Core-Shell Nanoparticles Based on Pullulan and Poly(β-amino) Ester for Hepatoma-Targeted Codelivery of Gene and Chemotherapy Agent Yuanyuan Liu, †,§ Yan Wang, †,§ Cong Zhang, † Ping Zhou, † Yang Liu, † Tong An, † Duxin Sun, ‡ Ning Zhang,* ,† and Yinsong Wang* ,† † Tianjin Cancer Institute and Hospital, Laboratory of Breast Cancer Prevention and Therapy, Ministry of Education, Research Center of Basic Medical Science & School of Pharmacy, Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), Tianjin Medical University, No. 22 Qixiangtai Road, Heping District, Tianjin 300070, People’s Republic of China ‡ Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States * S Supporting Information ABSTRACT: This study designs a novel nanoparticle system with core-shell structure based on pullulan and poly(β-amino) ester (PBAE) for the hepatoma-targeted codelivery of gene and chemotherapy agent. Plasmid DNA expressing green fluo- rescent protein (pEGFP), as a model gene, was fully condensed with cationic PBAE to form the inner core of PBAE/pEGFP polycomplex. Methotrexate (MTX), as a model chemotherapy agent, was conjugated to pullulan by ester bond to synthesize polymeric prodrug of MTX-PL. MTX-PL was then adsorbed on the surface of PBAE/pEGFP polycomplex to form MTX-PL/PBAE/pEGFP nanoparticles with a classic core-shell structure. MTX-PL was also used as a hepatoma targeting moiety, because of its specific binding affinity for asialoglycoprotein receptor (ASGPR) overexpressed by human hepatoma HepG2 cells. MTX-PL/PBAE/pEGFP nanoparticles realized the efficient transfection of pEGFP in HepG2 cells and exhibited significant inhibitory effect on the cell proliferation. In HepG2 tumor-bearing nude mice, MTX-PL/PBAE/pEGFP nanoparticles were mainly distributed in the tumor after 24 h postintravenous injection. Altogether, this novel codelivery system with a strong hepatoma- targeting property achieved simultaneous delivery of gene and chemotherapy agent into tumor at both cellular and animal levels. KEYWORDS: pullulan, poly(β-amino) ester, hepatoma, gene, chemotherapy agent ■ INTRODUCTION Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide, with the highest incidence rate reported in East Asia. 1,2 Surgical removal is believed as the optimal treatment for HCC, but most patients are diagnosed already at an advanced stage and are not suitable for hepatectomy. Traditional treatments, including chemotherapy and radio- therapy, have not been found to be effective in prolonging overall survival and often cause side effects. 3-6 Hence, exploring effective therapies against HCC is of great urgency and attracts wide atten- tion in medical and pharmaceutical field. Increasing investiga- tions have confirmed that the tumorigenesis and metastasis of HCC are a multigene-involving and multistep process; thus, gene therapy is currently believed to be a potent strategy for HCC treatments. In addition, combined gene therapy and chemotherapy often exhibit synergic effects on HCC 7-10 and are hoped to be an effective method in clinical practice. 11,12 One of the main challenges in advancing gene therapy technology is its effective delivery. Many viral vectors have been successfully used for ex vivo gene therapy, but their applications in vivo are greatly limited because of some intrinsic drawbacks, including the immunogenicity, low loading capacity for gene, and difficulty in mass production. 13,14 Nonviral vectors are therefore increasingly focused on because of their enhanced biosafety and biocompatibility. 15-17 A large number of cationic polymers have been reported to be capable of delivering genes. 18-20 Poly(β-amino) esters (PBAEs), first developed by the Langer group, exhibit great potential as gene delivery reagents because they are easily synthesized and exhibit the relatively low toxicities and high transfection efficiencies in a wide variety of cell types. PBAEs exhibit high efficiency of gene transfection because they can carry DNA to enter cells through endocytosis and then successfully escape from endosomal/ lysosomal compartment by the proton sponge effect. Thus, gene carriers based on PBAEs have attracted increased attentions recently. 21-25 Pullulan is a water-soluble, nontoxic, and nonimmunogenic nature polysaccharide that is produced by Aureobasidium Received: July 1, 2014 Accepted: October 7, 2014 Research Article www.acsami.org © XXXX American Chemical Society A dx.doi.org/10.1021/am504203x | ACS Appl. Mater. Interfaces XXXX, XXX, XXX-XXX