Liposomes: Structure, Composition, Types, and Clinical Applications PDF

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Heliyon 8 (2022) e09394 Contents lists available at ScienceDirect Heliyon journal homepage: www.cell.com/heliyon Review article Liposomes: structure, composition, types, and clinical applications☆ Hamdi Nsairat a, Dima Khater b, Usama Sayed c, Fadwa Odeh d, Abeer Al Bawab d, e, Walhan Alshaer f, * a Pharmacological and Diagnostic Research Center, Faculty of Pharmacy, Al-Ahliyya Amman University, Amman, 19328, Jordan b Department of Chemistry, Faculty of Arts and Science, Applied Science Private University, Amman, Jordan c Department of Biology, The University of Jordan, Amman, 11942, Jordan d Department of Chemistry, The University of Jordan, Amman, 11942, Jordan e Hamdi Mango Center for Scientific Research, The University of Jordan, Amman, 11942, Jordan f Cell Therapy Center, The University of Jordan, Amman, 11942, Jordan A R T I C L E I N F O A B S T R A C T Keywords: Liposomes are now considered the most commonly used nanocarriers for various potentially active hydrophobic Liposomes and hydrophilic molecules due to their high biocompatibility, biodegradability, and low immunogenicity. Lipo- Phospholipids somes also proved to enhance drug solubility and controlled distribution, as well as their capacity for surface Lamellarity modifications for targeted, prolonged, and sustained release. Based on the composition, liposomes can be Stealth liposomes Vaccinations considered to have evolved from conventional, long-circulating, targeted, and immune-liposomes to stimuli- responsive and actively targeted liposomes. Many liposomal-based drug delivery systems are currently clini- cally approved to treat several diseases, such as cancer, fungal and viral infections; more liposomes have reached advanced phases in clinical trials. This review describes liposomes structure, composition, preparation methods, and clinical applications. 1. Introduction nanoscale range to deliver a variety of active biomedical ingredients for the treatment, prevention, and diagnosis of many diseases [1, 9]. Drug delivery systems (DDSs) offer the potential to enhance the Despite the fast progress in this field, most nanoparticles-based drug therapeutic index of drugs by increasing the drug concentration, the delivery systems show improper loading capacity with a lack of speci- residence time in target cells and minimizing the side effects. DDSs ficity against their targets. As a result, the promising advances in the involve delivering the potentially active drug to the site of action via a drug delivery systems should involve designing high and regulated ca- nano-vehicle to enhance the pharmacological properties of free drugs pacity nanocarriers functionalized by recognition ligands that target and cover their undesirable features through improving drug pharma- specifically unique or overexpressed biomarkers. Liposomes are the cokinetics and biodistribution, as well as acting as drug reservoirs [2, 3]. most explored nanocarriers used in targeted drug delivery systems. Li- These nanoparticles (NPs) usually ranged from a few nanometers to posomes are spherical lipid vesicles (usually 50–500 nm in diameter several hundred nanometers according to their intended application. particle size) composed of one or more lipid bilayers, as a result of Different natural, organic and inorganic materials are used to create NPs emulsifying natural or synthetic lipids in an aqueous medium [12, 13] including ceramic, polymers, metals , and lipids that generate nano- (Figure 1). Liposomes were firstly discovered in the 1960's by Bengham particles like micelles and liposomes [5, 6, 7]. and later became among the most expansive drug delivery systems. Therapeutic drugs are incorporated into the NPs mainly by physical Liposomes nanoemulsions are widely used nanoparticles in nano- interactions including, entrapment, surface attachment, or encapsulation medicine mainly due to their biocompatibility, stability, ease to synthe-. These variations and unique properties of different NPs could be size and high drug loading efficiency [15, 16], high bioavailability , used to improve the characteristics of traditional therapeutics. and their safe excipients used in these formulations. Due to their Nanomedicine facilitates designing novel therapeutic options in the size, hydrophobic and hydrophilic characteristics and their ability to ☆ This article is a part of the "Lipid-Based Nanoparticles in Diagnosis and Treatment" Special issue. * Corresponding author. E-mail address: [email protected] (W. Alshaer). https://doi.org/10.1016/j.heliyon.2022.e09394 Received 31 December 2021; Received in revised form 19 February 2022; Accepted 6 May 2022 2405-8440/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). H. Nsairat et al. Heliyon 8 (2022) e09394 of cholesterol into liposomes is indispensable since cholesterol modulates membrane permeability, changes fluidity, and improves the stability of Hydrophobic bilayer membranes in the presence of biological fluids such as blood and bilayer plasma [31, 32]. Liposomal formulations may also contain polymers , and even membrane protein to prolong their circulation Cholesterol half-life, improve the biodistribution profile and enhance the encapsu- Hydrophilic lated drug effectiveness Moreover, Stealth stabilized liposomes, core Phospholipids incorporating phospholipids-attached polyethyleneglycol (PEG) into li- posomes infrastructure, has been shown to be a useful method for modifying liposomes pharmacokinetic properties and biodistribution profiles. The current review describes liposomes compositions, types, methods of preparation, and clinical applications. 2. Liposomes Figure 1. Schematic representation of liposomes. According to the liposomes structures, they are classified into four categories based on size and number of bilayers: small unilamellar ves- encapsulate drug molecules either in the aqueous interior of the vesicles icles (SUV), large unilamellar vesicles (LUV), multilamellar vesicle or in the lipophilic membrane , liposomes are considered promising (MLV), and multivesicular vesicles (MVV). Liposomes have mono phos- to be used effectively as drug delivery systems. Several liposomal-based pholipid bilayer in a unilamellar structure, while they have an onion-like drug delivery systems have been approved by Food and Drug Adminis- structure in a multilamellar structure. MVV form a multilamellar tration (FDA) for disease treatment in the market [20, 21]. Moreover, arrangement with concentric phospholipid spheres as many unilamellar liposomes are suitable for diagnostic and therapeutic applications using vesicles are produced within larger liposomes. Liposome encapsu- several routes of administration, including ocular , oral , pul- lation efficiency increases with liposome size and decreases with the monary , transdermal , and parenteral [26, 27, 28]. Liposomes number of bilayers for hydrophilic compounds only. The size of the are primarily created from phospholipids such as soybean phosphati- vesicles is an important factor that controls the circulation half-life of dylcholine or synthetic dialkyl or trialkyl lipids. Incorporation liposomes. Both the size and number of bilayers influence the amount of Figure 2. Natural phosphatides the most used to produce liposomes; A) Phosphatidylcholine, B) Phosphatidylethanolamine, C) Phosphatidylserine, D) Phosphati- dylinositol, E) Phosphatidylglecerol, and F) Phosphatidic acid. 2 H. Nsairat et al. Heliyon 8 (2022) e09394 Figure 3. Palmitic acid -based different synthetic phospholipids; A) 1,2-Dipalmitoyl-sn-glycero-3-phosphorylethanolamine, B) 1,2-Dipalmitoyl-sn-glycero-3-phospha- tidic acid sodium salt, C) 1,2-Dipalmitoyl-sn-glycero-3-phosphorylglycerol sodium salt, and D) 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). the encapsulated drug. When liposomes are employed for drug delivery, molecule). The charge of the hydrophilic group provides stability the desired vesicles usually extend from 50 nm to 150 nm. Liposomes through electrostatic repels. The hydrophobic group of lipids varies in the interaction with the cell membrane is represented by various theories: acyl chain length, symmetry, and saturation. The lipids that used in specific (modified with receptor-mediated) or nonspecific endocytosis liposomes preparation may be classified as: , local fusion (adhesion) , phagocytosis , and absorption into the cell membrane. Liposome-cell interactions are influenced 2.1.2. Natural lipids by a variety of factors, including composition , the diameters of li- The membrane bilayer of normal cells are mainly composed of glyc- posomes, surface charge , targeting ligand on the liposome surface, erophospholipids. Phospholipids are consist of a glycerol unit that is and biological environment. bonded to a phosphate group (PO2
4 ) and to two fatty acid molecules. The phosphate group can be also bonded to small, essential choline organic 2.1. Liposomes compositions molecule [21, 51] (Figure 2A). Natural phospholipids can be obtained from various sources such as soya bean, egg yolk. Phospholipids are 2.1.1. Lipids and phospholipids used for liposomes classified as phosphatidylcholine (PC), phosphatidylethanolamine (PE), Structurally, liposomes are spherical or multilayered spherical vesi- phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol cles made by the self-assembly of diacyl-chain phospholipids (lipid (PG), and phosphatidic acid (PA) regarding to the polar head groups. bilayer) in aqueous solutions. The bilayer phospholipid membrane Natural phospholipids are less stable than synthetic phospholipids in li- has a hydrophobic tail and a hydrophilic head [21, 47] that leads to the posomes preparation due to the unsaturated characteristics of the hy- formation of an amphiphilic structure. Liposomes can be made from both drocarbon chain [53, 54] (Figure 2). Natural phospholipids composed of natural and synthetic phospholipids. Lipid composition strongly a variety of fatty acids, one is a saturated fatty acid as palmitic acid affects liposome characteristics that include: particle size, rigidity, (hexadecanoic acid, H3C-(CH2)14-COOH); margaric acid (heptadecanoic fluidity, stability, and electrical charge [5, 49]. For example, liposomes acid, H3C-(CH2)15-COOH) and the other is an unsaturated fatty acid (here formulated from natural unsaturated phosphatidylcholine, as egg or oleic acid, or 9Z-octadecenoic acid that identified in egg yolk lecithin soybean phosphatidylcholine, provide highly permeable and low stable. The egg derived phospholipids and PCs are made of these fatty properties. Though, saturated-phospholipids-based liposomes such as acids patterns: palmitic acid (C16:0), stearic acid (C18:0), oleic acid dipalmitoyl phosphatidylcholine led to rigid and almost impermeable (C18:1), linoleic acid (C18:2), and arachidonic acid (C20:4). These fatty bilayer structures. acids are account for about 92% of the total fatty acid composition with a The hydrophilic group in the lipids may be negatively, positively typical presence of the polyunsaturated fatty acids C 20:4 (n-6) and C22:6 charged, or zwitterionic (both negative and positive charge in the same (n-3) in egg phospholipids. Egg PC contains about 40% 3 H. Nsairat et al. Heliyon 8 (2022) e09394 Figure 4. Stearic acid -based different synthetic phospholipids; A) 1,2-Distearoyl-sn-glycero-3-phosphorylethanolamine, B) 1,2-Distearoyl-sn-Glycero-3-Phosphatidic acid Na salt, C) 1,2-Distearoyl-sn-glycero-3-phosphorylglycerol sodium salt, and D) 1,2-Distearoyl-sn-glycero-3-phosphocholine. 1-palmitoyl-2-oleoylphosphatidylcholine. The principal saturated acid that both steroids reduce liposomes membrane fluidity, increase absolute was stearic in PE and PS, and palmitic in the other lipids. Furthermore, zeta potential, cause significant changes in particle size, and decrease the fatty acid pattern of the soybean derived account for about 95% DPPC phase transition temperature (Tm) and enthalpy. palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3). Since the unsaturated fatty 2.1.5. Surfactants acids of PE, PS, and PC amounted to over 50% of the total acids, they Surfactants were utilized in liposomes formulations to modify the must occur at both the α- and β-positions of the glycerol moiety of these encapsulation and release properties of liposomes through surface ten- phospholipids. sion reduction between different immiscible phases. Surfactants are single acyl-chain amphiphiles that destabilize the lipid bilayer of lipo- 2.1.3. Synthetic lipids somal nanoparticles (Figure 7), thus increasing nano-vessel deformability Synthetic phospholipids are made by specific chemical modifications [61, 62]. Commonly utilized surfactants in liposomes formulations are: to the non-polar and polar regions of the natural phospholipids. The sodium cholate, Span 60, Span 80, Tween 60, and Tween 80 [62, 63]. modification enables an unlimited variety of well-defined and catego- Various surfactants-containing liposomes have been widely used as a rized phospholipids. The major saturated synthetic phospholipids carrier in drug delivery to enhance skin penetration of encapsulated are based on either using stearic and/or palmitic fatty acid. Figures 3 and therapeutic agents. Ultra deformable liposomes, also called trans- 4 represent different possible and commercial, synthetic, and saturated fersomes, are a Surfactants-based nanovesicles with positive findings in phospholipids usually used to prepare liposomes. transdermal drug delivery [65, 66]. The key factor making liposomes Additionally, Synthetic phospholipids can be made from mixed fatty deformable is edge activator (surfactant). The edge activator can alter the acids, unsaturated fatty acids in both hydrocarbons or only in one hy- lipid bilayers of vesicles increasing the deformability of them. These drocarbon chain (Figure 5). nanovesicles are different from conventional liposomes in which they can respond to osmotic pressure by rapid shape transformations only by low 2.1.4. Steroid energy. Moreover, ultra deformable liposomes showed an increased Steroid are hydrophobic lipids consists of four-ring structure as in the drug transepidermal flow made them more suitable nanovehicle shown in Figure 6. Steroid's diversity comes from the various functional for the topical administration of antihypertensives. groups attached to those rings. Cholesterol is the major steroid usually used in liposomes preparation in a ratio less than 30 % of the total lipids 2.2. Liposomes types to improve liposomes rigidity and stability since its incorporated in the liposomes lipid bilayer [47, 58]. In a comparative study for cholesterol Based on their compositions and applications, liposmes can be classi- and β-Sitosterol effect on the liposome membrane features, they found fied into conventional liposomes , charged liposomes , stealth 4 H. Nsairat et al. Heliyon 8 (2022) e09394 Figure 5. Mixed and different types of synthetic phospholipids; A) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, B) 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt), C) L-a-phosphatidylcholine, D) 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine, E) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and F) 1,2-dimyr- istoyl-sn-glycero-3-phosphocholine. Figure 7. Chemical structure of surfactants A) sodium cholate, B) Sodium dodecyl sulfate (SDS). Figure 6. Chemical structure of A) cholesterol, B) β-sitosterol. 5 H. Nsairat et al. Heliyon 8 (2022) e09394 stable liposomes , actively targeted liposomes , stimuli-responsive specific receptors ligands to achieve narrowed distribution, and accu- liposomes , and bubble liposomes. mulation at the intended site. Huayluorinic acid , polyvinyl alcohol (PVA), and polyethylene glycol (PEG)) were considered the best 2.2.1. Conventional liposomes model for liposome steric protection. PEGylated liposomes are denoted as These liposomes were synthesized from natural or synthetic phos- stealth liposomes [97, 98]. Doxil® was the first successful pegylated pholipids with or without cholesterol as a liposomes first generation liposome-based product. Stealth stabilized liposomes showed longer. Cholesterol was added to improve liposomes fluidity, altering the circulation time, leading to a better target accumulation than conven- bilayer rigidity and liposomes stability [31, 32]. Wu et al hat revealed tional liposomal drugs. that liposomal membrane rigidity decreased with the addition of cholesterol into a liposomes composed of hydrogenated soybean phos- 2.2.4. Actively-targeted liposomes pholipids (HSPC) and DSPE-PEG2000. Moreover, liposomes with some Actively-targeted liposomes represent third-generation liposomes. rigidity showed excellent tumor penetration and enhanced anti-tumor Liposomes' active targeting increases the selectivity of liposome inter- activity. Kaddah et al investigated the cholesterol content on the action with diseased cells and triggers receptor-mediated endocytosis of permeability and fluidity of DPPC liposome membrane. High cholesterol the liposome and its payload into the desired cellular target [101, 102]. concentration increased the average liposomes size accompanying with a Many liposomes nanocarriers have been approved for anti-tumor shape transition from irregular to nanosized, regular and spherical ves- agents delivery by passive ways based on the enhanced permeability icles. In addition, cholesterol induced a decrease in the bilayer fluidity and retention (EPR) effect of cancerous cells. Passive targeting and modulating the release of hydrophilic molecules from lipid vesicles does not discriminate between normal and diseased cells ; there-. Jovanovi
c et al reported that increasing cholesterol content in the fore, cell-specific targeting liposomes have been developed to increase liposomes decreased the fluidity and enhanced the rigidity of liposomal the accumulation and localization of anti-tumor agents in diseased cells membranes. They verified that a stable liposome should have an optimal. Liposomes targeting can be enhanced by incorporating molecular 50 mol %. concentration of cholesterol to obtain and an appropriate recognition moieties, which can lead to drug transport with better effi- membrane fluidity. As a result, cholesterol plays a crucial role in cacy and low side effects. For example, liposomal targeting stra- liposomes bilayer fluidity and rigidity, but these properties affected by tegies have utilized simple peptides , proteins (including the cholesterol molar ratio with types of phospholipids used along with antibodies) or protein fragments , carbohydrates, nucleic acids, or the nature of the encapsulated drug. Conventional liposomes showed a vitamins [108, 109, 110, 111, 112]. short blood circulation time due to their susceptability to elimination by Both active (ligands-conjugated) and passive (‘non’-conjugated) tar- the mononuclear phagocyte system with rapid accumulation in liver geted liposomes are distributed to target cells via the same passive dis- and spleen. Hence, MPS obstructs the delivery of conventional liposomes tribution mechanism. The field of ligand-targeted liposomes has to the target region and restricts their distribution to other tissues of the expanded rapidly despite that several non-targeted liposomes have body. Conventional liposomes also showed relatively limited sta- reached the clinic or in clinical trials [114, 115]. bility in vitro. As a result, stealth stable liposomes were invented to New efforts in targeted drug delivery systems utilize polyunsaturated increase blood circulation and enhanced in vivo liposomes stability. fatty acids, folic acid, hyaluronic acid, or oligopeptides as tumor recog- nition moieties. These ligands encounter many discussion fields around 2.2.2. Charged liposomes their affinity and specificity with no detailed mechanism of tumor- Oleic acid and N-[1(2,3-dioleoyloxy) propyl]-N,N,N-trimethylam- targeting accompanied to limited success for certain small ligands monium chloride (DOTAP) are usually used to prepare anionic and , besides the enzymatic degradation in the systemic circulation, cationic liposomes, respectively. Charged liposomes showed higher making them inappropriate for many in vivo studies. Recently, liposomal stability during the storage, as charged particles repel each aptamers and aptamer-functionalized nanoparticles high affinity and other and reduce aggregation abilities. Cationic liposomes are used in specificity have great attention in targeted drug delivery systems [118, gene therapy due to their ability to successfully encapsulate nucleic acids 119, 120]. by electrostatic attractions. Active targeting of the nanocarriers can be achieved through non- Cationic liposomes are suitable for delivering various negatively covalent or covalent conjugation of targeting ligands to the drug mole- charged macromolecules such as DNA, RNA, and oligonucleotides cule or to the surface of nanocarrier to bind the overexpressed targeting because their negative charge and rather a large size restrict their passive biomarkers selectively on the tumor cells [110, 121]. The direct conju- diffusion into cells. Cationic liposomes also selectively target gation of drugs to the targeting ligand can disrupt the receptor/ligand angiogenic endothelial cells in tumors. Cationic liposomes are recognition and may alter the drug efficacy. Nano-carrier considered a potential tool for delivering therapeutics to the brain [6, active targeting enables drugs to be localized within the action site 86]. Cationic liposomes can cross the BBB by receptor-mediated trans- with higher effectiveness to reduce drug dose, minimize the drug cytosis or absorptive-mediated transcytosis. The higher posi- side effects and reduced drug variation in blood concentration. tive charge on the surface of cationic liposomes may affect their blood Stealth and conventional liposomes usually showed a slow release of the circulation and lead to due to electrostatic interactions with anionic loaded drugs and failed fusion with the endosome after internalization. species in the blood and increase liposomes aggregation that reduces Consequently, stimuli-responsive liposomes have been introduced to their localization site of action [89, 90]. Decorating the surface of these overcome these challenges. liposomes with poly ethylene glycol (PEG) protects the them from the circulating proteins, improving the drug efficiency through improving 2.2.5. Stimuli-responsive liposomes systemic circulation time and decrease immunogenicity [91, 92]. Stimuli-responsive liposomes are smart liposomal systems that Anionic liposomes are less stable in the bloodstream than neutral and display rapid release of their drug payload upon physicochemical or cationic liposomes; they showed a higher clearance rate [44, 93]. Anionic biochemical stimuli, such as pH, temperature, redox potentials, enzymes liposomes are usually utilized for transdermal drug delivery because they concentrations, ultrasound, electric or magnetic fields. improve penetration properties through the stratum corneum of the skin Stimuli-responsive liposomes should contain a certain constituent. that controls the lipid bilayer's stability and permeability. There are two basic kinds of inductions, remote and local. Remote inductions 2.2.3. Stealth stabilized liposomes respond to outside stimuli including, heat, magnetic field, light, electric These second-generation liposomes are characterized by surface field, and ultrasounds [73, 125]. Local triggering releases respond to decoration with synthetic polymers, glycoproteins, polysaccharides, or stimuli inside the target tissues, such as pH, redox potential [127, 128], 6 H. Nsairat et al. Heliyon 8 (2022) e09394 Table 1. The most common stimuli-responsive liposomes. Stimuli liposomes Stimuli Principle Advantages Reference Light-sensitive liposomes UV, near infrared or visible light Modification of fatty acyl chains of Controlling time, exposure, [131, 132] irradiation, the phospholipids with light- wavelength, and intensity sensitive functional groups and the resulting phospholipids have yielded photoactivable liposomes Thermosensitive (temperature- Radiofrequency or microwave Lipids with a transition Drug release at high-temperature [133, 134, 135, 136] sensitive) liposomes ablation temperature of 40–45  C, such as sites DPPC, have been employed to make these liposomes Redox-sensitive liposomes Reactive oxygen species (ROS) Depends on the redox potential ROS leads to high concentration [137, 138] peroxides, hydroxyl radicals, difference between the levels of glutathione (GSH) in singlet oxygen intracellular reducing space and tumor cells cleaving the liposomal oxidizing extracellular space that formulations occur during biological activities. Enzyme-responsive liposomes Protease, amidase, and esterase Based on amides or esters Decreases the adverse side effects [139, 140, 141] enzymes hydrolysis by protease or esterase of toxic drugs and enable enzymes release loaded drugs. encapsulation of prodrugs pH-sensitive liposomes pH change Cholesteryl hemisuccinate Liposomes with pH-dependent [142, 143, 144] (CHEMS) and 1,2-dioleoyl-sn- release features glycero-3-phosphoethanolamine (DOPE), were used to prepare pH- sensitive liposomes and enzymes. Table 1 represents the most common 2.3.1. Thin film hydration method (Bangham method) stimuli-responsive liposomes that respond to specific triggers that lead to In this method, all lipids and the hydrophobic drug are dissolved in a controlled release nanosystem, enhanced intracellular distribution suitable organic solvent using a round-bottom flask. The organic. solvent then evaporated gently under reduced pressure to create a thin film layer. The obtained thin film is then hydrated, at above the 2.2.6. Bubble liposomes transition temperature (Tm) of the used lipid, with an aqueous buffer Bubble liposomes (gas-encapsulated liposomes) are expected to solution. The hydration solution may contain a hydrophilic drug/s to be create new applications in the field of gene delivery and drug delivery loaded into the liposomes aqueous core. The rate of hydration determines systems. Recently, liposomes have been used to encapsulate the efficiency of drug encapsulation , which the slower the rate of bioactive gases and/or drugs for ultrasound-controlled drug release with hydration, the higher the encapsulation efficiency. Liposomes enhanced drug delivery. Nitric oxide (NO) bubble liposomes offer resizing, lamellarity types and particles distributions may be controlled a distinguishing NO intravenous therapeutics option overcome common by either extrusion through a polycarbonate membranes of specific pore microbubbles, in which liposomes shield NO from hemoglobin sizes or the use of bath or probe sonicators. Extrusion method ensures rummaging in vitro as usually occurred by free NO. Oxygen bubble stable liposomes with more encapsulation efficiency over sonication. liposome (OBL) enables high oxygen fixations with high pO2 conditions Sonication usually produce SUVs liposomes and may also degrade or of the lungs. This separates OBL from great fluorocarbon and hydrolyze encapsulated drugs and/or lipids. Probe sonication may sub- hemoglobin-based oxygen transporters and keeps their utilization as ject liposomes suspensions to potential metal contamination (Figure 8) supported oxygen conveyance stages.. 2.3. Methods of preparation 2.3.2. Reverse-phase evaporation method The reverse-phase evaporation method is usually used as an alterna- Liposomes can be formulated using different approaches. The process tive to thin-film hydration by forming a water-in-oil emulsion. of liposome manufacture and the phospholipids type critically affects the First, the lipids are dissolved in an organic solvent which is then directly final liposomes characteristics. Liposome's fabrication procedures mixed with an aqueous buffer containing the hydrophilic drug. The can be classified into: organic solvent then evaporated under a reduced pressure rotary Figure 8. Liposomes preparation via thin-film hydration extrusion technique. 7 H. Nsairat et al. Heliyon 8 (2022) e09394 Figure 9. Schematic representation of injection methods method. evaporator leading to form lipid vesicles dispersed in the aqueous solu- tion. The average size and polydispersity of the preformed vesicles can be reduced by extrusion. This method is suitable for high molecular Figure 10. Schematic representation of injection methods method. weight molecules, but therapeutic peptides may be denatured due to organic solvents and to sonication conditions. The hydrating agents act as a stabilizer and isotonizing additives that prevent nanoparticle coagulation and sedimentation. Moreover. The 2.3.3. Solvent injection methods hydration agents provide a cryoprotective effect that makes the heating The injection methods were classified according to the type of organic method an efficient method for the formulation of powder inhalable li- solvent used (Figure 9). An organic solvent dissolving the lipids posomes. and the hydrophobic active agents were rapidly injected into an aqueous phase. Diethyl ether enable direct solvent evaporation during mixing 2.3.7. pH jumping method process at a temperature above to the boiling point of the used solvent Another solvent-free method for liposomes preparation is the pH. Utilizing ethanol for injection required a 10-to-20-fold aqueous jumping method. In this method, the aqueous solution of phosphatidic solution and ethanol can be evaporated under vacuum using a rotary acid and phosphatidylcholine are exposed to almost four-fold increase in evaporator, dialysis, or filtering. This method mostly prepared liposomal pH over a short time to break down MLVs into SUVs [167, 168]. The ratio formulations with higher polydispersity indexes (PDI). In addition, of phosphatidic acid: phosphatidyl choline determine the percentage of continuous exposure to high temperature and organic solvent might SUVs versus LUVs produced. reduce drug and lipids stability. 2.3.8. Microfluidic channel method 2.3.4. Detergent removal method The microfluidic channel method (Figure 10) has been recently pro- In this method, lipids and a high critical micelle concentration (CMC) posed as a novel method for liposomes preparation. Microfluidics pro- surfactant were dissolved in a suitable organic solvent using a round vides a tool to employ liquids within microscopic channels. In this bottom flask. A thin film was obtained at the bottom of the flask after method, lipids are dissolved in ethanol or isopropanol, and the resultant solvent gentle evaporation. A mixed micelles solution then ob- solution is injected upright or in the opposite direction to the aqueous tained by hydrating the lipid film in an aqueous solution containing the medium within the micro-channels. This method involves continuous drug molecules. The surfactant is then removed by dialysis, axial mixing of the organic and aqueous solutions leads to liposomes size-exclusion chromatography, adsorption onto hydrophobic beads or formation. Liposomes are stabilized using surfactants to avoid coagula- dilution [157, 158, 159, 160]. Finally, a LUVs liposomes vesicle will be tion and separation. Microfluidic channel methods control the formulated after solution concentration. A main drawback of this mixing process of organic and aqueous phases to achieve reproducible method is that most hydrophilic drugs are separated from the liposomes liposomes with proper average size, polydispersity, morphology, and during detergent removal step. lamellarity. 2.3.5. Dehydration-rehydration method 2.3.9. Supercritical fluidic method It is an organic solvent free method to produce LUVs using sonication. This method utilized a supercritical fluid, carbon dioxide (CO2), to This method based on direct dispersing of the lipids at low concentrations dissolve lipids instead of using organic solvents. A high-performance into an aqueous solution containing the drug molecules followed by liquid pump provides a continuous flow of the aqueous phase into a sonication. First, the dehydration step to evaporate water under cell that contains the supercritical lipid solution, allowing phase transi- nitrogen to create multilayered film entrapping the drug molecules. tion of the dissolved phospholipids. Upon abrupt decrease in Then, a hydration step to form large vesicles encapsulating the drug pressure, liposomes will formed after completely removing of CO2. 5-fold molecules [50, 163]. This method is simple but with high heterogeneity higher encapsulation efficiencies were obtained by this method. This of the liposomes sizes. method suffers from high cost, low yield, and special infrastructures even with using the environmentally safe and cheap carbon dioxide. 2.3.6. Heating method It is also an organic solvent free technique. In this method, lipids are 2.4. Post preparation handlings hydrated directly with aqueous solution, and heated for not less than one hour above the Tm of the used phospholipids in the presence of a 3–5 % 2.4.1. Freeze-thaw cycles hydrating agent as glycerin or propylene glycol. The suspension can be This technique is usually used during liposomes preparations to in- heated up to 100  C when adding cholesterol to the formulation. crease the encapsulation efficiency and to enhance liposome lamellarity. 8 H. Nsairat et al. Heliyon 8 (2022) e09394 drugs are weak bases possessing primary, secondary, or tertiary amine Table 2. Represent different techniques used for the assessment of liposome functional groups that can be loaded in response to pH gradients. parameters. Drugs that are not weak bases, or do not have an ionizable functional Liposomes characteristics Characterization technique References group, can be converted to weak base prodrugs or encapsulated with Average particle size Dynamic light scattering (DLS) [179, 180] amino-modified carriers as cyclodextrins, therefore allowing encapsula- and microscope technology: tion and intraliposomal retention [193, 197, 198]. Scanning and transmission electron microscopy (SEM/TEM), cryogenic-TEM (Cryo-TEM), and 3. Protein corona fingerprints of liposomes atomic force microscopy (AFM) Zeta potential/Surface charge Electrophoretic mobility, DLS Liposomes have been used to overcome many problems associated Particle shape/morphology TEM, Cryo-TEM, and AFM with low efficiency of anticancer drugs. Recently, a concept is Lamellarity Cryo-TEM and 31P-NMR emerging that the limited success of liposomal drugs in clinical practice Phase behavior X-ray diffraction (XRD), [183, 184] due to poor knowledge of liposomes behavior in vivo. Lipid vesicles are differential scanning calorimetry usually covered by plasma proteins in vivo forming a biomolecular (DSC) and thermogravimetric coating, referred to as the protein corona (PrC). Recent studies analysis (TGA) verified that PrC fingerprints (PrCFs) enhanced liposome attachment Encapsulation efficiency/Drug Centrifugation, dialysis followed [185, 186] with cancer cells, triggering efficient particle localization and internali- release by drug content determination using chromatographic and/or zation. spectrophotometric methods Accordingly, enrichment in PrCFs was utilized to predict the targeting ability of synthesized liposomal formulations. Palchetti et al reported that the targeting capability of liposome–protein complexes clearly relate This approach utilized a freeze-thaw cycles between -196  C in liquid with cellular uptake in pancreatic adenocarcinoma (PANC-1) and insu- nitrogen and below the transition temperature of the used phospholipids linoma (INS-1) cells as quantified by flow-assisted cell sorting (FACS). lipids [175, 176]. The results showed that cellular uptake of the liposomal formulation with the highest abundance of PrCFs was much larger than that of Onivyde®, 2.4.2. Freeze-drying (lyophilization) an Irinotecan liposomal drug approved by the Food and Drug Adminis- This treatment is applied to preserve the liposomal products and tration in 2015 for the treatment of metastatic Pancreatic ductal improve their shelf stability. Freeze-drying involves deep freezing of the adenocarcinoma (PDAC). Furthermore, Digiacomo et al identified liposomes suspension after mixing with a cryoprotective, mainly 5–10 % a potential protein biomarker for pancreatic ductal adenocarcinoma sucrose or trehalose. Then, a sublimation step at very low tem- (PDAC) by utilizing liposomes to accumulate PrC coating layer from perature and a reduced vacuum was applied to convert the liquid samples human plasma proteins. These targeting liposomes may be used for the to fluffy solid particulates. Lyophilization becomes essential treatment early diagnosis of PDAC. This approach could open the interesting for liposomes encapsulating thermo-sensitive biomolecules. possibility to identify novel biomarkers for liposomes formulations in the context of personalized medicine. 2.5. Liposomes characterization 4. Liposomes in clinical applications Liposome physiochemical characterization include average size and size distribution (or polydispersity index (PDI)), surface charge (or Zeta Various liposomal-based formulations were successfully implemented potential), shape and morphology, lamellarity, encapsulation efficiency, in clinical fields as antitumor, anti-fungal therapies, analgesics. phase behavior (or polymorphism) and in vitro release profile (Table 2). Doxil® was the first approved clinical anticancer liposome drug in the USA (1995). It opened the way to several other liposomal formulations to 2.6. Liposomes drug loading get to the clinical application fields by innovating the pH gradient active loading and usage of PEGylation for stealth liposomes [203, 204]. Con- Liposomes drug loading can be attained by passive or active ap- ventional liposome without PEGylation, can be attractive when circula- proaches. Passive loading entraps hydrophilic drug in the lipo- tion half-life is not the goal. DepoFoam™ is mostly used for gradual somes aqueous core during lipid bilayer formation, while hydrophobic drug release, thus maintaining a continuous drug supply for long-lasting drugs accumulate in the small-sized hydrophobic lipid bilayer [103, 187, effect. 188, 189]. Passive loading suffers from bilayer destabilization, high drug/lipid ratio, and rapid drug release. Therefore, improving the 4.1. Marketed clinical liposomes aqueous solubility of these hydrophobic drugs by cyclodextrin host-guest complexation were successfully applied and permitliposomes aqueous 4.1.1. Cancer treatment core loading by forming drug-in-cyclodextrins-in-liposomes delivery Doxil® or Caelyx® was presented in 1995 by Sequus Pharmaceuticals. system. Doxil was designed as a polyethylene glycol coated doxorubicin (DOX) Active or remote loading has been developed to ensure high encap- liposome intended for the treatment of Kaposi's sarcoma. LipoDox® sulation efficiency of precious chemotherapeutic agents. Remote is another FDA approved PEGylated liposomal formulation encapsulating loading can be achieved into preformed liposomes by pH gradient and/or DOX manufactured by Sun Pharma in 2012. Daunorubicin was the potential ionic differences across liposomal bilayer membranes [101, second anthracycline antineoplastic drug loaded in liposomes to treat 187, 192]. The success of intraliposomal remote loading are govern by to acute myeloid leukemia (AML) under the generic name DaunoXome® main parameters, (i) drug aqueous solubility (ii) presence of an ionizable. Myocet® is a non-PEGylated liposomes encapsulating DOX that functional group in drug chemical [192, 193, 194]. showed a shorter circulation half-life with less cardiac side effects [205, Intraliposomal active loading of hydrophobic drugs in response to 209]. ionic and/or pH gradients across the liposomes bilayer was developed Depocyt® consists of Citarabine, a cell-cycle cytotoxic drug, enclosed [194, 195]. This procedure enables hydrophobic drugs to accumulate in the DepoFoam™ multivesicular enclosure, which allows a sustained inside the liposomes core after the vesicles are created. The advantage of two-week release. A new liposomes formulation called Mepact® this method is that the loading of the drug can be performed indepen- was globally approved for the treatment of osteosarcoma. dently of liposomes preparation conditions. Most potentially active Vincristine also incorporated into sphingomyelin/cholesterol-based 9 H. Nsairat et al. Heliyon 8 (2022) e09394 Table 3. Clinically used liposomes grouped by therapeutic usage. Usage Trade name Active ingredient(s) Liposome platform Manufacturer Year Administration References (Molar Ratio) Approved Route Anti-Cancer Doxil® Doxorubicin HSPC:Cholesterol:PEG Sequus Pharmaceuticals 1995 I.V 2000-DSPE (56:38:5) DaunoXome® Daunorubicin DSPC:Cholesterol (2:1) NeXstar Pharmaceuticals 1996 I.V Depocyt® Cytarabine DepoFoam™ SkyPharma Inc. 1999 Spinal Myocet® Doxorubicin Cholesterol:EPC (45:55) Elan Pharmaceuticals 2000 I.V Mepact® Mefamurtide DOPS:POPC (3:7) Takeda Pharmaceutical 2004 I.V Multilamellar liposome Limited Lipodox® Doxorubicin DSPC:Cholesterol:PEG Sun Pharma 2012 I.V [205, 207] 2000-DSPE (56:39:5) Marqibo® Vincristine SM:Cholesterol (60:40) Talon Therapeutics 2012 I.V Onivyde™ Irinotecan DSPC:Cholesterol:MPEG- Merrimack 2015 I.V 2000-DSPE (3:2:0.015) Pharmaceuticals Lipusu® Paclitaxel NA Luye Pharma Group 2006 I.V Vyxeos® Cytarabine:Daunorubicin DSPC:DSPG:Cholesterol Jazz Pharmaceuticals 2017 I.V [214, 221] 5:1 (7:2:1) Anti-Fungal Ambisome® Amphotericin B HSPC:Cholesterol:DSPG Astellas Pharma 1997 I.V (2:1:0.8) Fungisome® Amphotericin B PC:Cholesterol (7:3) Lifecare Innovations 2003 I.V Photodynamic Visudyne® Verteporphin Verteporphin:DMPC&EPG Novartis AG 2000 I.V therapy (1:8) Analgesic DepoDur™ Morphine sulfate DepoFoam™ SkyPharma 2004 Epidural ® Exparel Bupivacaine DepoFoam™ Pacira pharmaceuticals 2011 I.V liposome under the name of Marqibo®. This approved formula offered Lipusu® liposomes to treat gastric carcinoma efficiently with much less longer circulation time without surface-modified, resulting in a higher adverse effects. accumulation in target tissues in which vincristine is gradually released. Onivyde® is another PEGylated liposome carrying irinotecan and 4.1.2. Fungal treatment exhibits a long-acting, antitumor effect. In addition, Vyxeos® also A two major approved anti-fungal liposomes formulation were known as CPX-351, is composed of a combination of Cytarabine and Ambisome® and Fungisome®. They encapsulate Amphotericin B anti- daunorubicin, encapsulated in a liposome in a ratio of 5:1. This formu- fungal drug with many advantages compared free drug [216, 217]. lation reduced adverse effects with enhanced effectiveness [213, 214]. These Amphotericin B liposomes were stabilized in saline and have Finally, Paclitaxel, an anticancer drug, was also incorporated into longer bioavailability and less toxicity and side effects [218, 219]. Figure 11. Active clinical trials as per 28/dec/2021, source: https://ClinicalTrials.gov. 10 H. Nsairat et al. Heliyon 8 (2022) e09394 4.1.3. Photodynamic therapy Nevertheless, liposomes critical challenges are their physical and chem- Visudyne®: is the only liposomal drug delivery agent approved for ical stability. As a result, there are a n essential need to develop liposomes age-related macular degeneration therapy by inhibiting the generation of with high stability significantly impacts their clinical application. Thus, blood vessels in the eye. in silico simulation and computational investigations may enable approximate estimation for the best liposomal formulation in their con- 4.1.4. Pain management stituents and 3-D structure morphology. DepoDur™ is a morphine formulation using DepoFoam™ Technology that resulted in a sustained release formula with prolonging the clinical Declarations effect time. Exparel® also uses the DepoFoam™ technology to release Bupivacaine for sustained pain relief gradually. Author contribution statement Table 3 summarizes the different clinically approved liposomes for- mulations in terms of their purpose, lipid constituents, active ingredients, All authors listed have significantly contributed to the development and administration route. and the writing of this article. 4.2. Liposomes in clinical trials Funding statement From the 83316 active clinical trials registered, 511 liposomal clinical This research did not receive any specific grant from funding agencies trials investigating liposomal products which are distributed worldwide in the public, commercial, or not-for-profit sectors. as shown in Figure 11. The drugs being examined belong to anticancer drugs, analgesics, immune-modulators, anti-fungal, etc. Among these drugs, 121 of the 511 are in phase III testing, 236 are in phase II, 120 are Data availability statement in phase I, and 6 in early phase I. No data was used for the research described in the article. 4.3. Liposomes in vaccinations Declaration of interests statement Liposome formulations could protect DNA/RNA and proteins payload from biodegradation. Furthermore, their transfection efficiency could be The authors declare no conflict of interest. enhanced by modifying surface charge, size, and lipid structure. Two commercial vaccines based on virosome technology are currently on the market, Epaxal® and Inflexal® V (Berna Biotech Ltd, Bern, Switzerland), a Additional information hepatitis A vaccine. Virosomes are liposomal formulations that have viral envelope proteins anchored to their lipid membrane. No additional information is available for this paper. 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