Introduction and Objective: A novel strategy for selective delivery to malignant cells is to encapsulate the antitumor drug in nanoparticles (NPs) that are grafted with ligands with very high affinity for tumor cells. Biodegradable NPs <100 nm in size and surface-modified with hydrophilic polymers can evade the mononuclear phagocytes system (MPS), overcome resistance at the tumor level (EPR effect), and can be conjugated with ligands that bind with high specificity/affinity to the tumor cell surface to trigger the receptor-mediated endocytosis to localize the NPs (hence the antitumor drug) in the cell cytoplasm. Furthermore, it is well established that ligand-conjugated NPs display a stronger multivalent interaction and a much higher apparent affinity to cell surface receptors than the free ligand. Our laboratory has developed novel biodegradable poly(lactide-co-glycolide fumarate) (PLGF) and poly(lactide-co-ethylene oxide fumarate) (PLEOF) macromers that self-assemble into nanoparticles in the aqueous medium. After self-assembly, cell-responsive ligands can be conjugated to these NPs for targeted delivery of antitumor drugs. Cyclic RGD-based biomimetic peptides have high affinity for áíâ3 integrin receptor, which is implicated in tumor-induced angiogenesis and metastasis, and is up-regulated on both cancer cells and tumor-associated blood vessels. The objective of this study was to determine the effect of conjugation of cyclic RGD peptide [c(-RGDfk-)] conjugation on the uptake of PLGF-PLEOF NPs by tumor cells.

Methods: A 10% w/w solution of the PLGF-PLEOF macromers in dimethyl sulfoxide (DMSO) was dialyzed against distilled deionized (DDI) water to form a stable colloidal suspension of NPs. The morphology of the NPs was characterized by TEM and the size distribution of NPs was measured by dynamic light scattering. Degradation was measured as a function of time in vitro in primary culture media (CM) without fetal bovine serum (FBS) at 37°C. Dextran fluorescein isothiocyanate (FITC-dextran) and Paclitaxel were used as surrogate molecules for release studies. The release characteristic of Paclitaxel from the NPs was measured by HPLC. Near-infrared imaging was used for determination of in vivo distribution of NPs in mice with intestinal tumor. The MCF-7 and U87MG cancer cell lines with low and high ávâ3 integrin expression, respectively, were used for determination of in vitro cell binding affinity. The linear D-Phe-Cys-Arg-Gly-Asp peptide was cyclized in the solid-phase on the NovaSyn® TGA resin using Fmoc- chemistry. Cyclic (GRGfC) peptide was incubated for 10 h with NPs in PBS for conjugation.

Results: PLGF-PLEOF and PLAF-PLEOF NPs degraded in 15 and 28 days, respectively, which demonstrated that the release was dominated by hydrolytic degradation and erosion of the matrix. As the concentration of Paclitaxel was increased from 0 to 10, and 40 ìg/ml, cell viability with free Paclitaxel decreased from 100 to 65 and 40%, respectively, while those encapsulated in NPs decreased from 93 to 54 and 28%. Groups with Paclitaxel loaded NPs had higher cytotoxicity compared to Paclitaxel directly added to the media at the same concentration. Infrared image of mice injected with NPs showed significantly higher concentration of NPs in the tumor tissue. When cyclic RGD conjugated NPs were incubated with MCF-7 and U87MG cells, significantly higher uptake of NPs by U87MG cells with high ávâ3 integrin expression was observed.