(49e) Membrane-Active PSMA-Targeting Liposomes for Antivascular Chemotherapy

Locke, T., Rutgers University
Sofou, S., Rutgers University
Introduction: Cases of advanced solid cancer as of yet have no cure [1]. A challenge for the medical research community is the development of diagnostic and treatment protocols that significantly extend life expectancy and improve quality of life of these cancer patients. Based on the difficulties encountered in treatment, future effective interventions will likely require a combination of therapies to provide effective tumor control, of which antivascular therapy may be a key element. Antivascular therapy aims to kill tumor cells by cutting off blood flow, depriving tumors of nutrients they need to survive [2].

The objective of this study is to design a new class of liposomes that can target tumor vasculature via Prostate-Specific Membrane Antigen (PSMA) and rapidly and extensively release therapeutic agents intracellularly by using the pH-sensitive peptide GALA conjugated to lipid moieties to destabilize the integrity of intracellular endosomes and achieve better tumor killing outcomes.

GALA is a 30 amino acid peptide that forms an alpha helix at acidic pH [3]. Once formed, this helix can interact with and penetrate lipid bilayers (via pore formation) making it a candidate for inclusion in systems that desire endosomal escape [4, 5]. This study explores the mechanism of GALA anchored to gel-phase lipid vesicles in both clustered (pH and non-pH responsive) and uniform presentations with the goal of optimizing GALA presentation for improving therapeutic efficacy of tumor vasculature targeted liposome compositions.

PSMA is a cell surface glycoprotein receptor present on tumor vascular endothelium of many types of cancer [6, 7]. Although this target is also expressed in the healthy tissues of the prostate, gastrointestinal tract, kidneys, and brain [8-10], normal tissue vasculature is consistently PSMA-negative [11]. Therefore, tumor vasculature is directly and easily accessible to intravenously injected PSMA targeted treatments, whereas healthy vasculature and PSMA-expressing tissues are not expected to exhibit significant localization.

Methods: Liposomes were prepared by hydrating a thin lipid film with the desired aqueous phase buffer, incubating at 55° Celsius for 2 hours, and extruding the resultant suspension through 100 nm -pore polycarbonate membranes to generate unilamellar vesicles. GALA was synthesized by solid phase peptide synthesis (Anaspec) and was conjugated to a DPPE succinyl lipid using click chemistry by reacting the succinyl carbonyl group to the N-terminus amine of the peptide. Calcein at 75 mM was used as a tracking fluorophore for GALA-induced release. Leakage of calcein (EX/EM 495 nm/515 nm) from an endosome analogue EggPC membrane incubated with GALA-bearing liposomes resulting in self-quenching fluorescence release was monitored over time over the pH range from 7.4 to 4.0 (Qt). pH was adjusted with HCl. The two liposome compositions were incubated at a 1:1 mole ratio (equivalent to 1:100 GALA:lipid mole ratio). Triton-X 100 (5% w/v) was then added to induce the complete release of contents (Qmax). The ratio of these two measurements was used to determine the normalized change in self-quenching efficiency of calcein (Qt-1)/(Qmax-1) which is related to the extent of leakage. Calcein containing EggPC liposomes were also exposed to each pH without GALA present as a control.

For targeting experiments, the in vitro tumor analogue used consisted of HUVEC induced to express PSMA, as previously described [12]. PSMA induction was achieved by suspending HUVEC in tumor cell conditioned media (CCM; serum-free RPMI1640 incubated with MDA-MB-231 breast cancer cells for 24 hours) and seeding on MatrigelTM (Corning). Liposomes were prepared as described above. Liposomes were incubated with cells on ice for 6 hours at a 1:50 liposome:PSMA-receptor ratio. A peptidomimetic urea-based ligand was used for targeting [13].

Results: Gel-phase, GALA-bearing liposomes induced release of encapsulated contents from endosome analogue vesicles in a pH-dependent manner with clustered GALA inducing faster release at endosomal pH than uniform GALA. The apparent activity of liposome-grafted GALA is affected by the presentation of GALA-grafted peptides (clustered vs. uniformly distributed over the liposome surface).

PSMA-targeted liposomes exhibit increased specific binding to PSMA-positive HUVEC cells. Results showing the effect of targeted, GALA-bearing liposomes on PSMA-positive HUVEC cells under flow will be presented.

Conclusions: A clustered presentation of GALA liposomes has the potential to enhance the delivery of chemotherapeutics directly to the cytosol. It is possible that clustering of GALA allows for each peptide monomer to more readily interact with other peptide units, enhancing induced release in the range of endosomal pH. This is likely the primary variable determining the apparent reactivity of GALA as the comparison between the apparent reactivity of clustered GALA on the surface of non-charged liposomes (where clustering is pH-independent) and the apparent reactivity of uniformly distributed GALA-bearing liposomes showed that the absence of surface electrostatic effects alone does not account for differences in GALA reactivity.

PSMA can be specifically targeted by liposomes using a urea-based ligand as a targeting strategy.


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