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When gene therapy is performed for the treatment of malignant tumors, gene transfer efficiency and selectivity are highly important. Polymer vehicle microspheres are a novel type of therapy, which have been developed rapidly in recent years and are able to control drug release, prolong the biological half-life of drugs, decrease side effects and achieve targeted delivery. The present study was designed to construct a polymer microsphere-encapsulated recombinant adenovirus with human tissue inhibitors of the matrix metalloproteinase-1 (TIMP-1) gene, and to discuss its characterization for the purpose of liver cancer gene therapy. The microsphere was prepared from biodegradable poly-DL-lactide-poly(ethylene glycol) (PELA) encapsulating rAdTIMP-1, the recombinant adenovirus carrying TIMP-1, by a modified double-emulsion method. The particle morphology, diameter, virus encapsulation, loading rate and release kinetics of the rAd-microspheres were determined in vitro. Hepatocellular carcinoma (HCC) HepG2 cells were transfected with the rAd-microsphere and the efficiency of transfection was assessed by fluorescent microscopy. The production and expression of TIMP-1 was identified by gelatin zymography and western blot analysis, and the invasiveness was detected by a matrigel matrix invasion assay. The microsphere encapsulating rAdTIMP-1 was successfully constructed with a diameter of 1.965 μm, encapsulation efficiency of 60.0%, a viral load of 10.5 x 10(8)/mg, a virus release of ~60% within 120 h and a total release time of >240 h. The resultant rAd-microspheres were able to efficiently transfect HepG2 cells with the transfection efficiency enhanced by ~90%. As a result, the transfected HepG2 cells had significantly increased TIMP-1 enzyme activity and the expression of TIMP-1 was detected by western blot analysis. In addition, the proliferation and invasion ability of the HCC cells was markedly inhibited by the rAd-microspheres. The resultant rAd-microspheres, PELA-encapsulated recombinant TIMP-1 adenovirus, had enhanced transfection efficiency and were able to markedly inhibit the in vitro biological behavior of HepG2 cells. This provides an experimental basis for this polymer application and may pave the way for prospective in vivo clinical trials and further comprehensive therapy for liver cancer.