Different type of materials on the nanoscale were recently used in cancer therapy, including gold nanoparticles for radio/photodynaminc therapy [1
]; polymeric nanoparticles [3
], micelles and liposomes for chemotherapy [6
]; magnetic nanoparticles for hyperthermia [9
]; quantum dots [12
] and carbon nanotubes as theranostic agent [15
Calcium carbonate is an inorganic and a polymorphic material which represents a ubiquitous raw material in nature: it is widespread and continuously replenished by means of natural cycles in rivers, lakes, and oceans or formed as minerals in animals, in the form of shells and skeletons, and in caves as stalactites and stalagmites. The use of environmentally friendly nanomaterials is an essential issue for biomedical application [17
]. It is important to deeply investigate the possible interaction between nanomaterials with cells, tissue, and human organs. Experimental evidences, in vitro and in vivo, report that by pulmonary exposure, the nanomaterials localize in the lungs [19
], evoking biological and toxicological side effect [20
Calcium carbonate nanocrystals (CCNs) are appealing due to their applications in different fields since they are endowed with several attractive properties, such as a high surface area, a controllable pore size distribution, and a good biocompatibility [24
]. These features make CCNs an attractive biomaterial for various biomedical applications, especially in drug delivery systems [26
]. For these reasons, commercial CCNs are produced from natural sources which are widely available worldwide.
Many synthetic procedures were proposed in the last years, but all proposed methods are far from being challenge-free, requiring the use of surfactants or high temperature during the process [31
]. Furthermore, it is well known that the synthetical methodology directly affects the CCNs crystalline phase affording one of calcite, vaterite, aragonite, and amorphous calcium carbonate. In our previous work, a green, fast, and straightforward spray drying technique was developed to synthesize CCNs in the calcite phase [34
], suitable for drug delivery application [25
It is commonly known that usually any nanoparticles with diameter <200 nm are retained in tumors because of the enhanced permeability and retention (EPR) effect [35
], but in spite of extended investigations and developments in the field of cancer therapy, the use of nanoparticles in clinical applications is very limited. For instance, despite suitable features of CCNs as carrier systems [25
], they are unstable in biological environments, hindering their applications e.g., in long-term drug delivery.
However, intracellular trafficking and biological activity are influenced by the functionalization of the nanoparticles surface. The literature reports different protocols of nanoparticles functionalization with polymers, PEG, chemical functionalization with peptide, polysaccharides or other biomolecules [40
]. In principle, when exposed to a physiological environment, the NPs surface is coated by several biomolecules, mainly proteins, resulting in the formation of a “crown”, called protein corona. This corona can transform many of the physicochemical properties of NPs such as size, surface charge, surface composition, and functionality, therefore changing the molecular individuality of NPs [39
]. These modifications modulate the particle–cell interactions and could conspicuously influence the efficacy of the delivery and finally, the therapeutic effects [48
]. The protein corona has been reported to affect cellular interactions [51
], cytokine expression [52
], and protein function [53
], further interfering with the regular metabolism and organ function [54
]. For this reason, it is important to acquire an understanding of the many interactions occurring at the interface between NPs and the biological environment, to have a way to predict the fate of injected NPs. The adsorption of proteins on NPs was suggested as an essential toxicity mediation and its study is part of an ongoing efforts to establish the predictive tools needed for safety assessment. Another emerging important concept to obtain a “stealth effect”, is the requirement to mask NPs with proteins increasing systemic circulation time of NPs in the blood [55
Therefore, with the aim of enhancing the CCNs bioavailability and therapeutic response, we performed a protein functionalization with Human Serum Albumin (HSA), which allows initial protection from recruitment of blood proteins and from the following macrophages phagocytosis. Albumin forms a pure protein corona around CCNs by simple incubation.
To that end, we performed three steps: (i) synthesis of CNNs via an environmentally friendly spray drying process, followed by stabilization through an alcohol dehydration method, in order to avoid difficult to control factors affecting the nucleation process and subsequent crystal growth; (ii) CCNs functionalization by formation of a pure protein corona with Human Serum Albumin (HSA), allowing protection from recruitment of blood proteins and from the subsequent macrophages phagocytosis, followed by investigating physical changes in size, zeta potential, and morphology by TEM [56
]; and (iii) internalization kinetics of the modified CNNs in three human cancer cell lines, breast cancer (MCF7), cervical cancer (HeLa), and colon carcinoma (Caco-2), performed by cytofluorimetric assay through fluorophore functionalization, together with cellular localization obtained via confocal microscopy.
The results stemming from these studies will contribute to the future use of CCNs for biomedical applications, and could be adapted towards understanding the mechanism through which other inorganic NPs coated with proteins cross the cellular membrane.
2. Materials and Methods
The chemicals used were: calcium chloride dehydrate 99.99% (CaCl2·2H2O, Sigma Aldrich, Darmstadt, Germany), sodium hydrogen carbonate (NaHCO3) (pro analysis, Merck, Germany), fetal bovine serum (FBS, Sigma Aldrich, Darmstadt, Germany), penicillin–streptomycin solution (Sigma Aldrich, Darmstadt, Germany), sodium pyruvate (Sigma Aldrich, Darmstadt, Germany), Dulbecco’s modified eagle’s medium (DMEM) (Sigma Aldrich, Darmstadt, Germany), phosphate buffered saline, Dulbecco A (PBS, Oxoid). (3-Aminopropyl)triethoxysilane (APTES) (Sigma, USA), Hoechst 33342 (Sigma Aldrich, Darmstadt, Germany), Triton X-100 (Sigma Aldrich, Darmstadt, Germany). Human Serum Albumin (HSA), Fluorescein isothiocyanate isomer I (FITC), Thiazolyl blue formazan (MTT formazan) were purchased from Sigma Aldrich (Germany) and used without further purification.
2.2. CaCO3 Nanocrystals Synthesis and Bioconjugation
2.2.1. CaCO3 Nanocrystals Synthesis Procedure
nanocrystals were obtained by a literature method [2
]. The process was performed on a Büchi Mini Spray Dryer B-290. Two aqueous solutions of NaHCO3
(250 mL, 0.125 M) and CaCl2
(250 mL, 0.062 M) were drawn by two peristaltic pumps and mixed by means of a T junction. The flow rates (5.2 mL/min and 4.5 mL/min, respectively) were set in order to ensure a 1.2:1 volume mixing ratio. The resulting solution was allowed to flow into a water cooled two-fluid nozzle and sprayed (inlet temperature 140 °C, aspirator 100%, spray gas flow length 50 mm). Upon spraying, a white dry powder was quickly formed. The powder was recovered by the collecting vessel and washed thrice with deionized water and centrifuged to separate CaCO3
from the soluble reaction products or unreacted reagents. Eventually, to facilitate the water removal and to prevent the crystallization process, the powder was washed with isopropyl alcohol before drying at 50 °C with a rotavapor.
2.2.2. Amino-Functionalization of CaCO3 Nanocrystals
An amount of 0.150 g of CaCO3 powder was added to 6.25 mL toluene and 0.5 mL (3-aminopropyl)triethoxysilane (APTES). Then, the mixture was ultrasonic dispersed for 30 min and incubated under vigorous magnetic-stirring at 90 °C for 20 h. After stirring, the mixture was centrifuged three times with toluene, at 13,200 rpm for 10 min, to separate the amino-modified CaCO3 nanocrystals from the reaction medium. Eventually, the modified CaCO3 nanocrystals was repeatedly washed with water, dried in vacuo and stored for subsequent use.
2.2.3. Fluorescein Coupling of Amino-Functionalized CaCO3 Nanocrystals
Anamount of 20 mg of amino-functionalized CCNs was dispersed in 3 mL of distilled water and 5 mL of a FITC solution, prepared in absolute ethanol at a concentration of 0.3 mg/mL, and kept under stirring for 16 h at room temperature in a dark environment. After incubation, the obtained fluorescent nanocrystals were rinsed three times with ethanol and twice with deionized (DI) water to remove the physically adsorbed FITC molecules. Finally, the FITC-labeled CaCO3
nanocrystals were repeatedly washed with DI water, dried under vacuum and kept for application. The FITC labeled particles were used for fluorescence microscope observations and flow cytofluorimetry [57
2.2.4. Adsorption of HSA on CaCO3 nanocrystals
A weighted amount mass (25 mg) of CCNs (naked CaCO3, CaCO3-NH2, or CaCO3-NH2-FITC) was suspended in 5 mL of a 20 mg/mL solution of HSA in 10 mM phosphate buffer saline (PBS) solution (pH = 7.4 and 0.15 M NaCl) and left under stirring for 1 h at 37 °C. The powder was centrifuged, washed with PBS for three times and dried under vacuum at room temperature overnight. The HSA-loaded materials were characterized by DSC and light scattering.
2.3. Material Characterization
TEM images were collected with JEOL JEM 1400 with a LaB6 source at 120 kV for naked CaCO3, and CaCO3-NH2, and 80 kV for HSA-modified CCNs. The zeta potential and the hydrodynamic diameter of the nanoparticles were measured with a Malvern Zetasizer Nano ZS.
For fluorescent samples UV–Vis absorption spectra were obtained on a Varian-Cary 500 spectrophotometer (Agilent, Santa Clara, CA, USA). The excited fluorescence spectra measurements were performed using a Varian Cary Eclipse spectrofluorimeter. Absorption and fluorescence spectra were measured in DI water, with a concentration of 0.1 mg/mL. The quartz cuvettes used were of 1 cm path length.
In order to study cellular localization Zeiss LSM 700 confocal laser scanning microscope (Zeiss, Germany) was used. A 64× oil-immersion objective was used for the fluorescence measurements. The fluorescent samples were excited with a 0.5 mW laser of λ = 488 nm. The fluorescence intensity of samples was calculated from the microscopic images by the software of Zen 2009.
2.3.1. Fluorescence Stability of FITC-Coupled Nanocrystals
Procedure (1): stability in physiological solution (PS). FITC-coupled nanoparticles (1.0 mg/mL) were dispersed in PBS (pH = 7.4). The samples were incubated at 37 °C under dark. At fixed time intervals, a 0.5 mL amount of solution with the suspended nanoparticles was sampled. The supernatant solution was collected after centrifugation to analyze the amount of cleaved FITC by UV−vis (λem=493 nm). To avoid the effect of FITC protonation state on the extinction coefficient, the supernatant was diluted 1:5 with PBS before the measurement. The quantification of FITC detachment was based on the Beer−Lambert law in which the ε of FITC in PS was 80,300 M−1·cm−1.
Procedure (2): photostability tests. Photobleaching experiments were also conducted with a Zeiss LSM 700 microscope. FITC-coupled CCNs (1.0 mg/mL) were dispersed in HEPES buffer solution (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). A selected area was excited continuously with a 0.5 mW/488 nm laser. The intensity of the fluorescence was simultaneously recorded at 2.0 s intervals by the fluorescence microscope system. As the control sample, the corresponding photobleaching test of pure FITC in HEPES was also carried out with the same procedure. The measurement of each sample was repeated three times with the same parameters. To present the results with standard error marks more illustratively, the results of fluorescent intensity after bleaching for 5, 10, 15, 20, and 30 min were adopted.
2.3.2. Evaluation of HSA Adsorbed on CaCO3 Nanocrystals
After the adsorption protocol described in the previous section, nanocrystals powders were washed with DI water for three times. The residual concentration of HSA in the supernatant was determined spectrophotometrically (λ = 562 nm) by using the bicinchoninic acid (BCA) assay. This method, based on the formation of a violet complex between Cu+
ions and the protein, is highly sensitive and suitable for the determination of a wide range of different proteins [58
]. The amount of HSA adsorbed was calculated as a difference from the starting concentration. The results are reported as the mean value of three replicates, assuming the standard deviation as evaluation of the error.
2.3.3. TGA/DSC Measurements
Thermogravimetric analyses (TGA) and differential scanning calorimetry (DSC) measurements were carried out on a Q600 apparatus (TA Instruments, New Castle, DE, US). The measurements were performed under a nitrogen flow (100 mL·min−1
) with a heating rate of 10 °C/min [59
2.4. Internalization Assay
2.4.1. Cell Culture
Human breast cancer (MCF7), colon carcinoma cell line (Caco-2) and cervical cancer cell lines (HeLa) were cultured in Dulbecco’s modified eagle medium (DMEM; Sigma Aldrich, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich, Darmstadt, Germany), 1% glutamine, and 1% penicillin/streptomycin (Invitrogen, Carlsbad, California, USA) in a humidified incubator at 37 °C and 5% CO2 and 95% relative humidity.
2.4.2. Cell Proliferation Assay
MCF7, Caco-2 and Hela cells were treated with naked CCNs, CCNs-NH2-HSA and CCNs-HSA at a concentration of 100 µg mL−1 for 24 h. Cell proliferation was evaluated by MTT assay, which measures the conversion of a tetrazolium compound into formazan by a mitochondrial dehydrogenase enzyme in live cells. Briefly, 15 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well for 3 h at 37 °C. A solubilizing solution was added for 1 h, and the absorbance was then measured at 570 nm using a spectrophotometer. Each data point is the average of three independent determinations.
2.4.3. Flow Cytometry
Uptake kinetics. All cell lines at 2 × 105 cells were seeded in 6-well plates (2 mL per well) in DMEM with 10% FBS and cultured overnight. The following day, cells were washed twice with PBS (1 mL) and kept in DMEM without FBS for 2 h. Then, 100 µg of CCNs diluted in culture medium were incubated with cells for different times, from 0.5 to 24 h at 37 °C and 5% CO2. After the incubation period, cells were washed twice with PBS, detached with 0.08% w/v trypsin, washed twice again in order to perform analysis on an Attune Acoustic Focusing Cytometer (Thermo Fisher Scientific) equipped with a 488 nm laser. The BL1 (530/30nm) detector was used for detection of green fluorescence from FITC.
ROS production. All cell lines at 2 × 105 cells were seeded in 6-well plates (2 mL per well) in DMEM with 10% FBS and cultured overnight. The following day, cells were washed twice with PBS (1 mL) and kept in DMEM without FBS for 2 h. Then, 100 µg of CCNs diluted in culture medium were incubated with cells for 24 h at 37 °C and 5% CO2. After incubation period, cells were washed twice with PBS, and stained with a 10 µM dichorofluorescein (DCFA-DA) solution for 30 min and analyzed on a flow cytometer. As control cells were stained with propidium iodide (PI) 46 µM and a BL2 (575/24 nm) detector was used for detection of red fluorescence.
Confocal Microscopy. All cell lines were seeded on round coverslips at 6 × 104 cells per slide one day prior to the assay. Particles were diluted prior to the assay as described for flow cytometry. Cells were washed with PBS, and 100 µg of CCNs, diluted in culture medium, were incubated with cells for 6 h at 37 °C and 5% CO2.
After incubation, cells were washed with PBS, fixed in 0.25% glutaraldehyde in PBS for 10 min, and permeabilized with Triton X-100 (0.1% in PBS). The cells were then washed with PBS solution for 5 min and subsequently stained with 1 μg/mL of Hoechst 33342 for 5 min at room temperature.
Imaging tests were carried out with a Zeiss LSM700 (Zeiss, Germany) confocal microscope equipped with an Axio Observer Z1 (Zeiss, Goettingen, Germany) inverted microscope using a suitable oil-immersion objective (63X magnification, with 1.46 NA). Laser beams at λ = 405 nm and λ = 488 nm excitation wavelengths were used for imaging the nuclei and the fluorescent nanocrystals, respectively.
In order to improve targeting efficiency and limit the systemic toxicity effects of CCNs, we studied methods to graft HSA on nanocrystals and obtain a pure protein corona. Two pathways were compared: a simpler, direct method (using unmodified environmentally friendly CaCO3), and a surface functionalization with amino groups (using (3-aminopropyl)triethoxysilane to modulate the surface behavior of the nanocrystals). DLS, TEM, and TGA-DSC analysis were used to confirm the functionalization of CCNs and study the conjugates. HSA coating reduced the hydrodynamic diameter and prevented aggregation or agglomeration of the nanocrystals.
The amino groups proved essential to obtain the best bio-functionalization of CCNs with HSA, in comparison with pristine CCNs and HSA, as measured by the bicinchocinic assay. In fact, a larger amount of protein was observed in the amino-functionalized sample (75.8% bio-conjugation efficiency) with respect to the pristine CCNs (43.2% bio-conjugation efficiency). DSC confirmed the presence of a specific thermal event associated with the presence of HSA in the samples. Confocal microscopy on FITC-labeled samples confirmed that three human cancer cell lines MCF-7 (breast cancer), Caco-2 (epithelial colorectal adenocarcinoma), and HeLa (cervical cancer), efficiently internalize CCNs−NH2-FITC-HSA and CCNs-HSA-FITC. Moreover, while the fluorescence of non-HSA-functionalized samples was dispersed in the cytoplasm, z-stack sections confirmed the localization of the conjugates in the perinuclear region.
These results suggest that the CCNs-HSA conjugates show an improved uptake ability in cancer cell lines with respect to naked CCNs. The internalization studies verify that the endocytosis patterns depend on chemical functionalization and bioconjugation. The surface charge and the bio-functionalization affect the efficiency and the pathway of cellular uptake.
All these results indicate that CCNs-HSA conjugates deserve further insights as targeted nanocarriers of anticancer compounds.