Maximizing RNA Loading for Gene Silencing Using Porous Silicon Nanoparticles

Abstract

Gene silencing by RNA interference is a powerful technology with broad applications. However, this technology has been hampered by the instability of small interfering RNA (siRNA) molecules in physiological conditions and their inefficient delivery into the cytoplasm of target cells. Porous silicon nanoparticles have emerged as a potential delivery vehicle to overcome these limitationsbeing able to encapsulate RNA molecules within the porous matrix and protect them from degradation. Here, key variables were investigated that influence siRNA loading into porous silicon nanoparticles. The effect of modifying the surface of porous silicon nanoparticles with various amino-functional molecules as well as the effects of salt and chaotropic agents in facilitating siRNA loading was examined. Maximum siRNA loading of 413 μg/(mg of porous silicon nanoparticles) was found when the nanoparticles were modified by a fourth generation polyamidoamine dendrimer. Low concentrations of urea or salt increased loading capacity: an increase in RNA loading by 19% at a concentration of 0.05 M NaCl or 21% at a concentration of 0.25 M urea was observed when compared to loading in water. Lastly, it was demonstrated that dendrimer-functionalized nanocarriers are able to deliver siRNA against ELOVL5, a target for the treatment of advanced prostate cancer.

Patient‐Derived Prostate Cancer Explants: A Clinically Relevant Model to Assess siRNA‐Based Nanomedicines

“…Pivotal peaks were observed at both functionalization steps (Scheme S1, Supporting Information): 1) CO stretching vibrations at 1720 cm −1 confirmed the presence of carboxyl groups from undecylenic acid (UA‐pSiNPs) molecules, which were first reacted to freshly etched pSiNPs, and 2) peaks at 1650 and 1550 cm −1 from amide groups verified the successful attachment of PAMAM dendrimers to UA‐pSiNPs via carbodiimide crosslinking chemistry (Figure S1E, Supporting Information). Previous work done using PAMAM‐pSiNPs have demonstrated a siRNA loading efficiency of 74% and a sustained release profile over 72 h. [ 24 ] Furthermore, our previous work showed that exposure of naked siRNA to RNAse A resulted in complete degradation after 1 h at an RNAse A concentration of 0.01 ng mL −1 . At a tenfold higher concentration of RNAse A (0.1 ng mL −1 ), all siRNA loaded into PAMAM‐pSiNPs was recovered after enzyme incubation.…”

“…We have previously demonstrated that PAMAM‐pSiNPs are a potent non‐viral gene delivery vector for the delivery of siRNA. [ 24 ] PAMAM‐pSiNPs have been shown to both load and protect siRNA molecules. Representation of the functionalization steps followed and physicochemical characterization can be found in the Supplementary Information (Scheme S1, Figure S1, Supporting Information).…”

“…A 1000 × concentration (1 µg mL −1 ) was required in order to degrade all siRNA loaded in PAMAM‐pSiNPs. [ 24 ]…”

“…This pSi‐based delivery system has proven to be an efficacious platform in an in vitro setting, against prostate cancer cells. [ 24 ] However, traditional 2D cell culture fails to mimic the natural structures of tissues or tumors. Therefore, 3D growth of immortalized cancer cell lines, known as spheroids, is regarded as a more representative model in vitro than 2D cancer cell cultures.…”

“…We utilized dendrimer‐functionalized porous silicon nanoparticles (PAMAM‐pSiNPs) that have shown to be highly efficacious in the delivery of siRNA in a standard in vitro 2D monoculture model. [ 24 ] We aimed to silence the expression of the androgen receptor (AR), which drives the development of the normal prostate and progression of prostate cancer, [ 25 ] to determine the therapeutic efficacy of our nanoparticle delivery system. To the best of our knowledge, ex vivo culture of human tumor tissue has not been studied in the context of evaluating the efficacy of any form of nanomedicine.…”