PEGylated and Functionalized Aliphatic Polycarbonate Polyplex Nanoparticles for Intravenous Administration of HDAC5 siRNA in Cancer Therapy.

Guanidine and morpholine functionalized aliphatic polycarbonate polymers are able to deliver efficiently histone deacetylase 5 (HDAC5) siRNA into the cytoplasm of cancer cells in vitro leading to a decrease of cell proliferation were previously developed. To allow these biodegradable and biocompatible polyplex nanoparticles to overcome the extracellular barriers and be effective in vivo after an intravenous injection, polyethylene glycol chains (PEG750 or PEG2000) were grafted on the polymer structure. These nanoparticles showed an average size of about 150 nm and a slightly positive ζ-potential with complete siRNA complexation. Behavior of PEGylated and non-PEGylated polyplexes were investigated in the presence of serum, in terms of siRNA complexation (fluorescence correlation spectroscopy), size (dynamic light scattering and single-particle tracking), interaction with proteins (isothermal titration calorimetry) and cellular uptake. Surprisingly, both PEGylated and non-PEGylated formulations presented relatively good behavior in the presence of fetal bovine serum (FBS). Hemocompatibility tests showed no effect of these polyplexes on hemolysis and coagulation. In vivo biodistribution in mice was performed and showed a better siRNA accumulation at the tumor site for PEGylated polyplexes. However, cellular uptake in protein-rich conditions showed that PEGylated polyplex lost their ability to interact with biological membranes and enter into cells, showing the importance to perform in vitro investigations in physiological conditions closed to in vivo situation. In vitro, the efficiency of PEGylated nanoparticles decreases compared to non-PEGylated particles, leading to the loss of the antiproliferative effect on cancer cells.

Bioavailability enhancement of itraconazole-based solid dispersions produced by hot melt extrusion in the framework of the Three Rs rule.

Solid dispersion formulations made of itraconazole (ITZ) and Soluplus® (polyethylene glycol, polyvinyl acetate and polyvinylcaprolactame-based graft copolymer abbreviated SOL) were produced using hot melt extrusion. Since ITZ possesses a water solubility of less than 1ng/mL, the aim of this work was to enhance the aqueous solubility of ITZ, and thereby improve its bioavailability. The three formulations consisted of a simple SOL/ITZ amorphous solid dispersion (ASD), an optimized SOL/ITZ/AcDiSol® (super-disintegrant) ASD and an equimolar inclusion complex of ITZ in hydroxypropyl-ß-cyclodextrin (substitution degree=0.63, CD) with SOL. The three formulations were compared in vitro and in vivo to the marketed product Sporanox®. The in vitro enhancement of dissolution rate was evaluated using a biphasic dissolution test. In vitro dissolution results showed that all three formulations had a higher percentage of ITZ released than Sporanox® with the following ranking: SOL/ITZ/CD>SOL/ITZ/AcDiSol®>SOL/ITZ>Sporanox®. The bioavailability of these four formulations was evaluated in rats. The bioanalytical method was optimized so that only 10µL of blood was withdrawn from the rats using specific volumetric absorptive microsampling devices. This enabled to keep the same rats during the whole study, which was in accordance with the Three Rs rules (reduction, refinement and replacement). Furthermore, this technique allowed the suppression of inter-individual variability. Higher Cmax and AUC were obtained after the administration of all three formulations compared to the levels after the use of Sporanox® as follows: SOL/ITZ/AcDiSol®>SOL/ITZ/CD>SOL/ITZ>Sporanox®. The inversion in the ranking between SOL/ITZ/CD and SOL/ITZ/AcDiSol® made impossible the establishment of an in vitro-vivo correlation. Indeed, very different release rates were obtained in vitro and in vivo for the two optimized formulations. These results suggest that ITZ would be protected inside the core of the SOL micelles even during the absorption step at the intestine, while some agents present in the intestinal fluids could displace ITZ from the hydrophobic cavity of CD by competition.

Capillary electrophoresis method to determine siRNA complexation with cationic liposomes.

Small interfering RNA (siRNA) inducing gene silencing has great potential to treat many human diseases. To ensure effective siRNA delivery, it must be complexed with an appropriate vector, generally nanoparticles. The nanoparticulate complex requires an optimal physiochemical characterization and the complexation efficiency has to be precisely determined. The methods usually used to measure complexation in gel electrophoresis and RiboGreen® fluorescence-based assay. However, those approaches are not automated and present some drawbacks such as the low throughput and the use of carcinogenic reagents. The aim of this study is to develop a new simple and fast method to accurately quantify the complexation efficiency. In this study, capillary electrophoresis (CE) was used to determine the siRNA complexation with cationic liposomes. The short-end injection mode applied enabled siRNA detection in less than 5 min. Moreover, the CE technique offers many advantages compared with the other classical methods. It is automated, does not require sample preparation and expensive reagents. Moreover, no mutagenic risk is associated with the CE approach since no carcinogenic product is used. Finally, this methodology can also be extended for the characterization of other types of nanoparticles encapsulating siRNA, such as cationic polymeric nanoparticles.

Impact of the structure of biocompatible aliphatic polycarbonates on siRNA transfection ability.

RNAi therapeutics are promising therapeutic tools that have sparked the interest of many researchers. In an effort to provide a safe alternative to PEI, we have designed a series of new guanidinium- and morpholino-functionalized biocompatible and biodegradable polycarbonate vectors. The impact of different functions (morpholino-, guanidinium-, hydrophobic groups) of the architecture (linear homopolymer to dumbbell-shape) and of the molecular weight of these copolymers on their capacity to form polyplexes and to decrease the expression of two epigenetic regulators of gene expression, HDAC7 and HDAC5, was evaluated. The use of one of these polymers combining morpholine and guanidine functions at the ratio >1 and hydrophobic trimethylene carbonate groups showed a significant decrease of mRNA and protein level in HeLa cells, similar to PEI. These results highlight the potential of polycarbonate vectors for future in vivo application as an anticancer therapy.