一种新型设计肽作为药物载体的潜在应用研究

目前有大量关于全程电荷匹配设计肽的应用的研究,但是关于半程电荷匹配设计肽作为药物的应用研究却未见报道.本文对新型设计的半程电荷匹配肽P9（AC-Pro-Ser-Phe-Asn-Phe-Lys-Phe-Glu-Pro-NH2）的研究发现,P9可以成功地将作为疏水药物模型的芘稳定悬浮于水溶液中.大豆卵磷脂微囊被用以模拟细胞膜.荧光数据显示芘可以借助P9的包埋作用以晶体状态存在于水溶液中.并且当包埋体加入到脂质体微囊体系中时,芘可以从P9的包埋体系中以分子形态释放到膜的脂质层内.实验发现当包埋于芘上的P9较多,包埋壁较厚,芘的释放速度相对变缓.我们可以利用这一特性,通过调节加入的P9的浓度来控制包埋于芘上P9的厚度,从而控制芘的释放速率.研究表明,具有半程电荷匹配性质的P9肽可以作为运载疏水药物的载体.     

Self-assembling peptide as a potential carrier of hydrophobic compounds

Microcrystals of a hydrophobic cargo were stabilized by EAK16 II, a self-assembling oligopeptide, and suspended in aqueous solution. Pyrene was used as a model hydrophobic compound. Egg phosphatidylcholine (EPC) vesicles were prepared to mimic a cell membrane. Pyrene was released from its EAK16 II coating into EPC vesicles. The excimer decay profiles were acquired. They showed that pyrene is present in the crystalline form when stabilized by EAK16 II, it is molecularly dispersed in EPC vesicles, and it is completely released from its EAK16 II coating into the membrane bilayers. The release of pyrene from the microcrystals coated with EAK16 II into the EPC membrane was followed by fluorescence as a function of time. The amount of pyrene released into the EPC vesicles at a given time was quantified using a calibration curve. The concentration of pyrene released was determined as a function of time, and the concentration-versus-time profile was fitted with one exponential. The rate of pyrene release was found to depend on the peptide-to-pyrene molecular ratio. Higher peptide-to-pyrene ratios lead to slower transfer of pyrene to the lipophilic environment. Scanning electron micrographs demonstrated that a thicker coating on the pyrene crystals results in a slower release. The data presented in this work demonstrate that the self-assembling EAK16 II can stabilize a hydrophobic cargo in aqueous solution and deliver it into a lipophilic environment, and that the rate of transfer can be adjusted by tuning the peptide-to-pyrene ratio.     

Nitroanilines as Quenchers of Pyrene Fluorescence

The quenching of pyrene and 1‐methylpyrene fluorescence by nitroanilines (NAs), such as 2‐, 3‐, and 4‐nitroaniline (2‐NA, 3‐NA, and 4‐NA, respectively), 4‐methyl‐3‐nitroaniline (4‐M ‐3‐NA), 2‐methyl‐4‐nitroaniline (2‐M‐4‐NA), and 4‐methyl‐3,5‐dinitroaniline (4‐M‐3,5‐DNA), are studied in toluene and 1,4‐dioxane. Steady‐state fluorescence data show the higher efficiency of the 4‐NAs as quenchers and fit with a sphere‐of‐action model. This suggests a 4‐NA tendency of being in close proximity to the fluorophore, which could be connected with their high polarity/hyperpolarizability. In addition, emission and excitation spectra evidence the formation of emissive pyrene—NA ground‐state complexes in the case of the 4‐NAs and, in a minor degree, in the 2‐NA. Moreover, time‐resolved fluorescence experiments show that increasing amounts of NA decrease the pyrene fluorescence lifetime to a degree that depends on the NA nature and is larger in the less viscous solvent (toluene). Although the NA absorption and the pyrene (Py) emission overlap, we found no evidence of dipole–dipole energy transfer from the pyrene singlet excited state (¹Py) to the NAs; this could be due to the low NA concentration used in these experiments. Transient absorption spectra show that the formation of the pyrene triplet excited state (³Py) is barely affected by the presence of the NAs in spite of their efficiency in ¹Py quenching, suggesting the involvement of ¹Py—NA exciplexes which—after intersystem crossing—decay efficiently into ³Py.     

Self-aggregated nanoparticles of cholesterol-modified chitosan conjugate as a novel carrier of epirubicin

Cholesterol-modified chitosan conjugate with succinyl linkages (CHCS) was synthesized and characterized by fourier transform infrared (FTIR) and proton nuclear magnetic resonance (¹H NMR). The degree of substitution (DS) of cholesterol moiety determined by elemental analysis was 7.3%. The self-aggregation behavior of CHCS was evaluated by the fluorescence probe technique and the critical aggregation concentration (CAC) was 1.16 × 10⁻² mg mL⁻¹ in 0.1 M acetic acid solution. CHCS formed monodisperse self-aggregated nanoparticles with a roughly spherical shape and a mean diameter of 417.2 nm by probe sonication in aqueous media. Epirubicin (EPB), as a model anticancer drug, was physically entrapped inside CHCS self-aggregated nanoparticles by the remote loading method and the characteristics of EPB-loaded CHCS self-aggregated nanoparticles were analyzed using dynamic laser light scattering (DLLS), transmission electron microscopy (TEM) and fluorescence spectroscopy. EPB-loaded CHCS self-aggregated nanoparticles were almost spherical in shape and their size increased from 338.2 to 472.9 nm with the EPB-loading content increasing from 7.97% to 14.0%. The release behavior of EPB from CHCS self-aggregated nanoparticles was studied in vitro by dialysis method. The results showed that EPB release rate decreased with the pH increase of the release media. In phosphate buffered saline (PBS, pH 7.4), the EPB release was very slow and the total release amount was about 24.9% in 48 h.     

Synthesis and cell activity of novel galactosylated chitosan as a gene carrier

It is important for gene carrier to transport DNA into target cells. Although viral vectors are very efficient gene-transfer vehicles, significant drawbacks limit their applications. Chitosan (CS) has been researched widely as a non-viral vector. However, the low cell specificity and low transfection efficiency of chitosan need to be overcome. In order to conquer the drawback of chitosan, the present paper is concerned with the synthesis of novel galactosylated chitosan (GC) through etherization of chitosan and galactose in THF using BF₃·OEt₂ as promoter. The final product was characterized and confirmed by FT-IR and ¹H NMR. The degree of O-substitution (DS) of chitosan by galactose was measured to be 10.38% using anthrone-sulfuric acid colorimetric method. The mean particle diameter and average zeta potential of the GC/DNA complex were 350 nm and +22.1 mV, respectively. The GC/DNA nanoparticle was tested to transfect HEK293 cells, and the viability of HEK293 cells was not affected by the GC/DNA nanoparticle compared to that of the control.     

Synthesis,supramolecular complexation and DFTstudies of a bis(pyrene)-appended ‘capped’ triazole-linked calix[4]arene as Zn2+ and Cd2+ fluorescent chemosensors

ABSTRACT

A new bimodal triazole-bridged pyrene-appended calix[4]arene 2g has been synthesised and characterised and complexation studies showed that the binding of Cd²⁺ and Zn²⁺ occurs as demonstrated by enhanced monomer and declining excimer emission fluorescence spectral changes. With several other metal ions, quenching of both monomer and excimer emissions occurred. The observed monomer formation in the fluorescence spectra seen with Zn²⁺ and Cd²⁺ is possibly linked not just to the diminished parallel orientations of the pyrene rings but with the magnitude of the resulting HOMO-LUMO gaps and other parameters measured by DFT calculations.     

Diastereoselective synthesis of a novel phosphinic peptide as ACE inhibitor: Fragment-based design approach

In medicinal chemistry for the purpose of lead optimization, hit selection of new isofunctional chemotypes are crucial for the success of identifying novel chemical entities of increased potency. Using fragment-based design approach with the N-selective inhibitor RXP407, a novel phosphinic peptide scaffold that consisted of modified RXP407 fragments was generated. The presented synthetic route is straightforward and produces the desired product Z-RXP407 in moderate yield. The (S,R,S,S)-Z-RXP407 analog has been evaluated for the C- and N-domain constructs of angiotensin-converting enzyme. The potency of this analog has been much lower compared to the parent compound RXP407, providing thus valuable insights regarding further design based on structure–activity relationships.     

Stimuli‐responsive zein‐based nanoparticles as a potential carrier for ellipticine: Synthesis,release, and in vitro delivery

In the present research, we have investigated a drug delivery system based on the pH‐responsive behaviors of zein colloidal nanoparticles coated with sodium caseinate (SC) and poly ethylene imine (PEI). These systematically designed nanoparticles were used as nanocarriers for encapsulation of ellipticine (EPT), as an anticancer drug. SC and PEI coatings were applied through electrostatic adsorption, leading to the increased size and improved polydispersity index of nanoparticles as well as sustained release of drug. Physicochemical characteristics such as hydrodynamic diameter, size distribution, zeta potential and morphology of nanoparticles prepared using different formulations and conditions were also determined. Based on the results, EPT was encapsulated into the prepared nanoparticles with a high drug loading capacity (5.06%) and encapsulation efficiency (94.8%) under optimal conditions. in vitro experiments demonstrated that the release of EPT from zein‐based nanoparticles was pH sensitive. When the pH level decreased from 7.4 to 5.5, the rate of drug release was considerably enhanced. The mechanism of pH‐responsive complexation in the drug encapsulation and release processes was extensively investigated. The pH‐dependent electrostatic interactions and drug state were hypothesized to affect the release profiles. Compared to the EPT‐loaded zein/PEI nanoparticles, the EPT‐loaded zein/SC nanoparticles exhibited a better drug sustained‐release profile, with a smaller initial burst release and longer release period. According to the results of in vitro cytotoxicity experiments, drug‐free nanoparticles were associated with a negligible cytotoxicity, whereas the EPT‐loaded nanoparticles displayed a high toxicity for the cancer cell line, A549. Our findings indicate that these pH‐sensitive protein‐based nanoparticles can be used as novel nanotherapeutic tools and potential antineoplastic drug carriers for cancer chemotherapy with controlled release.     

Synthesis of N-methylene phosphonic chitosan （NMPCS） and its potential as gene carder

N-Methylene phosphoulc chitosan (NMPCS),an amphiphilic macromolecule with powerful chelating ability of Ca~(2 ) ions,was synthesized and characterized.The physicochemical properties of NMPCS and the interactions between NMPCS and plasmid DNA were investigated by FTIR,~(13)C NMR,X-ray,agarose gel electrophorcsis retardation assay,atomic force microscopy (AFM) and circular dichroism (CD).The results suggest that at charge ratio 2:1 or above,DNA could be completely entrapped and spherical complexes with mean size of 80-210 nm were formed.Taking HeLa as host cell,luciferase expression mediated by NMPCS improved about 100 times compared to the expression mediated by chitosan.     

Simultaneous Determination of Phenanthrene and Benzo(a)pyrene in Water Samples by Synchronous Fluorescence Spectroscopy

Abstract

A simple and sensitive method was developed to simultaneously determine phenanthrene and benzo(a)pyrene in water samples using synchronous fluorescence spectroscopy with a 56-nm Δλ. The method was used to simultaneously determine both compounds in samples of spiked surface water. Analytical recoveries were 96.9–101.1% for phenanthrene and 95.8–103.5% for benzo(a)pyrene. Analytical results were checked for precision with 15 measurements for each compound. Relative standard deviations were 1.9% for phenanthrene and 2.9% for benzo(a)pyrene, which shows that the proposed method is quite precise.     

Synthesis and characterization of a starch‐modified hydrogel as potential carrier for drug delivery system

Synthesis and characterization of a new hydrogel were carried out using a chemically modified starch (starch‐M) consisting of coupling C?C bounds coming from glycidil methacrylate (GMA) onto the polysaccharide structure. ¹³C NMR, ¹H NMR, and FT‐IR spectroscopies were used to confirm the incorporation of such groups onto the starch‐M. The hydrogel was prepared by a crosslinking polymerization of starch‐M using sodium persulfate as an initiating agent. The starch‐M hydrogel shows morphology clearly different from that of the raw starch film due to the presence of voids on its surface. The swelling process of the starch‐M hydrogel was not significantly affected by changes on the temperature or on pH of the surrounding liquid, indicating the such behavior can be then understood by a diffusional process, resulting from its physical–chemical interactions with the solvent. The values of the diffusional exponent n were on the order of 0.45–0.49 for the range of pHs investigated, demonstrating that the water transport mechanism of starch‐M hydrogel is more dependent on Fickian diffusion, that is, controlled by water diffusion. Such starch‐M hydrogel is a promising candidate to be used in transporting and in preserving acid‐responsive drugs, such as corticoids, for the treatment of colon‐specific diseases, for example, Crohn's disease. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 2567–2574, 2008     

Cyclic methacrylic acid oligomer as a novel pH‐sensitive carrier material

The pH effect of inclusion of methylene blue (MB) in cyclic methacrylic acid oligomer (CMAA) with degree of polymerization 14, which was a novel cationic host material with pK_a 4.83, was investigated in methanol. The self‐diffusion coefficients of MB in methanol at pH 7.0 and 4.0 with and without CMAA were measured by ¹H‐NMR spectrometry with pulsed‐field‐gradient stimulated‐echo pulse (PGSE) analysis. At both pH values, the addition of CMAA to MB solution resulted in the complex formation between CMAA and MB. MB was included in CMAA in methanol at a wide range of pH. The molar fraction of the slow diffusion component corresponding to the complex increased by varying the pH from 7.0 to 4.0. Then, SiO–CMAA was synthesized by immobilization of CMAA on silica gel beads. The time dependence of adsorption of MB to SiO–CMAA was investigated by UV–Vis spectrometry. At pH 7.0, the absorbance of MB–methanol solution gradually decreased by the addition of SiO–CMAA. The absorbance drastically decreased on varying the pH from 7.0 to 4.0, and the absorbance quickly increased on varying the pH from 4.0 to 7.0. The inclusion of MB in CMAA was fast and reversible on changing the pH of the solution. CMAA was the novel pH sensitive host material. Inclusion of MB in CMAA enhanced at pH 4.0 rather than at pH 7.0. Copyright © 2008 John Wiley & Sons, Ltd.     

Chemical conjugation of an mRNA cap analogue with a cell-penetrating peptide as a potential membrane permeable translation inhibitor

We present a versatile method for chemical conjugation of a dinucleotide cap analogue with a cell-penetrating peptide. The final coupling reaction is between an azide-modified peptide (MPS-N₃)—a fragment that is responsible for transport of the conjugate through the cell membrane, with a biologically active compound—and an alkynylated cap structure, using the Cu(I)-catalyzed click reaction.     

Solution structure of a designed cyclic peptide ligand for nickel and copper ions

Nuclear magnetic resonance (NMR) spectroscopy was used to study a cyclic peptide derived from the amino-terminal copper-and-nickel-binding (ATCUN) motif. The three-dimensional structure of the unliganded peptide in aqueous solution was solved by simulated annealing using distance constraints derived from Nuclear Overhauser Effects. A structural model for the Ni(II)-bound complex was also produced based on NMR evidence and prior spectroscopic data, which are consistent with crystal structures of linear ATCUN complexes. Structural interpolation, or ‘morphing’, was used to understand the transition of this highly structured cyclic peptide from its unliganded structure to its metal-ion-bound structure.     

多肽新药研发策略研究进展

多肽药物具有生物活性高、药用剂量小、产业化开发优势明显等诸多优点,已成为全球关注的创新药物研发热点之一.但是其代谢不稳定、半衰期短及较难通透组织屏障等缺点严重阻碍了多肽新药在临床治疗中的广泛应用.为了解决这些限制多肽药物的瓶颈,本课题组发展了一系列的改造策略.通过这些策略的应用,以期加快多肽药物临床应用的步伐.本文主要结合本课题组的工作对多肽新药创制过程中所遇到的关键问题及解决思路进行综述.     

Novel nanoparticles composed of chitosan and β-cyclodextrin derivatives as potential insoluble drug carrier

This research was aim to develop novel cyclodextrin/chitosan(CD/CS) nanocarriers for insoluble drug delivery through the mild ionic gelation method previously developed by our lab. A series of different bcyclodextrin(β-CD) derivatives were incorporated into CS nanoparticles including hydroxypropyl-bcyclodextrin(HP-β-CD), sulphobutylether-β-cyclodextrin(SB-β-CD), and 2,6-di-O-methy-β-cyclodextrin(DM-β-CD). Various process parameters for nanoparticle preparation and their effects on physicochemical properties of CD/CS nanoparticles were investigated, such as the type of CD derivatives,CD and CS concentrations, the mass ratio of CS to TPP(CS/TPP), and p H values. In the optimal condition,CD/CS nanoparticles were obtained in the size range of 215–276 nm and with the zeta potential from30.22 m V to 35.79 m V. Moreover, the stability study showed that the incorporation of CD rendered the CD/CS nanocarriers more stable than CS nanoparticles in PBS buffer at p H 6.8. For their easy preparation and adjustable parameters in nanoparticle formation as well as the diversified hydrophobic core of CD derivatives, the novel CD/CS nanoparticles developed herein might represent an interesting and versatile drug delivery platform for a variety of poorly water-soluble drugs with different physicochemical properties.     

色谱保留时间在蛋白质组研究中的应用

液相色谱与串联质谱联用(LC-MS/MS)技术是蛋白质组学研究中的常见方法。保留时间作为独立于质谱信息的参数已经被用于蛋白质的鉴定和定量工作中。在多肽鉴定领域,多肽的色谱保留时间预测与常规的二级串联质谱数据库搜索算法结合可以提高鉴定的可信度。鉴定的灵敏度也可以通过匹配多次LC-MS实验中具有相同精确质量数和保留时间的峰而提高。另一方面,由于色谱条件的微小改变即会引起保留时间的变化,因此对多次实验结果进行保留时间比对是进行非标记定量的不可或缺的步骤。另外,联合保留时间偏移和质量数信息还可以进行蛋白质翻译后修饰(post-translational modification, PTM)的鉴定。     

Tunable self-assembled stereocomplexed-polylactic acid nanoparticles as a drug carrier

In this study, a model hydrophilic drug (porphyrin) was encapsulated within hydrophobic polylactic acid (PLA) nanoparticles (NPs) with different crystallinity and the relevant release behaviors were investigated. The crystalline modification was done using a modified nanoprecipitation method, where homo and stereocomplexed PLA NPs with different average diameters based on varying polymer concentrations and solvent/nonsolvent ratios (S/N) were prepared. Entrapment efficiency and drug release of sterocomplexed-PLA NPs were compared with neat poly(l -lactic acid) (PLLA) NPs. Furthermore, to get the more sustained release, porphyrin-loaded NPs were immobilized within electrospun poly(d ,l -lactide-co-glycolide (PLGA) nanofibers (NFs). Outcomes revealed that solution concentration and solvent/nonsolvent ratio play significant roles in the formation of homo and stereocomplexed NPs. On the other hand, it was found that the formation of stereocrystals did not significantly affect the size and morphology of NPs compared with neat NPs. With regard to the entrapment efficiency and drug content, stereocomplexd-PLA NPs behave relatively the same as neat PLLA NPs while the more sustained release was observed for stereocomplexed NPs. Also, it was observed that electrospinning of PLGA solution loaded by NPs led to the uniform distribution of NPs into PLGA fibers. Encapsulating the drug-loaded NPs into nanofibers decreased the rate of drug release by 50% after 24 h, compared with direct loading of drug into PLGA NFs. We conclude that it is possible to tune the entrapment efficiency and modify the release rate of the drug by giving small changes in the process parameters without altering the physical properties of the original drug substance and polymer.     

Molecular dynamics of star‐shaped poly(L‐lactide)s in tetrahydrofuran as solvent monitored by fluorescence spectroscopy

Linear telechelic, α,ω‐ditelechelic, and star‐shaped tri‐, tetra‐, penta‐, and hexa‐arm poly(L ‐lactide)s (PLAs) fitted at every arm with pyrene end group have been prepared. Internal dynamics and mobility of the PLA chains in tetrahydrofuran solution at 25 °C, with regard to the number of PLA arms in one macromolecule and the individual arm average degree of polymerization, was followed by fluorescence spectroscopy. Analysis of both static and time‐resolved spectra of the star‐shaped polymers revealed dynamic segmental motion resulting in end‐to‐end cyclization, accompanied by an excimer formation. Probability and rate of the latter reaction increased with increasing number of arms and with decreasing their polymerization degree. Moreover, time‐resolved measurements revealed that for macromolecules containing few arms (2 or 3) the pyrene moieties are located in the interior of the star‐shaped PLAs, whereas in the instance of the higher number of arms (4–6) they are located at the periphery of the star‐shaped PLAs. Thus, increasing the number of arms leads to their stretching away from the center of the star‐shaped PLA macromolecule. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 4586–4599, 2005