CaCO3 as an Environmentally Friendly Renewable Material for Drug Delivery Systems: Uptake of HSA-CaCO3 Nanocrystals Conjugates in Cancer Cell Lines

Abstract

Chemical and biochemical functionalization of nanoparticles (NPs) can lead to an active cellular uptake enhancing their efficacy thanks to the targeted localization in tumors. In the present study calcium carbonate nano-crystals (CCNs), stabilized by an alcohol dehydration method, were successfully modified by grafting human serum albumin (HSA) on the surface to obtain a pure protein corona. Two types of CCNs were used: naked CaCO3 and the (3-aminopropyl)triethoxysilane (APTES) modified CaCO3-NH2. The HSA conjugation with naked CCN and amino-functionalized CCN (CCN-NH2) was established through the investigation of modification in size, zeta potential, and morphology by Transmission Electron Microscopy (TEM). The amount of HSA coating on the CCNs surface was assessed by spectrophotometry. Thermogravimetric analysis (TGA) and Differential scanning calorimetry (DSC) confirmed the grafting of APTES to the surface and successive adsorption of HSA. Furthermore, to evaluate the effect of protein complexation of CCNs on cellular behavior, bioavailability, and biological responses, three human model cancer cell lines, breast cancer (MCF7), cervical cancer (HeLa), and colon carcinoma (Caco-2) were selected to characterize the internalization kinetics, localization, and bio-interaction of the protein-enclosed CCNs. To monitor internalization of the various conjugates, chemical modification with fluorescein-isothiocyanate (FITC) was performed, and their stability over time was measured. Confocal microscopy was used to probe the uptake and confirm localization in the perinuclear region of the cancer cells. Flow cytometry assays confirmed that the bio-functionalization influence cellular uptake and the CCNs behavior depends on both cell line and surface features.

Insights into Characterization Methods and Biomedical Applications of Nanoparticle–Protein Corona

“…The size characterizations from DLS and TEM (or AFM) are helpful in eliminating ambiguities regarding the sizes of the NPs or the NPs' agglomeration(s). The advantage of TEM or AFM with respect to DLS is that they provide clear and intuitive information regarding NPs' sizes and shapes and the coating of the PC around the NP's surface [48][49][50]. However, the drying and staining during the sample preparation process for TEM may affect the morphology of the NP-PC complexes [26,51].…”

“…In addition to the above approaches, differential scanning calorimetry (DSC) can be used to detect conformational changes in PCs resulting from NP binding [49,105]. DSC can characterize the thermal denaturation of molecules and can provide information regarding the heat capacity of a solution as a function of temperature, by heating the reference and sample cells to the same temperature.…”

“…In comparison with bare NPs, human serum PCs have been proven to improve the uptake of β-cyclodextrin-pluronic modified iron oxide magnetic NPs (MNPs) by human pancreatic carcinoma (Panc-1) cells and human bone metastatic LNCaP-derivative cancer cell line (C4-2B) prostate cancer cells [93]. It is reported that HSA coronas can improve the uptake of calcium carbonate nanocrystals in breast-cancer (MCF7), cervical-cancer (HeLa), and colon-carcinoma (Caco-2) cells [49]. In addition, the cellular uptake of gold nanorods (AuNRs) in Cal 27 oral squamous-cell carcinoma has been enhanced by an apolipoprotein E (ApoE) corona [114].…”

Sonication-Assisted Production of Fosetyl-Al Nanocrystals: Investigation of Human Toxicity and In Vitro Antibacterial Efficacy against Xylella fastidiosa

“…actinidiae artificially infected kiwi plants [23]. Recently, new nanotechnological tools were proposed to control X. fastidiosa, investigating CaCO 3 nanocarriers [24,25] interaction with pathogen cells and olive plants [26]. CaCO 3 nanocrystals evidences have been proved the strategic role of nanopesticides to potentially control X. fastidiosa pathogen [26].…”

Inter-protein interactions govern protein loading into porous vaterite CaCO3 crystals

“…25,26 Recent studies also demonstrated the high cellular uptake of CaCO 3 . 27,28 In addition, CaCO 3 crystals can be used as sacrificial templates for the formulation of porous protein 29 and polymer beads, 30,31 and layer-by-layer assemblies to produce multilayer capsules. [32][33][34][35] All these findings indicate that CaCO 3 crystals are promising carriers for the encapsulation of protein-based therapeutics for oral, [36][37][38] intravenous, 17 intratumoral, 39 intranasal, 40 and mucosal drug delivery; 41,42 tissue engineering 43,44 and regeneration; 45,46 and diagnostics.…”