Osteotropicity of novel bone-targeted HPMA copolymer conjugates has been demonstrated previously with bone histomorphometric analysis. The pharmacokinetics and biodistribution of this delivery system were investigated in the current study with healthy young BALB/c mice. The 125 Ilabeled bone-targeted and control (non-targeted) HPMA copolymers were administered intravenously to mice and their distribution to different organs and tissues were followed using a gamma counter and single photon emission computed tomography (SPECT). Both the invasive and non-invasive data further confirmed that the incorporation of D-aspartic acid octapeptide (D-Asp 8 ) as bone-targeting moiety could favorably deposit the HPMA copolymers to the entire skeleton, especially to the high bone turnover sites. To evaluate the influence of molecular weight, three fractions (M w of 24, 46, and 96 kDa) of HPMA copolymer -D-Asp 8 conjugate were prepared and evaluated. Higher molecular weight of the conjugate enhanced the deposition to bone due to the prolonged half-life in circulation, but it weakened the bone-selectivity. A higher content of bonetargeting moiety (D-Asp 8 ) in the conjugate is desirable to achieve superior hard tissue selectivity. Further validation of the bone-targeting efficacy of the conjugates in animal models of osteoporosis and other skeletal diseases is needed in the future.

The polymer, OEI-HD, based on beta-propionamide-cross-linked oligoethylenimine and its chemical transferrin conjugate were evaluated for siRNA delivery into murine Neuro2A neuroblastoma cells in vitro and in vivo. An 80% silencing of luciferase expression in neuroblastoma cells, stably transfected with a luciferase gene, was obtained using standard OEI-HD polyplexes or transferrin-conjugated shielded OEI-HD polyplexes. The Ras-related nuclear protein Ran was selected as a therapeutically relevant target protein. Systemic delivery of transferrin-conjugated OEI-HD/RAN siRNA formulations (three intravenous applications at 3 days interval) resulted in >80% reduced Ran protein expression, apoptosis, and a reduced tumor growth in Neuro2A tumors of treated mice. The treatment was not associated with signs of acute toxicity or significant changes in weight, hematology parameters, or liver enzymes (AST, ALT, or AP) of mice. All our results demonstrate that OEI-HD/siRNA formulations can knockdown genes in tumor cells in vitro and in vivo in mice in the absence of unspecific toxicity.

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Finally, in vivo application studies confirmed the biocompatibility of PAHy-CPTA copolymers. With LPEI, clear signs of microvesicular fatty liver were observed and with LPEI polyplexes significant weight loss. In strong contrast, PAHy-CPTA did not induce histopathological changes or weight loss.