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WO2006080849A2 - Polymersomes de polyisocyanure - Google Patents

Polymersomes de polyisocyanure Download PDF

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Publication number
WO2006080849A2
WO2006080849A2 PCT/NL2006/000052 NL2006000052W WO2006080849A2 WO 2006080849 A2 WO2006080849 A2 WO 2006080849A2 NL 2006000052 W NL2006000052 W NL 2006000052W WO 2006080849 A2 WO2006080849 A2 WO 2006080849A2
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WO
WIPO (PCT)
Prior art keywords
piat
catalyst
polymersome
membrane
polymer
Prior art date
Application number
PCT/NL2006/000052
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English (en)
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WO2006080849A3 (fr
Inventor
Dennis Manuel Vriezema
Paula Maria Leandro Garcia
Jan Cornelis Maria Van Hest
Alan Edward Rowan
Roeland Johannes Maria Nolte
Original Assignee
Nederlandse Organisatie Van Wetenschappelijk Onderzoek
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Application filed by Nederlandse Organisatie Van Wetenschappelijk Onderzoek filed Critical Nederlandse Organisatie Van Wetenschappelijk Onderzoek
Publication of WO2006080849A2 publication Critical patent/WO2006080849A2/fr
Publication of WO2006080849A3 publication Critical patent/WO2006080849A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09J—ADHESIVES; NON-MECHANICAL ASPECTS OF ADHESIVE PROCESSES IN GENERAL; ADHESIVE PROCESSES NOT PROVIDED FOR ELSEWHERE; USE OF MATERIALS AS ADHESIVES
    • C09J2400/00—Presence of inorganic and organic materials
    • C09J2400/20—Presence of organic materials
    • C09J2400/26—Presence of textile or fabric
    • C09J2400/263—Presence of textile or fabric in the substrate

Definitions

  • the present invention relates to polyisocyanide polymersomes, optionally having substances such as catalysts or enzymes associated therewith, methods of making the same, and to methods of using the same.
  • Enzymes are essential catalysts in many processes both in nature and in industry. However, most enzymes are only active in a narrow environmental window, requiring buffered solutions, ambient temperatures and protection from harmful solutes. A number of research groups have adopted immobilization of enzymes as a means to stabilize them, but also to prepare recoverable and specific catalysts. The methods to immobilize enzymes are numerous and include covalent bonding to activated polymers, co-polymerization with multifunctional reagents, physical adsorption and entrapment in cross-linked polymer particles.
  • encapsulated compounds can either be enzymes, pharmaceuticals or genetic material. Vesicles are used also in the cosmetics industry.
  • This amphiphilic macromolecule contains a rigid helical polyisocyanide head group and a flexible polystyrene tail, making it a rod-coil type of a diblock copolymer.
  • the membrane thickness of the vesicle is approximately 30 nm.
  • the thiophene side groups present in the side chain can be further polymerized either electrochemically or by chemical oxidation, resulting in cross-linking of the polymersome membrane.
  • the present invention relates to PS-PIAT polymersom.es that are useful in a variety of applications. Accordingly, one aspect of the invention relates to a polymersome comprising a vesicular polymer membrane of a PS-PIAT polymer represented by the formula:
  • polymersomes of the invention are relatively stable against agglomeration and surprisingly can allow various compounds to enter and/or exit the polymersome. Accordingly, the polymersomes of the invention are useful as microcontainers, as they may retain an encapsulated substrate and, if needed, release it at the desired time in the desired place.
  • the polymersomes of the invention further contain a catalyst, such as an enzyme either encapsulated therein, i.e., inside the vesicular membrane of the polymersome, or in the polymeric membrane of the polymersome, or both.
  • a catalyst such as an enzyme either encapsulated therein, i.e., inside the vesicular membrane of the polymersome, or in the polymeric membrane of the polymersome, or both.
  • One or more kinds of catalysts can be present in the polymersome, e.g., one kind of enzyme encapsulated within the membrane and another kind contained in the membrane wall.
  • the catalyst-containing PS-PIAT polymersomes can be used as a "microreactor” or “nanoreactor", in which a chemical reaction proceeds whenever a suitable substrate reaches the enzyme in the active site of the "microreactor" by a diffusion process.
  • the product of the reaction leaves the "microreactor” and allows the next molecule of the substrate to come and react.
  • the possibility to serve as a microreactor is based on the fact that the membrane made from PS-PIAT is sufficiently permeable to the low molecular substrates.
  • the enzyme encapsulated within the PS-PIAT polymersome may be used as a "microsensor", i.e. within a process of testing based on a catalyzed conversion of a specific sensitive substrate.
  • Another aspect of the invention relates to processes for making the catalyst-containing polymersomes.
  • Two processes for providing the catalyst in the PS-PIAT polymersome were developed and are referred to hereinafter as the injection process and the lyophilization process.
  • the injection process comprises adding an organic solution of a PS-PIAT polymer of formula (1):
  • x represents a number between 30 and 50, preferably 35 to 45 and typically about 40 and n represents a number between 10 and 100; to an aqueous solution or dispersion of a catalyst to form PS-PIAT polymersomes containing said catalyst therein.
  • the injection process primarily yields polymersomes with enzymes located inside their water pool.
  • the catalyst is typically an enzyme, especially a water-soluble enzyme.
  • the organic solution of the PS-PIAT polymer can use any suitable organic solvent and typically uses tetrahydrofuran.
  • the lyophilization process comprises dissolving a PS-PIAT polymer of formula (1)
  • x represents a number between 30 and 50, preferably 35 to 45 and typically about 40 and n represents a number between 10 and 100, and a catalyst in a solvent to form a solution; lyophilizing said solution to form a catalyst coated PS-PIAT polymer; and contacting said catalyst coated PS-PIAT polymer with water or an aqueous solution to form PS-PIAT polymersomes having said catalyst in the polymeric membrane thereof.
  • the contacting with water or aqueous solution serves to form the polymersome or vesicular structure.
  • the coated catalyst is generally primarily located in the membrane. If the aqueous solution contains a second catalyst, then the polymersome generally will contain the second catalyst within the vesicular membrane, e.g. encapsulated.
  • two types of enzymes may be immobilized in the polymersome, one inside and the second one in the membrane.
  • the same type of catalyst can be placed in both locations.
  • the lyophilization process is used as a pre-treating to form a coated PS-PIAT polymer for the subsequent injection process.
  • the process of encapsulating and/or embedding a substrate, particularly a catalyst such as an enzyme, within the PS-PIAT polymersome has the advantage of providing a highly loaded and stable immobilized substrate/enzyme without the need of specific linking agents.
  • the immobilized substrate may be prepared in a simple method, safely stored for a prolonged period, used under controlled conditions, re-used after the reaction or used within a continual process.
  • the encapsulated compounds may be used in chemical, pharmaceutical, cosmetic and/or food industries.
  • Isocyanides may be polymerized with Ni(II) based agents in combination with the addition of nucleophiles, i.e. alcohols or amines, as initiators. Since the initiating nucleophile also becomes the first residue of the resulting polyisocyanide, block copolymers can be prepared merely by using a polymer with an alcohol or amine end group.
  • the PS-PIAT copolymers are derived from an amino-terminated polystyrene (PS) of formula (2) and L- isocyanoalanine(2-thiophen-3-yl-ethyl)amide (IAT) of formula (5).
  • PS-PIAT block copolymers are derived from an amino-terminated polystyrene (PS) of formula (2) and L- isocyanoalanine(2-thiophen-3-yl-ethyl)amide (IAT) of formula (5).
  • the starting polystyrene amine of formula (2) has typically from
  • the IAT may be synthesized by using known chemical transformations from beta-3- thienyl ethyl amine and a N-protected alanine.
  • the IAT may be, dependent mainly on the process of its production, either optically pure L-enantiomer of IAT, or may be partly or fully epimerised ( i.e. the ratio between L- and D- IAT is from 50 : 50 to 100:0 ). Pure L-enantiomer of IAT polymerizes with PS more rapidly.
  • the PS-PIAT polymer is preparable by the following process.
  • the PS of formula (2) e.g. PS40
  • the Ni(II) polymerization agent which is tetrakis(t-butylisocyanide)nickel(II) perchlorate of formula (3) (Cornelissen et al, Science 1998, 280,1427).
  • the formed Initiator Complex (IC) of formula (4) is stable and may be isolated and characterized.
  • the Initiator Complex is mixed with the IAT monomer (5) in a solvent and is allowed to react at ambient temperature, forming the PS-PIAT diblock copolymer.
  • the amount of units in the PS part is fixed by the nature of the starting material, the amount of isocyanide units (the variable "n" in the formula (I)) in the future polyisocyanide block may be modified, by simply changing the ratio of the Initiator Complex to the IAT monomer.
  • the resulted PS- PIAT copolymer is soluble in organic solvents such as dichloromethane, chloroform and tetrahydrofuran, and insoluble in water.
  • a molar ratio of IAT to IC 15:1 up to 100: 1 is possible in case of epimerised IAT, but max. molar ratio ofIAT to IC 20:1, preferable even 10: 1 is recommended for the optically pure L-IAT.
  • the n- value may vary from about 10 to about 100.
  • the exact polymer length in the PIAT part is however not always identical with the original molar excess of IAT. However, it may be determined by routine methods, e.g. by IH- NMR. For instance, when using a 17: 1 molar ratio between the IAT and IC, one may obtain the PS-PIAT copolymer with 17 units in the PIAT part. But when using a 100: 1 ratio, usually not more than 40 units in the PIAT part may be obtained.
  • the polymers were either made from optically pure L- IAT (i.e. the relative per cent amount of L- IAT within IAT is close to 100) or from mixtures comprising at least 50% of the L-IAT within the total IAT.
  • PS-PIAT possesses amphiphilic behavior, as a result of a difference in polarity of its two blocks. It thus forms aggregates both in solvents and upon precipitation from the solutions.
  • an organic solvent e.g. tetrahydrofuran
  • the solution of PS-PIAT in an organic solvent when contacted with water, forms a dispersion of the precipitated PS-PIAT polymer aggregates which have essentially a form of a spherical vesicle.
  • Micrographic analysis reveals that the average diameter of the spherical aggregates may vary from approx. 30 to approx. 150 nm. The size depends, i.e., on the length of the PIAT unit (the longer unit, the smaller the particles).
  • Most promising aggregates are formed from PS-PIAT 5 0 copolymer, i.e. the product made from the molar ratio IAT : IC of 50: 1.
  • Lipases have been immobilized in the prior art by anchoring them onto a solid support and by cross-linking them in order to increase their stability and to simplify recovery.
  • the activity of immobilized CAL-B is in general much higher than that of free enzyme due to better solvation and stabilization of the macromolecule.
  • the encapsulation of CAL B within PS-PIAT polymersome was carried out by applying two different methods. In the first one, injection was used as a tool, in the second one lyophilization was used.
  • the product of encapsulation of an enzyme within the polymersome is herein below called as a "biohybrid", hence PS-PIAT/CAL B biohybrid.
  • the starting PS-PIAT was prepared by polymerization of a mixture of 78% of L-IAT and 22% D-IAT. It was determined by IH-NMR that the PIAT block had a length of 31 units.
  • a solution of PS-PIAT in tetrahydrofuran was injected into an aqueous solution of CAL B.
  • the solution became turbid and a dispersion of PS-PIAT/CAL B biohybrid was formed.
  • the solid dispersion was removed by filtration. Under a TEM microscope, a population of spherical polymersomes is visible, some of them being dark and some of them light. It was concluded that the dark polymersomes are those that contain the enzyme encapsulated. This was confirmed by pre-labeling the CAL B enzyme with a fluorescent dye, wherein a similar population of fluorescent and non-fluorescent polymersomes was obtained.
  • the activity of the encapsulated enzyme may be tested by the external addition of a suitable substrate.
  • a suitable substrate 6,8-difluoro-4-methylumbeliferyl octanoate (DiFMU octanoate).
  • DiFMU octanoate 6,8-difluoro-4-methylumbeliferyl octanoate
  • This compound when decomposed by the CAL B catalyzed hydrolysis, exhibits strong fluorescence upon forming free DiFMU group.
  • the increase of fluorescence due to the enzyme-catalyzed hydrolytic reaction may be conveniently monitored by a fluorescence spectrophotometer.
  • the PS-PIAT polymersome comprises small pores, through which the substrate may diffuse. These pores are however smaller than the enzyme since no leakage of the enzyme was observed over a period of at least one week.
  • the enzyme compartmentalized with PS-PIAT polymersome within a biohybrid was found to be active even after being in the aqueous dispersion for 1.5 months.
  • the methods of varying cross-linking time or cross-linker concentration offer a way to tune the permeability of the PS-PIAT polymersomes. It is important particularly when the polymersomes are used as microcontainers, i.e. the incorporated substrate must not leak from the outer membrane unless being on the place when the container should be “opened". In this case, the degree of cross-linking may be very high.
  • the PS-PIAT/CAL B biohybrid may be prepared also by a lyophilization technique. Lyophilization, or freeze-drying, of enzymes with polymers and/or surfactants has been adopted as a technique to introduce enzymes in organic solvents or to protect the enzymes. After lyophilization, the enzyme becomes coated with the polymer or the surfactant, which has a stabilizing effect on the enzyme's conformation.
  • the THF solution was injected into pure water, resulting in the formation of a dispersion.
  • the polar and hydrophilic marker 5(6)-carboxyfluorescein (CF) was encapsulated.
  • the sample of the PS- PIAT dispersion was studied by fluorescence microscopy, which clearly revealed that the fluorescence came from the aggregate interior, precisely as expected for an aggregate with a vesicle architecture.
  • the spherical aggregates of the lyophilized PS-PIAT/CAL B biohybrids were much larger than the ones prepared by the injection process. Moreover, they contained may holes on their surface. An explanation for this phenomenon cannot be presently given. It is only obvious that the enzyme plays a role in the formation of the holes, since the aggregates of PS- PIAT formed in the absence of CAL B were perfectly spherical.
  • CAL B labeled with a fluorescent dye was used to visualize the enzyme within the biohybrid aggregate.
  • analysis of the sample by fluorescence microscopy revealed that the enzyme is accumulated within the membrane.
  • An explanation might be that the enzyme is coated by PS-PIAT during lyophilization. When these clusters are redissolved in THF, the amphiphilic diblock copolymer remains present around the enzyme. Injection of the THF solution into water will then lead to PS-PIAT spherical aggregates with membranes containing entrapped enzyme.
  • the difference in location of the enzyme within the biohybrid could result in a difference in activity of the enzyme.
  • biohybrids based on PS-PIAT polymersomes wherein one enzyme is present in the membrane and the second enzyme is encapsulated in the water pool inside.
  • This possibility was confirmed by encapsulating two types of CAL B lipase, one being labeled with Alexa488 fluorescent dye and the other with Alexa 633 dye.
  • the CAL B labeled with Alexa 488 was lyophilized together with PS-PIAT, while the CAL B labeled with Alexa-633 was encapsulated wherein the so prepared PS-PIAT/CAL B(Alexa 488) biohybrid was injected into an aqueous solution of this enzyme.
  • the location of the two differently labeled enzymes may be visualized by fluorescence microscopy using excitation wavelengths of 488 and 633 nm, resp. It was proven that the fluorescence at 488 nm was concentrated in the membrane, while the fluorescence at 633 nm was concentrated in the inner part of the polymersome.
  • the above approach allows to prepare a polymersome nanoreactor, wherein two completely different enzymes are incorporated. This way, one may perform cascade reactions within one polymersome nanoreactor. Interesting combinations may be created, capable to catalyze complex reactions. Immobilization of polymersomes on a surface and preparation of arrays of these systems with different enzymes opens the way to construct a "lab-on-a-chip" devices.
  • HRP horseradish peroxidase
  • HRP horseradish peroxidase
  • microsensor or “nanosensor”.
  • the PS-PIAT polymersome may encapsulate even large proteins having the mass of
  • glucose oxidase is a 160 kDa protein, which is highly specific for the oxidation of beta-D-glucose.
  • GOX is also generally used as a sensor, because the glucose conversion catalyzed by this enzyme may be easily monitored.
  • the encapsulated GOX may serve as the glucose sensor for patients with diabetes, or - together with co-encapsulated insulin - as an insulin release system.
  • the GOX was encapsulated within PS-PIAT aggregates in a similar manner as described above, by the injection method.
  • the used PS-PIAT was synthesized by polymerizing a mixture of 90% L-IAT and 10% D-IAT.
  • a solution of PS-PIAT in THF was injected into a solution of GOX in phosphate buffer ( 2OmM, ⁇ H7).
  • the not encapsulated enzyme was removed from the PS-PIAT/GOX aggregates by a Sephadex column.
  • Cross-linking of the PS-PIAT polymersome membrane results in systems in which the electronic resistance is three orders of magnitude lower than before cross-linking.
  • a "nanobattery" system might be developed, at least in principle, which is capable to generate electricity.
  • a working fuel cell might be possible.
  • aqueous phosphate buffer solution (0.50 ml, 20 mM, pH 7.5) was added to the filter and the eppendorf was centrifuged again until dryness. The step was repeated for the second time. The content of the filter was redispersed in a 0.50 ml aqueous phosphate buffer (20 mM, pH 7.5).

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne des polymersomes de polyisocyanure comportant éventuellement des matières associées, des procédés de fabrication de ceux-ci et des procédés d'utilisation de ceux-ci. Les polymersomes comprennent une membrane polymère vésiculaire constituée d'un polymère de PS-PIAT. Les matières éventuellement associées aux polymersomes comprennent des catalyseurs ou des enzymes, encapsulés dans ceux-ci, c.-à-d. à l'intérieur de la membrane vésiculaire du polymersome et/ou dans la membrane polymère du polymersome.
PCT/NL2006/000052 2005-01-31 2006-01-31 Polymersomes de polyisocyanure WO2006080849A2 (fr)

Applications Claiming Priority (2)

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US64779905P 2005-01-31 2005-01-31
US60/647,799 2005-01-31

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WO2006080849A3 WO2006080849A3 (fr) 2006-12-21

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1923701A1 (fr) * 2006-11-17 2008-05-21 Spinnovation Holding BV Méthode de criblage utilisant des microcapsules comme nanoréacteur
WO2012101587A1 (fr) 2011-01-28 2012-08-02 Koninklijke Philips Electronics N.V. Véhicules pour la libération locale de promédicaments hydrophiles
US10221445B2 (en) 2011-08-11 2019-03-05 Qiagen Gmbh Cell- or virus simulating means comprising encapsulated marker molecules
US10874611B2 (en) * 2016-02-25 2020-12-29 Ucl Business Ltd Chemotactic, drug-containing polymersomes
US10881613B2 (en) 2016-03-17 2021-01-05 Ucl Business Ltd Fumarate polymersomes
US12257344B2 (en) 2019-01-07 2025-03-25 Ucl Business Ltd Polymersomes functionalised with multiple ligands

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CORNELISSEN ET AL.: "Helical superstructures from charged poly(styrene)-polyisocanodipeptide) block copolymers" SCIENCE, vol. 280, 1998, pages 1427-1430, XP002402117 *
VRIEZEMA ET AL.: "Electroformed giant vesicles from thiophene-containing rod-coil diblock copolymers" MACROMOLECULES, vol. 37, no. 12, 2004, pages 4736-4739, XP002402111 *
VRIEZEMA ET AL.: "Vesicles and polymerized vesicles from thiophene-containing rod-coil block copolymers" ANGEWANDTE CHEMIE, vol. 42, no. 7, 2003, pages 772-776, XP002402110 cited in the application *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1923701A1 (fr) * 2006-11-17 2008-05-21 Spinnovation Holding BV Méthode de criblage utilisant des microcapsules comme nanoréacteur
WO2012101587A1 (fr) 2011-01-28 2012-08-02 Koninklijke Philips Electronics N.V. Véhicules pour la libération locale de promédicaments hydrophiles
US10221445B2 (en) 2011-08-11 2019-03-05 Qiagen Gmbh Cell- or virus simulating means comprising encapsulated marker molecules
US10874611B2 (en) * 2016-02-25 2020-12-29 Ucl Business Ltd Chemotactic, drug-containing polymersomes
US10881613B2 (en) 2016-03-17 2021-01-05 Ucl Business Ltd Fumarate polymersomes
US12257344B2 (en) 2019-01-07 2025-03-25 Ucl Business Ltd Polymersomes functionalised with multiple ligands

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Publication number Publication date
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