Integrin α_νβ₃ Targeted Gene Delivery Using RGD Peptidomimetic Conjugates with Copolymers of PEGylated Poly(ethylene imine)

Abstract

This study describes the synthesis and characterization of five conjugates of poly(ethylene glycol) modified polyethylenimine (PEG-PEIs) coupled in two different synthesis routes to a nonpeptidic pentacyclic RDG-mimetic for integrin receptor-targeted gene delivery. Synthesis of this panel of different conjugates allowed for systematic analysis of structure-activity relationships. Conjugates were therefore characterized regarding molecular composition, DNA condensation, size, and zeta potential of self-assembled polyplexes. In vitro characterization included investigation of blood compatibility, binding affinity to receptor-positive and receptor-negative cells measured by flow cytometry, cellular uptake quantified by scintillation counting, and efficiency and specificity of transfection assayed by reporter gene expression. In a first synthetic approach, low molecular weight PEI (LMW-PEI) was PEGylated using a heterobifunctional PEG linker and coupling of the RGD-mimetic was achieved at the distal end of PEG chains. In a second synthesis route, the RGD-mimetic was directly coupled to AB-block-copolymers of PEI (25 kDa) and PEG (30 kDa). Interactions of RGD-PEG-LMW-PEI conjugates with DNA were strongly impaired, whereas PEG-PEI-RGD conjugates were more promising candidates due to their physicochemical properties and higher receptor specificity. The binding, uptake, and transfection efficiency in receptor-positive cells was strongly increased upon conjugation of the RGD-mimetic to AB-block-copolymers of PEG-PEI and depended on the degree of peptide substitution. The conjugates of PEG-PEI AB-block-copolymers with low ligand density of the RGD-mimetic appear to be promising candidates for in vivo cancer gene therapy.

RGD-conjugated albumin nanoparticles as a novel delivery vehicle in pancreatic cancer therapy

“…The avβ3 receptor expression was determined by flow cytometric analysis. 30 Cells were cultured as monolayers in DMEM supplemented with 10% FBS (v/v) and maintained at 37°C in a humidified incubator (95% humidity) containing 5% CO 2 . Cultured cells were harvested and seeded with a concentration of 10 6 cells per well on the 24-well plates.…”

Solid-phase-assisted synthesis of targeting peptide–PEG–oligo(ethane amino)amides for receptor-mediated gene delivery

“…As toxicity is correlated with polymer size, biodegradable PEI analogs that degrade into non-toxic smaller fragments have been developed. [21][22][23] Other strategies focus on targeting moieties and surface shielding of polyplexes using polyethylene glycol (PEG) bound to PEI, 24,25 in some cases by pH-sensitive linkers. 26,27 Like many other conventional polymers, another limitation of PEI is the inherent polydispersity in molecular weight and the heterogeneity of modification sites in chemical conjugates.…”

Poly(Ethylene Glycol)-Based Backbones with High Peptide Loading Capacities

“…21 mg (18% yield) of a thick yellow gel was recovered. (8). Epichlorohydrin (1.24 g, 13.5 mmol) was cooled to 0°C.…”

“…Among them, the conjugation to polymers has resulted in multiple applications, not limited to clinical products [3], combining the advantages of these two classes of macromolecules [4]. Several polymer conjugates of low molecular weight drugs [5], peptides [3][4][5][6][7][8] and other macromolecules [9][10][11] based on water-soluble polymers have been developed.…”