Preparation Of PH-sensitive Doxorubicin-folate Conjugates And Its Antitumor Activity In Vitro

Objective:Doxorubicin is a kind of the anthracycline antitumor antibiotic, it is used to treatsolid tumors such as breast cancer, ovarian cancer, lung cancer, thyroid cancer,liver cancer and so on. But its side effects remain as the problem, such as bonemarrow suppression, chronic cardimyopathy, and even cause heart failure. In ourresearch, a series of folate receptor-targeted doxorubicin conjugates weresynthesized according to the tumor microenvironment characteristics. The drugrelease characteristics and the anti-tumor activity of the conjugates were exploredby in vitro experiments. This research tries to give a new idea for the tumortargeted drug delivery system.Methods:The aminocaproic acid and heterobifunctional polyethylene glycol (MW=5000)were choosed as a drug carrier, and folic acid as targeting ligand. First，poly(ethylene glycol)-folic acid conjugate (PEG-FA), and amino caproic acid-folicacid conjugate (AMA-FA) were synthesised by amide bond, then DOX wasconnected to the PEG-FA and AMA-FA by pH-sensitive hydrazone bond to get the targeted product of DOX-hyd-PEG-FA and DOX-hyd-AMA-FA. Meanwhile,DOX-ami-PEG-FA, DOX-ami-PEG, DOX-hyd-PEG and DOX-hyd-AMA werepreparaed as control. The structure of conjugates was characterized by LC/MS,FT-IR and1H NMR spectroscopy. The drug loading of the conjugates wasdetected by HPLC method, and the drugs release characteristic was performancedin different pH media in vitro. The KB and HepG2cells which are folatereceptor-positive expression and A549cells which is folate receptor-negativeexpression were selected to study the cytotoxicity of the conjugates, meanwhilethe effect of exogenous folic acid on the cytotoxicity of the conjugates was alsoobserved. The uptake of DOX and its conjugates by tumor cells were analyzed byflow cytometry and fluorescence microscope. The intracellular distribution ofconjugates was studied by using laser scanning confocal microscope.Results:1. The structural characterization showed that the conjugates complied with thedesign requirements.2. The regression equation of standard curve to detect DOX wasy=47555x-19758， R2=0.9997. The drug loading of DOX-ami-PEG,DOX-ami-PEG-FA, DOX-hyd-PEG and DOX-hyd-PEG-FA was12.8%,13.2%,11.7%and12.6%, respectively.3. When DOX-hyd-PEG-FA was incubated in pH5.0PBS medium for50h,94%of conjugated DOX was released. However, at pH6.5and7.4, the release rateswere43%and11%in50h; DOX-hyd-AMA-FA had the same releasecharacteristics. When DOX-ami-PEG-FA was incubated in pH5.0for50h,12%of conjugated DOX was released; in pH6.5and7.4PBS medium, the release ratewas7%and5%in50h.4. The MTT results showed that the toxicity of DOX-hyd-PEG-FA on KB cells was increased in dose-depend manner, and the toxicity was much higher than thatof DOX-hyd-PEG and DOX-ami-PEG-FA. The cytotoxicity ofDOX-hyd-PEG-FA can be inhibitited by exogenous folic acid. TheDOX-hyd-AMA-FA had the same characteristics. While on A549cells, there wasno significant difference between the non-folate conjugates and folate conjugates.But the toxicity of hydrazone bond conjugate is greater than the amide bondconjugate.5. The flow cytometry results showed that the fluorescence intensity ofDOX-hyd-PEG-FA in KB cells was stronger than that of DOX-hyd-PEG, andincreased in time-depend manner.6. The fluorescence microscopy results showed that the uptake ofDOX-hyd-AMA-FA was much more than DOX-hyd-AMA in KB cells; however,in A549cells, the uptake of DOX-hyd-AMA-FA and DOX-hydAMA had nosignificant difference. The uptake of DOX-hyd-PEG-FA in KB cells wasobviously more than DOX-hyd-PEG. Compared with DOX-hyd-PEG,fluorescence intensity in the nucleus was much greater after KB cell incubatedwith DOX-hyd-PEG-FA.7. The laser scanning confocal microscope results showed thatDOX-hyd-PEG-FA firstly went into the lysosome, and then DOX distributed innucleus.Conclusion:DOX-hyd-AMA-FA and DOX-hyd-PEG-FA can be selectively transported intothe tumor cells which folate receptors are highly expressed via folatereceptor-mediated endocytosis, and then the DOX is released and exertanti-tumor effect. However, the tumor targeting characteristic and anti-tumoractivity of DOX-hyd-PEG-FA and DOX-hyd-AMA-FA in vivo still need further study.