Fabrication and intracellular delivery of siRNA/carbonate apatite nano-composites for effective knockdown of cyclin B1 gene

Abstract

<p>Gene therapy through intracellular delivery of a functional gene or a gene-silencing element is a promising approach to properly treat critical human diseases like cancer. The ability of synthetically designed small interfering RNA (siRNA) to effectively silence genes<em> </em>at post-transcriptional level has made them attractive options in targeted therapeutics. However, naked siRNA being unable to passively diffuse through cellular membranes, poses difficulty in fully exploiting the potential of the technology. pH-sensitive carbonate apatite has been developed as an efficient tool to deliver siRNA into the mammalian cells by virtue of its high affinity interaction with the siRNA and effective cellular endocytosis. Moreover, internalized siRNA has been found to escape from the endosomes in a time-dependent manner and effectively silenced reporter gene expression. Knockdown of cyclin B1 gene with only 10 nM of siRNA delivered by carbonate apatite has resulted in significant death of cervical cancer cells. Moreover, delivery of siRNA against cyclin B1 gene has led to the sensitization of the cancer cells to both cisplatin and doxorubicin at a particulat drug concentration. Thus, the new method of siRNA delivery is highly promising for pre-clinical and clinical cancer therapy using siRNA therapeutics.</p

Carbonate Apatite Nanoparticles-Facilitated Intracellular Delivery of siRNA(s) Targeting Calcium Ion Channels Efficiently Kills Breast Cancer Cells

“…Thus, 3–6 µL of 1 M exogenous calcium chloride (i.e., 15–30 mM) is necessary to induce particles with an average size range of 125–225 nM in 200 µL DMEM. Interestingly, in our earlier studies particle fabrication in 1 mL DMEM led to an increase in both turbidity and particle diameter upon an increase in the concentration of exogenous Ca 2+ (1–6 µL of 1 M Ca 2+ ), with the highest particle size observed at 6 mM of Ca 2+ [ 22 , 23 , 24 ]. This could be explained by the notion that a relatively lower concentration of exogenous Ca 2+ (1–6 mM) in 1 mL DMEM, unlike 200 µL DMEM with 5–30 mM of exogenous Ca 2+ , is unable to react all inorganic phosphate (Pi) in the solution, with the remaining Pi stimulating further growth and consequently the aggregation of particles.…”

“…On the other hand, cells treated with NPs-siRNA showed a diffusive pattern of fluorescence ( Figure 6 h) after the washing, thus confirming the internalization of NPs-siRNA in breast cancer cells, as shown previously in breast tumor studies [ 22 , 26 ]. In this study, CA NPs with smaller particle sizes seemed to be capable of carrying electrostatically associated siRNA inside cancer cells through endocytosis, eventually releasing the bound siRNA from the endosome to the cytosol through pH-responsive self-dissolution to induce death in breast cancer cells [ 22 , 23 ].…”

Nanoparticle-facilitated Intratumoral Delivery of Bcl-2/IGF-1R siRNAs and p53 Gene Synergistically Inhibits Tumor Growth in Immunocompetent Mice

“…It should be noted that we had extensively analyzed the morphology, size distribution and zeta potential of carbonate apatite nanoparticles by transmission electron microscope (TEM), scanning electron microscope (SEM), dynamic light scattering (DLS) and zeta sizer in our earlier publications. Moreover, we had reported on siRNA binding affinity towards the nanoparticles, cellular uptake and knockdown efficacy using reporter gene assays [32][33][34][35][36][37][38]. We have therefore not included those data in the current manuscript.…”

“…Recently, we developed pH-responsive carbonate apatite nanoparticles to efficiently deliver siRNA as well as DNA across the cell membrane and facilitate them to release from the particles and endosomal vesicles to carry out knockdown of a specific mRNA transcript or expression of a desirable protein, respectively [32,33]. Moreover, we demonstrated nanoparticleassisted delivery of the siRNAs targeting cyclin B1, PLC-gamma-2/ calmodulin 1, HER2/ErbB2, ABCG2/ABCB1 and cROS1 mRNAs sensitizes cervical adenocarcinoma and breast cancer cells towards traditional anti-cancer drugs [34][35][36][37][38]. Here, we report that combined delivery of the siRNA against IGF-1R and Bcl-2 gene transcripts along with p53 gene using carbonate apatite nanoparticles synergistically induces death or growth inhibition of breast cancer cell lines as well as regression of the breast tumor induced in Balb/c mice.…”

Knockdown of PLC-gamma-2 and calmodulin 1 genes sensitizes human cervical adenocarcinoma cells to doxorubicin and paclitaxel

“…Recently, we have developed an efficient siRNA delivery system based on some unique properties of carbonate apatite- electrostatic affinity for binding anionic siRNA, ability of preventing crystal growth for generation of nano-size particles for efficient endocytosis and fast dissolution kinetics in endosomal acidic compartments to facilitate the release of siRNA from the particles as well as from the endosomes, leading to the efficient silencing of reporter gene expression. Moreover, nanoparticle-assisted delivery of validated siRNA against cyclin B1 resulted in the significant inhibition of cancer cell growth [ 18 , 19 ].…”