INTRODUCTION:

The goal of any drug delivery system is to provide a therapeutic amount of drug to the target site in the body to promptly achieve and then maintain the desired drug concentration¹. An ideal drug delivery system (DDS) delivers drug at rate decided by the need of the body throughout the period of treatment and provides the active entity solely to the site of action³. There are various approaches in delivering a therapeutic substance to the target site, one such approach is liposome. Liposome, as carrier for drug is one such approach which can be used in a sustained controlled release fashion.

The range of techniques for the preparation of liposome offers a variety of opportunities to control drug administration issue. Liposome ensures the accurate delivery of small quantity of the potent drugs, reduced drug concentration at the site other than the target site and the protection of the labile compound before and after the administration.²

Liposomes are artificially prepared, spherical, self-enclosed microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of phospholipid molecule. Phospholipids, when dispersed in an aqueous environment at a concentration above their critical micelle concentration (CMC), tend to form these closed vesicles spontaneously and encapsulate some of the aqueous environment. The drug molecules can either be encapsulated in aqueous space or intercalated into the lipid bilayer.³

Fig 1: Structure of liposome

Stealth liposomes are surface modified liposomes in which polymer units are attached to the surface of liposomes. The liposomes are camouflaged to fool phagocytosis by ignoring them. They are called as “stealth” because of the fact that they increase circulation time of these liposomes by avoiding detection by our body’s immune system.⁴

Fig. No. 2: Structure of Stealth Liposomes

Stealth Liposome Manufacturing Technique⁵

There are three ways to modify a liposome surface with lipopolymers:

1. Incorporating an amphiphilic conjugate of the polymer during liposome formation (pre-insertion).

2. Inserting the polymer conjugate onto the surface of pre-formed liposomes (post-insertion).

3. Post-modification by chemically reacting a polymer to the exposed functionalities on the liposome surface.

A) Pre-insertion:

In this process, the lipopolymer is added to the lipid phase before the liposomes are formed by hydration with an aqueous phase. This is the most commonly used and easy technique for the formulation of stealth liposomes. The lipopolymer gets incorporated into the liposome structure, with a lipid portion inserted into the bilayer and the hydrophilic part point towards aqueous surfaces. The common draw backs are, (1) it requires an excess amount of lipopolymers, makes the extrusion process challenging (2) both the inner and outer sides of the lipid bilayer membrane are modified and occupy the valuable interior space in the vesicle (3) the internal lipopolymers (PEG-phospholipids) are susceptible to acid/base-catalyzed hydrolytic degradation in liposomes prepared for pH gradient-based active loading.

B) Post-insertion:

Lipopolymers are slowly added to the dilute suspension of the pre-formed liposomes at temperatures close to the transition temperature of the constituent lipids. In order to prevent the self-assembly of the amphiphilic lipopolymers, their concentration is maintained at a level lower than their critical micellar concentration. The advantage of the post-insertion method is that the lipopolymers modify only the outer surface of liposome, thereby keeping the internal space of the vesicle available for the accommodation of drugs or biomolecules.

C) Post-modification by chemical reaction:

The technique based on a chemical reaction between the polymer and liposome surface has been mostly used to modify liposomes for the purposes of targeted drug delivery, but not so much for enabling long-circulating liposomes.

MATERIALS AND METHODS:

Preformulation Study:

Identification of Drug:

The monograph of Ketoprofen signified that the substance under examination was intimately mixed with potassium bromide. FTIR spectrum of the sample was taken using potassium bromide pellet method. The spectrum of test specimen was recorded over the range from 4000cm^-1 to 500cm^-1 and compared with the corresponding USP reference standard⁶.

Organoleptic Evaluation:

Organoleptic properties of drug like color, appearance and odor was observed and recorded⁷.

Determination of Melting Point:

The melting point of drug was determined by capillary tube method. The drug was filled to capillary tube which has one end sealed. The filled capillary tube was placed inside the melting point apparatus and the temperature at which drug melted was noted⁸.

Determination of Solubility of Ketoprofen⁹

Solubility of Ketoprofen was checked in various solvents like water, phosphate buffer saline pH 7.4, methanol, dichloromethane, acetone and chloroform.

100mg of drug was accurately weighed and transferred into a stoppered tube containing 0.1ml of solvent. If completely dissolved, the drug is said to be very soluble. If insoluble, added 0.9ml of solvent to it and is said to be freely soluble on complete dissolution. Otherwise, added 2ml of solvent to the same. The drug, if completely dissolved in the solvent, then it is said to be soluble. If insoluble, further 7ml of solvent was added and observed to be sparingly soluble on complete dissolution. On further addition of 10ml of solvent it is said to be slightly soluble, if completely dissolved.

If it is not completely dissolved in the above solution, accurately weighed 1mg of drug and added 10ml of solvent. If the solvent dissolves the drug, it is said to be very slightly soluble¹⁰.

Analytical Method Used in the Determination of Ketoprofen:

UV spectrophotometry method was developed for the analysis of drug using double beam Systronics-2202 spectrophotometer.

Determination of λmax of Ketoprofen:

Absorption maximum of pure Ketoprofen was determined by dissolving Ketoprofen in phosphate buffer saline pH 7.4. A sample of 10µg/ml was prepared and scanned for maximum absorbance using UV Visible spectrophotometer in the range from 200 - 400nm using phosphate buffer saline pH 7.4 as blank¹¹.

Preparation of calibration curve of Ketoprofen¹²

10mg of ketoprofen was accurately weighed and transferred into 100ml volumetric flask. The drug was dissolved and made up to the volume with phosphate buffer saline pH 7.4. It was further diluted with same buffer to get concentration of 2, 4, 6, 8 and 10µg/ml. The absorbance of solution was measured spectrophotometrically at 259nm using buffer as blank. The absorbance values were plotted against concentration to obtain the standard graph.

Compatibility studies:

Excipients are any substance other than active or prodrug included in the manufacturing process or contained in the finished product¹³.

FTIR Study:

The IR spectra were recorded using FTIR spectrophotometer. The samples were prepared by mixing the drug and the excipients in 1:1 ratio and the mixtures were stored in closed containers for 1 month. FTIR spectrum of the samples was taken using potassium bromide pellet method. The physical mixtures of Ketoprofen and excipients were scanned in the wavelength region between 4000 and 500 cm^-1 and compared to check compatibility of drug with excipient¹⁴.

Preparation of Stealth Vesicular Dispersions:

1ml of 5%, 7.5% and 10% w/v of PEG 4000 polymeric aqueous solution was used for the preparation of stealth liposomes. Stealth liposomes were prepared by injecting 1ml of 5, 6 and 10% w/v of PEG 4000 to the vesicular dispersion of liposomes that was being stirred at 100 rpm slowly to ensure uniform coating of PEG around the vesicles.

Evaluation of Stealth Liposomes:

Optical Microscopy

The stealth liposomes prepared were observed under binocular compound microscope at 10X and 40X magnification for studying the shape and surface morphology.¹⁵

Particle Size and Polydispersity Index:

The mean particle size and particle size distribution of stealth liposome was determined by Malvern nano zeta sizer instrument. The vesicles after diluted with distilled water were considered for the measurement of size.¹⁶

Drug entrapment of stealth liposome ¹⁷

The %EE of the vesicles was determined using centrifugation technique. The vesicular dispersion was centrifuged for 20 min. Supernatant containing unentrapped drug was withdrawn and measured UV spectrophotometricaly at 259nm against phosphate buffer saline pH 7.4. The sediment also measured spectrophotometrically. All the determinations were made in triplicate. The amount of drug entrapped in liposomes was determined by:

T-C

%EE = ----------- X 100

Where,

T =Total amount of drug calculated in both supernatant and sediment. C =Drug in supernatant

Determination of pH of Vesicular Dispersion:

The pH of the stealth vesicular dispersion was measured by pH meter.

In Vitro Drug Release¹⁸

In vitro drug release was measured using Franz diffusion cell. 50mg ketoprofen containing liposome suspension was placed on one side of egg membrane in a vertical franz diffusion cell. Other side of membrane was in contact with the dissolution medium of 22ml of phosphate buffer saline of pH 7.4. Entire dissolution assembly was placed on a magnetic stirrer at temperature of 37°C. Aliquots of dissolution medium was withdrawn at different time intervals for 8hr. Drug concentration in the dissolution medium were determined by UV spectrophotometry at 259nm.

Zeta Potential¹⁹

Zeta potential of the stealth liposomal formulation was determined using Zeta sizer. 1ml of stealth liposome was diluted with water and the sample taken in a clear and cleaned cuvette was placed inside the sample holder for measurement of size.

TEM:

A drop of stealth vesicular dispersion was applied on a carbon film-covered copper grid. Excess dispersion was blotted from the grid with filter paper to form a thin film specimen. The sample was then examined under TEM.

Release Kinetics¹⁹

Kinetic study was carried out by fitting the in vitro drug release data into Zero order, First order, Higuchi model, Hixon-Crowell Cube Root Law model and Korsmeyer-peppas models. The best outfit model was confirmed by the value of R²which is near to 1.

Stability Studies:

The stability studies were carried out as per ICH guidelines. A sufficient quantity of optimized stealth liposome and liposome was kept in sealed glass vials. It was subjected to stability study at 2-8°C for 3 months. The physical stability of liposome and stealth liposome was inspected at initial and one month by checking particle size, % drug content, zeta potential, surface pH and in vitro drug release.

RESULTS AND DISCUSSION:

Preformulation Study:

Identification of Drug:

Fig No 3: FTIR spectrum of Ketoprofen

The sample spectrum was compared with the reference spectrum. There were no significant changes in the functional groups. The frequency of observed functional groups C=O, C=C and O-H are within the standard limits. The finger print region has not changed significantly. So the drug was identified as Ketoprofen.

Organoleptic Evaluation:

The Colour was found to be White or almost white crystalline powder with Odorless or almost odorless

Determination of Melting Point:

The standard melting point of Ketoprofen is in the range of 94-97°C.

Determination of Solubility of Drug:

The solubility was determined by dissolving the drug in different solvents like water, phosphate buffer saline 7.4, methanol, chloroform and acetone. It was very slightly soluble in water, freely soluble in methanol and soluble in acetone, phosphate buffer saline 7.4 and chloroform.

Analytical Method for The Determination of Ketoprofen

Determination of λ max of Ketoprofen in phosphate buffer saline pH 7.4

The 10µg/ml sample was prepared and scanned between 200 to 400nm. The drug showed maximum absorption at 259nm. So, the λ max of Ketoprofen was found to be 259nm.

Preparation of Calibration Curve of Ketoprofen in Phosphate Buffer Saline 7.4

Table No. 1: Standard calibration curve data of Ketoprofen in buffer

Sl. No.	Concentration (µg/ml)	Absorbance
1	0	0.00
2	2	0.182
3	4	0.322
4	6	0.458
5	8	0.584
6	10	0.729

FTIR:

The FTIR spectrum of Ketoprofen exhibited peak signals at 1699 cm-1, 1655 cm-1, 1597 cm-1 and 2978 cm-1 due to stretching vibrations of C=O stretching of acid, C=O stretching of ketone, aromatic C=C stretching and O-H stretching of acid. There were no significant changes in the frequency of the functional groups of Ketoprofen. So, the drug was compatible with Soya lecithin, Cholesterol and PEG 4000.

Evaluation of ketoprofen stealth liposomes:

Optical Microscopy:

Fig. No. 4: Microscopic view of Stealth liposome

The microscopic view of stealth liposomes of Ketoprofen was shown in the Fig. No.4. The microscopic images obtained under an optical microscope confirmed the coating of PEG around the liposome. It was observed that the coated liposomes showed no aggregation.

Particle Size and Polydispersity Index:

Particle size of the stealth liposomal suspension was measured in Malvern nano zetasizer instrument. The size of stealth liposomes was slightly enhanced compared to conventional liposomes due to PEG 4000 forming thick surface layer on surface of the liposome vesicles. As the concentration of PEG 4000 increases, size of stealth liposome also increases.

Drug Entrapment Studies of Stealth Liposome:

The drug entrapment of stealth liposome was given in the Table No. 18. It was observed that the % drug entrapment of stealth liposomes were similar as that of liposomes. It was revealed that there was no drug loss while coating of liposomes using PEG 4000.

Determination of pH of stealth liposomes:

The pH of stealth liposomes was also found to be around pH 7. The PEG 4000 does not change the pH of the stealth vesicular suspensions and are suitable for parenteral drug delivery.

In Vitro Drug Release Studies of Stealth Liposomes

Table No. 2: In vitro drug release of Stealth Liposome of Ketoprofen

Time (hr)	%CDR
Time (hr)	SL1	SL2	SL3
0	0	0	0
1	20.12	17.80	15.21
2	28.21	24.60	19.35
3	35.17	30.45	24.82
4	41.45	37.88	30.61
5	49.28	42.70	39.24
6	55.80	49.6	46.85
7	61.32	56.12	54.23
8	69.70	65.91	60.12
12	74.30	73.10	71.98
16	80.17	79.36	79.17
20	84.60	81.67	86.56
24	90.03	92.58	94.03

The results of the in vitro drug release studies revealed that with increasing concentration of PEG 4000 the stealth liposomes showed more sustained drug release profile. The result showed that the stealth liposomes had the ability to extend the release of Ketoprofen for duration of 24 hr [Table No. 2]. The maximum drug diffused at the maximum time for conventional liposome was 86.80% for 8hr and for stealth liposomes it was 94.03% for 24hrs. This result indicated that the stealth liposomes showed sustained release profile than conventional liposomes.

Zeta Potential:

The formulations were subjected to zeta potential evaluation. The zeta potential of stealth liposomes was found to be -42 mV. The value of zeta potential showed that stealth liposomes had sufficient charge to inhibit aggregation of liposomes due to electric repulsion. When compared to conventional liposomes, stealth liposomes showed more zeta potential values due to the effect of PEG 4000.

TEM:

TEM of Ketoprofen loaded stealth liposome is given in Fig. No.5. TEM confirmed the presence of PEG coating around the liposomes.

Fig No 5: TEM of Stealth Liposomes

Kinetic Study of The Liposome and Stealth Liposome:

To determine the release mechanism that gives the best description to the pattern of drug release, the in vitro release data were fitted to zero-order, first-order, Hixson Crowell equation and Higuchi matrix model. The release data were also kinetically analyzed using the Korsmeyer– Peppas model.

The release kinetics data indicates that the release of drug from stealth liposome best fits to zero order release kinetics. R²values of zero order kinetic equations were found to be close to unity indicating that the release from the films was not dependent on the concentration of drug present in the formulation.

The data was fitted with Higuchi equation which gave almost a linear plot with highest R²indicating the mechanism of drug release was diffusion. The dissolution data was also plotted in accordance with Hixon- crowell cube root law.

To determine whether fickian or non-fickian diffusion existed, data was analyzed using the Korsmeyer Peppas equation. The n value determined lies between 0.5 and 1.0 indicates it follows non-fickian diffusion. These observations showed that mechanism of drug release for all the formulations were non-fickian diffusion following Higuchi model of drug release. The formulation stealth liposome showed better results when compared to other formulations.

Stability Study:

The selected formulations of liposome and stealth liposome were subjected to stability study. Initial and one month studies were done and results were mentioned in Tables 32,33. There were no significant changes in the particle size, zeta potential, % drug content, surface pH and in vitro drug release for stealth liposomes when compared to that of liposomes. So, stealth liposomes were more stable than liposomes. The stability studies will be continued further up to 3 months.

CONCLUSION:

Stealth liposome is a novel dosage form that has prolonged release than conventional oral dosage forms and improves the stability of the drug. In certain situations like arthritis, sustained drug release can be achieved through parenteral administration of stealth liposomes. The liposome was prepared by thin film hydration method with different concentration of cholesterol. The concentration of soyalecithin, methanol, chloroform and phosphate buffer saline pH 7.4 were made constant. The best formulated liposomes were coated with 10% PEG to form stealth liposomes.

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Received on 07.03.2020 Modified on 13.04.2020

Research J. Pharm. and Tech 2021; 14(3):1313-1318.

DOI: 10.5958/0974-360X.2021.00233.X