Suchergebnisse

Qunyou Tan,1,* Dan He,2,* Mingjun Wu,2,* Lin Yang,3 Yong Ren,4 Juan Liu,2 Jingqing Zhang,21Department of Thoracic Surgery, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, 2Medicine Engineering Research Center, Chongqing Medical University, Chongqing, 3Chongqing Institute for Food and Drug Control, Chongqing, 4Center of Drug Discovery, Nanjing Normal University, Nanjing, People's Republic of China*These authors contributed equally to this workBackground: The purpose of this study was to develop, characterize, and investigate a molecular inclusion complex containing rifaldazine with good solubility and antibacterial activity.Methods: Rifaldazine, a lipophilic molecule, was encapsulated into the hydrophobic cavity of ß-cyclodextrin to form a molecular inclusion complex (RAABCD) with good solubility. RAABCD was prepared in a short time using a solid-state grinding method. The inclusion ratio, binding constant, and change in Gibbs free energy were determined by a phase solubility diagram and/or ultraviolet-visible spectroscopy. Differential scanning calorimetry and Fourier transform infrared spectroscopy of RAABCD were performed. Morphological features of RAABCD were observed by photomicroscopy. The most likely optimal configuration for RAABCD was simulated by computer modeling. Broth macrodilution testing was done to investigate the antibacterial activity of RAABCD.Results: The inclusion ratio, binding constant, and change in Gibbs free energy, determined by a phase solubility diagram and/or ultraviolet-visible spectroscopy were 1:1, 288.33/261.33 L/mol, and 32.29/31.73 kJ/mol, respectively. Differential scanning calorimetry and Fourier transformed infrared spectra of RAABCD confirmed the molecular interaction between rifaldazine and ß-cyclodextrin. The morphological difference between irregular and amorphous-shaped RAABCD and columnar-shaped rifaldazine further confirmed the molecular encapsulation of rifaldazine. The most likely optimal configuration for RAABCD was confirmed by computer modeling. Broth macrodilution testing indicated that RAABCD had good antibacterial activity.Conclusion: RAABCD had improved solubility and good activity, and might be a promising alternative for treatment of a range of bacterial infections.Keywords: rifaldazine, cyclodextrin inclusion complex, stoichiometric relationships, differential scanning calorimetry, Fourier transform infrared spectra, computer modeling

In 1999, a company in Israel did what no one thought could be done – it struck natural gas and lots of it. Since then, two of the largest offshore natural gas fields have been found in Israel's waters, disproving the belief that Moses led the Jews to the only Middle Eastern country to not have petroleum. In 2011, it found what is believed to hold 250 billion barrels of shale oil – an amount that rivals the 260 billion barrels of crude oil in Saudi Arabia. Most economists argue, however, that this is not good news for Israel due to the "resource curse." The concept claims that finding an abundant amount of natural resources actually harms the local economy, politics and society as a whole through means of abuse of power, manipulation and pure disregard for societal welfare. This usually is applied to small, poor and previously corrupt governments. Currently, Israel is in a good position to avoid the resource curse. It is a militarily and economically strong, democratic nation. It has many resources available to it as well as lessons from the past to help it avoid the turmoil that historically faces nations with newfound petroleum wealth. This paper argues that not only can Israel beat the resource curse, but that the concept of the resource curse itself is flawed. Through historical examples of Nigeria and Canada, it is proven that not only small and weak governments fall victim to the greed and temptation that follows new resource wealth. The United States and Norway will show that with correct policy response, governments can avoid the curse highlighting the fact that an avoidable "curse" is, in fact, no "curse" at all. Finally, this paper will outline the appropriate political response for Israel containing several policies that limit sector transfers and exports as well as outlines the establishment of two sovereign wealth funds. Israel, like others before, can avoid the resource curse and boost all parts of its economy by taking several intricate steps.

Qunyou Tan,1,* Rong Jiang,3,* Meiling Xu,2,4,* Guodong Liu,5,* Songlin Li,1 Jingqing Zhang21Department of Thoracic Surgery, Institute of Surgery Research, Daping Hospital, Third Military Medical University, 2Medicine Engineering Research Center, Chongqing Medical University, 3Stem Cells and Tissue Engineering Research, Chongqing Medical University, 4Department of Pharmacy, Chongqing Emergency Medical Center, 5Eighth Department, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China*These authors contributed equally to this workBackground: Pyridostigmine bromide (3-[[(dimethylamino)-carbonyl]oxy]-1-methylpyridinium bromide), a reversible inhibitor of cholinesterase, is given orally in tablet form, and a treatment schedule of multiple daily doses is recommended for adult patients. Nanotechnology was used in this study to develop an alternative sustained-release delivery system for pyridostigmine, a synthetic drug with high solubility and poor oral bioavailability, hence a Class III drug according to the Biopharmaceutics Classification System. Novel nanosized pyridostigmine-poly(lactic acid) microcapsules (PPNMCs) were expected to have a longer duration of action than free pyridostigmine and previously reported sustained-release formulations of pyridostigmine.Methods: The PPNMCs were prepared using a double emulsion-solvent evaporation method to achieve sustained-release characteristics for pyridostigmine. The preparation process for the PPNMCs was optimized by single-factor experiments. The size distribution, zeta potential, and sustained-release behavior were evaluated in different types of release medium.Results: The optimal volume ratio of inner phase to external phase, poly(lactic acid) concentration, polyvinyl alcohol concentration, and amount of pyridostigmine were 1:10, 6%, 3% and 40 mg, respectively. The negatively charged PPNMCs had an average particle size of 937.9 nm. Compared with free pyridostigmine, PPNMCs showed an initial burst release and a subsequent very slow release in vitro. The release profiles for the PPNMCs in four different types of dissolution medium were fitted to the Ritger-Peppas and Weibull models. The similarity between pairs of dissolution profiles for the PPNMCs in different types of medium was statistically significant, and the difference between the release curves for PPNMCs and free pyridostigmine was also statistically significant.Conclusion: PPNMCs prepared by the optimized protocol described here were in the nanometer range and had good uniformity, with significantly slower pyridostigmine release than from free pyridostigmine. This novel sustained-release delivery nanosystem for pyridostigmine might alleviate the need to identify new acetylcholinesterase inhibitors.Keywords: nanosized microcapsules, process optimization, characteristics, sustained-release, pyridostigmine bromide

Guichen Zhou,1,2,* Ying Lu,1,* He Zhang,1,* Yan Chen,1 Yuan Yu,1 Jing Gao,1 Duxin Sun,3 Guoqing Zhang,2 Hao Zou,1 Yanqiang Zhong1 1Department of Pharmaceutical Science, Second Military Medical University, Shanghai, People's Republic of China; 2Department of Pharmacy, East Hospital of Hepatobiliary Surgery, Shanghai, People's Republic of China; 3Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI, USA*These authors contributed equally to this workPurpose: The aim of this report was to introduce a novel "core-membrane" microgel drug-delivery device for spontaneously pulsed release without any external trigger.Methods: The microgel core was prepared with alginate and chitosan. The semipermeable membrane outside the microgel was made of polyelectrolytes including polycation poly(allylamine hydrochloride) and sodium polystyrene sulfonate. The drug release of this novel system was governed by the swelling pressure of the core and the rupture of the outer membrane.Results: The size of the core-membrane microgel drug-delivery device was 452.90 ± 2.71 µm. The surface charge depended on the layer-by-layer coating of polyelectrolytes, with zeta potential of 38.6 ± 1.4 mV. The confocal microscope exhibited the layer-by-layer outer membrane and inner core. The in vitro release profile showed that the content release remained low during the first 2.67 hours. After this lag time, the cumulative release increased to 80% in the next 0.95 hours, which suggested a pulsed drug release. The in vivo drug release in mice showed that the outer membrane was ruptured at approximately 3 to 4 hours, as drug was explosively released.Conclusion: These data suggest that the encapsulated substance in the core-membrane microgel delivery device can achieve a massive drug release after outer membrane rupture. This device was an effective system for pulsed drug delivery.Keywords: polyelectrolyte, chitosan–alginate, microgels, layer-by-layer, pulsed drug delivery

Shuang Jiang,1 Xiaobo Wang,1 Ronggang Xi,1 Yingge Zhang2 1210th Hospital of People Liberation Army, Dalian, People's Republic of China; 2Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing, People's Republic of China Abstract: Atomic force microscopy (AFM) was applied for obtaining structural information about acetylcholinesterase (AChE) tetramer (AChE G4) before and after reaction with S-acetylcholine iodide (S-ACh), in the presence or absence of propidium iodide (PI), an inhibitor for peripheral anionic sites (PAS). An iced-bath ultrasound was used to prepare the phospholipid membrane. Ves-fusion technique was applied for incorporating AChE G4 in a lipid layer on mica. Before reaction with substrates, the single AChE G4 particle was ellipsoid in shape with a clear border. It had a smooth surface with a central projection. The four subunits of a single enzyme particle were arranged tightly (no separated subunits being found, with an average size of 89 ± 7 nm in length, 68 ± 9 nm in width, and 6 ± 3 nm in height). After reaction with S-ACh in the absence of PI, the loose arrangement of subunits of AChE G4 was seen, with an average size of 104 ± 7 nm in length, 91 ± 5 nm in width, and 8 ± 2 nm in height. Also there was free-flowing space amongst the four subunits of the AChE G4. This was consistent with the results of the X-ray diffraction crystallography and molecular dynamics studies. The apparent free space was the central path of AChE G4, changing from small to big, to small, to lateral door appearance, with an average size of 60 ± 5 nm in length and 51 ± 9 nm in width. The size of lateral door was 52 ± 5 nm in width and 32 ± 3 nm in depth on average. In the presence of PI, S-ACh could not cause topological structure changes of AChE G4. AFM verified that the central path might govern the turnover of the enzyme morphologically, and the interactions between PI and S-ACh might gate the creation of a central path and the opening of ACG in monomer; and the combination of S-ACh with peripheral anionic sites is conducive to the opening of ACG while PI can inhibit this action. Resolution at the inframolecular level is favorable in providing substantial information on how the spatial structure is adapted to the high efficiency of AChE molecules. Keywords: AChE, AFM, ACG, Ves-fusion, phospholipid membrane

Qingyi He,2,* Xiaohui Wang,3,* Xing Zhang,4 Huifang Han,4 Baosan Han,5 Jianzhong Xu,2 Kanglai Tang,2 Zhiren Fu,1 Hao Yin1,4 1Department of Surgery, Organ Transplant Center, Shanghai Changzheng Hospital, Shanghai Second Military Medical School, 2Department of Orthopedic Surgery, Southwest Hospital, Third Military Medical University, Chongqing, 3Department of General Surgery, Bayinguoleng Mengguzizhizhou People's Hospital, Korla, Xinjiang, People's Republic of China; 4Department of Surgery, The University of Chicago, Chicago, IL, USA; 5Department of Surgery, Shanghai Xinhua Hospital, Shanghai, People's Republic of China*These authors contributed equally to this workAbstract: The traditional xenograft subcutaneous pancreatic cancer model is notorious for its low incidence of tumor formation, inconsistent results for the chemotherapeutic effects of drug molecules of interest, and a poor predictive capability for the clinical efficacy of novel drugs. These drawbacks are attributed to a variety of factors, including inoculation of heterogeneous tumor cells from patients with different pathological histories, and use of poorly defined Matrigel®. In this study, we aimed to tissue-engineer a pancreatic cancer model that could readily cultivate a pancreatic tumor derived from highly homogenous CD24+CD44+ pancreatic cancer stem cells delivered by a well defined electrospun scaffold of poly(glycolide-co-trimethylene carbonate) and gelatin. The scaffold supported in vitro tumorigenesis from CD24+CD44+ cancer stem cells for up to 7 days without inducing apoptosis. Moreover, CD24+CD44+ cancer stem cells delivered by the scaffold grew into a native-like mature pancreatic tumor within 8 weeks in vivo and exhibited accelerated tumorigenesis as well as a higher incidence of tumor formation than the traditional model. In the scaffold model, we discovered that oxaliplatin-gemcitabine (OXA-GEM), a chemotherapeutic regimen, induced tumor regression whereas gemcitabine alone only capped tumor growth. The mechanistic study attributed the superior antitumorigenic performance of OXA-GEM to its ability to induce apoptosis of CD24+CD44+ cancer stem cells. Compared with the traditional model, the scaffold model demonstrated a higher incidence of tumor formation and accelerated tumor growth. Use of a tiny population of highly homogenous CD24+CD44+ cancer stem cells delivered by a well defined scaffold greatly reduces the variability associated with the traditional model, which uses a heterogeneous tumor cell population and poorly defined Matrigel. The scaffold model is a robust platform for investigating the antitumorigenesis mechanism of novel chemotherapeutic drugs with a special focus on cancer stem cells.Keywords: pancreatic cancer, cancer stem cell, electrospun scaffold, gemcitabine, oxaliplatin

Jingjing Dong,1,* Geng Cui,2,* Long Bi,1,* Jie Li,3 Wei Lei11Institute of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, People's Republic of China; 2Institute of Orthopedics, General Hospital of PLA, Beijing, People's Republic of China; 3Institute of Gynecology and Obstetrics, General Hospital of PLA, Beijing, People's Republic of China*These authors contributed equally to this workAbstract: In order to improve the mechanical and biological properties of calcium phosphate cement (CPC, nanometer-biomaterial) for bone reconstruction in the rabbit femoral defect model, fibrin glue (FG, the natural product, purified from the blood) was introduced at three different ratios. The CPC powder and the FG solution were mixed, respectively, at the powder/liquid (P/L) ratios (g/mL) of 1:1, 3:1, and 5:1 (g/mL), and pure CPC was used as a control. After being implanted into the femoral defect in rabbit, the healing process was evaluated by micro-computed tomography scan, biomechanical testing, and histological examination. By micro-computed tomography analysis, the P/L ratio of 1:1 (g/mL) group indicated the largest quantity of new bone formation at 4 weeks, 8 weeks, and 12 weeks after implantation, respectively. Bone volume per trabecular volume of the 1:1 group was highest in the four groups, which was 1.45% ± 0.42%, 7.35% ± 1.45%, and 29.10% ± 1.67% at 4 weeks, 8 weeks, and 12 weeks after the operation, respectively. In the biomechanical tests, the compressive strength and the elastic modulus of the three CPC–FG groups were much higher than those of the pure CPC group at the determined time point (P < 0.05). The histological evaluation also showed the best osseointegration in the 1:1 group at 4 weeks, 8 weeks, and 12 weeks after the operation, respectively. In the 1:1 group, the bone grew into the pore of the cement in the laminar arrangement and connected with the cement tightly at the 12th week after the operation. This present study indicated that the CPC–FG composite at the P/L ratio of 1:1 (g/mL) stimulated bone regeneration better than any other designed group, which suggested that CPC–FG at the P/L ratio of 1:1 has significant potential as the bioactive material for the treatment of bone defects.Keywords: calcium phosphate cements, fibrin glue, bone reconstruction, micro-CT, biomechanical testing, histological examination

Yan Chen,* Ji Sun,* Ying Lu, Chun Tao, Jingbin Huang, He Zhang, Yuan Yu, Hao Zou, Jing Gao, Yanqiang Zhong Department of Pharmaceutical Science, School of Pharmacy, The Second Military Medical University, Shanghai, People's Republic of China *These authors contributed equally to this work Abstract: pH-sensitive liposomes represent an effective gene vector in cancer therapy. However, their use is greatly hampered by their relatively low transfection efficiency. To improve the transfection efficiency of pH-sensitive liposomes, we prepared complexes containing 3β-[N-(N',N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidyl ethanolamine (DOPE) liposomes and pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and DOPE, and evaluated the influence of various factors on plasmid DNA (pDNA) transfection efficiency. All DC-Chol/DOPE liposome/pDNA and pH-sensitive liposome complexes showed similarly potent pH sensitivity. In the presence of serum-containing medium, two optimized complexes of DC-Chol/DOPE liposomes/pDNA and pH-sensitive PEGylated liposomes showed high transfection efficiency of 22.94% and 20.07%, respectively. Notably, DC-Chol/DOPE (2:3) liposomes/pH-sensitive PEGylated (1%) liposome complexes with a charge ratio of 1:1 (m/m [+/-]) showed enhanced accumulation in tumors in vivo. Our results show the influence of various factors on pDNA transfection efficiency in complexes of DC-Chol/DOPE liposomes and pH-sensitive PEGylated liposomes. Understanding of such mechanisms will lead to better design of complexes of DC-Chol/DOPE liposomes and pH-sensitive liposomes for gene therapy. Keywords: cationic liposomes, pH-sensitive liposomes, pDNA, transfection, PEGylated

Kaimin Wu,1,* Jie Xu,2,* Mengyuan Liu,1 Wen Song,1 Jun Yan,1 Shan Gao,3 Lingzhou Zhao,2 Yumei Zhang1 1Department of Prosthetic Dentistry, 2Department of Periodontology and Oral Medicine, School of Stomatology, The Fourth Military Medical University, Xi'an, People's Republic of China; 3The Interdisciplinary Nanoscience Center and Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark; School of Stomatology, Tianjin Medical University, Tianjin, People's Republic of China*Both authors contributed equally to this workAbstract: MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 µL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and -20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall, the miRNA reverse transfection formulation developed in this study is a promising approach for miRNA transfection which can control stem cell fate and is suitable for loading miRNAs onto various biomaterials.Keywords: microRNAs, lyophilization, tissue culture plate, mesenchymal stem cells, osteogenic differentiation

De-Xin Wang,1,* Yao He,2,* Long Bi1,* Ze-Hua Qu,2 Ji-Wei Zou,1 Zhen Pan,2 Jun-Jun Fan,1 Liang Chen,2 Xin Dong,1 Xiang-Nan Liu,2 Guo-Xian Pei,1 Jian-Dong Ding,21Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi'an, People's Republic of China; 2State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai, People's Republic of China*These authors contributed equally to this workPurpose: Poly(lactic-co-glycolic acid) (PLGA) is excellent as a scaffolding matrix due to feasibility of processing and tunable biodegradability, yet the virgin scaffolds lack osteoconduction and osteoinduction. In this study, nano-hydroxyapatite (nHA) was coated on the interior surfaces of PLGA scaffolds in order to facilitate in vivo bone defect restoration using biomimetic ceramics while keeping the polyester skeleton of the scaffolds.Methods: PLGA porous scaffolds were prepared and surface modification was carried out by incubation in modified simulated body fluids. The nHA coated PLGA scaffolds were compared to the virgin PLGA scaffolds both in vitro and in vivo. Viability and proliferation rate of bone marrow stromal cells of rabbits were examined. The constructs of scaffolds and autogenous bone marrow stromal cells were implanted into the segmental bone defect in the rabbit model, and the bone regeneration effects were observed.Results: In contrast to the relative smooth pore surface of the virgin PLGA scaffold, a biomimetic hierarchical nanostructure was found on the surface of the interior pores of the nHA coated PLGA scaffolds by scanning electron microscopy. Both the viability and proliferation rate of the cells seeded in nHA coated PLGA scaffolds were higher than those in PLGA scaffolds. For bone defect repairing, the radius defects had, after 12 weeks implantation of nHA coated PLGA scaffolds, completely recuperated with significantly better bone formation than in the group of virgin PLGA scaffolds, as shown by X-ray, Micro-computerized tomography and histological examinations.Conclusion: nHA coating on the interior pore surfaces can significantly improve the bioactivity of PLGA porous scaffolds.Keywords: PLGA, nano-hydroxyapatite, bone tissue engineering, BMSCs, bone defect

Cheng-Wei Chen,1 Ming-Kung Yeh,2 Chia-Yang Shiau,3 Chiao-Hsi Chiang,4,* Da-Wen Lu5,*1Chengwei Biotechnology Co, Ltd, 2Bureau of Pharmaceutical Affairs, Military of National Defense Medical Affairs Bureau, 3Graduate Institute of Medical Sciences, 4School of Pharmacy, National Defense Medical Center, 5Department of Ophthalmology, Tri-Service General Hospital, Taipei, Taiwan *These authors contributed equally to this workBackground: The purpose of this study was to demonstrate the effectiveness of an integrin peptide ligand-labeled liposomal delivery system loaded with vascular endothelial growth factor (VEGF)-siRNA in a model study of gene therapy for retinopathy using human retinal pigment epithelial cells.Methods: Arg(R)-Gly(G)-Asp(D) motif peptide conjugating polyethylene glycol modified (RGD-PEGylated) liposomes were prepared using a thin-film hydration method and optimized for surface charge, particle size, small interfering RNA (siRNA) load, and entrapment efficiency. Reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays were used to determine VEGF levels in retinal pigment epithelial cells. Cytotoxicity was determined using the 3-[4, 5-dimethylthiazol-2-yl]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and flow cytometry.Results: Physicochemical properties, including particle size, zeta potential, and siRNA load, of the prepared RGD-PEGylated liposomes and their entrapment efficiency were determined to be within the following ranges: 123.8–234.1 nm, 17.31–40.09 mV, 5.27%–6.33%, and >97%, respectively. RGD-PEGylated liposome-mediated fluorescent-labeled siRNA delivery demonstrated significantly enhanced cellular uptake, and 3 mol% RGD-PEGylated liposomes (having 3β-[N-(N´, N´-dimethylaminoethane) carbamoyl] cholesterol (DC-cholesterol) DSPE and DSPE-PEG(2000)-RGD with molar ratio of 50/47/3) were shown to have better efficacy with regard to specificity for retinal pigment epithelial cells, reduced cytotoxicity, and knockdown of the target molecule.Conclusion: By integrin receptor-mediated endocytosis, 3 mol% RGD-PEGylated liposomes were shown to be a suitable vector when loaded with VEGF-siRNA for efficient downregulation of VEGF in retinal pigment epithelial cells at both the protein and gene levels. This integrin ligand-modified liposomal delivery system has therapeutic potential for ocular gene therapy.Keywords: vascular endothelial growth factor, siRNA delivery, liposome, retinal pigment epithelial cellsA Letter to the Editor has been received and published for this article.

Shih-shiung Huang,1 I-Hsun Li,2,3 Po-da Hong,1 Ming-kung Yeh1,2 1Biomedical Engineering Program, Graduate Institute of Applied Science and Technology, and Department of Materials Science and Engineering, National Taiwan University of Science and Technology, Taiwan; 2School of Pharmacy, National Defence Medical Center and Bureau of Pharmaceutical Affairs, Military of National Defence Medical Affairs Bureau, Taipei, Taiwan; 3Department of Pharmacy Practice, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan Abstract: Dengue virus results in dengue fever or severe dengue hemorrhagic fever/dengue shock syndrome in humans. The purpose of this work was to develop an effective antidengue virus delivery system, by designing poly (dl-lactic-co-glycolic) acid/polyethylene glycol (PLGA/PEG) microspheres using a double-emulsion solvent extraction method, for vaccination therapy based on locally and continuously sustained biological activity. Nonstructural protein 1 (NS1) in deoxyribonucleic acid (DNA) vaccine–loaded PLGA/PEG microspheres exhibited a high loading capacity (4.5% w/w), yield (85.2%), and entrapment efficiency (39%), the mean particle size 4.8 µm, and a controlled in vitro release profile with a low initial burst (18.5%), lag time (4 days), and continued released protein over 70 days. The distribution of protein on the microspheres surface, outer layer, and core were 3.0%, 28.5%, and 60.7%, respectively. A release rate was noticed to be 1.07 µg protein/mg microspheres/day of protein release, maintained for 42 days. The cumulative release amount at Days 1, 28, and 42 was 18.5, 53.7, and 62.66 µg protein/mg microspheres, respectively. The dengue virus challenge in mice test, in which mice received one dose of 20 µg NS1 protein content of microspheres, in comparison with NS1 protein in Al(OH)3 or PBS solution, was evaluated after intramuscular immunization of BALB/c mice. The study results show that the greatest survival was observed in the group of mice immunized with NS1 protein–loaded PLGA/PEG microspheres (100%). In vivo vaccination studies also demonstrated that NS1 protein–loaded PLGA/PEG microspheres had a protective ability; its steady-state immune protection in rat plasma changed from 4,443 ± 1,384 pg/mL to 10,697 ± 3,197 pg/mL, which was 2.5-fold higher than that observed for dengue virus in Al(OH)3 at 21 days. These findings strongly suggest that NS1 protein–loaded PLGA/PEG microspheres offer a new therapeutic strategy in optimizing the vaccine incorporation and delivery properties of these potential vaccine targeting carriers. Keywords: dengue virus, adjuvant, NS1 protein–loaded PLGA/PEG microspheres, vaccination

Li Deng,1,* Yingying Zhang,1,* Lulu Ma,1,5,* Xiaolong Jing,1,3 Xingfa Ke,1,3 Jianhao Lian,1,3 Qiang Zhao,1,3 Bo Yan,1,3 Jinfeng Zhang,4 Jianzhong Yao,2 Jianming Chen1 1Department of Pharmaceutical Science, 2Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai, People's Republic of China; 3Department of Pharmacy, Fujian University of Traditional Chinese Medicine, Fujian, People's Republic of China; 4Shanghai TCM Integrated Hospital, Shanghai, People's Republic of China; 5Department of Pharmacy, First Affiliated Hospital of Bengbu Medical College, Bengbu, People's Republic of China *These authors contributed equally to this paper Background: Targeted liposome-polycation-DNA complex (LPD), mainly conjugated with antibodies using functionalized PEG derivatives, is an effective nanovector for systemic delivery of small interference RNA (siRNA). However, there are few studies reporting the effect of different conjugation linkers on LPD for gene silencing. To clarify the influence of antibody conjugation linkers on LPD, we prepared two different immunoliposomes to deliver siRNA in which DSPE-PEG-COOH and DSPE-PEG-MAL, the commonly used PEG derivative linkers, were used to conjugate anti-EGFR Fab' with the liposome. Methods: First, 600 µg of anti-EGFR Fab' was conjugated with 28.35 µL of a micelle solution containing DSPE-PEG-MAL or DSPE-PEG-COOH, and then post inserted into the prepared LPD. Various liposome parameters, including particle size, zeta potential, stability, and encapsulation efficiency were evaluated, and the targeting ability and gene silencing activity of TLPD-FPC (DSPE-PEG-COOH conjugated with Fab') was compared with that of TLPD-FPM (DSPE-PEG-MAL conjugated with Fab') in SMMC-7721 hepatocellular carcinoma cells. Results: There was no significant difference in particle size between the two TLPDs, but the zeta potential was significantly different. Further, although there was no significant difference in siRNA encapsulation efficiency, cell viability, or serum stability between TLPD-FPM and TLPD-FPC, cellular uptake of TLPD-FPM was significantly greater than that of TLPD-FPC in EGFR-overexpressing SMMC-7721 cells. The luciferase gene silencing efficiency of TLPD-FPM was approximately three-fold high than that of TLPD-FPC. Conclusion: Different conjugation linkers whereby antibodies are conjugated with LPD can affect the physicochemical properties of LPD and antibody conjugation efficiency, thus directly affecting the gene silencing effect of TLPD. Immunoliposomes prepared by DSPE-PEG-MAL conjugation with anti-EGFR Fab' are more effective than TLPD containing DSPE-PEG-COOH in targeting hepatocellular carcinoma cells for siRNA delivery. Keywords: liposome-polycation-DNA, anti-EGFR Fab', immunoliposomes, small interfering RNA delivery, conjugation technology, hepatocellular carcinoma

Yang Guo,1 Zhuoli Zhang,1 Dong-Hyun Kim,1,5 Weiguo Li,1 Jodi Nicolai,1 Daniel Procissi,1 Yi Huan,2 Guohong Han,3 Reed A Omary,1,4,5 Andrew C Larson1,4,5 1Department of Radiology, Northwestern University, Chicago, IL, USA; 2Department of Radiology, Xijing Hospital, Fourth Military Medical University, Xi'an, People's Republic of China; 3Department of Digestive Interventional Radiology, Xijing Hospital, Fourth Military Medical University, Xi'an, People's Republic of China; 4Department of Biomedical Engineering, Northwestern University, Chicago, IL, USA; 5Robert H Lurie Comprehensive Cancer Center, Chicago, IL, USA Purpose: Photothermal ablation is a minimally invasive approach, which typically involves delivery of photothermal sensitizers to targeted tissues. The purpose of our study was to demonstrate that gold nanoparticles are phagocytosed by pancreatic cancer cells, thus permitting magnetic resonance imaging (MRI) of sensitizer delivery and photothermal ablation. Patients and methods: Iron-oxide core/gold-shell nanoparticles (GoldMag®, 30 nm diameter; Xi'an GoldMag Biotechnology Co, Xi'an, People's Republic of China) were used. In a 96-well plate, 3 × 104 PANC-1 (human pancreatic cancer cell line) cells were placed. GoldMag (0, 25, or 50 µg/mL) was added to each well and 24 hours allowed for cellular uptake. Samples were then divided into two groups: one treated with photothermal ablation (7.9 W/cm2) for 5 minutes, the other not treated. Photothermal ablation was performed using laser system (BWF5; B&W Tek, Inc, Newark, DE, USA). Intraprocedural temperature changes were measured using a fiber optic temperature probe (FTP-LN2; Photon Control Inc, Burnaby, BC, Canada). After 24 hours, the remaining number of viable cells was counted using trypan blue staining; cell proliferation percentage was calculated based on the total number of viable cells after treatment compared with control. MRI of GoldMag uptake was performed using a 7.0T ClinScan system (Bruker BioSpin, Ettlingen, Germany). Results: Temperature curves demonstrated that with increased GoldMag uptake, laser irradiation produced higher temperature elevations in the corresponding samples; temperature elevations of 12.89°C, 35.16°C, and 79.51°C were achieved for 0, 25, and 50 µg/mL GoldMag. Without photothermal ablation, the cell proliferation percentage changed from 100% to 71.3% and 47.0% for cells treated with 25 and 50 µg/mL GoldMag. Photothermal ablation of PANC-1 cells demonstrated an effective treatment response, specifically a reduction to only 61%, 21.9%, and 2.3% cell proliferation for cells treated with 0, 25, and 50 µg/mL GoldMag. MRI was able to visualize GoldMag uptake within PANC-1 cells. Conclusion: Our findings suggest that photothermal ablation may be effective in the treatment of pancreatic cancer. GoldMag nanoparticles could serve as photothermal sensitizers, and MRI is feasible to quantify delivery. Keywords: photothermal ablation therapy, hybrid nanoparticles, magnetic resonance imaging