Polymeric Prodrugs Conjugated With Reduction-sensitive Dextran-camptothecin And PH-responsive Dextran-doxorubicin:An Effective Combinatorial Drug Delivery Platform For Cancer Therapy

Multiple drugs in combination therapy can improve the treatment of cancer, because it can overcome efficiently multidrug resistance(MDR) of tumor cells efficiently. camptothecin(CPT) and doxorubicin(DOX) are common topoisomerase(Top) inhibitors, which interfere with action of Top Ⅰ and Ⅱ, respectively. The combine use of Top Ⅰ and Ⅱ inhibitors might exhibit synergistic effects and deter the drug resistance. However, both of them suffer from poor water solubility, rapid blood/renal clearance, nonspecific selectivity, and severe adverse side effects for healthy tissues. Dextran, a kind of polysaccharide, is a good choice as drug delivery, which is a natural analog of PEG. In addition, dextran can be easily chemically modified because of its 5% branching structure and plentiful hydroxyl groups on the chain.To this end, this paper prepard reduction-sensitive dextran-ss-camptothecin(Dex-ss-CPT) and p H-responsive dextran-hydrazone-doxorubicin(Dex-hyd-DOX) conjugates for combinational therapy. The chemical structures and properties of the two prodrugs were studied. Simultaneously, their biological properties were also studied.The work is mainly divided into the following three parts:(1) Preparation and characterization of reduction-sensitive dextran-camptothecin(Dex-ss-CPT) and p H-responsive dextran-doxorubicin(Dex-hyd-DOX) conjugatesFirst, alkyne-modified dextran(Dex-C ≡ C), CPT-ss-N3 and DOX-hyd-N3 were prepared. Then polymeric prodrug Dex-ss-CPT and Dex-hyd-DOX were synthesized by Cu AAC “click” reaction. The chemical structures and properties of the two prodrugs were studied by 1H NMR, LC/MS, FT-IR, UV-Vis and HPLC. The results confirm their structure.(2) Performance and Application of reduction-sensitive dextran-camptothecin(Dex-ss-CPT) and p H-responsive dextran-doxorubicin(Dex-hyd-DOX) conjugatesThe nanoparticles self-assembled from prodrugs were prepared by dialysis. The morphologies of the nanoparticles self-assembled from Dex-ss-CPT or Dex-hyd-DOX were observed by transmission electron microscope(TEM) instrument. The average particles sizes( Dz) and size polydispersity indices(size PDIs) of the self-assembled prodrug nanoparticles in Milli-Q water were measured by dynamic light scattering(DLS) instrument. The results show the diameters of both the prodrug are less than 200 nm. To study the trigger-controlled release behavior of the prodrugs, DLS was carried out to monitor the size changes of Dex-ss-CPT micelles with 2 ?M and 10 m M of GSH and Dex-hyd-DOX micelles with different p H conditions at different time intervals. In vitro drug release also demonstrats the behavior. The cytotoxicity of Dex-C?C20 against L929 cells and 4T1 cells was studied by MTT assay, which suggests excellent biocompatibility of the polymer. Furthermore, we get the optimal formulation of the prodrugs via MTT assay of single prodrug and polymer prodrug combines with different drug molar ratios. To evaluate the extents of internalization of the prodrug micelles, the cellular internalization of the optimal combination was the examined against 4T1 cells.(3) In vivo study of the combinational therapy of the reduction-sensitive dextran-camptothecin(Dex-ss-CPT) and p H-responsive dextran-doxorubicin(Dex-hyd-DOX) conjugatesTo evaluate the prodrugs show a longer retention time in the bloodstream, the pharmacokinetic study was undertaken by i.v. injection of the free CPT/DOX(4:1) and Dex-CPT/Dex-DOX(4:1) particles to BALB/c mice(~20 g)(dosage 5 mg kg-1). Furthermore, to examine the amount of Dex-CPT/Dex-DOX(4:1) particles in the tumor and other organs, the 4T1 tumor-bearing mice were sacrificed after intravenous injection with different time intervals. In order to evaluate whether efficient accumulation and improved biodistribution results in the enhancement of therapeutic efficacy, 4T1 tumor-bearing mice were randomly divided into different groups, and treated with various formulations(the carrier Dex-C?C, low and high concentrations of free CPT/DOX(4:1) mixture, low and high concentrations of Dex-CPT/Dex-DOX(4:1) mixture, and Milli-Q water as control) via intraperitoneal administration. The immunohistochemical analysis was adopted to assess the different antitumor efficacy after different treatments. The in vivo pharmacokinetics and biodistribution studies indicate that the therapeutic efficacy of high-concentration prodrug mixture(5 mg kg-1) is the highest among the therapeutic groups(other groups are low-concentration prodrug mixture(2.5 mg kg-1), low(2.5 mg kg-1) and high concentrations(5 mg kg-1) of free drug mixture). Overall, we believe that if the solubility of the prodrug in water can be improved, the therapeutic efficacy will be better and would eventually be applied in clinic to treat many kinds of cancers.