α-Ketoglutaric Acid-Modified Carbonate Apatite Enhances Cellular Uptake and Cytotoxicity of a Raf-Kinase Inhibitor in Breast Cancer Cells through Inhibition of MAPK and PI-3 Kinase Pathways

Abstract

AZ628 is a hydrophobic Raf-kinase inhibitor (rapidly accelerated fibrosarcoma) currently in clinical trial of various cancer. The physicochemical properties of hydrophobic drugs that affect the drug-particle interactions and cause aggregation of drugs and particles might be the key aspect to impede effective drug delivery. Retaining smaller particle size is the prerequisite to overcome the opsonization and improve cytotoxicity in the targeted region. Carbonate apatite (CA), an attractive biodegradable vector, has been used to carry both hydrophilic and hydrophobic drugs and release the payloads inside the cells following endocytosis. We incorporated AZ628 into CA and also modified it with α-ketoglutaric acid (α-KA) for reducing particle growth kinetics and increasing total surface area to improve the delivery of AZ628 by enhancing cellular uptake by breast cancer cells. AZ628-loaded nanoparticles of CA and α-KA-modified CA (α-KAMCA) were synthesized and evaluated in MCF-7 and 4T1 cell lines by measuring cytotoxicity and cellular uptake analysis. HPLC (high-performance liquid chromatography) assay was performed to quantify the binding affinity of the nanocarriers towards the drug. Western blot analysis was done to see the activation and expression levels of Akt, MAPK (mitogen-activated protein kinase) pathways and Caspase-3. Zetasizer was used to measure the particle size along with the surface charge. α-KAMCA showed almost 88% encapsulation efficacy for AZ628 with around 21% enhanced cellular uptake of the drug in two different breast cancer cell lines. These findings suggest that α-KAMCA could be a promising therapeutic tool to carry AZ628 for breast cancer treatment.

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

“…The release of drug from the nanoparticle-based formulation depends on many factors including pH, temperature, drug solubility, desorption of the surface-bound or adsorbed drug, drug diffusion through the nanoparticle matrix, nanoparticle matrix swelling and erosion, and the combination of erosion and diffusion processes [57][58][59]. The influence of the pH-sensitive dissolution of CA, CMCA, and α-KAMCA NPs ensured drug release in a acidic microenvironment [28,40] in our former study. An in vitro turbidity test ( Figure 11) was conducted by modeling physiological pH (7.4) and endosomal acidic pH (6.5-5.0), and the NPs were subjected to these environments to ensure pH sensitivity.…”

“…CMCA, SMCA (succinate-modified carbonate apatite), and α-KAMCA NPs were prepared by modifying CA with citrate, containing three carboxyl groups, succinate, containing two carboxyl groups, and ketoglutarate, containing one ketone group and two carboxylic groups, respectively. In our previous experiment [28,39,40], we showed that the presence of carboxylic and ketone groups in the chemical structure has an influential role in determining the particle size and the binding affinity of the drugs toward the NPs. The α-KAMCA NPs exhibited the desired particle size with a large number of resultant particles and a folded larger surface morphology with rapid dissolution at acidic pH in the microenvironment, resulting in more significant cytotoxicity than the CA NPs.…”

“…Drug-loaded NPs showed a particle size ranging from 180-436 nm (Figure 9). In our previous study [28,39,40], we recorded the surface charges for all NP formulations within the range of −9 to −13 mV with a PDI value of 0.553, 0.787, and 0.322 for CA, CMCA, and α-KAMCA NPs, respectively. The PDI values of the nanocarriers pointed out that α-KAMCA NPs are homogeneous (monodisperse) compared to CA and CMCA NPs.…”

“…Previously, we successfully synthesized and characterized CA NPs, citrate-modified CA (CMCA) NPs [28,39], succinate-modified CA (SMCA) NPs, and α-ketoglutaric acid-modified CA (α-KAMCA) NPs [40], and we evaluated their particle size, drug-loading efficiency, enhancement in cytotoxicity, and cell viability in a human breast cancer cell line (MCF-7 cells) and a murine breast cancer cell line (4T1 cells). Here, we examined CA, CMCA, and α-KAMCA NPs for in vivo tumor regression, biodistribution, plasma concentration, and toxicology study depending on their promising in vitro results, especially through a cytotoxicity assay in the 4T1 cell line.…”

Optimization and Formulation of Nanostructured and Self-Assembled Caseinate Micelles for Enhanced Cytotoxic Effects of Paclitaxel on Breast Cancer Cells

“…The cells were seeded in 24-well plates with a density of the 50,000 cells per well and were incubated for 24 h [ 41 ]. After 24 h incubation, the media was removed and was replaced with the free PTX and PTX-NaCNs with final concentrations of 5 and 10 µM.…”