Lyposomal as Drug Delivery System

Abstract

The liposomes were the first Nano medicine to be accepted for clinical use. They are the spherical vesicles that possess mid empty aqueous space, which is encircled by a phospholipid’s bilayer. Liposomes have immense capability to prevent the degradation of drugs, reduce side effects and are thus increasingly used for targeted drug delivery. The drugs can either be incorporated inside the aqueous space (hydrophilic drugs) or inside the phospholipid’s bilayer (hydrophobic drugs) of liposomes for the targeted drug delivery. Considering the importance of liposomes as a drug delivery system, the present review paper tries to look into its details. The entire paper is classified into six parts. The first part is introductory. The second part discusses the classification of liposomes. In the third segment, the structural components of liposomes are detailed. The fourth portion of the paper talks about methods of preparation of liposomes. In the fifth segment, the characterization of liposomes is discussed. The sixth part discusses the application of liposomes and the last part is given to concluding observation. Literature shows distinct types of liposomes, categorized based on size, number of lipid layers, composition and preparation method. They are recently used for various nanoscale drugs formulation and a piece of concrete evidence was seen recently in recommended drug for black fungus i.e., Liposomal Amphotericin B. Although, their development and application are remaining the challenge due to costly and tedious processes involved in their production and development. Therefore, further research and development are required to perform to overcomes these challenges.

Keywords

Liposome, characterization comparison to conventional

Introduction

2) Based on Method of Preparation

Advantages of liposomes:-

• Liposomes  are  biocompatible,  completely  biodegradable,  non- toxic, flexible, and nonimmunogenic for systemic and nonsystemic administrations.
• Provide controlled and sustained release.
• It carries both water and lipid-soluble drugs.
• The drug can be stabilized from oxidation
• Targeted drug delivery or site – specific drug delivery

Disadvantages of liposome:-

• Less stability
• Low solubility.
• Short half-life.
• High production cost.
• Leakage and fusion of encapsulated drug/molecules.

1. Technique of passive loading

1. Mechanical dispersion

Lipid Hydration Method

This is perhaps the most well-known and widely utilized approach to make MLVs. The round bottomed flask can be utilised for its formulation. The procedure entails forming a thin coating by dehydrating the lipophilic mixture and on hydrating the membrane by introducing an aqueous buffer as well as thoroughly mixed the dispersion. The hydration stage is performed at a temperature higher than the gel -liquid-crystalline (Lβ→Lα) transition temperatures of the lipid or just above the transition temperature of the lipid mixture's maximal melting part. The pharmaceuticals somehow been enclosed are introduced to an aqueous buffer or into an organic solvent comprising lipids solvents depending on their solubilities. The method's drawbacks include low internal volume, lower efficiency of encapsulation and differing size. Diethyl ether overcomes the poor encapsulation ability of lipids by hydrating them in the presence of immiscible organic solvents like petroleum ether. After that, sonication is used to emulsify the mixture. By removing organic layer by passing nitrogen, MLVs are developed.

Micro emulsification

This technique is used in commercial scales to prepare small lipid vesicles. For the preparation of tiny vesicles somewhat from concentrated lipid suspension, an apparatus called a micro fluidizer is used. As a suspension of large MLVs, the lipids should be taken into the fluidizer. This machine pumps the fluid into a 5μm panel at very high pressure.17 Then a long micro channel is forced, which drives two fluid streams colliding at very high velocity at the right angle together. Micro emulsion can be developed for biological applications by regulating rotation speeds from 20 to 200.

Sonication 

This technique decreases the size of the vesicles and provides energy to lipid mixture. This can be accomplished by irradiating the MLV with ultrasonic waves. Sonication can be done in two different ways (i) using bath sonicator (ii) using probe sonicator. The probe sonicator is often used for suspensions that demand a lot of energy in a little amount of volume.19-21. For substantial volumes of dilute lipids, the bath sonicator is used. The primary disadvantages of this technique are its poor internal encapsulation efficacy, phospholipid disintegration, elimination of particularly huge molecules, metal impurities from the probe tip, and the coexistence of MLV and SUV.

French Pressure Cell Method

MLV is extruded via a tiny hole at 20,000 pressure and 4°C during the process. In comparison to sonication techniques, the method offers numerous advantages. The approach is simple, quick, and repeatable, yet it entails carrying hazardous materials with care. Liposomes produced as a result are considerably bigger than those produced by sonicated SUVs. The drawbacks comprise difficulties in attaining temperature and the working volumes are comparatively smaller (a maximum of 50 mL).

Membrane extrusion

A heterogeneous liposomal suspension is processed via a polymer sieve with a web like fabrication that develops a tortuous-path capillary pore, an interconnected network having not less than 100 micron thick membrane. The liposomes that were treated had a small distribution of size and a chosen mean size of less than about 0.4 microns. Both LUVs and MLVs can be processed using this approach.

Dried reconstituted vesicles

In this approach, liposomes are combined with lyophilized protein or introduced to an aqueous solution containing pharmaceutical and after that dehydrated.

Freeze-Thaw Method

In this procedure, SUVs are rapidly frozen then gradually thawed. Aggregated compounds are dispersed to LUV by sonication. The formation of unilamellar vesicles in the freezing and thawing processes is a result of SUV17. This form of fusion is highly hindered by enhancing the medium's ionic strength and by enhancing the amount of phospholipids. This technique was used to achieve entrapment efficiency of about 20 to 30%.

b) Solvent dispersion

Ethanol Injection Method

This is simple technique. In this approach, an ethanol lipid solution is quickly incorporated directly through a fine needle into an excess of saline or other aqueous media. In water, ethanol is dissolved and phospholipid molecules are uniformly distributed through the medium. The main disadvantage of this approach is that the particles can have a heterogeneous particles size (30-110 nm). Another major downside is that it is difficult to remove all ethanol, which may lead to the formation of azeotrope with water.

Double emulsification

In this approach, a primary emulsion is produced by mixing the therapeutics in an aqueous phase (w1), which is further emulsified to produce a primary w1/o emulsion in an organic polymer solvent. The w1/o/w2 double emulsion is created by combining the foremost emulsion with an emulsifier containing aqueous solution (w2). In the aqueous continuous stage, when the solvent is removed, microspheres are left behind that are further separated by filtering/centrifuging.

Reverse-phase evaporation

In an Erlenmeyer bulb the lipid mixture is taken and the solvent is removed at decreased pressure using a rotary evaporator. The lipids are disintegrated in the organic phase once the nitrogen has been removed. This phase will see the development of reverse phase vesicles. The standard solvents used are isopropyl ether and diethyl ether. Just after the lipids have been re-dispersed among this phase, an aqueous phase containing the pharmaceutical to be encapsulated is added. The all-aqueous systems are sonicated till the combination generates a distinct one-phase dispersion and the system is preserved under nitrogen pressure at all times. The combination is further transferred to arotovap and organic solvent removal is carried out until a gel is developed followed by non-encapsulated substance removal.. Reverse-phase evaporation vesicles are the resulting liposomes that emerge from this process. The method's major benefit is its extremely high encapsulation efficiency.

c) Detergent removal

At their optimum micellar concentrations, detergents solubilize lipids. The micelles became more enriched in phospholipids when the detergent was eliminated using dialysis and eventually fused to create LUVs. High reproducibility as well as the development of homogenous liposome populations are two of the positive effects of the detergent dialysis approach. The primary downside of the technique is that detergent residues are retained.

 2. Active loading technique

Proliposome

In this technique lipid and drug were coated onto a soluble carrier to develop free flowing granular component in pro liposome which when hydrated forms an isotonic liposomal solution. The pro-liposome strategic approach will offer an incentive for large-scale, cost-effective manufacturing of liposomes incorporating lipophilic drugs in particular.

Lyophilization

The separation of water from substances at highly reduced pressure in the frozen state is termed cryodesiccation (freeze drying). The technique is extensively employed to dry thermolabile materials that can be ruined by heat-drying. With regard to liposomal stability, this procedure serves as an important tool for resolving long-term stability issues. During the freeze-drying and reconstitution process, leakage of trapped materials can take place.

SIZING OF LIPOSOME

The size of the liposome seems to have a significant impact on their fate or the purpose for which it may be employed. The functional viability and uniformity of phospholipids bilayer are required for therapeutic uses of liposomes. Therefore, with particle size distribution within a certain size range, the liposome processing process must be predictable and reproducible. The typical methods of sizing liposomes are sequential extrusion, gel chromatography and sonification.

However, these methods have the following disadvantages in the long run:

1. 1. It's difficult to keep oxygen out, which leads to a per oxidation process.
   2. Metal particles are shed by titanium probes, resulting in toxicity.
   3. They will develop aerosols that restrict them from the use of such agents.

These issues are primarily due to the probe's sonication, but using the bath sonication will eliminate these problems.

CHARACTERIZATION OF LIPOSOMES

The liposome must be characterized after preparation and before use in immunoassays. The assessment may be categorized into three broad categories: physical, chemical and biological methods.

The physical methods require different criteria, such as shape, size, surface features, lamellarity phase behaviors and drug release profile.

Chemical characterization refers to research that have determined the purity and efficiency of different liposomal components.

Biological characterization aids in the analysis of a formulation's effectiveness and viability for in vivo pharmacological intervention.

1. Visual Appearance

The appearance of the liposomal suspension can vary from transparent to milky depending on the particle size and composition. If the turbidity has a bluish shade, the samples are homogeneous; non-liposomal dispersion occurs in a flat, grey color and is most usually a distributed inverse hexagonal phase or dispersed micro crystallites. In addition to contamination of larger particles, an optical microscope can detect liposomes larger than 0.3 μm.

2. Determination of Liposomal Size

The size distribution is of primary importance when liposomes are intended for inhalation or parenteral administration, as it regulates the in vivo fate of liposomes along with the encapsulated drug molecules. Liposome is usually measured by dynamic light scattering. For this method, liposomes with a relatively homogeneous distribution of size are reliable. Gel exclusion chromatography is a direct way of detecting a completely hydrodynamic radius. In the 30-300nm size range, sephacryl-S100 will separate liposomes. SUVs can be isolated from micelles by sepharose columns -4B and -2B.

3. Determination of lamellarity

The determination of lamellarity is important in defining the liposome structure and it’s in vivo efficiency. The efficacy of encapsulation and the kinetics of drug release are greatly affected by the amount of lipid bilayers in the liposome. Lamellarity influences the uptake of liposomes and intercellular fate. The Liposomal lamellarity varies greatly depending on lipid selection and methods of preparation. Electron microscopy or spectroscopic techniques may calculate the lamellarity of liposomes. The NMR liposome spectrum is most commonly reported with and without the addition of a paramagnetic agent that changes or bleaches the signal of the observed nuclei detected on the outer liposome surface.

4. Liposome Stability

Stability is an essential factor to consider when using liposomal drug delivery systems. The processing system and storage conditions have an impact on the physical stability and chemical purity of a drug molecule. The liposomal stability of the drug molecules regulates their therapeutic function. Size, size distribution, composition and drug retention are correlated with the physical integrity of liposomes, whereas Chemical equilibrium struggles with phospholipid oxidation and hydrolysis, as well as drug degradation. A safe liposomal medicinal product has a qualified physical, chemical and microbial stability that guarantees the integrity of the products during its storage time. Therefore, a given stability study protocol emphasizes its significance in deciding the products physical and chemical integrity having a well-established methods for development, characterization, effectiveness and stability testing.

5. Entrapped Volume

This is an important parameter that regulates vesicle morphology. The entrapped liposome volume (in μL/mg phospholipids) can also be deduced from measurements of the cumulative amount of solute trapped within liposomes, ensuring that the concentration of solute within liposomes in the aqueous medium is the same after isolation from the untrapped material. During the drying down process, water from the inner compartment may be lost in two phases of preparation to eliminate organic solvent.

This can be calculated by a given formula

6. Surface Charge

The nature and density of charge on the liposome surface will regulate the lipid-cell interaction. Liposomes are typically prepared by means of charge imparting/constituting lipids and hence the charge on the surface of the vesicle is examined. In general, two procedures are employed to analyse the charge: free flow electrophoresis and zeta potential testing.

APPLICATION OF LIPOSOME

Liposome research has progressed dramatically in the previous 30 years. A large variety of liposomes of variable sizes, phospholipid constituents, cholesterol constituents and surface morphologies can now be developed suitable for a wide range of applications.

The liver and spleen may be targeted using the liposome carrier, and tomography can easily differentiate between malignant and non - malignant tissue. Liposome has a great use in the case of the transdermal drug delivery system. In the treatment of tumor cells the liposomal drug delivery mechanism contributes to a decrease in the toxic effect and increases drug efficacy. Liposomes are targeted at the region of operation by binding a fragment of amino acid to particular cell receptors.

DNA vaccination and increased gene therapy success is only a couple of the latest liposomal treatment applications. Many sorts of drug administration implementations have been suggested for the liposomal drug delivery, some of which are mentioned below:

1. Enhance drug solubilisation (Cyclosporins, Minoxidil, Amphotericin-B and Paclitaxels)
2. Drug molecules that are sensitive are protected (Cytosine arabinosa, Ribozymes, DNA, Anti-sense oligo nucleotides and RNA)
3. Enhance intracellular uptake (Anti-microbial, Anti tumor and anti-viral drugs)
4. Modification in pharmacokinetics and bio distribution(prolonged or sustained released drugs with short circulatory half-life)

A. Liposome for Respiratory Drug Delivery System

Liposomes are typically used in many forms of respiratory illnesses. It is possible to formulate liposomal aerosols to develop prolonged release, avoid local inflammation, minimize side effects and improve stability throughout the vast aqueous core.

Numerous injectable liposome-based drugs, including ambisome, Fungisome and Myocet, are now available in the market. To be successful, the delivery mechanism of liposomal drugs to the lungs depends on lipid composition, charge, size, drug & lipid ratio and delivery techniques. The emerging application of liposomes for DNA transmission to the lungs indicating that a better knowledge of their usage in macromolecules administration by inhalation is now developing. All of this new expertise can be used to improve liposome-based protein formulations. The liquid or dried form is utilized for liposome inhalation and the pharmaceutical is released during nebulization. Milling or sprays drying have been used to develop drug powder liposomes.

B. Liposome in Eye Disorders

Liposomes have long been utilised to treat both the anterior and posterior regions of the eye. Dryness, keratitis, proliferative vitreoretinopathy, endophthalmitis, and corneal graft rejection are all eye diseases. In developing nations, retinal abnormalities constitute the single biggest cause of blindness. Liposomes are employed as a genetic transfection vector and a carrier for monoclonal antibodies. In the therapy of targeted tumours and neovascular artery occlusion, angiography, retinal and choroidal blood vessel stasis, recent treatment strategies such as the application of heat-activated liposomes in focused lasers, as well as heat-induced release of liposomal therapeutics and dyes for targeted delivery. Too far, two patent medicines have received approval for liposomal drug formulations, and many more are undergoing scientific testing. 'Verteporfin' is a commercially endorsed liposomal medication for ocular usage. The relevance of the liposome will be expanded in the future to include the therapeutic, diagnostic, and research aspects of ophthalmology

C. Liposome as Vaccine Adjuvant

Liposomes are a well-known immune adjuvant that enhances both cell-mediated and non-cell-mediated immunity. Liposomal immune adjuvants work by releasing encapsulated antigen slowly and passively into the localized lymph node after intramuscular injection. Accumulation of liposomes into lymphoid is accomplished by targeting liposomes with the aid of phosphotidyl serine. It is possible to prepare a liposomal vaccine by immunizing bacteria, soluble antigen and deoxyribonucleic acid cytokinesis with liposomes. However, it induces immunological reaction to antigenic protein expression. Antigens will further combine with the liposomal membrane in a covalent manner. Liposomal vaccines can be kept for around 12 months in a refrigerated state.

D. Liposomes for Brain Targeting

Liposomes have lately gained popularity as a brain drug delivery mechanism due to their biocompatibility and biodegradability. Liposomes with small (100 nm) or even big diameters diffuse freely across the Blood Brain Barrier (BBB). SUVs linked to cognitive drug carriers, on the other hand can be carried across the BBB by receptor-mediated or absorptive-mediated transcytosis. Absorptive mediated endocytosis of cationic liposomes occurs in cells, however it has yet to be proven that absorptive induced transcytosis occurs across the BBB .The mannose-coated liposomes reach the brain and help in drug transport across the BBB. When systemically administered, the Leu-enkephalin, mefenkephalinkyoforphin and neutropeptides do not typically penetrate the blood-brain barrier. Due to the versatility of this approach, the anti-depressant amitriptyline usually penetrates the BBB. Nanoparticles (NP) have been developed from various stabilizers. The amount of amitriptyline was observed to be substantially increased in the brain when the drug was adsorbed to the NP and wrapped or particle remained stable with polyoxyethylene 20 sorbitan trioleate.

E. Liposome as Anti-Infective Agents

The intracellular pathogen such as protozoa, bacteria and fungi resides in the liver and spleen and thus the therapeutic substances can be administered to such organs using liposomes as the transport system to remove these pathogen. It is possible to treat diseases such as leishmaniasis, histoplasmosis, candidiasis, erythrococosis, aspergelosis, gerardiasis, tuberculosis and malaria by integrating and targeting the medication with the liposomal carrier.

Amphotericin B, a polyene antibiotic is associated with significant kidney damage when used to treat systemic fungal infection. Amphotericin B acts by binding to sterol in the membrane of susceptible fungi, thereby enhancing the permeability of the membrane. Because of its non-specificity and affinity to cholesterol in mammalian cells this substance is hazardous. The first liposome formulation of Amphotericin B has recently been performed in all clinical tests and is now commercialized fortreating various fungal diseases such as Mucormycosis (Black Fungus), aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, and cryptococcosis. Liposomal Amphotericin B decreases renal and general toxicity at the usual dosage by passively targeting the liver and spleen, but renal toxicity usually happens whenever drug is delivered at a high dose due to liver and spleen macrophage saturation. By coating the vesicle using o stearoyl amylopectin, polyoxyl ethylene, or mono sialogangliocyte liposomes can also be specifically targeted to the lungs. The encapsulation in the lung-targeted liposome of ant tuberculous agents such as isoniazid and rifampicin modulates toxicity and increases the effectiveness of these products. Varieties of formulations of amphotericin have been approved in many clinical trials and are now being sold in numerous European countries.

F. Liposome in Tumor Cell Therapy

Anti-cancer medicines have serious hazardous side effects when taken for a long time. The liposomal treatment for tumor cell targeting has enhanced the field of tumor treatment by reducing side effects to a minimum. Although the tiny and stable liposome are supposed to be passively targeted to various tumors although they can circulate for prolonged periods of time and can additionally vasate in tissues with increased vascular permeability. The intake of liposome macrophages by the liver and spleen has impeded liposome growth as a drug delivery for more than 20 years. Many herbal anticancer medications have now been incorporated into liposome to provide improved targeting with increased bioavailability.

ADVANCEMENTS IN LIPOSOMES

• Ethosomes: They are effective in delivering soya phosphatidylcholine and 30% ethanol to the skin.
• Immuno liposomes: They were modified with antibodies.
• Niosomes: They are small unilamellar vesicles composed of surfactants that are non-ionic.
• Stealth liposomes: These are new types of liposome designed to enhance stability and extend their half-life in circulation. Coating of liposomes should be done by poly ethylene glycol (PEG) for preparing these liposomes.

CONCLUSION

The development of numerous types of liposomes as a targeted drug delivery system for treating various diseases has exploded in the last decade. This review discussed liposomes as a targeted drug delivery method, including their current status, limitations, and future prospects. The versatility of their behavior could also be used to administer drugs by any route and for every drug ingredient regardless of solubility capabilities. Liposome’s effectiveness as therapeutic agents has led to the development of a variety of liposome-based formulations that provide greater concentration of drug. The liposomal method can be used to increase pharmacokinetics and therapeutic effect while lowering the toxic effects of a variety of enormously potent therapeutics. Without a doubt the liposomal delivery method has the ability to transform conventional therapy for the cure of a wide range of life-threatening illnesses including cancer. Liposome’s usage in pharmaceutical and gene delivery is promising and it will undoubtedly be further developed in near future. The development of liposome’s as a drug carrier will remain to be a priority in the coming years with a higher potential to be transferred into the therapy.

REFERENCE

1. Mishra H, Chauhan V, Kumar K, Teotia D. A comprehensive review on Liposomes: a novel drug delivery system. Journal of Drug Delivery and Therapeutics. 2018; 8(6):400-404. https://doi.org/10.22270/jddt.v8i6.2071
2. Lasic D, Papahadjopoulos D. Liposomes revisited. Science. 1995; 267(5202). https://doi.org/10.1126/science.7871422
3. Vyas SP, Khar RK. Targeted And Controlled Drug Delivery: Novel Carrier Systems. 2006:421-427.
4. Samad A, Sultana Y, Aqil M. Liposomal Drug Delivery Systems:An Updated Review. Current Drug Delivery. 2007; 4:297-305. https://doi.org/10.2174/156720107782151269
5. Riaz M. Liposomes preparation methods. Pakistan journal of pharmaceutical sciences. 1996:65-77.
6. Gregoriadis G, Florence AT. Liposomes in Drug Delivery. Drugs. 1993:15-28. https://doi.org/10.2165/00003495-199345010 00003
7. Dunnick JK, Rooke JD, Aragon S, Kriss JP. Alteration of Mammalian Cells by Interaction with Artificial Lipid Vesicles. Cancer Research. 1976(36):2385-2389.
8. Fujisawa T, Miyai H, Hironaka K, et al. Liposomal diclofenac eye drop formulations targeting the retina: formulation stability improvement using surface modification of liposomes. Int J Pharm. 2012; 436:564-567. https://doi.org/10.1016/j.ijpharm.2012.07.024
9. Bangham AD, Standish MM, Watkins JC. Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol. 1965; 13:238-252. https://doi.org/10.1016/S0022-2836(65)80093-6
10. McIntosh TJ. The effect of cholesterol on the structure of phosphatidylcholine bilayers. Biochim Biophys Acta. 1978; 513:43-58. https://doi.org/10.1016/0005-2736(78)90110-4
11. Cullis PR. Lateral diffusion rates of phosphatidylcholine in vesicle membranes, effects of cholesterol and hydrocarbon phase transitions. FEBS Letters. 1976; 70:223-228. https://doi.org/10.1016/0014-5793(76)80762-4
12. Li X, Chen D, Le C, et al. Novel mucus-penetrating liposomes as a potential oral drug delivery system: preparation, in vitro characterization, and enhanced cellular uptake. Int J Nanomedicine. 2011(6):3151--3162. https://doi.org/10.2147/IJN.S25741
13. Specialized Drug Delivery System,Manufacturing and Production Technology. In: Tyle P, ed. Pharmaceutical manufacturing of liposomes: Marcell Dekker; 1990.
14. Sharma A, Sharma US. Liposomes in drug delivery: Progress and limitations. International Journal of Pharmaceutics. 1997; 154(2):123-140. https://doi.org/10.1016/S0378 5173(97)00135-X
15. Gregoriadis G. Engineering liposomes for drug delivery,progress and problems. Trends in Biotechnology. 1995; 13(12):527-537. https://doi.org/10.1016/S0167-7799(00)89017-4
16. Deshmukh R, Gawale S, Bhagwat M, Ahire P, Derle N. A Review on: liposomes. World journal of pharmacy and pharmaceutical sciences, (03):506-517.
17. Gaurav R, Tejal S. Liposomal drug delivery syst em: an overview. IJPBA. 2011; 2(6):1575-1580.
18. Dua J, Rana PA, Bhandari DK. Liposome: methods of preparation and applications. IJPSR. 2012; 3(2):14-20.
19. Hwang T, Lee WR, Hua SC, Fang JY. Cisplatin encapsulated in phosphatidylethanolamine liposomes enhances the in vitro cytotoxicity and in vivo intratumor drug accumulation against melanomas. J Dermatol Sci. 2007; 46:11-20. https://doi.org/10.1016/j.jdermsci.2006.12.011
20. Prabhu P, Kumar N, Dhondge G, et al. Preparation and evaluation of liposomes of brimonidine tartrate as an ocular drug delivery system. International Journal of Research in Pharmaceutical Sciences. 2010; 1(4):502-508. https://doi.org/10.4103/0975 1483.71623
21. Lopes L, Scarpa M, Pereira N, Oliveira LCd, Oliveira A. Interaction of sodium diclofenac with freeze-dried soya phosphatidylcholine and unilamellar liposomes. Braz J Pharm Sci. 2006; 42(4):497 504. https://doi.org/10.1590/S1516-93322006000400004
22. Sipai A, Vandana Y, Y M, V.V P. Liposomes: an overview. JPSI. 2012; 1(1):13-21.
23. Jadhav MP, Nagarsenker MS, Gaikwad RV, Samad A, Kshirsagar NA. Formulation and Evaluation of Long Circulating Liposomal Amphotericin B: A Scinti-kinetic Study using 99mTc in BALB/C Mice. Indian J Pharm Sci. 2011; 73(1): 57-64. https://doi.org/10.4103/0250-474X.89757
24. Chauhan T, Arora S, Parashar B, Chandel. Liposome Drug Delivery. IJPCS. 2012; 1(3):1103-1113.
25. Nidhal K, Athmar D. Preparation and evaluation of salbutamol liposomal suspension using chloroform film method. Mustansiriya Medical Journal. 2012; 11(2):39-44.
26. Anwekar H, Patel S, Singhai AK. Liposome- as drug carriers. Int J of Pharm & Life Sci. 2011; 2(7): 945-951.
27. Traïkia M, Warschawski DE, Recouvreur M, Cartaud J, Devaux PF. Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and 31P-nuclear magnetic resonance. Eur Biophys J. 2000; 29(3):184-195. https://doi.org/10.1007/s002490000077
28. Kumar A, Badde S, Kamble R, Pokharkar V. Development and characterization of liposomal drug delivery system for nimesulide. Int J Pharm Pharm Sci. 2010; 2(4):87-89.
29. Da Costa C, Moraes A. Encapsulation of 5-fluorouracil. Maringá. 2003; 25(1):53-61.
30. Niu M, Lu Y, Hovgaard L, Wu W. Liposomes containing glycocholate as potential oral insulin delivery systems: preparation, in vitro characterization, and improved protection against enzymatic degradation. Int J Nanomedicine. 2011; 6:1115-1166.