WO2009149883A1 - Nanoéponges de cyclodextrine comme transporteurs pour des biocatalyseurs, et dans l'administration et la libération d'enzymes, de protéines, de vaccins et d'anticorps - Google Patents

Nanoéponges de cyclodextrine comme transporteurs pour des biocatalyseurs, et dans l'administration et la libération d'enzymes, de protéines, de vaccins et d'anticorps Download PDF

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WO2009149883A1
WO2009149883A1 PCT/EP2009/004098 EP2009004098W WO2009149883A1 WO 2009149883 A1 WO2009149883 A1 WO 2009149883A1 EP 2009004098 W EP2009004098 W EP 2009004098W WO 2009149883 A1 WO2009149883 A1 WO 2009149883A1
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enzyme
enzymes
nanosponges
activity
proteins
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Inventor
Gianfranco Gilardi
Francesco Trotta
Roberta Cavalli
Paolo Ferruti
Elisabetta Ranucci
Giovanna Di Nardo
Carlo Maria Roggero
Vander Tumiatti
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SEA MARCONI TECHNOLOGIES Sas DI VANDER TUMIATTI
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SEA MARCONI TECHNOLOGIES Sas DI VANDER TUMIATTI
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Priority to EP09761446A priority Critical patent/EP2294190A1/fr
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    • 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
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • 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
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/091—Phenol resins; Amino resins
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • 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
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic 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
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • 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
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • 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
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/096—Polyesters; Polyamides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals

Definitions

  • the present invention relates to the use of cyclodextrin nanosponges as carriers for enzymes, proteins, vaccines or antibodies, a process for the preparation of the enzymes carried by said nanosponges, and catalysts obtainable by said process.
  • Proteins perform numerous functions, such as oxygen transport, maintenance of the structure of some tissues, reception and transduction of signals in the cells, immune response, and catalysis in metabolic processes.
  • Enzymes operate under mild reaction conditions, have a high reaction speed, and are highly specific. In view of their efficiency, only small amounts of enzyme are needed to transform large volumes of reagents. They have a beneficial effect on the environment because they reduce energy consumption and reduce the production of pollutants.
  • Examples of industrially useful enzymes include alpha amylase, trypsin, cellulase and pectinase for fruit juice clarification processes, ligninase to break down lignin, lipase in the detergent industry and biodiesel production, etc.
  • Enzymes like all catalysts, can be used in homogeneous phase (homogeneous catalysis) or heterogeneous phase (heterogeneous catalysis).
  • the catalyst is in the same phase (liquid) as the reagents and the reaction products.
  • Homogeneous catalysis is characterised by high selectivity, high activity, and constant reproducibility.
  • the main disadvantages of this technique are the short life of the catalyst, its possible poisoning, and the difficulty of separating the reaction products from the catalyst and recycling the catalyst, which is always problematic.
  • the catalyst is in a different phase from the one that contains the reagents and reaction products. In this case, separation of the reaction products is easy, and work can be performed in continuous flow.
  • the catalytic performance of the heterogenised enzyme depends on the substrate used and the immobilisation techniques. As catalytic activity depends mainly on the correct orientation of the active site, the enzyme must be immobilised in such a way as to minimise modifications to the catalytic site.
  • the surface on which the enzyme is carried is responsible for retention of the enzyme structure, and the reaction micro-environment created can modify the fields of use of the enzyme, for example by extending the operational pH range.
  • proteins, peptides, enzymes and derivatives thereof can be used in the biomedical and therapeutic field.
  • Proteolytic enzymes can be used to treat cancer or type I mucopolysaccharidosis, while DNA and oligonucleotides are used in gene therapy, for example.
  • the administration of these molecules presents various problems and limitations.
  • Most protein drugs are poorly absorbed through the biological membranes due to a combination of factors such as large molecular size, hydrophilic nature, ionicity, high surface charge, chemical and enzymatic instability, and low permeability through the mucous membranes.
  • protein molecules Following intravenous administration, protein molecules present rapid blood clearance, a significant ability to bond to the plasma proteins, and considerable sensitivity towards proteolytic enzymes.
  • Oral administration presents the drawback of low bioavailability.
  • Various approaches exist for therapeutic use such as increasing the dose or using absorption promoters, which can cause toxicity problems, or using alternative administration routes.
  • a number of systems for carrying enzymes and proteins have been developed, such as nano- and micro- particles, liposomes and hydrogels. Carriage in a particulate system can protect proteins from breakdown, modify their pharmacokinetics and improve their stability in vivo.
  • Cyclodextrins are non-reducing cyclic oligosaccharides consisting of 6-8 glucose molecules bonded with a 1,4- ⁇ -glucoside bond, which have a characteristic cone-frustum structure.
  • the arrangement of the functional groups of the glucose molecules is such that the surface of the molecule is polar, while the inner cavity is relatively lipophilic.
  • the lipophilic cavity enables the CDs to form inclusion complexes which are also stable in solution with organic molecules of suitable polarity and dimensions.
  • CDs have therefore been studied, and present numerous applications in various fields (pharmaceuticals, analysis, catalysts, cosmetics, etc.) in which the characteristics of the inclusion compounds are exploited.
  • Nanosponges are crosslinked polymers of cyclodextrins with various bonds which have proved very useful in various applications ranging from environmental decontamination to controlled drug delivery and release.
  • cyclodextrin nanosponges are a particularly suitable carrier to adsorb proteins, enzymes, antibodies and macromolecules in general.
  • enzymes it is possible to maintain their activity and efficiency, prolong their operation and extend the pH and temperature range of activity as well as allowing the conduct of continuous- flow processes.
  • proteins and other macromolecules can be carried by adsorbing or encapsulating them in cyclodextrin nanosponges. The interaction between proteins and nanosponges allows the formulation of slow, controlled-release systems.
  • nanosponges usable according to the invention are described, for example, in WO 2006/002814, and prepared by performing a solvent- free reactions between cyclodextrins and organic carbonates, with or without ultrasound irradiation. It has surprisingly been found that structurally amorphous nanosponges and nanosponges with a significant degree of crystallinity can be obtained ( Figure Ia and Ib). Other types of nanosponges, obtainable by reacting a cyclodextrin with a multifunctional polyisocyanate crosslinker, as described in WO 98/22197, can also be used.
  • a new class of nanosponges containing polyamidoamine bonds have also been used as carriers; they can be obtained, for example, by reacting cyclodextrins with bis-acrylamides, the synthesis of which is described in detail in example 1. Synthesis is preferably conducted in the presence of ultrasound.
  • the immobilisation of enzymes on said nanosponges involves placing an aqueous solution of enzyme in contact with the crosslinked nanosponges for a time ranging between 10 and 720 minutes at temperatures of between 4 and 40 0 C.
  • the aqueous solution is suitably buffered at a pH of between 5 and 9.
  • the invention relates both to the immobilisation process and to the enzymes immobilised on cyclodextrin crosslinked nanosponges.
  • enzymes which can be advantageously immobilised according to the invention include oxidoreductase, transferase, hydrolase, lyase, isomerase and ligase, and in particular amylase, trypsin, lipase, catalase, cellulase, ligninase, pectinase, protease and other enzymes of industrial interest.
  • the amount of enzyme immobilised on the nanosponges depends mainly on the incubation time of the polymer solution containing the nanosponges, as well as their nature and temperature. Broadly speaking, the immobilisation yields can be regulated within various limits, such as 1-50 mg per gram of nanosponges.
  • the enzyme activity is maintained for several days, and is more resistant to temperature and pH conditions, thus allowing more efficient enzymatic reactions to be performed.
  • Example 1 Synthesis of polyamidoamine nanosponges ( Figure 2) 0.66 ml of distilled water, 339 mg of 2,2'-bis(acrylamido) acetic acid
  • the resin was swollen again in an excess of water and then acidified with IM HCl to pH 2.5.
  • the acidified resin was purified by washing three times in distilled water under vigorous stirring, followed by centrifugation. The traces of water were extracted with ethanol and by leaving the product under vacuum for two days. The end product took the form of an odourless fine white powder.
  • the degrees of swelling of the resin in three buffer solutions at pH 2, 4.5 and 7 were evaluated, and are shown in Table 1.
  • the same reaction can be conducted with other stoichiometric ratios and other types of crosslinker such as methylenebisacrylamide, methylpiperazine or mixtures thereof, and using other natural cyclodextrins (alpha and gamma) or derivatives thereof (e.g. monotosyl cyclodextrin, monoazidocyclodextrin, etc.) Synthesis takes place more rapidly if the operations are ultrasound- assisted. Nanosponges with magnetic properties can also be obtained if synthesis is performed in the presence of magnetic particles, such as magnetite.
  • Example 2 Loading of enzymes onto nanosponges The best immobilisation yield was obtained from the crosslinked carrier described in WO 2006/002814, with a 1 :2 molar ratio between ⁇ -cyclodextrins and the crosslinking agent.
  • 1,2- dioxygenase enzyme was immobilised on 1.0 g of both types of crosslinked carrier (1 :2 and 1:4) at two different temperatures (4°C and 22°C).
  • the carrier was pre-washed with the same solution in which immobilisation took place
  • the quantity of immobilised enzyme was evaluated by calculating the difference between the total amount loaded onto the nanosponges and the amount not bonded to the resin which was present in the washing solutions.
  • the quantity of enzyme was calculated by measuring the activity on catechol and calculating the quantity of enzyme in mg from the specific activity (activity /mg of protein).
  • the experiment was conducted by incubating the enzyme and the nanosponges for different times and calculating the immobilisation yield.
  • Figure 3 shows the immobilisation yield obtained after incubation of the enzyme and crosslinked carrier 1 :2 at different times at 22°C.
  • the enzyme When the enzyme loading conditions had been established, the enzyme could be immobilised by absorption to a loading capacity of 28.9 mg of protein per gram of carrier.
  • the enzyme 1,2-dioxygenase catalyses the reaction that transforms catechol into muconic acid. This reaction was exploited to evaluate the activity of the immobilised enzyme. The reaction was monitored for 60 seconds by means of the increase in absorption at 260 nm due to the formation of the reaction product, namely muconic acid.
  • the reaction mixture contained
  • reaction product was monitored by separation in HPLC, as shown in Figure 5.
  • Reverse-phase separation with LiChrospher 100 RP- 18 5 ⁇ m 250x4 (Merck) column was used.
  • the mobile phase consisted of water and acetonitrile in the ratio of 60:40.
  • a column (10 x 2.5 mm) was packed with 1 g of the hyper-crosslinked carrier described in WO 2006/002814, on which 0.3 mg of catechol 1,2- dioxygenase were immobilised.
  • a 1 mM catechol solution was injected into the bioreactor at regular intervals (once a day), and the column was washed with 50 mM Hepes buffer, pH 8.0, after 1 hour.
  • the presence of the reaction product in the output of the bioreactor was monitored spectrophotometrically and by HPLC analysis.
  • the reaction product was quantitated with a molar extinction coefficient at 260 nm Of HOOO M- 1 Cm "1 .
  • the immobilised enzyme shows its greatest activity around 50 0 C, and still maintains 70% of its activity at 60 0 C.
  • the optimum temperature range for the enzyme in solution is between 30 0 C and 40 0 C. Almost total loss of activity of the enzyme in solution was observed after incubation at 60 0 C.
  • catechol 1,2 dioxygenase at two different temperatures, 40 0 C and 6O 0 C, the activity of the immobilised protein and that of the protein in solution were measured at different incubation times at the two selected temperatures ( Figures 8a and 8b).
  • the immobilised enzyme and the enzyme in solution were incubated in
  • the immobilisation process makes the enzyme more heat stable.
  • the enzyme in solution completely loses its activity after incubation of the enzyme for 90 minutes at 40 0 C, whereas the immobilised enzyme still retains 50% of its initial activity after 90 minutes.
  • Complete loss of the enzyme activity of the immobilised protein only takes place after 3 hours.
  • the activity loss profile of the immobilised enzyme at 40 0 C shows a two-phase behaviour which is probably due to the presence of two catalytic sites stabilised in different ways by the immobilisation process. As no cases of allosterism between the two monomers of the enzyme catechol 1,2-dioxygenase have so far been reported in the literature, this behaviour is probably due to the immobilisation process, which can create two non- equivalent iron centres.
  • Figure 8b shows the enzyme activity profile after incubation at 60 0 C. Complete loss of activity takes place after 15 minutes for the enzyme in solution, whereas at the same time, the immobilised enzyme still retains 70% of its initial activity.
  • Example 6 Influence of pH on enzyme activity
  • the enzyme (20 ⁇ l of wet resin described in example 1 with the immobilised enzyme) was incubated for 3 minutes in the different buffer solutions at a given pH value at 30 0 C, and the residual activity was then evaluated.
  • the buffers used were: Mes (for pH 5.5 to 7), Hepes (for pH 7 to 8.5), and Ches (for pH 8.5 to 10). The concentration of the buffer was selected in such a way as to maintain a constant ionic strength.
  • the activity profiles are different, which demonstrates that the immobilisation process influences the catalytic site of the enzyme.
  • the optimum pH for the enzyme activity of the immobilised protein is
  • the immobilised enzyme already has an activity of 40% at pH 6.5, whereas the protein in solution only has 8% of its peak activity.
  • the concentration of the protein was estimated by absorption at 402 nm using a molar extinction coefficient of 93500 M- I cm “1 (Keilin and Hartree, 1951), and the immobilisation yield was calculated as the difference between the quantity of protein incubated and the quantity of protein not bonded to the carrier.
  • the enzyme activity was measured on the substrate 2,2-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS), following the formation of the cation radical ABTS.+ in the presence Of H 2 O 2 and peroxidase at 414 nm ( Figure 10).
  • the reaction mixture consisted of 20 ⁇ l of wet resin with the immobilised enzyme, 1 mM ABTS and 15 ⁇ M H 2 O 2 in 50 mM phosphate buffer, pH 7.4. Table 1. Loading of HRP onto different NSs

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Abstract

La présente invention concerne des éponges de cyclodextrine réticulée cristalline ou amorphe comme transporteurs pour des enzymes, des anticorps, des protéines, des vaccins et des macromolécules en général.
PCT/EP2009/004098 2008-06-10 2009-06-08 Nanoéponges de cyclodextrine comme transporteurs pour des biocatalyseurs, et dans l'administration et la libération d'enzymes, de protéines, de vaccins et d'anticorps Ceased WO2009149883A1 (fr)

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EP09761446A EP2294190A1 (fr) 2008-06-10 2009-06-08 Nanoéponges de cyclodextrine comme transporteurs pour des biocatalyseurs, et dans l'administration et la libération d'enzymes, de protéines, de vaccins et d'anticorps

Applications Claiming Priority (2)

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IT001056A ITMI20081056A1 (it) 2008-06-10 2008-06-10 Nanospugne a base di ciclodestrine come supporto per catalizzatori biologici e nella veicolazione e rilascio di enzimi, proteine, vaccini ed anticorpi
ITMI2008A001056 2008-06-10

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102140178A (zh) * 2010-12-28 2011-08-03 重庆工商大学 环糊精-聚酰胺胺交联聚合物及其制备方法与应用
WO2013046165A1 (fr) 2011-09-30 2013-04-04 Sea Marconi Technologies Di Vander Tumiatti S.A.S. Utilisation de nano-éponges fonctionnalisées pour la croissance, la conservation, la protection et la désinfection d'organismes végétaux
WO2013158710A3 (fr) * 2012-04-18 2014-01-03 Cerulean Pharma Inc. Procédés et systèmes de précipitation de polymère et de génération de particules
CN105924496A (zh) * 2016-06-06 2016-09-07 西北工业大学 一种通过环糊精表面接枝来提高蛋白质结晶成功率的方法
CN108743961A (zh) * 2018-05-29 2018-11-06 暨南大学 具有化疗自增敏效果的药物载体、包含该载体的化疗药物及其制备方法
CN115006549A (zh) * 2022-06-16 2022-09-06 湖南科技大学 一种粒径可控及可控降解的酸敏型交联环糊精纳米水凝胶药物递送系统的制备方法

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WO2006002814A1 (fr) * 2004-06-25 2006-01-12 Sea Marconi Technologies Di W. Tumiatti S.A.S. Synthese assistee par ultrasons de nanoeponges a base de cyclodextrine

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US5718905A (en) * 1992-06-16 1998-02-17 Centre National De La Recherche Scientifique (C.N.R.S.) Preparation and use of novel cyclodextrin-based dispersible colloidal systems in the form of nanospheres
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TSUTSUMI T, HIRAYAMA F, UEKAMA K, ARIMA H.: "Potential use of polyamidoamine dendrimer/alpha-cyclodextrin conjugate (generation 3, G3) as a novel carrier for short hairpin RNA-expressing plasmid DNA.", JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 97, no. 8, 11 October 2007 (2007-10-11) - 11 October 2007 (2007-10-11), pages 3022 - 3034, XP002547969 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102140178A (zh) * 2010-12-28 2011-08-03 重庆工商大学 环糊精-聚酰胺胺交联聚合物及其制备方法与应用
WO2013046165A1 (fr) 2011-09-30 2013-04-04 Sea Marconi Technologies Di Vander Tumiatti S.A.S. Utilisation de nano-éponges fonctionnalisées pour la croissance, la conservation, la protection et la désinfection d'organismes végétaux
WO2013158710A3 (fr) * 2012-04-18 2014-01-03 Cerulean Pharma Inc. Procédés et systèmes de précipitation de polymère et de génération de particules
US9598503B2 (en) 2012-04-18 2017-03-21 Cerulean Pharma Inc. Methods and systems for polymer precipitation and generation of particles
CN105924496A (zh) * 2016-06-06 2016-09-07 西北工业大学 一种通过环糊精表面接枝来提高蛋白质结晶成功率的方法
CN105924496B (zh) * 2016-06-06 2019-04-19 西北工业大学 一种通过环糊精表面接枝来提高蛋白质结晶成功率的方法
CN105924496B9 (zh) * 2016-06-06 2019-06-11 西北工业大学 一种通过环糊精表面接枝来提高蛋白质结晶成功率的方法
CN108743961A (zh) * 2018-05-29 2018-11-06 暨南大学 具有化疗自增敏效果的药物载体、包含该载体的化疗药物及其制备方法
CN108743961B (zh) * 2018-05-29 2021-05-28 暨南大学 具有化疗自增敏效果的药物载体、包含该载体的化疗药物及其制备方法
CN115006549A (zh) * 2022-06-16 2022-09-06 湖南科技大学 一种粒径可控及可控降解的酸敏型交联环糊精纳米水凝胶药物递送系统的制备方法

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