Liposomes containing alkylated methotrexate analogues for phospholipase A(2) mediated tumor targeted drug delivery.

Two lipophilic methotrexate analogues have been synthesized and evaluated for cytotoxicity against KATO III and HT-29 human colon cancer cells. Both analogues contained a C16-alkyl chain attached to the gamma-carboxylic acid and one of the analogues had an additional benzyl group attached to the alpha-carboxylic acid. The cytotoxicity of the gamma-alkylated compound towards KATO III (IC(50) = 55 nM) and HT-29 (IC(50) = 400 nM) cell lines, was unaffected by the alkylation, whereas the additional benzyl group on the alpha-carboxyl group made the compound nontoxic. The gamma-derivative with promising cytotoxicity was incorporated into liposomes that were designed to be particularly susceptible to a liposome degrading enzyme, secretory phospholipase A(2) (sPLA(2)), which is found in high concentrations in tumors of several different cancer types. Liposome incorporation was investigated by differential scanning calorimetry (DSC), and sPLA(2) hydrolysis was examined by fluorescence spectroscopy and high performance liquid chromatography (HPLC). The results showed that the methotrexate (MTX)-analogue could be incorporated into liposomes that were degradable by sPLA(2). However, the in vitro cytotoxicity of the MTX-liposomes against KATO III and HT-29 cancer cells was found to be independent of sPLA(2) hydrolysis, indicating that the alkylated MTX-analogue was available for cancer cell uptake even in the absence of liposome hydrolysis. Using a DSC based method for assessing the anchoring stability of alkylated compounds in liposomes, it was demonstrated that the MTX-analogue partitioned into the water phase and thereby became available for cell uptake. It was concluded that liposomes containing alkylated MTX-analogues show promise as a drug delivery system, although the MTX-analogue needs to be more tightly anchored to the liposomal carrier. Also, the developed DSC-assay for studying the anchoring stability of alkylated drugs will be a useful tool in the development of liposomal drug delivery systems.

Increased expression and activity of group IIA and X secretory phospholipase A2 in peritumoral versus central colon carcinoma tissue.

Secretory phospholipase A2 (sPLA2) type IIA and X was analyzed in tumors from 22 patients with colon adenocarcinomas in order to determine the involvement and activity of sPLA2 in colon cancer. Evaluation of immunoreactive sPLA2 IIA by Western blotting showed a significantly higher level in the periphery of the tumors, compared to central tumor regions. Increased levels of sPLA2 IIA protein correlated with a two-fold increase in sPLA2 enzymatic activity in the peripheral regions compared to central regions. Nineteen out of 22 tumors showed high levels of sPLA2 IIA, whereas 7 out of the 22 tumors showed sPLA2 type X. These data demonstrate that both sPLA2 type IIA and X are present in human colon cancer and suggest a role for sPLA2 in colon cancer tumor immunology and tumorigenesis.

Synthesis and biological activity of anticancer ether lipids that are specifically released by phospholipase A2 in tumor tissue.

The clinical use of anticancer lipids is severely limited by their ability to cause lysis of red blood cells prohibiting intravenous injection. Novel delivery systems are therefore required in order to develop anticancer ether lipids (AELs) into clinically useful anticancer drugs. In a recent article (J. Med. Chem. 2004, 47, 1694) we showed that it is possible to construct liposome systems composed of masked AELs that are activated by secretory phospholipase A2 in cancerous tissue. We present here the synthesis of six AELs and evaluate the biological activity of these bioactive lipids. The synthesized AEL 1-6 were tested against three different cancer cell lines. It was found that the stereochemistry of the glycerol headgroup in AEL-2 and 3 has a dramatic effect on the cytotoxicity of the lipids. AEL 1-4 were furthermore evaluated for their ability to prevent phosphorylation of the apoptosis regulating kinase Akt, and a correlation was found between their cytotoxic activity and their ability to inhibit Akt phosphorylation.

Triggered activation and release of liposomal prodrugs and drugs in cancer tissue by secretory phospholipase A2.

The selectivity of anticancer drugs in targeting the tumour tissue presents a major problem in cancer treatment. In this article we review a new generation of smart liposomal nanocarriers that can be used for enhanced anticancer drug and prodrug delivery to tumours. The liposomes are engineered to be particularly degradable to secretory phospholipase A2 (sPLA2), which is a lipid hydrolyzing enzyme that is significantly upregulated in the extracellular microenvironment of cancer tumours. Thus, when the long circulatory liposomal nanocarriers extravasate and accumulate in the interstitial tumour space, sPLA2 will act as an active trigger resulting in the release of cytotoxic drugs in close vicinity of the target cancer cells. The sPLA2 generated lysolipid and fatty acid hydrolysis products will furthermore be locally released and function as membrane permeability promoters facilitating the intracellular drug uptake. In addition, the liposomal membrane can be composed of a novel class of prodrug lipids that can be converted selectively to active anticancer agents by sPLA2 in the tumour. The integrated drug discovery and delivery technology offers a promising way to rationally design novel tumour activated liposomal nanocarriers for better cancer treatment.

Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C14-peptides.

The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated peptide, which is a synthetic decapeptide N-terminally linked to a C14 acyl chain (C14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0-20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C14-peptide on the lipid bilayer thermodynamics. This is manifested as a concentration-dependent downshift of both the main phase transition and the pretransition. In addition, the main phase transition peak is significantly broadened, indicating phase coexistence. In the AFM imaging scans we found that the C14-peptide, when added to supported gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10 A height difference. The AFM images also show that the appearance of the ripple phase of the DPPC lipid bilayers is unaffected by the C14-peptide. The experimental results are supported by molecular dynamics simulations, which show that the C14-peptide has a disordering effect on the lipid acyl chains and causes a lateral expansion of the lipid bilayer. These effects are most pronounced for gel-like bilayer structures and support the observed downshift in the phase-transition temperature. Moreover, the molecular dynamics data indicate a tendency of a tryptophan residue in the peptide sequence to position itself in the bilayer headgroup region.

Advanced strategies in liposomal cancer therapy: problems and prospects of active and tumor specific drug release.

Tumor specific drug delivery has become increasingly interesting in cancer therapy, as the use of chemotherapeutics is often limited due to severe side effects. Conventional drug delivery systems have shown low efficiency and a continuous search for more advanced drug delivery principles is therefore of great importance. In the first part of this review, we present current strategies in the drug delivery field, focusing on site-specific triggered drug release from liposomes in cancerous tissue. Currently marketed drug delivery systems lack the ability to actively release the carried drug and rely on passive diffusion or slow non-specific degradation of the liposomal carrier. To obtain elevated tumor-to-normal tissue drug ratios, it is important to develop drug delivery strategies where the liposomal carriers are actively degraded specifically in the tumor tissue. Many promising strategies have emerged ranging from externally triggered light- and thermosensitive liposomes to receptor targeted, pH- and enzymatically triggered liposomes relying on an endogenous trigger mechanism in the cancerous tissue. However, even though several of these strategies were introduced three decades ago, none of them have yet led to marketed drugs and are still far from achieving this goal. The most advanced and prospective technologies are probably the prodrug strategies where non-toxic drugs are carried and activated specifically in the malignant tissue by overexpressed enzymes. In the second part of this paper, we review our own work, exploiting secretory phospholipase A2 as a site-specific trigger and prodrug activator in cancer therapy. We present novel prodrug lipids together with biophysical investigations of liposome systems, constituted by these new lipids and demonstrate their degradability by secretory phospholipase A2. We furthermore give examples of the biological performance of the enzymatically degradable liposomes as advanced drug delivery systems.

Secretory phospholipase A2 as a tumor-specific trigger for targeted delivery of a novel class of liposomal prodrug anticancer etherlipids.

The use of many common clinically relevant chemotherapeutics is often limited due to insufficient delivery to the tumor and dose-limiting systemic toxicities. Therefore, therapeutics that specifically target tumor cells and are nontoxic to normal cells are required. Here, we report the development of a novel class of liposomes composed of lipid prodrugs, which use the increased secretory phospholipase A2 type IIA (sPLA2) activity of the tumor microenvironment as a trigger for the release of anticancer etherlipids (AEL). Treatment of sPLA2-secreting tumor cells in vitro with liposomes consisting of proAELs resulted in growth inhibition comparable with addition of the AELs alone. Using a specific sPLA2 inhibitor, we showed the low cytotoxicity of the nonhydrolyzed proAEL liposomes and have proven the sPLA2 dependency of the activation of proAELs to cytotoxic AELs. In addition, we showed that our proAEL liposomes circumvent the inherent hemolytic toxicities associated with the use of etherlipids, thereby allowing i.v. administration of such therapeutics as nontoxic prodrug liposomes. Furthermore, using a sPLA2-secreting human colon cancer xenograft model, we showed that the proAEL liposomes are capable of inducing a tumor growth delay in vivo. Taken together, these data support the validity of this novel tumor-selective liposomal prodrug delivery strategy. This new approach also provides a promising system for tumor-selective delivery and release of conventional chemotherapeutics encapsulated in the sPLA2-degradable prodrug liposomes.

Enzymatic release of antitumor ether lipids by specific phospholipase A2 activation of liposome-forming prodrugs.

An enzymatically activated liposome-based drug-delivery concept involving masked antitumor ether lipids (AELs) has been investigated. This concept takes advantage of the cytotoxic properties of AEL drugs as well as the membrane permeability enhancing properties of these molecules, which can lead to enhanced drug diffusion into cells. Three prodrugs of AELs (proAELs) have been synthesized and four liposome systems, consisting of these proAELs, were investigated for enzymatic degradation by secretory phospholipase A(2) (sPLA(2)), resulting in the release of AELs. The three synthesized proAELs were (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphocholine (1-O-DPPC), (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol)(350) (1-O-DPPE-PEG(350)), and 1-O-DPPE-PEG(2000) of which 1-O-DPPC was the main liposome component. All three phospholipids were synthesized from the versatile starting material (R)-O-benzyl glycidol. A phosphorylation method, employing methyl dichlorophosphate, was developed and applied in the synthesis of two analogues of (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol). Differential scanning calorimetry has been used to investigate the phase behavior of the lipid bilayers. A release study, employing calcein encapsulated in non-hydrolyzable 1,2-bis-O-octadecyl-sn-glycero-3-phosphocholine (D-O-SPC) liposomes, showed that proAELs, activated by sPLA(2), perturb membranes because of the detergent-like properties of the released hydrolysis products. A hemolysis investigation was conducted on human red blood cells, and the results demonstrate that proAEL liposomes display a very low hemotoxicity, which has been a major obstacle for using AELs in cancer therapy. The results suggest a possible way of combining a drug-delivery and prodrug concept in a single liposome system. Our investigation of the permeability-enhancing properties of the AEL molecules imply that by encapsulating conventional chemotherapeutic drugs, such as doxorubicin, in liposomes consisting of proAELs, an increased effect of the encapsulated drug might be achievable due to an enhanced transmembrane drug diffusion.

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.

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.