Efficient Click Synthesis of a Protonized and Reduction-Sensitive Amphiphilic Small-Molecule Prodrug Containing Camptothecin and Gemcitabine for a Drug Self-Delivery System.

Drug self-delivery systems consisting of small-molecule active drugs with nanoscale features for intracellular delivery without the need for additional polymeric carriers have drawn much attention recently. In this work, we proposed a highly efficient strategy to fabricate protonized and reduction-responsive self-assembled drug nanoparticles from an amphiphilic small-molecule camptothecin-ss-1,2,3-triazole-gemcitabine conjugate (abbreviated as CPT-ss-triazole-GEM) for combination chemotherapy, which was prepared via a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" reaction. To obtain this drug-triazole-drug conjugate, we first prepared a CPT derivate containing a propargyl group linked with a disulfide group and a GEM derivate attached to an azide group. Subsequently, the two kinds of modified drugs were connected together through a CuAAC reaction between the alkynyl and azide groups to yield the CPT-ss-triazole-GEM prodrug. The characterizations of chemical structures of these intermediates and the final product were performed by H NMR, Fourier transform infrared, and liquid chromatography/mass spectrometry measurements. This amphiphilic small-molecule drug-triazole-drug conjugate displayed a high drug loading content, that is, 36.0% of CPT and 27.2% of GEM. This kind of amphiphilic small-molecule prodrugs could form spherical nanoparticles in an aqueous solution in the absence of any other polymeric carriers, in which the hydrophobic CPT formed the core of the nanoparticles, whereas the hydrophilic GEM and protonated 1,2,3-triazole group yielded the shell. In the tumor microenvironment, the prodrug nanoparticles could release both pristine drugs simultaneously. Under the conditions of pH 7.4, and pH 7.4 and 2 μM glutathione (GSH), the prodrug nanoparticles could maintain stability and only 7% of CPT was leaked. However, in a high-GSH environment (pH 7.4 and 10 mM GSH) with the same incubation time, the disulfide linkage would be dissociated and lead to about 34% of CPT release. The results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test demonstrated that these prodrug nanoparticles showed a higher cytotoxicity toward HepG2 cells than free CPT and free GEM on both 48 and 72 h of incubation. Both in vitro cellular uptake and flow cytometry results implied that these prodrug nanoparticles could be internalized by HepG2 cells with efficient drug release inside cells. The pharmacokinetics and tissue distribution of the prodrug showed a moderate half-life in vivo, and the prodrug peak concentration in most of the collected tissues appeared at 0.25 h after administration. In addition, the CPT-ss-triazole-GEM prodrug could not cross the blood-brain barrier. Even more important is the fact that there is no accumulation in tissues and a rapid elimination of this small-molecule prodrug could be achieved. In brief, this protonized and reduction-sensitive prodrug simultaneously binds both antitumor drugs and has good self-delivery behavior through the donor-acceptor interaction of the H-bonding ligand, that is, the 1,2,3-triazole group. It provides a new method for combined drug therapy.