Functionalization of MWCNTs with HSA
In order to obtain a directly targeted delivery of MWCNTs into the cancer cells and to visualize and detect the localization of the nanotubes inside the cell, the FITC–HSA system was preformed and noncovalently labeled on the oxidized surface of MWCNTs.
To provide clues regarding the success of noncovalent HSA–MWCNT functionalization, confocal microscopy was proposed for the identification of FITC-labeled CNTs in solution. As shown in , globular green CNTs corresponding to large molecules of fluorescent albumin were observed.
Figure 1 A) Illustration of the covalent labeling of HSA with FITC. B) The formation of oxidized MWCNTs–HSA–FITC. C) A typical fluorescent image of HSA–MWCNTs (100 mg/L): globular fluorescent CNTs corresponding to attached large molecules (more ...)
The oxidation of the nanotubes using a 3:1 (v/v) mixture of concentrated sulfuric and nitric acid gave them hydrophilicity and stability in aqueous systems due to the formation of –COOH, OH groups at the end and along the sidewalls of the tubes.21
FTIR spectra from confirm successful oxidation. Comparing the FTIR spectra of pristine MWCNTs (black) with those of oxidized MWCNTs (red), the characteristic bands of the oxygen-containing groups appear at 3422 cm−1
, corresponding to the stretching vibration of O–H and water,33
a band at 1721 cm−1
, corresponding to the carbonyl and carboxyl C=O stretching vibration, at 1582 and 1380 cm−1
, corresponding to the O–H deformation vibration, and the band at 1117 cm−1
, corresponding to the C–O stretching vibration. The band at 620 cm−1
corresponds to the CO out-of-plane deformation.34
Figure 2 FTIR spectra of A) pristine MWCNTs (black) and oxidized MWCNTs (red); B) HSA–FITC (black) and HSA–FITC-coated oxidized MWCNTs (red); C) UV–Vis adsorption spectra of HSA–FITC (black), oxidized MWCNTs (blue), oxidized MWCNTs–HSA–FITC (more ...)
Further, we conjugated the HSA–FITC system noncovalently on the surface of oxidized MWCNTs. First, we covalently labeled HSA with FITC at an increased pH (above pH = 9), as shown schematically in .35
FITC covalently attached to the protein through the alpha-amino group. Second, HSA– FITC complex was adsorbed on the nanotubes, presumptively, through electrostatic interactions between the functional groups of MWCNTs and the protein-positive domains (). Considering the fact that not all the surface of the nanotubes is oxidized, hydrophobic interactions can also occur.36
UV–Vis spectroscopy is a simple but efficacious method that confirms the formation of the oxidized MWCNT–HSA– FITC complex. The nanotubes solutions give an adsorption band at 295.7 cm−1
, which corresponds to the +-plasmon transition of MWCNT.37
The yellowish HSA–FITC solution has the characteristic adsorption band at 489 cm−1 and a second adsorption band at 292 cm−1, suggesting the existence of aromatic amino acids from HSA. Comparing the aforementioned spectra, the formation of the MWCNTs–HSA–FITC complex becomes obvious due to the appearance of the oxidized MWNT band and the HSA–FITC band at 475.6 cm−1, which is shifted and has low intensity ().
The conjugation of HSA–FITC onto the surface of the nanotubes is also confirmed by FTIR spectroscopy as seen in . No similarity can be observed when comparing the spectra of HSA–FITC with those of the nanotube-conjugated HSA–FITC. All the corresponding peaks had shifted their position, and some even disappeared. In the higher region, the stretching vibration band of the N–H groups at 3409 cm−1 changed their shape in a broad band that included two peaks: one at 3389 cm−1 (N–H groups stretching vibration) and the second at 3303 cm−1, which is the pyridine aromatic C–H vibrations band. The aliphatic C–H stretching vibration at 2929 and 2873 cm−1 moved at 2922 and 2865 cm−1, such that these groups were involved in electrostatic bonds. In addition, the amide I and II are shifted to low frequency: amide I, from 1656 to 1649 cm−1; amide II, from 1544 to 1532 cm−1. The asymmetric and symmetric deformations of CH3 have changed their bands from 1459 to 1447 cm−1 and 1416–1389 cm−1, respectively. The region in between has dramatically changed their intensity. This is due to the spontaneous adsorption of the crystalline HSA–FITC complex on the MWCNTs and the formation of a well-organized oxidized MWCNT–HSA–FITC.
To that end, atomic force microscopy (AFM) analysis of the HSA–MWCNTs solution was performed. Representative AFM evidence of the successful attachment of HSA molecules onto the surface of the nanotubes is shown in . By AFM, analysis at the nanometric scale of the two HSA molecules (black arrows in ) attached at the end of the nanotubes (white arrows) was carried out. A single HSA molecule (red arrow) has also been observed in the topographic image shown here. The length of the CNTs was estimated as being <200 nm. The lateral resolution of an AFM image is determined by the tip of the object that is imaged. In the presented image, the width of the nanotube appears to be >2 nm, as we used an AFM tip with a ~15 nm radius of curvature.
The ability of an FITC-labeled bioconjugate of HSA– MWCNTs to internalize inside an HepG2 cell was evaluated by confocal fluorescence microscopy imaging. The results presented in show that at low concentration and short exposure time, HSA–MWCNT accumulates inside HepG2 cells. Thus, we provided imaging evidence that HSA can act as a carrier for MWCNTs, and because we were unable to identify any fluorescence in the epithelial cells in similar conditions () we reasoned that HSA–MWCNT bioconjugates exhibit specific affinity for liver cancer cells.
Figure 3 Selective nanophotothermolysis of HepG2 cells. A) Confocal image of human hepatocytes incubated for 30 min with 5 mg/L FITC–HSA–MWCNTs. (The nucleus was stained with DRAQ5-red.) B) Confocal detection of MWCNT–HSA–FITC (green) (more ...)
Furthermore, phase contrast microscopy was used to demonstrate the presence of CNTs inside HepG2 cells following HSA–MWCNT administration. As seen in (red arrows), intracellular aggregates of MWCNTs appear as dark, optically dense signals that associate with a refringent signal under phase contrast. Once more, we were unable to identify any aggregates inside the epithelial cells that have been similarly treated. () Moreover, the cellular areas that appeared to contain MWCNTs were further subjected to TEM analysis. When these regions were observed under TEM, MWCNTs could be clearly identified in the form of intracellular aggregates, as shown by the red arrows in .
The mechanism of selective internalization of HSA–MWCNTs inside the malignant liver cells
In order to shed light on the molecular mechanisms involved in the specific uptake of HSA–MWCNTs in HepG2 cells, we investigated the possibility that a 60 kDa glycoprotein, Gp60, which is known to function in albumin transcytosis in malignant cells,38
was involved in the selective uptake of albumin bound to CNTs. To accomplish this, we allowed the cells treated with 5 mg/L HSA–MWCNTs for 1 h to incorporate cy3–anti-Gp60 Ab for 30 min at 37°C. To that end, we obtained fluorescent images demonstrating the internalized cy3 fluorescence (, first panel).
Figure 4 HSA–MWCNTs in vitro endocytosis mechanism in human liver cancer cells. A) Colocalization of Cy-Gp60 antibody and FITC–HSA–MWCNTs in HepG2 cells. B) Colocalization of Cy-Gp60 antibody and FITC–HSA–MWCNTs in hepatocyte (more ...)
Also, we showed that HepG2 cells internalized with albumin-bound MWCNTs (fluorescently labeled with FITC) were distributed into the punctate structure inside the cells (, 2nd panel). DAPI, which is known to form fluorescent complexes with natural double-stranded DNA, was used for nuclei staining. In , fourth panel, nearly complete colocalization of the FITC fluorescence (green image) and cy3 fluorescence (red image) was evident by yellow in the merged image. This finding suggests that albumin bound to MWCNTs was incorporated into plasmalemmal vesicles containing Gp60 as a membrane protein, further validating HSA–MWCNT specificity for Gp60 receptors. Importantly, as seen in , no significant colocalization in the hepatocyte cells (CRL-4020) was observed for cy3–Gp60 Ab and HSA–FITC–MWCNTs incubated under same circumstances.
Therefore, based on these data, we showed that HSA– MWCNTs can act as specific and sensitive site-targeted nanosystems against Gp60 receptor located on the liver cancer cell membrane.
Association of caveolin-1 with FITC–HSA–MWCNTs-containing vesicles
Most data indicate that caveolae-mediated endocytosis in cells is stimulated by the binding of albumin to Gp60, a receptor located in the caveolae.38
Given these data and the described role of caveolin in albumin endocytosis, we reasoned that the mechanism of HSA–MWCNT internalization in HepG2 cells was similar. To test this hypothesis, we immunostained the HepG2 cells with Cy3–anti-caveolin-1 Ab. As shown in , confocal imaging revealed that the majority of FITC–HSA–MWCNT-containing plasmalemmal vesicles stained for caveolin-1 used this fluorescent anti-caveolin-1 monoclonal Ab. Taken together, all these data demonstrate that HSA–MWCNTs selectively internalize in human hepatocellular cancer cells via caveolae-mediated endocytosis by the binding of the albumin carrier to Gp60, a specific albumin-binding protein.
Cytotoxicity induced by laser irradiation or by the administration of HSA–MWCNTs
Before testing the in vitro response of HSA–MWCNT-treated cells to laser irradiation, we investigated the possible effect of cytotoxicity induced by the administration of CNTs in the cells. HepG2 cells and the epithelial cells were treated with various concentrations of HSA–MWCNT at various incubation periods. Cell Death Detection ELISAPLUS was used to evaluate the effect of MWCNT bioconjugates on cell viability.
After 24 h of incubation, HepG2 exposed to 50 mg/L of HSA–MWCNT showed a 5.71% decrease in viability compared with 1.6% (P < 0.02) (). For human hepatocytes exposed to 50 mg/L of HSA–MWCNT, the decrease in viability was 6.23% compared with the nontreated sample, in which the percentage of viable cells was 98.7% (P < 0.001). The statistical data showed that nanomaterial exposure per se induced no significant cytotoxic effects at small and medium concentrations (P > 0.05 for all comparisons).
Cytotoxic-induced effects on HepG2 and CRL-4020 cells by various concentrations of bionanomaterial at various incubation times
The next step in order to eliminate any potential errors was represented by a 2 minutes irradiation of a sample of cells without nanoparticles, using a 2 W, 808 nm laser beam. There was no lysis among the cells after irradiation. The process demonstrates the transparency of HepG2 for NIR beam.
Assessment of cellular necrosis after laser treatment and administration of HSA–MWCNTs
The postirradiation lysis rate of HepG2 cells treated with HSA–MWCNTs ranged from 35.45% (for 1 mg/L) to 88.24% (for 50 mg/L) at 60 sec (P < 0.001), whereas at 30 min the necrotic rate increased from 59.34% (1 mg/L) to 92.34% (50 mg/L), P value <0.001. Significantly lower apoptotic rates were obtained in irradiated epithelial cells treated for 60 sec and 30 min at concentrations ranging from 1 mg/L to 50 mg/L (6.78%–64.32% for 60 sec; 9.89%–70.78% for 30 min). As can be observed, the optimal apoptotic effect of malignant cells after incubation with HSA–MWCNT was obtained at a concentration of 5 mg/L (HepG2/CRL-4020: 65.79%/11.34% at 60 sec, and 75.34%/14.67% at 30 min) (). After 60 min of incubation, the difference among the apoptotic rates was also statistically significant among the two cell lines for low/medium concentrations of HSA– MWCNT (78.92%: 1 mg/L, 88.34%: 5 mg/L, 87.88%: 20 mg/L, for HepG2; 15.56%: 1 mg/L, 21.34%: 5 mg/L, 52.14%: 20 mg/L, for CRL-4020). P values were <0.001 for comparisons between various forms of nanomaterials. No significant differences (P = 0.143) among the apoptotic rates of HepG2 and CRL-4020 treated with HSA–MWCNT could be observed (100%: HepG2; 84.13%: CRL-4020) for a high concentration of nanomaterials (50 mg/L).
Results of experimental seriate exposure to nanomaterials (control vs MWCNTs–HSA) in different concentrations, followed by laser irradiation. Bars represent the average percentage of dead cells (%).
After 3–5 h of incubation, a significant apoptotic rate of the two cell lines was obtained only when the cells were treated with low concentrations of nanomaterials (<20 mg/L). Elevated concentrations recorded a nonsignificant difference in the cell lysis effect of the two cell lines (P = 0.256–20 mg/L; P = 0.296–50 mg/L).
After 24 h of incubation, the HepG2 cells treated with 1 mg/L HSA–MWCNT were 100% necrotic after laser irradiation, as compared with 52.2% of the CRL-4020 cells similarly treated. For very low concentrations of HSA–MWCNTs, we could observe a difference among the percentage of dead cells of the two cell lines. However, the difference reached only a marginal significance (P = 0.07). The lysis rate of the irradiated cells incubated with more than 5 mg/L nanomaterials for 24 h was almost similar for the two cell lines (100% vs 85.94%).
In contrast, no significant differences in the percentage of nonviable cells were obtained between the two cell lines when the nonfunctionalized MWCNT solution was used for treatment (P > 0.05 for all comparison and each exposure interval). Moreover, for HepG2 cells, the results showed a significant difference between MWCNTs and MWCNT– HSA-exposed groups for low concentrations (1, 5, and 20 mg/L) and short exposures (60 sec, 30 min, 1 h, 3 h, and 5 h) ().