W_T is the weight of drug added in the system, W_S is the analytical weight of drug in the supernatant after centrifugation and W_L is the weight of lipid added in the system.

In-vitroDrug Release Study:

The in vitro drug release studies of all formulations were carried out using Franz diffusion cell. The dialysis membrane (Mol. Wt. 12,000, Hi Media, Mumbai) after treatment, was mounted in Franz diffusion cell, formulations were placed in donor compartment, and phosphate buffer (pH 7.4 along with 1.5% SLS), as a dissolution medium was placed in the receptor compartmentof Franz diffusion cell at 37°C temperature.The assembly was kept on a magnetic stirrer and solution was stirred continuously (100rpm) using a magnetic bead. The sample (1.0ml) was withdrawn at a predetermined timeinterval (0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h) and replaced with an equal amount of fresh dissolution media to maintain the sink condition. The samples were analyzed spectrophotometrically (UV-1800, Shimadzu, Japan) at 238nm²³the cumulative percentage drug release was calculated. The experiments were performed in triplicate and SD was determined^24,25.

Release Kinetic Study:

In vitro release data of optimized formulation (formulation LSLN3) was analyzed by various kinetic models like zero order (C = k₀t), first order (LogC = LogC₀- k_ft/2.303), Higuchi’s model (Q = Kt^1/2) and Korsmeyer-Peppas’ model (M_t/M_N = Ktⁿ) to check the release pattern and mechanism of drug release²¹.

RESULTS AND DISCUSSION:

Amongst the three lipids used glyceryl monostearate was having maximum solubilizing power for lovastatin (70mg per 1000mg of GMS) followed by glyceryl behanate (63mg) and least solubility was observed in stearic acid (50mg) (Table 2). GMS was selected to fabricate the SLNs as it has a maximum solubilizing capacity which affects drug loading in SLNs²⁶.

Table 2: Solubility of lovastatin loaded solid lipid.

Lipid	Solubility (mg of Lovastatin per 1000 mg of lipid
Glyceryl monostearate	70
Glyceryl behenate	63
Stearic acid	52

Solvent emulsification diffusion technique was used since the technique is simple,a robust and requires less surfactant, and easily performed in the laboratory as compared to other techniques^27,28. A totalof eight formulations with different compositions were prepared (Table 2). The prepared LSLNs are spherical and having a smooth surface (Figure 2). The minimum particle size was 288.8±0.94 nm for LSLN3 and the maximum particle size was 401.2±1.81nm (Table 3). The observation revealed that increase in drug to lipid ratio from 1:1 to 1:3 leads to increase in particle size at different surfactant concentration (1.5 to 2.0% w/v) the result is in accordance with previous literature¹⁸.

Figure 2 TEM image for the best selected formulation (formulation LSLN3).

The polydispersity index (PDI) was a parameter to predict about physical stability of SLNs ²⁹ minimum PDI was 0.32±0.06 for LSLN5, while maximum PDI was 0.84±0.089 for LSLN9 (Table 3). Zeta potential is the surface charge on particle responsible for preventing particle aggregation and thus ensure physical stability, the value of ZP for better stability should be in higher ranges either positive or negative³⁰ the value of zeta potential showed variation in magnitude from -13.2±0.73 MeV for LSLN5 to -24.5±0.91 MeV for LSLN8 but all the values are in negative it may be due to the percent drug loading of prepared SLNs were found in a range of 35±1.2 for LSLN2 to 49.81±0.87 for LSLN6. It was observed that as the drug to lipid ratio increases the percent drug loading also increases but at the same time surfactant concentration also has an impact on percent drug loading.

Table 3: Characterization of various formulations

Formulation code	Particle size (nm)	Poly dispersity index (PDI)	Zeta potential	Drug Loading (%)
LSLN1	Lump Formation
LSLN2	328.2 ± 1.4	0.39 ± 0.04	-22.2± 0.93	35.4 ± 1.2
LSLN3	288.8 ± 0.94	0.43 ± 0.07	-21.8± 0.99	39.41 ± 0.99
LSLN4	382.3 ± 0.42	0.81 ± 0.41	-22.3± 0.89	41.32 ± 0.93
LSLN5	338.1 ± 0.98	0.32 ± 0.06	-13.2± 0.73	44.12 ± 0.9
LSLN6	298.9 ± 1.1	0.42 ± 0.09	-19.1± 0.81	49.81 ± 0.87
LSLN7	401.2 ± 1.8	0.79 ± 0.29	-18.9± 0.23	46.07 ± 0.98
LSLN8	324.4 ± 1.2	0.76 ± 0.34	-24.5± 0.91	48.4 ± 0.44
LSLN9	378.5±0.92	0.84 ± 0.89	-20.1± 0.54	49.6 ± 0.39

In-vitro release study suggests that formulations can release the drug upto 24 h and almost all formulation except LSLN4, LSLN7 and LSLN9 showed more than 90% drug release. Release of drug is least for LSLN4 which is 79.57±6.74 and maximum was 98.06±2.63 for LSLN6 (Figure 3). Based onabove-mentioned observation, the formulation LSLN6 was selected as an optimized formulation although some formulation like LSLN3 is having a smaller size and PDI than formulation LSLN6. However, it was not taken as optimized one as it is less percent drug loading as compared to LSLN6. since the formulation is designed for transdermal delivery, so the size is not concerned in the study so LSLN6 was taken as optimized formulation and possible release kinetic study was performed for single optimized formulation LSLN6.

Figure 3: In-vitro release profile of lovastatin fromsolid lipid nanoparticles.

Release kinetic study was performed to know the order and mechanism of in vitro release^32,33. The maximum value of correlation coefficient r² was found to be 0.894 for Higuchi kinetics (Table 4), followed by 0.865 for first order and least correlation was 0.743 for zero order suggested that the release kinetics followed mixed order kinetics so for mechanism of drug release Korsmeyer Peppas’ model was applied and the value of r²was 0.862 and release exponent n was found to be 0.416 reveals that release followed Fickian diffusion.

Table 4: Release kinetic parameters of LSLN6

Zero order	First order	Higuchi’s kinetics	Korsmeyer Peppas’
Y = 3.415X+ 11.73	Y = -0.471X + 2.346	Y= 22.82X − 0.763	Y = 0.312X + 2.122
r² = 0.743	r² = 0.865	r² = 0.894	r² = 0.862n = 0.416

CONCLUSIONS:

Lovastatin is hypolipidemic drug used to lower the cholesterol level in the blood, poor bioavailability of the drug makes it a good candidate for transdermal delivery, moreover the lipid nanoparticles specially SLNs are good carrier for delivery of actives through various routes like transdermal, nasal, oral, i.v. etc. in this study lovastatin loaded SLN was fabricated by using a simple robust and versatile technique named as solvent emulsification diffusion technique. The prepared SLN were evaluated for various parameters like particle size, PDI, percent drug loading and in vitro release profile. All the formulations were spherical and below 500nm particle size and show PDI in acceptable limit with good drug loading percentage. The SLNs were also showing sustained release up to 24 hwith mixed order kinetics. So, the present study concluded to fabricate the SLNs loaded with lovastatin and can be further incorporate into suitable Gel or any cream for application to skin and may provide a good alternative to the oral route of lovastatin.

DISCLOSURE:

The authors report no conflicts of interest in this work.

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Received on 01.01.2021 Modified on 20.02.2021

Research J. Pharm.and Tech 2022; 15(3):1085-1089.

DOI: 10.52711/0974-360X.2022.00181