Studies On The Tumor Targeting Of RGD Peptide Modified Stearic Acid-g-Chitosan Oligosaccharide Polymeric Micelles

Amphiphilic block copolymer could self-aggregate into spherical micelles in an aqueous medium, which emerged as a potential tumor targeting drug delivery system. PEGylation of micelles has been proven to be an effective approach for extending circulation in the blood stream and accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect, which termed as tumor passive targeting. The active targeting to tumor cells of micelles can be reached by further ligand or antibody modification.Stearic acid grafted chitosan oligosaccharide copolymer (CSO-SA) was synthesized by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction of amino groups of low-molecular-weight chitosan and carboxyl group of stearic acid (SA).In order to reduce the uptake of CSO-SA micelles by macrophage and prolong the cirulation time in plasma, polyethylene glycol (PEG) was grafted onto CSO-SA, the PEG modified CSO-SA (PEG-CSO-SA) was obtained. To reach the active targeting of micelles, RGD peptide was also coupled to PEG-CSO-SA, the RGD modified PEG-CSO-SA (RGD-PEG-CSO-SA) was obtained. Substitute degrees of amino groups (SD%) of micelles, defined as the graft number of SA groups per 100 amino groups of CSO, was measured by TNBS method. The critical micelle concentration (CMC) of copolymers in water was determined using pyrene as fluorescent probe. Size and zeta potencial of copolymers in water were determined using ZETASIZER (3000HS, Malvern Co., UK).The BEL-7402 (avβ3 integrin over-expressing) and Hela (not over-express avβ3 integrin) were chosen as model tumor cells. The cell viabilities of blank micelles against the test cells were evaluated using the MTT test. The cellular uptakes of fluorescein isothiocyanate (FITC) labeled micelles were investigated. Using doxorubicin (DOX) as a model drug, and the DOX-loaded micelles were prepared by dialysis method. The size and zeta potential of DOX-loaded micelles were determined by Zetasizer(3000HS, Malvern Co., UK). The doxorubicin content was measured by fluorescence spectrophotometer. The drug encapsulation efficiency (EE) and drug loading (DL) of DOX-loaded micelles were measured by centrifugal-ultrafiltration method. Phosphate-buffered saline (PBS), pH 7.4, was used as the dissolution medium for the in vitro DOX release from the micelles. A comparison of cytotoxicity of DOX-loaded micelles and free DOX against the test cells were determined by MTT method. To study the difference in uptake of DOX-loaded micelles, qualitative and quantitative analysis of cellular uptake were conducted, by using free DOX as control. To further elucidate the role of RGD peptide in the cellular uptake of RGD-modified micelles, competitive cellular uptake test was conducted. BEL-7402 cells were pretreated with free RGD peptide to block the integrin, and then quantitative analysis of cellular uptake was conductedIn this study, CSO-SA was synthesized by the coupling reaction of SA and CSO. The substitute degrees of amino groups (SD%) was determined as 19.6±0.60%, and the CMC value was determined to be 25.0±0.325.0±0.3μg/ml in DI water. The CMC value increased after the modification of PEG and RGD peptide, varying from 20.0μg/ml to 50.0μg/ml. It was found that the size of micelles increased after the modification of PEG and RGD peptide, while the zeta potential decreased. Using MTT method, the IC50 values of PEG-CSO-SA and RGD-PEG-CSO-SA micelles were vary from 290μg/ml to 500μg/ml, indicating the micelles can be used as a safe drug carrier. The cellular uptake of FITC labeled micelles showed that the modification of RGD peptide could significantly increased the uptake of micelles in BEL-7402 cells, while the cellular uptake of micelles were almost same in Hela cells.The doxorubicin loaded micelles were prepared and the properties were characterized. The drug encapsulation efficiency (EE) was above 90%, and the in vitro drug release profiles from the micelles suggested that the micelles could be used as a controlled release carrier for the poorly soluble drug. The inhibitor effects of DOX-loaded PEG-CSO-SA and RGD-PEG-CSO-SA micelles indicated that the cytotoxicity of DOX against the BEL-7402 cells could be improved significantly via the modification of RGD peptide, while the cytotoxicity of micelles did not changed obviously against the Hela cells. Qualitavie and Quantitative analysis of cellular uptake indicated that RGD-modifided micelles could significantly enhanced the DOX concentration in BEL-7402 cells, while did not affect the cellular uptake of micelles in Hela cells. The competitive cellular uptake test showed that the cellular uptake of RGD-modified micelles by BEL-7402 cells was significantly inhibited in the presence of excess free RGD peptide. The free RGD peptide could prevented the cellular uptake of the RGD-modified micelles by competitive binding to integrin on the cell surface of BEL-7402 cells. Overall, these results demonstrate that integrin is responsible for the binding and uptake of RGD-modified micelles in integrin over-expressing BEL-7402 cells, and the cell uptake was governed by a receptor-mediated process.