[Peptides analysis in digested edible bird's nest by HPLC-MS].

Edible bird's nest contains lots of glycoproteins. The glycosylation inhomogeneity for glycoprotein often results in wide range of molecular weight and the difficulty for protein separation and charaterization. In this paper, proteins in the edible bird's nest were extracted using multiple extractions, and then digested by PNgase F and trypsin. The digest mixture was separated with HPLC, and peptides were identified based on MS/MS data searching. The results indicated that the extracted proteins were amount to 79.7% of total protein in the edible bird's nest. More than 20 species of peptides in the digested mixture were identified. The sequences of these peptides showed similarity with some proteins from Swiss-prot. The research indicated that deglycosylation, tryptic digestion coupled with HPLC-MS/MS is a proper strategy for characterization of proteins in the edible bird's nest.

Microcosmic mechanisms for protein incomplete release and stability of various amphiphilic mPEG-PLA microspheres.

The microcosmic mechanisms of protein (recombinant human growth hormone, rhGH) incomplete release and stability from amphiphilic poly(monomethoxypolyethylene glycol-co-D,L-lactide) (mPEG-PLA, PELA) microspheres were investigated. PELA with different hydrophilicities (PELA-1, PELA-2, and PELA-3) based on various ratios of mPEG to PLA were employed to prepare microspheres exhibiting a narrow size distribution using a combined double emulsion and premix membrane emulsification method. The morphology, rhGH encapsulation efficiency, in vitro release profile, and rhGH stability of PELA microspheres during the release were characterized and compared in detail. It was found that increasing amounts of PLA enhanced the encapsulation efficiency of PELA microspheres but reduced both the release rate of rhGH and its stability. Contact angle, atomic force microscope (AFM), and quartz crystal microbalance with dissipation (QCM-D) techniques were first combined to elucidate the microcosmic mechanism of incomplete release by measuring the hydrophilicity of the PELA film and its interaction with rhGH. In addition, the pH change within the microsphere microenvironment was monitored by confocal laser scanning microscopy (CLSM) employing a pH-sensitive dye, which clarified the stability of rhGH during the release. These results suggested that PELA hydrophilicity played an important role in rhGH incomplete release and stability. Thus, the selection of suitable hydrophilic polymers with adequate PEG lengths is critical in the preparation of optimum protein drug sustained release systems. This present work is a first report elucidating the microcosmic mechanisms responsible for rhGH stability and its interaction with the microspheres. Importantly, this research demonstrated the application of promising new experimental methods in investigating the interaction between biomaterials and biomacromolecules, thus opening up a range of exciting potential applications in the biomedical field including drug delivery and tissue regeneration.

[Inhibitory effect of folic acid/polyamide-amine as a miR-7 vector on the growth of glioma in mice].

OBJECTIVE: To explore if folic acid/polyamide-amine (FA/PAMAM) enhances the therapeutic effect of miR-7gene therapy for glioma in vivo. METHODS: The miR-7 gene was transfected into U251 glioma cells by FA/PAMAM. The efficiency of gene transfection was assessed by fluorescence microscopy. The miR-7 level was detect by quantitative RT-PCR. Intracranial glioma models were established in thymectomized mice, and FA/PAMAM nanoparticles were transplanted into the tumors in situ 3 days later. The animal survival was recorded and the gross tumor volume and degree of edema were observed by MRI. Apoptosis in the glioma cells and expression of proliferating cell nuclear antigen (PCNA), matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) were assessed by immunohistochemistry, and EGFR and AKT-2 protein expression was detected by Western blot assay. RESULTS: Compared with the liposomes, the FA/PAMAM nanoparticles were more efficient to transfer miR-7 gene into U251 glioma cells, MRI showed that the tumor growth was much slower in the FA/PAMAM/miR-7 group, and the animal survival time was longer. The apoptosis rate was (5.3 ± 0.9)% in the control group, (11.4 ± 2.4)% in the liposome/miR-7 group, and (17.7 ± 3.7)% in the FA/PAMAM/miR-7 group. The immunohistochemical assay showed that the levels of PCNA, MMP-2 and MMP-9 protein in the FA/PAMAM/miR-7 group were (34.6 ± 5.4)%, (24.5 ± 4.1)%, (25.4 ± 5.1)%, respectively, significantly lower than those in the liposome/miR-7 group (49.3 ± 5.9)%, (31.7 ± 7.1)% and (39.4 ± 6.4)%, respectively, and those in the control group (57.3 ± 7.4)%, (45.4 ± 6.9)% and (55.1 ± 7.3)%, respectively (all P < 0.05). The expressions of EGFR and AKT-2 proteins were 1.09 ± 0.12 and 0.62 ± 0.10 in the control group, 0.63 ± 0.11 and 0.43 ± 0.07 in the liposome/miR-7 group, and significantly deceased (0.47 ± 0.09 and 0.31 ± 0.04, respectively) in the FA/PAMAM/miR-7 group (all P < 0.05). CONCLUSION: Compared with the liposomes, FA/PAMAM can transfect miR-7 into glioma cells with a higher efficiency in vivo, makes a longer time of the drug action, and shows a certain inhibitory effect on the growth of glioma, therefore, might become a new drug targeting agent in gene therapy forglioma.

[Effects of Wastewater Discharge on Antibiotic Resistance Genes and Microbial Community in a Coastal Area].

Wastewater treatment plants (WWTPs) are important sources of antibiotic resistance genes (ARGs) in aquatic environments. Mobile genetic elements (MGEs) and microbial communities are key factors that affect the proliferation of ARGs. To reveal the effects of WWTPs effluent discharge on the ARGs and microbial community in a coastal area, the structure and distribution of ARGs, MGEs, and microbial community in Shangyu (SY) and Jiaxing (JX) effluent receiving areas (ERAs) and the offshore area of Hangzhou Bay (HB) were investigated via high-throughput quantitative PCR and 16S rRNA high-throughput sequencing. The results showed that multidrug resistance genes were the most abundant ARGs across all the sampling sites. The diversity and abundance of ARGs and MGEs in the ERAs were much higher than those in the HB. Additionally, the diversities of the microbial community in the JX-ERA were higher than those in the SY-ERA and HB. PCoA showed that the distribution of ARGs, MGEs, and microbial communities in the ERAs and HB were significantly different, indicating that the long-term wastewater discharge could alter the distribution of ARGs, MGEs, and microbial communities in the coastal area. The co-occurrence pattern among ARGs, MGEs, and microbial communities revealed that 12 bacterial genera, such as Psychrobacter, Pseudomonas, Sulfitobacter, Pseudoalteromonas, and Bacillus, showed strong positive correlations with ARGs and MGEs. Most potential hosts carried multidrug and ß-lactamase resistance genes.

Salt induced irreversible protein adsorption with extremely high loadings on electrospun nanofibers.

LiCl is a kosmotrope that generally promotes protein salvation in aqueous solutions. Herein we report that LiCl embedded in electrospun polymeric nanofibers interestingly induced an abnormal protein adsorption and substantially augmented the adsorption capacity of the fibers. As a result, equilibrium protein loadings reached over 64% (w/w) of the dry mass of fibers, 9-fold higher than that observed in the absence of the salt. The adsorption appeared to be irreversible such that little protein loss was observed even after washing the fibers vigorously with fresh buffer solutions. We further examined the application of such intensified protein adsorption for enzyme immobilization. Proteins including bovine serum albumin (BSA) and protamine were first adsorbed, followed by covalent attachment of an outer layer of an enzyme, α-chymotrypsin. Such a multilayer-structured nanofibrous enzyme exhibited extremely high stability with no obvious activity loss even after being incubated for 8 months at 4 °C in aqueous buffer solution. The LiCl induced irreversible protein adsorption, which has been largely ignored in previous studies with electrospun materials, rendering an interesting scenario of interfacial protein-material interactions. It also reveals a new mechanism in controlling and fabricating molecular interactions at interfaces for development of a broad range of biomaterials.

Surface charge affects cellular uptake and intracellular trafficking of chitosan-based nanoparticles.

Chitosan-based nanoparticles (NPs) are widely used in drug delivery, device-based therapy, tissue engineering, and medical imaging. In this aspect, a clear understanding of how physicochemical properties of these NPs affect the cytological response is in high demand. The objective of this study is to evaluate the effect of surface charge on cellular uptake profiles (rate and amount) and intracellular trafficking. We fabricate three kinds of NPs (∼ 215 nm) with different surface charge via SPG membrane emulsification technique and deposition method. They possess uniform size as well as identical other physicochemical properties, minimizing any differences between the NPs except for surface charge. Moreover, we extend our research to eight cell lines, which could help to obtain a representative conclusion. Results show that the cellular uptake rate and amount are both positively correlated with the surface charge in all cell line. Subsequent intracellular trafficking indicates that some of positively charged NPs could escape from lysosome after being internalized and exhibit perinuclear localization, whereas the negatively and neutrally charged NPs prefer to colocalize with lysosome. These results are critical in building the knowledge base required to design chitosan-based NPs to be used efficiently and specifically.

Simultaneous solid-liquid separation and primary purification of clavulanic acid from fermentation broth of Streptomyces clavuligerus using salting-out extraction system.

Clavulanic acid (CA) is usually used together with other ß-lactam antibiotics as combination drugs to inhibit bacterial ß-lactamases, which is mainly produced from the fermentation of microorganism such as Streptomyces clavuligerus. Recently, it is still a challenge for downstream processing of low concentration and unstable CA from fermentation broth with high solid content, high viscosity, and small cell size. In this study, an integrated process was developed for simultaneous solid-liquid separation and primary purification of CA from real fermentation broth of S. clavuligerus using salting-out extraction system (SOES). First, different SOESs were investigated, and a suitable SOES composed of ethanol/phosphate was chosen and further optimized using the pretreated fermentation broth. Then, the optimal system composed of 20% ethanol/15% K2HPO4 and 10% KH2PO4 w/w was used to direct separation of CA from untreated fermentation broth. The result showed that the partition coefficient (K) and recovery yield (Y) of CA from untreated fermentation broth were 29.13 and 96.8%, respectively. Simultaneously, the removal rates of the cells and proteins were 99.8% and 63.3%, respectively. Compared with the traditional method of membrane filtration or liquid-liquid extraction system, this developed SOES showed the advantages of simple operation, shorter operation time, lower process cost and higher recovery yield of CA. These results demonstrated that the developed SOES could be used as an attractive alternative for the downstream processing of CA from real fermentation broth.

Three-liquid-phase salting-out extraction of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)-rich oils from Euphausia superba.

The TLPSOES parameters were optimized by response surface methodology using Box-Behnken design, which were 16.5% w/w of ammonium citrate, 17.5% w/w of ethanol, and 46% w/w of n-hexane at 70 min of stirring time. Under optimized conditions the extraction efficiency attained was 90.91 ± 0.97% of EPA, 90.02 ± 1.04% of DHA, and 91.85 ± 1.11% of KO in the top n-hexane phase. The highest extraction efficiency of proteins and flavonoids, i.e. 88.34 ± 1.35% and 79.67 ± 1.13%, was recorded in the solid interface and ethanol phase, respectively. The KO extracted by TLPSOES system consisted of lowest fluoride level compared to the conventional method and whole wet krill biomass. The TLPSOES is a potential candidate for nutraceutical industry of KO extraction from wet krill biomass.

[Molecular imprinting-flow injection chemiluminescence method for determination of doxycycline hydrochloride].

Doxycycline hydrochloride can enhance the chemiluminescence of potassium ferricyanide and luminol in alkaline medium. So a molecular imprinting-flow-injection chemiluminescence method for the determination of doxycycline hydrochloride was established by using doxycycline hydrochloride-imprinted polymers as recognition material and potassium ferricyanide and luminol as detection system. Doxycycline hydrochloride-imprinted polymer was synthesized using methacrylic acid as functional monomer and ethylene glycol dimethacrylate as cross-linker. The linear range is 9.0 x 10(-7)-6.0 x 10(-5) g x mL(-1) and the detection limit is 3.2 x 10(-7) g x mL(-1). The relative standard deviation for 6.0 X 10(-6) g x mL(-1) of doxycycline hydrochloride was 3.5% (n = 9). This method has been successfully applied to the determination of doxycycline hydrochloride in tablets and in urine samples.

Fabrication of rigid and macroporous agarose microspheres by pre-cross-linking and surfactant micelles swelling method.

A novel combined method of pre-cross-linking and surfactant micelles swelling was proposed in this study to fabricate highly cross-linked and macroporous agarose (HMA) microspheres. Agarose was chemically modified by allylglycidyl ether (AGE) as heterobifunctional cross-linker via its active glycidyl moieties before gel formation and pre-cross-linking was achieved. By this means, the effective concentration of cross-linker presented in agarose gel increased significantly, and thus cross-linking with a high-efficiency was achieved. Further to enhance the intraparticle mass transfer of agarose microspheres, the surfactant micelles swelling method was utilized to create interconnected macropores. Under the optimal condition, HMA microspheres with homogeneous reticular structure and pore size of hundreds nanometers were successfully prepared. They exhibited a low backpressure with a flow velocity as high as 1987 cm/h, which was much higher than that of commercial Sepharose 4 F F. HMA microspheres were then derivatized with carboxymethyl (CM) groups and applied in ion-exchange chromatography. As expected, CM-HMA column separated model proteins effectively even at a flow velocity three times higher than that of commercial CM-4 F F. Visualization of dynamic protein adsorption by confocal laser scanning microscope (CLSM) revealed that the intraparticle mass transfer of CM-HMA microspheres was intensified due to its macroporous structure. All of the results indicated the newly developed agarose microspheres were a promising medium for high-speed chromatography.

Preparation of hierarchical hollow CaCO3 particles and the application as anticancer drug carrier.

One-pot approach to couple the crystallization of CaCO(3) nanoparticles and the in situ symmetry-breaking assembly of these crystallites into hollow spherical shells was developed under the templating effect of a soluble starch. Further functional study using HP-a as an anticancer drug carrier (DOX) demonstrated its advantages for localizing drug release by the pH value-sensitive structure and enhancing cytotoxicity by increasing cellular uptake, perinuclear accumulation, and nuclear entry.

Bioprocess of uniform-sized crosslinked chitosan microspheres in rats following oral administration.

Chitosan microspheres have a great potential in pharmaceutical application. In this study, uniform-sized chitosan microspheres crosslinked with glutaraldehyde (CG microspheres) were prepared by Shirasu Porous Glass (SPG) membrane emulsification technique. Based on the characterizations of uniform size and autofluorescence, it was possible to develop a new detecting system for observing and quantifying the CG microspheres in rats with three different diameters (2.1, 7.2 and 12.5 microm) synchronously after oral administration. This system was a combination of scanning electron microscopy (SEM), laser scanning confocal microscope (LSCM) and flow cytometer technique, which showed the advantages of being simple, intuitionistic, repeatable and sensitive. After oral administration of three kinds of particles with different diameters, bioadhesion in gastrointestinal tract, absorption in gastrointestinal tract, distribution in systemic tissues, and biodegradation in reticuloendothelial system (RES) were studied firstly in detail. The CG microspheres showed different fates in bioadhesion, absorption and distribution according to their diameters, while the biodegradation also varied due to the different locations in RES. These original results would indicate a better way for the CG microspheres in the clinical application.

Preparation and characterization of thermo-responsive albumin nanospheres.

Thermo-responsive poly(N-isopropylacrylamide-co-acrylamide)-block-polyallylamine-conjugated albumin nanospheres (PAN), new thermal targeting anti-cancer drug carrier, was developed by conjugating poly(N-isopropylacrylamide-co-acrylamide)-block-polyallylamine (PNIPAM-AAm-b-PAA) on the surface of albumin nanospheres (AN). PAN may selectively accumulate onto solid tumors that are maintained above physiological temperature due to local hyperthermia. PNIPAM-AAm-b-PAA was synthesized by radical polymerization, and AN was prepared by ultrasonic emulsification. AN with diameter below 200 nm and narrow size distribution was obtained by optimizing the preparative conditions. Rose Bengal (RB) was used as model drug for entrapment into the AN and PAN during the particle preparation. The release rate of RB from PAN compared with AN in trypsin solution was slower, and decreased with the increase of PNIPAM-AAm-b-PAA molecular weight, which suggested that the existence of a steric hydrophilic barrier on AN made digestion of AN more difficult. Moreover, the release of RB from PAN above the cloud-point temperature (T(cp)) of PNIPAM-AAm-b-PAA became faster. This was because the density of temperature-responsive polymers on AN was not so high, so that the interspace between the polymer chains increased after they shrunk due to the high temperature. As a result, the biodegradable AN was attacked more easily by trypsin. The design of PAN overcame the disadvantages of temperature-responsive polymeric micelles.

Uniform-sized PLA nanoparticles: preparation by premix membrane emulsification.

Nanoparticle size is crucial to drug release behavior and biodistribution in vivo, but few studies have been performed on biodegradable nanoparticles with narrow size distribution. In this note, uniform-sized nanoparticles were prepared by a facile method combining emulsion-solvent removal and premix membrane emulsification for the first time. After preparation of coarse emulsions, additional premix membrane emulsification with very high pressure was occupied to achieve uniform-sized nanodroplets, and nanoparticles were formed by further solidification. Polylactide (PLA) was selected as a model polymer. Several factors played key roles to obtain uniform-sized PLA nanoparticles, including type of organic solvent, the volume ratio of oil phase and external water phase, pore size of the microporous membrane and transmembrane pressure. The coefficient of variation (CV) value of PLA nanoparticles could be controlled below 16.9% under an optimum condition. The novel method also has the advantages of high productivity, simplicity and easy scale-up. The uniform-sized nanoparticles prepared by this novel method have great potentials in drug delivery.

Preparation of uniform-sized pH-sensitive quaternized chitosan microsphere by combining membrane emulsification technique and thermal-gelation method.

In this study, uniform-sized pH-sensitive quaternized chitosan microsphere was prepared by combining Shirasu porous glass (SPG) membrane emulsification technique and a novel thermal-gelation method. In this preparation process, the mixture of quaternized chitosan solution and alpha-beta-glycerophosphate (alpha-beta-GP) was used as water phase and dispersed in oil phase to form uniform W/O emulsion by SPG membrane emulsification technique. The droplets solidified into microspheres at 37 degrees C by thermal-gelation method. The whole process was simple and mild. The influence of process conditions on the property of prepared microspheres was investigated and the optimized preparation condition was obtained. As a result, the coefficient of variation (C.V.) of obtained microspheres diameters was below 15%. The obtained microsphere had porous structure and showed apparent pH-sensitivity. It dissolved rapidly in acid solution (pH 5) and kept stable in neutral solution (pH 7.4). The pH-sensitivity of microspheres also affected its drug release behavior. Bovine serum albumin (BSA) as a model drug was encapsulated in microspheres, and it was released rapidly in acid solution and slowly in neutral medium. The novel quaternized chitosan microspheres with pH-sensitivity can be used as drug delivery system in the biomedical field, such as tumor-targeted drug carrier.

Preparation of uniform-sized PELA microspheres with high encapsulation efficiency of antigen by premix membrane emulsification.

Relatively uniform-sized poly(lactide-co-ethylene glycol) (PELA) microspheres with high encapsulation efficiency were prepared rapidly by a novel method combining emulsion-solvent extraction and premix membrane emulsification. Briefly, preparation of coarse double emulsions was followed by additional premix membrane emulsification, and antigen-loaded microspheres were obtained by further solidification. Under the optimum condition, the particle size was about 1 mum and the coefficient of variation (CV) value was 18.9%. Confocal laser scanning microscope and flow cytometer analysis showed that the inner droplets were small and evenly dispersed and the antigen was loaded uniformly in each microsphere when sonication technique was occupied to prepare primary emulsion. Distribution pattern of PEG segment played important role on the properties of microspheres. Compared with triblock copolymer PLA-PEG-PLA, the diblock copolymer PLA-mPEG yielded a more stable interfacial layer at the interface of oil and water phase, and thus was more suitable to stabilize primary emulsion and protect coalescence of inner droplets and external water phase, resulting in high encapsulation efficiency (90.4%). On the other hand, solidification rate determined the time for coalescence during microspheres fabrication, and thus affected encapsulation efficiency. Taken together, improving the polymer properties and solidification rate are considered as two effective strategies to yield high encapsulation.

Porogen effects in synthesis of uniform micrometer-sized poly(divinylbenzene) microspheres with high surface areas.

A new method of synthesizing uniform poly(divinylbenzene) (polyDVB) microspheres with high specific surface areas was designed by combining Shirasu porous glass (SPG) membrane emulsification, suspension polymerization, and post-crosslinking techniques. It was shown that the physicochemical properties of porogens have a great influence on the size distribution and porous features of microspheres. The low aqueous solubility of porogen facilitated preparation of uniform emulsions and microspheres, and high aqueous solubility led to polydispersed emulsions and poor microsphere yields. Such aqueous solubility effects can be tailored by adding a low molecular weight polystyrene (LPST) as costabilizer in porogen, thus improving the uniformity of microspheres. Moreover, different affinities of porogens for copolymers demonstrate various contributions to specific surface areas of microspheres in suspension polymerization especially post-crosslinking. Solvating porogen requires a much higher addition than nonsolvating porogen to obtain equal specific surface areas in polymerization, but has more potential to enhance the specific surface area in post-crosslinking. Two kinds of uniform microspheres were obtained with high specific surface areas, up to 706.6 m2/g by heptane and 937.5 m2/g by toluene.

[Primary structure determination of hirudin and reteplase fusion protein by LC/ESI-MS/MS spectrometry].

The aim is to determine the primary structure of a new hirudin and reteplase fusion protein (HV12p-rPA) by LC-ESI-MS/MS spectrometry. The molecular weight of the hirudin and reteplase fusion protein (HV12p-rPA) was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HV12p-rPA was digested with trypsin and chymotrypsin separately and the peptides in the digest mixtures were identified by LC-ESI-MS/MS. The molecular weight of HV12p-rPA was 41,472 Da, which was in accordance with the theoretical value. The peptide fragments of HV12p-rPA digested with trypsin were identified by LC-ESI-MS/MS spectrometry and the results indicated that the fusion protein contained r-PA. Then, the peptides of HV12p-rPA digested with chymotrypsin were identified by the same method. The results indicated that the fusion protein contained HV12p and the linker-containing peptide, DEGGGSY. MASDF and LDWIRDNMRP were identified as the N-terminal and C-terminal containing peptides in the chymotryptic digest mixture of the fusion protein. All of the Xcorr values exceeded 1.5, some of which were above 3.0, showing that the results were correct and credible and a sequence coverage of 85% was achieved. HPLC/MS analysis coupled with uncompleted digestion indicated that all these peptides were arranged with the correct order as expected. Thus, sequence of the fusion protein was confirmed and it was consistent with our design in upstream construction.

A thermosensitive hydrogel based on quaternized chitosan and poly(ethylene glycol) for nasal drug delivery system.

A new thermosensitive hydrogel was designed and prepared by simply mixing N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) and poly(ethylene glycol) (PEG) with a small amount of alpha-beta-glycerophosphate (alpha-beta-GP). The optimum preparative condition was investigated, and the obtained formulation underwent thermal transition from solution below or at room temperature to non-flowing hydrogel around 37 degrees C in several minutes. As a new formulation, its potential use as nasal drug delivery system was studied. It can be dropped or sprayed easily into nasal cavity and spread on the nasal mucosa in solution state. After being administered into nasal cavity, the solution transformed into viscous hydrogel at body temperature, which decreased nasal mucociliary clearance rate and released drug slowly. Morever, quaternized chitosan as absorption enhancer has been studied extensively in several reports and proved its non-toxicity, mucoadhesivity and the capacity to open the tight junctions between epithelial cells. Therefore, in this study insulin as a model drug was entrapped in this formulation and its release behavior in vitro was also investigated. The enhancement of absorption of fluorescein isothiocyanate (FITC)-labeled insulin in rat nasal cavity by this formulation was proved by confocal laser scanning microscopy (CLSM). The cytoxicity and the change of the blood glucose concentration after nasal administration of this hydrogel were also investigated. The hydrogel formulation decreased the blood glucose concentration apparently (40-50% of initial blood glucose concentration) for at least 4-5h after administration, and no apparent cytoxicity was found after application. These results showed that HTCC-PEG-GP formulation can be used as nasal drug delivery system to improve the absorption of hydrophilic macromolecular drugs.

Modeling of alphak/gamma2 (k=1, 2, 3 and 5) interface of GABA A receptor and docking studies with zolpidem: implications for selectivity.

The three-dimensional models of the alphak/gamma2 (k=1, 2, 3 and 5) interface of GABA(A) receptors, which included the agonist-binding site, were constructed and validated by molecular modeling technology. To investigate the mechanism of alpha subunit selectivity of zolpidem, docking calculations were used to illustrate the potential binding modes of zolpidem with different alpha subtypes. The results revealed that there were three reasons resulting in the distinct binding affinity of zolpidem to different alpha subtype. Firstly, the number of hydrogen bonds of agonist-receptor complex would determine the magnitude of binding affinity. Secondly, the His residue in loop A of alpha subunit was indicated as a key role of benzodiazepine binding. Thirdly, the side chain of Glu in loop C reduced the affinity of zolpidem to those receptors containing alpha2, alpha3 or alpha5 subunits.