Pharmaceutical NanotechnologyUptake of folate-conjugated albumin nanoparticles to the SKOV3 cells
Introduction
Site-specific delivery of drug receives a lot of attention because it can reduce drug toxicity and increase therapeutic effects (Allen et al., 1995). Nanoparticles have been considered as effective delivery systems for many reasons including: (i) sufficient physical and biological stability that may facilitate drug entrapment and controlled release (Yang et al., 1999, Maia et al., 2000), (ii) good tolerability of the components (Maia et al., 2000), (iii) simplicity of the formulation processing (Oyewumi and Mumper, 2002) and (iv) possibility of scaling up the formulation process (Muller et al., 2000). Therefore, nanoparticles have been extensively employed to deliver drugs, genes, vaccines and diagnostics into specific cells/tissues (Coester et al., 2000, Jenning et al., 2000, Stella et al., 2000).
Among these colloidal systems those based on proteins may be of promising because of their biodegradability, lack of toxicity and antigenicity, stability, shelf life, controllable drug-release properties and high loading capacity for hydrophilic molecules (Rubino et al., 1993).
To solve the problem of site-specific targeting for the colloidal systems, some authors have attempted to increase the tissue specificity of colloidal drug carriers by coupling targeting agents. Among the possible targeting agents, folic acid could be exploited to realize delivering drugs into cancer cells. Folic acid is a low molecular weight (441 Da) vitamin whose receptor is frequently overexpressed in human cancer cells (Weitman et al., 1992, Franklin et al., 1994). This receptor has been identified as a tumor marker, especially in ovarian carcinomas (Campbell et al., 1991), and it is highly restricted in most normal tissues. Folic acid presents advantages as a targeting device to the tumor cells as follows. First, it is stable, inexpensive, and nonimmunogenic compared with proteins such as monoclonal antibodies. Second, folic acid binds to the folate receptors at cell surfaces with very high affinity (Kd = ∼1 nM) and is internalized by receptor-mediated endocytosis (Kamen and Caston, 1986, Lee and Low, 1994, Lee and Low, 1995, Lee and Huang, 1996, Atkinson et al., 2001). The feasibility of tumor-targeting via folate receptors has been reported (Lee and Low, 1995). It has also been shown that folate-conjugated macromolecules can be specifically taken up by the tumor cells (Lee and Low, 1995, Lee and Huang, 1996, Stella et al., 2000, Oyewumi and Mumper, 2003).
In this study, bovine serum albumin nanoparticles (BSANPs) were prepared and conjugated with folic acid via amino groups. Furthermore, uptake of folate-conjugated albumin nanoparticles in SKOV3 cells, human ovarian cancer cells, was examined.
Section snippets
Materials
Bovine serum albumin (BSA), folic acid, dicyclohexylcarbodiimide (DCC), fluorescein isothiocyanate (FITC), 2,4,6-trinitrobenzenesulfonic acid (TNBS) and N-hydroxysuccinimide were all purchased from Sigma. SKOV3, a human ovarian cancer cell line, was a gift from immunity laboratory of Sichuan University. RPMI-1640 medium without folic acid was obtained from Gibco. All other reagents were of analytical grade.
Preparation of albumin nanoparticles
The albumin nanoparticles were prepared by a desolvation technique. BSA (10 mg) was
Cell culture
Cell uptake studies were performed using SKOV3 cells, a human ovarian cancer cell line. The cells were cultured in T-75 flasks at 37 °C in a humidified atmosphere containing 5% CO2 using folate-free RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum. The only source of folic acid in the media was due to the presence of the 10% fetal calf serum.
Cell uptake studies
BSA was labeled with FITC to investigate uptake of nanoparticles to SKOV3 cancer cells according to the method by Leamon and Low (1991). After the labeling reaction, the BSA-FITC conjugates were purified from unreacted FITC with Sephadex G-50 column and nanoparticles were prepared using the BSA-FITC conjugates following the process described in the preparation of BSANPs.
FITC-labeled folate-conjugated BSANPs or BSANPs suspensions in folate-free RPMI-1640 were added to monolayers of SKOV3 cells
Preparation and purification of folate-conjugated BSANP
Folic acids were covalently linked to BSANPs by the method described above. To activate the folate carboxylic groups for coupling with amino groups of the BSANPs, the NHS-ester of folic acid was prepared. Then, the NHS-folate was added to the BSANPs suspension. The reaction of NHS-folate with amino groups of BSANPs led to formation of an amide bond. The folate-conjugated nanoparticles were then purified from unreacted folic acid by a gel chromatography, using bicarbonate buffer as an eluent (
Conclusions
In this study, the folate-conjugated BSANPs were prepared and characterized. Furthermore, folate-conjugated BSANPs were taken up to human ovarian cancer cells (SKOV3). The level of uptake of folate-conjugated BSANPs increased with a saturation manner. The uptake of folate-conjugated BSANPs to SKOV3 cells could be inhibited by folic acid, suggesting that the internalization could be mediated by the folate receptor.
The folate-conjugated BSANPs represent a new potential delivery system for
Acknowledgement
This project was supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20020610092).
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