Integrin alphaVbeta3 targeted gene delivery using RGD peptidomimetic conjugates with copolymers of PEGylated poly(ethylene imine).

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.

Synthesis and characterization of chemically condensed oligoethylenimine containing beta-aminopropionamide linkages for siRNA delivery.

Polyethylenimines (PEI) are often inefficient in gene knockdown experiments with small interfering RNA (siRNA), presumably due to the strong complexing properties. A more efficient and potentially degradable oligoethylenimine-based carrier was synthesized by the condensation of 800 molecular weight PEI oligomers with hexanedioldiacrylate. Reaction conditions were chosen such that Michael reaction occurs followed by complete N-acylation of all residual ester bonds resulting in beta-aminopropionamide linkage sites and an average molecular weight of 30,000. Based on NMR analysis, these conditions produced 38% tertiary amides and 62% secondary amides, with about 2% residual carboxylate, presumably from hydrolysis. The ionizable equivalent weight of the carrier increased to 51, compared to a value of 43 for standard PEI. Sensible in vitro knockdown of the luciferase gene in stably transfected HUH7 cells, up to 80% in comparison to non-specific siRNA, demonstrated its suitability for siRNA delivery.

The application of ink-jet technology for the coating and loading of drug-eluting stents.

The combination of drugs with devices, where locally delivered drugs elute from the device, has demonstrated distinct advantages over therapies involving systemic or local drugs and devices administered separately. Drug-eluting stents are most notable. Ink jet technology offers unique advantages for the coating of very small medical devices with drugs and drug-coating combinations, especially in cases where the active pharmaceutical agent is very expensive to produce and wastage is to be minimized. For medical devices such as drug-containing stents, the advantages of ink-jet technology result from the controllable and reproducible nature of the droplets in the jet stream and the ability to direct the stream to exact locations on the device surfaces. Programmed target deliveries of 100 microg drug, a typical dose for a small stent, into cuvettes gave a standard deviation (SD) of dose of 0.6 microg. Jetting on coated, uncut stent tubes exhibited 100% capture efficiency with a 1.8 microg SD for a 137 microg dose. In preliminary studies, continuous jetting on stents can yield efficiencies up to 91% and coefficients of variation as low as 2%. These results indicate that ink-jet technology may provide significant improvement in drug loading efficiency over conventional coating methods.

Combined tumor therapy by using radiofrequency ablation and 5-FU-laden polymer implants: evaluation in rats and rabbits.

PURPOSE: To evaluate the use of 5-fluorouracil (5-FU)-laden polymer implants as an adjunct to radiofrequency (RF) ablation for tumor treatment. MATERIALS AND METHODS: All animal studies were performed in compliance with the Case Western Reserve University Institutional Animal Care and Use Committee guidelines. Three studies were performed to investigate (a) in vitro dissolution of 5-FU-laden polymer implants in saline and bovine serum, (b) tissue distribution of 5-FU and its metabolite, 5-fluorouridine (5-FUrd), in the ablated liver tissue of rats (n = 4), and (c) efficacy of combined approach (n = 4) compared with that of ablation alone (n = 6) for VX2 liver tumor model in rabbits. Characterization of 5-FU release in vitro and distribution of 5-FU in rat liver tissue were analyzed by using high performance liquid chromatography; in vivo efficacy was assessed by using computed tomography and pathologic examination. RESULTS: Results of the in vitro dissolution study showed that a 75% release of 5-FU occurred in 2 days when exposed to bovine serum and in 9 days when exposed to phosphate-buffered saline. In the ablated rat liver, the 5-FU level was higher at the center and lower at the periphery of the tissue both at 24 hours (41.0 mg per kilogram tissue vs 15.0 mg per kilogram tissue, respectively) and at 48 hours (8.0 mg per kilogram tissue vs 2.0 mg per kilogram tissue, respectively). The 5-FUrd concentration was twofold higher peripherally than centrally and was higher at 48 hours than at 24 hours. In rabbits, local delivery of 5-FU immediately after RF ablation provided a significant (P < .05) reduction in tumor size compared with ablation alone (1.80 cm3 +/- 0.28 [standard error] vs 3.53 cm3 +/- 0.52, respectively; P = .034) and a more than 20-fold reduction in tumor size compared with the control (1.80 cm3 +/- 0.28 vs 41.95 cm3 +/- 11.58, respectively; P = .018). CONCLUSION: Combined treatment by using 5-FU polymer implants and RF ablation shows uniform sustained release of 5-FU for 48 hours at least 8 mm from the edge of the ablation zone and appears to be successful at controlling the growth of an experimental tumor in rabbits appreciably better than does ablation alone.