Pharmaceutical liposomal drug delivery: a review of new delivery systems and a look at the regulatory landscape

Abstract

Liposomes were the first nanoscale drug to be approved for clinical use in 1995. Since then, the technology has grown considerably, and pioneering recent work in liposome-based delivery systems has brought about remarkable developments with significant clinical implications. This includes long-circulating liposomes, stimuli-responsive liposomes, nebulized liposomes, elastic liposomes for topical, oral and transdermal delivery and covalent lipid-drug complexes for improved drug plasma membrane crossing and targeting to specific organelles. While the regulatory bodies’ opinion on liposomes is well-documented, current guidance that address new delivery systems are not. This review describes, in depth, the current state-of-the-art of these new liposomal delivery systems and provides a critical overview of the current regulatory landscape surrounding commercialization efforts of higher-level complexity systems, the expected requirements and the hurdles faced by companies seeking to bring novel liposome-based systems for clinical use to market.

Keywords:

• Bioengineering
• drug delivery
• liposomes
• regulatory landscape
• synthetic biology

Introduction

Since first being described by English hematologist Alec Bangham in 1961 (Bangham et al., 1965), artificial lipid vesicles (also called liposomes) have been recognized and extensively used as delivery vehicles for pharmaceuticals (Karmali & Chaudhuri, 2007), as chemical microreactors (Deepthi & Kavitha,, 2014; Lemière et al., 2015; Sercombe et al., 2015), and as model biomembrane systems (Mouritsen, 2011). The phospholipid bilayer envelope is a cell-like boundary appropriate for cellular investigations and affords liposomes a functional scaffold suitable for fundamental cellular functions such as motility and shape change (Sharma et al., 2014), not to mention the ability to mimic the biophysical properties of living cells (Hua & Wu, 2013). These “dynamic” behaviors refer to functions such as membrane deformation and actin polymerization which impart cell-like kinetic behavior to liposomes (Lemière et al., 2015).

In the past 15 years, some major breakthroughs in liposome technology have fueled the rapid development of new pharmaceutical liposomal applications. In order to optimize the delivery of factors for maximum efficacy, novel methods have been proposed to increase the permeation rate of drugs temporarily and deliver the desired target compound in a time regulated and locally restricted manner to the target site. New approaches to construct improved liposomes for therapeutic delivery have addressed, on one end, biophysical parameters (one common example is charge (Sercombe et al., 2015)) which can be manipulated by altering the constituent bilayer phospholipids to better tailor the liposome to the required application. Other parameters that can and have been manipulated include lamellarity (Deepthi & Kavitha,, 2014), bilayer curvature (Mouritsen, 2011), bilayer fluidity (Sharma et al., 2014), as well as surface modification for active or passive targeting approaches (Hua & Wu, 2013). Assembly methods play a key role in defining final liposome characteristics, including encapsulation efficiency and drug release profiles.

Some of these new approaches include modifying the liposome bilayer with suitable amphiphiles to increase the circulation time of liposomes (e.g. stealth liposomes), improve their elasticity (e.g. transferosomes) or develop covalent drug–lipid complexes for improved delivery of drugs (e.g. pharmacosomes). These systems have significantly advanced the scope of drug delivery available to traditional liposome systems, but have also introduced turmoil into the regulatory space. Triggered-release approaches based on molecular motors are another area of considerable interest, and have enjoyed a favorable regulatory opinion. For instance, a number of systems have combined external energy sources such as pH, ultrasound, heat or light (Yudina et al., 2011; Bibi et al., 2012) with appropriate lipid compositions in order to improve controlled drug release at the tumor site. They have, however, been largely disappointing in practice, mostly due to difficult engineering. For instance, it has been technically difficult to design liposomes that can be stable at physiological pH 7.4, but leaky at pH 6.5 for tumor targeting (Andresen et al., 2005). Another area of research has focused on developing new engineering tools to achieve improved physicochemical features (e.g. encapsulation efficiency) of liposomes. These strategies have focused on developing automated, programable and controlled delivery systems for the assembly of liposomes of controllable physicochemical characteristics using microfluidic technology, which has emerged as a robust alternative for the assembly of vesicles that reigns in some of the weaknesses associated with traditional liposome assembly methods. In addition to this, it is important to mention that microfluidics has been recognized as an enabling new technology providing a controlled environment to address issues of size and structure heterogeneity (Matosevic, 2012; Matosevic & Paegel, 2013). Recently, a number of microfluidic approaches have been described aimed at circumventing some of the main drawbacks encountered with traditional lipid assembly techniques. Unilamellar vesicles have been formed by microfluidic inkjet printing (Federman & Denny, 2010), T-shaped junction induced membrane deformation (Zheng et al., 2015), hydrodynamic focusing (Gabizon et al., 2003), while oligolamellar vesicles were formed by lipid-coated ice droplet hydration (Chang et al., 2009). The main drawback of microfluidic approaches is the technical know-how needed to achieve their assembly. However, while the elegance of some of these approaches should not be understated, simultaneous control over encapsulation efficiency, size and lamellarity has been difficult to achieve. Nonetheless, remarkable advances in recent years have fueled exponential growth in liposomal delivery systems and have opened up opportunities for brand new medical applications. These developments have given rise to more complex and sophisticated delivery systems which are both tissue- as well as application-specific than traditional first-generation liposomes. The Food and Drug Administration (FDA) has been keenly interested in liposomes as nanocarriers, and the clearest path to approval has so far been achieved with traditional, “bulk” scale systems.

Moreover, targeting to specific sites (such as cells or organelles) is a key functional aspect of liposomes as drug delivery systems. This has traditionally been achieved by so-called active or passive targeting. Technical requirements for the two approaches are different: In passive targeting, liposome transport and delivery is guided by the natural distribution patterns of liposomes. PEGylated (stealth) liposomes are an example of vehicles that have been used in passive-targeting approaches. Active targeting, on the other hand, refers to the modification of the liposomes’ natural distribution patterns (Federman & Denny, 2010). One way of achieving this is by attaching ligands to liposomes that recognize and bind to specific molecular and macromolecular cues on the surface of target cells. Ligands that have been used to achieve active targeting include the glycoprotein transferrin (Zheng et al., 2015), the organic compound folic acid (Gabizon et al., 2003) and peptides of tryoptophan, threonine and tyrosine (Chang et al., 2009).

This review presents the current state-of-the art in the new, higher-level complexity “bulk-scale” liposomal systems – including ethosomes, pharmacosomes, transferosomes nebulized liposomes and more – for delivery of encapsulated drugs to therapeutic sites with a particular focus on liposome biophysics and the interaction of liposome membranes with encapsulated cargo, and outlines a path forward towards smarter systems for therapeutic use. A critical look at the requirement, hurdles, challenges and shortcomings surrounding the regulatory landscape for the approval of such products is also given.

Improving circulation time: stealth liposomes

Traditional “first-generation liposomes” based on phospholipid bilayer membranes displayed poor stability and rapid clearance after injection. This is because conventional liposome membranes are strongly affected by physical interactions with circulating proteins in the blood (opsonization) and protein adsorption, which contribute to their clearance. In order to improve on these shortcomings, longer-circulating liposomes were developed by modulating the composition, size and charge of regular liposomes. It is important to mention here that increased circulation time may not always be desired, such as in applications where an encapsulated drug is needed to reach the target site as quickly as possible.

By coating the liposome shell with inert hydrophilic polymers such as polyethylene glycol (PEG), longer-circulating liposomes were produced that were shown to reduce adsorption of various blood proteins and hence extend their circulation time. These were called stealth liposomes. Long-circulating liposomes demonstrate dose-independent, non-saturable, log-linear kinetics and increased bioavailability (Van Slooten et al., 2001). Stealth liposomes can be prepared with various lengths of PEG chains covalently attached to various hydrocarbon chain anchors. Improved properties of stealth liposomes are in large part due to the favorable properties of PEG, which has excellent biocompatibility, is nonionic, low fouling and possesses high solubility in both aqueous and organic media.

Studies have indicated that stealth liposomes are also controlled by their physical characteristics. Based on in vitro laboratory work, it has been reported that there exists a maximum liposome size (∼275 nm) beyond which the stealth property of PEG-liposomes is significantly compromised, and its distribution is characterized by high mononuclear phagocyte system (MPS) accumulation (Nag & Awasthi, 2013).

Other advances based on in vitro and in vivo work have sought to further enhance the physical properties of stealth liposomes and overcome issues of loss of nanoparticle integrity upon systemic injection. Pasut et al. (2003) described new PEG-dendron-phospholipid constructs that they assembled into structures called super stealth liposomes (SSLs). This was achieved by using a β-glutamic acid dendron to anchor a PEG chain to distearoyl phosphoethanolamine lipids. The new structures displayed increased stability and prolonged circulation half-life compared to regular stealth liposomes in in vitro studies involving CaCo-2 cells and in vivo pharmacokinetic studies involving BALB/c mice.

It was recently reported, in an in vivo research study involving tumor-bearing mice, that stealth liposomes with a slightly negative surface charge (zeta potential − 7.6 mV) have longer circulation times and a higher tumor accumulation in vivo than stealth liposomes with more negative zeta potentials (Lee et al., 2011). Also, detachable PEG conjugates have been described in vitro (Zalipsky et al., 1999), in which the detachment process is based on the thiolysis of the dithiobenzylurethane linkage between PEG and an amino-containing substrate (such as PE [phosphoethanolamine]). This allows for the loss of the polymer coating under conditions similar to those in vivo which causes rapid delivery of encapsulated cargo after the liposomes have reached their target site.

“Stealth” liposomes are suitable delivery vehicles for active targeting to target cells, with the PEG coating providing prolonged circulation time and a protective hydrophilic layer. Active targeting ligands coupled to these carriers have included small-molecule ligands, peptides and monoclonal antibodies (Byrne et al., 2008). While challenging (Sawant & Torchilin, 2012), such active targeting has gained therapeutic traction. Actively targeted liposomes have been used in cancer therapy, owing to the fact that receptors such as folate and transferrin receptors are overexpressed on many cancer cells, making transferrin and folic acid suitable ligands (Torchilin, 2007). Other ligands used in cancer therapy include RGD, peptides and antibodies against VEGF, VCAM, matrix metalloproteases (MMPs) and integrins (Liechty & Peppas, 2012). Examples of such targeting based on in vitro cell-based studies wherein monoclonal antibodies (mAb) that selectively bind to internalizing receptors on cancer cells are attached to the distal ends of PEG chains grafted to the liposomal surface (Allen et al., 1995) have shown the therapeutic potential of these systems. Zhu et al. (2012) recently developed a multifunctional PEGylated liposome made from a mixture of phosphatidylcholine and phosphoethanolamines with an MMP2-cleavable octapeptide (Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln) conjugated between the lipids and the liposomal PEG coat for active tumor targeting. When inside the cell (the authors used an in vitro system of 4T1 and H9C2 cells to demonstrate functionality), the peptide is cleaved by the highly expressed extracellular MMP2, leading to exposure and enhanced intracellular penetration of cell-penetrating peptide TATp.

Elsewhere, ligands used for targeting to lungs for treatment of TB include maleylated bovine serum albumin (MBSA) and O-steroyl amylopectin (O-SAP) (Pinheiro & Lúcio, 2011), both of which have high specificity towards alveolar macrophages. For such active targeting applications, negatively charged lipids such as phosphatidylserine (PS) and phosphatidylglycerol (PG) are preferentially used (Ahsan et al., 2002).

Another area of interest for targeted liposomes includes angiogenic therapy, with recent examples including the development of prohibitin-targeted liposomes via KGGRAKD peptides on the surface of PEGylated liposomes to deliver cytochrome C to adipose endothelial cells (Sakurai et al., 2015).

Some actively targeted liposomal formulations have also progressed into the clinic, though the number is limited. Merrimack Pharmaceuticals’ targeted liposomal formulation MM-302 for breast cancer announced positive Phase I trial results in 2015. MM-302 is based on a PEGylated liposome targeting ErbB2 (HER2) using an antibody fragment as a ligand to deliver doxorubicin. The company is currently engaged in efforts for accelerated approval of the therapy.

Passively targeted stealth liposomes have enjoyed more success in the clinical setting. A recent Phase I/II study evaluating the safety and efficacy of a novel neoadjuvant combination treatment of paclitaxel, pegylated liposomal doxorubicin, and hyperthermia to treat locally advanced breast cancer patients proved successful in the subjects treated (Vujaskovic et al., 2010). The study showed that the treatment was well-tolerated in patients with locally advanced breast cancer.

A single-arm Phase II safety and efficacy trial of pegylated doxorubicin and carboplatin (Nakanishi et al., 2015) combination chemotherapy to treat patients with platinum-sensitive recurrent ovarian cancer showed positive results with an overall objective 51.5% response rate, deeming the trial successful and the therapy safe and effective.

Improving elasticity: transferosomes

Transdermal drug delivery is a significant therapeutic need that is stymied by the low-permeation efficiency of therapeutic drugs across the skin (Langer, 2004). The reason for using vesicles in transdermal drug delivery is based on the fact that they act as drug carriers to deliver entrapped drug molecules across the skin, as well as penetration enhancers because of their composition. In addition, these vesicles enable the sustained release of active compounds in the case of topical formulations, as well as rate limiting membrane barrier for the modulation of systemic absorption in the case of transdermal formulations. Moreover, in many cases topical administration of drugs encapsulated inside liposomes is a desired alternative to intravenous injection owing to its local mode of action and numerous advantages and convenience of treatment.

The major advantages of topical liposomal formulations stem from their demonstrated ability to reduce serious side effects that may arise from undesirably high systemic absorption of a drug and to significantly enhance the accumulation of drug at the site of administration as a result of the high substantivity of liposomes with biological membranes. Other advantages of dermal liposomal delivery include avoidance of first pass metabolism, minimization of unwanted side effects, an improved utility of short half-life drugs, an improved physiological and pharmacological response as well as an avoidance of fluctuations in drug levels and, finally, convenience and safety to patients (Bouwstra et al., 2003; Lee et al., 2005). While the interaction of nanoparticles, including liposomes, and human skin remains a controversial topic, scientific evidence suggest these particles do not, in fact, penetrate skin layers themselves (Watkinson et al., 2013).

As the outer layer of the epidermis, the stratum corneum (SC), covers the entire outside of the body and is the rate-limiting barrier to absorption, liposomes having deformable properties have especially drawn considerable attention for improved capacity to delivery active drugs across skin layers (Isayed et al., 2006). It has been reported, through in vitro work, that the high elasticity of vesicles could result in enhanced drug transport across the skin as compared to vesicles with rigid membranes. This deformability can be imparted on the liposomes by decorating the outer surface of the liposomes with elasticity-imparting substances. Specifically, chemical permeation enhancers (Karande & Mitragotri, 2009) (as well as some phospholipids) fluidize the stratum corneum lipid bilayers and facilitate the transdermal diffusion of drugs.

In order to meet this need for improved elasticity, a new family of liposomal structures was invested, called transferosome. Transferosomes were developed for improved skin permeation following topical delivery. They are ultradeformable vesicles with increased permeation capacity of active drugs across skin layers (Darwhekar et al., 2012). This is achieved by the addition of membrane-modifiers called edge activators (Duangjita et al., 2011). The edge activators are typically single-chain surfactants with a high radius of curvature which destabilizes the lipid bilayers and increases the deformability of the liposomal membranes, in turn allowing them to squeeze between the skin layers.

However, liposomes vary greatly in size, lamellarity, charge, membrane fluidity and encapsulation efficiency, which influences their functions during transdermal delivery. This results in different mechanisms of action, so it is very important that assembly techniques used to make these vesicles account for the variability by including a sizing step, such as extrusion, to minimize polydispersity of the liposomal population.

There have been numerous in vitro – and some ex vivo – studies involving transferosomes. Elastic liposomes have been reported to improve skin penetration of various drugs (Mohammed et al., 2005), and they have been demonstrated for the delivery of drugs such as ibuprofen (Irfan et al., 2012), terbinafine (Ghannouma et al., 2012) and emodin (Lu et al., 2014). Transferosomes have also been reported in clinical studies to varying degrees of success. A recent highlight is a 12-week randomized human trial evaluating safety and efficacy of ketoprofen in transferosomes to treat patients with ostheoartritis. The purpose of this study was to evaluate transferosomes as a delivery route for ketoprofen and assess whether the transdermal penetration of the drug is improved in such a vehicle. The study indicated that two different dosages (50 mg and 100 mg) of ketoprofen encapsulated in transferosomes were comparable to free ketoprofen in their ability to reduce the pain associated with knee osteoarthritis (Kneer et al., 2013). Both routes of delivery were well-tolerated while no significant improvement was seen with the transferosome system, the authors accepted transferosomes as a promising and viable mode of delivery.

Improving skin penetration: ethosomes

In order to improve the penetration efficiency of liposomes across the skin, a novel formulation of liposomes, called ethosomes, has been developed by utilizing ethanol’s penetrating properties. Ethosomes are phospholipid-based elastic nanovesicles containing a high content of ethanol (20–45%), which is the main way in which they differ from regular liposomes. They were first described by Touitou et al. (1997), and they range in size from a few tens of nanometer to a few microns depending on method of preparation. The mechanism of ethanol’s skin penetration efficiency has been well documented (Kalbitz et al., 1996; Lachenmeier, 2008). Ethanol can interact with the polar head group region of the lipids, thereby increasing lipid fluidity and cell membrane permeability.

As ethosomes show improved skin penetration to ethanol alone, the mechanism by which ethosomes are able to penetrate the skin has been suggested to be synergistic between ethanol, vesicles and skin lipids (Touitou et al., 2000). It is thought that the first part of the mechanism is due to the “ethanol effect” (Elsayed et al., 2007), where ethanol interacts with the lipid molecules in the polar head group region resulting in an increase in the membrane fluidity and decreasing the density of the lipid multilayer. Following this initial interaction, the ‘ethosome effect’ is thought to take place, wherein lipid penetration and permeation occurs due to the fusion of ethosomes with skin lipids, resulting in the release of the drug into the skin layers (Fan et al., 2013). Ethanol may also provide vesicles with soft flexible characteristics (Sun & Sun, 1985), which allow them to penetrate more easily into the deeper layers of the skin. The release of the drug in the deep layers of the skin and its transdermal absorption could then be the result of a fusion of ethosomes, with skin lipids and drug release at various points along the penetration pathway (Verma & Pathak, 2010).

The general approach for making ethosomes involves dissolving lipids and drugs in ethanol. There are two main ways in which they can be assembled: the so-called hot and cold method (Chandel et al., 2012). Despite the names, both approaches involve a heating step; in the cold method the temperature is brought up to 40 °C, while the hot method usually employs a heating step at 40 °C. By far the most common way of obtaining ethosomes is the cold method.

Numerous in vitro and ex vivo studies have demonstrated the improved permeation efficiency of ethosomes compared to ethosome-free formulations. Xu et al. (2007) demonstrated, in in vitro percutaneous absorptions studies using a Franz diffusion cell using human skin, an 11.68-higher increase in steady-state flux compared to phosphate buffer solution (PBS) of naloxone, an opiod antagonist. This was achieved with ethosomes containing propylene glycol, N,N-dimethyl formamide, N,N-dimethyl acetamide, dimethyl sulfoxide, azone and polyethylene glycol 400 as chemical enhancers.

Elsewhere, other in vitro and ex vivo permeation studies using a Franz diffusion cell showed the effectiveness of these delivery systems as well. In one such study, Bendas & Tadros (2007) formulated ethosomes of salbutamol sulfate (SS); a hydrophilic drug used as bronchodilator, and compared its transdermal delivery potential with classic liposomes containing different cholesterol and dicetylphosphate concentrations. The study showed a significant decrease in vesicle size by decreasing cholesterol concentration and increasing dicetylphosphate and ethanol concentrations. They also prepared an ethosomal gel formulation by incorporating the optimized SS ethosomal dispersion into Pluronic F 127 gel. In vitro/ex vivo permeation studies via synthetic semipermeable membrane or skin from newborn mice showed that both formulations (SS ethosomal dispersion and gel) were much more efficient at delivering SS than conventional liposomes.

In a recent ex vivo permeation study through excised human skin comparing conventional liposomes, transferosomes and ethosomes for the topical delivery of celecoxib, ethosomes with Tween 20 showed the highest liposomal homogeneity, the highest encapsulation efficacy (54.4%), and enabled the highest increase in drug penetration through the skin (Bragagni et al., 2012) when compared to either transferosomes or conventional phospholipid bilayer-based liposomes. At the time of this writing, no completed clinical trials involving ethosomes are available.

Improving delivery of poorly soluble drugs: pharmacosomes

First described in the 1980s, pharmacosomes have received less attention than their counterparts, partly due to the more targeted nature of their applications as well as more laborious assembly techniques. Pharmacosomes are amphiphilic phospholipid complexes of drugs that bind to phospholipids through covalent, electrostatic or hydrogen bonding (Semalty et al., 2009). They are thought to improve the bioavailability of poorly water soluble as well as poorly lipophilic drugs. Based on their chemical structure, pharmacosomes can exist either as ultrafine micelle or hexagonal aggregates (Shivhare et al., 2013).

While other vesicles, such as ethosomes and transferosomes, can be made by straightforward liposomes assembly approaches, pharmacosomes often require complexation steps in order to achieve the necessary drug-lipid complex. Often, methods of assembly are specific to the type of drug that is complexed to the phospholipid. Once prepared, however, pharmacosomes tend to show significantly better dissolution profile than free drug, such as a recent example where aceclofenac pharmacosomes showed an almost 10% better dissolution profile than aceclofenac acid (Semalty et al., 2010) based on a number of analytical in vitro measurements, including in vitro dissolution. The solubility of the pharmacosomes was also improved compared to free acid. Moreover, other reported benefits of pharmacosomes over traditional liposomes include improved stability due to covalent linkages, reduced leakage of the entrapped drugs and the lack of the need to remove non-entrapped drug. Application-wise, pharmacosome-encapsulated drugs can be administered orally, topically, extra-or intravascularly.

Most commonly, pharmacosomes are prepared by solvent evaporation following the drug complexation step. Following solvent evaporation, the dried powder, which represents the pharmacosomes, is rehydrated in aqueous buffer to generate the aqueous phase pharmacosomes. Pharmacosomes have been prepared for a number of non-steroidal antiinflammatory, cardiovascular and antineoplastic drugs. After absorption, the pharmacosomes’ in vivo pharmacokinetic behavior and rate of delivery of active drug molecule without the lipid carrier depends primarily on the size and functional groups of the drug molecule, the lipid chain length, and the spacer (Sharma et al., 2014).

Drugs such as Ketoprofen (Kamalesh et al., 2014) have recently been encapsulated inside pharmacosomes with remarkable results in in vitro laboratory studies: the dissolution of ketoprofen was 93.3% compared to that of free ketoprofen (49.77%) based on analytical measurements. Elsewhere, furosemide-bound pharmacosomes were shown to have increased permeability of the encapsulated drug by over 28% when compared to free furosemide in in vitro studies (Chatap et al., 2014).

Stimuli-responsive liposomes and nebulized liposomes

Two main types of triggers have been explored: remote triggers such as heat, ultrasound and light, and local triggers such as enzymes and pH (Bibi et al., 2012; Puri, 2014). Such stimuli-responsive liposomes have been extensively studied and despite a growing body of work, have been fairly disappointing in practice. Despite that, there have been some systems that have progressed to clinical trials: these are mostly thermo-sensitive liposomes.

The thermo-sensitive liposomal formulation ThermoDox® (Celsion Corporation, Lawrenceville, NJ), which contains lysophosphatidylcholine and is intended for the treatment of various cancers including primary liver cancer, recurrent chest wall breast cancer, colorectal, pancreatic and metastatic liver cancer, is currently in various stages of human clinical trials (Phase II/III) with studies ongoing to further improve the performance of the drug and increase its chance of clinical trial success.

Elsewhere, nebulized liposomes have also found a way into the clinic. Insmed’s liposomal amikacin (Arikace®) is one such formulation for the delivery of nebulized amikacin to the lungs (Li et al., 2008). The formulation is based on sustained-release liposomes of phospholipids (DPPC) and cholesterol encapsulating amikacin. The clinical trials for the use of these liposomes for the treatment of cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection are complete, and the formulation is currently undergoing clinical trials for the treatment of M. avium infections.

Targeting liposomes to specific organelles

Related to active targeting discussed in the section on stealth liposomes, a sub-area of growing interest is the ability to target drugs to specific organelles. While specific subcellular targeting is still a significant challenge, efforts have so far been most successful with targeting drugs to lysosomes or mitochondria. Most of these systems are still at the in vitro research phase. In one such in vitro demonstration, drugs encapsulated in liposomes modified with various lysosomotropic ligands, such as octadecyl-rhodamine B (RhB), were successfully delivered to lysosomes (Meerovich et al., 2011). Elsewhere, mitochondria-targeting was achieved in vitro with the polymer (Rh123)-PEG-DOPE (Rhodamine 123-Polyethylene glycol-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) which contains mitochondriotropic dye rhodamine (Biswas et al., 2011). The polymer, incorporated into the lipid bilayer of the liposomes, showed good uptake by cells (HeLa, B16F10) with a high degree of accumulation in the mitochondria. When these mitochondrial-targeted liposomes were loaded with paclitaxel (PCL), they showed enhanced cytotoxicity toward cancer cells compared with non-targeted liposomes or free PCL.

Such subcellular targeting is highly pharmaceutically desirable and efficiently developing new delivery systems that can successfully achieve such targeting will require understanding the microenvironment of the diseased site in order to design drugs and delivery vehicles with robust stability, pharmacokinetic and pharmacodynamics profiles, and good biocompatibility and biodegradability.

Liposomes in clinical use

The function of liposomes in vivo is directly related to their composition. Liposomes can be classified according to their lamellarity (unilamellar liposomes contain a single phospholipid bilayer, while multilamellar liposomes are composed of multiple liposomal membranes), size (liposomes are defined as small [≤100 nm], intermediate [100–250 nm], large [≥250 nm] or giant [>1 um]), and surface charge (anionic, cationic, or neutral). Generally, assembly methods can be used to control the physical characteristics of liposomes. For instance, hydration of a thin lipid film generates liposomes that are largely oligolamellar, while extrusion is used to further size these liposomes and obtain monodisperse populations of unilamellar structures. Practically, a population of monodisperse, unilamellar liposomes is always preferred. Such liposomes behave more uniformly in a way that can be aligned with the behavior of cellular systems and exhibit physical characteristics that are more predictable and statistically comparable. It is important to mention that cellular organelles with a high number of bilayers exist (mitochondria and the nucleus are two examples of double-bilayer systems), and synthetic systems mimicking their behavior need to account for such structural specificities. Such structural specificities are critical and should not be understated: in mitochondria, for instance, the two membranes create distinct compartments within the organelle, and differ significantly in structure and in function. The outer membrane contains porins, while the inner membrane’s structure is highly complex with multiple transport membrane proteins. Mimicking such complexity on liposomes is not simple.

Clinically, liposomes are used as carriers for biologically active molecules and are nontoxic to humans. Two delivery areas where liposomes have shown most promise are drug delivery (Allena & Cullis, 2013) and gene therapy (Tseng & Huang, 1998), owing to the advantages that their use brings over traditional methods. Their unique chemical composition allows them to encapsulate hydrophilic biomolecules or drugs in the aqueous core and increase penetration through lipophilic membranes (Fahr et al., 2005). On the other hand, the lipid bilayer can entrap lipophilic drugs and thus increase their solubility in aqueous body fluids. In immunotherapy, for instance, the use of liposomes is preferred to viral gene delivery methods for mesenchymal stem cell-based cell therapy due to its safety, lack of immunogenicity, negligible toxicity, and the ability to carry larger therapeutic genes (Madeira et al., 2010). As a result, an increased number of studies have focused on developing improved liposomal transfection agents.

Drug delivery has been the area where the use of liposomes have shown most promise. These efforts are well-documented in the scientific literature (Samad et al., 2007). In clinical studies, liposomes have shown improved pharmacokinetics and biodistribution of therapeutic agents which minimizes toxicity by their accumulation at the target tissue (Park, 2002). Currently, there are approximately a dozen liposome-based drugs approved for clinical use with more in various stages of clinical trials (Table 1). While most liposomal drug formulations are approved for intravenous application, intramuscular (Koshkina et al., 1999) and oral (Rogers & Anderson, 1998) delivery have also been examined. Although the mechanism of drug release in tumors from liposomes is not fully understood, it is thought to depend on three main factors: the mechanism of drug loading, lipid composition of the membrane, and the tumor microenvironment (Torchilin, 2010). All of the systems currently approved are based on either simple liposomes or PEGylated variants to improve circulation time. New, higher complexity systems – as discussed at length in this review and which include transferosomes and ethosomes – that have emerged in the past few years, have not been met with the same kind of success in the clinic and in the commercial space.

Table 1. FDA-approved liposome-based drugs on the market.

Product	Administration	Drug	Particle type	Lipid composition	Indication
Ambisome	Intravenous	Amphotericin B	Liposome	HSPC, DSPG, cholesterol and amphoteracin B in 2:0.8:1:0.4 molar ratio	Sever fungal infections
Abelcet	Intravenous	Amphotericin B	Lipid complex	DMPC and DMPG in 7:3 molar ratio	Sever fungal infections
Amphotec	Intravenous	Amphotericin B	Lipid complex	Cholesteryl sulfate	Sever fungal infections
DaunoXome	Intravenous	Daunorubicin	Liposome	DSPC and cholesterol (2:1 molar ratio)	Blood tumors
DepoCyt	Spinal	Cytarabine	Liposome	DOPC, DPPG, cholesterol and triolein	Neoplastic meningitis and lymphomatous meningitis
DepoDur	Epidural	Morphone sulfate	Liposome	DOPC, DPPG, cholesterol and triolein	Pain
Doxil	Intravenous	Doxorubicin	PEGylated liposome	HSPC:cholesterol:PEG 2000- DSPE (56:39:5 molar ratio)	Kaposi’s sarcoma, Ovarian/Breast Cancer
Epaxal	Intramuscular	Inactivated hepatitis A virus (strain RGSB)	Liposome	DOPC:DOPE (75:25 molar ratio)	Hepatitis A
Inflexal	Intramuscular	Inactivated hemaglutinine of Influenza virus strains A and B	Liposome	DOPC:DOPE (75:25 molar ratio)	Influenza
Lipodox	Intravenous	Doxorubicin	PEGylated liposome	HSPC:cholesterol:PEG 2000- DSPE (56:39:5 molar ratio)	Kaposi’s sarcoma, Ovarian/Breast Cancer
Myocet	Intravenous	Doxorubicin	Liposome	EPC:cholesterol (55:45 molar ratio)	Combination therapy with cyclophosphamide in metastatic breast cancer
Marqibo	Intravenous	Vincristine	Liposome	Sphingomyelin, cholesterol	Acute lymphoblastic leukemia
Visudyne	Intravenous	Verteporphin	Liposome	EPG and DMPC	Macular degeneration

In line with this, a significant hurdle in developing liposomal systems for delivery of active biomaterials has been the development of robust formulations that overcome the traditional limitations associated with traditional liposomal delivery systems. The complexity of some of these systems is increased because of the physicochemical changes to the liposomes that are brought about by the addition of components that interact with the membrane, such as ethanol or covalent complexing agents. Moreover, the development of most new liposomal drug products is challenged by a lack of biologically relevant in vitro release methods. For those reasons, regulatory bodies rely on various academic and industry developers to provide timely and thorough information on these new systems to support and advance the regulatory process. The issues that are the core of development studies to achieve robust liposome-based drug delivery systems can be broadly summarized as having to achieve the following: (a) Controlled release rate of encapsulated material, (b) Prolonged lifetime/reduced clearance of liposomes, (c) Intracellular delivery of encapsulated material to the target site. This is part of the reason why more complex systems, which are based on agents other than lipids to achieve the delivery system’s biophysical function, still face significant hurdles to regulatory acceptance.

In the cell therapy space, liposomes were shown to be efficient for the targeted delivery of growth factors such as vascular endothelial growth factor (VEGF (Tang et al., 2014)), VEGF aptamers (Willis et al., 1998) and endothelial growth factor (EGF (Alemdaroğlu et al., 2008)) as well as small interfering RNA (siRNA (Wang et al., 2013)). Tang et al. (2014) recently developed VEGF-encapsulated immunoliposomes targeting myocardial infarction (MI). By injecting the VEGF-immunoliposomes together with mesenchymal stem cells (MSCs) into a rat immediately following MI, the authors observed a remarkably high attenuation in cardiac function loss, together with an 80% increase in blood vessel density.

While the FDA has been increasing its efforts in soliciting new research and development in these areas in order to ultimately develop robust methods for the delivery of drugs, there are significant challenges in current landscape that will be addressed in this review.

The regulatory landscape: hurdles and challenges to approval of new delivery systems

Since the first liposomal system for drug delivery, Doxil®, was approved in 1995, there have been many technological improvements in liposomal delivery systems which have led to delivery systems with vastly improved physicochemical properties. The three guiding principles that were true at the time of Doxil®’s approval, however, ring true today as well and can be applied to all liposomal delivery systems, regardless of type. Liposomal drug delivery systems promise (i) prolonged drug circulation time and avoidance of the reticuloendothelial system due to the use of PEGylated nano-liposomes; (ii) high and stable remote loading of the encapsulate drug; and (iii) optimizing the composition of the phospholipid membrane to achieve improved stability.

Nowadays, Doxil® generates sales of over $500 million annually (Reuters, 2011). Currently, there are around a dozen liposome-based drugs approved for clinical use and five in clinical development (Chang & Yeh, 2012), with many more in various early stages of development. However, despite the commercial success of some of these products, the regulatory landscape has not been able to keep up with the rapid pace of development of new liposome-based delivery systems. This likely stems from the sheer number of new products and the lack of robust efforts on the part of academic and private-sector developers to keep an open dialog with the FDA. As of 2015, most of the FDA-approved delivery vehicles are either fully liposomal or PEG-ylated, and none have addressed compositions of higher-level complexity, such as ethosomes or pharmacosomes, demonstrating that either more data is needed from the scientific community to prove efficacy of such formulations, or more urgency is required on the part of the FDA. There is growing consensus that the burden of proof rests on academic as well as private and public sector research and development scientists to identify the critical attributes related to the safety and efficacy of their new products and technologies, and inform and educate the FDA of such. Indeed, the scientific community should accept the responsibility for advocating these new technologies and promoting them to the appropriate regulatory bodies.

The FDA generally classifies liposomes as “nanocarriers,” alongside dendrimers, quantum dots and fullerenes. Among these, combination nanocarrier products have grown in interest in recent years (Parhi et al., 2012), such as the co-encapsulation of synergistic anti-cancer drug combinations (Shaikh et al., 2013) inside liposomes. Celator Pharmaceuticals Inc. (Princeton, NJ) recently developed a PEGylated liposomal formulation of cytarabine:daunorubicin (CPX-351, 5 : 1 molar ratio), loaded with the weak acid drug 5-fluoroorotic acid (FOA) and the amphiphatic drug, irinotecan (CPT-11) at a 5 : 1 ratio (Feldman et al., 2012). The co-encapsulated drugs showed a synergistic therapeutic effect when delivered together. Celator is currently undergoing Phase III trials of their CPX-351 technology. Combination products typically have more hurdles on the path to wide acceptance by the FDA, despite the agency’s full inclusion of such products in regulatory evaluations.

Over the last decade, US and European regulatory agencies began to publish draft guidance documents containing non-binding recommendations on liposome-based products; the US Food and Drug Administration (FDA) published the first draft “Guidance for industry on liposome drug products” in August 2002 (US FDA, 2010). However, this document did not provide information on bioequivalence assessment methods. In February 2010, FDA published a product-specific guidance to abbreviate new drug applications for pegylated liposomal doxorubicin (PLD) injectable formulations (Meng et al., 2013). This was the first, and till then, the only attempt to guide developers when designing liposome bioequivalence by carefully defining the reference product. For demonstrating bioequivalence, this guidance mentions using human pharmacokinetic (PK) studies and in vitro dissolutions studies. Earlier in 2007, the FDA released a Nanotechnology Task Force report which outlined scientific and regulatory challenges surrounding commercialization of nanotechnology-based products. These have to deal with issues that are relevant to product safety, and effectiveness may change as size varies within the nanoscale.

In November 2015, the FDA issued a revised draft guidance, “Liposome Drug Products Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation”, which addresses a number of topics for liposome drug products: chemistry, manufacturing, and controls (CMC); (B) human pharmacokinetics and bioavailability or, in the case of an ANDAs (abbreviated new drug applications), bioequivalence; and labeling in new drug application (NDAs) and ANDAs. It replaces the draft guidance for industry on “Liposome Drug Products, Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation” that was published in August 2002.

It is important to highlight that FDA guidance are not requirements. They provide recommendations for meeting the regulatory requirements and are welcome efforts on the part of the FDA. More generally, FDA draft guidance documents recommend that companies developing novel drug delivery systems carry out in vivo and in vitro studies in order to demonstrate bioequivalence. They also recommend the type and number of studies, the population to be studied, blood sampling time points and analytes to be measured in blood samples. Moreover, when developing new liposomal drug delivery systems, the composition and psysicochemical characteristics of the systems are of paramount importance, which speaks of the high degree of characterization necessary for approval. Generally, for new liposomal formulations, pharmacokinetic dose-dependent behavior must be evaluated against a reference standard and equivalence in the recommended dose range must be demonstrated, while any validated bioanalytical method should reliably quantify encapsulated and non-encapsulated drug substance. For liposomes with extended or sustained release, typical parameters to be evaluated include clearance, volume of distribution, terminal half-life and partial AUCs (area under curves) over time. For those liposomes, three criteria must be taken into account: the rate of clearance of the liposomal carrier itself, the rate of release of entrapped drug from the liposomal carrier, and the rate of clearance and metabolism of free drug upon its release. Moreover, profiles of their in vivo fate as well as the safety and efficacy are also required by the FDA for approval. These bioequivalence tests should not be confused with release tests and criteria set out by the individual manufacturer for a particular product. Because of the limited number of liposomes-based drugs approved for clinical use, there are many indications that have one or no liposomal delivery systems approved for use. Visudyne, marketed by Bausch + Lomb and based on egg phosphatidylglycerol and dimyristoyl phosphatidyl choline in a 3:5 molar ratio, is the only drug approved by the FDA for the photodynamic treatment of age-related macular degeneration. To assure the quality of liposomal formulations, a number of evaluation tests are usually performed (Table 2).

Table 2. Analytical assays for liposome characterization.

Assays	pH, phospholipid concentration and composition, cholesterol concentration, drug concentration
Chemical stability	Phospholipid peroxidation, phospholipid hydrolysis, cholesterol autooxidation, antioxidant degradation
Physical stability	Vesicle size distribution, number of bilayers, encapsulation efficiency, dilution-dependent drug release, relevant body fluid-induced leakage, electrical surface potential,
Biological  characterization	Sterility, pyrogenicity, animal toxicity

Moreover, it is important to mention that, as the complexity of the liposomal systems increases – such as the case of ethosomes or pharmacosomes – so do the practical hurdles which accompany establishing release criteria for such products. For instance, ligand-targeted liposomes have different penetration and targeting properties to ethanol-complexed liposomes, and require release tests that are more appropriate to their mode of action. Regulatory and legislative frameworks, as well as requirements, vary by country. As new systems attempt to enter the commercial space, new paradigms for approval will need to be established, making the regulatory process more difficult.

As briefly mentioned above, for new liposome products, release testing is required to demonstrate claimed release characteristics of the encapsulated drugs. In vitro release tests should be predictive of in vivo release. In general, an in vitro release test is expected to be performed for each novel dosage form, formulation development, investigations to support post-approval changes and for batch-to-batch quality control. For in vitro liposome release tests, paddle apparati, flow-through cells, and modified flow-through cells have been typically used. Challenges of these tests are to determine the appropriate duration of the test and the time points at which samples are drawn in order to characterize the release profile. The FDA expects that release profiles to be quantified and validated during early testing, in the middle of the release testing and one near the end (>80% total release) with in vivo data. The choice of release criteria is often challenging, and it requires careful assessment of expected in vivo release profiles.

When generic, or biosimilar, encapsulated liposome-based drug release products are being developed, the FDA also recommends dissolution testing to be carried out as part of the demonstration of bioequivalence between test and reference products. Drug release assays can be used to identify liposome formulations that are indistinguishable in drug release and physical properties from the reference standard, and are routinely carried out for biosimilar evaluation.

In vivo stability of any type of liposome is particularly important, as they should remain stable from loss of integrity or content leakage until being taken up by the target site. Permeability and encapsulation efficiency are part of the integrity tests carried out on new liposome products. Such characteristics are affected, among other factors, by the size and composition of the liposomes. Larger liposomes, such as LUVs, tend to be less rigid, while membrane components such as cholesterol may reduce their permeability. Moreover, storage conditions (frozen in liquid form or freeze-died) may affect their size distribution. Drug complexation changes the biophysical characteristics of the liposomes and may, in some cases, limit the handling that these liposomes can go through, and these changes must be taken into account.

As mentioned above, the composition of the lipid membrane is critical in controlling size distribution, permeability, rigidity and physicochemical properties of the liposomes. Raw materials used in the assembly of these systems may include natural or synthetic lipids, or completely natural lipid mixtures, such as egg lecithin. Additional complexation agents further change these parameters and may affect the fluidity of the membranes around their phase transition temperatures. For instance, it was reported that the interaction between ethanol and the lipid of the stratum corneum reduces the phase transition temperature of ethosomes, thus promoting mobility and drug penetration (Meng et al., 2013). The drawback of using lipids from natural sources is the poor control of composition, as different preparations may contain different fatty acid compositions. For that reason, manufacturers have opted for synthetic versions of natural lipids, such as dioleoylphospocholine (DOPC) or dimyristoylphosphocholine (DMPC), which are significantly cheaper to manufacture and source.

The European Medicines Agency (EMA, 2011) published its first draft guidance in July 2011. This document aims to provide guidance in generating relevant clinical and non-clinical data to support their application for market approval of intravenous liposomal products. However, it does not define a specific analytical, non-clinical or clinical strategy and provides only general principles for assessing liposomal products (EMA, 2009). Using this guidance, the extent and complexity of non-clinical and clinical studies would be defined on a case-by-case basis.

The various guidances are provided as recommendations to facilitate meeting regulatory requirements. In the US, the FDA’s efforts should be matched by greater engagement from industry and academia with regard to identifying critical parameters related to safety, efficacy and mode of action for new liposome-based products. The private sector and academia should accept the burden of providing timely information and data about new products and technologies to regulatory bodies.

An open dialog between industry, academia and regulators in strongly encouraged to advance faster development and approval of new liposomal products.

Conclusion

Liposomal drug delivery systems have come of age since their humble beginnings over 5 decades ago. Over a dozen liposome-based drug delivery systems is currently approved by the FDA, with many more in various stages of development. The FDA’s positive opinion on liposomes alongside other clinically approved particulate drug delivery systems has further helped commercialization efforts. However, regulatory guidance have not kept up with the rapid pace of development of novel liposome-based drug delivery systems, and regulatory demands are, by and large, still outdated. This in turn placed the burden of responsibility on industrial and academic entities who are in the process of taking new systems through the development process of educating regulatory bodies and providing them with timely updates about issues of safety and efficacy of these products in order to progress regulatory approval. Concerted effort on the part of regulatory bodies as well as industry and academia to maintain an open dialog is needed to facilitate the full medical potential of liposomal drug delivery systems and keep transparency in the approval process.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.