Vladimir P. Torchilin

Recent advances with liposomes as pharmaceutical carriers

Liposomes — microscopic phospholipid bubbles with a bilayered membrane structure — have received a lot of attention during the past 30 years as pharmaceutical carriers of great potential. More recently, many new developments have been seen in the area of liposomal drugs — from clinically approved products to new experimental applications, with gene delivery and cancer therapy still being the principal areas of interest. For further successful development of this field, promising trends must be identified and exploited, albeit with a clear understanding of the limitations of these approaches.

Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes

Incorporation of dioleoyl N‐(monomethoxy polyethyleneglycol succinyl)phosphotidylethanolamine (PEG‐PE) into large unilamellar liposomes composed of egg posphatidylcholine:cholesterol (1:1) does not significantly increase the content leakage when the liposomes are exposed to 90% human serum at 37°C, yet the liposomes show a significant increase in the blood circulation half‐life (t = 5 h) as compared to those without PEG‐PE(t <30 min). The PEG‐PEs activity to prolong the circulation time of liposomes is greater than that of the ganglioside GM1, awell‐described glycolipid with this activity. Another amphipathic PEG derivative, PEG stearate, also prolongs the liposome circulation time, although its activity is less than that ofGM1. Amphipathic PEGs may be useful for the sustained release and the targeted drug delivery by liposomes.

Micellar Nanocarriers: Pharmaceutical Perspectives

Micelles, self-assembling nanosized colloidal particles with a hydrophobic core and hydrophilic shell are currently successfully used as pharmaceutical carriers for water-insoluble drugs and demonstrate a series of attractive properties as drug carriers. Among the micelle-forming compounds, amphiphilic copolymers, i.e., polymers consisting of hydrophobic block and hydrophilic block, are gaining an increasing attention. Polymeric micelles possess high stability both in vitro and in vivo and good biocompatibility, and can solubilize a broad variety of poorly soluble pharmaceuticals many of these drug-loaded micelles are currently at different stages of preclinical and clinical trials. Among polymeric micelles, a special group is formed by lipid-core micelles, i.e., micelles formed by conjugates of soluble copolymers with lipids (such as polyethylene glycol–phosphatidyl ethanolamine conjugate, PEG–PE). Polymeric micelles, including lipid-core micelles, carrying various reporter (contrast) groups may become the imaging agents of choice in different imaging modalities. All these micelles can also be used as targeted drug delivery systems. The targeting can be achieved via the enhanced permeability and retention (EPR) effect (into the areas with the compromised vasculature), by making micelles of stimuli-responsive amphiphilic block-copolymers, or by attaching specific targeting ligand molecules to the micelle surface. Immunomicelles prepared by coupling monoclonal antibody molecules to p-nitrophenylcarbonyl groups on the water-exposed termini of the micelle corona-forming blocks demonstrate high binding specificity and targetability. This review will discuss some recent trends in using micelles as pharmaceutical carriers.

Structure and design of polymeric surfactant-based drug delivery systems.

The review concentrates on the use of polymeric micelles as pharmaceutical carriers. Micellization of biologically active substances is a general phenomenon that increases the bioavailability of lipophilic drugs and nutrients. Currently used low-molecular-weight pharmaceutical surfactants have low toxicity and high solubilization power towards poorly soluble pharmaceuticals. However, micelles made of such surfactants usually have relatively high critical micelle concentration (CMC) and are unstable upon strong dilution (for example, with the blood volume upon intravenous administration). On the other hand, amphiphilic block co-polymers are also known to form spherical micelles in solution. These micelles have very high solubilization capacity and rather low CMC value that makes them very stable in vivo. Amphiphilic block co-polymers suitable for micelle preparation are described and various types of polymeric micelles are considered as well as mechanisms of their formation, factors influencing their stability and disintegration, their loading capacity towards various poorly soluble pharmaceuticals, and their therapeutic potential. The basic mechanisms underlying micelle longevity and steric protection in vivo are considered with a special emphasis on long circulating drug delivery systems. Advantages and disadvantages of micelles when compared with other drug delivery systems are considered. New polymer-lipid amphiphilic compounds such as diacyillipid-polyethylene glycol, are described and discussed. These compounds are very attractive from a practical point of view, since they easily micellize yielding extremely stable micelles with very high loading capacity. Micelle passive accumulation in the areas with leaky vasculature (tumors, infarct zones) is discussed as an important physiology-based mechanism of drug delivery into certain target zones. Targeted polymeric micelles prepared by using thermo- or pH-sensitive components or by attaching specific targeted moieties (such as antibodies) to their outer surface are described as well as their preparation and some in vivo properties. The fast growing field of diagnostic micelles is analyzed. Polymeric micelles are considered loaded with various agents for gamma, magnetic resonance, and computed tomography imaging. Their in vitro and in vivo properties are discussed and the results of the initial animal experiments are presented.

Tumor delivery of macromolecular drugs based on the EPR effect

Enhanced permeability and retention (EPR) effect is the physiology-based principal mechanism of tumor accumulation of large molecules and small particles. This specific issue of Advanced Drug Delivery Reviews is summing up multiple data on the EPR effect-based drug design and clinical outcome. In this commentary, the role of the EPR effect in the intratumoral delivery of protein and peptide drugs, macromolecular drugs and drug-loaded long-circulating pharmaceutical nanocarriers is briefly discussed together with some additional opportunities for drug delivery arising from the initial EPR effect-mediated accumulation of drug-containing macromolecular systems in tumors.

TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors

To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway. TAT peptides derived from the HIV-1 TAT protein facilitate intracellular delivery of proteins and small colloidal particles. We demonstrated that relatively large drug carriers, such as 200-nm liposomes, can also be delivered into cells by TAT peptide attached to the liposome surface. Liposomes were fluorescently labeled with membranotropic rhodamine-phosphatidylethanolamine or by entrapping FITC-dextran. Incubation of fluorescent TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocyte H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide⋅cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW polyethylene glycol on the liposome surface) abolish liposome internalization, evidencing the importance of direct contact of TAT peptide with the cell surface. Low temperature or metabolic inhibitors, sodium azide or iodoacetamide, have little influence on the translocation of TAT liposomes into cells, confirming the energy-independent character of this process. The approach may have important implications for drug delivery directly into cell cytoplasm.

Targeted pharmaceutical nanocarriers for cancer therapy and imaging

The use of various pharmaceutical nanocarriers has become one of the most important areas of nanomedicine. Ideally, such carriers should be specifically delivered (targeted) to the pathological area to provide the maximum therapeutic efficacy. Among the many potential targets for such nanocarriers, tumors have been most often investigated. This review attempts to summarize currently available information regarding targeted pharmaceutical nanocarriers for cancer therapy and imaging. Certain issues related to some popular pharmaceutical nanocarriers, such as liposomes and polymeric micelles, are addressed, as are different ways to target tumors via specific ligands and via the stimuli sensitivity of the carriers. The importance of intracellular targeting of drug- and DNA-loaded pharmaceutical nanocarriers is specifically discussed, including intracellular delivery with cell-penetrating peptides.

Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery

The use of nanoparticulate pharmaceutical drug delivery systems (NDDSs) to enhance the in vivo effectiveness of drugs is now well established. The development of multifunctional and stimulus-sensitive NDDSs is an active area of current research. Such NDDSs can have long circulation times, target the site of the disease and enhance the intracellular delivery of a drug. This type of NDDS can also respond to local stimuli that are characteristic of the pathological site by, for example, releasing an entrapped drug or shedding a protective coating, thus facilitating the interaction between drug-loaded nanocarriers and target cells or tissues. In addition, imaging contrast moieties can be attached to these carriers to track their real-time biodistribution and accumulation in target cells or tissues. Here, I highlight recent developments with multifunctional and stimuli-sensitive NDDSs and their therapeutic potential for diseases including cancer, cardiovascular diseases and infectious diseases.

Immunomicelles: Targeted pharmaceutical carriers for poorly soluble drugs

To prepare immunomicelles, new targeted carriers for poorly soluble pharmaceuticals, a procedure has been developed to chemically attach mAbs to reactive groups incorporated into the corona of polymeric micelles made of polyethylene glycol–phosphatidylethanolamine conjugates. Micelle-attached antibodies retained their ability to specifically interact with their antigens. Immunomicelles with attached antitumor mAb 2C5 effectively recognized and bound various cancer cells in vitro and showed an increased accumulation in experimental tumors in mice when compared with nontargeted micelles. Intravenous administration of tumor-specific 2C5 immunomicelles loaded with a sparingly soluble anticancer agent, taxol, into experimental mice bearing Lewis lung carcinoma resulted in an increased accumulation of taxol in the tumor compared with free taxol or taxol in nontargeted micelles and in enhanced tumor growth inhibition. This family of pharmaceutical carriers can be used for the solubilization and enhanced delivery of poorly soluble drugs to various pathological sites in the body.

Activity of amphipathic poly(ethylene glycol) 5000 to prolong the circulation time of liposomes depends on the liposome size and is unfavorable for immunoliposome binding to target

Dioleoyl-N-(monomethoxy polyethyleneglycol succinyl)-phosphatidylethanolamine (PEG-PE) (mol. wt. of PEG = 5000), an amphipathic polymer, can be incorporated into the liposome membrane and significantly prolong the blood circulation time of the liposome. As little as 3.7 mol% of PEG-PE in liposome resulted in maximal enhancement of liposome circulation time. However, this activity of PEG-PE was only seen with relatively small liposomes (d less than or equal to 200 nm); larger liposomes containing PEG-PE showed an unusually high level (approx. 35% injected dose) of accumulation in the spleen. We have tested whether the small, PEG-PE containing liposomes are suitable for immuno targeting by incorporating a lung-specific monoclonal antibody on the liposome surface. While another amphiphile, ganglioside GM1, which is well known for its activity to prolong the liposome circulation time, significantly enhanced the lung binding of the immunoliposomes, PEG-PE incorporation of immunoliposomes resulted in a low level of target binding. To test if the reduced target binding is due to a steric barrier effect of the surface PEG polymer, we have incorporated a small amount of N-biotinaminocaproylphosphatidylethanolamine into the PEG-PE containing liposomes and examined the liposome agglutination induced by the addition of streptavidin. As little as 0.72 mol% PEG-PE in these liposomes completely abolished agglutination. In contrast, incorporation of GM1 in liposomes only reduced the rate, but not the extent, of liposome agglutination. These results strongly support the hypothesis that PEG-PE prolongs liposome circulation time by providing a strong steric barrier which prevents close contact with another liposome or cell. Since GM1 provides only a weak steric barrier effect, its activity to prolong the liposome circulation time must involve another yet unknown mechanism.