Abstract
Breast cancer is the abnormal, uncontrollable proliferation of cells in the breast. Conventional treatment modalities like chemotherapy induce deteriorating side effects on healthy cells. Non-viral inorganic nanoparticles (NPs) confer exclusive characteristics, such as, stability, controllable shape and size, facile surface modification, and unique magnetic and optical properties which make them attractive drug carriers. Among them, carbonate apatite (CA) particles are pH-responsive in nature, enabling rapid intracellular drug release, but are typically heterogeneous with the tendency to self-aggregate. Here, we modified the nano-carrier by partially substituting Ca2+ with Mg2+ and Fe3+ into a basic lattice structure of CA, forming Fe/Mg-carbonate apatite (Fe/Mg-CA) NPs with the ability to mitigate self-aggregation, form unique protein corona in the presence of serum and efficiently deliver doxorubicin (DOX), an anti-cancer drug into breast cancer cells. Two formulations of Fe/Mg-CA NPs were generated by adding different concentrations of Fe3+ and Mg2+ along with a fixed amount of Ca2+ in bicarbonate buffered DMEM (Dulbecco’s Modified Eagle’s Medium), followed by 30 min incubation at 37 °C. Particles were characterized by turbidity analysis, z-average diameter and zeta potential measurement, optical microscopy, field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), energy dispersive X-ray (EDX), flame atomic absorption spectroscopy (FAAS), pH dissolution, drug binding, cellular uptake, thiazolyl blue tetrazolium bromide (MTT) assay, stability analysis, and protein corona study by LCMS (Liquid chromatography-mass spectrometry). Both formulations of Fe/Mg-CA displayed mostly uniform nano-sized particles with less tendency to aggregate. The EDX and FAAS elemental analysis confirmed the weight (%) of Ca, Fe and Mg, along with their Ca/P ratio in the particles. A constant drug binding efficiency was noticed with 5 μM to 10 μM of initial DOX concentration. A pH dissolution study of Fe/Mg-CA NPs revealed the quick release of DOX in acidic pH. Enhancement of cytotoxicity for the chemotherapy drug was greater for Fe/Mg-CA NPs as compared to CA NPs, which could be explained by an increase in cellular internalization as a result of the small z-average diameter of the former. The protein corona study by LCMS demonstrated that Fe/Mg-CA NPs exhibited the highest affinity towards transport proteins without binding with opsonins. Biodistribution study was performed to study the effect of DOX-loaded Fe/Mg-CA NPs on the tissue distribution of DOX in Balb/c 4T1 tumor-bearing mice. Both formulations of Fe/Mg-CA NPs have significantly increased the accumulation of DOX in tumors. Interestingly, high Fe/Mg-CA NPs exhibited less off-target distribution compared to low Fe/Mg-CA NPs. Furthermore, the blood plasma analysis revealed prolonged blood circulation half-life of DOX-loaded low and high Fe/Mg-CA NPs compared to free DOX solution. Modifying CA NPs with Fe3+ and Mg2+, thereby, led to the generation of nano-sized particles with less tendency to aggregate, enhancing the drug binding efficiency, cellular uptake, and cytotoxicity without hampering drug release in acidic pH, while improving the circulation half-life and tumor accumulation of DOX. Therefore, Fe/Mg-CA which predominantly forms a transport protein-related protein corona could be a proficient carrier for therapeutic delivery in breast cancer.
Highlights
Breast cancer is one of the major lethal diseases with an occurrence rate of 1 in 35 [1]
We report on the development of pH-responsive Fe/Mg-carbonate apatite (Fe/Mg-Carbonate Apatite (Mg-CA)) NPs that formed unique transport protein-related corona and enhanced the cellular uptake of DOX with the consequence of much higher cytotoxicity compared to CA NPs in breast cancer cells
CA NPs synthesized with 40 mM Ca2+, low Fe/Mg-CA NPs generated with 40 mM Ca2+, 10 μM Fe3+ and 40 mM Mg2+, and high Fe/Mg-CA formulated with 40 mM Ca2+, 50 μM Fe3+ and 120 mM Mg2+ were prepared in bicarbonate buffered Dulbecco’s modified eagle medium (DMEM) through incubation at 37 ◦C for 30 min, followed by supplementation with 10% fetal bovine serum (FBS)
Summary
Breast cancer is one of the major lethal diseases with an occurrence rate of 1 in 35 [1]. NPs used in drug delivery are usually in the range of 100–1000 nm [8] In cancer therapy, they can mitigate adverse side effects altering the stability, absorption, and release profile of drugs. They can mitigate adverse side effects altering the stability, absorption, and release profile of drugs They can enhance the site targeted delivery and cellular uptake in tumors through receptor-mediated endocytosis that may enable them to escape P-glycoprotein recognition [9] and overcome MDR (multidrug resistance), and reduce the off-target effects and systemic clearance of drugs. We report on the development of pH-responsive Fe/Mg-carbonate apatite (Fe/Mg-CA) NPs that formed unique transport protein-related corona and enhanced the cellular uptake of DOX with the consequence of much higher cytotoxicity compared to CA NPs in breast cancer cells. Fe/Mg-CA which forms a transport protein-related protein corona could be a promising tool for efficient therapeutic delivery in breast cancer patients
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