Liposomal Delivery Systems
The versatility of liposomal carriers does not just simply rely on their capability to encapsulate various types of therapeutic substances, but also on the large array of components used for constructing liposome-based nanoformulations that allow for a straightforward control over targeting and the release of the encapsulated contents. This leads to a wide array of design strategies which can be easily adapted to any desired theraupetic effect, rendering liposomes one of the most promising systems for drug delivery.
1. Three Main Features of Liposomal Delivery Systems
Liposomal systems can be characterized by three main features that influence their physicochemical properties, which have a direct impact on the pharmacokinetics and pharmacodynamics of the therapeutic compounds upon administration into the bloodstream:
Lipid composition: The diversity and molar ratio of lipids present in the bilayer directly impact membrane fluidity, permeability, and surface charge, as well as the loading capacity of drugs.
Drug loading and release: The nature of the encapsulated drug, which can be either hydrophilic or lipophilic. The inclusion of stimuli-sensitive lipids or other components allows for a triggered drug release under specific conditions.
Targeting methods: Active targeting by the attachment of ligands/molecules on the vesicle surface, which are preferentially (or exclusively) recognizable by target cells/tissues, and passive targeting through usage of the enhanced permeability and retention effect (EPR) effect. The vast majority of liposomal drug formulations contain “PEGylated lipids” (lipids with attached polyethylene glycol (PEG) chains) that affect the clearance of liposomes.
One of the most well-known liposomal formulations available in clinical practice is Doxil, which was created to overcome the cardiotoxicity of doxorubicin and clearly shows reduced cytotoxicity when compared to the free drug. At the same time, Myocet, which is another liposomal formulation of doxorubicin, displays vastly different pharmacokinetics in comparison with Doxil, which may be partly due to the lack of PEGylated lipids in the liposomal shell. For those reasons, these two formulations are used in treatment of different types of cancer, despite encapsulating the same type of drug (see Table 1) .
Table 1. List of liposomal drug products for injection clinically approved by European Medicines Agency (EMA) and Food and Drug Administration (FDA).
|Drug||Product Name||Route of Administration||Lipid Composition (Molar Ratio 1)||Treatment||Ref.|
|Amphotericin B||Abelcet||Intravenous||DMPC, DMPG (7:3)||Systemic fungal infections|||
|Ambisome||Intravenous||HSPC, DSPG, cholesterol (2:0.8:0.4)||Systemic fungal infections|||
|Bupivacaine||Exparel||Supraperiosteal Injection||DEPC, DPPG, cholesterol, tricaprylin||Postsurgical local analgesia|||
|Nocita||Supraperiosteal Injection||DEPC, DPPG, cholesterol, tricaprylin||Postsurgical local analgesia (for dogs only)|||
|Cytarabine||Depocyt||Spinal||DOPC, DPPG, cholesterol, triolein (7:1:11:1)||Lymphomatous meningitis|||
|Daunorubicin||DaunoXome||Intravenous||DSPC, cholesterol (2:1)||Kaposi’s sarcoma|||
|Doxorubicin||Doxil/Caelyx 2||Intravenous||HSPC, cholesterol, DSPE-PEG (2000) (56:39:5)||Kaposi’s sarcoma|||
|Lipodox||Intravenous||DSPC, cholesterol, DSPE-PEG (2000) (56:39:5)||Kaposi’s sarcoma, ovarian/breast cancer|||
|Myocet liposomal 3||Intravenous||EPC, cholesterol (55:45)||Metastatic breast cancer|||
|Inactivated hepatitis A virus||Epaxal||Intramuscular||DOPC, DOPE (75:25)||Hepatitis A|||
|Inactivated hemagglutinin of influenza virus strains A and B||Inflexal V||Intramuscular||DOPC, DOPE (75:25)||Influenza|||
|Irinotecan||Onivyde||Intravenous||DSPC, MPEG-2000-DSPE||metastatic adenocarcinoma of the pancreas|||
|Mifamurtide||Mepact 2||Intravenous||POPC, DOPS (7:3)||High-grade non-metastatic osteosarcoma|||
|Morphine sulfate||DepoDur||Epidural||DOPC, DPPG, cholesterol, triolein (7:1:11:1)||Pain management|||
|Verteporfin||Visudyne||Intravenous||DMPC, EPG (5:3)||Age-related macular degeneration, pathologic myopia, ocular histoplasmosis|||
|Vincristine sulfate||Marqibo||Intravenous||Sphingomyelin, cholesterol (6:4)||Acute lymphoblastic leukaemia|||
HSPC—hydrogenated soya bean phosphatidylcholine; DSPG—1,2-distearoyl-sn-glycero-3-phosphoglycerol; DEPC—1,2-dierucoyl-sn-glycero-3-phosphocholine; DSPE-PEG(2000)—1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]; EPC—egg phosphatidylcholine; MPEG-2000-DSPE—1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000]; DOPS—1,2-dioleoyl-sn-glycero-3-phospho-L-serine; EPG—egg phosphatidylglycerol. 1 If available. 2 Outside the United States, Doxil is known as Caelyx. 3 These formulations are only approved by EMA and not by FDA.
2. Drug Loading and Release
There are many ways to encapsulate drugs into liposomes, but all these methods generally fall under one of two categories. Passive loading is carried out during the formation of the liposomes where either the dry lipid film is formed in the presence of a hydrophobic drug or the lipid film is rehydrated with the use of a hydrophilic drug solution. Unfortunately, the encapsulation efficacy of hydrophilic drugs is usually low. This method can also cause a rapid, uncontrolled release of entrapped contents from the liposomes . Active loading often depends on either an ion or a pH gradient across the membrane of already preformed liposomes. The properties of an encapsulated drug make a major difference in their liposome-modulated bioavailability. For example, the release rate of the hydrophobic drug dibucaine is much lower than that of the hydrophilic 5-fluorouracil when encapsulated within multilamellar liposomes formed with egg phosphatidylcholine and cholesterol. This difference was further increased in the case of negatively charged liposomes where the hydrophobic drug release was inhibited due to the charge on the liposomal surface . It is possible to achieve tighter control over the release of the liposomal cargo through the use of stimulus-responsive liposomes, which become metastable under certain conditions, such as pH, redox potential or temperature . These aspects are unique to a disease condition and pathological state of tissues, because inflammation is always accompanied by local hyperthermia. For the design of thermosensitive liposomes lipids such as 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC) (Tm = 35 °C) and 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC) (Tm = 40 °C) are commonly used . The Tm of these lipids is around the physiological temperature, rendering the lipid bilayer more permeable during the phase transition temperature, which occurs in the tissue in which the inflammation takes place . Local hyperthermia can also be artificially induced by near-infrared (NIR) radiation that is able to penetrate into deep tissues. This was the case with thermosensitive liposomes carrying both the NIR-absorbing dye indocyanine green and the anticancer drug doxorubicin. This synergic solution allowed for the effective release of encapsulated contents into the cancer cells . Yet another way of facilitating drug release from thermosensitive liposomes is with the use of radiofrequency ablation (RFA). This procedure involves high-frequency electrical pulses which pass through an electrode, creating a small region of heat in a selected area. A phase III clinical study was conducted on a combination of lyso-thermosensitive liposomal doxorubicin (ThermoDox) with RFA. ThermoDox contains a lysolipid monostearoyl-phosphatidylcholine (MoSPC) that forms defects above its Tm (40 °C) that aid the release of encapsulated contents . An animal study was conducted in order to optimize heating time and then this combinational therapy was tested on patients with hepatocellular carcinoma, and was found to significantly improve their overall survival . Chen et al. developed a thermoresponsive liposomal system for extracellular delivery of doxorubicin. Its key component, ammonium bicarbonate, which is used in generating a transmembrane gradient for the encapsulation of the drug, decomposes to carbon dioxide bubbles upon heating. This process generates defects in the lipid bilayer, leading to the quick release of the encapsulated doxorubicin. These liposomes are also more stable in blood plasma and have a longer circulation time when compared to lysolipid liposomes, such as ThermoDox . In solid tumors, the intratumoral pH value is slightly lower than the pH of blood and surrounding tissues, which is taken into consideration when composing pH-sensitive liposomal formulations. Those liposomes enter the tumor tissue and quickly become destabilized while releasing the encapsulated contents. However, pH values differ in endosomes and in the tumoral environment. All these elements must be taken into account when designing a pH-sensitive liposomal formulation and choosing lipids with the desired Tm . DOPE is the most popular choice as thanks to its cone shape it forms hexagonal phases. However, due to this, DOPE cannot form lipid bilayers by itself in neutral pH and requires the presence of weakly acidic amphiphilic lipids such as cholesteryl hemisuccinate or cylindrically shaped lipids such as PC . Redox potential can also be used as a stimulus, as in the case of liposomes designed for the treatment of human osteosarcoma. The surface of liposomes was coated with chitooligosaccharides via disulfide bonds. The intracellular environment (especially in cancer tissues) is much more reductive than the extracellular environment. This means that liposomes did not show any unwanted drug leakage in physiological conditions but were destabilized by reducing agents such as dithiothreitol or glutathione .
3. Targeting and Clearance
Liposomes can be the subject of active and passive targeting. The latter depends on a phenomenon called the enhanced permeability and retention effect (EPR) in the environment of tumors. Rapid tumor vascularization leads to the formation of immature tumor vessels inside the tumoral mass characterized by high permeability, which leads to the accumulation of nanoparticles smaller than 150 nm which are able to cross these vessels. Moreover, due to the abrupt ending of these vessels, there is a lack of functional lymphatic drainage, so the clearance of any accumulated particles is hindered (Figure 1) . The EPR effect does not occur only in tumors, as inflamed tissues are also characterized by enhanced vascular permeability, as for example in the case of rheumatoid arthritis. Jia et al. tested a liposomal formulation of the hydrophobic drug dexamethasone on an adjuvant-induced arthritis (AIA) rat model. While the free drug showed a decrease in inflammation, it also led rats to develop hyperglycemia. The liposomes seemed not to have such a strong side effect and also showed better accumulation in inflamed tissues . Targeting via size is also effective when the target is a part of the RES. Particles in the range of 100 nm to 150 nm are preferentially taken up by phagocytes and accumulate in the liver . Liposomes that extend beyond 150 nm are characterized by rapid uptake by the mononuclear phagocytic system (MPS), which matters in the treatment of such diseases as leukemia and rheumatoid arthritis .