Systhesis And Antitumor Acitvity Of10-Hydroxycamptothecin-adenovirus Derivative

Adenovirus (Ad) vectors have received an extensive evaluation, and constituted a quarterof all clinical gene therapy trials. Compared with other virus vector, Ad vectors don’t insert theirDNA into host genome, posesses low immunogenicity and low toxicity. The gene deleted Adlacks of the replication potential. Furher more, Ad vector has higher infection ability than thenon-virus particles. With the development of nano-biotechnology, Ad is treated as a new carrierfor delivering the drugs, antigen, peptides other than DNA. The new vehicles have the ability ofimaging, drug targeting and sustained-release. Camptothcin derivatives have an extensiveapplication in cancer clinical therapy. Because of the complex structure, most of the derivatives’solubility are poor. In addition, the derivatives have low issue targeting and more side effection.Here, to improve the water-solubility and stability of10-Hydroxy camptothecin, the10-OH and20-OH are esteryfied by the succinic anhydride, the production is conjugated on the surface ofadenovirus capsid protein. The effections are evaluted with the colon cancer cell.Firstly，10-Hydroxy camptothecin derivative—(10,20(S)-O-disuccinyl-Hydroxycam-ptothecin) is synthesized via the catalysising of4-dimethylamiopryidine and identified by1H-NMR. The water-solublity of derivative is tested in the water bath. The cytotoxicities ismeasured by MTT assay, agarose gel electrophoresis and Hoechst33342staining are performedfor cell apoptosis. The result show that the derivative has a higher water-solublity(25℃):2.3mg; the inhibition is time/dose-depended, the IC50is700nM, the reason is inferenced that thesuccinic C-20pevent the attachment between and Topo I; ladder-like DNA fragment in agarosegel and the Hoechst33342staining prove that the derivative may induce cell apoptosis.Secondly，to get more adenovirus: Ad5-EGFP,293A cells are used to amplificated thevectors. CsCl and TCID50is applied for the purification and the detection of titer. Theexpression of fluorescent protein was surveyed by inverted fluorescent microscope. The resultshow that, the vector titer is1×1010pfu, the purificated virus have a high effection to the tumorcellFinally, carboxyl of DSH is activated by N-hydroxy succiimide (NHS) and1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride)(EDAC) for3h at roomtemperature, the complex DSH-Ad is synthesised with the activated DSH and adenovirus inPBS for12h at4℃. The DSH-Ad is identified by the UV absorption spectrum analysis, the number of DSH molecules conjugated to the Ad surface is tested by the UV. The invertedfluorescent microscope was used for the observation of the fluorescent protein expressed in thecell. Hoechst33342staining is performed for cell apoptosis. The result showed that, the DSHsare successfully conjugated to Ad, for the absorption spectra of DSH-Ad have displayed theabsorption peak in260nm,375nm; the number of DSH molecules conjugated to the Ad surfaceis363±24(n=4); The the inhibition of DSH-Ad to SW480was time/dose-depended, and theeffective rate is simlar to HCPT, on the other hands, the DSH have no effetion at the sameconcentration, it is guessed that the succinic ester is hydrolysised; the DSH-Ad restrains theexpression of fluorescent protein, but the other molecules like NHS,EDAC do not; theHoechst33342staining proved that the derivative may induce cell apoptosis. This stduy willlay a solid fundation for the further research in derivatives of camptothecin-other modifiedadvenovirus conjugation.