聚乙二醇化聚氰酯毫微粒作为salvicine载体：合成、制备和体外特性

目的：合成和鉴定聚乙二醇化聚氰酯共聚物,制备聚乙二醇化聚氰酯共聚物和聚氰酯聚合物的毫微粒,测定二种毫微粒的体外特性。方法：用^1H-NMR,^13C-NMR和FTIR测定聚乙二醇化聚氰酯共聚物的结构,用凝胶渗透色谱法测定共聚物的分子量,用乳化／蒸发法制备毫微粒。结果：^1H-NMR,^13C-NMR和FTIR光谱与聚乙二醇化聚氰酯共聚物的结构相符,合成共聚物的分子量是6680,用HPLC测定毫微粒的包封效率时,共聚物对salvicine的测定无干扰,聚乙二醇化聚氰酯共聚物毫微粒的包封率是92．6％,聚氰酯聚合物的包封效率是98．9％。二种毫微粒的粒径均为250nm左右。Zeta电位值受聚合物结构的影响,与聚氰酯毫微粒比较（－23．1mV),聚乙二醇化聚氰酯毫微粒显示低的Zeta电位值（－9.6mV)。毫微粒的体外释放显示一个开始的突释效应,然后缓慢释放达28天。结论：聚乙二醇化聚氰酯毫微粒可能是salvicine体内抗肿瘤作用的一个有效载体。     

新型安眠药加波沙朵的合成

目的合成新型安眠药加波沙朵。方法以甘氨酸乙酯盐酸盐、氯化苄和γ-丁内酯为原料,合成N-苄基甘氨酸乙酯和4-溴丁酸乙酯,经环合得到N-苄基-3-氧代-4-甲酸乙酯哌啶盐酸盐,该盐经氢化、基团保护,再与羟胺反应环合得到3-羟基-6-甲酸乙酯-4,5,6,7-四氢异唑[5,4-c]吡啶,最后去保护得到目标产物。结果与结论中间体及产物结构经IR、^1H-NMR和^13C-NMR确认。     

微生物转化植物甾醇产物20-羟基-23,24-二降胆-4-烯-3-酮的鉴定

目的：鉴定微生物转化植物甾醇过程中一未知产物的结构。方法：将待鉴定产物用柱色谱、制备HPLC分离纯化后送质谱、^1H-NMR、^13C-NMR、Dept、Cosy和Noesy分析。结果：待鉴定产物为20-羟基-23，24-二降胆-4-烯-3-酮。结论：此产物为胆固醇边链切除过程中一中间产物的类似物。     

β-乳香酸-3-O-β-D-半乳糖苷的合成

目的从我国传统中药资源中寻求和开发更好的抗肿瘤药物。方法通过糖化学手段和有机合成方法修饰和改性中药化学成分的结构。结果合成出β-乳香酸-3-O-13-D-半乳糖苷。结论^1H-NMR和^13C-NMR结构表征。     

肝靶向前药胆酸拉米夫定酰胺的合成及其稳定性初步研究

目的设计合成具有人体胆酸转运体结合活性的肝靶向前体药物.方法运用活泼酯法原理,设计合成新化合物胆酸拉米夫定酰胺,使用1H-NMR、13 C-NMR、IR、MS确认其结构,并对其稳定性进行初步研究.结果合成了目标产物,初步稳定性结果显示其在各种溶剂中较稳定.结论得到了稳定的目标化合物胆酸-拉米夫定酰胺,为进一步体内肝脏靶向性研究奠定了基础.     

N-辛基-N’-(2-羧基环己甲酰基)-壳聚糖的制备、表征与pH敏感性

目的以pH敏感的酰胺键为连接臂,制备两亲性壳聚糖衍生物。方法通过在壳聚糖2-NH2上引入疏水辛基和pH敏感的酰胺键,制备两亲性壳聚糖接枝共聚物,用FTIR、1H-NMR和13C-NMR对其结构进行表征,使用XRD、DSC对其物理性质进行分析,采用紫外-可见分光光度计和粒径仪评价其pH敏感性。结果合成了7种取代度的N-辛基-N’-(2-羧基环己甲酰基)-壳聚糖,在pH 5.0~6.0内具有pH敏感性。结论合成的pH敏感衍生物有望用于智能型药物释放系统,在体内特定部位(肿瘤、梗塞)或细胞内(内涵体、细胞质)释放包载的难溶性药物。     

罗库溴铵的合成工艺研究

目的研究罗库溴铵的合成工艺。方法以2β-（4-吗啉基）-16β-（1-毗咯烷基）-3α,17β-二羟基-5α-雄甾烷为原料,经乙酰化,再选择性水解得到17p乙酰化物,然后与烯丙基溴成盐得到罗库淡铵。考察了乙酰化、选择性水解的条件,同时分离得到3a,17β-二乙酰化物。结果与结论罗库决铵及其中间体的结构经核磁共振氢谱（1H-NMR）、碳谱（^13C-NMR）,质谱（ESI-MS）确证,总收率59．6％。     

药用1,3-二甲基戊胺盐酸盐的合成工艺改进

目的合成1,3-二甲基戊胺盐酸盐并改进其工艺。方法 以乙酰乙酸乙酯和2-溴丁烷为原料,经4步合成反应得到1,3-二甲基戊胺盐酸盐,并采用单因素考察法对合成工艺进行改进。结果1,3-二甲基戊胺盐酸盐各步反应的收率均>40%,产物总收率达到12.3%。结论产物结构经1 H-NMR和13 C-NMR确认为1,3-二甲基戊胺盐酸盐。     

聚乙二醇单甲醚接枝壳聚糖自组装纳米球的制备*

目的:合成聚乙二醇单甲醚接枝壳聚糖(monomethoxy poly(ethylene glycol)-grafted-chitosan,mPEG-g- CS),并制备自组装纳米球。方法:利用甲醛连接法将聚乙二醇单甲醚(monomethoxy poly(ethylene glycol),mPEG)接枝干壳聚糖(ehitosan,CS)分子,得到聚乙二醇(poly(ethylene glycol),PEG)改性的壳聚糖衍生物,并通过傅立叶红外光谱仪(Fourier transform infrared spectroscopy,FT-IR),核磁共振仪(proton nuclear magnetic resonance,~1H-NMR)对产物进行结构表征;采用超声透析法制备自组装纳米球,并通过透射电镜(transmission electron microscopy,TEM),动态激光粒度分析仪(dynamic laser light scattering,DLLS)表征了纳米球的形态和粒径;以芘为荧光探针,通过荧光检测分析测定了mPEG-g-CS的临界胶束浓度(critical micellar concentration,CMC)。结果:通过FT-IR,~1H- NMR确证了接枝产物的存在;mPEG-g-CS在水溶液中能够自组装形成球状纳米胶束,平均粒径为250 nm。结论:通过甲醛连接法制备mPEG-g-CS,具有制备方法简捷、反应周期短、易操作的优点。利用该产物制备的纳米球有望成为长循环纳米药物载体。     

利拉萘酯乳膏中杂质的分析、合成和结构鉴定

目的研究利拉萘酯乳膏中杂质的结构和产生原因。方法采用LC/MS与定向合成的方式获得杂质粗品,再通过硅胶柱层析法进行分离纯化获得纯品。采用MS、^1H-NMR、^13C-NMR对杂质进行结构鉴定。结果该杂质为N-（6-甲氧基-2-吡啶基）-N-甲基氨基甲酸-（5,6,7,8-四氢）-2-萘酯,为利拉萘酯氧化降解产物。结论本研究为利拉萘酯乳膏质量评价提供了依据。     

Synthesis,characterization, and kinetic release study of methotrexate loaded mPEG–PCL polymersomes for inhibition of MCF-7 breast cancer cell line

In this study, we designed a polymersome system for the controlled release of methotrexate (MTX) as an anticancer drug with the objective of improving the loading efficiency of the drug in polymersomes as well as achievement of an efficient control on the release rate of drug from nanocarriers. We synthesized mono methoxy poly(ethylene glycol)–poly(e-caprolactone) (mPEG–PCL) diblock copolymers. The structure of the copolymers was characterized by proton nuclear magnetic resonance spectroscopy (¹H NMR), Fourier transform infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC) techniques. MTX was encapsulated within nanoparticles (NPs) through multiple emulsion method. The resulting NPs were characterized further by various techniques such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Next, the various kinetic equations were fitted to the release data of MTX from MTX-loaded mPEG–PCL polymersomes. The results showed that the zeta potential of MTX-loaded mPEG–PCL polymersomes was about –5.49?mV and the average size was 49.18?nm. MTX was encapsulated into polymersomes loading capacity of 12?±?0.09% and encapsulation efficiency of 45.5?±?0.41%. The metabolic activity assays of void of MTX, mPEG–PCL polymersomes, and MTX-loaded mPEG–PCL polymersomes were compared to each other by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of the treated MCF-7 cell lines. It can be concluded that application of NPs is a better and more effective strategy for controlled and slow release of MTX in the treatment of cancer.     

Affinity chromatography of maltase on aliphatic amines as ligands.

The primary amino group, the competitive inhibitor of maltase, was used as ligand and the enzyme was purified to homogeneity in a single step. The amino group affiants of ethylene (C2-NH2) and hexamethylene (C6-NH2) diamines were prepared by coupling to cyanogen bromide activated Sepharose CL-4B. The enzyme was quantitatively adsorbed at alkaline pH (pH 8.2), while the elution could be effected only in presence of maltose at acidic pH. The elution of enzyme by maltose was independent of spacer arms (C2 and C6) which suggests specific binding of the enzyme through inhibitor site.     

Toll-like receptor-7 agonist decoration enhances the adjuvanticity of chitosan-DNA nanoparticles

In order to provide an adjuvant-equipped carrier system for plasmid deoxyribonucleic acid (pDNA) vaccines, we grafted for the first time a Toll-like receptor (TLR)-7 agonistic moiety [9-benzyl-8-hydroxyadenine (HA)] through a poly(ethylene glycol) (PEG) spacer onto a water-soluble chitosan derivative [final copolymer: 6-0-carboxymethyl-N,N,N-trimethylchitosan (CTC)-graft-PEG-HA (CTCPHA)]. Successful grafting was confirmed by spectroscopic (H NMR, mass, ultraviolet-visible, and Fourier transform infrared spectroscopy) and chromatographic (size-exclusion chromatography-multi-angle laser light scattering) methods. In this article, TLR-7 agonist-decorated CTCPHA nanoparticles (NPs) were formulated by complex coacervation with pDNA expressing the green fluorescence protein. Resulting NPs had a size of around 200 nm with a positive surface charge and high DNA encapsulation efficiency. In contrast to the use of DNA alone, NP protected DNA against enzymatic degradation and enabled transfection of alveolar A549 cells. Interestingly, TLR-7 agonist decoration increased significantly the interleukin-8-related immune stimulatory capacity of polymeric chitosan and chitosan-based NP in human THP-1 macrophages when compared with controls. In summary, we demonstrate here the proof-of-principle that covalent TLR-7 agonist functionalization of chitosan-DNA NPs enhances the carrier's adjuvanticity, representing a valuable concept for future polymer-based DNA vaccination.     

Effects of PEG conjugation on insulin properties

The goal of this research was to determine whether the site-specific attachment of poly(ethylene glycol) to insulin could enhance the physical and pharmacological properties of insulin without negatively affecting its biological activity or immunological properties. Electrophilically activated derivatives of low-molecular-weight monomethoxypoly(ethylene glycol) (mPEG) were chemically coupled to insulin via its amino groups at positions phenylalanine-B1 or lysine-B29, with an amide bond being formed between the polymer and protein. The site-specific attachment of mPEG to insulin did not substantially alter insulin's secondary/tertiary structure, self-association behavior, or potency in vivo. However, mPEG attachment did significantly enhance insulin's resistance to aggregation. In addition, the pegylation of insulin almost completely eliminates the resultant conjugate's immunogenicity, allergenicity, and antigenicity. Finally, the conjugates were observed to remain in the systemic circulation for longer periods of time than unmodified insulin after subcutaneous administration.     

Facile synthesis of a chitosan hybrid of a laminin-related peptide and its antimetastatic effect in mice

Laminin, a cell adhesion protein, consists of three peptide chains (alpha-1, beta-1 and gamma-1). The beta-1 chain contains a Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence that has been found to inhibit experimental metastasis in mice. We have prepared a hybrid of a water-soluble chitosan and a laminin-related peptide, and have examined its inhibitory effect on experimental metastasis in mice. A laminin-related peptide, acetyl-Tyr-Ile-Gly-Ser-Arg-betaAla-OH (Ac-YIGSRbetaA-OH), was prepared by a solid-phase method. Ac-YIGSRbetaA-OH was then reacted with a water-soluble chitosan. BetaAla is a spacer and was placed to avoid racemization of the Arg residue when the peptide was coupled with chitosan. Although chitosan has amino groups, they did not react with the peptide. Four methods were tried to achieve a coupling reaction, the diphenylphosphoryl azide method, the diisopropylcarbodiimide/1-hydroxybenzotriazole method, the water-soluble carbodiimide (WSC), and the 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) method, but all four methods were unsuccessful. Therefore, a small spacer, tert-butyloxycarbonyl-Gly, was intercalated in chitosan, by the TBTU method, to facilitate its coupling with the peptide. After removal of the protecting group, the Gly-chitosan was coupled with Ac-YIGSRbetaA-OH by the water-soluble carbodiimide method to give Ac-YIGSRbetaAG-chitosan. Conjugation of the peptide with the larger chitosan molecule did not reduce the inhibitory effect of the peptide on experimental metastasis in mice, it actually potentiated the antimetastatic effect, demonstrating that chitosan may be effective as a drug carrier for peptides.     

Polyion complex micelles composed of all-trans retinoic acid and poly (ethylene glycol)-grafted-chitosan

The goal of this study is to develop novel types of polyion complex micelles for the drug delivery to brain tumor. Methoxy poly(ethylene glycol) (mPEG)-grafted chitosan (CP) was synthesized in order to make polymeric micelles encapsulating all-trans retinoic acid (ATRA) based on polyion complex formation. Polyion complex micelles were found to have spherical shapes with sizes of about 50 approximately 200 nm. The loading efficiency of micelle was higher than 80% (w/w) for all formulations. 1H nuclear magnetic resonance (NMR) spectra confirmed the formation of polymeric micelles. The CP graft copolymer and ATRA have distinguishing peaks in their 1H NMR spectra. The specific peaks of ATRA disappeared in D2O or DMSO while it appeared at mixtures of D2O/DMSO, indicating that ATRA and chitosan formed ion complex inner-core. In the cell cytotoxicity study using U87MG cells in vitro, polyion complex micelles showed similar cytotoxicity to that of free ATRA. A migration test was performed to investigate the inhibition of tumor cell invasion in vitro. The results suggested that the polyion complex micelles was more effective at inhibiting tumor cell migration than free ATRA.     

聚乙二醇-壳聚糖共聚物作为基因传递载体的体外研究

本文通过将单甲氧基聚乙二醇(mPEG)的端羟基氧化为醛基，进而与壳聚糖(CS)链节上的氨基反应，合成了聚乙二醇-壳聚糖(mPEG-CS)共聚物。用MTT法检验不同浓度共聚物对HeLa细胞和A549细胞的毒性，结果显示5~100 μg·mL^-1聚合物的细胞毒性较低。通过考察不同PEG取代度的共聚物与质粒DNA所形成复合物的粒径、zeta电位及凝胶阻滞分析，筛选出最佳共聚物为取代度3.55%的mPEG(3.55)-CS。将mPEG(3.55)-CS作为基因传递载体，介导绿色荧光蛋白基因(pEGFP-C1)转染HeLa细胞和A549细胞，荧光显微镜下观察到荧光蛋白的表达，流式细胞仪测定HeLa细胞与A549细胞的最高转染率分别为8.1%和4.8%，证实了mPEG-CS共聚物是一种有效的非病毒类基因传递载体。     

beta-Lactam derivatives as enzyme inhibitors: 1-peptidyl derivatives of 4-phenylazetidin-2-one as inhibitors of elastase and papain

N-Peptidyl substituted azetidin-2-ones were synthesized and evaluated as inhibitors of the serine protease elastase, and the cysteine protease papain. All compounds were synthesized from 4-phenylazetidin-2-one, either from the racemate or from the pure enantiomers. The (S)-enantiomer was prepared by enantioselective synthesis from (S)-beta-phenyl-beta-alanine, while the (R)-enantiomer was obtained by enzymatic resolution with alpha-chymotrypsin. N-Alkylation with bromoacetates introduced a spacer group which, after hydrolysis to the free acid, was acylated with amino acid esters or di- or tripeptide esters. The enzymatic assays proved some derivatives to be effective inhibitors of PPE and/or papain. N-BOC protected amino acid derivatives without a spacer group inhibited PPE reversibly, while derivatives with spacer group showed either weak or no inhibitory properties. On the other hand, papain was inactivated irreversibly by ethyl (RS)-2-oxo-4-phenylazetidin-1-acetate. The highest inhibitory activity against papain was found for the diastereomers of N-(2-oxo-4-phenylazetidin-1-acetyl)-L-alanyl-L-valine benzyl ester, a compound with a spacer group.     

甲基聚乙二醇修饰HCG后对表位的影响

采用活化酯法用甲基聚乙二醇(mPEG)对高比活的人绒毛膜促性腺素(HCG)纯品进行化学修饰,利用放射免疫测定法研究了HCG结合mPEG后对HCG免疫反应性的影响,发现mPEG对HCG的修饰位点结合可影响抗体对抗原表位的识别结合,间接证明mPEG-HCG比天然HCG降低了免疫原性。     

Characterization, efficacy, pharmacokinetics, and biodistribution of 5kDa mPEG modified tetrameric canine uricase variant

PEGylated uricase is a promising anti-gout drug, but the only commercially marketed 10kDa mPEG modified porcine-like uricase (Pegloticase) can only be used for intravenous infusion. In this study, tetrameric canine uricase variant was modified by covalent conjugation of all accessible ? amino sites of lysine residues with a smaller 5kDa mPEG (mPEG-UHC). The average modification degree and PEGylation homogeneity were evaluated. Approximately 9.4 5 kDa mPEG chains were coupled to each monomeric uricase and the main conjugates contained 7-11 mPEG chains per subunit. mPEG-UHC showed significantly therapeutic or preventive effect on uric acid nephropathy and acute urate arthritis based on three different animal models. The clearance rate from an intravenous injection of mPEG-UHC varied significantly between species, at 2.61 mL/h/kg for rats and 0.21 mL/h/kg for monkeys. The long elimination half-life of mPEG-UHC in non-human primate (191.48 h, intravenous injection) indicated the long-term effects in humans. Moreover, the acceptable bioavailability of mPEG-UHC after subcutaneous administration in monkeys (94.21%) suggested that subcutaneous injection may be regarded as a candidate administration route in clinical trails. Non-specific tissue distribution was observed after administration of (125)I-labeled mPEG-UHC in rats, and elimination by the kidneys into the urine is the primary excretion route.