Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me(2+)) ions, with the level of activity decreasing in the order Mn(2+)> or =Mg(2+)>Ca(2+)> or =Cu(2+)>Co(2+)> or =Ni(2+)> or =Zn(2+), whereas Fe(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me(2+) ions in the 0.1-12 mM range, depending on the particular metal ion. In the presence of all Me(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu(2+), Zn(2+), or Ca(2+) inhibited the Mg(2+)-dependent hydrolysis of scDNA, while Ni(2+), Co(2+), and Mn(2+) activated this reaction. The Mn(2+)-dependent hydrolysis of scDNA was activated by Ca(2+), Ni(2+), Co(2+), and Mg(2+) ions but was inhibited by Cu(2+) and Zn(2+). After addition of the second metal ion, only in the case of Mg(2+) and Ca(2+) or Mn(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0-100%) in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.     

Adsorption of toxic mercury(II) by an extracellular biopolymer poly(gamma-glutamic acid)

Adsorption of mercury(II) by an extracellular biopolymer, poly(gamma-glutamic acid) (gamma-PGA), was studied as a function of pH, temperature, agitation time, ionic strength, light and heavy metal ions. An appreciable adsorption occurred at pH>3 and reached a maximum at pH 6. Isotherms were well predicted by Redlich-Peterson model with a dominating Freundlich behavior, implying the heterogeneous nature of mercury(II) adsorption. The adsorption followed an exothermic and spontaneous process with increased orderliness at solid/solution interface. The adsorption was rapid with 90% being attained within 5 min for a 80 mg/L mercury(II) solution, and the kinetic data were precisely described by pseudo second order model. Ionic strength due to added sodium salts reduced the mercury(II) binding with the coordinating ligands following the order: Cl(-) >SO(4)(2-) >NO(3)(-). Both light and heavy metal ions decreased mercury(II) binding by gamma-PGA, with calcium(II) ions showing a more pronounced effect than monovalent sodium and potassium ions, while the interfering heavy metal ions followed the order: Cu(2+) > Cd(2+) > Zn(2+). Distilled water adjusted to pH 2 using hydrochloric acid recovered 98.8% of mercury(II), and gamma-PGA reuse for five cycles of operation showed a loss of only 6.5%. IR spectra of gamma-PGA and Hg(II)-gamma-PGA revealed binding of mercury(II) with carboxylate and amide groups on gamma-PGA.     

A new metal chelate affinity adsorbent for cytochrome C

We have prepared a novel metal-chelate adsorbent utilizing N-methacryloyl-L-histidine methyl ester (MAH) as a metal-chelating ligand. MAH was synthesized by using methacryloyl chloride and l-histidine methyl ester dihydrochloride. Spherical beads with an average diameter of 75-125 microm were produced by suspension polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAH carried out in an aqueous dispersion medium. Then, Cu(2+) ions were chelated directly on the chelating beads. Cu(2+)-chelated beads were used in the adsorption of cytochrome c (cyt c) from aqueous solutions. The maximum cyt c adsorption capacity of the Cu(2+)-chelated beads (658.2 micromol/g Cu(2+) loading) was found to be 31.7 mg/g at pH 10 in phosphate buffer. The nonspecific cyt c adsorption on the naked PHEMA beads was 0.2 mg/g. Cyt c adsorption increased with increasing Cu(2+) loading. Cyt c adsorption capacity was demonstrated for the buffer types with the effects in the order phosphate > HEPES > MOPS > MES > Tris-HCl. Cyt c molecules could be adsorbed and desorbed five times with these adsorbents without noticeable loss in their cyt c adsorption capacity.     

Effect of divalent metal cations on the dimerization of OXA-10 and -14 class D beta-lactamases from Pseudomonas aeruginosa

The factors influencing the oligomerization state of OXA-10 and OXA-14 class D beta-lactamases in solution have been investigated. Both enzymes were found to exist as an equilibrium mixture of a monomer and dimer, with a K(d) close to 40 microM. The dimeric form was stabilized by divalent metal cations. The ability of different metal ions to stabilize the dimer was in the following order: Cd(2+) > Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) > Mn(2+) > Ca(2+) > Mg(2+). The apparent K(d)s describing the binding of Zn(2+) and Cd(2+) cations to the OXA-10 dimer were 7.8 and 5.7 microM, respectively. The metal ions had a profound effect on the thermal stability of the protein complex observed by differential scanning calorimetry. The enzyme showed a sharp transition with a T(m) of 58.7 degrees C in the absence of divalent cations, and an equally sharp transition with a T(m) of 78.4 degrees C in the presence of a saturating concentration of the divalent cation. The thermal transition observed at intermediate concentrations of divalent metal ions was rather broad and lies between these two extremes of temperature. The equilibrium between the monomer and dimer is dependent on pH, and the optimum for the formation of the dimer shifted from pH 6.0 in the absence of divalent cations to pH 7.5 at saturating concentrations. The beta-lactamase activity increased approximately 2-fold in the presence of saturating concentrations of zinc and cadmium ions. Reaction with beta-lactams caused a shift in the equilibrium toward monomer formation, and thus an apparent inactivation, but the divalent cations protected against this effect.     

Studies of Laryngotracheitis Virus in Avian Tissue Cultures: III. Enhancement of Infectivity by Diethylaminoethyl-Dextran

Diethylaminoethyl-dextran (DEAE-D) enhanced the infectivity of laryngotracheitis virus (LTV) for chicken kidney (CK) cells when cultures were treated before inoculation with virus and when DEAE-D was present in the inoculum. Infectivity was not increased when cultures were treated after virus had adsorbed to cells; since infection was not synchronized, most of the virus had probably already penetrated the plasma membrane by the time DEAE-D was added. Maximal enhancement occurred when DEAE-D was present in the inoculum. Enhancement of a lesser degree occurred when virus and DEAE-D were mixed, diluted, and inoculated onto cultures. Adsorption of LTV at 37 C as compared to that at 5 C usually yields about a threefold greater number of plaques after a 2-hr adsorption period. However, when DEAE-D was incorporated in the inoculum, greater enhancement occurred at 5 C than at 37 C, and the number of plaques produced at both adsorption temperatures was about equal. Results are compatible with the hypothesis that increased adsorption is a factor in enhancement of infectivity of LTV by DEAE-D.     

Adsorption of Hg2+, Cu2+ and Zn2+ ions from aqueous solution using formaldehyde cross-linked modified chitosan-thioglyceraldehyde Schiff's base

A chitosan-thioglyceraldehyde Schiff's base cross-linked magnetic resin (CSTG) was prepared and characterized using various instrumental methods. Then, the prepared resin was used for comparative studies on the removal of toxic metal ions like: Hg(2+), Cu(2+) and Zn(2+) from aqueous solutions. The effects of the initial pH value of the solution, contact time, the initial metal ion concentration and temperature on the adsorption capacity of the composite were investigated. The kinetics data were analyzed by pseudo-first order and pseudo-second order equations. The adsorption kinetics was well described by the pseudo-second order equation, and the adsorption isotherms were better fitted by the Langmuir equation. The maximum theoretical adsorption capacities of the CSTG resin for Hg(2+), Cu(2+) and Zn(2+) were found to be 98±2, 76±1 and 52±1 mg g(-1), respectively. The negative values of Gibbs free energy of adsorption (ΔG(ads°) indicated the spontaneity of the adsorption of all metal ions on the novel resin.     

Multiple-point adsorption of terbium ions by lead ion templated thermosensitive gel: elucidating recognition of conformation in gel by terbium probe

Lead ion templated thermosensitive heteropolymer gel which has recognition ability of methacrylate pairs has been synthesized and characterized. The gel consists of a main monomer component, N-isopropylacrylamide (NIPA), responsible for volume phase transition, methacrylic acid (MAA) moieties imprinted as pairs to adsorb terbium ions and cross-links. An imprinting technique was applied using lead ion complex with methacrylate ligands in dioxane media. After gel was obtained, lead ions were removed by washing and the imprinted gel showed strong binding ability to terbium ions, comparable with that of the non-imprinted gel prepared without lead ions. It was found that the Tb(3+) fluorescence intensity was considerably increased upon binding this ion to both imprinted and non-imprinted gels, but the largest enhancement of fluorescence intensity was observed when Tb(3+) was bound to imprinted gel in shrunken state. This is because of the decrease of coordinated water molecules on Tb(3+) and the strong binding of this ion to methacrylate pairs which are encoded within the weakly cross-linked network of imprinted gel.     

Dual on-off and off-on switchable oligoaziridine biosensor

A water-soluble biocompatible aziridine-based biosensor with pendant anthracene units was synthesized by radicalar polymerization of N-substituted aziridines in supercritical carbon dioxide. The binding ability of the sensor towards a series of metal ions was examined by comparing the fluorescence intensities of the solutions before and after the addition of 100 equivalents of a solution of the metal ion chloride salt. A fast, simple and highly optical sensitive dual behavior, "off-on" and "on-off" response, was observed after the biosensor was exposed to the metal cations in aqueous solution. Zinc presented the highest fluorescence enhancement (turn-on) and copper presented the highest fluorescence quenching (turn-off). The response time was found to be instantaneous and the detection limit was achieved even in the presence of excess metal cation competitors. By using immunofluorescence microscopy it was also shown that oligoaziridine acts as an "on-off" probe through highly sensitive (detection limit of 1.6nM), selective and reversible binding to copper anions under physiologic conditions using living Human Fibroblast cells. The stoichiometry for the reaction of the biosensor with Cu(2+) was determined by a Job plot and indicates the formation of an oligoaziridine-Cu(2+) 1:2 adduct.     

A heavy metal biotrap for wastewater remediation using poly-gamma-glutamic acid

Poly-gamma-glutamic acid (gamma-PGA) obtained from Bacillus licheniformis ATCC 9945 was evaluated as a potential biosorbent material for use in the removal of heavy metals from aqueous solution. Copper (Cu(2+)) was chosen as the model heavy metal used in these studies since it is extensively used by electroplating and other industries, has been the model for many other similar studies, and can be easily assayed through a number of convenient methods. Cu(2+)-gamma-PGA binding parameters under varying conditions of pH, temperature, ionic strength, and in the presence of other heavy metal ions were determined for the purified biopolymer using a specially designed dialysis apparatus. Applying the Langmuir adsorption isotherm model showed that gamma-PGA had a copper capacity approaching 77.9 mg/g and a binding constant of 32 mg/L (0.5 mM) at pH 4.0 and 25 degrees C. Cu(2+)-gamma-PGA adsorption was relatively temperature independent between 7 and 40 degrees C, while an increase in ionic strength led to a decrease in metal ion binding. Cd(2+) and Zn(2+) ions compete with Cu(2+) for binding sites on the gamma-PGA biopolymer. Metal uptake by gamma-PGA was further tested using a tangential flow filtration apparatus in a diafiltration mode in which metal was continually processed through a dilute solution of gamma-PGA without allowing for equilibrium to be established. The circulating polymer solution was able to complex metal as well as successfully prevent passage of unbound copper ions present in solution through the membrane. Using 500 mL of a 0.2% gamma-PGA solution, up to 97% of a 50 mg/L copper sulfate solution processed at a flow rate of 115 mL/min was retained by the polymer. For a 10 mg/L solution of Cu(2+) as copper sulfate, filtrate concentrations of Cu(2+) never rose above 0.6 mg/L while processing 2.5 L of dilute copper sulfate.     

Mechanism of the transition-metal-catalyzed mutarotation reaction of N-(p-chlorophenyl)-beta-D-glucopyranosylamine in methanol

Rate constants for the mutarotation reaction of N-(p-chlorophenyl)-beta-D-glucopyranosylamine (NGlc) in methanol have been determined in the presence of transition metal chlorides (MCl(2)), at 25 degrees C. The activity of the metal ions catalyzing the alpha-pyranoside<-->beta-pyranoside interconversion has been found to increase in the following series: Mn(2+)相似文献   

Chelation ability of spironaphthoxazine with metal ions in silica gel

Spironaphthoxazine (SNO) and three metal ions, Mg(2+), Zn(2+), and Al(3+), were dispersed in silica gels by the sol-gel method. The chelation ability of SNO with the metal ions in silica gels was investigated by measuring the fluorescence spectra and was compared to that of 8-hydroxyquinoline (8-HQ) in ethanol and silica gels. A merocyanine-type isomer photoderived from SNO as well as 8-HQ easily formed complexes of the metal ions in the order of Al(3+), Zn(2+), and Mg(2+) because the coordination ability of the metal ions to such ligands depended on their electron affinity. The changes in the fluorescence spectra of the silica gel samples during light irradiation were also investigated. The relative band intensity due to the intermediate species between the original SNO and the merocyanine species decreased and that of the complex increased with the UV irradiation time. The reverse process was observed during visible irradiation. The UV irradiation effects on the chelation of SNO and its photochromic property also depended on the electron affinity of the metal ions.     

Effects of some metal ions on human erythrocyte glutathione reductase: an in vitro study

In this study, we investigated inhibitory effects of some metal ions on human erythrocyte glutathione reductase. For this purpose, initially human erythrocyte glutathione reductase was purified 1051-fold in a yield of 41% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis was done in order to control the purification of enzyme. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Hg(2+), Cd(2+), Pb(2+), Cu(2+), Fe(3+) and Al3+ exhibited inhibitory effects on the enzyme in vitro. K(i) constants and IC(50) values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I]. IC(50) values of Pb(2+), Hg(2+), Cu(2+), Cd(2+), Fe(3+) and Al(3+) were 0.011, 0.020, 0.0252, 0.0373, 0.209 and 0.229 mM, and the Ki constants 0.0254+/-0.0027, 0.0378+/-0.0043, 0.0409+/-0.0048, 0.0558+/-0.0083, 0.403+/-0.043 and 1.137+/-0.2 mM, respectively. While Pb(2+), Hg(2+), Cd(2+) and Fe(3+) showed competitive inhibition, others displayed noncompetitive inhibition.     

Enterovirus 71 adsorption on metal ion-composite chitosan beads

In this study, we developed composite chitosan beads combining various metal ions, including Ni(2+), Cu(2+), Zn(2+), and Fe(2+), for direct adsorption of enterovirus 71 (EV71). The metal-ion species had significant effects on the adsorption capacity of beads. Among these metal ion-composite chitosan beads, Ni(2+)-chitosan beads exhibited the best adsorption capacity of EV71. Using a concentration of 0.01-M Ni(2+) was found to best provide for bead formation and EV71 adsorption. The adsorption of EV71 for Ni(2+)-chitosan beads at neutral or alkaline pH was favored. Under a competitive condition with albumin proteins, Ni(2+)-chitosan beads exhibited significant capacity of EV71 adsorption in culture media. The adsorption of EV71 on the Ni(2+)-chitosan beads was attributed to the strong binding between Ni(2+) ions chelated to the surface amino acid of EV71 capsids and Ni(2+) ions chelated on the chitosan materials. Moreover, the adsorbed EV71 retained its antigenicity and infectivity after desorption. The Ni(2+)-chitosan beads exhibit a promising application to EV71 adsorption and removal.     

Metal ions binding to NAD-glycohydrolase from the venom of Agkistrodon acutus: regulation of multicatalytic activity

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(2+) ions that are essential for its multicatalytic activity. In this study, the interactions between divalent metal ions and AA-NADase and the effects of metal ions on its structure and activity have been investigated by equilibrium dialysis, isothermal titration calorimetry, fluorescence, circular dichroism, dynamic light scattering and HPLC. The results show that AA-NADase has two classes of Cu(2+) binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu(2+) ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn(2+) binding site, one Zn(2+) binding site, one strong and two weak Co(2+) binding sites, and two strong and six weak Ni(2+) binding sites. Metal ion binding affinities follow the trend Cu(2+) > Ni(2+) > Mn(2+) > Co(2+) > Zn(2+), which accounts for the existence of one Cu(2+) in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu(2+), and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn(2+) > Mn(2+) > Cu(2+) ~Co(2+) > Ni(2+). Metal ions serve as regulators for its multicatalytic activity. Although all tested metal ions have no obvious effects on the global structure of AA-NADase, Cu(2+)- and Zn(2+)-induced conformational changes around some Trp residues have been observed. Interestingly, each tested metal ion has a very similar activation of both NADase and ADPase activities, suggesting that the two different activities probably occur at the same site.     

Binding of divalent metal ions to calcium-free peroxidase: thermodynamic and kinetic studies

Thermodynamics of binding of divalent metal ions including Ca(2+) , Mg(2+) , Ba(2+) , and Cd(2+) to Ca-free horseradish peroxidase (HRP) enzyme was investigated using UV/VIS spectrophotometry and molecular-mechanic (MM) calculations. According to the obtained binding and thermodynamic parameters, trend of the relative binding affinities of these divalent metal cations was found to be: Ca(2+) >Cd(2+) >Mg(2+) >Ba(2+) . Binding analysis based on Scatchard and Hill models showed positive cooperativity effect between the two distal and proximal binding sites. Furthermore, kinetics of binding and reconstitution process was examined (using relaxation-time method) for binding of Ca(2+) (as the typical metal ion) to Ca-free HRP, which was found a second-order type having a two-step mechanism involving fast formation of Ca-free HRP/1?Ca(2+) as the kinetic intermediate in step 1. Finally, by means of MM calculations, the comparative stability energies were evaluated for binding of M(2+) metal cations to Ca-free HRP. Based on MM calculations, preferential binding of Ca(2+) ion was occurred on distal and proximal binding sites of Ca-free HRP associated with higher stability energies (E(total) ). Indeed, among the divalent metal ions, Ca(2+) with the highest binding affinity (maximum value of K(bin) and minimum value of ΔG$\rm{{_{bin}^{0}}}$), maximum value of exothermic binding enthalpy, and stability energies stabilizes the HRP structure along with an optimized catalytic activity.     

壳聚糖-g-N.羧甲基-二(2-苯并眯唑)-1,2-乙二醇的制备及其性能

以2-溴乙酸、壳聚糖、二（2-苯并咪唑）-1,2-乙二醇为原料,利用接枝作用将化学修饰后的小分子药物二（2-苯并咪唑）-1,2-乙二醇连接在天然高分子壳聚糖（CTS）上。并以。HNMR,IR,热分析及XRD等方法对其结构进行表征并研究接枝聚合物的理化性质。本文采用络合滴定法测定了接枝聚合物对一系列重金属离子的吸附作用;采用震荡法进行悬菌定量杀菌实验;还以经典的静态失重法研究了合成的聚合物在腐蚀介质中对N80钢片腐蚀的抑制作用。结果表明：小分子药物-（2-苯并咪唑）-1,2-乙二醇在接枝到天然高分子壳聚糖后热稳定性提高,在酸中具有良好的溶解度,对金属离子吸附能力在一个较宽温度范围内得以保持;同时增强了抑菌力,降低了最小抑菌浓度;利用BBIE与CTS韵协同作用提高了聚合物对金属腐蚀的抑制能力。     

Adsorption of heavy metal ions from aqueous solutions by activated carbon prepared from apricot stone

Apricot stones were carbonised and activated after treatment with sulphuric acid (1:1) at 200 degrees C for 24 h. The ability of the activated carbon to remove Ni(II), Co(II), Cd(II), Cu(II), Pb(II), Cr(III) and Cr(VI) ions from aqueous solutions by adsorption was investigated. Batch adsorption experiments were conducted to observe the effect of pH (1-6) on the activated carbon. The adsorptions of these metals were found to be dependent on solution pH. Highest adsorption occurred at 1-2 for Cr(VI) and 3-6 for the rest of the metal ions, respectively. Adsorption capacities for the metal ions were obtained in the descending order of Cr(VI) > Cd(II) > Co(II) > Cr(III) > Ni(II) > Cu(II) > Pb(II) for the activated carbon prepared from apricot stone (ASAC).     

Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes.

A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.     

Preparation and characterization of a novel pachyman-based pharmaceutical aid. II: A pH-sensitive,biodegradable and biocompatible hydrogel for controlled release of protein drugs

In this investigation, the fabrication, physico-chemical and biological characterization of a novel smart hydrogel had been evaluated for its potentials in effective controlling protein delivery. The hydrophilic pachyman-based hydrogel was generated facilely by crosslinking hydrosoluble carboxymethyl pachyman (CMP) with epichlorohydrin (ECH). The ECH concentration possessing maximum (99.7%) encapsulation efficiency and the most appropriate swelling characteristics was found to be 1.25% (w/v). The resultant hydrogel exhibited swelling ratios most favorable for drug release in simulated intestinal media. It could release two model protein drugs (bovine serum albumin and lysozyme) in the controlled manner and with full preservation of the protein stability and enzymatic activity. Importantly, the ECH-CMP hydrogel was confirmed to be biocompatible and biodegradable. From these findings, we were able to conclude that the synthesized pachyman-based hydrogel would be a promising delivery carrier candidate for site-specific delivery of protein drugs.