WO2002010218A1 - Encapsulation directe de biomacromolecules dans des materiaux mesoporeux et nanoporeux a matrice tensioactive - Google Patents

Encapsulation directe de biomacromolecules dans des materiaux mesoporeux et nanoporeux a matrice tensioactive Download PDF

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Publication number
WO2002010218A1
WO2002010218A1 PCT/US2001/023979 US0123979W WO0210218A1 WO 2002010218 A1 WO2002010218 A1 WO 2002010218A1 US 0123979 W US0123979 W US 0123979W WO 0210218 A1 WO0210218 A1 WO 0210218A1
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WIPO (PCT)
Prior art keywords
sol
hrp
gel
surfactant
mesoporous
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PCT/US2001/023979
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English (en)
Inventor
Yen Wei
Jigeng Xu
Hua Dong
Qiuwei Feng
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Drexel University
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Publication date
Application filed by Drexel University filed Critical Drexel University
Priority to US10/332,423 priority Critical patent/US6989254B2/en
Priority to AU2001280918A priority patent/AU2001280918A1/en
Publication of WO2002010218A1 publication Critical patent/WO2002010218A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier

Definitions

  • the present invention relates to direct immobilization of enzymes and other bioactive agents to surfactant-templated mesoporous or nanoporous materials.
  • enzymes encapsulated within mesoporous or nanoporous materials can be obtained via surfactant-templated sol-gel reactions.
  • the encapsulated enzymes exhibit much higher catalytic activity than conventional microporous, materials.
  • enzyme-immobilization can be achieved by surfactant-templated sol-gel synthesis.
  • Encapsulation of enzymes and other biomacromolecules in sol-gel materials has drawn great interest in recent years because of the various potential applications in, for example, biocatalysis, biosensor, and drug release vehicles.
  • Encapsulated enzymes retain the same functionality but usually have higher thermal, storage and operational stability in comparison with their counterparts in solution.
  • the apparent activity of an entrapped enzyme is often hindered by internal diffusion, and sometimes, by reduced accessibility in microporous sol-gel matrixes even if the synthesis is optimized to preserve the labile biomolecules .
  • An object of the present invention is to provide biomacromolecules encapsulated or immobilized within mesoporous or nanoporous materials via surfactant-templated sol-gel reactions.
  • Another object of the present invention is to provide a method for encapsulating or immobilizing biomacromolecules in mesoporous or nanoporous materials via surfactant-templated sol-gel reactions conducted at near neutral pH and near room temperature.
  • surfactant-templated mesoporous and nanoporous materials can serve as host matrixes for immobilizing biomacromolecules.
  • biomacromolecule it is meant to include any biologically active macromolecule . Examples include, but are not limited to, enzymes, other proteins, cells, organelles and nucleic acids.
  • nonionic surfactant copolymers such as poly (ethylene oxide) (PEO) copolymers can be employed as the template for the synthesis of mesostructured enzyme-containing sol-gel matrixes.
  • PEO poly (ethylene oxide) copolymers
  • HRP horseradish peroxidase
  • GOx glucose oxidase
  • enzymatic assay data show that HRP alone or HRP with GOx in the mesoporous silica or organically modified hybrid silica matrixes have significantly greater enzymatic activities compared to the same enzymes encapsulated in microporous matrixes .
  • HRP-containing sol-gel materials were synthesized in the presence of various nonionic PEO copolymer surfactants .
  • relatively low ratios of water to surfactant were employed as precipitation was found to occur at higher ratios of 10:1.
  • the reaction systems were homogeneous at various stages of the sol-gel process from sol to gel.
  • the synthesized HRP- containing sol-gels were mostly homogeneous and transparent or, in some samples, translucent.
  • the compositions of the PEO surfactant-templated samples are summarized in Table 1.
  • the apparent initial activity (V of the sol-gel samples were evaluated at room temperature at
  • the host sol-gel matrixes were characterized by nitrogen adsorption measurements as described by Wei et al . (Adv. Mater. 1998 3:313-316), Wei et al . (Chem. Mater. 1999 11:2023-2029) and Sing et al . (Pure Appl. Chem. 1985 57:603-619).
  • the pore structure parameters of the silica matrixes are summarized in Table 1. The results indicate that the host silica materials are mesoporous with large pore volumes. The pore size distributions are relatively narrow.
  • the enzymatic activity of the sol-gel samples is believed to be associated, to some extent, with the pore microstructure parameters of the host matrix.
  • the activity of HRP encapsulated in the non- templated microporous sol-gel matrix, prepared in the absence of templates under otherwise identical conditions was found to be small (V ⁇ ⁇ 0.1 unit mg" 1 min" 1 ; Avnir et al . Chem. Mater. 1994 6:1605-1614; and Dave et al . Anal. Chem. 1994 66:1120A- 1127A) .
  • the HRP in the nonionic surfactant-templated mesoporous matrixes exhibits remarkable improvements in the enzymatic activity (see Table 1) , which could be attributed to the reduced internal diffusion resistance and improved accessibility.
  • Glucose oxidase is one of the most widely studied enzymes because of its utility in the selective determination of D-glucose, an analyte of broad analytical and pharmaceutical interest.
  • the use of insoluble glucose oxidase provides some advantages that overcome limitations encountered when using soluble enzyme in solution.
  • use of insoluble glucose results in an increase in the retention of enzyme activity with time and easy separation and recovery with minimum contamination.
  • the insoluble enzyme preparations are ubiquitous in the design of reactor/ sensing units and are adaptable to continuous flow sample and reagent processing.
  • PEO surfactant templates co-immobilized with glucose oxidase (GOx) and horseradish peroxidase (HRP) in silica sol-gel matrixes under near neutral pH conditions were also prepared.
  • the activities of the two enzymes in their catalysis of consecutive reactions, along with structure characterization of the materials were evaluated.
  • the change in absorbance of quinoneimine, a red dye, at 510 nm was used to evaluate the enzymatic activity.
  • the relationship between the ratio of HRP/GOx and the activity of free and immobilized HRP/GOx is that the higher the ratio of HRP/GOx, the higher the apparent activity.
  • the product hydrogen peroxide, generated in the GOx catalyzed glucose oxidation can be consumed more efficiently in the HRP catalyzed reaction.
  • the response time for the surfactant-templated HRP/GOx-containing sol-gel samples was less than 5 minutes, while the nontemplated controls derived from TMOS (control-B) and 95 mol%-TMOS/5 mol%-PhTMS (control-A) had no visible color change for 3 hours after adding glucose .
  • the nonionic surfactant-templated HRP/GOx-containing sol-gel samples were mesoporous materials, as evidenced by the nitrogen adsorption isotherms and BJH pore size distributions. Accordingly, these experiments demonstrate that enzyme immobilization or encapsulation can be achieved by surfactant- templated sol-gel synthesis conducted at near neutral pH and near room temperature.
  • the enzyme-containing sol-gels produced via this method have structural characteristics of mesoporous materials after removing the surfactants and exhibit relatively high remaining enzyme activities.
  • the template synthesis of the present invention provides a useful means for the in si tu immobilization of biomacromolecules in highly ordered mesoporous or nanoporous sol-gel materials or molecular sieves.
  • nonionic surfactant copolymer PEO as the template or pore forming agent
  • other polyethers or polyamines can be used.
  • the methods disclosed herein are useful with ionic surfactants as well.
  • ionic surfactants which can be used in the mesoporous or nanoporous templates include, but are not limited to, cationic surfactants such as alkyl and aryl sulfates, zwitter-ionic surfactants containing both cations and anions, and mixtures thereof.
  • Mesoporous or nanoporous materials suitable for encapsulation of biomacromolecules include, but are not limited to, inorganic materials such as silica, aluminosilicates, titania, zirconia, and alumina, organic-modified materials such as alkyl, aryl, and vinyl modified materials, and inorganic, polymer-modified materials such as polymethacrylate and polystyrene.
  • inorganic materials such as silica, aluminosilicates, titania, zirconia, and alumina
  • organic-modified materials such as alkyl, aryl, and vinyl modified materials
  • polymer-modified materials such as polymethacrylate and polystyrene.
  • HRP (EC 1.11.1.7) Type II; 200 purpurogallin units/mg; Sigma, lot 16119522), GOx (E.G. 1.1.3.4, from Aspergillus niger; 200 Fluka units/mg) , tetramethyl orthosilicate (TMOS) , phenyltrimethoxysilane (PhTMS) , IGEPAL CO-890 (denoted as surfactant-A) , and BRIJ 78 (denoted as surfactant-B) were purchased from Aldrich (Milwaukee, WI) .
  • Example 3 HRP Activity assay A colorimetric method using phenol, 4-aminoantipyrine and hydrogen peroxide (H 2 0 2 ) as the dye-generating compounds was used to evaluate the initial activity of HRP, as described in Xu et al . Polym. Prep. (Am. Chem. Soc. Div. Polym. Chem. 2000, 41(1), 1042-1043), Xu et al . Polym. Prep. (Am. Chem. Soc. Div. Polym. Chem. 2000, 41(1), 1046-1047), Xu et al . Polym. Prep. (Am. Chem. Soc. Div. Polym. Chem. 2000, 41(1), 1044-1045) , and Worthington, V. (Worthington Enzyme Manual; Worthington Biochemical Co.; Lakewood, NJ 1993; pp. 293-299).
  • Example 4 Characterization of HRP-containing sol-gels The content of Si0 2 in as-synthesized sol-gels was estimated from weight loss at 750°C in the air by thermogravimetric analysis (TGA) .
  • TGA thermogravimetric analysis
  • Nonionic surfactants were removed from as- synthesized sol-gels by calcination at 600°C.
  • Fine powder from as-synthesized sol-gel samples was heated to 600°C at a heating rate of 2°C/minute in the air flow and kept at 600°C for 4 hours before cooling to room temperature.
  • the calcined silica sol-gel material was characterized by the BET method as described by Wei et al . (Adv. Mater. 1993 3:313-316), Wei et al. (Chem. Mater. 1999 11:2023-2029) and Sing et al . (Pure Appl. Chem. 1985 57:603-619).
  • each surfactant solution was mixed with either of the above two sols under stirring at room temperature, cooled to 0°C and, then, mixed with an appropriate amount of HRP and GOx solutions. After gelation, the enzyme-containing gels were dried under room temperature until constant weight was reached. The final enzyme-containing dry gels were ground into fine powders and stored under -15°C.
  • the surfactant- template was extracted from as-synthesized sol-gels with phosphate buffer before the enzymatic activity assay and with
  • a colorimetric method using glucose, phenol and 4- aminoantipyrine as the dye-generating compounds, was used to evaluate the activity of both free and immobilized GOx at room temperature (23°C) with slight modifications of the protocol described by Worthington, V. (Worthington Enzyme Manual; Worthington Biochemical Co.; Lakewood, NJ, 1993; pp. 293-299).
  • the activity of GOx in the coupling of HRP/GOx was determined from the absorbance change at 510 nm, due to the formation of N-antipyryl-p-benzoquinoneimine, by using a UV-Vis spectrophotometer (Perkin Elmer Lambda 2, Norwalk, CT) .

Abstract

La présente invention concerne des biomacromolécules encapsulées ou immobilisées dans des matériaux mésoporeux ou nanoporeux via des réactions sol-gel à matrice tensioactive, ainsi que des méthodes de production de ces biomacromolécules encapsulées ou immobilisées.
PCT/US2001/023979 2000-07-31 2001-07-31 Encapsulation directe de biomacromolecules dans des materiaux mesoporeux et nanoporeux a matrice tensioactive WO2002010218A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US10/332,423 US6989254B2 (en) 2000-07-31 2001-07-31 Direct encapsulation of biomacromolecules in surfactant templated mesoporous and nanoporous materials
AU2001280918A AU2001280918A1 (en) 2000-07-31 2001-07-31 Direct encapsulation of biomacromolecules in surfactant templated mesoporous andnanoporous materials

Applications Claiming Priority (3)

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US22226600P 2000-07-31 2000-07-31
US60/222,266 2000-07-31
US22226601P 2001-07-31 2001-07-31

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1852393A1 (fr) * 2003-06-27 2007-11-07 K.U.Leuven Research & Development Matériaux à base d'oxyde mésoporeux cristallin utiles pour la fixation et la libération contrôlée de médicaments
US8273371B2 (en) 2003-06-27 2012-09-25 Johan Adriaan Martens Crystalline mesoporous oxide based materials useful for the fixation and controlled release of drugs
US8871999B2 (en) 2004-10-29 2014-10-28 Board Of Trustees Of Michigan State University Protection against herbivores
EP2677870A4 (fr) * 2011-02-22 2015-04-29 Univ Minnesota Biomatériaux encapsulés dans de la silice
CN105169398A (zh) * 2014-06-12 2015-12-23 华东理工大学 基于介孔氧化硅纳米粒子的控释***及其制备方法
US9534236B2 (en) 2013-03-08 2017-01-03 Regents Of The University Of Minnesota Membranes for wastewater-generated energy and gas
EP1585783B1 (fr) * 2003-01-23 2018-06-06 Commissariat à l'Énergie Atomique et aux Énergies Alternatives Materiau hybride organique-inorganique comprenant une phase minerale mesoporeuse et une phase organique, membrane et pile a combustible
US10035719B2 (en) 2014-10-15 2018-07-31 Regents Of The University Of Minnesota System and membrane for wastewater-generated energy and gas
CN114652819A (zh) * 2022-03-21 2022-06-24 滨州医学院 一种靶向肿瘤微环境可降解的多功能纳米材料及其制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999022227A2 (fr) * 1997-10-29 1999-05-06 Yizhu Guo Applications electroanalytique avec tensioactif pour composites de graphite sol-gel a serigraphie
WO1999036357A1 (fr) * 1998-01-20 1999-07-22 Drexel University Matieres mesoporeuses et leurs procedes de fabrication

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999022227A2 (fr) * 1997-10-29 1999-05-06 Yizhu Guo Applications electroanalytique avec tensioactif pour composites de graphite sol-gel a serigraphie
WO1999036357A1 (fr) * 1998-01-20 1999-07-22 Drexel University Matieres mesoporeuses et leurs procedes de fabrication

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XU ET AL.: "Use of poly(ethylene oxide) nonionic surfactants as template for enzyme-containing mesoporous sol-gel materials", POLYMER PREPRINTS, vol. 41, no. 2, August 2000 (2000-08-01), pages 1673 - 1674, XP002949270 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1585783B1 (fr) * 2003-01-23 2018-06-06 Commissariat à l'Énergie Atomique et aux Énergies Alternatives Materiau hybride organique-inorganique comprenant une phase minerale mesoporeuse et une phase organique, membrane et pile a combustible
AU2004251792B2 (en) * 2003-06-27 2009-08-13 K.U. Leuven Research & Development Crystalline mesoporous oxide based materials useful for the fixation and controlled release of drugs
US7749521B2 (en) 2003-06-27 2010-07-06 K.U. Leuven Research & Development Crystalline mesoporous oxide based materials useful for the fixation and controlled release of drugs
EP2336086A1 (fr) * 2003-06-27 2011-06-22 K.U.Leuven Research & Development Matériaux à base d'oxyde mésoporeux cristallin utiles pour la fixation et la libération contrôlée de médicaments
US8273371B2 (en) 2003-06-27 2012-09-25 Johan Adriaan Martens Crystalline mesoporous oxide based materials useful for the fixation and controlled release of drugs
EP1852393A1 (fr) * 2003-06-27 2007-11-07 K.U.Leuven Research & Development Matériaux à base d'oxyde mésoporeux cristallin utiles pour la fixation et la libération contrôlée de médicaments
US9796984B2 (en) 2004-10-29 2017-10-24 Board Of Trustees Of Michigan State University Protection against herbivores
US8871999B2 (en) 2004-10-29 2014-10-28 Board Of Trustees Of Michigan State University Protection against herbivores
EP2677870A4 (fr) * 2011-02-22 2015-04-29 Univ Minnesota Biomatériaux encapsulés dans de la silice
US9790484B2 (en) 2011-02-22 2017-10-17 Regents Of The University Of Minnesota Silica encapsulated biomaterials
US9534236B2 (en) 2013-03-08 2017-01-03 Regents Of The University Of Minnesota Membranes for wastewater-generated energy and gas
CN105169398A (zh) * 2014-06-12 2015-12-23 华东理工大学 基于介孔氧化硅纳米粒子的控释***及其制备方法
CN105169398B (zh) * 2014-06-12 2020-08-18 华东理工大学 基于介孔氧化硅纳米粒子的控释***及其制备方法
US10035719B2 (en) 2014-10-15 2018-07-31 Regents Of The University Of Minnesota System and membrane for wastewater-generated energy and gas
CN114652819A (zh) * 2022-03-21 2022-06-24 滨州医学院 一种靶向肿瘤微环境可降解的多功能纳米材料及其制备方法

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