The method by which the drug is delivered can have a significant effect on its efficacy. Certain drugs have an optimum concentration range within which maximum benefit is derived, and concentrations above or below this range can be toxic or produce no therapeutic benefit at all¹.

On the other hand, the very slow progress in the efficacy of the treatment of severe diseases, has suggested a growing need for a multifaceted approach to the delivery of therapeutics to targets in tissues. From this, new ideas on controlling the pharmacokinetics, pharmacodynamics, non-specific toxicity, immunogenicity, biorecognition and efficacy of drugs were developed. These new approaches, often called drug delivery systems (DDS), which are based on interdisciplinary approaches that combine polymer science, pharmaceutics, bioconjugate chemistry and molecular biology. An ideal drug delivery system (DDS) delivers the drug at a rate decided by the need of the body throughout the period of treatment and provides the active entity solely to the site of action². There are various approaches in delivering a therapeutic substance to the target site and one such approach is liposome. Liposome, as a 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 administration².

1.1 Liposomes:

Liposomes were first described by British hematologist Dr Alec D Bangham FRS at the Babraham Institute, in Cambridge. The name liposome is derived from two Greek words: 'Lipos' which means fat and 'Soma' which means body. A liposome is a small bubble (vesicle), made out of the same material as a cell membrane. They are usually made up of phospholipids, which are molecules that comprise a tail and a head group. The head is hydrophobic, whereas the tail is made up of a long hydrocarbon chain and is hydrophilic^3,4.

1.2 Stealth Liposome:

Stealth liposome is a long circulating liposome which are obtained by modulating the lipid composition, size, and charge of the vesicle. 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 system⁵.

Fig No 1: Structure of Stealth liposome

1.2.1 Manufacturing techniques of Stealth: Liposome^6,7:

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.

1.2.2 Antibiotic as Stealth Liposome:

Bacteria are living things that they have only one cell. Under a microscope, they look like balls, rods, or spirals. Infectious bacteria reproduce quickly in body. Many give off chemicals called toxins which can damage tissues. The severity of bacterial infection is based largely on the type of bacteria involved, the general health of the affected individual and other factors that can either enhance or minimize infection.

Bacterial infection is mainly treated using antibiotics. Fluroquinolones are large group of broad-spectrum bactericides which are effective against both gram negative and gram positive. Quinolones inhibit the bacterial DNA gyrase or the topoisomerase enzymes, thereby inhibiting DNA replication and transcription⁸.

Levofloxacin is a fluroquinolone antibiotic which are used to treat number of bacterial infections including acute bacterial sinusitis, pneumonia, urinary tract infections, chronic prostatitis and some type of gastroenteritis. In this study Levofloxacin is formulated as stealth liposome to increase the efficacy and reduce the toxicity and target the organs RES and thus prolonging its circulation time thereby reducing clearance. Two polymers are used to prepare Levofloxacin as stealth liposome i.e., PEG and PVP and their effects are compared.

2. MATERIALS AND METHODS:

2.1 Materials:

Levofloxacin was obtained from Tablet Private limited Chennai. The soyalecithin and PVP K 30 was obtained from Chemco Rajasthan. The cholesterol was received from fortune chemicals Malapuram. The methanol and PEG 4000 was obtained from Nice chemicals Kottayam. The Chloroform was obtained from spectrum chemicals, Kochi.

2.2 Methodology:

2.2.1 Preformulation Studies:

2.2.1.1 Identification of Drug:

The monograph of levofloxacin 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 IP reference standard⁹.

2.2.1.2 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 noted¹⁰.

2.2.1.3 Determination of Solubility of levofloxacin:

Solubility of levofloxacin 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 1 mg of drug and added 10ml of solvent. If the solvent dissolves the drug, it is said to be very slightly soluble¹¹.

2.2.1.4 Determination of λmax of levofloxacin:

Standard stock solution of levofloxacin was prepared by dissolving 50mg of drug in 100ml methanol. This is then further diluted with methanol to get standard solution concentration of 10mcg/ml. The resulting solution was then scanned between 200-400nm.

2.2.1.5 Preparation of calibration curve of levofloxacin:

Accurately weighed 100mg of levofloxacin was dissolved in 100ml of pᴴ 7.4 phosphate buffer to give a solution of 1mg/ml (1000mcg/ml). From this first stock solution 1ml was taken and diluted to 100ml using pᴴ 7.4 buffer to get a solution 10mcg/ml. This serve as the second stock solution into a series of 10ml volumetric flask, aliquots of second standard solution 2ml, 4ml, 6ml, 8ml, 10ml was added and volume made up to 10ml using pᴴ 7.4 buffer. The absorbance values were plotted against concentration to obtain the standard graph¹².

2.2.1.6 FTIR Study¹²:

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 levofloxacin and excipients were scanned in the wavelength region between 4000 and 400 cm-1 and compared to check compatibility of drug with excipient.

2.2.1.7 DSC¹²:

DSC study was carried out using DSC-60 instrument to check the compatibility of ingredients. The samples were prepared by mixing the drug and the excipients in 1:1 ratio. Accurately weighed samples were sealed in aluminum pans and analyzed in an inert atmosphere of nitrogen at flow rate of 25ml/min. A temperature range of 0°C to 300°C was used, and the heating rate was 10°C/min. DSC thermograms of pure drugs and physical mixtures of drugs and excipients were studied for their interactions.

2.3 Preparation of Stealth Vesicular Dispersions:

1ml of 5%, 7.5% and 10% w/v of PEG 4000polymeric aqueous solution and 1ml of 0.5%, 1% and 2% w/v of PVP K30 polymeric aqueous solution was used for the preparation of stealth liposomes. Stealth liposomes were prepared by injecting 1ml of 5, 7.5 and 10% w/v of PEG 4000 and 1ml of 0.5,1and 2% w/v of PVP K 30 to the vesicular dispersion of liposomes that was being stirred at 100rpm slowly to ensure uniform coating of PEG and PVP around the vesicles.

2.4 Evaluation of Stealth Liposomes^13,14,15:

2.4.1 Optical Microscopy:

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

2.4.2 Particle Size and Polydispersity Index:

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

2.4.3 Drug entrapment of stealth liposome:

The %EE of the vesicles was determined using centrifugation technique. The vesicular dispersion was centrifuged for 20 min. Supernatant containing entrapped drug was withdrawn and measured UV spectrophotometrically at 293nm 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:

%𝐸𝐸=𝑇_𝐶𝐶×100

where,

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

C =Drug in supernatant

2.4.4 In Vitro Drug Release:

In vitro drug release was measured using Franz diffusion cell. 50mg levofloxacin 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 293nm.

2.4.5 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.

3. RESULTS AND DISCUSSIONS:

3.1 Preformulation Study:

3.1.1 Identification of Drug:

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-H, O-H and F are within the standard limits. The finger print region has not changed significantly. So, the drug was identified as Levofloxacin.

3.1.2 Determination of melting point:

The standard melting point of Levofloxacin is in the range of 225-227°C. The observed value was 225°C which is within the range as per official monograph. So, the drug was identified as Levofloxacin.

3.1.3 Determination of Solubility of Drug:

The drug was very slightly soluble in water, freely soluble in methanol and soluble in acetone, phosphate buffer saline 7.4 and chloroform.

3.1.4 Determination of λ max of Levofloxacin 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 293nm. So, the λ max of Levofloxacin was found to be 293nm.

3.1.5 Preparation of Calibration Curve of Levofloxacin in Phosphate Buffer Saline 7.4

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

Sl No	Concentration (μg/ml)	Absorbance
1	0	0.00
2	2	0.100
3	4	0.182
4	6	0.279
5	8	0.377
6	10	0.461

Various concentrations [2, 4, 6, 8, 10 μg/ml] of the drug were prepared as shown in Table No. 1 and the standard graph was plotted. The y- intercept and R2 values were found to be 0.046, 0.998 respectively.

The FTIR spectrum of Levofloxacin exhibited peak signals at 3260 cm-1, 2935 cm-1, 1724.39 cm-1, 1291.39 cm-1 and 1089.92 cm-1 due to carboxylic acid, alkane group stretching, C=O stretching of carbonyl group, stretching of amines and presence of halogen. There were no significant changes in the frequency of the functional groups of Levofloxacin. So, the drug was compatible with Soya lecithin, Cholesterol, PEG 4000, and PVP K30.

3.1.7 DSC STUDIES:

The DSC studies were carried out for drug [Levofloxacin] and drug-excipients physical mixtures. The recorded DSC thermograms showed the profile of Levofloxacin with melting point at 227.96ºC. Drug with excipients, Soya lecithin, Cholesterol and PEG 4000 showed melting point at 227.9ºC and 225.1ºC. The melting point remains almost the same, indicated that the drug and excipients are compatible with each other.

3.2 Evaluation of Levofloxacin Liposomes:

3.2.1 Optical Microscopy:

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

a) b)

Fig. No. 4: Microscopic view of Stealth liposome

a) PEG b) PVP:

3.2.2 Particle Size and Polydispersity Index Measurement:

Particle size of the stealth liposomal suspension was measured in Malvern Nano zeta sizer instrument. The vesicle size of Levofloxacin loaded stealth liposomes was shown in the Table No. 2. The size of stealth liposomes was slightly enhanced compared to conventional liposomes due to PEG 4000 and PVP forming thick surface layer on surface of the liposome vesicles. As the concentration of PEG 4000 and PVP increases, size of stealth liposome also increases.

Table No 2: Particle size and polydispersity of stealth liposome

Formulation Code	Particle size (nm)	PDI
SL1	209	0.25
SL2	220	0.25
SL3	245	0.24
SLA	220	0.31
SLB	230	0.25
SLC	250	0.27

3.2.3 Drug Entrapment Studies of Stealth Liposome:

The drug entrapment of stealth liposome was observed that the % drug entrapment of stealth liposomes was similar as that of liposomes. It was revealed that there was no drug loss while coating of liposomes using PEG 4000 and PVP.

3.2.5 In Vitro Drug Release Studies of Stealth Liposomes:

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 than that of PVP K30. The result showed that the stealth liposomes with PEG 4000 had the ability to extend the release of Levofloxacin for duration of 24 hr than PVP K30. The maximum drug diffused at the maximum time for conventional liposome was 85.1% for 8hr and for stealth liposomes it was 93.7% (PEG4000) for 24hrs. This result indicated that the stealth liposomes with PEG 4000 showed sustained release profile than conventional liposomes.

3.2.6 TEM:

TEM confirmed the presence of PEG coating around the liposomes.

3.2.7 Kinetic Study of the Liposome and Stealth Liposome

The release kinetics data indicates that the release of drug from stealth liposome best fits to zero order release kinetics. R2 values 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 R2 indicating 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.

3.2.8 Stability Study:

The selected formulations of liposome and stealth liposome were subjected to stability study. Initial and three-month studies were done and results were mentioned in Tables 32, 33, 34, 35. There were no significant changes in the particle size, zeta potential, % drug content, surface pH and in vitro drug release for stealth liposomes at 4±1ºC when compared to that of liposomes stored at 40ºC. So, stealth liposomes were more stable at 4±1ºC temperature. The stability studies will be continued further up to 6 months.

4. SUMMARY:

Stealth liposome is a novel dosage form that has prolonged release than conventional oral dosage forms and improves the stability of the drug. The liposome was prepared by thin film hydration method with different concentration of cholesterol. The concentration of soya lecithin, methanol, chloroform and phosphate buffer saline pH 7.4 were made constant. The best formulated liposomes were coated with 10% PEG and 1% PVP K30 to form stealth liposomes and compared the study. The drug was identified by comparing the sample spectrum with that of the reference spectrum. In FTIR spectra, Levofloxacin exhibited peak signals at 1724 cm-1 due to C=O stretching, 2935 cm-1 due to -CH3 stretching, 3260 cm-1 due to Carboxylic acid and 1294cm-1 due to stretching of amines, 1089.92 due to presence of halogen group. The DSC thermogram indicated that melting point of drug was 227.96°C, which remained almost same in the presence of polymers. FTIR and DSC studies revealed that the drug and polymers were compatible without any significant changes in the nature of drug.

The stealth liposomes were also evaluated for surface morphology, particle size, polydispersity index, % EE, surface pH, in vitro drug release and zeta potential. Images obtained under an optical microscope confirmed the coating of PEG and PVP around the liposome. Particle size and polydispersity index of stealth liposome was found to be 244nm and 0.24 respectively and with that of PVP it was found to be 230nm and 0.25 respectively. The size of stealth liposomes was slightly enhanced compared to conventional liposomes due to the polymers forming thick surface layer on surface of the vesicles. The in vitro drug release indicated that the stealth liposomes with PEG 4000 showed more sustained release profile than conventional liposomes as well as with PVP K30. The kinetic data analysis revealed that formulated stealth liposomes best fit to Higuchi model and follows zero order kinetics. The mechanism of drug release from the stealth liposomes followed non-fickian diffusion. The stability studies of the optimized stealth liposome were investigated as per ICH guidelines.

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Received on 30.01.2020 Modified on 27.03.2020

Research J. Pharm. and Tech 2021; 14(3):1493-1498.

DOI: 10.5958/0974-360X.2021.00265.1

Formulation Code	Zero Order R²	First Order R²	Higuchi Model R²	Hixon-Crowell Model R²	Korsmeyer- Peppas N
SL1	0.874	0.980	0.981	0.965	0.664
SL2	0.773	0.961	0.940	0.914	0.604
SL3	0.984	0.884	0.987	0.977	0.729
SLA	0.906	0.972	0.953	0.990	0.616
SLB	0.853	0.977	0.974	0.949	0.655
SLC	0.792	0.949	0.952	0.906	0.623