The history of modern psychiatry in China began at the end of the nineteenth century, as a result of the work of missionaries. Soochow was one of the first cities to establish a hospital for the treatment of mental patients, but historians knew little about it. It provided a valuable service from 1898 to 1937. In the 1930s, there were 200 beds in the psychiatry and neurology section, making it the most influential psychiatric hospital in East China. After Soochow was occupied by the Japanese army in 1937, the hospital was destroyed and shut down.

Aim: To develop a nanocarrier for targeted delivery of agents to the cartilage. Materials & methods: Chondrocyte affinity peptide modified PEGylated polyamidoamine conjugates (CAP-PEG-PAMAM) were prepared and rhodamine B isothiocyanate (RB) fluorophore was linked on them for comparative biological tracing and profiling. Results: CAP4-PP-RB exhibited much more efficient cellular uptake in vitro than that of PEG-PAMAM-RB. Both the conjugates were likely internalized by chondrocytes via clathrin and caveolin co-mediated endocytosis, and delivered to lysosomes. In vivo imaging demonstrated the fluorescein-labeled nanocarrier was capable to persist in the joint cavity of rats for a prolonged time. Furthermore, the CAP4-PEG-PAMAM showed a good biocompatibility and enhanced penetration effects in vivo. Conclusion: CAP-PEG-PAMAM could be an effective nanocarrier for intra-articular delivery of agents to cartilage.

目的:构建双胍基(biguanidine,BIG)和聚乙二醇修饰的pH/还原敏感聚酰胺-胺(PAMAM)聚合物(PSSPG),并考察其对阿霉素(DOX)细胞内递送的影响.方法:合成3种不同双胍基比例修饰的双胍基化聚酰胺-胺聚合物,物理包载抗肿瘤药物阿霉素制得3种载药复合物(PG25/DOX,PG50/DOX,PG100/DOX),考察双胍基化修饰程度对双胍基修饰聚酰胺-胺/阿霉素复合物细胞摄取的影响.随后制备PEG化聚酰胺-胺/阿霉素复合物和双胍基修饰PEG化聚酰胺-胺/阿霉素复合物,并对其粒径电位、载药量、包封率、细胞摄取、体外释放以及细胞毒性等进行考察.结果:3种双胍基修饰聚酰胺-胺/阿霉素复合物的粒径均在40 ～ 50 nm,具有较高的包封率和载药量;随双胍基修饰程度的增加,复合物摄取略有增加;体外释放结果表明二硫键修饰复合物具有明显pH/还原敏感性;与PEG化聚酰胺-胺/阿霉素复合物相比,双胍基修饰PEG化聚酰胺-胺/阿霉素复合物组体外细胞实验摄取增加,细胞毒性亦随之增加.结论:双胍基和PEG修饰的pH/还原敏感聚酰胺-胺载体可用于细胞对于阿霉素的高效转运.

In this study,a T7 modified redox-sensitive polyamide amine (T7-PEG-SS-PAMAM,T7-PSSP) conjugate with potential for liver cancer targeting was constructed for pyruvate kinase M2 (PKM2)-siRNA delivery.1H NMR was used to confirm the structural characters of the polymer.The particle size,polydispersity index and ξ potential of the T7-PSSP/siRNA complex were (119.8±0.76) nm,0.19±0.12 and (8.92±0.38) mV,respectively.The results of agarose gel electrophoresis indicated that the carrier-gene complex had a good stability and could protect siRNA from RNase degradation.Flow cytometry quantitative analysis confirmed that the modification of T7 peptide could significantly increase the uptake of siRNA in HepG2 cells.According to the semi-quantitative analysis results,siRNA was rapidly released from the PSSP/siRNA complex under the action of glutathione reductant,indicating that the PSSP/siRNA complex could efficiently release the delivered siRNA in a reducing environment.Real-time fluorescence quantitative detection results showed that T7-PSSP/siRNA complex displayed the highest PKM2 gene transfection and silencing efficiency compared to the negative control and other complexes groups.The above results showed that this bifunctional siRNA delivery system,T7-PSSP/siRNA complex,had both tumor-targeted and redox-sensitive property,it could effectively release siRNA in the intracellular environment to enhance siRNA knockout effect,and thus could be used as a potential nanocarrier for efficient siRNA delivery.

Partly PEGylated polyamidoamine (PAMAM) dendrimer was used as the nanocarrier for the cytoplasmic delivery of kartogenin (KGN) to induce chondrogenic differentiation of mesenchymal stem cells (MSCs). Here, KGN was conjugated to the surface of PAMAM and the end group of polyethylene glycol (PEG) to obtain PEG-PAMAM-KGN (PPK) and KGN-PEG-PAMAM (KPP) conjugate, respectively. The effects of PPK and KPP on the in vitro chondrogenic differentiation of MSCs were evaluated. KPP induced higher expression of chondrogenic markers than PPK and free KGN. In particular, after treatment of KPP, CBF β nuclear localization intensity was significantly increased, indicating enhanced efficacy of chondrogenesis. The fluorescein labeled PEG-PAMAM was capable to persist in the joint cavity for a prolonged time of both healthy and osteoarthritis (OA) rats. Thus, PEG-PAMAM could be a useful nanocarrier for intra-articular (IA) delivery of drug to treat OA.

目的 制备pH、还原双重敏感的基于聚酰胺-胺树状大分子(PAMAM)的纳米载体PSSP,用于联合携载化疗药物多柔比星(DOX)及耐药逆转剂依克立达(ELC),同时对其逆转乳腺癌多药耐药的效果进行体外评价.方法 采用红外光谱FTIR对载体进行结构表征;共聚焦显微镜考察载药纳米粒在细胞内的释药情况;流式细胞术和MTT实验分别考察联合载药纳米粒对乳腺癌多药耐药的逆转作用以及体外抗肿瘤活性.结果 成功制备联合载药PSSP/多柔比星/依克立达纳米粒,并通过细胞实验证实pH、还原双重敏感性.共聚焦定位实验表明载体进入细胞后主要集中在溶酶体,在其酸性条件下促发释药并扩散入核.罗丹明123外排实验表明,依克立达可显著增加MCF-7/ADR细胞内罗丹明123的蓄积量.并且PSSP/多柔比星/依克立达纳米粒对MCF-7/ADR细胞的细胞毒性显著大于游离药多柔比星以及PSSP/多柔比星纳米粒.结论 多柔比星和依克立达联载纳米粒逆转乳腺癌多药耐药的效果显著提高,且对肿瘤细胞的毒性增强,是一种有应用前景的抗肿瘤药物递送系统.

目的：构建组氨酸（His）修饰pH敏感响应的载抗肿瘤药物阿霉素（DOX）复合物PAMAM-His/DOX,并对其进行表征及初步评价。方法：通过控制His和PAMAM的投料比,合成3种不同His修饰程度的PAMAM-His载体;采用物理包埋的方式将DOX包载在PAMAM的疏水空腔内制备系列载药复合物。对各载药复合物的粒径、电位、包封率、载药量、体外释放行为等进行考察,并采用MTT法评价各复合物对乳腺癌细胞MCF-7的细胞毒性作用。结果：PAMAM-His/DOX复合物的粒径均在10nm左右;随着His修饰程度的增加,其Zeta电位降低,且对抗MCF-7的细胞毒性增加;体外释放实验结果表明复合物具有明显的pH酸敏感特性,并且在酸性条件下（pH5.0）,药物累积释放量与His修饰程度成正比。结论：本实验成功制备并初步评价了3种His修饰程度的PAMAM-His/DOX,为下一步的体内外抗肿瘤研究奠定了理论基础。

To solve the contradiction between long circulation time and effective intracellular drug release, redox and pH-responsive drug delivery system was developed by incorporated redox-sensitive disulfide linkage between poly(amidoamine) dendrimers (PAMAM) and poly(ethylene glycol) (PEG). Doxorubicin (DOX) was loaded into the hydrophobic core of the conjugates to prepare PAMAM-SS-PEG/DOX complexes (PSSP/DOX). In vitro release studies suggested that DOX release from PSSP/DOX complexes followed an redox and acid-triggered manner and increased with increasing PEGylation degree. In vitro cytotoxicity of PSSP/DOX complexes against B16 tumor cells increased with, while cellular uptake decreased with increasing PEGylation degree. Further, intracellular DOX release observation and measurement indicate that the intracellular DOX release played a critical role for the cytotoxicity of DOX-loaded PSSP conjugates. In addition, cellular entry mechanism of the PSSP/DOX study demonstrated that both clathrin- and caveolae-mediated endocytosis were the primary pathways for cellular entry of PSSP/DOX. Finally, in vivo study of PSSP/DOX complexes in B16 tumor-bearing mice indicate that PSSP/DOX could significantly improve antitumor efficiency and present a good safety. The redox and pH-responsive drug delivery system has been demonstrated to be a promising candidate for solid tumor therapy.Statement of SignificanceIn previous research, pH-sensitive diblock polymer of poly(ethylene glycol)-poly(2,4,6-trimethoxybenzy lidene-pentaerythritol carbonate) (PEG-PTMBPEC) was synthesized to facilitate the intracellular anticancer drug release. However, the nanoparticles based on PEG-PTMBPEC get into the tumor cells just relying on the EPR-mediated passive targeting resulting in the low drug accumulation. Therefore, cRGD peptide modified PEG-PTMBPEC polymeric micelles were developed for specific targeted delivery of doxorubicin (DOX) to neovascular cells and tumor cells simultaneously. The precise intracellular target site and effective drug concentration will contribute to enhancing the antitumor toxicity and reducing the systematic toxicity of DOX. The cRGD modified pH-sensitive micellar system is a promising vehicle for intracellular drug delivery to alpha v beta 3 integrin receptor overexpressed tumor cells and neovascular cells. (C) 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

An ideal drug delivery system for cancer therapy should be equipped with extended circulation, improved tumor targeting and controlled drug release, as well as low toxicity from the carrier. In this study, a multifunctional drug delivery system based on the PEGylated poly(amidoamine) (PAMAM) dendrimer was designed, and folate-PEGylation was applied to modify the dendrimer in order to enhance tumor selectivity. A series of acid-labile PAMAM-DOX conjugates (FPP-hyd-DOX) with different FA ligand ratios were successfully constructed. H-1 NMR, FTIR, DLS and TEM were used to describe the physicochemical characterization of PAMAM-DOX conjugates. Both in vitro drug release assay and subcellular localization, the conjugates exhibited an obvious pH-triggered drug release. The FPP-hyd-DOX 16/1 displayed much lower IC50 value than that of non-targeted PP-hyd-DOX. Through fluorescence microscopy and flow cytometry investigations, the cellular uptake of FPP-hyd-DOX 16/1 was obviously enhanced, compared with that of PP-hyd-DOX. The cellular uptake mechanism and subcellular localization study revealed that the conjugates were internalized by KB cells via FA receptor and clathrin co-mediated endocytosis, delivered to acidic lysosomes and triggered the release of DOX into nuclei to exert its cytotoxicity. These obtained results showed that FPP-hyd-DOX conjugations would be a promising drug delivery carrier for targeted cancer therapy. (C) 2015 Published by Elsevier B.V.

Stimuli-responsive nanocarriers attract wide attention because of the unique differences in microenvironment between solid tumors and normal tissues. Herein, we reported a novel cRGDyK peptide modified pH-sensitive nanoparticle system based on poly(ethylene glycol)-poly(2,4,6-trimethoxy benzylidene-pentaerythritol carbonate) (PEG-PTMBPEC) diblock copolymer, which was expected to destroy tumor angiogenesis and kill tumor cells simultaneously. Doxorubicin (DOX)-loaded nanoparticles (NPs) were characterized to have a uniform size distribution, high entrapment efficiency, good stability in plasma as well as a pH dependent drug release pattern. Blank NPs were non-toxic to both tumor cells and normal cells, while DOX-loaded cRGDyK peptide modified NPs (cRGDyK-NPs) exhibited the pronounced cytotoxicity against B16 cells and human umbilical vein endothelial cells (HUVEC) overexpressing alpha(v)beta(3) integrin receptors. Cellular uptake studies revealed that the highly efficient uptake of cRGDyK-NPs was attributed to the receptor-mediated endocytosis and acidic-triggered drug release. Importantly, cRGDyK-NPs could dramatically reduce the systemic toxicity of DOX and exert excellent tumor killing activity in vivo. The cRGDyK modified pH-sensitive nanocarrier is a promising vehicle for intracellular drug delivery to alpha(v)beta(3) integrin receptor overexpressed tumor cells and neovascular cells.Statement of SignificanceSlow intracellular drug release and poor tumor targeting capacity are still the critical barriers of polymeric nanoparticles (NPs) for the treatment efficiency of chemotherapy. In the present study, we designed cRGDyK peptide modified poly(ethylene glycol)-poly(2,4,6-trimethoxybenzylidene-pentaery thritol carbonate) (cRGDyK-PEG-PTMBPEC) NPs with active targeting and fast pH-triggered drug release. Doxorubicin (DOX)-loaded cRGDyK-PEG-PTMBPEC NPs exhibited pronounced cytotoxicity and enhanced cellular uptake against B16 cells and human umbilical vein endothelial cells overexpressing alpha v beta 3 integrin receptors. Moreover, the active targeted pH-sensitive NPs can enhance the antitumor activity and reduce the systematic toxicity of DOX in vivo. (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

PURPOSE:Using different chain lengths of PEG as linkers to develop a novel folate (FA) and TAT peptide co-modified doxorubicin (DOX)-loaded liposome (FA/TAT-LP-DOX) and evaluate its potential for tumor targeted intracellular drug delivery.METHODS:FA/TAT-LP-DOX was prepared by pH gradient method and post-insertion method and the optimal ligand density was screened by MTT assay. In vitro evaluation was systematically performed through cytotoxicity assay, cellular uptake studies, subcellular localization and cellular uptake mechanism in folate receptor (FR) over-expressing KB tumor cells. In vivo tumor targeted delivery of FA/TAT-LP-DOX was also studied by in vivo fluorescence imaging in a murine KB xenograft model.RESULTS:The particle size and zeta potential determination indicated that FA and TAT were successfully inserted into the liposome and cationic TAT peptide was completely shielded. With the optimal ligand density (5% of FA and 2.5% TAT), the FA/TAT-LP-DOX exhibited improved cytotoxity and cellular uptake efficiency compared with its single-ligand counterparts (FA-LP-DOX and PEG/TAT-LP-DOX). Competitive inhibition and uptake mechanism experiments revealed that FA and TAT peptide played a synergistic effect in facilitating intracellular transport of the liposome, and association between FA and FA receptors activated this transport process. In vivo imaging further demonstrated the superiority of FA/TAT-LP in tumor targeting and accumulation.CONCLUSIONS:Folate and TAT peptide co-modified liposome using different chain lengths of PEG as linkers may provide a useful strategy for specific and efficient intracellular drug delivery.

Non-small cell lung cancer (NSCLC) is a serious threat to human health, and 40%-80% of NSCLCs express high levels of epidermal growth factor receptor (EGFR). GE11 is a novel peptide and exhibits high affinity for EGFR binding. The aim of this study was to construct and evaluate GE11-modified liposomes for targeted drug delivery to EGFR-positive NSCLC. Doxorubicin, a broad-spectrum antitumor agent, was chosen as the payload. GE11 was conjugated to the distal end of DSPE-PEG(2000)-Mal by an addition reaction with a conjugation efficiency above 90%. Doxorubicin-loaded liposomes containing GE11 (GE11-LP/DOX) at densities ranging from 0% to 15% were prepared by combination of a thin film hydration method and a post insertion method. Irrespective of GE11 density, the physicochemical properties of these targeted liposomes, including particle size, zeta potential, and drug entrapment efficiency, were nearly identical. Interestingly, the cytotoxic effect of the liposomes on A549 tumor cells was closely related to GE11 density, and liposomes with 10% GE11 had the highest tumor cell killing activity and a 2.6-fold lower half maximal inhibitory concentration than that of the nontargeted counterpart (PEG-LP/DOX). Fluorescence microscopy and flow cytometry analysis revealed that GE11 significantly increased cellular uptake of the liposomes, which could be ascribed to specific EGFR-mediated endocytosis. It was found that multiple endocytic pathways were involved in entry of GE11-LP/DOX into cells, but GE11 assisted in cellular internalization mainly via the clathrin-mediated endocytosis pathway. Importantly, the GE11-modified liposomes showed enhanced accumulation and prolonged retention in tumor tissue, as evidenced by a 2.2-fold stronger mean fluorescence intensity in tumor tissue than the unmodified liposomes at 24 hours. In summary, GE11-modified liposomes may be a promising platform for targeted delivery of chemotherapeutic drugs in NSCLC.

Objective: To prepare and evaluate the ligand peptide-GE11 modified doxorubicin-loaded liposome.Methods: The DSPE-PEG 2000 and DSPE-PEG 2000-GE11 were synthesized by substitution reaction and addition reaction,and the composition and purity of the products were characterized by TLC,FTIR,1H-NMR and HPLC,respectively.After doxorubicin-loaded liposome was prepared by the thin film hydration and the pH loading method,the GE11 conjugated doxorubicin liposome was prepared by the post insertion method.The particle size,zeta potential,morphology and encapsulation efficiency of the targeting liposome were evaluated by DLS,TEM and HPLC.Results: The DSPE-PEG 2000 and DSPE-PEG 2000-GE11 were successfully synthesized,and the purity of the two products was 85.7% and 90%.The mean particle size of the liposome was 126 nm and the zeta potential was-13 mV.The morphology of the liposome was regularly spherical in shape.The encapsulation efficiency of doxorubicin in the liposome was about 91%.Conclusion: The GE11 modified liposome has been successfully prepared and this drug delivery device may provide a novel strategy for treatment of non-small cell lung cancer.

OBJECTIVE:To prepare and characterize the cleavable PEG and TAT peptide co-modified doxorubicin liposomes(C-TAT-DOX-LP).METHODS:DSPE-S-S-PEG5000 and DSPE-PEG2000-TAT were synthesized and characterized by TLC,IR,and 1 H-NMR.Interconnection rate of TAT peptide was determined by HPLC.DOX-LP was prepared by pH gradient method,C-TATDOX-LP was prepared by post-insertion method.Particle sizes,Zeta potential,morphology,encapsulation efficiency and the in vitro release rate within 48 h of DOX from C-TAT-DOX-LP were investigated.RESULTS:DSPE-S-S-PEG5000 and DSPE-PEG2000-TAT were successfully synthesized and characterized,and interconnection rate of TAT peptide was 96.3%.Mean particle size of C-TAT-DOX-LP was 152.1 nm and encapsulation efficiency was 91.8%,Zeta potential was-5.4 mV.Prepared liposomes were well-distributed and regularly spherical in shape.By DTT reaction,the particle size of C-TAT-DOX-LP decreased from 155.1 nm to 140.5 nm,while Zeta potential increased from-5.9 mV to 7.8 mV,the disulfide bonds were cleaved.Accumulative release rate of DOX from C-TAT-DOX-LP was 40% within 48 h,and was relatively enhanced after PEG was cleaved.CONCLUSIONS:C-TATDOX-LP is successfully prepared,showing certain sustained-release property.

OBJECTIVE: To synthesize and characterize folic acid (FA) modified pH-sensitive PAMAM doxorubicin conjugates. METHODS: FA, PEG and DOX were connected to PAMAM by amidation and hydrazinolysis reaction of ester. The polymer was characterized by TLC, IR and 1H-NMR, respectively. Drug-loading amount, particle size, Zeta potential, shape and morphology of conjugates were investigated, and the dissolution behavior of DOX in vitro was also investigated. RESULTS: 8 FA and 10 DOX were bonded on the periphery of PAMAM-DOX. The structure of conjugates was firmed as the target conjugates by TLC, IR and 1H-NMR successfully. The particle size and Zeta potential for conjugates were (75.2±0.25) nm and (7.04±0.11) mV, respectively. The morphology of the conjugates was regularly spherical shape. 40% of DOX of conjugates was released at pH 4.5, 29% of DOX was released at pH 5.5 and 13% of DOX was released at pH 7.4 during 48 h. CONCLUSIONS: FA-modified pH-sensitive conjugates are successfully synthesized, which release in pH dependent manner in vitro.