Construction And Cancer Therapy Applications Of Fuctionalized Dendrimer-based Drug Delivery System

The construction and application of drug delivery system is expected to bring significant change to the pharmaceutical and biotechonogly industries, and now the dendrimer-based nano drug delivery system has already become a hot pot of molecular pharmaceutics. In this research, we used G5Poly(amidoamine)(PAMAM) dendrimer as drug vehicle to develop multiple approaches towards improved cancer therapeutic efficiency. Different functional groups needed for selective drug delivery and locating are covalently conjugated onto the surface of dendrimer to form multifunctional "nanodevices". Then the functionalized dendrimers were used to covalently conjugate drug molecules or physically encapsulate or complex drug molecules for cancer therapeutics. The characterizations and biomedical applications of functionalized dendrimers and dendrimer/drug complex were investigated in detail.In this research, firstly, G5PAMAM dendrimers were modified with different surface functional groups to form fully acetylated dendrimer (G5.NHAc), fully hydroxyl-terminated dendrimer (G5.NGlyOH) and fully carboxyl-terminated dendrimer (G5.SAH) in complex with an anticancer agent doxorubicin (DOX) for the studies of influence of dendrimer surface group on the bioacitivity of dendrimer/DOX complex.4-9drug molecules can be trapped by one dendrimer molecule for every type of functionalized dendrimer. Multiple NMR techniques including one-dimensional NMR (1D-NMR) and two-dimensional NMR (2D-NMR) studies show that different interaction intensities between dendrimers and drug molecules can be used to explain the difference of in vitro drug release rate from functionalized dendrimers. Both MTT assay and cell morphological observation show that the bioactivity of DOX complexed with dendrimers was solely dependent on DOX. The relatively stronger interactions of G5.NHAc and G5.NGlyOH dendrimers with DOX enable them to be more ideal drug delivery vehicles: however, the weaker interaction between carboxyl-terminated G5dendrimers and DOX may not allow this type of dendrimer/DOX complex for further in vivo antitumor research. Our findings indicate that the surface modification of dendrimers with different functional groups is crucial for the development of drug delivery vehicles in therapeutic applications.In addition, we show the synthesis of multifunctional dendrimer. Amine-terminated G5PAMAM dendrimers were sequentially modified with fluorescein isothiocyanate (FI) as an imaging agent and folic acid (FA) as a targeting ligand, then the functionalized dendrimers were partially or fully acetylated to neutralize a significant portion or total of the terminal amines. Both UV-vis and1H NMR results reveal that G5.NHAc, G5.NHAc-FA, G5.NHAc-FI and G5.NHAc-FA-FI are successfully synthesized.The next part focuses on the encapsulation of novel anticancer drug combretastatin A4(CA4) into G5.NHAc-FA-FI and in vitro release studies together with targeted cancer therapy studies of formed G5.NHAc-FA-FI/CA4into cancer cells overexpressing folic acid receptors (FAR). The formed inclusion complexes of G5.NHAc-FA-FI/CA4are able to significantly improve the water solubility of CA4from11.8to240μg/mL. In vitro release studies show that CA4complexed with the multifunctional dendrimers can be released in a sustained manner. Both MTT assay and cell morphological observation reveal that the inhibitory effect of the G5.NHAc-FA-FI/CA4complex is similar to that of free CA4at the same drug concentration. More importantly, the complexes are able to selectively target and display specific therapeutic efficacy to cancer cells overexpressing high-affinity FAR.We finally covalently conjugated DOX onto the surface of G5PAMAM dendrimer for pH-induced release and targeted cancer therapy. We used cis-aconitic acid to bond DOX and dendrimer to form a much stable dendrimer-DOX conjugates (G5.NHAc-DOX and G5.NHAc-FA-DOX). In vitro release studies show that G5.NHAc-FA-DOX conjugates rarely release DOX at pH7.4condition; while in the acid condition, the release of drug increases with the decrease of pH value. MTT assay, cell morphological observation, flow cytometry assay and confocal microscopy imaging not only reveal that the therapeutic activity of dendrimer-DOX conjugates solely depends on the drug DOX, but also indicate that G5.NHAc-FA-DOX conjugates can specifically target KB cells overexpressing high-affinity FAR and display targeted cancer theraputic efficiacy.