| Objective:The objective of this study is to develop a breast cancer cell targeted nanoparticle delivery system for TAM using a novel polymer material, genistein modified chitosan(GEN-CS) as materials, TAM as model drug, sodium tripolyphosphate(TPP) as the coagulant. Genistein, acting as a targeted therapeutic compound, competes with estradiol or TAM selectively binding to GPR30. Ionic crosslinking method is used to construct TAM nanoparticles. Further the morphology of nanoparticles, particle size, Zeta potential, drug loadings and the envelopment rate and the in vitro release are evaluated. Cells uptake of nanoparticles are observed by the Laser confocal microscope. MTT method is used to compare drug-loaded nanoparticles with free drugs to MCF-7 cells proliferation inhibition.Methods:Genistein(GEN) and chitosan(CS) were coupled by the double carboxyl of succinic acid through the catalytic reaction of 1-(3-dimethyl amino propyl)-3-ethyl carbodiimide/N-hydroxy succinimide(EDC/NHS), it got carrier material, genistein modified chitosan(GEN-CS). The material was characterized by FT-IR and other technology.Through ionotropic gelation method which combined amino on chitosan and sodium tripolyphosphate(TPP) to form nanoparticles under suitable conditions, wraped TAM in nanoparticles.The nanoparticles were characterized by transmission electron microscopy(TEM), Melvin ZS90 granularity instrumen. HPLC method was developed for the determination of drug loadings and envelopment rate of nanoparticles. The constant temperature water bath oscillation method examined drug release of nanoparticles in vitro.FITC labeled TAM chitosan nanoparticles(TAM-CS-NPs) and TAM-GEN-CS-NPs were prepared. The uptake and distribution of different nanoparticles in the MCF-7 cell were carried out using laser confocal microscope.MCF-7 cells were cultured in vitro, then the cytotoxicities in vitro of free TAM,TAM-GEN-CS-NPs suspension, GEN-CS-NPs suspension were determined by MTT assay.And when cell growth inhibitory rate was 50%, the required drug concentration was calculated, namely IC50.Results:The percentage of GEN group in GEN-CS carrier material was 1.16%, the characteristic peaks of GEN-CS were observed through FT-IR. Contrast infrared spectra of GEN-CS with infrared spectra of CS, found that 1600 cm-1、1456 cm-1as the characteristic absorption peak of benzene ring, 1668 cm-1 for the amide I band, these confirmed GEN grafted on CS successfully.The nanoparticles were round or oval in shape by Transmission electron microscope(TEM). Their average particle size was 299.8 nm, PDI was 0.181, Zeta potential was 27.6m V, drug loading was 14.02, envelopment rate was 85.77%. Compared with TAM,TAM-GEN-CS-NPs had obvious sustained release performance.MCF-7 cell and FITC labeled CS-NPs and GEN-CS-NPs were incubated jointly for 4h(37℃), the uptake of FITC GEN-CS NPs by MCF-7 cells was obviously higher than FITC CS-NPs under laser confocal microscopy.The variance of factorial design analysis showed that there were interaction effects between drug factors and concentration factors(F = 31.04, P = 31.04), different groups on tumor cell proliferation inhibition rates were drug concentration-dependented. Under the same condition, the strongest MCF-7 cell proliferation inhibition was induced by TAM-GEN-CS-NPs, but the weakest was induced by GEN-CS-NPs. It had a significant difference(P<0.05). The IC50 of TAM, TAM-GEN-CS-NPs were 10.25 μg/m L, 7.22μg/m L respectively.Conclusions:Genistein modified chitosan(GEN-CS) as the carrier, the nanoparticles were formed through ionotropic gelation method. Its shape was round, size distribution was uniform,drug loading was high and slow-release performance was good, targeted to the MCF-7cells. Its inhibition to MCF-7 cells was strong. TAM-GEN-CS-NPs are expected to become an promising targeting drug carrier for breast cancer. |
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