INTRODUCTION:

Pulmonary drug delivery systems have achieved tremendous scientific interest in recent years. The administration of drugs by the pulmonary route, particularly for drug targeting into the lung, provides promise for the treatment of a broad spectrum of pulmonary disorders like lung cancer, tuberculosis and offers numerous advantages over other conventional formulations.^1,2 Lungs are an attractive target for the pulmonary drug delivery of active pharmaceutical ingredients in the form of various drug delivery approaches.³ Additionally, pulmonary route offers many systemic advantages over other conventional drug delivery systems, such as a high surface area with rapid absorption due to high vascularization and circumvention of the first pass effect.⁴

A majority of anti-tubercular drugs, which are highly effective towards the affected cells in the lung and it also, affect the normal cells. This is due to their low therapeutic index of such drugs. That is the dose required to achieve the anti-tubercular effect turns into the toxic effect on normal cells.^5,6Antitubercular therapy by oral administration leads to high systemic doses over a longer time. Thus the long duration of antitubercular therapy is related to various adverse effects and poor patient compliance. Particle deposition at the site of action is a serious drawback for inhaled therapeutics. The residential time at the site of action of the deposited drug will be minimal due to the mucociliary clearance. The barriers like lung lining fluid, airway macrophages, and lung epithelial cells minimizes the duration of action of such therapies.⁷

To overcome the above-mentioned drawbacks, targeted drug delivery to the lungs is possible by the various novel approaches like polymeric micro-nano particles, niosomes, liposomes, and dendrimers represent a promising plan to deliver the drug at site of action. The novel drug carrier plays a supreme role to deliver the dose uniformly among the alveoli and controls the release of the drug in a sustained manner at the site of action. It also improves the solubility and stability of the active drug substance against metabolism and degradation.⁸

Figure 1 Structure of a Liposome

Among the several approaches used for targeting the lungs, liposomes are one of the most convenient and extensively investigated systems due to its high biocompatibility behavior. Liposomes are tiny vesicles, made out of the same material as a cell membrane. They can be filled with drugs and used to deliver drugs for various major diseases like cancer and tuberculosis.⁹ Liposome facilitates intracellular drug delivery and improves the residential time of encapsulated drugs for a longer period. The term liposome is derived from Greek words: “Lipos” which means fat and “Soma” meaning body. Liposomes are bi-layered, colloidal, microspherical vesicles consisting of an aqueous core enclosed in phospholipid molecules. It has potential advantages like both hydrophilic and hydrophobic nature of drugs that can be encapsulated within the liposomal vesicles and the active drugs can be targeted to the desired site of action. The size range of such carriers is about 0.01 – 5.0µm. The liposomal vesicles are formed when the phospholipids are hydrated more than aqueous medium.¹⁰

Figure 2 Observation of liposomal vesicle under optical microscope

Mechanism of liposomal vesicle formation:

The backbone of the liposome is phosphatidylcholine, which has a hydrophilic head and hydrophobic tail. The hydrophobic part contains two fatty acid chains with 10 – 24 carbon atoms and 0 – 6 double bonds in each chain and the hydrophilic part consists of phosphoric acid, which is bound to a water-soluble molecule.

Phospholipids will form lamellar sheets after the dispersion in aqueous medium. The sheets are formed by organizing in such a manner that, the polar head part faces outwards to the aqueous region, while the hydrophobic part face each other to form liposomal vesicles. The polar part remains in contact with the aqueous region along with shielding of the non- polar part. External energies like sonication, heating, and homogenization have also influenced the formation of the vesicles by maintaining the thermodynamic equilibrium between lipid and water molecules.¹¹

Figure 3 Mechanism of liposomal vesicle formation

Figure 4 Influence of external energies to form liposomal vesicle

Classification of Liposome:

Liposomes are can be classified according to their vesicular size as follows:

Type of liposomes	Size ranges
Multilamellar Vesicles (MLV)	>0.5 µm
Oligolamellar Vesicles (OLV)	0.1 – 1.0 µm
Multivesicular Vesicles (MVV)	>0.1 µm
Small Unilamellar Vesicles (SUV)	20- 100 nm
Giant Unilamellar Vesicles (GUV)	>0.1 µm
Large Unilamellar Vesicles (LUV)	>100 nm

The present study provides a review of the pulmonary delivery of liposomes and indicates that liposomes can be effectively deposited in the human respiratory tract, wherein they may remain for longer time period and the technological aspects of delivering liposomes to the lung are discussed, including the characterization of liposome-containing aerosols and the potential advantages and disadvantages of the various methods which have been employed for their production.¹

Pulmonary drug delivery can be attained by the following three types of devices:

Pressurized Metered Dose Inhalers (pMDI):

In this approach drug solution or suspension are incorporated to liquefied propellants. Mostly hydrofluroalkanes are used as a propellant instead of chlorofluorocarbons because hydrofluroalkanes are non-ozone depleting propellant.

Dry Powder Inhalers (DPIs):

Liposome formulations are dried to achieve in dry powder form. The several benefits like stability improvement of liposomal formulations by the dry powder inhalers method. Spray-dried liposomes entrapped dapsone dry powder inhalers have prolonged therapeutic action of a drug in the lungs to treat Pneumocystis carinii pneumonia. In vitro studies showed 16 hours of prolonged drug release was achieved by the liposome approach.

Soft Mist Inhalers (SMIs):

Soft mist inhalers are the hand-held propellant-free metered-dose inhalers that produce slow-moving aqueous aerosols for deep-lung expulsion. Large doses are required for the treatment of many diseases in the lung (e.g. cancers, tuberculosis, etc.); however, all the above-mentioned devices (i.e. pMDIs and DPIs) can deliver only small amounts of aerosol, and thus are more appropriate for treating diseases that require small doses of the therapeutic agent.

Nebulizers:

It is one of the simplest methods for delivering liposomes to the respiratory tract but poor drug stability can occur due to liposome breakage. Dry powder formulations are used to improve this stability problem.¹¹

Methods of Liposome Preparation:

The conventional methods for preparing liposomes include dissolving the lipids in organic solvent, drying down the lipids from organic solutions to get a thin lipid film under vacuum, finally the dispersion of lipids film in aqueous media to form liposomal vesicles, purification of prepared liposomes and analysis of the final product. If the drug is hydrophilic it is entrapped in the aqueous buffer and if the drug is hydrophobic, it can be encapsulated in the lipid film. The main drawback of this method is poor encapsulation efficiency (5 – 15% only) for hydrophobic drugs. The percentage of encapsulation efficiency can be improved by hydrating the lipids in presence of organic solvent. Large unilamellar vesicles (LUV) can be prepared by solvent injection method, detergent dialysis method; calcium induced fusion and revese phase evaporation techniques. Small unilamellar vesicles (SUV) can be prepared by the extrusion or sonication of MLV or LUV. Some of the methods are discussed below.

Thin-Film Hydration Method:

All the methods used for preparing liposomes, thin-film hydration method are the simplest and widely used. Multi lamellar vesicles (MLV) are produced by this method within a size range of 1–5µm. In this method, lipid mixtures are dissolved in organic solvent mixture generally chloroform and methanol in a ratio of 2:1 is used. The organic solvent part is then removed in a rotary evaporator flask and a dried thin film of lipid is produced using a rotary evaporator under reduced pressure. Then the lipid film is hydrated by adding an adequate amount of aqueous buffer containing drug to be encapsulated and again use of rotary evaporator for making homogeneous milky white suspension. The liposomal suspension is then sonicated by using a bath sonicator or probe sonicator for the sizing of the vesicles. ^[12,13]

Figure 5 Thin- film hydration method of liposome preparation

French Pressure Cell Method:

The French pressure cell method involves the extrusion of MLVs at 20,000 psi at 4°C through a small orifice. The method has many advantages like it is the simplest and reproducible method than the sonication method. Also, it involves the gentle handling of unstable materials. The liposomes are produced by this method somewhat larger than sonicated SUVs. The drawbacks of the method are that the optimum temperature is difficult to control and the working volumes are relatively very less.¹⁴

Figure 6 French pressure cell and parts used for preparation of liposomal vesicles.

Ether Injection Method:

A solution of lipid is firstly prepared by dissolving lipids in an organic solvent (Ether). The lipid solution is then slowly injected into an aqueous buffer containing drug to be encapsulated at 55-65°C or under vacuum. The removal of organic solvent under vacuum leads to the formation of liposomal vesicles. The main disadvantage of this method is the exposure of compounds to be encapsulated to organic solvents and decomposition of heat-sensitive drugs can occur.^14,15

Ethanol Injection Method:

A lipid solution of ethanol is injected into a buffer solution containing an active pharmaceutical agent. The liposomal vesicles are immediately formed after injecting the organic solution into the aqueous phase. The drawbacks of the ethanol injection method are the same as the ether injection method.¹⁶

Figure 7 Method of vesicles formations by solvent dispersion methods.

Reverse Phase Evaporation Method:

In this method water in oil emulsion is prepared by sonication of a two-phase system containing phospholipids in the organic solvent phase and aqueous buffer solution phase. After that the organic solvents are removed under vacuum, resulting in the formation of a thick gel. The liposomes are formed when residual solvent is removed by continued rotary evaporation under vacuum. With this method, high encapsulation efficiency up to 70% can be achieved. The method has been used to encapsulate various active drug substances. The actual drawback of the method is the exposure of the drug to be encapsulated to organic solvents which can cause toxicity.¹⁶

Figure 8 Formation of liposomes using reverse-phase evaporation method.

Characterization Methods of Liposomes^{17 -21}:

Liposomes produced by various methods have varying physicochemical properties, which causes differences in their in vitro and in vivo executions. It is important to characterize the liposomes after their formulation and upon storage. A liposomal formulation can be evaluated for some of the parameters that are discussed below.

Size and Size Distribution:

Size and size distribution is the primary parameter when the liposomes are used for pulmonary drug delivery or parenteral administration. The various methods including optical microscopy, scanning electron microscopy, laser light scattering method, photon correlation spectroscopy are used to determine the size and size distribution of liposomal vesicles.

Percent Drug Encapsulation:

The amount of drug encapsulated within liposomal vesicles is determined by the percentage of drug encapsulation. The formulation contains both free and entrapped drug. To determine the actual amount of drug entrapped, the free drug is needed to separate from the entrapped drug. After that, the fraction of liposomal vesicles containing the entrapped drug is treated with a detergent, so as to achieve lysis of the vesicles, which allows to the discharge of the drug from the liposomal vesicles into the surrounding buffer medium. This exposed drug is assayed by suitable techniques like spectrophotometry and column chromatography which gives the percent drug entrapped.

Surface Charge:

The surface charge of liposomes can be determined by free-flow electrophoresis and zeta potential. Since the charge on the liposome surface plays an important role in the in vivo disposition, it is mandatory to know the surface charge on the vesicle surface. It can be defined as the potential difference between the electroneutral region of the solution and surface of tightly bound layer (shear place). The surface charge can be determined by using the Helmholtz–Smolochowski equation.

Vesicle Shape and Lamellarity:

Various electron microscopic techniques can be used to determine the shape of the liposomal vesicles. Lamellarity is the number of bilayers present in the liposome. It can be determined using freeze-fracture electron microscopy and 31P-Nuclear magnetic resonance analysis. The surface morphology of liposomal vesicles can be determined by using freeze-fracture and freeze-etch electron microscopy.

Stability of Liposomes:

During the formulation of liposomal drug products, the stability of the prepared formulation is a major factor. The therapeutic action of the drug substance is related by the stability of the liposomes right from the preparing steps to storage to its delivery. A stable dosage form should maintain the physical stability as well as the chemical integrity of the active drug substance during its formulation procedure and storage. A well-planned stability study includes the determinations of its physical, chemical and microbial parameters throughout its entire storage period. So, the stability study is very important to study the physical and chemical properties of the drug product in its storage.

Therapeutic Applications of Liposomes:

Novel drug delivery systems are developed to provide a desired therapeutic action and to minimize some severe adverse drug reactions. Liposomes are such novel drug carriers that provide an optimum therapeutic response as well as safety in comparison to conventional formulations. Some of the major therapeutic applications of liposomal formulations in drug delivery are as follows:

Site-avoidance Delivery:

The anti-tubercular drugs cause cytotoxicity to normal tissues due to their narrow therapeutic index. Liposome encapsulated drug minimizes the delivery of drug to normal cells. So, the therapeutic index of an antitubercular drug can be improved by the liposomal drug delivery system. Free rifampicin has some serious side effects, but when formulated as liposomes, the toxic effect of rifampicin was reduced without any change in the therapeutic activity.

Site Specific Targeting:

Liposomal drug carrier allows delivery of a larger fraction of drug to the desired site of action also it reduces the exposure of drugs to normal cells. Entrapping the drug in liposomal vesicles can be used for both active and passive targeting of drugs in order to get a safe and efficacious drug therapy during disease condition.

Intracellular Drug Delivery:

Increased delivery of potent drugs to the receptor site can be achieved by the liposome approach. Several anti-cancer drugs showed greater activity against ovarian tumor cell lines in comparison to free drugs.

Sustained Release Drug Delivery:

Liposomes can be used to provide a sustained release manner of drugs, which require a prolonged plasma drug concentration at therapeutic levels to achieve the optimum therapeutic action of medicaments. The release profile of drugs can be improved by the liposomal drug delivery approach.

CONCLUSION:

Antimicrobial agents can be encapsulated in liposomes for two reasons. Primarily reason is the liposome can protect the entrapped active drug substance against enzymatic degradation and the secondary reason is liposomes can promote enhanced cellular uptake of the antibiotics. The drug substances which are highly potent and have a low therapeutic index can be targeted to the required site of action using the liposome approach. Liposomes can alter the pharmacokinetic parameters of a drug substance. Poorly soluble drugs can be encapsulated in lipoidal vesicles. The effectiveness of the liposomal formulation totally depends on its ability to deliver the drug to the desired site of action over a longer period of time, simultaneously reducing the adverse effects of drugs. The drugs are encapsulated within the liposomal vesicles and are expected to diffuse out from the liposomal vesicles for a prolonged period of time. The parameters like concentration of drug, the molar ratio of drug and lipid, percentage encapsulation efficiency and in vivo and in vitro drug release must be determined during the formulation of liposomal drug delivery systems. Thus liposomal formulations can be successfully utilized to improve the pharmacokinetics and therapeutic index of a drug substance by minimizing the adverse effects of various highly potent drugs.

ACKNOWLEDGEMENT:

We acknowledge the support of the faculty members of Department of Pharmaceutical Technology, JIS University, Kolkata-700109.

CONFLICTS OF INTERESTS:

The authors confirm that this article content has no conflicts of interest.

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Received on 06.03.2020 Modified on 15.04.2020

Research J. Pharm. and Tech 2021; 14(3):1791-1796.

DOI: 10.5958/0974-360X.2021.00318.8