Ming Zhao

PDF - 295K, Table S1. In vitro effects of CLO prodrug in NCI-60 cell screen. Table S2. In vitro effects of the CLO prodrug on SK-Mel-5 and UACC-62 melanoma cells. Figure S1. Bisphosphonamidate prodrugs are cell permeable and undergo intracellular activation in UACC-62 cells. Figure S2. CLO prodrug does not affect the viability of human keratinocytes and peripheral blood mononuclear cells. Figure S3. Intratumoral delivery of clodronate has no antitumor activity.

The most concerning adverse effects of thrombolytic agents are major bleeding and intracranial hemorrhage due to their short half-life, low fibrin specificity, and high dosage. To alleviate bleeding side effects during thrombolytic therapy which would bring about the risk of aggravation, we try to find a novel biodegradable delivery nanosystem to carry drugs to target the thrombus, reduce the dosage of the drug, and system side effects. A novel urokinase/poly-α, β-d, l-aspartyl-Arg-Gly-Asp-Ser complex (UK/PD-RGDS) was synthesized and simply prepared. Its thrombolytic potency was assayed by the bubble-rising method and in vitro thrombolytic activity by the thrombus clot lysis assay separately. The in vivo thrombolytic activity and bleeding complication were evaluated by a rat model of carotid arteriovenous bypass thrombolysis. The thrombolytic potency (1288.19 ± 155.20 U/mg) of the UK/PD-RGDS complex nano-globule (18–130 nm) was 1.3 times that of commercial UK (966.77 ± 148.08 U/mg). In vivo, the UK/PD-RGDS complex (2000 IU/kg) could reduce the dose of UK by 90% while achieving the equivalent thrombolysis effect as the free UK (20,000 IU/kg). Additionally, the UK/PD-RGDS complex decreased the tail bleeding time compared with UK. The organ distribution of the FITC-UK/PD-RGDS complex was explored in the rat model. The UK/PD-RGDS complex could provide a promising platform to enhance thrombolytic efficacy significantly and reduce the major bleeding degree.

Background: The discovery of novel derivative of berberine (BBR) having higher anti-tumor activity in vivo is of clinical importance. In this profile, 13-[CH₂CO-Cys-(Bzl)-OBzl]-berberine (13-Cys-BBR) was prepared for related assays. Purpose: The object of preparation and evaluation is to show the advantages of 13-Cys-BBR over BBR in both in vitro and in vivo anti-tumor actions, furthermore to correlate the proliferation of cancer cells with ROS formation and anti-apoptosis protein (XIAP) expression inside cancer cells. Methods: Transwell chamber was used to simulate the intestinal and cell wall for bioavailability evaluation; MTT assay was used to evaluate the in vitro anti-proliferation activity; fluorescein isothiocyanate content was used to represent ROS level in HCT-8 cells; Western blot assay was used to quantify the expression of XIAP, caspase-3, and poly ADP-ribose polymerase in HCT-8 cells; and S180 mouse model was used to evaluate the in vivo anti-tumor activity. Results: In vitro the IC₅₀ values (∼15–40 μM) of 13-Cys-BBR against the proliferation of eight cancer cell lines were significantly lower than those of BBR (∼25–140 μM); the content of ROS formed inside HCT-8 cells treated by 13-Cys-BBR was ∼3.44-folds higher than that inside HCT-8 cells treated by BBR; the expression of XIAP in HCT-8 cells treated by 13-Cys-BBR was ∼1.21-folds lower than that in HCT-8 cells treated by BBR; the tumor weight of S180 mice orally treated by 2 μmol/kg/day of 13-Cys-BBR (∼1.5 g) was significantly lower than that of S180 mice orally treated by 2 μmol/kg/day of BBR (∼2.5 g); and the active pocket of XIAP was more suitable for 13-Cys-BBR than for BBR. Conclusion: The anti-tumor action correlates with ROS and apoptosis protein, which suggests 13-Cys-BBR is a promising candidate for preclinical study.

Background: Gemcitabine has been widely used as a chemotherapeutic drug. However, drug resistance, short half-life and side effects seriously decrease its chemotherapeutic efficacy. Purpose: The object of preparing RGDV-gemcitabine was to prolong the half-life, to overcome drug resistance and to eliminate bone marrow toxicity of gemcitabine. Methods: Arg-Gly-Asp-Val was coupled with gemcitabine, forming 4-(Arg-Gly-Asp-Val-amino)-1-[3,3-difluoro-4-hydroxy-5-(hydroxylmethyl)oxo-lan-2-yl]pyrimidin-2-one (RGDV-gemcitabine) involving 9-step reactions. The advantages of RGDV-gemcitabine to gemcitabine were demonstrated by a series of assays, such as in vitro half-life assay, in vitro drug resistance assay, in vivo anti-tumor assay, in vivo kidney toxicity assay, in vivo liver toxicity assay and in vivo marrow toxicity assay. The nano-features of RGDV-gemcitabine were visualized by TEM, SEM and AFM images. The tumor-targeting action and release of RGDV-gemcitabine were evidenced by FT-MS spectra. Results: Half-life and anti-tumor activity of RGDV-gemcitabine were 17-fold longer and 10-fold higher than that of gemcitabine, respectively. RGDV-gemcitabine, but not gemcitabine, showed no kidney toxicity, no liver toxicity, no marrow toxicity and no drug resistance. The advantages attributed to the nanofeatures of RGDV-gemcitabine were targeting tumor tissue and releasing gemcitabine in tumor tissue. Conclusion: RGDV-gemcitabine successively overcame the defects of gemcitabine and provided a practical strategy of nano-medicine.

Background: In vitro (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val (MTCA-KKV) adheres activated platelets, targets P-selectin and GPIIb/IIIa. This led to the development of MTCA-KKV as thrombus targeting nano-medicine. Methods: MTCA-KKV was characterized by nano-feature, anti-thrombotic activity, thrombolytic activity, thrombus target and targeting release. Results: In vivo 0.01 μmol/kg of MTCA-KKV formed nano-particles less than 100 nm in diameter, targeted thrombus, released anti-thrombotic and thrombolytic pharmacophores, prevented thrombosis and dissolved blood clots. Conclusion: Based on the profiles of targeting thrombus, targeting release, inhibiting thrombosis and dissolving blood clots MTCA-KKV is a promising nano-medicine.

Background: In the discovery of DNA intercalators, β-carbolines compose one member of the most interesting alkaloid family and are of clinical importance. In the efforts, N-(3-benzyloxycarbonyl-β-carboline-1-yl)ethyl-Ser-Ala-OBzl (BCESA) was designed as a nano-scaled DNA intercalator without Dox-like toxicity. Methods: Based on the structural analysis and CDOCKER energy comparison, BCESA was rationally designed as such a nano-scaled intercalator. The anti-tumor activity, the toxicity and the tumor targeting action of BCESA were evaluated on mouse models. Results: The in vitro proliferation of cancer cells, but not non-cancer cells, was effectively inhibited by BCESA. On S180 mouse model BCESA dose-dependently slowed the tumor growth, and 0.01 μmol/kg/day was found as a minimal effective dose. Both BCESA and its moiety were found in the tumor tissue, but not in the organs and the blood, of S180 mice. Conclusion: BCESA should be a nano-scaled intercalator capable of targeting tumor tissue to release anti-tumoral β-carboline-3-carboxylic acid and its 1-methyl derivative, while Ser-Ala-OBzl is a simple and desirable carrier.

Background: Due to the discovery that deep venous thrombosis (DVT) inhibitor is of chemotherapeutic importance, the nano-property of dimethyl 2,2'-[2,2'-(ethane-1,1-diyl)bis(1H-indole-3,2-diyl)]-diacetate (DEBIC), a recently reported antitumor agent, is worthy of characterization. Materials and methods: One-pot reaction was used to prepare DEBIC. Electrospray Ionization (+/-)-Fourier Transform-Ion Cyclotron Resonance-Mass Spectrometer (ESI(+/-)-FT-ICR-MS), quadrupole Collision Induced Dissociation (qCID) and nuclear overhauser effect spectroscopy spectra were used to present the assembly of DEBIC. Transmission electron microscopy, scanning electron microscopy, atomic force microscopy and Faraday–Tyndall effect were used to visualize the nano-property of DEBIC. Rat models were used to evaluate DVT inhibition and the bleeding reaction of DEBIC. Results: One-pot reaction can provide DEBIC in acceptable yield and high purity. In water, rat plasma and lyophilized powders of DEBIC existed as particles of small nano-size. In vivo DEBIC inhibited DVT in a dose-dependent manner. The minimal effective dose of DEBIC was 1.7 µmol/kg. Even the dose of 36 µmol/kg/day DEBIC did not induce bleeding side effect in DVT rats like in warfarin (0.82 µmol/kg/day). Conclusion: DEBIC is a small molecule capable of nano-scale assembly, inhibiting venous thrombosis and inducing no bleeding side effect.

Aim: The modification of platelet inhibitor to enhance its targeting capacity toward platelets is of clinical importance. Thus, (1R, 3S)-1-methyl-1, 2, 3, 4-tetrahydro-β-carboline-3-carboxylic acid (MTCA), a platelet inhibitor, was modified with Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val (KKV), platelet targeting peptide, to form MTCA-KKV. Materials & methods: MTCA and MTCA-KKV were synthesized to identify the effect of KKV modification on MTCA and platelets. Results: Atomic force microscopy imaged MTCA-KKV effectively accumulated on activated platelets. UV spectra showed that MTCA-KKV concentration dependently changed P-selectin and GPIIb/IIIa conformations. For platelet aggregation, the IC50 of MTCA-KKV was approximately 1/10 folds of MTCA. Conclusion: KKV modification led to forming MTCA-KKV that is superior to MTCA in terms of accumulating on activated platelets, targeting P-selectin and GPIIb/IIIa and inhibiting platelet aggregation. MTCA-KKV could be a promising lead for further investigation. Graphical abstract