Tumor-specific gene transfer with receptor-mediated nanocomplexes modified by polyethylene glycol shielding and endosomally cleavable lipid and peptide linkers.

Synthetic nanoparticle formulations have the potential for tumor-targeted gene delivery. Receptor-targeted nanocomplex (RTN) formulations comprise mixtures of cationic liposomes and targeting peptides that self-assemble on mixing with nucleic acids. RTN formulations were prepared containing different polyethylene glycol (PEG)ylated lipids with esterase-cleavable linkers (e.g., ME42) to promote intracellular PEG detachment and nanoparticle disassembly. In addition, integrin-targeting peptides (peptide ME27) were tested with endosomal furin- and cathepsin B-cleavable peptide linkers located between the integrin-binding ligand and the K(16) nucleic acid-binding domain to promote intracellular disengagement from the receptor. ME42/ME27 RTNs formed stable particles of <200 nm in isotonic salt buffers, compared with 4-microm particles formed by un-PEGylated RTNs. Transfection efficiency by PEG-modified, cleavable RTNs improved approximately 2-fold in 4 different cell lines, with 80% efficiency in murine neuroblastoma cells. In an in vivo model of neuroblastoma, ME42/ME27 RTNs delivering luciferase genes were tumor specific, with little expression in other organs tested. PEGylation of the RTNs enhanced luciferase transfection 5-fold over non-PEG formulations, whereas the cleavability of the peptide ME27 enhanced transfection 4-fold over that of RTNs with noncleavable peptides. Cleavability of the lipid for in vivo transfections had no effect. PEGylated, cleavable RTN formulations offer prospects for tumor-specific therapeutic gene transfer.

Stabilized integrin-targeting ternary LPD (lipopolyplex) vectors for gene delivery designed to disassemble within the target cell.

Recent research in the field of nonviral gene delivery vectors has focused on preparing nanoparticles that are stabilized by the incorporation of a PEG coating and where one of the vector components is also cleavable. Here,we describe the synthesis, formulation, transfection properties, and biophysical studies of a PEG-stabilized ternary lipopolyplex vector in which, for the first time, both the lipid and peptide components are designed to be cleaved once the vector has been internalized. A series of cationic lipids, bearing short tri- or hexaethylene glycol groups, attached to the headgroup via an ester linkage, has been prepared. Trifunctional peptides have also been prepared, consisting of a Lys(16) sequence at the N-terminus (to bind and condense plasmid DNA); a spacer group (containing a sequence recognized and cleaved by endosomal enzymes) and an optional PEG4 amino acid; and an integrin-targeting cyclic peptide sequence (allowing the resulting nanoparticle to be internalized via receptor-mediated endocytosis). Differing combinations of these lipids and peptides have been formulated with DOPE and with plasmid DNA, and complex stability, transfection, and cleavage studies carried out. It was shown that optimal transfection activities in a range of cell types and complex stabilities were achieved with lipids bearing short cleavable triethylene glycol moieties, whereas the incorporation of PEG4 amino acids into the cleavable peptides had little effect. We have synthesized appropriate fluorescently labeled components and have studied the uptake of the vector, endosomal escape, peptide cleavage, and plasmid transport to the nucleus in breast cancer cells using confocal microscopy. We have also studied the morphology of these compact, stabilized vectors using cryo-EM.

Integrin-targeted nanocomplexes for tumour specific delivery and therapy by systemic administration.

Nanoparticle formulations offer opportunities for tumour delivery of therapeutic reagents. The Receptor-Targeted Nanocomplex (RTN) formulation consists of a PEGylated, endosomally-cleavable lipid and an RGD integrin-targeting, endosomally-cleavable peptide. Nancomplexes self-assemble on mixing with plasmid DNA to produce nanoparticles of about 100 nm. The environmentally-sensitive linkers promote intracellular disassembly and release of the DNA. RTNs carrying luciferase genes were administered intravenously to mice carrying subcutaneous neuroblastoma tumours. Luciferase expression was much higher in tumours than in liver, spleen and lungs while plasmid biodistribution studies supported the expression data. Transfection in tumours was enhanced two-fold by integrin-targeting peptides compared to non-targeted nanocomplexes. RTNs containing the interleukin-2 (IL-2) and IL-12 genes were administered intravenously with seven doses at 48 h intervals and tumour growth monitored. Tumours from treated animals were approximately 75% smaller on day 11 compared with RTNs containing control plasmids with one third of treated mice surviving long-term. Extensive leukocyte infiltration, decreased vascularization and increased necrotic areas were observed in the tumours from IL2/IL12 treated animals. Splenocytes from re-challenged mice displayed enhanced IL-2 production following Neuro-2A co-culture, which, combined with infiltration studies, suggested a cytotoxic T cell-mediated9 tumour-rejection process. The integrin-targeted RTN formulation may have broader applications in the further development of cancer therapeutics.