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Lijuan Zhang,1,* Shengan Zhang,2,* Min Jiang,1 Lu Lu,3 Yue Ding,3 Ninghui Ma,1 Yuan Zhao,4 Sihan Xuchen,1 Nailian Zhang1 1School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China; 2School of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China; 3Experiment Center for Teaching and Learning, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China; 4Center of Science and Technology, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Lu Lu; Yue DingExperiment Center for Teaching and Learning, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of ChinaEmail yjll66@outlook.com; dingyue-2001@hotmail.comIntroduction: As high cholesterol level has been reported to be associated with cancer cell growth and cholesterol is vulnerable to oxidation, the conventional liposomes including cholesterol in the formulation seem to be challenged. Timosaponin AIII (TAIII), as a steroid saponin from Anemarrhena asphodeloides Bunge, possesses a similar structure with cholesterol and exhibits a wide range of antitumor activities, making it possible to develop a TAIII-based liposome where TAIII could potentially stabilize the phospholipid bilayer as a substitution of cholesterol and work as a chemotherapeutic drug as well. Meanwhile, TAIII could enhance the uptake of doxorubicin hydrochloride (DOX) in human hepatocellular carcinoma (HCC) cells and exhibit synergistic effect. Thus, we designed a novel thermally sensitive multifunctional liposomal system composed of TAIII and lipids to deliver DOX for enhanced HCC treatment.Methods: The synergistic effects of DOX and TAIII were explored on HCC cells and the tumor inhibition rate of TAIII-based liposomes carrying DOX was evaluated on both subcutaneous and orthotopic transplantation tumor models. TAIII-based multifunctional liposomes were characterized.Results: Synergistic HCC cytotoxicity was achieved at molar ratios of 1:1, 1:2 and 1:4 of DOX/TAIII. TAIII-based liposomes carrying a low DOX dose of 2 mg/kg exhibited significantly enhanced antitumor activity than 5 mg/kg of DOX without detected cardiotoxicity on both subcutaneous and orthotopic transplantation tumor models. TAIII-based liposomes were characterized with smaller size than cholesterol liposomes but exhibited favorable stability. Mild hyperthermia generated by laser irradiation accelerated the release of DOX and TAIII from liposomes at tumor site, and cell permeability of TAIII enhanced uptake of DOX in HCC cells.Conclusion: The innovative application of TAIII working as bilayer stabilizer and chemotherapeutic drug affords a stable multifunctional liposomal delivery system for synergistic therapy against HCC, which may be referred for the development of other types of saponins with similar property.Keywords: doxorubicin, timosaponin AIII, liposomes, hepatocellular carcinoma, cholesterol

Quan Chen,1 Siheng Li,2 Zixiong Feng,1 Meng Wang,3 Chengzhi Cai,2 Jufang Wang,3 Lijuan Zhang1 1School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, People’s Republic of China; 2Department of Chemistry, University of Houston, Houston, TX, USA; 3School of Bioscience & Bioengineering, South China University of Technology, Guangzhou, People’s Republic of China Abstract: We have demonstrated a novel drug delivery system to improve the selectivity of the current chemotherapy by pH-responsive, polymeric micelle carriers. The micelle carriers were prepared by the self-assembly of copolymers containing the polybasic poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA) block. The mixed copolymers exhibited a comparatively low critical micelle concentration (CMC; 1.95–5.25mg/L). The resultant mixed micelles were found to be

Xiaoyu Wang,1,2* Zongguang Tai,1* Jing Tian,1* Wei Zhang,3 Chong Yao,1 Lijuan Zhang,1 Yuan Gao,1 Quangang Zhu,1 Jing Gao,4 Shen Gao1 1Department of Pharmaceutics, Changhai Hospital, Second Military Medical University, Shanghai, 2Department of Pharmaceutics, ChengDu Military General Hospital, ChengDu, 3Department of Pharmaceutics, Shanghai Pulmonary Hospital, Tongji University, Shanghai, 4Department of Pharmaceutical Science, School of Pharmacy, Second Military Medical University, Shanghai, People’s Republic of China *These authors contributed equally tothis work Abstract: Cationic oligopeptide as a nonviral gene delivery vector has aroused much research interest recently, but its further application is limited by its low transfection efficiency. In the present study, we have created a high-efficiency gene vector by using octa-d-arginine and tetra-l-histidine to form a disulfide cross-linked chimeric polypeptide and used this vector to deliver the therapeutic gene tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) to see whether the gene could be transferred and could exert antitumor effects in vitro and in vivo. The result showed that the newly designed vector was able to condense DNA into nanosized polyplexes effectively, thus facilitating its transmembrane transport, promoting its endosomal escape, and finally enabling degradation within the cell. Our study has demonstrated that this chimeric polypeptide is an effective gene carrier in cancer therapy. Keywords: reducible polypeptide, disulfide bond, octa-d-arginine, tetra-l-histidine, TRAIL

Xin Wu,1,2,* Zongguang Tai,1,* Quangang Zhu,3,* Wei Fan,4 Baoyue Ding,5 Wei Zhang,1,6 Lijuan Zhang,1 Chong Yao,1 Xiaoyu Wang,1 Xueying Ding,2 Qin Li,2 Xiaoyu Li,2 Gaolin Liu,2 Jiyong Liu,1 Shen Gao1 1Department of Pharmaceutics, Changhai Hospital, Second Military Medical University, 2Department of Pharmaceutics, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, 3Department of Pharmaceutics, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China; 4Department of Pharmaceutics, The 425th Hospital of PLA, Sanya, People’s Republic of China; 5Department ofPharmaceutics, Medical College of Jiaxing University, Jiaxing, People’s Republic of China; 6Department ofPharmaceutics, The 522nd Hospital of PLA, Luoyang, People’s Republic of China *These authors contributed equally tothis work Abstract: Ligand-mediated prostate cancer (PCa)-targeting gene delivery is one of the focuses of research in recent years. Our previous study reported the successful preparation of aptamer-modified nanoparticles (APT-NPs) in our laboratory and demonstrated their PCa-targeting ability in vitro. However, the mechanism underlying this PCa-targeting effect and their anticancer ability in vivo have not yet been elucidated. The objective of this study was to assess the feasibility of using APT-NPs to deliver micro RNA (miRNA) systemically to PCa cells, to testify their tumor-targeting efficiency, and to observe their biodistribution after systemic administration to a xenograft mouse model of PCa. In addition, the effect of APT depletion and endocytosis inhibitors on cellular uptake was also evaluated quantitatively in LNCaP cells to explore the internalization mechanism of APT-NPs. Finally, blood chemistry, and renal and liver function parameters were measured in the xenograft mouse model of PCa to see whether APT-NPs had any demonstrable toxicity in mice in vivo. The results showed that APT-NPs prolonged the survival duration of the PCa tumor-bearing mice as compared with the unmodified NPs. In addition, they had a potential PCa-targeting effect in vivo. In conclusion, this research provides a prototype for the safe and efficient delivery of miRNA expression vectors to PCa cells, which may prove useful for preclinical and clinical studies on the treatment of PCa. Keywords: miRNA, aptamer, polyamidoamine, prostate-specific membrane antigen, targeted delivery, prostate cancer

Lijuan Zhang,1 Thomas J Webster21Department of Chemistry, 2School of Engineering, Brown University, Providence, RI, USABackground: The aim of this study was to investigate the effects of poly-lactic-co-glycolic acid (PLGA) nanotopographies with alginate or chitosan protein preadsorption on the functioning of healthy and cancerous lung and breast cells, including adhesion, proliferation, apoptosis, and release of vascular endothelial growth factor (VEGF), which promotes tumor angiogenesis and secretion.Methods: We used a well established cast-mold technique to create nanoscale surface features on PLGA. Some of the nanomodified PLGA films were then exposed to alginate and chitosan. Surface roughness and the presence of protein was confirmed by atomic force microscopy. Surface energy was quantified by contact angle measurement.Results: Nanostructured PLGA surfaces with 23 nm features decreased synthesis of VEGF in both lung and breast cancer cells compared with conventional PLGA. Preadsorbing alginate further decreased cancer cell function, with nanostructured PLGA preadsorbed with alginate achieving the greatest decrease in synthesis of VEGF in both lung and breast cancer cells. In contrast, compared with nonmodified smooth PLGA, healthy cell functions were either not altered (ie, breast) or were enhanced (ie, lung) by use of nanostructured features and alginate or chitosan protein preadsorption.Conclusion: Using this technique, we developed surface nanometric roughness and modification of surface chemistry that could selectively decrease breast and lung cancer cell functioning without the need for chemotherapeutics. This technique requires further study in a wide range of anticancer and regenerative medicine applications.Keywords: breast, lung, cancer, nanotechnology, alginate, chitosan

Yongchen Wang,1 Lijuan Zhang,1 Linlin Sun,2 Thomas J Webster2,31Department of Chemistry, 2School of Engineering, 3Department of Orthopedics, Brown University, Providence, RI, USAAbstract: Poly(lactic-co-glycolic acid) ([PLGA] 50:50 wt% PLA:PGA) films with a flat surface and with 27 nm, 190 nm, 300 nm, 400 nm, and 520 nm nanopatterns were fabricated using a cast-mold process. The nanopatterns were transferred from self-assembled polystyrene (PS) beads to PLGA films through polydimethylsiloxane (PDMS) molds. The surface features, root-mean-square (RMS) roughness, and wettability of these PLGA surface features were studied by atomic force microscope (AFM) height scans, AFM z-sensor scans, and water contact angles, respectively. In order to evaluate the influence of the material topography alone (without changes in chemistry) for bone-cancer applications, both human healthy osteoblasts and human cancerous osteosarcoma cells were cultured on these PLGA surface features, and their densities were determined. Most importantly, compared to all other substrates, it was found that the 27 nm PLGA nanopatterns significantly increased the healthy osteoblast-to-osteosarcoma cell-density ratio. For these reasons, and since previous studies have highlighted that similar nanometer PLGA surface features decreased functions of other types of cancerous cells (specifically lung and breast), this study suggests that 27 nm PLGA nanopatterns should be further studied for a wide range of bone-cancer applications, particularly where healthy bone-cell functions need to be promoted over cancerous bone-cell functions.Keywords: polymers, cancer, anti-cancer, surface nanometer features, bone, surface wettability

Lijuan Zhang1, Young Wook Chun2, Thomas J Webster21Department of Chemistry and 2Division of Engineering, Brown University, Providence, RI USAAbstract: Poly(lactic-co-glycolic) acid (PLGA) has been widely used as a biomaterial in regenerative medicine because of its biocompatibility and biodegradability properties. Previous studies have shown that cells (such as bladder smooth muscle cells, chondrocytes, and osteoblasts) respond differently to nanostructured PLGA surfaces compared with nanosmooth surfaces. The purpose of the present in vitro research was to prepare PLGA films with various nanometer surface features and determine whether lung cancer epithelial cells respond differently to such topographies. To create nanosurface features on PLGA, different sized (190 nm, 300 nm, 400 nm, and 530 nm diameter) polystyrene beads were used to cast polydimethylsiloxane (PDMS) molds which were used as templates to create nanofeatured PLGA films. Atomic force microscopy (AFM) images and root mean square roughness (RMS) values indicated that the intended spherical surface nanotopographies on PLGA with RMS values of 2.23, 5.03, 5.42, and 36.90 nm were formed by employing 190, 300, 400, and 530 nm beads. A solution evaporation method was also utilized to modify PLGA surface features by using 8 wt% (to obtain an AFM RMS value of 0.62 nm) and 4 wt% (to obtain an AFM RMS value of 2.23 nm) PLGA in chloroform solutions. Most importantly, lung cancer epithelial cells adhered less on the PLGA surfaces with RMS values of 0.62, 2.23, and 5.42 nm after four hours of culture compared with any other PLGA surface created here. After three days, PLGA surfaces with an RMS value of 0.62 nm had much lower cell density than any other sample. In this manner, PLGA with specific nanometer surface features may inhibit lung cancer cell density which may provide an important biomaterial for the treatment of lung cancer (from drug delivery to regenerative medicine).Keywords: nanotechnology, cancer, nanotopography, lung carcinoma

Chong Yao,1,* Zongguang Tai,1,* Xiaoyu Wang,1,* Jiyong Liu,1 Quangang Zhu,1,2 Xin Wu,3 Lijuan Zhang,1 Wei Zhang,4 Jing Tian,1 Yuan Gao,1 Shen Gao1 1Department of Pharmaceutics, Changhai Hospital, Second Military Medical University, 2Department of Pharmacy, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, 3Department of Pharmaceutics, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, 4Department of Pharmaceutics, Shanghai Pulmonary Hospital, Shanghai Tongji University, Shanghai, People’s Republic of China *These authors contributed equally tothis work Abstract: Low efficiency and significant toxicity are the main obstacles to successful gene delivery. We have developed a cationic reduction-responsive vector based on a disulfide cross-linked stearylated polyarginine peptide modified with histidine (C-SHR) for DNA delivery. The structure of the C-SHR was characterized, and the in vitro and in vivo transfection efficiency and cytotoxicity of C-SHR/plasmid DNA complexes were examined. Compared with non-cross-linked stearylated polyarginine peptide (SHR), C-SHR increased the intracellular uptake and dissociation behavior of the complexes. In addition, the gene transfection efficiency of C-SHR/plasmid DNA complexes in HEK293 and HeLa cells was improved and was comparable with that of bPEI-25K/plasmid DNA complexes, and the cytotoxicity of C-SHR was significantly less than that of bPEI-25K. Importantly, the in vivo gene transfection efficiency of C-SHR/plasmid DNA complexes was five fold higher than that of SHR/plasmid DNA complexes, suggesting that C-SHR is an efficient non-viral vector for DNA delivery.Keywords: polyarginine, histidine, stearyl, reduction-responsive, peptide, DNA delivery

Can Yang Zhang, Di Xiong, Yao Sun, Bin Zhao, Wen Jing Lin, Li Juan Zhang School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, Guangdong Province, People’s Republic of China Abstract: A novel amphiphilic triblock pH-sensitive poly(ß-amino ester)-g-poly(ethylene glycol) methyl ether-cholesterol (PAE-g-MPEG-Chol) was designed and synthesized via the Michael-type step polymerization and esterification condensation method. The synthesized copolymer was determined with proton nuclear magnetic resonance and gel permeation chromatography. The grafting percentages of MPEG and cholesterol were determined as 10.93% and 62.02%, calculated from the area of the characteristic peaks, respectively. The amphiphilic copolymer was confirmed to self-assemble into core/shell micelles in aqueous solution at low concentrations. The critical micelle concentrations were 6.92 and 15.14 mg/L at pH of 7.4 and 6.0, respectively, obviously influenced by the changes of pH values. The solubility of pH-responsive PAE segment could be transformed depending on the different values of pH because of protonation–deprotonation of the amino groups, resulting in pH sensitivity of the copolymer. The average particle size of micelles increased from 125 nm to 165 nm with the pH decreasing, and the zeta potential was also significantly changed. Doxorubicin (DOX) was entrapped into the polymeric micelles with a high drug loading level. The in vitro DOX release from the micelles was distinctly enhanced with the pH decreasing from 7.4 to 6.0. Toxicity testing proved that the DOX-loaded micelles exhibited high cytotoxicity in HepG2 cells, whereas the copolymer showed low toxicity. The results demonstrated how pH-sensitive PAE-g-MPEG-Chol micelles were proved to be a potential vector in hydrophobic drug delivery for tumor therapy. Keywords: micelle, pH-sensitive, cholesterol, poly(ß-amino ester), drug delivery