Nanomaterials As SiRNA Delivery Vehicle And Their Interaction With Mammalian Cells In Vitro

The RNAi-based therapy has displayed great potentials in fighting against various diseases such as cancer, infections and diabetes. Although the principle of RNAi is well-understood, there is still a lack of effective carriers for siRNA delivery which largely hinder the clinical application of siRNA. The virus vectors have a high RNAi efficiency with specificity; application of them also brings the risk of mutagenesis and immunogenicity. In comparison with the virus vectors, nonviral vectors are strongly attracting attentions and research interests. One typical nonviral vector is nanoparticles, which have distinctive size effects as well as facile modification strategies and are capable of mediating effective RNAi with targeting potential. However, nanomaterials have different degree of cytotoxicity. Therefore researches are focusing on the interaction between nanoparticles and cells including uptake, traffic and fate of them. Addressing above issues can be helpful in designing more effective RNAi vectors and alleviating associated side effects. In this study, gold nanorods (AuNRs) and spermine-modified pullulan (Ps) are employed as siRNA-carrier models to silence targeted proteins in breast cancer MDA-MB-231cells; meanwhile the interaction between the carriers and cells was comprehensively examined. Main results are listed below:(1) Extensive transmission electron microscopy (TEM) observation together with other experimental results demonstrates that the AuNRs are internalized through receptor-meditated endocytosis (RME) pathway. After internalized, AuNRs enter into lysosome maturation process and finally reside in the residual body (RB) in aggregation status. Parts of the AuNRs restrained in vesicular system are exocytosed by the cells and can be re-uptaken by the cells. Although a few of AuNRs escape from the lysosomes, autophagy process recycles them into the lysosomes, which plays a cytoprotective role when the cells are encountered with AuNRs in cytoplasm. A long-time chase of AuNRs-incubated cells reveals that dilution of AuNRs in the cells is mainly dependent on the cell proliferation as the endocytosed AuNRs can re-enter cells. Above observations well explain the minimal cytotoxicity of AuNRs, as AuNRs are restricted in vesicular system, which guarantee the other important organelles such as nucleus, mitochondria and endoplasmic reticulum (ER) are exempted from the direct contact with AuNRs.(2). In order to characterize AuNRs in living cells, a quantitative method to measure the number of AuNRs in cells based on UV-Vis-NIR absorption spectrum is established. Compared with inductively coupled plasma mass spectrometry (ICP-MS), the proposed method realizes quantification of AuNRs in living cells through a simple and cost-effective way.(3). The AuNRs as siRNA carriers successfully down-regulate the expression of PAR-1(cancer metastasis-associated protein) in breast cancer cells and inhibit the cells migration in vitro. Althouth the efficiency of AuNRs-mediated RNAi is between that of commercialized vectors Lipofectamine2000and TurboFect, cytotoxicity of AuNRs is minimal in comparison to the commercialized vectors. The finding suggests that combination of AuNRs and PAR-1siRNA could be a potential therapeutic strategy towards breast cancer metastasis.(4). Using Ps as siRNA vector, effects of serum on the size of Ps and siRNA complex (Ps-siRNA) are examined; meanwhile the associated effects on RNAi efficiency are probed. The Ps-siRNA is aggregated in low-serum condition. After internalization by the cells in culture medium with low serum content, Ps-siRNA forms aggregates and locates in lysosomes. Because the aggregates have large amounts of imino groups, they could induce a stronger proton sponge effect and subsequent lysosomal swelling, which facilitates the lysosomal escape of siRNA molecules. However, Ps-siRNA exhibits severer cytotoxicity under the culture condition with low serum content. Compared with that in low-serum condtion, Ps-siRNA in high-serum medium is well-dispersed with a compromised ability of escaping from lysosomes. The well-dispersed Ps-siRNA also displays better biocompatibility. After the post-treatment with chloroquine, the RNAi efficiency of well-dispersed Ps-siRNA is largedly elevated. Above results suggest a simple way to balance RNAi efficiency and cytotoxicity by controlling serum contents in the culture medium.In conclusion, we have explored potentials of two kinds of nanomaterials as RNAi vectors and probed the interaction between the vectors and the breast cancer cells. Our scientific findings provide deep insights in improving performance of nanomaterials in siRNA delivery as well minimizing their associated side effects.