Studies On The Construction And In Vitro Anti-tumor Efficiency Of Multifunctional PAMAM-DOX Conjugates Drug Delivery System

Objective: Based on the specific physiological and pathological characteristics of thetumor, this study aimed to construct a drug delivery system with superior tumorrecognition and intracellular acid-triggered drug release properties. The characterization ofconjugates and in vitro evaluations was performed. Here, we evaluated it as a promisingcandidate for targeted drug delivery.Methods: For the construction of this polymeric drug delivery system,Polyamidoamine (PAMAM) dendrimer was modified with polyethylene glyeol (PEG) atfirst, then doxorubicin (DOX) was conjugated to each PAMAM by acid-sensitivehydrazone bond, finally Folate was applied to modify the PEG-PAMAM in order toenhance tumor selectivity. By controlling the feed ratio of starting materials (FA/PAMAM),PAMAM-DOX conjugates were synthesized with different numbers of FA ligands butsimilar PEGylation degree and drug loading.1H NMR, TLC, FTIR, DLS and TEM wereused to confirm and describe the PAMAM-DOX conjugates separately. UV-Vis spectraand dialysis bag methods were applied to evaluate the total drug content and in vitro drugrelease behavior, respectively. The MTT assay was used to optimize the number of FAligands and evaluate the IC50for PAMAM-DOX conjugates. Fluorescence microscope andflow cytometry were applied to qualitative and quantitative analyses of DOX cellularuptake by KB cells. The endocytic routes of PP-hyd-DOX and FPP-hyd-DOX16/1wereconfirmed by the cellular uptake mechanism test. Confocal laser scanning microscopy wasused to visualize the subcellular localization of conjugates.Results: Acid-labile PAMAM-DOX conjugates with different numbers of FA ligandsbut similar PEGylation degree and drug loading were successfully synthesized(PP-hyd-DOX, FPP-hyd-DOX4/1, FPP-hyd-DOX8/1and FPP-hyd-DOX16/1). Theresults of1H NMR, FTIR, DLS and TEM have verified the constitutive property ofPAMAM-DOX conjugates. The morphology of conjugates was regularly spherical with thesize ranging from~20nm to~30nm and the zeta potential was about+5mv. Thesubstitution level of DOX on the NH2of PAMAM was determined as6.11±0.12(wt.%), which corresponds with10DOX molecules per PAMAM on average. In vitro drug releasestudy of the conjugates revealed a pH-triggered manner. FPP-hyd-DOX16/1wasdetermined to be the formulation with optimal number for FA targeting ligands and theIC50value of FPP-hyd-DOX16/1was shown to be2.2-fold lower than that of non-targetedPP-hyd-DOX. Fluoresence microscopy and flow cytometry experiments displayed thatcellular uptake of FPP-hyd-DOX16/1was a time-dependent mode and the fluorescenceintensity of KB cells treated with FPP-hyd-DOX16/1was higher than that treated withPP-hyd-DOX at either1h or2h. The cellular uptake mechanism indicated thatFPP-hyd-DOX16/1and PP-hyd-DOX mainly interacted with plasma membrane byelectrostatic interaction, and then was internalized by KB cells via clathrin-mediatedendocytosis. Confocal laser scanning microscopy further confirmed the conjugates weredelivered to acidic lysosomes and triggered the release of DOX into nuclei to exert itscytotoxicity.Conclusion: FPP-hyd-DOX16/1with superior tumor recognition and intracellularacid-triggered drug release property was constructed and the obtained results demonstratedthat it would be a promising drug delivery carrier for targeted cancer therapy.