Hepatocellular carcinoma (HCC) is a public health concern because HCC is fifth in the occurrence rate for all cancers, and second only to lung cancers in mortality rate [1
]. HCC incidence continues to increase with approximately 700,000 cases per year worldwide. The issue of greatest concern is the poor survival rate [3
]. HCC is usually diagnosed in the advanced stages when prognosis for the disease recovery is meager, and survival is one to two months [5
]. Until now, conventional treatment has been unsatisfactory for patients in this stage. Therefore, the development of effective therapies is urgently needed [2
]. Recent reports suggest that targeting drugs to a specific tissue allows high concentrations of the drug within a tumor; this results in high efficacy and low toxicity in the organism [2
]. In this sense, nanovehicles targeted to tumor sites could potentially be used as anticancer therapies with greater safety and efficacy [1
In targeting drugs to a specific site, the selection of an appropriate surface receptor is crucial [1
]. Furthermore, if receptor-mediated endocytosis follows receptor binding, this entry method could be used to direct drug-containing nanovehicles to any type of target cell [8
]. The asialoglycoprotein receptor (ASGPR) is abundantly expressed on hepatocyte membranes (500,000 ASGPR/hepatocyte) and minimally expressed in extrahepatic tissues [4
]. In vivo
, the ASGPR recognizes and captures proteins and peptides that carry exposed galactose residues [4
] and the receptor cargo enters the cell via receptor-mediated endocytosis.
In addition, enhanced permeability of the tumor vasculature allows nanovehicles to move in the tumor, whereas the suppressed lymphatic filtration allows them to be retained [9
]. Hence, the nanoparticles retained in the tumor will interact with the ASGPR and initiate the process of endocytosis, achieving a targeted drug delivery.
The material of choice for the formation of nanostructures for human cell targeting is not a trivial issue, since it must comply with several safety requirements. Albumin is a globular protein that has been used as a therapeutic vehicle for anticancer drugs (such as doxorubicin) and that, due to effective permeability and retention (EPR), accumulates efficiently in tumors, improving the pharmacokinetic profile of anticancer agents [11
]. Abraxane® is an example of this type of drug; it is an albumin conjugate linked to paclitaxel that has been approved by the Food and Drug Administration (FDA) for the treatment of metastatic breast cancer [14
]. Thus, albumin is considered potentially useful for the synthesis of drug nanovehicles.
Previously, we reported the modification of bovine serum albumin (BSA) with lactose through thermal glycation. Lactose was coupled to the amino groups of lysine residues present in the BSA, while galactose remained unchanged. Using this method, we obtained lactosylated bovine serum albumin (BSA-Lac) as the product [15
]. Lactose has several advantages; it lacks immunogenicity, it is highly stable, and is easy to use for modification. Lactose is considered an excellent ligand for several artificial systems [1
] and a promising candidate for the nanovehicle targeting of drugs to the ASGPR [8
]. The aim of the present work was to synthesize BSA-Lac NPs and test their specific biorecognition by ASGPRs present on HepG2 cells, which is a line derived from an HCC.
3. Materials and Methods
All of the reagents used were analytical-grade and, unless specified, all of the other reagents and chemicals were purchased from Sigma-Aldrich. Bovine serum albumin (BSA; 66.5 kDa and ∼96%), D-lactose monohydrate (Lac), glutaraldehyde (25%), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S), fluorescein isothiocyanate (FITC), and [3-5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ricinus communis agglutinin I (RCA I) was obtained from the Vector Lab (Burlingame, CA, USA). HepG2 (human liver cancer) and HeLa (human cervical carcinoma) cells were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA).
3.2. Lactosylation of Albumin
The modification of albumin with lactose was carried out according to Sarabia-Sainz et al. (2011) [22
]. BSA-Lac samples were frozen at −40 °C and freeze-dried (Virtis Benchop 6.6, NY, USA), and subsequently incubated at 100 °C for 30 min. Finally, unreacted lactose was removed by dialysis. Thermally treated albumin without lactosylation was used as a negative control. Samples were freeze-dried and stored at −20 °C until later use.
3.3. Characterization of BSA-Lac
3.3.1. Enzyme-linked Lectin Recognition Assays (ELLA)
The biorecognition of galactose was evidenced by the interaction of BSA-Lac with biotin-labeled RCA I. Briefly, 5 μg/100 μL BSA-Lac and negative controls (BSA) and BSA thermally treated in the absence of lactose (tBSA) were immobilized in a 96-well plate. Following adsorption, wells were blocked for 3 h with 20 mM of phosphate-buffered saline (PBS) containing, 0.05% Tween 20, pH 7.5 (PBS-T), and 1.5% BSA, to prevent nonspecific interactions. Next, 100 μL of biotinylated RCA was added at a concentration of 2.5 μg/mL, and incubated at room temperature for 1.5 h. After washed with PBS-T, samples were incubated with 100 μL of streptavidin-peroxidase (1:2000) in PBS for 40 min. The color of the reaction was developed using SIGMA FAST OPD, following the manufacturer’s instructions. Absorbance at 450 nm was read in an ELISA reader (Anthos Zenyth 340st, Alcobendas, Spain) at 10 min [15
3.3.2. SDS-PAGE Electrophoresis
BSA-Lac was analyzed by electrophoresis under denaturing and reducing conditions using 8% polyacrylamide gels (SDS-PAGE) according to Laemmli (1970) [33
]. Controls included untreated BSA and tBSA. Each sample containing 5 µg of protein was loaded onto the gel and after running stained with 1% Coomassie blue. The relative molecular mass of the sample was estimated by comparison with molecular weight standards and documented using a Molecular Imager® Gel Doc™ XR+ System with Image Lab™ Software (Image Lab 3.0, BioRad, Hercules, CA, USA).
3.3.3. Lectin-blotting Assay
Lectin-blotting assay was performed as described by Lagarda-Díaz et al. (2009) [34
]. Proteins (BSA, tBSA, and BSA-Lac) previously separated by SDS-PAGE were transferred onto a nitrocellulose membrane at a rate of 0.8 mA/cm2 for 45 min, using a semi-dry blotter (LABCONCO, Kansas City, MO). Membranes were blocked for 1.5 h with PBS containing 2% BSA. BSA-Lac incubated with biotinylated RCA I (5 μg/mL for 3 h), followed by an incubation with streptavidin peroxidase (1: 2000) for 1.5 h. The color reaction was developed at room temperature by the addition of peroxidase substrate, 0.075% 3,3′-diaminobenzidine-4HCl (DAB).
3.3.4. Fourier-transform Infrared Spectroscopy
The presence of functional groups in BSA-Lac and their absence in negative controls (BSA and tBSA) was evaluated by Fourier transform infrared (FT-IR) spectroscopy [17
]. Infrared spectra were recorded using Agilent Cary 630 FTIR Spectrometer (Agilent, Cary 630 FTIR Spectrometer, Santa Clara, CA, USA) at a resolution of 4 cm−1 in the range of 650 to 4000 cm−1
3.4. Synthesis of BSA-Lac Nanoparticles (BSA-Lac NPs)
BSA-Lac NPs were prepared using a desolvation system to form nanoparticles as described by Gallegos-Tabanico et al. (2017) [14
] with minor modifications. Briefly, 10 mg of BSA-Lac was dissolved in 1 mL of deionized water. Ethanol (3 mL) was added slowly dropwise into the BSA-Lac solution under constant stirring. Then, 5 or 10 μL of glutaraldehyde (8%) was added to each sample to induce the crosslinking of the BSA-Lac molecules and give stability to the formed nanoparticles. The mixture was stirred for 5 h and the nanoparticles were then washed three times with deionized water and recovered by centrifugation (1644× g for 10 min). Thus, low-crosslinking (5 μL of glutaraldehyde) BSA-Lac nanoparticles (LC BSA-Lac NPs), high-crosslinking (10 μL of glutaraldehyde) BSA-Lac nanoparticles (HC BSA-Lac NPs) were obtained. Also, for synthesis control, LC tBSA NPs, and HC tBSA NPs were prepared.
3.5. Characterization of BSA-Lac NPs
3.5.1. Particle Size and Zeta Potential
The particle size (mean particle diameters and size distributions) and zeta potential of NPs were measured at 25 °C using dynamic light scattering (DLS) at a scattering angle of 90° with Zetasizer Nano ZS90 (Malvern Instruments Ltd, Malvern UK) with a doppler anemometry laser. Samples were diluted in PBS (1mg/mL) at pH 7.2. All of the measurements were done in triplicate.
3.5.2. Nanoparticle Morphology
The morphology of NPs was characterized by scanning electron microscopy (SEM, JEOL JSM-7800F, Akishima, Tokyo, Japan) and atomic force microscopy (AFM, XE-Bio system, Park Systems Corp, Su-won, Korea). SEM images were obtained with a magnification of 30,000× using an acceleration voltage of 5.0 kV. AFM images were reconstructed in the non-contact mode using Nanosensors: NCHR cantilevers (force constant 10 to 130 N/m). The analysis was performed using 5 × 5 μm scanning images. The three-dimensional (3D) images were analyzed with the software Gwyddion version 2.49 available online at http://gwyddion.net/
3.6. Cellular Uptake Evaluation and Specificity of Recognition
Prior to evaluate the cellular interaction, NPs were labeled with FITC according to Huang et al. (2017) [4
] with modification. Briefly, 0.5 mL of FITC (0.4 mg/mL DMSO) was mixed with 2 mL of NPs suspensión (5 mg). After 3 h of reaction in the dark at room temperature, the FITC-labeled NPs were washed using water and several centrifugation cycles (2422 ×g, 10 min at 25 °C). The conjugation of FITC with NPs was evaluated by the measurement of excitation and emission (λexc 480 and λemi 520 nm, respectively) spectra using a Fluorolog (Horiba JobinYvon, Palaiseau France) with the software FluorEssence 18.104.22.168.
NPs labeled with FITC were evaluated using Hep G2 (ASGPR+) cells and HeLa cells (ASGPR–) as control. Cells were seeded in 48-well plates at a density of 10,000 cells/200 µL per well using DMEM containing 10% FBS. After incubation (5% CO2 at 37 °C for 24 h), cells were washed three times with physiological saline solution (200 μL) and subsequently incubated with PBS containing BSA-Lac nanoparticles (LC BSA-Lac NPs, HC BSA-Lac NPs, LC tBSA NPs, or HC tBSA NPs) at a 10 μg/200 µL at 37 °C for 30 min. Cells were rinsed three times with physiological saline solution (200 μL), and then observed by confocal microscopy (the images dimensions were 1024 × 1024 pixels). For competition assays, cells were incubated simultaneously with NPs (10 μg/100 µL) plus or minus free lactose (10 μg/100 µL); for uptake inhibition assays, cells were preincubated with lactose (20μg/200µL) before the NPs (10 μg/200 µL) were added. The fluorescence intensity was quantified taking the cell body regions in the visual field that were selected as the regions of interest (ROI). The images and mean fluorescence intensity were obtained by confocal microscopy (Nikon TiEclipse C2+, Japan) with 488-nm lasers at 20x magnification.
3.7. Statistical Analysis
The results are presented as mean ± standard deviation. For ELLA, BSA, and BSA-Lac assay, the Student’s t-test was used. Statistical analysis of the fluorescence intensity of the cellular interaction was performed using a one-way ANOVA followed by the application of Tukey’s test. p ≤ 0.05 were considered statistically significant.