Control of aggregation temperatures in mixed and blended cytocompatible thermoresponsive block co-polymer nanoparticles.

A small library of thermoresponsive amphiphilic copolymers based on polylactide-block-poly((2-(2-methoxyethoxy)ethyl methacrylate)-co-(oligoethylene glycol methacrylate)) (PLA-b-P(DEGMA)-co-(OEGMA)), was synthesised by copper-mediated controlled radical polymerisation (CRP) with increasing ratios of OEGMA : DEGMA. These polymers were combined in two ways to form nanoparticles with controllable thermal transition temperatures as measured by particle aggregation. The first technique involved the blending of two (PLA-b-P(DEGMA)-co-(OEGMA)) polymers together prior to assembling nanoparticles (NPs). The second method involved mixing pre-formed nanoparticles of single (PLA-b-P(DEGMA)-co-(OEGMA)) polymers. The observed critical aggregation temperature Tt did not change in a linear relationship with the ratios of each copolymer either in the nanoparticles blended from different copolymers or in the mixtures of pre-formed nanoparticles. However, where co-polymer mixtures were based on (OEG)9MA ratios within 5-10 mole%, a linear relationship between (OEG)9MA composition in the blends and Tt was obtained. The data suggest that OEGMA-based copolymers are tunable over a wide temperature range given suitable co-monomer content in the linear polymers or nanoparticles. Moreover, the thermal transitions of the nanoparticles were reversible and repeatable, with the cloud point curves being essentially invariant across at least three heating and cooling cycles, and a selected nanoparticle formulation was found to be readily endocytosed in representative cancer cells and fibroblasts.

Extracellular vesicles at the cross-line between basic science and clinical needs.

MiRNAs are small noncoding RNAs vital for protein regulation and gene expression. Since their discovery in the early nineties, many of their intracellular roles have been characterized. However, it is only recently that EVs loaded with miRNAs and other molecular types have started to be appreciated for their substantial involvement in cell-to-cell communication and signaling in physiological and pathological processes. EVs cell-to-cell signaling functions are complex and largely unknown, which still hampers the direct use of endogenous engineered EVs as therapeutics. However, ad hoc engineered synthetic EVs could represent new therapeutics. The potential of EV-inspired delivery carriers has now attracted the interest of the pharmaceutical industry and has challenged drug delivery researchers with new questions. This review will focus on EVs and EV-inspired drug delivery carriers, on their potential and on the challenges involved in the use of EV-inspired drug delivery systems.

Systemic in vivo delivery of siRNA to tumours using combination of polyethyleneimine and transferrin-polyethyleneimine conjugates.

Materials for delivery of oligonucleotides need to be simple to produce yet effective in vivo to be considered for clinical applications. Formulations of biomaterials based on combinations of existing demonstrated polymeric gene carriers with targeted derivatives are potential candidates for rapid translation but have not been fully explored for siRNA applications. Here we investigated formulations based on derivatised PEI for delivery of siRNA to gastrointestinal cancer cells. siRNA was complexed with linear PEI alone or with a mixture of linear PEI and transferrin-conjugated branched PEI (TfPEI), and knockdown of reporter genes was investigated. Overall, the in vitro use of complexes containing TfPEI resulted in up to 93% knockdown at 72 h post-transfection. Sustained knockdown was also achieved in a bioluminescent xenograft model. When complexes were delivered intratumorally, a 43% reduction in luminescence was achieved in the treated group compared with the control group 48 h after treatment. For systemic administration, only the intraperitoneal route, and not the intravenous route was effective, with 49% knockdown achieved at 72 h and sustained up to 144 h (44%) after a single administration of TfPEI-complexed siRNA. No toxicity or induction of the interferon response was observed. These findings demonstrate that simple formulations of transferrin-conjugated PEI with a 'parent' polymer such as linear PEI have potential as a method for therapeutic delivery of siRNA when administered either intratumorally or systemically.

Triblock Copolymer Nanovesicles for pH-Responsive Targeted Delivery and Controlled Release of siRNA to Cancer Cells.

New pH-responsive polymersomes for active anticancer oligonucleotide delivery were prepared from triblock copolymers. The delivery systems were formed by two terminal hydrophilic blocks, PEG and polyglycerolmethacrylate (poly-GMA), and a central weakly basic block, polyimidazole-hexyl methacrylate (poly-ImHeMA), which can complex with oligonucleotides and control vesicle formation/disassembly via pH variations. Targeted polymersomes were prepared by mixing folate-derivatized and underivatized copolymers. At pH 5, ds-DNA was found to complex with the pH-responsive copolymers at a N/P molar ratio above ∼2:1, which assisted the encapsulation of ds-DNA in the polymersomes, while low association was observed at pH 7.4. Cytotoxicity studies performed on folate receptor overexpressing KB and B16-F10 cells and low folate receptor expressing MCF-7 cells showed high tolerance of the polymersomes at up to 3 mg/mL concentration. Studies performed with red blood cells showed that at pH 5.0 the polymersomes have endosomolytic properties. Cytofluorimetric studies showed a 5.5-fold higher uptake of ds-DNA loaded folate-functional polymersomes in KB cells compared to nontargeted polymersomes. In addition, ds-DNA was found to be localized both in the nucleus and in the cytosol. The incubation of luciferase transfected B16-F10 cells with targeted polymersomes loaded with luciferase and Hsp90 expression silencing siRNAs yielded 31 and 23% knockdown in target protein expression, respectively.