Elevation of Transfection Efficiency by Conjugation of Poly(amindoamine)-diethylenetriamine (PAM-DET) with Dexamethasone

Abstract

A number of modifications and functionalizations of dendrimers as non-viral vectors of gene delivery system have been attempted by many researchers in order to solve the general problem associated with these structures, i.e., the high cytotoxicity of polymers such as the 25-kDa poly(ethylenimine) (PEI) and poly(amidoamine) dendrimer (PAMAM). As a result of these attempts, PAMAM-R and PAM-DET, which are respectively arginineand diethyltriamine (DETA)-modified PAMAM dendrimers, were synthesized by our group and have shown high transfection efficiency and low cytotoxicity. In this study, we tried to conjugate the nontoxic and hydrophobic group, dexamethasone (DX) with the ends of PAM-DET and synthesized PAM-DET-DX. We introduced DX group specially due to both its nuclear localization effect in gene-transfection and its biocompatibility as a glucocorticoid analog. In comparison with PAM-DET, this conjugated PAM-DET dendrimers showed polyplexes (gene-polymer complexes) with better and longer size-stability in aqueous solutions with high ion strength. The PAM-DET-DX gene carriers showed higher transfection efficiency than PAM-DET and cytotoxicity level equivalent to that shown by PAM-DET in HeLa and U87MG Cell lines. PAM-DET was synthesized as previously described. The conjugation reaction was performed as previously reported with some modification. The overall synthesis scheme is displayed in Figure 1(a). Briefly, dexamethasone coupling to PAM-DET was performed with 4 equivalents of Traut’s reagent and 4 equivalents of dexamethasone mesylate in anhydrous DMSO for 4 h at room temperature. An equal volume of water was added to the reaction mixture, and it was dialyzed against pure water and filtered through a 0.45μm syringe filter to remove insoluble impurities. The product was then obtained after freeze-drying. The resulting PAM-DET-DX product was analyzed by H NMR at 40 °C to calculate the actual degree of conjugation (Figure 1(b)). Each PAM-DET-DX conjugate was named based on its actual degree of DX conjugation (Table 1). The pDNA/PAM-DET and pDNA/PAM-DET-DX polyplexes were prepared in 10 mM HEPES buffer (pH 7.4) by incubation at 25 °C for 30 min. To prepare each polyplex sample with PAM-DET and PAM-DET-DX (2), (5), and (10) (their charge ratio of cationic polymer/pDNA was commonly 16) as the first group, 1 mg/mL of pDNA was used. For the second group, pDNA/PAM-DET and pDNA/ PAM-DET-DX (10) polyplexes with charges of 4, 8, and 16 were also prepared, taking into consideration the molecular weight and actual degree of conjugation, as shown in Table 1.