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

Abstract

pH sensitive nanoparticles of carbonate apatite (CA) have been proven to be effective delivery vehicles for DNA, siRNAs and proteins. More recently, conventional anti-cancer drugs, such as doxorubicin, methotrexate and cyclophosphamide have been successfully incorporated into CA for intracellular delivery to breast cancer cells. However, physical and chemical properties of drug molecules appeared to affect their interactions with CA, with hydrophillic drug so far exhibiting better binding affinity and cellular uptakes compared to hydrophobic drugs. In this study, anastrozole, a non-steroidal aromatase inhibitor which is largely hydrophobic, and gemcitabine, a hydrophilic nucleoside inhibitor were used as solubility models of chemotherapy drug. Aggregation tendency of poorly soluble drugs resulting in larger particle-drug complex size might be the main factor hindering their delivery effectiveness. For the first time, surface modification of CA with poly(ethylene glycol) (PEG) has shown promising result to drastically reduce anastrozole- loaded CA particle size, from approximately 1000 to 500 nm based on zeta sizer analysis. Besides PEG, a cell specific ligand, in this case fibronectin, was attached to the particles in order to facilitate receptor mediated endocytosis based on fibronectin–integrin interaction. High-performance liquid chromatography (HPLC) was performed to measure uptake of the drugs by breast cancer cells, revealing that surface modification increased the drug uptake, especially for the hydrophobic drug, compared to the uncoated particles and the free drug. In vitro chemosensitivity assay and in vivo tumor regression study also showed that coated apatite/drug nanoparticle complexes presented higher cytotoxicity and tumor regression effects than uncoated apatite/drug nanoparticles and free drugs, indicating that surface modification successfully created optimum particles size with the consequence of more effective uptake along with favorable pharmacokinetics of the particles.

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

“…DOX-loaded SMCA NPs demonstrated 17% (CI 95%, p < 0.0001 vs. same treatment of free drug) at 10 pM concentration and 22% (CI 95%, p < 0.0001 vs. same treatment of free drug) enhanced cytotoxicity at 1 µM concentration of DOX in MCF-7 cells and 4T1 cells, respectively. This enhancement in cytotoxicity could be as a result of the endocytosis-facilitated cellular uptake of DOX [ 12 , 13 ].…”

“…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.…”

“…Inorganic NPs are most popular for their physiochemical properties [ 12 ], such as the ease of formulation, controlled particle size and a very narrow size distribution. Inorganic NPs are advantageous over organic NPs in terms of site-specific drug delivery, stability and rapid release [ 13 , 14 , 15 ]. Liposomes, the most commonly used lipid nanoparticles, face some major difficulties including instability, poor batch-to-batch reproducibility, difficulties in sterilization and low drug loading.…”

Krebs Cycle Intermediate-Modified Carbonate Apatite Nanoparticles Drastically Reduce Mouse Tumor Burden and Toxicity by Restricting Broad Tissue Distribution of Anticancer Drugs

“…Among the wide variety of NPs, carbonate apatite (CA) NPs and surface-modified CA NPs attracted increasing attention due to their auspicious therapeutic outcomes in nucleic acid and anticancer drug delivery by virtue of a high drug/nucleic acid encapsulation capacity, favorable pharmacokinetics, enhanced cellular uptake, pH-sensitive payload release, efficient transgene expression or gene silencing, and eventually a significant tumor-inhibitory effect both in vitro [23][24][25][26][27][28] and in vivo [29][30][31][32][33][34][35].…”

Cytotoxicity Enhancement in Breast Cancer Cells with Carbonate Apatite-Facilitated Intracellular Delivery of Anti-Cancer Drugs

“…With the molecular formula of Ca 10 ·(PO 4 ) 6 − X ·(CO 3 ) X ·(OH) 2 particles are formed through a flexible process by incubating a bicarbonate-buffered medium containing appropriate concentrations of phosphate and calcium salts in presence of a therapeutic agent. The required chemical reaction takes place with development of solution supersaturation with respect to Ca 2+ , PO 4 3− and HCO 3 − , and the therapeutic agent is electrostatically associated either with the cationic (Ca 2+ -rich) domains or anionic sites (PO 4 3− or HCO 3 − -rich) of the particles depending on their net charges [ 21 , 22 , 23 , 24 , 25 ]. The structural role of bicarbonate, though to a minor amount, is to control the size of the particles and inhibit aggregation [ 22 ].…”