INTRODUCTION:

Wound healing is a normal biological process in the human body and is attained through four exceptionally programmed phases Hemostatis, Inflammation, Proliferation and Remodelling. For a wound to get better successfully all the above four mentioned phases must take place in the correct sequence and time frame. The wounds which fails to progress through normal stages of healing are said to exhibit impaired healing.¹ One of the major causes of impaired wound healing is Diabetes mellitus. Among all the wound problems in Diabetes mellitus the most of them are associated with feet. The global prevalence of diabetes is currently approaching in the year 2014, it was estimated 422million people were suffering from diabetes worldwide², among which 90% of the cases were of type 2 DM.²

Nanoparticles are particles between 1 and 100nm in size. Only the advancement of new drugs is not fulfilment in drug therapy. The parameters like poor solubility in water and poor bioavailability of the new drug substance is the main issue which is to be found out. Hence, there is a need for developing a carrier for the drug.

SLN are the alternative carrier scheme to liposomes, polymeric nano particles and emulsions.³ Formulation of SLN is done by from solid lipids only.SLN are relatively new pharmaceutical delivery systems made of sub-micron (between 10 and 1000 nm) colloidal lipid drug carriers with relatively above ambient melting points which remain solid at room temperature as well as body temperature.⁴ The main advantages of SLN include high drug loading capacity, high biocompatibility, more resistant to chemical degradation, and enhanced stability of liable pharmaceuticals.

On the other hand, SLN possess some limitations including; possibility of particle growth, high burst release, and compromised stability in tropical climates.^5,6 NLC were introduced to avoid such limitations.⁷ Incorporation of oil that solubilises the drug and using a combination of lipids with different hydrocarbon chain length are the main approaches employed in the design of NLC system.^8,9 NLC were successfully used to enhance the solubility and/or intestinal permeability of many Biopharmaceutical Classification System (BCS) class II drugs

Valsartan drug is a non-peptide, orally active and specific angiotensin II antagonist. Which acting on the AT1 receptor subtypes. It is the category of angiotensin II receptor blocker. According to the Biopharmaceutical Classification (BCS) Valsartan is a class-II category which is having less water solubility and high permeability.¹⁰

MATERIAL AND METHODS:

Valsartan obtained from Sigma Aldrich, Mumbai. Palmitic acid, Polysorbate 80, Soya oil and Span 80 obtained from SD fine chemicals, Mumbai, India. Dialysis bags obtained from Himedia labs, Mumbai, India. All the chemicals and reagents obtained are of analytical grade.

Method:

Preparation of valsartan loaded nano structured lipid carrier (VNLCs)¹¹:

In the preparation of Valsartan loaded nano structure lipid carrier, two phases were prepared. Oil phase and aqueous phase is prepared. In the oil phase different ratio of solid lipid and liquid lipid was added and heated up to 70°c. Then after that 10 mg of valsartan is added and again heated up to 70°c. In aqueous phase, different ratio of surfactant is dissolved into the solvent and heated up to 70°c. In both the phases temperature should be same. After maintaining the temperature at 70°c aqueous phase is added drop wise to oil phase. Pre emulsion was formed then continuous stirring at 5000rpm was done using magnetic stirrer. After the stirring, probe sonication was done for 4 minutes at amplitude 100 and temperature 37°c. Then 100 ml of cold water was added for the solidification of lipid nanoparticles.

CHARATERIZATION:

Solubility studies:

10mg of Valsartan was dissolved in 10ml of different solvents i.e. water, ethanol, acetonitrile, glacial acetic acid, ether. Solubility was determined on the basis of physical appearance. Valsartan is slightly soluble in water, soluble in ethanol, acetonitrile, glacial acetic acid and ether.

Fourier transformer infrared (FTIR) spectroscopy¹²:

The FTIR Study was performed to examine the drug, physical mixture and formulation (Shimadzu FTIR8400 S, Japan). Potassium bromide (KBr) pellets containing weight ratio is 4:1 of KBr, respectively. Result was obtained within the range of 4000cm⁻¹ - 400cm⁻¹ and showed as the FTIR spectra.

Differential Scanning Calorimetric (DSC)^13,14:

Differential scanning calorimetry was used to determine the thermal behavior of the drug (Valsartan), Palmitic acid and their physical mixtures. It was done by taking sample into the aluminum pans which was sealed after loading the samples. Both the Sample pan and reference pan were fitted into the DSC chamber and then lid was closed, then the sample was run and was placed into the chamber, heated up to 20-400^°C. The heating rate was 10^°C/min. DSC thermograms were acquired using automatic thermal analyzer system. The thermograms were obtained. DSC was used to compare and to find the compatibility of the samples taken.

Particle size (PS) and zeta potential (ZP) analysis^9,15:

Size distribution of mean size of VNLCs and zeta potential of formulation were analyzed using a Zeta Sizer Nano Series ® (Malvern Instruments, UK) at a wavelength of 633nm. Every measurement was finished in triplex manner and the data were shown as mean ± standard deviation.

Scanning electron microscopy (SEM):

The Scanning electron microscopy of the nano structured lipid carriers is used to find out the surface morphology. To acquire the proper concentration of the sample, it was diluted using ultra-purified water. After that samples were placed on a sample holder and dried using vacuum. Then they were coated with gold and sample was tested through SEM. It was scanned using refractive index at 30second run time.

Lyophilisation of NLCs:

Lyophilisation of the optimized formulation was performed to concentrate nanoparticulate and improving the stability of the NLCs. The Sample was lyophilized using cryoprotectant (mannitol). Before freezing of the different-different concentration of mannitol (1%, 2%, 3%) was added to the NLC dispersion. Then dispersion was added and dispersed in the big petridish and kept for deep freezing for 20hrs. A frozen thin film of drug loaded NLCs were obtained. These NLCs films were frozen in lyophilizer at -60°C and 0.01bar pressure for 6hours. The concentrations of cryoprotectant (mannitol) were selected according to the redispersibility, particle size and flow property of reconstituted NLCs.

Drug Loading and Entrapment Efficiency:

To separate nanoparticles from the nanosuspension, the nanosuspension was ultra-centrifuged at 8,000rpm for 1h. After removing the supernatant, the nanoparticles were collected. Then the Nanoparticles were dissolved in 5ml of phosphate buffer and make up to 10ml. After that the samples were passed by way of millipore membrane filter 0.22μm and the content of drug in the sample was analysed by UV-VISIBLE Spectrophotometer.¹⁶ The drug loading and entrapment efficiency was calculated by these equations:

Content of the drug in nanoparticle

Drug loading (%w/w)^{-
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------} = * 100

Content of nanoparticle recovered

Content of the drug in nanoparticle

Entrapment efficiency = ^{----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------}* 100

Content of the drug used in the formulation

In-vitro drug release¹⁷:

Levis – beaker method was used to measure the drug release profile from NLCs. The NLCs formulation was filled in dialysis tube which was knotted at one and the other ends with clips. Then dialysis bag was dipped in 80ml of HCL of pH 1.2 combined with phosphate buffer of pH 6.8. At an time break of 15 minutes, 3 ml of buffer solution was withdrawn for a time period of 1.15 hours and also at an period of 1 hour for12 hours and was substituted instantly with same volume of buffer solution. The concentration of drug in the solution was identified by using UV Visible method. At 250nm the absorbance was measured by using buffer solution as blank. Various mathematical models (Zero order, first order, Higuchi, Krosmeyer Peppas) were used to determine the drug release profile of the optimized formulation which was used to explain the kinetics of drug release. The Best result of fit test (R²) was taken as standard for selecting the most suitable model.

Release kinetics:

In-vitro dissolution is a main parameter in the development of drug. During definitive situations it can be applied as the evaluation of bioequivalence. There are many systems to stand for the dissolution of drug and drug profiles where, ft denotes the function of t (time) which related to the amount of drug decomposed from the pharmaceutical dosage system. In order to possibility of dissolution profiles amongst two drug products model dependent (curve fitting), statistical analysis and model independent methods also can be used.

To illuminate the method and mechanism of drug release, the in vitro statistics was altered and interpreted at graphical interface made using dissimilar kinetic models. Zero order release Eq. (1) describes the drug dissolution. There are numerous types of modified release pharmaceutical dosage forms, such as transdermal systems, matrix tablets with less soluble drugs, osmotic systems and coated forms etc., where the release of drug is independent of concentration.

Q_t = Q_o + K_ot (1)

Where, Qt denotes amount of drug released in time t, Qo shows the initial amount of the drug in the solution and Ko denotes the zero order release constant

The first order Eq. (2) demonstrates that the drug release from the formulation where the release is concentration dependent e.g. pharmaceutical dosage forms containing water soluble drugs in porous matrices.

log Q_t = log Q_o + K1 t/ 2.303 (2)

Where Qt which shows the amount of drug released with respect to time t, Q showing the initial amount of drug in the solution and K denotes first order release constant.

Higuchi explained that the drug release from insoluble matrix as a square root of time.

Where,

Qt = the amount of drug release in time t,

KH =is Higuchi’s dissolution constant

Korsmeyer-Peppas model derived a trouble-free correlation of drug which describes about drug release from a polymer matrix system equation. To estimate the mode of drug release, 1^st60% drug release data were put into Korsmeyer-Peppas model.

Mt / M∞ = Ktn

Where,

Mt/M∞= fraction of released drug with time t,

k = release rate constant and

‘n’ = release exponent.

The ‘n’ value is elaborating various releases for cylindrical shaped matrices. In this model, the value of ‘n’ characterizes the drug release mechanism as showed in Table 4. For in the case of cylindrical tablets, 0.45 ≤ n match to a Fickian diffusion mechanism, 0.45 < n < 0.89 to non-Fickian transport, n = 0.89 to Case II (relaxation) transport, and n > 0.89 to super case II transport. To appraise the exponent of n the portion of the release curve, where Mt / M∞ < 0.6 should only be used. To inspect the release kinetics, result was plotted as log cumulative percentage drug release vs. log time

Table 1: Interpretation of diffusion release mechanisms from polymeric films

Release exponent (n)	Drug transport Mechanism	Rate as a function of time
0.45	Fickian diffusion	_t - 0.5
0.45 < n = 0.89	Non -Fickian transport	_t n-1
0.89	Case II transport	Zero order release
Higher than 0.89	Super case II transport	_t n-1

In vivo studies:

Induction of diabetes and Experimental design:

The animal model (Albino wistar male rats) weight of 180–230gm was maintained at a constant temperature 23±1°C on a 12hours in light and dark cycle with food and water. After that Diabetes was induced by a single intra peritoneal injection of 55 mg/kg streptozotocin (STZ), in 0.1M sodium-citrate buffer (pH 4.4). Age-matched control rat received an equivalent amount of the sodium-citrate buffer. Then Blood samples were collected from vein of tail 48 hours after STZ administration. The rat with blood glucose more than 250mg/dl were considered as diabetic and were further considered for study The treatment was started 6 weeks after diabetes induction and was continued daily for period of 4 weeks. The functional and behavioral experiments were performed 24h after administration of last dose.

Grouping of Animals-:

Albino wistar rats of weight of 180 – 240g were taken from the JSS College of Pharmacy, Ooty. The In-vivostudieswere approved by IAEC (Institutional animal ethics committee) for the animal care and use. The animals were divided into 3 groups. Group 1 received control, group 2 was treated with drug (valsartan) and the group 3 was treated with Placebo.

Stability study¹⁸:

The NLC dispersion was stored in refrigerator temperature (4-10°C) and at room temperature (approximately 25°C) and for 1 month and then examined for particle size, polydispersity index and zeta potential.

RESULT AND DISCUSSION:

Solubility Studies:

Solubility analysis of Valsartan was done with various solvents and results are shown in table Valsartan was found to be slightly soluble in water; it was freely soluble in ethanol, acetonitrile, glacial acetic acid and ether.

Table 2: Solubility analysis of Valsartan

Solvent	Solubility
Water	0.0234mg/ml	Sparingly soluble
Ethanol	0.89mg/ml	Freely Soluble
Acetonitrile	0.78mg/ml	Freely Soluble
Glacial acetic acid	0.81mg/ml	Freely Soluble

Fourier transformer infrared spectroscopy:

FT-IR study was done to check the compatibility within the selected lipid palmitic acid, and the drug Valsartan. The FT-IR study was also helpful to assure the complete physical adsorption of the drug into the lipid matrix without any mutual interaction. The spectra obtained from IR studies at wave length from 4000cm^-1 to 400cm^-1 are depicted in Figure and data was shown in table 8-10. The drug valsartan had shown a significant peak at 1602.90 (C=O), 2838.35 (-OH), 1469.81(C=C), 1274.03 (C-N) and the physical mixture has shown a peak at 1272.10 (C-N), 1602.90 (C=O), 1098.50 (-OH), 1188.19 (C-O), 1464.02 (C=C). Through the spectra results were that, lipid palmitic acid was compatible with the selected drug Valsartan.

Figure 1: IR spectrum of Valsartan

Figure 2: Infra-red spectrum of Valsartan + palmitic acid

Differential scanning calorimeter (DSC):

Compatibility studies between the drug and lipid was done using DSC. DSC thermogram of Valsartan, lipid and formulation are shown in Figure 3-4. DSC thermogram of lipid palmitic acid showed sharp peak. However, the endothermic peak of drug in the drug loaded nano particles has not appeared and it showed that the drug was dispersed uniformly in the amorphous state in the lipid matrix. Hence, the result of the thermogram clearly revealed that there is no physical interaction among the lipid and the VNLCs.

Figure 3: Differential Scanning Calorimetric Thermogram of Valsartan

Figure 4: DSC Thermogram of Valsartan Formulation

Formulation and optimization of Nano structured lipid carrier:

Melt Emulsification was used for the preparation of valsartan loaded nano lipid carrier. This is very simple and suitable method. The carrier capacity of lipid contacting to valsartan was examined by preparing six batches of VNLCs, by altering the concentration of lipid to a constant amount of drug and varying the concentration of surfactant (Table 3). During the study it was observed that the batch F1 1:9 ratios of solid lipid and liquid lipid with 0.5% w/w surfactant concentration, it was the considered the best batch based on particle size, entrapment efficiency and zeta potential reports.

Table 3: Valsartan loaded NLCs with different concentrations of lipid and surfactant

Formulation	Drug	Solid	Liquid Lipid	Surfactant
Code	Added (mg)	Lipid (w/w)	(w/v)	%(w/v)
F1	10	90	10	0.5
F2	10	90	10	1
F3	10	90	10	1.5
F4	10	70	30	0.5
F5	10	70	30	1
F6	10	70	30	1.5

Drug- Valsartan, *Solid Lipid- Palmitic acid, *Liquid Lipid- Soya oil, *Surfactant - Tween80

Determination of particle size and zeta potential:

The Particle size of Valsartan NLCs was found to be 125.3nm and the zeta potential was found to be -29mV. Poly dispersity index (PDI) values were found to be 0.310. As all the characterization results are within the limit so VL-NLCs Formulation-1is having nanoparticles.

Table 4: Zeta Potential report for VNLCs

Sl. No	Parameters	Results	Limits
1.	Particle size	125.3nm	≤ 200nm
2.	PDI	0.310	<30
3.	Zeta potential	-29.0mV	-2 to -5.98mV

Scanning electron microscopy (SEM)

The Size and uniformity of the Particles of the NLCs formulation is viewed by Scanning electron microscopy. SEM microphotographs of valsartan loaded nano structured lipid carrier revealed that the nano particles were not in spherical shape but it comes under nano size range having rough surface shown in Figure 5.

Figure 5: Scanning Electron Microscopy of VNLC-1 Formulation

Drug Loading and Entrapment Efficiency (EE):

The % EE for the optimized formulation was obtained as 88% ±0.05%. The High % Entrapment Efficiency value denotes that the drug is lipophilic in nature and has a high affinity to lipid matrix. Drug loading for optimized NLCs formulation was found to be 8%±0.05%.

In-vitro drug release studies:

The In-vitro drug release studies of the valsartan formulation are done in the two pH solution, intestinal and gastric pH. Valsartan is a very weakly acidic drug so it is rapidly absorbed in the small intestine. The solubility of valsartan is increased due to presence of lipid leading to formation of micelles. So in these studies the drug was solubilised in lipid matrix as it helps in enhancement of the absorption and bioavailability of the drug. For the NLCs loaded Valsartan the absorption takes place in the gastric lumen. Hence the absorption window of NLCs loaded with valsartan is broadened. Release of drug was examined for 12 hours in both buffer solutions. The percentage of content of drug release acquired in Phosphate buffer pH 6.8 was 70.1% whereas in HCL pH 1.2 buffer as lower as 16%.

Figure 6: Drug release profiles of F1 formulation and pure drug in pH 1.2

* (n=5) Average± S.D

Release kinetics:

The release kinetics data was found out for Valsartan loaded NLC formulation and it was putted into different kinetic models. Outcomes are mention in Table 5. The value of regression coefficient R2 determined which is given into the table. Based on the R2 values acquired for the release of drug in phosphate buffer pH 6.8 follow first order kinetics. Then (n>1) values derived from krosmeyer peppas equation and the value derived was n=0.647 which is <0.45, so it follows Fickian diffusion mechanism.

Table 5: Regression value for various kinetic models

Formulation	kinetic models	R² value	Slope(n)
Valsartan NLCs	ZERO	0.943	5.903
	HIGUCHI	0.985	22.344
	PEPPAS	0.997	0.647
	FIRST	0.9811	-0.008
	Hixson Crowell	0.972	0.134

The best linearity was obtained in First order plot for VNLC formulation which indicated that release from matrix as a square root of time dependent process.

Stability Study:

Aim of Stability study was to give the Stability of the product remains stable for the given period. The Particle size, Poly Dispersity Index and Zeta potential were determined (table 6) to assure that the formulation qualities and mass of drug into the product remain unaltered. The variation in the Particle size, Poly dispersity index and Entrapment Efficiency were found to be non-significant. At normal room temperature, the valsartan loaded NLCs were found to be stable for 7days and after 7 days clumps were observed. Therefore, Nano lipid Carriers suspension is more stable at cold conditions in comparison with room temperature. The stability study results showed that the formulation was greatly stable at refrigerator temperature.

Table6: Stability study data for optimized batch

Storage condition

(nm)

PDI

Zeta Potential

At the time of Preparation

125.3

0.310

-29mV

Refrigerator temperature (4°C)

(1 month)

125.9

0.313

-25mV

Histopathological study:

Histopathological study was done on the day 2, 12 and 24. In the figure 6, Control group showed medium inflammation, leukocyte movement followed by medium neovascularization. In Placebo group, animals were treated with palcebo and showed leukocyte movement, inflammation and epithelialization in comparison with that of control. In Placebo group, animals were treated with valsartan loaded NLC ointment, which showed that there was no inflammation andre-epithelialization in comparison with control and placebo.

Figure 6: Comparison of wound healing property of control, placebo and formulation at2, 12 and 24 days on rat.

CONCLUSION:

All the above investigations we conclude that melt emulsification method was suitable for producing valsartan loaded nano structured lipid carriers. Lipophilic drugs like Valsartan can be easily incorporated into the lipid matrix. VNLCs provide sustained drug release. The formulated Valsartan loaded nano structured lipid carriers showed better protection from diabetic wound healing compared to pure drug, increased bioavailability.

ABBREVIATIONS:

DM = Diabetes Mellitus

SLN = Solid lipid nanoparticles

NLC = Nano structured lipid carrier

FTIR = Fourier transformer infrared Spectroscopy

PS = Particle size

ZP = Zeta Potential

SEM = Scanning electron microscopy

DSC = Differential Scanning calorimetric

PDI = Poly dispersity index

EE = Entrapment Efficiency

STZ = Streptozotocin

CONFLICT OF INTEREST:

The authors declare that there are no conflicts of interest involved in this study. The authors alone are responsible for the content and writing of the paper.

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Received on 31.01.2019 Modified on 28.02.2019

Research J. Pharm. and Tech. 2019; 12(6):2922-2928.

DOI: 10.5958/0974-360X.2019.00492.X