Efficient and Tumor Targeted siRNA Delivery by Polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol)-folate (PEI-PCL-PEG-Fol).

Efficient delivery of functional nucleic acids into specific cells or tissues is still a challenge for gene therapy and largely depends on targeted delivery strategies. The folate receptor (FR) is known to be overexpressed extracellularly on a variety of human cancers and is therefore an outstanding gate for tumor-targeted Trojan horse-like delivery of therapeutics. In this study, an amphiphilic and biodegradable ternary copolymer conjugated with folate as ligand, polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol)-folate (PEI-PCL-PEG-Fol) was synthesized and evaluated for targeted siRNA delivery via folate-FR recognition. The amphiphilic character of similar polymers was shown previously to support endosomal release of endocytosed nanocarriers and to promote formation of long circulating micelles. The obtained PEI-PCL-PEG-Fol exhibited less cytotoxicity in comparison with the corresponding ternary copolymer without folate (PEI-PCL-PEG) and with unmodified PEI25kDa. Stable micelle-like polyplexes with hydrodynamic diameters about 100 nm were found to have a zeta potential of +8.6 mV, which was lower than that of micelleplexes without folate-conjugation (+13-16 mV). Nonetheless, increased cellular uptake and in vitro gene knockdown of PEI-PCL-PEG-Fol/siRNA micelleplexes were observed in SKOV-3 cells, an FR overexpressing cell line, in comparison with the nonfolate-conjugated ones. Moreover, PEI-PCL-PEG-Fol/siRNA micelleplexes exhibited excellent stability in vivo during the analysis of 120 min and a longer circulation half life than hyPEI25kDa/siRNA polyplexes. Most interestingly, the targeted delivery system yielded 17% deposition of the i.v. injected siRNA per gram in the tumor after 24 h due to the effective folate targeting and the prolonged circulation.

In situ forming nimodipine depot system based on microparticles for the treatment of posthemorrhagic cerebral vasospasm.

The present study was conducted to examine the feasibility of nimodipine-loaded PLGA microparticles suspended in Tisseel fibrin sealant as an in situ forming depot system. This device locally placed can be used for the treatment of vasospasm after a subarachnoid hemorrhage. Microparticles were prepared via spray-drying by using the vibration mesh spray technology of Nano Spray Dryer B-90. Spherically shaped microparticles with different loadings and high encapsulation efficiencies of 93.3-97.8% were obtained. Depending on nimodipine loading (10-40%), the particle diameter ranged from 1.9 ± 1.2 µm to 2.4 ± 1.3 µm. Thermal analyses using DSC revealed that nimodipine is dissolved in the PLGA matrix. Also, fluorescent dye loaded microparticles were encapsulated in Tisseel to examine the homogeneity of particles. 3D-pictures of the in situ forming devices displayed uniform particle homogeneity in the sealant matrix. Drug release was examined by fluorescence spectrophotometry which demonstrated a drug release proportional to the square root of time. A prolonged drug release of 19.5h was demonstrated under in vitro conditions. Overall, the nimodipine in situ forming device could be a promising candidate for the local treatment of vasospasm after a subarachnoid hemorrhage.

Poly(ethylene carbonate) nanoparticles as carrier system for chemotherapy showing prolonged in vivo circulation and anti-tumor efficacy.

The aim of this study is to investigate the feasibility and efficacy of PEC nanoparticles as delivery system for cancer chemotherapy. Assembly of paclitaxel-loaded nanoparticles with high loading efficiency and narrow-size distribution is successful. For non-invasive in vivo tracing, nanoparticle blends of chelator bearing poly(lactide) with PEC and PLGA are successfully prepared. Pharmacokinetic studies in mice reveal a twofold higher circulation time of PEC as compared to PLGA. A tumor model shows an accumulation of PEC NPs in cancerous tissue and a higher anti-tumor efficiency compared to the standard Taxol™, which is reflected in a significantly slower tumor growth compared to the NaCl control group.

Modular synthesis of folate conjugated ternary copolymers: polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol)-folate for targeted gene delivery.

Folate receptor (FR) is overexpressed in a variety of human cancers. Gene delivery vectors conjugated with folate as a ligand could possibly deliver gene materials into target tumor cells via FR-mediated endocytosis. This study addresses novel folate-conjugated ternary copolymers based on polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) (PEI-g-PCL-b-PEG-Fol) as targeted gene delivery system using a modular synthesis approach including "click" conjugation of folate moieties with heterobifunctional PEG-b-PCL at PEG terminus and subsequently the introduction of PEI by a Michael addition between folate-PEG-b-PCL and PEI via active PCL terminus. This well-controlled synthetic procedure avoids tedious separation of byproduct. The structure of PEI-g-PCL-b-PEG-Fol was confirmed by (1)H NMR and UV spectra. DNA condensation of PEI-g-PCL-b-PEG-Fol was tested using a SYBR Gold quenching assay and agarose gel electrophoresis upon heparin competition assay. Although PEI-g-PCL-b-PEG-Fol could condense DNA completely at N/P ratio >2, polyplexes of N/P ratio 10 with sizes of about 120 nm and positive zeta potentials were selected for further biological evaluations due to polyplex stability. An enhancement of cellular uptake of PEI-g-PCL-b-PEG-Fol/pDNA polyplexes was observed in FR overexpressing KB cells in comparison to unmodified PEI-g-PCL-b-PEG, through flow cytometry analysis and confocal laser scanning imaging. Importantly, this enhanced cellular uptake could be inhibited by free folic acid and did not occur in FR-negative A549 cells, demonstrating specific cell uptake by FR-mediated endocytosis. Furthermore, the transfection efficiency of PEI-g-PCL-b-PEG-Fol/pDNA polyplexes was increased approximately 14-fold in comparison to folate-negative polyplexes. Therefore, the PEI-g-PCL-b-PEG-Fol merits further investigation under in vivo conditions for targeting FR overexpressing tumors.

Self-assembled biodegradable amphiphilic PEG-PCL-lPEI triblock copolymers at the borderline between micelles and nanoparticles designed for drug and gene delivery.

Amphiphilic PEG-PCL-PEI triblock copolymers self-assemble into nano-scaled, positively charged, multifunctional carriers, suitable for drug and gene delivery. A set of block copolymers with varying hydrophilic/hydrophobic ratio (systematically altered at the borderline of micelle and particle forming polymers) was synthesized, characterized and assembled into carriers. A detailed structural characterization in the liquid state of these assemblies was carried out: carrier size was determined using dynamic light scattering, cryogenic scanning electron microscopy and atomic force microscopy. Nuclear magnetic resonance analyses elucidated carrier's core-shell structure. ζ-potential and thickness of the hydrophilic outer polymer shell were determined by laser Doppler anemometry. Subsequently the impact of carrier's structure on its features (stability and toxicity) was investigated. Polymers hydrophilic in nature formed small (<40 nm) micelle-like carriers, whilst hydrophobic polymers aggregated to larger particle-like assemblies (>100 nm). Monitoring carrier size as a function of initial polymer concentration clarified different assembly mechanisms. Shell thickness, colloidal stability and toxicity were found to depend on the length of the hydrophilic polymer block. Due to controllable size, charge, stability and toxicity, this class of novel carriers is a promising candidate for prospective co-delivery of drugs and nucleic acids.

Biodegradable poly(ethylene carbonate) nanoparticles as a promising drug delivery system with "stealth" potential.

The goal of this study was to investigate the suitability of poly(ethylene carbonate) (PEC) nanoparticles as a novel drug delivery system, fulfilling the requirements for a long circulation time. Particles were obtained with a narrow size distribution and nearly neutral zeta potential. Adsorption studies with human plasma proteins revealed that PEC nanoparticles bind much less proteins in comparison to polystyrene (PS) nanoparticles. Cell experiments with fluorescently labeled PEC showed no uptake of the nanoparticles by macrophages. These novel PEC nanospheres with their unique surface properties are a promising candidate for long circulating drug delivery systems in vivo.

Nanoparticles for paclitaxel delivery: a comparative study of different types of dendritic polyesters and their degradation behavior.

The aim of this study was to formulate nanoparticles from three different hyperbranched polymers, namely an unmodified dendritic polyester (Boltorn H40™), a lipophilic, fatty acid modified dendritic polymer (Boltorn U3000™) and an amphiphilic dendritic polymer (Boltorn W3000™) for drug delivery of paclitaxel and to investigate their properties. A solvent displacement method allowed preparation of nanoparticles from all three hyperbranched polymers. Nanoparticle sizes ranged from 70 to 170 nm. The lipophilic Boltorn U3000™ formed the biggest nanoparticles and the amphiphilic Boltorn W3000™ formed the smallest ones. Nanoparticles of amphiphilic Boltorn W3000™ displayed only a slightly negative zeta potential, while more negative zeta potentials were measured for nanoparticles based on the other two polymers. Degradation profiles were investigated by short time pH-stat titration. Boltorn H40™ showed a faster degradation rate then the two other fatty acid containing polymers. For Boltorn H40™, degradation rate was also investigated in longer term mass loss studies resulting in 30% degradation during 3 weeks. Cytotoxicity of the nanoparticles was studied by MTT assay displaying low cytotoxicity for all three polymers. All three types of nanoparticles were loaded with paclitaxel and their release profiles were studied. Sizes and zeta potentials remained stable after loading and did not change significantly. These three types of hyperbranched polymers show potential as nanoparticulate delivery systems and should be further studied. Due to their high loading efficiency, Boltorn U3000 and W3000 represent the most interesting candidates.