Biomedical subjects

Zhi-Guo Su

Publications and source records attributed to Zhi-Guo Su.

At least 19 recordsLinked to original sources

PubMed2006

A newly proposed mechanism for arginine-assisted protein refolding--not inhibiting soluble oligomers although promoting a correct structure.

Arginine has been demonstrated to be capable of suppressing aggregation during protein refolding. However, the pathway and the mechanism for arginine to participate in and to assist refolding process still remains unclear. In this study, arginine-assisted refolding of recombinant consensus interferon (rIFN-con1) was investigated. It was found that although arginine minimized the formation of protein precipitate, it failed to prevent the formation of the soluble oligomeric species. The amount of the oligomers increased with the increase in arginine concentration. This phenomenon has not been reported. On the other hand, arginine was able to promote the yield of correctly refolded rIFN-con1, which was more than 2 times higher than that in the absence of arginine. A proposed mechanism is the stabilization of different soluble species by arginine, which slowed down the conformational movement. The stabilization effect on native-like structure formation overwhelmed the oligomeric promotion effect, which resulted in a composite effect of increased refolding yield for rIFN-con1 when arginine concentration was below 0.5M.

Arginine↗

PubMed2006

Preparation of insulin-loaded PLA/PLGA microcapsules by a novel membrane emulsification method and its release in vitro.

Uniform-sized biodegradable PLA/PLGA microcapsules loading recombinant human insulin (rhI) were successfully prepared by combining a Shirasu Porous Glass (SPG) membrane emulsification technique and a double emulsion-evaporation method. An aqueous phase containing rhI was used as the inner water phase (w1), and PLA/PLGA and Arlacel 83 were dissolved in a mixture solvent of dichloromethane (DCM) and toluene, which was used as the oil phase (o). These two solutions were emulsified by a homogenizer to form a w1/o primary emulsion. The primary emulsion was permeated through the uniform pores of a SPG membrane into an outer water phase by the pressure of nitrogen gas to form the uniform w1/o/w2 droplets. The solid polymer microcapsules were obtained by simply evaporating solvent from droplets. Various factors of the preparation process influencing the drug encapsulation efficiency and the drug cumulative release were investigated systemically. The results indicated that the drug encapsulation efficiency and the cumulative release were affected by the PLA/PLGA ratio, NaCl concentration in outer water phase, the inner water phase volume, rhI-loading amount, pH-value in outer water phase and the size of microcapsules. By optimizing the preparation process, the drug encapsulation efficiency was high up to 91.82%. The unique advantage of preparing drug-loaded microcapsules by membrane emulsification technique is that the size of microcapsules can be controlled accurately, and thus the drug cumulative release profile can be adjusted just by changing the size of microcapsules. Moreover, much higher encapsulation efficiency can be obtained when compared with the conventional mechanical stirring method.

Capsules↗

PubMed2006

Preparation and characterization of uniform-sized chitosan microspheres containing insulin by membrane emulsification and a two-step solidification process.

Chitosan microsphere has important application in controlled release of protein and peptide drug, because it shows excellent mucoadhesive and permeation enhancing effect across the biological surfaces. In the conventional preparation methods of chitosan microsphere, the W/O emulsion was usually prepared by mechanical stirring method, and then the droplets were solidified by glutaraldehyde. There existed limitation and shortage such as broad size distribution, de-activity of bio-drug and difficulty in drug release because protein and peptide drug have the same amino group as chitosan. In this study, we established a method to prepare uniform-sized microsphere, and solve above problems by combining a special membrane emulsification technique and a step-wise crosslinking method. That is, the chitosan/acetic acid aqueous solution was pressed through the uniform pores of a porous glass membrane into a paraffin/petroleum ether mixture containing PO-500 emulsifier, to form a W/O emulsion with uniform droplet size. Then, the uniform droplets were solidified by a two-step crosslinking method. At the first step, tripolyphosphate (TPP) solution was dropped gradually in the emulsion, TPP diffused into the droplet to crosslink chitosan by an ionic linkage, generating a microgel. At the second step, an adequate amount of glutaraldehyde was added. The solidification conditions of the two-step process were optimized by investigating the effects of solidification conditions on morphology of microspheres, encapsulation efficiency (EE), drug activity and release profile in vitro. The suitable preparative conditions were determined as follows: pH value of aqueous phase and TPP solution was 3.5-4.0, the molar ratio of amino group of chitosan to aldehyde group of glutaraldehyde was 1:1 and the crosslinking time of glutaraldehyde was 60 min.

Biocompatible Materials↗

PubMed2006

A thermo- and pH-sensitive hydrogel composed of quaternized chitosan/glycerophosphate.

The quaternized chitosan was synthesized by the reaction of chitosan and glycidyltrimethylammonium chloride (GTMAC) and named as N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC). A novel hydrogel system composed of HTCC/glycerophosphate (HTCC/GP) with thermo- and pH-sensitivity was synthesized and used as an intelligent drug carrier. The formulation was solution below or at room temperature, which allowed it injectable and to incorporate living cells, proteins, enzymes or other therapeutic drugs easily. Once the surrounding temperature was up to 37 degrees C, the system was transformed to a non-flowing hydrogel, and the formed hydrogel can release the trapped drug as a function of pH values. The swelling behavior of the system and the release profiles of doxorubicin hydrochloride (DX) as a model drug at different pH values were investigated. At acidic condition the hydrogel dissolved and released drug quickly, while it absorbed water and released drug slowly at neutral or basic conditions. Hydrogel composed of chitosan hydrochloride and glycerophosphate (CS/GP) was also prepared to compare with HTCC/GP hydrogel. The HTCC/GP hydrogel in this study was transparent which made it suitable for some specific uses such as ocular drug formulation.

Chitosan↗

PubMed2006

Preparation and improvement of release behavior of chitosan microspheres containing insulin.

Chitosan microsphere has potential applications in orally and other mucosally administration of protein and peptide drug, because it shows excellent mucoadhesive and permeation enhancing effect across the biological surfaces. The control of the size and size distribution of chitosan microsphere is necessary in order to improve its reproducibility, bioavailability and repeatable release behavior. Furthermore, it is a big challenge how to maintain the chemical stability of protein drug and improve its release behavior in the preparation of chitosan microspheres, because conventional crosslinking method by glutaraldehyde cannot be used in encapsulation of protein drug containing amino group. In this study, we established a method to prepare uniform-sized microsphere, and solve above problems by combining a special membrane emulsification technique and a step-wise crosslinking method. The preparative condition was optimized, and the chemical stability of protein, encapsulation efficiency, and release behavior were compared with conventional preparative method of drug-loaded chitosan microspheres. As a result, fairly uniform chitosan microspheres were obtained with a coefficient of variation (C.V.) value less than 11%, and the step-wise crosslinking method developed specially for membrane emulsification method provided the microspheres with higher encapsulation efficiency (80%), higher chemical stability of insulin (>95%), lower burst release and steady release behavior.

Chemistry, Pharmaceutical↗

PubMed2006

[Development of a new hydrophobic interaction chromatography absorbent and its application to the purification of recombinant hepatitis B surface antigen].

A new hydrophobic absorbent based on homemade highly cross-linked agarose beads was synthesized by immobilizing butyl derivative onto the matrix linkage. The density of ligand was controlled by adjusting the concentration of butanethiol and the synthesis route was optimized by evaluating the purification efficiency of recombinant Hepatitis B surface antigen (HBsAg) expressed by Chinese hamster ovary (CHO) cell line. A high performance absorbent was finally screened out with up to 80% of HBsAg recovery and purification-fold (PF) about 20. Furthermore, the column pressure was about 0.06 MPa under the flow rate of 500cm/h, and no leaked butyl were detected after exposing the gel in common buffers, chaotropic agents, high concentrations of denaturing agents such as guanidine hydrochloride, urea and polar organic solvents. These results demonstrated that the absorbent have high physico-chemical stability, so it was available for the downstream process. Finally, after scaled up to 2L wet gel/batch, the absorbent was applied to the integration of three-step chromatography and obtained the purified CHO-HBsAg with 95% purity by SDS-PAGE and HPLC, which meet the requirements of SFDA. The purification efficiency and the reproducible ability of the absorbents were also evaluated from batch-to-batch. The results demonstrated that the absorbent met the requirement of scalable, reproducible, economic effect as well. This absorbent is a promising alternative exported HIC gel for wildly being used in Chinese pharmaceutical industries.

Animals↗

PubMed2005

Influence of process parameters on the size distribution of PLA microcapsules prepared by combining membrane emulsification technique and double emulsion-solvent evaporation method.

Relatively uniform-sized biodegradable poly(lactide) (PLA) microcapsules with various sizes were successfully prepared by combining a glass membrane emulsification technique and water-in-oil-in-water (w1/o/w2) double emulsion-solvent evaporation method. A water phase was used as the internal water phase, a mixture solvent of dichloromethane (DCM) and toluene dissolving PLA and Arlacel 83 was used as the oil phase (o). These two solutions were emulsified by a homogenizer to form a w1/o primary emulsion. The primary emulsion was permeated through the uniform pores of a glass membrane into the external water phase by the pressure of nitrogen gas to form the uniform w1/o/w2 double emulsion droplets. Then, the solid polymer microcapsules were obtained by simply evaporating solvent. The influence of process parameters on the size distribution of PLA microcapsules was investigated, with an emphasis on the effect of oil-soluble emulsifier. A unique phenomenon was found that a large part of emulsifier could adsorb on the interface of internal water phase and oil phase, which suppressed its adsorption on the surface of glass membrane, and led to the successful preparation of uniform-sized double emulsion. Finally, by optimizing the process parameters, PLA microcapsules with various sizes having coefficient of variation (CV) value under 14.0% were obtained. Recombinant human insulin (rhI), as a model protein, was encapsulated into the microcapsules with difference sizes, and its encapsulation efficiency and cumulative release were investigated. The result suggested that the release behavior could be simply adjusted just by changing precisely the diameters of microcapsule, benefited from the membrane emulsification technique.

Capsules↗

PubMed2005

Preparation of uniform sized chitosan microspheres by membrane emulsification technique and application as a carrier of protein drug.

The control of size and size distribution of microspheres is necessary for obtaining repeatable controlled release behavior. The chitosan microspheres were prepared by a membrane emulsification technique in this study. Chitosan was dissolved in 1 wt.% aqueous acetic acid containing 0.9 wt.% sodium chloride, which was used as a water phase. A mixture of liquid paraffin and petroleum ether 7:5 (v/v) containing PO-500 emulsifier was used as an oil phase. The water phase was permeated through the uniform pores of a porous glass membrane into the oil phase by the pressure of nitrogen gas to form W/O emulsion. Then GST (Glutaraldehyde Saturated Toluene) as crosslinking agent was slowly dropped into the W/O emulsion to solidify the chitosan droplets. The preparation condition for obtaining uniform-sized microspheres was optimized. The microspheres with different size were prepared by using the membranes with different pore size, and there was a linear relationship between the diameter of microspheres and pore size of the membranes when the microspheres were in the range of micron size. The smallest chitosan microspheres obtained was 0.4 mum in diameter. This is the first report for preparing the uniform-sized chitosan microspheres by membrane emulsification technique. Uniform chitosan microspheres were further used as a carrier of protein drug. Bovine serum albumin (BSA) as a model drug was loaded in the microspheres and released in vitro. The effects of pH value, diameter and crosslinking degree of microspheres, and BSA concentration on loading efficiency and release behavior were discussed.

Adsorption↗

PubMed2005

Immobilized beta-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle.

A novel artificial chaperone system using a combination of interactions between the unfolded protein, a detergent and a chromatographic column packed with immobilized beta-cyclodextrin (beta-CD) polymer coupled to an agarose gel, was introduced to refold recombinant Staphylococcus aureus elongation factor-G (EF-G). Pre-mixing of 10% Triton X-100 and unfolded EF-G at 24 mg/ml followed by a 20-fold dilution into refolding buffer led to successful capturing of EF-G by Triton X-100 resulting in formation of a detergent-protein complex at 1.2mg/ml of final protein concentration. The complex was subsequently applied to the immobilized beta-CD polymer column resulting in correct refolding of EF-G at a concentration of 530 microg/ml with 99% mass recovery. Detergent concentrations above critical micelle concentration were required for efficient capturing of EF-G at high protein concentration. Other detergents with hydrophile-lipophile-Balance values similar to that of Triton X-100 (Triton N-101, Noindet P40 (NP40), and Berol 185) also produced similar result. Soluble polymerized beta-CD was more efficient than the monomer to remove the detergent from the protein complex in a batch system. Immobilized beta-CD polymer column further improved the capability of detergent removal and was able to prevent aggregation that occurred with the addition of soluble beta-CD polymer at high protein concentration in the batch system. The mechanism for this system-assisted refolding was tentatively interpreted: the released protein could correctly refold in an enclosed hydrophilic environment provided by the integration of matrix and beta-CD polymer, and thus avoided aggregation during detergent removal.

Electrophoresis, Agar Gel↗

PubMed2005

A cyanopropyl reversed phase chromatography enables resolution of huperzine A and B on analytical and preparative scales.

Huperzine A (HupA) and huperzine B (HupB) are two medically important components of Huperzia serrata. It is difficult to obtain high yields of the separation from the plant using conventional liquid extraction and chromatography. However, this study has found that RP chromatography with cyanopropyl (CN) medium was able to separate these two analogs simultaneously from the plant extract with higher resolution. Comparison between a CN medium and a popular C18 medium demonstrated the superiority of the CN over the C18 in resolution for both analytical and preparative separation of HupA and HupB. A preparative process was developed for simultaneous purification of HupA and HupB from H. serrata. The yields on the basis of the mass of the herbal powder for HupA and HupB were 0.019 and 0.008% respectively, which were about 1.9 and 10 times of those reported in the literature.

Alkaloids↗

PubMed2005

A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding recombinant Staphylococcus aureus elongation factor G.

Recombinant Staphylococcus aureus elongation factor G (EF-G) is difficult to refold by dilution due to the formation of large amounts of misfolded structures. However, refolding of EF-G by adsorption to a chromatographic column packed with immobilized polyethylene glycol 20,000 (PEG 20 K) followed by pulse elution with 8 M urea resulted in 88% mass recovery and 80% of correctly refolded structure. The PEG 20 K was coupled to brominated allyl group derivatized Sepharose High Performance to construct a mild hydrophobic adsorbent. Various other hydrophobic interaction adsorbents were also attempted to refold EF-G. However, ligands with high hydrophobicity tended to misfold EF-G, resulting in irreversible adsorption. Various solvents, detergents, and low temperature as well as 8 M urea were tried to release bound EF-G. Only pulse elution with 8 M urea was efficient. Urea concentrations favorable for efficiently refolding EF-G were investigated. Low urea concentration produced more misfolded structures.

Bacterial Proteins↗

PubMed2005

Conjugate of bovine hemoglobin and human serum albumin as a candidate for blood substitute: characteristics and effects on rats.

Conjugate of bovine hemoglobin (bHb) and human serum albumin (HSA) was prepared. The product was simply composed of 89.7% one-to-one Hb-HSA conjugate, 6.0% oligomer of Hb and HSA, 3.5% unconjugated HSA and 0.8% unconjugated Hb, with an average molecular weight of 157 kD. The physicochemical characteristics were determined. Effects of single replacement on blood pressure and long-term survival of rats with 30% and 60% acute blood loss were studied, in comparison with Ringer-lactate solution, stroma-free hemoglobin (SFHb), 5% HSA in Ringer-lactate, whole blood and no resuscitation fluid. Results showed that Hb-HSA conjugate maintained the mean arterial pressure of rats to initial level with no pressor effect. Long-term effects of the replacement fluids on 30% bleeding rats showed that, for the group infused with Hb-HSA conjugate, histology of five major organs, heart, kidney, liver, spleen and lung, were essentially normal, similar to that of whole blood, while obviously renal side-effects appeared in other groups. The efficacy of the conjugate was further demonstrated by the resuscitation of lethal hemorrhagic shock rats (60% acute blood loss) with 100% survival rate (followed for 14 days), the same result as whole blood. The Hb-HSA conjugate can thus be another candidate for blood substitute in emergency.

Animals↗

PubMed2005

[Separation and purification of PEGylated rhG-CSF by two-step ion-exchange chromatography].

In order to separate and purify the PEGylated recombinant human granulocyte stimulating factor (rhG-CSF) at large laboratory-scale level, a two-step ion-exchange chromatographic separation procedure was designed. Cation-exchange chromatography was applied first to separate PEGylated rhG-CSF from un-reacted rhG-CSF, followed by anion-exchange chromatography to dissolve individual PEG-rhG-CSF species (mono-, di- and tri-PEGylated rhG-CSF) and remove the free PEG. The molecular weight of individual PEGylated rhG-CSF was determined by MALDI-TOF and SDS-PAGE. MALDI-TOF mass spectrometry revealed that the molecular weights of mono-, di- and tri-PEGylated rhG-CSF are 23.8 kD, 28.6kD and 33.8kD, respectively. Cell proliferation activity was detected by MTT assay using NFS-60 cell. The in vitro residual bioactivity of mono-, di- and tri-PEGylated rhG-CSF were 90%, 75% and 43% respectively, comparing with the un-conjugated rhG-CSF. These results indicated that the un-conjugated rhG-CSF and excess free PEG can be removed completely and the three conjugate species can be purified into homogeneity by the two consecutive ion-exchange chromatographic steps. The purification procedure is easy to scale-up, high in performance and recovery.

Chromatography, Ion Exchange↗

PubMed2005

[Separation of correctly refolded and mis-refolded consensus interferon by hydrophobic interaction chromatography].

Hydrophobic interaction chromatography was used to separate correctly refolded and mis-refolded consensus interferon. The effects of ligand types, salt concentration, pH and flow rate were investigated. The best result could be obtained by using Butyl Sepharose 4 Fast Flow, 0.8 mol/L of ammonium sulfate, pH 8.3 and 90cm/h of linear flow rate. Reverse-phase HPLC analysis showed the purity of the pooled fraction was as high as 99.6%. The specific activity of purified consensus interferon was 2.3 x 10(9) IU/mg, The mass recovery of targeth protein was 36.7%.

Chromatography, Liquid↗

PubMed2005

[Simultaneous purification of superoxide dismutase, catalase and hemoglobin from bovine erythrocyte lysate with PEG 600 as chaperon].

Superoxide dismutase, catalase and hemoglobin were purified simultaneously from the same batch of bovine erythrocyte lysate. The process involves an initial anion exchange chromatography, followed by a hydrophobic interaction chromatography and gel filtration chromatography. 0.75% polyethylene glycol 600 was added as a purification chaperon before the anion exchange chromatography. The hemoglobin fraction passed through the ion exchange column without being retained. The superoxide dismutase and catalase were adsorbed by the column and were eluted separately during elution. The two eluted fractions containing crude superboxide dismutase and catalase were further purified with hydrophobic interaction chromatography and gel filtration chromatography in sequence. The specific activities of superoxide dismutase and catalase were 15932u/mg and 65918u/mg, respectively. SDS-polyacrylamide gel electrophoresis and gel filtration chromatography were used to analyze the purity of the proteins. The purity of superoxide dismutase, catalase and hemoglobin were 77.6%, 81.9% and 99.9%, respectively. The total recoveries for superoxide dismutase, catalase and hemoglobin were 47.4%, 29.6% and 88.7%, respectively.

Animals↗

PubMed2005

[Analyses of Streptomyces coelicolor inner membrane proteome by multidimentional protein identification technology].

Streptomyces coelicolor is the model species among streptomycetes. Until now, proteomic analyses of S. coelicolor have been conducted using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry method, few integral membrane proteins were identified due to the hydrophobic and low-abundance nature of these proteins. In this work, 154 possible inner membrane proteins from S. coelicolor were identified using high pH-proteinase K sample preparation method and multidimensional protein identification technology, among them 44 are integral membrane proteins containing at least one transmembrane domain, most peptides and their corresponding proteins were identified experimentally for the first time.

Bacterial Proteins↗

PubMed2005

[Polyethylene glycol-accompanied ion-exchange chromatography to purify recombinant hepatitis B virus surface antigen].

The dissociation of virus-like particles of Hepatitis B surface antigen (HBsAg) during the adsorption-desorption on the solid-phase of chromatography is a main challenge for its purification. Herein, poly (ethylene glycol) (PEG) was applied as an additive during the purification of HBsAg from recombinant Chinese hamster ovary (CHO) cell culture to improve the HBsAg recovery and protect its structural assembly. The presence of 1% of PEG10000 in the mobile phase of ion-exchange chromatography (IEC) of DEAE-Sepharose FF column could increase the recovery of HBsAg from about 55% to 80%, with a similar purification (-fold) (about 12) compared with the absence of PEG. Importantly, glycosylated protein forms of HBsAg were reserved well by PEG-accompanied chromatography. Furthermore, size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) analysis was performed on line to monitor the aggregates, particle size and molecular weight distribution of HBsAg. The results demonstrated that the HBsAg particle size and assembly are more homogenous after adding PEG in the mobile phase of IEC than no PEG added in the mobile phase.

Animals↗

PubMed2005

[Study on rhG-CSF modified with polyethylene glycol].

Monomethoxy Polyethylene Glycol(mPEG20000) was activated by N-hydroxysuccinimede and analyzed by infrared spectrum and hydrolysis kinetics. In order to propose the optimized reaction conditions of mono-PEGylated rhG-CSF, orthogonal design of the experiment was investigated. Ion exchange chromatography was used to separate and purify PEGylated rhG-CSF from unPEGylated rhG-CSF. The purity of mono-PEGylated rhG-CSF was analyzed by high performance liquid chromatography (HPLC) to be 97%.

Granulocyte Colony-Stimulating Factor↗