Methotrexate- and Cyclophosphamide-embedded Pure and Strontiumsubstituted Carbonate Apatite Nanoparticles for Augmentation of Chemotherapeutic Activities in Breast Cancer Cells

Abstract

Most of the classical drugs used today to destroy cancer cells lead to the development of acquired resistance in those cells by limiting cellular entry of the drugs or exporting them out by efflux pumps. As a result, higher doses of drugs are usually required to kill the cancer cells affecting normal cells and causing numerous side effects. Accumulation of the therapeutic level of drugs inside the cancer cells is thus required for an adequate period of time to get drugs' complete therapeutic efficacy minimizing the side effects on normal cells. In order to improve the efficacy of chemotherapeutic drugs, nanoparticles of carbonate apatite and its strontium (Sr(2+))-substituted derivative were used in this study to make complexes with three classical anticancer drugs, methotrexate, cyclophosphamide and 5-flurouracil. The binding affinities of these drugs to apatite were evaluated by absorbance and HPLC analysis and the therapeutic efficacy of drug-apatite complexes was determined by cell viability assay. Carbonate apatite demonstrated significant binding affinity towards methotrexate and cyclophosphamide leading to more cellular toxicity than free drugs in MCF-7 and 4T1 breast cancer cells. Moreover, Sr(2+) substitution in carbonate apatite with resulting tiny particles less than 100 nm in diameter further promoted binding of methotrexate to the nanocarriers indicating that Sr(2+)-substituted apatite nanoparticles have the high potential for loading substantial amount of anti-cancer drugs with eventual more therapeutic effectiveness.

Citrate- and Succinate-Modified Carbonate Apatite Nanoparticles with Loaded Doxorubicin Exhibit Potent Anticancer Activity against Breast Cancer Cells

“…Different parameters were optimized in earlier studies to improve the growth kinetics of CA NPs such as salt concentration, pH of the media, incubation time and temperature, and replacement of Ca 2+ , a divalent cation, with another divalent cation such as magnesium (Mg 2+ ) [ 30 ] or strontium (Sr 2+ ) [ 20 ] to form smaller NPs. However, a recent study on intracellular transport of Anastrozole and Gemcitabine into breast cancer cells using CA NPs [ 13 ] has shown limited enhancement in drug-induced cytotoxicity, particularly owing to the lack of proper control on growth kinetics of the particles, motivating us to further improve the current technology.…”

“…Drug-containing NPs can be internalized into the tumor cell by endocytosis, resulting in particle dissolution and release of the entrapped drug inside the endosomal acidic environment [ 18 , 19 ]. However, CA particles formed by exogenously adding Ca 2+ tend to aggregate and form larger particles through ionic interactions [ 20 ], which can be stabilized by adding serum proteins [ 21 ]. Since citrate, a tricarboxylic acid of the Krebs’ cycle, is known to bind with free calcium and reduce platelet aggregation and fibrinogen binding [ 22 , 23 ], we hypothesized that it might electrostatically interact with Ca 2+ -rich domains of CA NPs during the fabrication process and thus limit their uncontrolled growth.…”

Surface-Modification of Carbonate Apatite Nanoparticles Enhances Delivery and Cytotoxicity of Gemcitabine and Anastrozole in Breast Cancer Cells

“…This process is likely a time-consuming process unlike the passive diffusion of free drugs. We carried our another study [31] which indicated that the length of incubation period had a significant effect on cytotoxicity, with 48 h incubation of cells with NP-bound drugs and free drugs resulting in more significant cytotoxicity for NP–drugs than free drugs, compared to the shorter exposure periods (4 or 24 h). In addition to the incubation time, number of cells seeded in a well of a dish also influences cell viability, with longer drug exposure time and lower number of cells eventually leading to higher cytotoxicity.…”

Carbonate apatite nanoparticles carry siRNA(s) targeting growth factor receptor genes egfr1 and erbb2 to regress mouse breast tumor

“…For in vitro study, NPs were prepared as described earlier (Tiash et al, 2014). Briefly, NPs were formed in 1 mL of freshly prepared bicarbonated (44 mM) DMEM media (pH 7.4) by mixing 3 mM of exogenous CaCl 2 (3 mL from 1 M stock), followed by incubation of the mixture for 30 min at 37 C. For making complexes with siRNA(s) (NPs-siRNA(s)), 1 nM of either allstars negative control siRNA or functionally validated siRNA(s) against single or multiple GFR genes were added to 1 mL of DMEM media along with 3 mM of CaCl 2 prior to 30 min incubation at 37 C. For making NPs-complexes of fluorescent negative siRNA, 5 nM or 10 nM of allstars negative control siRNA AF488 (3 0 ) was used for the studies of siRNA binding affinity for NPs and cellular uptake.…”