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Transfection Ability and Intracellular Pathway of LbL Nanostructured siRNA Delivery Systems

Final Report Summary - NANOSIRNA (Transfection Ability and Intracellular Pathway of LbL Nanostructured siRNA Delivery Systems.)



Executive Summary:

The project entitled “Transfection Ability and Intracellular Pathway of LbL Nanostructured siRNA Delivery Systems” (acronym: Nanosirna) was a three years (2011-2013) joint research project supported by the IRSES International Research Staff Exchange Funding Scheme. The partnership was composed by the Fondazione IRCCS Istituto Nazionale dei Tumori of Milan (INT, IT), the University of Bayreuth (UBT, DE), the University of Rome “Tor Vergata” (UTV, IT) and the University of Melbourne (UoM, AU). The Fondazione IRCCS Istituto Nazionale dei Tumori of Milan was responsible for the coordination of the project activities.

The main objective of Nanosirna was the development of a new technology to effectively deliver siRNA to cancer cells by embedding a polyplex into the multilayers and multifunctional nanocapsules. Specifically, we proposed to target pro-survival and anti-apoptotic factors in human cancer cells by using siRNA encapsulated into polyelectrolytes nanocapsules. In fact, when siRNAs are formulated into degradable polymer nanocapsules they may be protected from nuclease digestion and last longer than naked siRNAs improving their efficacy and therapeutical properties. The capsules have been prepared by using the Layer-by-Layer (LbL) strategy, which is based on the deposition of interacting polymers onto a sacrificial porous colloidal template followed by core removal. Nanoengineered micro/nanocapsules composed of sequentially assembled polymer layers hold immense promise for a variety of biomedical applications. As the challenge of siRNA delivery by nanocapsules is met, it will be possible to advance RNAi therapeutics rapidly into clinical studies for many diseases, including some which remain untreatable or poorly treated by conventional drugs.

The outcomes and achievements of scientific results (36 months) of Nanosirna project are briefly outlined:

1) Preparation of nanoengineered capsules;
2) Characterization of structural and functional properties of nanocapsules;
3) Assessment of in vitro biocompatibility of siRNA-loaded nanocapsules;
4) Evaluation of cancer target expression levels following the exposure of tumor cells to siRNA-loaded nanocapsules;
5) Determination of the effects of siRNA/nanocapsules-dependent knockdown of specific targets on tumor cell growth and induction of apoptosis.

Different types of microcapsules (C), including PMASS µC, PMASS-PLL Cs,PVPON, PDPA, PMPC µC,pHPMASS-PLL Cs, PLL-PEG microsponges (S) were successfully designed and synthesized by the LbL and porous silica infiltration technology, and subsequently characterized for their structural and functional properties. Once defined the physico-chemical properties of our capsules, the loading with siRNA cargo, designed to target the messenger RNA encoding for the anti-apoptotic factor survivin, was pursued. Results showed that under different experimental conditions capsules were able to retain up to 85% of siRNA cargo and to efficiently release it in a simulated intracellular environment. In addition, capsules displayed good biocompatibility properties, as assessed by viability assay carried out on prostate cancer cells grown in vitro and exposed to different amounts of empty and siRNA-loaded capsules as well as free polymers. Our data show that PMASS µC loaded with siRNA targeting survivin, were able to efficiently release a fully active cargo, as gauged by the marked down-regulation of the target gene in siRNA/capsule-treated androgen-independent prostate cancer PC-3 cells compared to cells exposed to the same amount of siRNA delivered by means of a commercially available transfection agent. However, down-regulation of survivin also occurred in PC-3 cells exposed to PMASS µC embedded with a scrambled siRNA as well as in cells treated with empty microcapsules. These findings indicate a capsule-dependent off-target effect, which is supported by a reduction in the expression of other survivin-unrelated proteins. In addition, in PC-3 cells exposed to microcapsules, a marked induction of autophagy (i.e. self-digestion), a degradation pathway involved in the maintenance of cell homeostasis in response to different stresses, was observed. This evidence suggests that empty micro-nanocapsules can induce a perturbation of the intracellular environment, which causes the activation of a cell safeguard mechanism that may limit the therapeutic effect of the microcapsules in tumor cells.

Hence, our preliminary data showed that micro/nanocaspule may represent a superior delivery system for siRNAs compared to lipid-based transfection agents and highlight the need of a careful evaluation of possible off-target effects to unravel the possible interference with cell survival pathways that may have a detrimental outcome on the therapeutic exploitation of such nanodevices.

We further investigated the mechanisms by which PMASS µC trigger survivin down-regulation and authophagy induction by focusing on the possible correlations between the observed off-target effects and (i) the chemical and functional properties of the µC building blocks, (ii) the physico-chemical properties of the µCs, and (iii) the cellular entry and intracellular trafficking of the µCs. We show that, depending on the capsules’ physicochemical properties as well as their polymeric components, PMASS µC can elicit a number of cellular responses. We have demonstrated that surface charge, stiffness, thickness and chemical composition of PMASS-PLL and pHPMASS-PLL Cs influence cellular uptake and the efficiency of siRNA delivery. Our data suggest a faster but less efficient cellular uptake for microcapsules bearing a higher positive surface charge. Thicker and more rigid microcapsules are more efficiently internalized in PC-3cells.

Finally, we optimized the design of multifunctional poly-L-lysine-PEG (PLL-PEG) microsponges, PLL-PEG S, with unique properties for sustained and efficient siRNA delivery. The sequential treatment of PC-3 cells with PLL-PEG S and paclitaxel synergistically improved the therapeutic efficiency of the antineoplastic drug.

The success of Nanosirna project has led to the publication of four papers (Becker AL et al., ACS Nano. 5(2):1335-44,2011 (IF 9.8); Ng SL et al Biomaterials, 32, 6277-6284, 2011 (IF 7.4) Best, J. P et al Langmuir 2013, 29, 9824. Best, J. P et al. Langmuir 2013, 29, 9814.) and three submitted paper on peer-reviewed international journals with high impact factor.

A certain number of oral and poster communications were presented at international conferences. In addition to achieving scientific results, this joint research project also generated additional benefits for the partners both in terms of transfer of knowledge and generation of a basis for sustainable cooperation. The IRSES Funding Scheme has allowed to strengthen a research partnerships through the exchanges of seven researchers and networking activities (seminars, conferences) between the UTV, UBT UoM and INT. European and Australian researchers (experienced and early stage researchers) have been introduced to the new technologies of Layer-by-Layer deposition of materials and biomaterials broadening their research and academic skills.

The IRSES Mobility Programme provided support to the European research organisations to establish and reinforce the long-term research co-operation with Australia.

The australian partner was supported by the Australian Academy of Science according the S&T agreement EU-AU. During the last three years, the partnership has put effort in applying for Collaborative research projects within 7th Framework Programme and Australian Research Council Funding Schemes to continue the long-term collaborative research activities. In addition, a five-year Memorandum of Understanding between UTV and UoM focused to the exchange MSc and PhD students has been signed by the two educational Institutions. Socio-economic impact of the overall project is described in the attached document (point 5. Additional Information).