Carbonate Apatite and Hydroxyapatite Formulated with Minimal Ingredients to Deliver SiRNA into Breast Cancer Cells In Vitro and In Vivo

Abstract

Introduction: Cancer is one of the top-ranked noncommunicable diseases causing deaths to nine million people and affecting almost double worldwide in 2018. Tremendous advancement in surgery, chemotherapy, radiation and targeted immunotherapy have improved the rate of cure and disease-free survival. As genetic mutations vary in different cancers, potential of customized treatment to silence the problem gene/s at the translational level is being explored too. Yet delivering therapeutics at the required dosage only to the affected cells without affecting the healthy ones, is a big hurdle to be overcome. Scientists worldwide have been working to invent a smart drug delivery system for targeted delivery of therapeutics to tumor tissues only. As part of such an effort, few organic nanocarriers went to clinical trials, while inorganic nanoparticles (NPs) are still in development stage despite their many customizable properties. Carbonate apatite (CA), a pH sensitive nanocarrier has emerged as an efficient delivery system for drugs, plasmids and siRNAs in preclinical models of breast and colon cancers. Like hydroxyapatite (HA) which serves as a classical tool for delivery of genetic materials such as siRNA and plasmid, CA is an apatite-based synthetic carrier. We developed simplified methods of formulating CA-in-DMEM and a DMEM-mimicking buffer and HA in a HEPES-buffered solution and characterized them in terms of size, stability, protein corona (PC) composition, cytotoxicity, siRNA delivery efficiency in breast cancer cells and siRNA biodistribution profile in a mouse model of breast cancer. Methods: Particle growth was analyzed via spectrophotometry and light microscopy, size was measured via dynamic light scattering and scanning electron microscopy and confirmation of functional groups in apatite structures was made by FT-IR. siRNA-binding was analyzed via spectrophotometry. Stability of the formulation solutions/buffers was tested over various time points and at different temperatures to determine their compatibility in the context of practical usage. Cellular uptake was studied via fluorescence microscopy. MTT assay was performed to measure the cytotoxicity of the NPs. Liquid chromatography—mass spectrometry was carried out to analyze the PC formed around all three different NPs in serum-containing media. To explore biodistribution of all the formulations, fluorescence-labeled siRNA-loaded NPs were administered intravenously prior to analysis of fluorescence intensity in the collected organs and tumors of the treated mice. Results: The size of NPs in 10% serum-containing media was dramatically different where CA-in-DMB and HA were much larger than CA-in-DMEM. Effect of media was notable on the PC composition of all three NPs. All three NPs bound albumin and some common protease inhibitors involved in bone metabolism due to their compositional similarity to our bone materials. Moreover, CA also bound heme-binding proteins and opsonins. Unlike CA, HA bound different kinds of keratins. Difference in PC constitution was likely to influence accumulation of NPs in various organs including those of reticuloendothelial system, such as liver and spleen and the tumor. We found 10 times more tumor accumulation of CA-in-DMB than CA-in-DMEM, which could be due to more stable siRNA-binding and distinct PC composition of the former. Conclusion: As a nanocarrier CA is more efficient than HA for siRNA delivery to the tumor. CA prepared in a buffer containing only the mere constituents was potentially more efficient than classical CA prepared in DMEM, owing to the exclusion of interference attributed by the inorganic ions and organic molecules present in DMEM.

Inhibition of Breast Tumour Growth with Intravenously Administered PRKCA siRNA- and PTEN Tumour Suppressor Gene-Loaded Carbonate Apatite Nanoparticles

“…A study by Islam et al demonstrated that upon systemic delivery, NPs come into interaction with serum proteins, forming a protein corona (PC). The composition of PC depends on size, charge and other physicochemical features of NPs, and in in vivo mouse studies, the PC can be remodeled with the change of proteome in different treatment formulations [28,52,53]. Another possibility of epigenetic changes in in vivo environment may affect gene expression and protein interaction in different treatment groups; hence, future study might focus on the in vivo mechanism of the combined therapy.…”

“…As our NPs contain both positive and negative ions, such as Ca 2+ , PO 4 3− , OH − , the negative charge may come from the protein corona formed around the NPs in serum-containing media. In fact, a recent study revealed that at physiological pH, CA NPs attach to both positive and negative proteins; however, the negative proteins outnumbered the positive ones, which could contribute to the negative zeta potential [28]. Negative zeta potential is advantageous in terms of longer retention in blood stream [34].…”

“…Next, FBS is added to stop further nanoparticle complex formation [27]. In Field Emission Scanning Electron Microscope (FE-SEM) images, CA NPs displayed spherical shape, rough texture, and an average diameter of 50-300 nm [28][29][30]. With molecular formula Ca 10 (PO 4 ) 6−x (CO 3 ) x (OH) 2, the small sized nanoparticles have durable binding affinity towards the siRNA/DNA; the positively charged Ca 2+ present in the CA NPs can electrostatically combine with the negatively charged siRNA/DNA molecules and come close to the cell membrane through ionic interaction, ultimately transporting the siRNA/DNA into cytoplasmic acidic compartments (endosomes) by endocytosis [27].…”

“…With molecular formula Ca 10 (PO 4 ) 6−x (CO 3 ) x (OH) 2, the small sized nanoparticles have durable binding affinity towards the siRNA/DNA; the positively charged Ca 2+ present in the CA NPs can electrostatically combine with the negatively charged siRNA/DNA molecules and come close to the cell membrane through ionic interaction, ultimately transporting the siRNA/DNA into cytoplasmic acidic compartments (endosomes) by endocytosis [27]. Upon cellular internalization by endocytosis, particles are quickly dissolved into component ions such as Ca 2+ , PO 4 3− and HCO 3 − in endosomal acidic pH, resulting in particle dissolution, swelling and breakage of the endosomes following development of osmotic pressure across the endosomal membrane, and release of the siRNA/DN A in cytosol [27,28]. In the cytoplasm, the released siRNA is merged into a RISC (RNAinduced silencing complex) and relaxed into single-stranded RNAs by Argonaute-2.…”

One-Step Synthesis of Magnetic Nanocomposite with Embedded Biologically Active Substance

“…HAP/magnetic NP composites are promising biomaterials for targeted drug delivery systems [ 15 ], for example, for orthopedic drugs, and the protein adsorption/controlled pH-dependent release of antitumor drugs [ 16 , 17 , 18 ]. New areas of potential application are gene therapy [ 19 , 20 ], antibacterial nanocomposites, and composites for implants [ 21 ]. The combined effect of magnetic and hydroxyapatite nanostructures provides a high loading capacity of a drug compound and its efficient delivery to targets under the influence of external magnetic fields.…”