Formulation, optimization, characterization and in vitro-ex vivo evaluation of atorvastatin loaded solid lipid nanoparticles using quality by design approach

 

Nilesh B Chaudhari¹*, Amar G Zalte², Vishal S Gulecha³

^1,2 Department of Pharmaceutics, School of Pharmaceutical Sciences, Sandip University, Nashik, India.

³Department of Pharmacology, School of Pharmaceutical Sciences, Sandip University, Nashik, India.

 

ABSTRACT:

Solid lipid nanoparticles (SLN) have engrossed snowballing consideration throughout recent years. This paper depicts an impression about the quality by design approach on formulation, optimization, characterization of solid lipid nanoparticles (SLN) loaded with Atorvastatin (AT). A unique microwave-assisted microemulsion technique of preparation was contemplated to formulate AT SLN. Optimization was executed using 3² response surface methodology where optimized the effect of Glyceryl monostearate (GMS) (X1) and Poloxamer 407 (X2) concentration as an independent variable on % entrapment efficiency (EE) (Y1) and particle size (Y2) as dependent variables. Characterization of optimized formulation was done by employing EE, particle size, Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), Differential Scanning Calorimetry (DSC), scanning electron microscopy (SEM), in vitro and ex vivo drug release. The percentages of EE fell within the range of 55 to 82%, particle size was in between the range of 290.3 to 890.4 nm and in vitro drug release was found to be 63.8% also ex vivo absorption was found to be 59.8 % for the duration of 8 hours. The AT SLN was successfully prepared and effective in releasing the drug in sustained mode.

 

 

 

INTRODUCTION: 

Voluminous research groups are presently considering at the feasibility of employing SLN as a delivery system. In order to resolve demerits of conventional colloidal drug delivery systems like non-biodegradability and instability, the SLN was initially explored. To upsurge bioavailability and accomplish sustained drug delivery ¹, SLN is often employed. AT is used to lower the increased levels of total cholesterol since it is a specific and competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase ². AT's enormously poor water solubility is a noteworthy obstacle to the therapeutic use and effectiveness of AT as an oral dose form. It crafts variance in the drug's bioavailability by instigating fluctuating drug dissolution and absorption in the gastrointestinal tract³.

 

Consequently, there is a requisite to develop a formulation of AT which increases oral bioavailability. One of the eminent strategy for increasing the bioavailability of highly lipophilic molecules is to employ lipid-based drug carriers³. Extensive research revealed that high potential is observed in lipid-based formulations to improve drug bioavailability and drug therapeutic efficacy via various mechanisms namely increasing luminal solubilization, bypassing first-pass metabolism of the drug and inhibiting the Cytochrome P450^{4, 5,6}.

 

Nano sized SLN is prepared using lipids and surfactants which offer certain merits like higher surface area, increased interface, more bioavailability and absorption^5,6, drug targeting, and more half-life⁷. Which eventually results in lower drug dose, toxicity and improved safety and protection to non-targeted tissues^8,9. 

 

A significant limitation of SLN is drug loading, particularly for drugs with low aqueous solubility^9,10. The current study aims to formulate and optimize ATSLN by using a novel microwave-assisted microemulsion method ¹¹. where an aqueous surfactant phase containing poloxamer 407 is mixed with a lipid phase containing glyceryl monostearate (GMS). 3² full factorial design was applied to optimize SLN. EE, particle size, FTIR, DSC, XRD, scanning SEM, in vitro and  Ex vivo drug release were used to characterize optimized SLN formulation.

 

MATERIALS AND METHODS:

Materials

A gift sample of Atorvastatin calcium was obtained from Enaltec Labs PVT Limited, Mumbai, India, and Poloxamer 407 was provided as a gift sample by Corel Pharma, Ahmedabad. Glyceryl Monostearate was purchased from Research lab fine chem, Mumbai. Analytical grade reagents and solvents were used for the study.

 

Selection of Lipid

The lipid in which active ingredient has highest solubility was selected. The solubility of atorvastatin was determined in different lipids such as glyceryl monostearate , stearic acid and gelucire. In this determination, accurately weighed atorvastatin was transferred in melted lipids until the clear and transparent solution was obtained.

 

Selection of Surfactant

Poloxamer 188, poloxamer 407, various grades of tween and span were examined for appropriate selection of surfactant.

 

Method of Preparation

AT SLN was prepared by a novel microwave-assisted microemulsion technique followed by probe sonication¹¹. GMS was melted above its melting point in a microwave oven with a power of 320W. Continuous stirring was used to dissolve AT in the lipid (molten state), after which the addition of Poloxamer 407 aqueous surfactant solution. The aqueous phase with Poloxamer 407 was also maintained at a similar temperature as that of the molten lipid phase. The aqueous phase was added slowly into the hot oil phase, and the resulting dispersion was constantly stirred (temperature was maintained above the melting point of GMS) until it get clear microemulsion. After that, the hot oil in water (o/w) microemulsion was added to cold water at 2-4⁰ C which was stirred with a magnetic stirrer to obtain SLN. Formulated SLN was sonicated for 20 min. with probe sonicator (PCI analytics)^12,13. A resulted colloidal dispersion of SLN was cooled at room temperature and lyophilized. Southern Scientific freeze dryer was used to get the lyophilized product. The Lyophilization procedure was initiated after 24 hrs of refrigeration of formulation. The freeze drying of formulation was performed  at -40°C and under 0.5 lbs pressure^12-18 .

 

Factorial experimental design for optimization

To investigate the impact of formulation variables, a 3² full factorial optimization design was used with 13 trial runs which include two factors and three levels. The lipid concentration (X1) and surfactant concentration (X2) were an independent variable. However, % Entrapment efficiency (Y1) and Particle size (nm) (Y2) as dependent variables or responses (Table 1). 2D plots & 3D Counter Surface response plots were designed to analyze the effect of independent variables. Version 13 of design Expert software was used to fit the obtained data moreover to analyse ANOVA. Collected data was also investigated to response surface methodology   to explain the effects of concentration of lipid and surfactant on the dependent variable. Table 1 indicates 13 batch runs of AT SLN prepared using 3² full factorial designs ^19.

 

CHARACTERIZATION

Entrapment Efficiency

Ultracentrifugation at 10,000 rpm for 1 hour was used to separate the drug-loaded SLN from the dispersion, with the lipid nanoparticles settling at the bottom and separated. To solubilize the lipid, pour 5 ml of chloroform into the separated samples which result in lipid precipitation, moreover, chloroform was removed by evaporation to dryness. Add methanol and bath sonicate for 10 min. and finally filtered through wattman filter paper (0.45 um). Dilute with methanol, if necessary. Measured the absorbance at 245.5nm and the concentration of Atorvastatin was calculated using the calibration equation. The dilution factor was used to determine the amount of the drug ^{15, 16, 20}.

 

                 The actual amount of drug entrapped in SLN

% Entrapment efficiency = -------------------------   X 100

                        The total amount of drugs added in SLN

 

Particle Size

To determine the particle size of AT-loaded SLN liquid samples, nanoplus particulate system instrument was used to find out particle size. The dispersion of ATSLN was used to record the average particle size and size distribution of formulation^{15, 16, 20}.

 

Zeta Potential and Polydispersity index

Double distilled water was used to dispersed  freeze dried nanoparticles. Zeta potential was measured using a NanoPlus Particulate System. Polydispersity index (PDI) was used to determine the particle size distribution. Zeta potential was used to study the surface charge of SLNs. The zeta potential was studied with the help of electrophoretic light scattering (ELS) at 25 °C with electric field strength of 23 V/cm using NanoPlus Particulate System. All the measurements were taken out in triplicate^15,16,20.

 

Fourier Transforms Infrared Spectroscopy (FTIR)

Shimadzu FTIR-8400 was used to record IR spectra of AT and the AT SLN. The sample disc was prepared by mixing sample of AT and ATSLN with KBr and compressed using KBr press. A range of wave number 4000 to 400 cm^-1 was used to examine         transmission^{15, 16, 20}.

 

Differential Scanning Calorimetry (DSC)

DSC 3 Star system, Mettler Toledo was used to record the DSC thermogram of AT.  20ml/min Nitrogen flow at a scanning rate of 100c/min. was used to analyze the samples in sealed aluminum pans^{15, 16, 20}.

 

X-ray Diffraction (XRD) Analysis

A Bruker AXS D-8 XRD was used to ascertain the crystalline state of pure atorvastatin and optimized formulation of atorvastatin loaded SLN. The formulation and drug were analyzed over the angle (2Ɵ) range from 0⁰ to 80⁰ and scanned with with filter Ni, Cu- Kά radiation, 20mA current and voltage            40kV ^15,16,17,20.

 

Field Emission Scanning Electron Microscopy (FE-SEM)

The surface morphology of optimized lyophilized SLN formulation of atorvastatin was studied under field emission scanning electron microscope. SEM study was conducted using Olympus Corporation FV3000 ^[22]. Before studying morphology, lyophilized powder samples were placed on metal stub by using double adhesive tape. To make the nanoparticle conductive gold was used to coat the samples under the vacuum. Finally 8-20 kV accelerating voltage was used to analyze samples. ^{21, 22.}

 

In vitro Drug Release Study

Calibrated dissolution test apparatus (Electrolab Mumbai) was used to conduct  In vitro release at 37±0.5⁰C. pH 6.8 phosphate buffer used as a dissolution medium for  in vitro drug release of ATSLN. 10 mg equivalent 5 ml formulation was placed inside the dialysis bag which was soaked in dissolution medium prior 12 hours and tied at both ends to the paddle rod. The paddle was placed in a dissolution medium with constant stirring speed. Aliquots of 5 ml dissolution medium were withdrawn every after certain intervals while fresh to keep sink volume constant, the dissolution medium was replaced. UV wavelength of 245.5 nm was used to analyze the sample solution.^{9, 19, 20, 23}.

Ex vivo Absorption Study

Formulation of the optimized batch was used to conduct absorption study on everted chicken ileum using ex vivo absorption method. U shaped perfusion glass apparatus with 25 ml volume capacity used to tie chicken ileum. After mounting everted segment on glass apparatus was filled with 25 ml tyroid solution. Now, place the assembly into the beaker having 250 ml of phosphate buffer (pH 6.8) containing SLN formulation 10 ml and constant supply with aeration. Mount the assembly over magnetic stirrer and maintain 37±0.5°C temperature. The serosal side becomes an internal part of the tube whereas mucosal side would be buffer solution   in beaker.  Sample for analysis were collected from tyroid solution after every 1 hour. % Permeation was assessed using UV wavelength at 245.5 nm. Replacement of tyroid solution with fresh one is mandatory to compensate sampling loss ^24-27.

 

Countour plots and surface response plots

The values of the response are diagrammatic represented using contour plots and surface response plots. This helps in explaining the relationship between independent and dependent variables. Response surface methodology (RSM) exhibits how an experimental response and a set of input variables are related. RSM sets a mathematical trend in the experimental design for determining the optimum level of experimental factors required for a given response. The reduced models were used to plot two dimension contour plots and three dimensions RSM using Design of Expert^24-29.

 

RESULT AND DISCUSSION:

Selection of Lipid

Lipid was selected depending on the highest solubility criterion of active ingredient in lipid. GMS was found to be  suitable for the formulation of ATSLN.

 

Selection of Surfactant

The choice of surfactant based on the production of nanosized particle, smaller polydispersity index, safety and stability. Poloxamer 407 satisfactorily complied with the characteristics stated above.

 

Entrapment Efficiency

3D counter-plot from design of expert software was used, to predicts the effects of the amount of GMS and concentration of poloxamer 407 on factor EE (Fig. 8b). The range of 13 experimental batches for EE were from 55% to 82%. At a higher (6%) concentration of GMS and 0.5 % concentration of poloxamer 407, the formulation has shown 82% EE. Therefore, we draw the conclusion that %EE increased with the highest concentrations of GMS and poloxamer 407.

 

Particle Size

Table 1 shows the size of prepared ATSLN varied within the ranges of 290.3 to 890.4 nm. A particle size increases depending on GMS and poloxamer 407 concentration. When the GMS concentration increase, particle size also increases while the increase in poloxamer 407 concentration causes a drop in particle size, this could be due to more solubilization of the drug in lipid. Figure 1 shows particle size of optimized batch which is 290.3 nm.

 

Figure 1: Particle size of ATSLN formulation

 

Polydispersity Index (PDI)

The physical stability of SLNs dispersion is govern by  PDI, which  is an important parameter for the long term stability of SLNs dispersion. The PDI should be as low as possible, has the range of 0 to 1 which defines the dispersion homogeneity. Value near to 1 shows heterogeneity and less than 0.5 shows homogeneity. The PDI value shown in table 2 for all formulation and which was found within the range of 0.159 to 0.451 which was less than 0.5, indicating their homogeneity.

Zeta Potential

Stability of colloidal dispersions upon storage depends on Zeta potential. The repulsion effect between charged nanoparticles causes better colloidal suspension stability if there is the larger zeta potential value of a nanoparticulate system. The zeta potential valueshas a range  between -24 to -40 mV.

 

The surfactant concentration has potential impact on the charge on the particle. It was observed that, as the concentration of surfactant was increased from 0.1 to 0.5%, there was a decrease in the value of zeta potential. This is because the surfactant is non- ionic and increasing its concentration lowers the total charge on the particle. The optimized batch of atorvastatin calcium loaded SLNs (Batch AT4) was found to have Zeta potential of -27.80±(-1.79) mV. For well stabilized nanoparticles Zeta potential values must present in the ± 15 mV to ± 50 mV hence, it was concluded that the nanoparticles would remain stable.

 

Table 2: PDI and Zeta potential of  AT1-AT13 batch (Mean ± SD, n=3).

Experimental Run	PDI	Zeta Potential (mV)
AT1	0.159±0.039	-35.05±(-2.12)
AT 2	0.403±0.069	-24.38±(-2.59)
AT 3	0.350±0.038	-26.41±(-1.85)
AT 4	0.451±0.031	-27.80±(-1.79)
AT 5	0.343±0.040	-24.08±(-2.35)
AT 6	0.330±0.025	-34.64±(-0.21)
AT 7	0.355±0.049	-26.92±(-2.44)
AT 8	0.254±0.037	-24.44±(-2.08)
AT 9	0.454±0.034	-23.89±(-)2.89
AT 10	0.339±0.042	-30.24±(-2.64)
AT 11	0.330±0.025	-25.93±1.87
AT 12	0.352±0.046	-26.54±(-2.31)
AT 13	0.251±0.035	-29.11±(-0.53)

 

Infrared Spectroscopy

The FTIR spectra of pure drug and optimized formulation were recorded. Characteristic peak of AT observed at 3363.97 cm-1 for O-H, 1595.18 cm-1 for C=C, 1651.12 cm-1 for C=O and N-H also confirmed. ATSLN peak observed at  1734.06 cm-1  for C=O, 1473.66 for C-H. The drug entrapment in lipid matrix was confirmed from FTIR of lipid GMS and formulation ATSLN, the disappearance of the distinguishing peaks of drug and replacement by the peak of GMS. While other peaks replaced or shifted in the IR spectra of formulation (Figure 2).

 

 

Figure 2: FTIR of AT and ATSLN formulation

 

X-ray Diffraction (XRD) Analysis

Intense peak in XRD were observed at 17⁰, 19.41⁰, 21.57⁰ and 21.95⁰ indicating crystalline nature of Atorvastatin. The lyophilized formulation of ATSLN did not show any sharp peak characteristics of AT which indicates the amorphous nature. This amoprhinization of formulation indicated the large amount of active ingredient was entrapped in the lipid and eventually homogeneously dispersed  at molecular level (Figure 3).

 

 

Figure 3: XRD of AT and ATSLN formulation

 

Differential Scanning Calorimetry

A 60⁰ C endothermic peak in the DSC analysis of the SLN formulation indicated the presence of GMS. In the formulation of AT SLN, there was no observable AT peak. This shows that the AT has completely dissolved in the lipid matrix of the SLN (Figure 4).

 

 

Figure 4: DSC of AT and ATSLN formulation

 

Scanning Electron Microscopy (SEM)

The SEM image showed the drug particles were irregular and non smooth surface and then transform into spherical solid lipid nanoparticles. The micrograph indicate that the optimized formulation of solid lipid nanoparticles has sphere shape of  ATSLN formulation (Figure 5).

 

 

Figure 5: SEM of AT and ATSLN formulation

 

In vitro Drug Release Study

AT SLN in vitro release was assessed using phosphate buffer (pH 6.8)  for 8 hours using a dialysis membrane. AT SLN formulation showed 63.8 % of drug release. The initial rapid release may be due to free drugs from the SLN. Release from SLN include complex mechanism which is based on diffusion from the lipid matrix (Figure 6).

 

 

Figure 6: ATSLN release profile in vitro

 

Ex vivo Absorption Study

Figure 7 shows the result of AT SLN for ex vivo absorption through everted chicken ileum. Permeation absorption for AT SLN formulation was 59.8 %.

 

Figure 7: ATSLN release profile ex vivo

 

Contour Plots and Response Surface Plot by using 3² Full Factorial Design

A 3² response surface optimization methodology was used to detect the influence of independent variables (X1, X2) on dependent variables (Y1, Y2). To analyze the effect of independent variables, plot 2D and 3D counter surface response graph. In order to understand interaction effects of independent variables, the 3D response surface graph is very important ^[22]. Lipid & poloxamer 407 concentration were selected as the independent variables, whereas % entrapment efficiency & particle size (nm) were dependent variables for the study. The 2 dimensional & 3 dimensional plots for entrapment & particle size predict that, as the concentration of GMS increases EE of SLN. While decrease in the particle size because of the increased concentration of poloxamer 407. As increase in the the lipid concentration causes increase in particle size increases while decrease in particle size as increase in Poloxamer 407 concentration. This is because reduction in surface tension which benefits to stabilize the newly generated  surface and prevents particle clump. The quadratic model for Y1 and Y2 responses, R² (correlation coefficient) value was 0.99 and 0.97 (Table 3). The EE was in the range 55 to 82 % and particle size 290.3 to 890.4 nm. For EE (Y1) and particle size (Y2) response following equations are obtained,

 

%Entrapment= 72.52+9.5X₁+4X₂-3.81X12-0.31X₂²   .1

Efficiency             

 

Particle    =565.6-228X₁-100.45X₂                                     ….2        

Size

 

Equation mentioned above signifies, positive value for synergistic while negative values represent antagonistic effect. ANOVA responses for model Y1 and Y2 is shown in Tables 4. The quadratic equation explains how the independent variables X1, X2, X12, X22, and X1X2 affected the entrapment (Y1) and particle size (Y2) responses. These independent variables had a significant impact on EE and particle size at P < 0.05. Both models had F values of 129.24 and 88.16 at P < 0:0 that indicated they were significant. Table 5 shows the diagnostic case statistics. Model was significantly fit as the difference between actual and predicted values was less ^[26].

 

Table 3: Model summary of results of regression analysis for responses Y1 & Y2

Model Summary Statistics: EE (Y1)
Source	Std Dev	R2	Adjusted R2		Predicted R2	Press
Linear	2.39	0.91	0.90		0.84	105.37	Suggested
2FI	2.52	0.91	0.88		0.69	208.97
Quadratic	1.03	0.99	0.98		0.98	12.20
Cubic	1.22	0.98	0.97		0.94	39.36
Model Summary Statistics: Particle size (Y2)
Source	Std Dev	R2		Adjusted R2	Predicted R2	Press
Linear	45.95	0.94		0.93	0.87	48284.8	Suggested
2FI	46.71	0.95		0.93	0.73	103242
Quadratic	35.38	0.97		0.96	0.78	84830.9
Cubic	16.13	0.99		0.99	0.61	151294

 

 

 

Table 4: ANOVA of models for Y1 and Y2

Source	Sum of Squares	DF	Mean square	F Value	Prob > F
Model for Y1
	687.62	5	137.52	129.24	< 0.0001	significant
A	541.5	1	541.5	508.90	< 0.0001
B	96	1	96	90.22	< 0.0001
A2	40.09	1	40.09	37.68	0.0005
B2	0.26	1	0.26	0.25	0.6324
AB	0	1	0	0	1.0000
Model for Y2
	372445.22	2	186223	88.16	< 0.0001	significant
A	311904	1	311904	147.66	< 0.0001
B	60541.21	1	60541.2	28.66	0.0003
Residual	21121.98	10	2112.2

 

Table 5: Diagnostics Case Statistics for various response variables.

 

Fig. 8 indicates, the 2D contour plot (fig. 8a) and 3D response surface plot (Fig 8b) indicated the relative effect of increasing Glyceryl monostearate and poloxamer 407 concentration on % entrapment efficiency of Atorvastatin SLN (Y1). With increase in GMS concentration (X1) and Poloxamer 407 concentration (X2), % entrapment efficiency was increased and this can be confirmed from equation1. At different levels (-1, 0, 1) of GMS concentration, was increased, the % entrapment efficiency of AT SLN was increased for each level of GMS concentration (55 to 82%).

 

(a)

 

(b)

Figure 8: Various plots showing influence of GMS and Poloxamer 407 concentration on the % drug entrapment a) 2D Contour plot and b) 3D Response surface plot

 

Figure 9 indicates, effect of variables X1 & X2 on particle size (Y2) can be explained with 2-dimensional contour plot (fig. 9a) and 3-dimensional response surface plot (Fig 9b). Increase in GMS concentration (X1) increases particle size (Y2). Increase in Poloxamer 407 concentration (X2) showed the slight decrease in response (Y2).

 

 

(a)

 

(b)

Figure 9: Various plots showing influence of GMS concentration and Poloxamer 407 concentration on the particle size a) 2D Contour plot and b) 3D Response surface plot

 

CONCLUSION:

The AT SLN were developed successfully in the current study. The newly created AT SLN formulations exhibit an 8-hour prolonged drug release. The 3² where three level and two factors optimization was preferred to study the effects of dependant variables (EE and particle size) on the responses of independent variables (GMS and poloxamer 407). The results of EE were in the range from 55 % to 82%, while particle size was in the range of 290.3 to 890.4 nm, in vitro and ex vivo drug release of the optimized batch was in a sustained manner which was found to be of 63.8 % and 59.5% after 8 hours respectively. In FTIR, XRD, and DSC spectra, no interactions were reported. SEM shows the spherical SLNs. Microwave microemulsion method of formulation of SLN overcomes the drawback of conventional heating used in microemulsion SLN formulation method.

 

ACKNOWLEDGMENT:

The authors are grateful to Enaltec Labs PVT Ltd, Mumbai and Corel Pharma, Ahmedabad providing gift samples of Atorvastatin and Poloxamer 407 respectively.

 

CONFLICT OF INTEREST:

No potential conflict of interest was reported by the authors.

 

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Accepted on 23.03.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1433-1441.