Anticancer double lipid prodrugs: liposomal preparation and characterization

The escape of encapsulated anticancer drugs from liposomes by passive diffusion often leads to suboptimal drug concentrations in the cancer tissue, therefore calling for effective trigger mechanisms to release the drug at the target. We investigated mixtures of lipid components that not only form stable liposomes, but also can be turned into active drugs by secretory phospholipase A? (sPLA?), an enzyme that is upregulated in various cancer cells, without the necessity for conventional liposome drug loading. The liposomes are composed of a novel lipid-based retinoid prodrug premixed with saturated phospholipids. The prodrug is found to be miscible with phospholipids, and the lipid mixtures are shown to form liposomes with the desired size distribution. The preparation procedure, phase behavior, and physicochemical properties of the formed liposomes are described as a function of lipid composition. We show that the premixing of the prodrug with phospholipids can be used to modify the physicochemical properties of liposomal formulations. The results should prove useful for further exploration of the potential for using these novel lipid prodrugs in liposomal formulations for cancer treatment.     

Biodistribution and Therapeutic Utility of Liposomal Drug Carrier Systems

Abstract

Delivery of the drug at a specific site (drug targeting) or controlled and prolonged release of the liposome-bound drug are the two major considerations for adding liposomes to the existing arsenal of drug delivery systems. In particular the concept of liposomal drug targeting has been evolving rapidly in the past 10 years with the development of 'second generation' carriers such as immunoliposomes (liposomes bearing covalently coupled antibodies as homing device) and, more recently, the long-circulating liposomes. In this contribution novel approaches in the field of liposomal drug targeting will be briefly described: (1) immunoliposomes for chemotherapy of intraperitoneal malignancies, such as ovarian carcinoma, (2) a new type of immunoliposomes for mediating the targeting of enzymes to be used for site-specific prodrug activation (immuno-enzymosomes), (3) long-circulating liposomes for the targeting of antibiotics to sites of bacterial infection, and (4) polyethyleneglycol (PEG)-modified proteoliposomes with the homing device coupled to the ends of the long PEG chains for achieving effective target binding along with prolonged circulation times.     

Phospholipase A2 sensitive liposomes for delivery of small interfering RNA (siRNA)

Small interfering RNA (siRNA) is potent and highly specific for gene silencing and there is currently a lot of enthusiasm for developing siRNA into a drug. However, for most therapeutic applications of siRNA, delivery systems are needed. These delivery systems have multiple requirements and should on one hand ideally be stable carriers protecting the siRNA from degradation and on the other hand assist the siRNA in overcoming membrane barriers for intracellular delivery to the cytosol. Long-circulating liposomes, which are sensitive to secretory phospholipase A(2) (sPLA(2)) are feasible delivery systems for systemic administration of drugs due to their passive targeting to pathological tissue via the enhanced permeability and retention (EPR) effect and their site-specific, enzyme-triggered release of encapsulated drug in response to sPLA(2) which exists locally at elevated levels at, e.g,. sites of inflammation. However, recent data suggest that endosomal membrane destabilizing approaches could be addressed to design sPLA(2)-sensitive liposomes as successful delivery systems for siRNA to the RNA interference pathway in the cytoplasm upon systemic administration.     

Enzyme-triggered nanomedicine: drug release strategies in cancer therapy

Nanomedicine as a field has emerged from the early success of nanoparticle-based drug delivery systems, in particular for treatment of cancer, and the advances made in nano- and biotechnology over the past decade. A prerequisite for nanoparticle-based drug delivery systems to be effective is that the drug payload is released at the target site. A large number of drug release strategies have been proposed that can be classified into certain areas. The simplest and most successful strategy so far, probably due to relative simplicity, is based on utilizing certain physico-chemical characteristics of drugs to obtain a slow drug leakage from the formulations after accumulation in the cancerous site. However, this strategy is only applicable to a relatively small range of drugs and cannot be applied to biologicals. Many advanced drug release strategies have therefore been investigated. Such strategies include utilization of heat, light and ultrasound sensitive systems and in particular pH sensitive systems where the lower pH in endosomes induces drug release. Highly interesting are enzyme sensitive systems where over-expressed disease-associated enzymes are utilized to trigger drug release. The enzyme-based strategies are particularly interesting as they require no prior knowledge of the tumour localization. The basis of this review is an evaluation of the current status of drug delivery strategies focused on triggered drug release by disease-associated enzymes. We limit ourselves to reviewing the liposome field, but the concepts and conclusions are equally important for polymer-based systems.     

Evaluation of tetraether lipid-based liposomal carriers for encapsulation and retention of nucleoside-based drugs

Although liposomal nanoparticles are one of the most versatile class of drug delivery systems, stable liposomal formulation of small neutral drug molecules still constitutes a challenge due to the low drug retention of current lipid membrane technologies. In this study, we evaluate the encapsulation and retention of seven nucleoside analog-based drugs in liposomes made of archaea-inspired tetraether lipids, which are known to enhance packing and membrane robustness compared to conventional bilayer-forming lipids. Liposomes comprised of the pure tetraether lipid generally showed improved retention of drugs (up to 4-fold) compared with liposomes made from a commercially available diacyl lipid. Interestingly, we did not find a significant correlation between the liposomal leakage rates of the molecules with typical parameters used to assess lipophilicity of drugs (such logD or topological polar surface area), suggesting that specific structural elements of the drug molecules can have a dominant effect on leakage from liposomes over general lipophilic character.     

Enzyme-triggered nanomedicine: Drug release strategies in cancer therapy (Invited Review)

Abstract

Nanomedicine as a field has emerged from the early success of nanoparticle-based drug delivery systems, in particular for treatment of cancer, and the advances made in nano- and biotechnology over the past decade. A prerequisite for nanoparticle-based drug delivery systems to be effective is that the drug payload is released at the target site. A large number of drug release strategies have been proposed that can be classified into certain areas. The simplest and most successful strategy so far, probably due to relative simplicity, is based on utilizing certain physico-chemical characteristics of drugs to obtain a slow drug leakage from the formulations after accumulation in the cancerous site. However, this strategy is only applicable to a relatively small range of drugs and cannot be applied to biologicals. Many advanced drug release strategies have therefore been investigated. Such strategies include utilization of heat, light and ultrasound sensitive systems and in particular pH sensitive systems where the lower pH in endosomes induces drug release. Highly interesting are enzyme sensitive systems where over-expressed disease-associated enzymes are utilized to trigger drug release. The enzyme-based strategies are particularly interesting as they require no prior knowledge of the tumour localization. The basis of this review is an evaluation of the current status of drug delivery strategies focused on triggered drug release by disease-associated enzymes. We limit ourselves to reviewing the liposome field, but the concepts and conclusions are equally important for polymer-based systems.     

Secreted phospholipase A(2) as a new enzymatic trigger mechanism for localised liposomal drug release and absorption in diseased tissue

Polymer-coated liposomes can act as versatile drug-delivery systems due to long vascular circulation time and passive targeting by leaky blood vessels in diseased tissue. We present an experimental model system illustrating a new principle for improved and programmable drug-delivery, which takes advantage of an elevated activity of secretory phospholipase A(2) (PLA(2)) at the diseased target tissue. The secretory PLA(2) hydrolyses a lipid-based proenhancer in the carrier liposome, producing lyso-phospholipids and free fatty acids, which are shown in a synergistic way to lead to enhanced liposome destabilization and drug release at the same time as the permeability of the target membrane is enhanced. Moreover, the proposed system can be made thermosensitive and offers a rational way for developing smart liposome-based drug delivery systems. This can be achieved by incorporating specific lipid-based proenhancers or prodestabilisers into the liposome carrier, which automatically becomes activated by PLA(2) only at the diseased target sites, such as inflamed or cancerous tissue.     

Phospholipase A2 Sensitive Liposomes for Delivery of Small Interfering RNA (siRNA)

Small interfering RNA (siRNA) is potent and highly specific for gene silencing and there is currently a lot of enthusiasm for developing siRNA into a drug. However, for most therapeutic applications of siRNA, delivery systems are needed. These delivery systems have multiple requirements and should on one hand ideally be stable carriers protecting the siRNA from degradation and on the other hand assist the siRNA in overcoming membrane barriers for intracellular delivery to the cytosol. Long-circulating liposomes, which are sensitive to secretory phospholipase A₂ (sPLA₂) are feasible delivery systems for systemic administration of drugs due to their passive targeting to pathological tissue via the enhanced permeability and retention (EPR) effect and their site-specific, enzyme-triggered release of encapsulated drug in response to sPLA₂ which exists locally at elevated levels at, e.g,. sites of inflammation. However, recent data suggest that endosomal membrane destabilizing approaches could be addressed to design sPLA₂-sensitive liposomes as successful delivery systems for siRNA to the RNA interference pathway in the cytoplasm upon systemic administration.     

Secreted phospholipase A2 as a new enzymatic trigger mechanism for localised liposomal drug release and absorption in diseased tissue

Polymer-coated liposomes can act as versatile drug-delivery systems due to long vascular circulation time and passive targeting by leaky blood vessels in diseased tissue. We present an experimental model system illustrating a new principle for improved and programmable drug-delivery, which takes advantage of an elevated activity of secretory phospholipase A₂ (PLA₂) at the diseased target tissue. The secretory PLA₂ hydrolyses a lipid-based proenhancer in the carrier liposome, producing lyso-phospholipids and free fatty acids, which are shown in a synergistic way to lead to enhanced liposome destabilization and drug release at the same time as the permeability of the target membrane is enhanced. Moreover, the proposed system can be made thermosensitive and offers a rational way for developing smart liposome-based drug delivery systems. This can be achieved by incorporating specific lipid-based proenhancers or prodestabilisers into the liposome carrier, which automatically becomes activated by PLA₂ only at the diseased target sites, such as inflamed or cancerous tissue.     

Liposomal systems as viable drug delivery technology for skin cancer sites with an outlook on lipid-based delivery vehicles and diagnostic imaging inputs for skin conditions'

Skin cancer is among one of the most common human malignancies wide-spread world-over with mortality statistics rising continuously at an alarming rate. The increasing frequency of these malignancies has marked the need for adopting effective treatment plan coupled with better and site-specific delivery options for the desired therapeutic agent's availability at the affected site. The concurrent delivery approaches to cancerous tissues are under constant challenge and, as a result, are evolving and gaining advancements in terms of delivery modes, therapeutic agents and site-specificity of the therapeutics delivery. The lipid-based liposomal drug delivery is an attractive and emerging option, and which is meticulously shaping up beyond a threshold level to a promising, and viable route for the effective delivery of therapeutic agents and other required injuctions to the skin cancer. An update on liposomal delivery of chemotherapeutic agents, natural-origin compounds, photosensitizer, and DNA repair enzymes as well as other desirable and typical delivery modes employed in drug delivery and in the treatment of skin cancers is discussed in details. Moreover, liposomal delivery of nucleic acid-based therapeutics, i.e., small interfering RNA (siRNA), mRNA therapy, and RGD-linked liposomes are among the other promising novel technology under constant development. The current clinical applicability, viable clinical plans, future prospects including transport feasibility of delivery vesicles and imaging techniques in conjunction with the therapeutic agents is also discussed. The ongoing innovations in liposomal drug delivery technology for skin cancers hold promise for further development of the methodology for better, more effective and site-specific delivery as part of the better treatment plan by ensuring faster drug transport, better and full payload delivery with enough and required concentration of the dose.     

Comparison of Conventional Chemotherapy,Stealth Liposomes and Temperature-Sensitive Liposomes in a Mathematical Model

Various liposomal drug carriers have been developed to overcome short plasma half-life and toxicity related side effects of chemotherapeutic agents. We developed a mathematical model to compare different liposome formulations of doxorubicin (DOX): conventional chemotherapy (Free-DOX), Stealth liposomes (Stealth-DOX), temperature sensitive liposomes (TSL) with intra-vascular triggered release (TSL-i), and TSL with extra-vascular triggered release (TSL-e). All formulations were administered as bolus at a dose of 9 mg/kg. For TSL, we assumed locally triggered release due to hyperthermia for 30 min. Drug concentrations were determined in systemic plasma, aggregate body tissue, cardiac tissue, tumor plasma, tumor interstitial space, and tumor cells. All compartments were assumed perfectly mixed, and represented by ordinary differential equations. Contribution of liposomal extravasation was negligible in the case of TSL-i, but was the major delivery mechanism for Stealth-DOX and for TSL-e. The dominant delivery mechanism for TSL-i was release within the tumor plasma compartment with subsequent tissue- and cell uptake of released DOX. Maximum intracellular tumor drug concentrations for Free-DOX, Stealth-DOX, TSL-i, and TSL-e were 3.4, 0.4, 100.6, and 15.9 µg/g, respectively. TSL-i and TSL-e allowed for high local tumor drug concentrations with reduced systemic exposure compared to Free-DOX. While Stealth-DOX resulted in high tumor tissue concentrations compared to Free-DOX, only a small fraction was bioavailable, resulting in little cellular uptake. Consistent with clinical data, Stealth-DOX resulted in similar tumor intracellular concentrations as Free-DOX, but with reduced systemic exposure. Optimal release time constants for maximum cellular uptake for Stealth-DOX, TSL-e, and TSL-i were 45 min, 11 min, and <3 s, respectively. Optimal release time constants were shorter for MDR cells, with ∼4 min for Stealth-DOX and for TSL-e. Tissue concentrations correlated well quantitatively with a prior in-vivo study. Mathematical models may thus allow optimization of drug delivery systems to achieve a better therapeutic index.     

Development of Nanoparticles Incorporating a Novel Liposomal Membrane Destabilization Peptide for Efficient Release of Cargos into Cancer Cells

In anti-cancer therapy mediated by a nanoparticle-based drug delivery system (DDS), overall efficacy depends on the release efficiency of cargos from the nanoparticles in the cancer cells as well as the specificity of delivery to tumor tissue. However, conventional liposome-based DDS have no mechanism for specifically releasing the encapsulated cargos inside the cancer cells. To overcome this barrier, we developed nanoparticles containing a novel liposomal membrane destabilization peptide (LMDP) that can destabilize membranes by cleavage with intramembranous proteases on/in cancer cells. Calcein encapsulated in liposomes modified with LMDP (LMDP-lipo) was effectively released in the presence of a membrane fraction containing an LMDP-cleavable protease. The release was inhibited by a protease inhibitor, suggesting that LMDP-lipo could effectively release its cargo into cells in response to a cancer-specific protease. Moreover, when LMDP-lipo contained fusogenic lipids, the release of cargo was accelerated, suggesting that the fusion of LMDP-lipo with cellular membranes was the initial step in the intracellular delivery. Time-lapse microscopic observations showed that the release of cargo from LMDP-lipo occurred immediately after association of LMDP-lipo with target cells. Consequently, LMDP-lipo could be a useful nanoparticle capable of effective release of cargos specifically into targeted cancer cells.     

Biophysical mechanisms of phospholipase A2 activation and their use in liposome-based drug delivery

Secretory phospholipase A2 (PLA2) is a ubiquitous water-soluble enzyme found in venom, pancreatic, and cancerous fluid. It is also known to play a role in membrane remodeling processes as well as in cellular signaling cascades. PLA2 is interfacially active and functions mainly on organized types of substrate, e.g. micelles and lipid bilayers. Hence the activity of the enzyme is modulated by the lateral organization and the physical properties of the substrate, in particular the structure in the nanometer range. The evidence for nano-scale structure and lipid domains in bilayers is briefly reviewed. Results obtained from a variety of experimental and theoretical studies of PLA2 activity on lipid-bilayer substrates are then presented which provide insight into the biophysical mechanisms of PLA2 activation on lipid bilayers and liposomes of different composition. The insight into these mechanisms has been used to propose a novel principle for liposomal drug targeting, release, and absorption triggered by secretory PLA2.     

Factors affecting the pharmacokinetics and pharmacodynamics of liposomal drugs

Various attempts to increase the therapeutic index of the drug while minimizing side effects have been made in drug delivery systems. Among several promising strategies, liposomes represent an advanced technology to target active molecules to the site of action. Rapid clearance of circulating liposomal drugs administered intravenously has been a critical issue because circulation time in the blood affects drug exposure at the target site. The clinical use of liposomal drugs is complicated by large intra- and interindividual variability in their pharmacokinetics (PK) and pharmacodynamics (PD). Thus, it is important to understand the factors affecting the PK/PD of the liposomal formulation of drugs and to elucidate the mechanisms underlying the variability in the PK/PD of liposomal drugs. In this review article, we describe the characteristics of liposome formulations and discuss the effects of various factors, including liposome-associated factors, host-associated factors, and treatment on the PK/PD of liposomal agents.     

Factors affecting the pharmacokinetics and pharmacodynamics of liposomal drugs

Various attempts to increase the therapeutic index of the drug while minimizing side effects have been made in drug delivery systems. Among several promising strategies, liposomes represent an advanced technology to target active molecules to the site of action. Rapid clearance of circulating liposomal drugs administered intravenously has been a critical issue because circulation time in the blood affects drug exposure at the target site. The clinical use of liposomal drugs is complicated by large intra- and interindividual variability in their pharmacokinetics (PK) and pharmacodynamics (PD). Thus, it is important to understand the factors affecting the PK/PD of the liposomal formulation of drugs and to elucidate the mechanisms underlying the variability in the PK/PD of liposomal drugs. In this review article, we describe the characteristics of liposome formulations and discuss the effects of various factors, including liposome-associated factors, host-associated factors, and treatment on the PK/PD of liposomal agents.     

Adhesion characteristics and stability assessment of lectin-modified liposomes for site-specific drug delivery

Carbohydrate moieties of the cellular glycocalyx have been suggested to play an important role in biological recognition processes during pathologic conditions, such as inflammation and cancer. Herein, we describe lectin-modified liposomes which might have potential for site-specific drug delivery during the therapy of such diseases. Specific interactions of plain (i.e., unmodified) and PEGylated, lectin-grafted liposomes with model membranes were investigated under real-time flow conditions using a quartz crystal microbalance. In addition, the morphology of the liposomal systems was assessed by atomic force microscopy. Plain liposomes exhibited only unspecific adhesion to glycolipid membranes and had a tendency to coalesce. The degree of membrane interaction was significantly increased when plain liposomes were modified with the lectin, Concanavalin A. However, vesicle fusion also markedly increased as a result of lectin modification. Additional PEGylation of liposomes reduced unspecific adhesion phenomena, as well as coalescence. Moreover, our studies enabled us to establish quartz crystal microbalance and atomic force microscopy as powerful and complementary methods to characterize adhesion properties of targeted drug delivery systems.     

Liposomes of terbutaline sulphate: in vitro and in vivo studies

In vitro studies were conducted to understand the comparative drug diffusion pattern, across artificial membrane, of the drug and of the prepared liposomes of different liposomal membrane composition. In vivo studies were carried out to determine the extent and time-course of pulmonary tissue uptake of administered liposomes containing terbutaline sulphate(TER) on rat lungs. In vitro studies revealed that the drug released from the prepared liposomes obeys Higuchi's diffusion controlled model. Different loading doses and release patterns of drug from the liposomes can be obtained by altering the PC:CHOL ratio and incorporation of cholesterol was found to reduce permeability of the membrane. Similarly drug absorption in vivo in rat's lung following intratracheal instillation, prolonged over 12 hr by liposomal entrapment of TER. The findings of present investigation indicated that liposomally encapsulated TER can be used for pulmonary delivery for maximizing the therapeutic efficacy and reducing undesirable side effects.     

Adhesion characteristics and stability assessment of lectin-modified liposomes for site-specific drug delivery

Carbohydrate moieties of the cellular glycocalyx have been suggested to play an important role in biological recognition processes during pathologic conditions, such as inflammation and cancer. Herein, we describe lectin-modified liposomes which might have potential for site-specific drug delivery during the therapy of such diseases. Specific interactions of plain (i.e., unmodified) and PEGylated, lectin-grafted liposomes with model membranes were investigated under real-time flow conditions using a quartz crystal microbalance. In addition, the morphology of the liposomal systems was assessed by atomic force microscopy. Plain liposomes exhibited only unspecific adhesion to glycolipid membranes and had a tendency to coalesce. The degree of membrane interaction was significantly increased when plain liposomes were modified with the lectin, Concanavalin A. However, vesicle fusion also markedly increased as a result of lectin modification. Additional PEGylation of liposomes reduced unspecific adhesion phenomena, as well as coalescence. Moreover, our studies enabled us to establish quartz crystal microbalance and atomic force microscopy as powerful and complementary methods to characterize adhesion properties of targeted drug delivery systems.     

Interaction of liposomes bearing a lipophilic doxorubicin prodrug with tumor cells

When used as nanosized carriers, liposomes enable targeted delivery and decrease systemic toxicity of antitumor agents significantly. However, slow unloading of liposomes inside cells diminishes the treatment efficiency. The problem could be overcome by the adoption of lipophilic prodrugs tailored for incorporation into lipid bilayer of liposomes. We prepared liposomes of egg yolk phosphatidylcholine and yeast phosphatidylinositol bearing a diglyceride conjugate of an antitumor antibiotic doxorubicin (a lipophilic prodrug, DOX-DG) in the membrane to study how these formulations interact with tumor cells. We also prepared liposomes of rigid bilayer-forming lipids, such as a mixture of dipalmitoylphosphatidylcholine and cholesterol, bearing DOX in the inner water volume, both pegylated (with polyethylene glycol (PEG) chains exposed to water phase) and non-pegylated. Efficiency of binding of free and liposomal doxorubicin with tumor cells was evaluated in vitro using spectrofluorimetry of cell extracts and flow cytometry. Intracellular traffic of the formulations was investigated by confocal microscopy; co-localization of DOX fluorescence with organelle trackers was estimated. All liposomal formulations of DOX were shown to distribute to organelles retarding its transport to nucleus. Intracellular distribution of liposomal DOX depended on liposome structure and pegylation. We conclude that the most probable mechanism of the lipophilic prodrug penetration into a cell is liposome-mediated endosomal pathway.     

Effect of Fluidizing Agents on Paclitaxel Penetration in Cervical Cancerous Monolayer Membranes

The aim of this study was to compare modulation of paclitaxel penetration in cancerous and normal cervical monolayers by four fluidizing agents: PCPG (9:1 DPPC:PG), PCPE (9:1 DPPC:DOPE), ALEC (7:3 DPPC:PG) and Exosurf (13.5:1.5:1.0 DPPC:hexadecanol:tyloxapol). Presence of the fluidizing agents improved drug penetration significantly. PCPG and PCPE were promising penetration enhancers. PCPG 0.1% caused 3.8– and 1.7-fold higher maximum increments in surface pressure due to drug penetration, (Δπ)_max, than the control in cancerous and normal monolayers, respectively, at 20 mN/m. In cancerous monolayer at 20 mN/m, presence of 0.1%, 0.5%, 1%, 5% and 10% PCPE produced 3.4-, 5.7-, 7.4-, 9.6- and 9.8-fold higher drug penetration compared to the control monolayer without PCPE, respectively. In cancerous monolayer at 20 mN/m, PCPG and PCPE liposomes having 1 mg lipid gave 2.1 and 3.6 times higher (Δπ)_max compared to the control, respectively. Further, the liposomal drug penetration was found to be directly proportional to the liposomal lipid content. The effect of the fluidizing agents was confirmed by increased calcein release from model cervical cancer liposomes. These results may have implications in using the above biocompatible lipids and surfactants as penetration enhancers along with anticancer drugs or as carriers for liposomal formulations of anticancer drugs for improved membrane penetration.