A Review of Liposomes as a Drug Delivery System: Current Status of Approved Products, Regulatory Environments, and Future Perspectives

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Abstract

Liposomes have been considered promising and versatile drug vesicles. Compared with traditional drug delivery systems, liposomes exhibit better properties, including site-targeting, sustained or controlled release, protection of drugs from degradation and clearance, superior therapeutic effects, and lower toxic side effects. Given these merits, several liposomal drug products have been successfully approved and used in clinics over the last couple of decades. In this review, the liposomal drug products approved by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) are discussed. Based on the published approval package in the FDA and European public assessment report (EPAR) in EMA, the critical chemistry information and mature pharmaceutical technologies applied in the marketed liposomal products, including the lipid excipient, manufacturing methods, nanosizing technique, drug loading methods, as well as critical quality attributions (CQAs) of products, are introduced. Additionally, the current regulatory guidance and future perspectives related to liposomal products are summarized. This knowledge can be used for research and development of the liposomal drug candidates under various pipelines, including the laboratory bench, pilot plant, and commercial manufacturing.

1. Introduction

Liposomes are self-assembled (phospho)lipid-based drug vesicles that form a bilayer (uni-lamellar) and/or a concentric series of multiple bilayers (multilamellar) enclosing a central aqueous compartment [1]. The size of liposomes ranges from 30 nm to the micrometer scale, with the phospholipidbilayer being 4–5 nm thick [2]. The field of liposomology was launched by the British scientist Alec Bangham and colleagues at Babraham Cambridge in the mid-1960s [3], and they first published the structure of liposomesin 1964 [4]. Since then, liposomes have been widely investigated as delivery vehicles for small molecular drugs, protein, nucleic acid, and imaging agents [5,6,7,8,9]. Different administration routes, such as parenteral, pulmonary, oral, transdermal, ophthalmic, and nasal routes, have been developed to improve therapeutic efficacy and patient compliance [10,11,12,13,14]. In addition, liposomes have been widely applied in the fields of food [15] and cosmetics [16].

As drug vehicles, liposomes exhibit outstanding properties, such as protecting the encapsulated substances from physiological degradation [17], extending the half-life of the drug, controlling the release of drug molecule s [18], and excellent biocompatibility and safety. Furthermore, liposomes can selectively deliver their payload to the diseased site through passive and/or active targeting, thus decreasing the systemic side-effect, elevating the maximum-tolerated dose, and improving therapeutic benefits [19,20].

Unlike normal tissue with tight intracellular junctions (2–6 nm) between endothelial cells [21], abnormal tissues such as a solid tumor or inflammatory site have highly porous capillaries (100 nm–2 µm depending upon the size and type of tumor tissue [22]). Liposomes can cross over the discontinuous neovasculature and be passively accumulated and detained at the abnormal tissues, which is called the enhanced permeability and retention (EPR) effect. Actively targeting employs specific interactions between the ligands and receptors on the surface of liposomes and tumor cells, respectively. Tumor cells may overexpress specific receptors, such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), folic acid (FA), integrin, CD44 (a cell surface glycoprotein), CD13, and prostate-specific membrane antigen [23]. According to these receptors, specific ligands, such as antibody [24], nuclear acid (e.g., aptamers [25]), protein (e.g., transferrin [26]), peptides (e.g., iRGD [27], iNGR [28]), small molecules (folic acid [29]), and carbohydrates (e.g., dextran, mannose, and galactose [30], targeting macrophages) were proposed for the surface modification of liposomes.

Besides the specific medicines, liposomes stand as an excellent technique for drug delivery. However, only 14 types of liposomal products are available on the market, which means the advantages of liposomes have not been fully exploited. Therefore, in this review, we summarized the knowledge about commercial liposomal products approved by the FDA and EMA. Attention is paid to the composition and manufacturing technologies adopted in commercial products. In addition, the CQAs of liposomes, the current regulatory environment, and future perspectives are introduced. The purpose of this review is to provide important reference information to accelerate the development of liposomes.

2. The Marketed Liposomal Products

We searched the approved drug database published on the website of the FDA and EMA, and found that 14 types of liposomal products have been authorized ( Table 1 ). It should be noted that this list excludes generics, lipid complexes (e.g., Abelcet, Amphotec, and Onpattro), and nationally authorized liposomal products in Europe. Doxil(Doxorubicin HCl liposome injection) was the first liposomal product approved by the FDA in 1995. Among these marketed products, 43% of products were approved before the year 2000, and 57% of products were approved before the year 2010. The therapeutic area mainly focuses on cancer therapy but also involves other areas, such as infection, anesthesia, vaccine, lung disease, and photodynamic therapy. The dosage forms are mainly focused on sterile suspension and lyophilization powder. The administration routes include intravenous infusion, intramuscular and intrathecal injection, epidural, local infiltration, and oral inhalation.

Table 1

Summary of liposomal products approved by FDA and EMA.

This list is only for liposomal forms approved by FDA and EMA, excludes generics (e.g., doxorubicin hydrochloride (liposomal), lipid complexes (e.g., Abelcet, Amphotec, and Onpattro), and also excludes the nationally authorized liposomal products in Europe. Abbreviations: intravenous infusion (IV), intramuscular injection (IM), intrathecal injection (IT), lyophylization (Lyo), muramyl tripeptide phosphatidyl ethanolamine (MTP-PE).

3. Structures and Main Components of Liposomes

3.1. Structures of Liposomes

Liposomes can be classified as unilamellar vesicles (ULVs), oligolamellar vesicles (OLVs), multilamellarvesicles (MLVs), and multivesicular liposomes (MVLs) depending on the compartment structure and lamellarity ( Figure 1 ) [31]. OLVs and MLVs show anonion-like structure but present 2–5 and >5 concentric lipid bilayers, respectively. Different from MLVs, MVLs include hundreds of non-concentric aqueous chambers bounded by a single bilayer lipid membrane and display a honeycomb-like structure [32]. Based on the particle size, ULVs can be further divided into small unilamellar vesicles (SUVs, 30–100 nm), large unilamellar vesicles (LUVs, >100 nm), and giant unilamellar vesicles (GUVs, >1000 nm) [33]. Different size range of ULVs was reported, i.e., SUVs with a size of less than 200 nm and LUVs with a size of 200–500 nm [34].

Categories and structures of liposomal drug delivery system. (a) Structural illustration of liposome composition. The size of a typical phospholipid bilayer is 4.5 nm, which is much smaller than the one of the inner aqueous core; (b) Classification of liposomal vesicles according to their lamellarity/compartment and particle size; (c) The size and lamellarity of different types of liposomes; (d,e) The cryo-transmission electron microscopy of Doxil [35] and Vyxeos [36]; (f,g) The electron micrographs of DepoFoam TM particles with a typical diameter of 1–100 μm (e.g., DepoCyt) and MLVs with a typical diameter of 0.2–5 μm (e.g., Mepact) [37].

The particle size and the structures of the commercial products are concluded in Table 2 . Most of the current commercial products are SUVs (e.g., Doxil ( Figure 1 d) because of the long circulation time and ability to passively target the diseased site. Arikaye (amikacin liposome inhalation suspension) is considered an LUV because of its large particle size (200–300 nm). Vyxeos (daunorubicin: cytarabine liposome for injection) is a bilamellar liposome system ( Figure 1 e), which is generated during the first drug cytarabine loading procedure. The mechanism of internal lamella formation is explained as a thermodynamic response of the lipid bilayer to decrease the surface area-to-volume ratio of the liposomes caused by water egress in response to an external osmotic challenge [36]. Myocet (liposomal doxorubicin) and Mepact (liposomal mifamurtide powder for concentrate for dispersion for infusion) ( Figure 1 g) are MLVs. The abundant lamellae provide a large space for the encapsulation of lipophilic compounds [38].

Table 2

The particle size, structure, and lipid components of the commercial liposomal products.

Abbreviations: fully hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dierucoyl phosphatidylcholine (DEPC), palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoyl phosphatidylserine(DOPS), dioleoylphosphatidylserine (OOPS), cholesterol (Chol), sphingomyelin (SM), N-(carbonyl-methoxypolyethlyeneglycol-2000)-distearolyphosphatidylethanolamine (MPEG-2000-DSPE).

There are four products with micron diameters, i.e., Mepact, DepoCyt (cytarabine liposome suspension), DepoDur (morphine sulfate extended-release liposome injection), and Exparel (bupivacaine liposome injectable suspension). Mepactis a sterile and lyophilized cake, and will form multilamellar liposomes with a particle size of 2.0–3.5 µm after reconstituted with 0.9% saline solution. This size is ideal for recognition and phagocytosis by monocytes/mocrophages, and triggers the macrophages and the immunomodulatory effects for cancer therapy. DepoCyt, DepoDur, and Exparel are manufactured by the same DepoFoam technique. The MVLs structure ( Figure 1 f) can load a large volume of drug–aqueous solution because of the numerous chambers and provide sustained release due to the erosion/degradation of liposomes and diffusion of drug molecules [39,40].

3.2. Main Components of Liposomes

Table 2 shows that glycerolphospholipid (GP), sphingomyelin (SM), and cholesterol (Chol) are the basic components used in the marketed products. GP contains glycerol, which links a pair of hydrophobic fatty acid chains and a hydrophilic polar head group [48]. The types of fatty acid and polar heads are described in Figure 2 a. Under the physiological pH, different head groups provide liposomes with negative (PA, PS, PG, and cardiolipin) or neutral (PC and PE) charges [49].

(a) Structural illustration of glycerolphospholipid. R1 and R2 can be saturated or unsaturated fatty acids, such as decanoic acid, lauric acid, palmitic acid, oleic acid, myristic acid, stearic acid, and erucic acid. R3 can be phosphatidylcholine (PC), phosphatidyl ethanolamine (PE), phosphatidyl serine (PS), phosphatidyl inositols (PI), phosphatidic acid (PA), phosphatidylglycerol (PG), and cardiolipin; (b) Structure of sphingomyelin. (c) Structure of cholesterol.

Negatively charged DSPG used in AmBisome (ambisome liposome for injection) can interact with the positively charged amine group of AmpB to form a stable ionic complex [50], while DSPG used in Vyxeos minimize liposome aggregation by a strong Coulombic repulsive force [51]. DSPC used in DaunoXome (daunorubicin citrate liposome injection), Onivyde (irinotecan liposome injection), and Vyxeos is a neutral and synthetic lipid with well-defined fatty acid composition (two molecules of stearic acid), high purity, and a relatively high phase transition (T_m of 55 °C).

EPC is adopted as an excipient in Myocet and Visudyne (verteporfin powder for solution for infusion). EPC is a naturally sourced phospholipid (NPL) purified from egg yolk. Compared to semi-synthetic and synthetic lipids, NPL exhibit a low production cost, but a broad transition temperature, problematic to obtain completely identical NPL and potential batch variation of liposomes [52]. In addition, the unsaturated fatty acid of EPC leads to a low phase transition temperature of −15~−5 °C [53], indicating the liposome bilayer is in a disorder and drug “leaky” state in the body temperature. Myocet, composed of EPC, is unstable in blood, and most drugs are released after 24 h [54]. Visudyne composed by EPC and DMPC also exhibits less stability in the presence of serum. The verteporfin rapidly transfers from the disordered liposome membrane and associates with the plasma lipoproteins, then reaches higher levels in the neovasculature since low-density lipoprotein (LDL) receptors are abundant in neovascular tissue [55,56].

MethoxyPEG (Mw 2000 Da), covalently attached to DSPE (MPEG-DSPE) used in Doxil and Onivyde, provides “stealth” and sterically stabilized liposomes. The molecular weight of PEG and the mole percentage of PEG-DSPE in lipid composition play important roles on the bilayer packing, circulation time, and thermodynamic stability. The high molecular weight of PEG (>2000 Da) grafted to the lipid headgroup exhibits repulsive forces from the liposome surface, as well as protects liposomes from binding serum proteins and avoids further clearance by the mononuclear phagocytic system (MPS), but also decreases the interaction and endocytosis of liposomes by targeted cells [57]. The low molecular weight of PEG (

DepoCyte, DepoDur, and Exparel have special structures and similar lipid components. A minimum of two types of lipids in the formulation are required for MVLs formation, the amphipathic lipid and the neutral lipid (e.g., diglycerides, triglycerides, vegetable oil) [61]. DOPC and DEPC are amphipathic zwitterionic phospholipids that form the walls of honeycomb-like chambers. DPPG with a negative charge prevents the MVLs from aggregation [62]. Neutral lipids (e.g., triolein and triglycerides) act as a hydrophobic space filler at bilayer intersection points and stabilize these membrane junctions [63]. Without the neutral lipids, conventional ULVs or MLVs will be formed instead of MVLs. The amount of neutral lipids used in formulation decides the capture volume and encapsulation efficiency of MVLs [63].

GPs play a key role in formulation since they affect the biophysical properties of liposomes (e.g., drug encapsulation, stability, and drug release) and further influence the pharmacokinetic behavior and pharmacodynamics in vivo [64]. The length, symmetry, inter- and intra-molecular interactions, branching, and unsaturation degree of hydrocarbon chains decide the thickness and fluidity of the bilayer, phase transition temperature, and drug release rate [49,65]. In brief, the longer hydrocarbon chain could induce a tighter membrane packing and increase drug retention, whereas the higher degree of unsaturation or branching of the hydrocarbon chain could result in looser membrane packaging, which is probably caused by the preferential interaction of cholesterol with saturated phospholipids in comparison with unsaturated ones [66,67].

Sphingomyelin (SM) ( Figure 2 b) has a similar structure toa glycerolphospholipid, except that glycerol is replaced by sphingosine [68]. Marqibo (vincristine sulfate liposome injection) uses SM to form the bilayer membrane, which significantly decreases the lipid hydrolysis in an acidic environment and prompts the stability of liposomes. An acid environment (pH 2.0–4.0) is routinely used for producing a transmembrane pH gradient for active drug loading. Under the condition of 37 °C and pH 2.0, the rate of hydrolysis for liposomes was approximately 100-fold slower in SM/Chol (55/45, mol/mol) liposomes than in DSPC/Chol liposomes [69]. In addition, liposomes with SM/Chol showed optimal pharmacokinetic properties, i.e., increased circulation time and enhanced delivery of the drug to target tissues [70].

Cholesterol (Chol) ( Figure 2 c) is another main component of the liposome bilayer and can be used in almost all commercial products ( Table 2 ). The addition of Chol can promote the packing of lipid chains and bilayer formation [71], modulate the fluidity/rigidity of membrane [72], and further affect the drug release [73,74], stability of liposomes [75], and the kinetics of exocytosis [76]. For the product of Shingrix (herpes zoster vaccine, contains glycoprotein E antigen and AS01_B liposomal adjuvant system), Chol can avoid the hydrolysis of QS21 (one of the immunoenhancers in the AS01_B adjuvant system) at a ratio of 2:1 (Chol: QS21, w/w) [77]. For the product of AmBisome, Chol reduces the toxicity of the liposomal formulation compared with the non-sterol one [41]. The effect of Chol on the bilayer’s properties is concentration-dependent. It was reported that [71] the low (2.5 mol%) and high (>30 mol%) concentrations of Chol showed little effect on the properties of the lipid bilayer. The “condensing effect” or “ordering effect” of Chol with the content of 5 < Chol mol% < 30 led to a gradual increase in particle size from 220 nm to 472 nm, a decrease in the fluidity of membrane, and a decrease in the release of drug. Besides Chol, other sterols, such as progesterone, ergosterol, and lanosterol, with a similar structure to Chol were also investigated to modulate the membrane rigidity and stability [74,78].

4. Manufacturing Process

Various preparation methods of liposomes have been developed. The potential manufacturing processes of the marketed liposomal products summarized based on the related patents and publications are shown in Figure 3 [79]. The commonly used manufacturing processes include the thin-film hydration, ethanol injection, and double emulsion methods. The processes routinely include (1) the preparation of MLVs or ULVs depending on the choice of methods; (2) size reduction if necessary; (3) preparation of the drug solution(s) and drug loading, while this step is combined with step 1 in the case of passive drug loading; (4) buffer exchange and concentration if necessary; (5) sterile filtration or aseptic processing; (6) lyophilization, if needed, and packaging.