WO2006080849A2

WO2006080849A2 - Polyisocyanide polymersomes

Info

Publication number: WO2006080849A2
Application number: PCT/NL2006/000052
Authority: WO
Inventors: 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
Current assignee: NEDERLANDSE ORGANISATIE VAN WETENSCHAPPELIJK ONDERZOEK
Priority date: 2005-01-31
Filing date: 2006-01-31
Publication date: 2006-08-03
Anticipated expiration: 2007-07-31
Also published as: WO2006080849A3

Abstract

The present invention relates to polyisocyanide polymersomes, optionally having substances associated therewith, methods of making the same, and to methods of using the same. The polymersomes comprise a vesicular polymer membrane of a PS-PIAT polymer. The substances optionally associated with the polymersomes may comprise catalysts or enzymes, either encapsulated therein, i.e., inside the vesicular membrane of the polymersome, or in the polymeric membrane of the polymersome, or both.

Description

POLYISOCYANIDE POLYMERSOMES Background of the Invention

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.

Additionally, it has been proposed to encapsulate enzymes inside vesicles to mimic cell functions. Most applications utilize the vesicle as a container to release enzymes in a controlled manner. Especially in the field of the drug delivery much research has been carried out in the use of vesicular architectures. The encapsulated compounds can either be enzymes, pharmaceuticals or genetic material. Vesicles are used also in the cosmetics industry.

Much research has been focused on the use of artificial cell membranes to encapsulate enzymes and other biomolecules. In most studies involving the encapsulation of enzymes, phospholipid-based vesicles, or liposomes, have been used. However, some studies also involved vesicles constructed from polymers. These so called polymersomes (or polymerosomes) possess a higher membrane toughness than liposomes. Furthermore, the rich diversity in available monomers and polymerization methods make it possible to precisely tune the properties of the resulting polymersomes. For instance, Nardin et al. prepared polymersomes of crosslinkable triblock copolymers with incorporated channel proteins in their membrane and encapsulated lactamase enzymes inside their water pool. [Nardin et al. , Chem. Commun. 2000, 1433] They showed that an externally added substrate was converted inside these polymersomes and that the channel protein activity was retained upon cross- linking of the membrane. In a second example [Napoli et al., Langmuir 2004, 20,3487], the copartmentalization of glucose oxidase enzymes within polyethyleneglycol-b- polypropylenesulfide-b-polyethyleneglycol (PEG-PPS-PEG) polymersomes led to a formation of oxidation-responsible capsules. The hydrogen peroxide which is produced during the oxidation of glucose, oxidized the thioether group in the PPS block resulting in destabilization of the membrane and thus release of its content.

One drawback in the use of polymersomes is the increased membrane thickness which drastically reduces the permeability and thus the activity of the enzyme entrapped in the polymersome.

It was recently found out that the diblock copolymer polystyrene-b-poly (L- isocyanoalanine(2-thiophen-3-yl-ethyl)amide) - in further PS-PIAT - is able to form very stable and well defined spherical polymersomes in water and organic solvents [Vriezema et al. , Angew.Chem. Int.Ed. 2003, 42, 772 ]. Schematic structure formula of PS-PIAT of formula (1) is shown below.

(1) 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. Summary of the Invention

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:

(1) wherein 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, typically 10 to 40 especially 15 to 40 and more typically 20 to 35. The 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. In many embodiments, 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. 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. In any event, 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. By the same diffusion, 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. Similarly, 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):

(1) wherein 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)

(1) wherein 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. In an advantageous way, two types of enzymes may be immobilized in the polymersome, one inside and the second one in the membrane. Alternatively, the same type of catalyst can be placed in both locations. In this way, 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. Detailed Description of the Invention

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. Thus, 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). The process leading to PS-PIAT block copolymers is illustrated on the following scheme.

(4)

(D Within our invention, the starting polystyrene amine of formula (2) has typically from

30 to 50 styrene units ( x = 30 to 50) , preferably 40 units (PS40).

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.

In the first step, the PS of formula (2) , e.g. PS40, is mixed with an equimolar amount of 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. In the second step, 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. While 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. For the purposes of this invention, it is important that the resulted PS- PIAT copolymer is soluble in organic solvents such as dichloromethane, chloroform and tetrahydrofuran, and insoluble in water. For to obtain such copolymer, 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. Thus, in the above structural formula of PS-PIAS, 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. Within the present invention, 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. What is the most important, the solution of PS-PIAT in an organic solvent (e.g. tetrahydrofuran), 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-PIAT₅0 copolymer, i.e. the product made from the molar ratio IAT : IC of 50: 1.

The polymersome in combination with a catalyst will be further described with respect to the preferred catalyst type, namely en2ymes. It be understood that other catalysts or substrates can be incorporated by similar procedures as desired.

The possibility to encapsulate enzymes inside aggregates of PS-PIAT was confirmed by preparing and testing the polymersome complex with the enzyme lipase B from Candida antarctica (CAL/B). This enzyme of 33 kDa has been well studied in aqueous solutions, where it hydrolyses esters, and in anhydrous organic solvents, in which it performs amidation, esterification and transesterifϊcation reactions in an enantioselective manner. Most lipases display almost no hydrolytic activity when they are in a dissolved state. In the absence of a lipid interface several varieties of the enzyme are in an inactive state, since a part of the enzyme, an alpha-helix, covers the active site. Upon contact with the hydrophobic interface the alpha-helix is folded back, thus allowing substrates to reach the active site. The crystal structure of CAL B has revealed that this enzyme does not have a lid that shields the active site, making it suitable for the studies illustrating this invention.

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.

Within the injection-type encapsulation experiments, 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. In the case of the encapsulated lipase within the PS-PIAT/CAL B biohybrid, the activity was tested by 6,8-difluoro-4-methylumbeliferyl octanoate (DiFMU 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.

Experiments showed that the entrapped en2yme not only catalyses the hydrolytic reaction inside the polymersome but also that the product of the hydrolysis may easily leak out from the polymersome and thus may be separated from the polymersome reactor. This is a surprising finding in comparison with the known polymersomes prepared by Nardin et al, which require a channel protein in order to obtain diffusion of the substrate.

It is tentatively proposed that 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.

In a further study, an effect of cross-linking the membrane of PS-PIAT polymersomes containing CAL-B, was determined. The cross-linking was achieved by a bis(2,2'- bipyridine)ruthenium(II)bis(pyrazolyl) complex (BRP). This complex is known that it protonates in water and the resulting cation has a high oxidation potential of 1.60V, making it suitable to polymerize the thiophene groups in PS-PIAT. It was found out that the crosslinking is possible, however the temperature during the crosslinking reaction must be kept as close to the ambient as possible due to the undesired denaturation of the CAL-B enzyme at elevated temperatures.

The study of influence of the degree of cross-linking on the enzyme-catalyzed hydrolysis of DiFMU octanoate was investigated and was proven that that the activity of the enzyme, expressed as the rate of hydrolysis, decreases either with the prolonged cross-linking time or with the extended cross-linker concentration. It is tentatively proposed that this effect is caused by closing the pores present in the polymersome membrane by the cross-linking reaction.

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.

Interestingly, we have found out that 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.

Furthermore, the solubility and hence the activity in organic solvents is significantly increased. In a study of a co-lyophilization of CAL B with PS-PIAT, it was found out that not only the solubility of CAL B in organic solvents has been improved, but it turned out that the formed biohybrid displayed interesting aggregation behavior itself in aqueous solvents. For these experiments, the PS-PIAT that was synthesized from 100% L-IAT was used. The length of the PIAT block was determined by IH NMR to be 4 units. A solution of PS-PIAT in tetrahydrofuran was introduced in an aqueous solution of

CAL B by injection, in a similar way as described above. The mixture was then lyophilized, resulting in a fluffy off-white powder, which was re-dissolved in THF to give a clear solution.

The THF solution was injected into pure water, resulting in the formation of a dispersion. To confirm the vesicular nature of the PS-PIAT aggregates by lyophilization, 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.

In the same way as described above, CAL B labeled with a fluorescent dye was used to visualize the enzyme within the biohybrid aggregate. Quite surprisingly, 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 fact that the enzyme is located in the membrane, and not in the water pool inside the PS-PIAT microsphere, was also confirmed by studying the hydrolytic activity of air-dried PS-PIAT/CAL B biohybrids to aqueous solutions of DiFMU octanoate. While the injection- made biohybrids showed no activity, the lyophilized biohybrids exhibited hydrolytic activity, indicating that the enzyme is present in the membrane.

In principle, the difference in location of the enzyme within the biohybrid, either in the membrane or in the inner aqueous compartment of the polymersome, could result in a difference in activity of the enzyme.

Moreover, it is also possible to prepare 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.

Some enzymes, which have a high affinity to the PS-PIAT polymer, may be encapsulated within the polymersome membrane even by the simple injection technique disclosed above. For instance, horseradish peroxidase (HRP) is a 44 kDa enzyme found in plants, which catalyses oxidation of substrates using hydrogen peroxide. The HRP is widely used in immunodiagnostic assays, together with suitable color-forming substrates.

Encapsulation of HRP inside PS-PIAT polymersomes was performed in the same injection process as disclosed above. The PS-PIAT used in the process was prepared by polymerization of 100% L-IAT. In a phosphate-buffered solution of HRP (HRP is quite prone to denaturation, hence the buffer present), a solution of PS-PIAT in THF was injected. The resulting dispersion was isolated by filtration. TEM microscopy and fluorescence microscopy proved that the resulting aggregates were vesicular in nature. However, the most of the fluorescence, when used HRP labeled with rhodamine isocyanate, appears to be concentrated within the membrane.

The activity of so encapsulated HRP was tested in a colorimetric assay at 420 nm using the 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid ) (ABTS) as the color-forming substrate in a reaction with hydrogen peroxide. The test has proven that the encapsulated HRP is active for catalyzing the reaction and that the substrate and the reaction product can migrate through the polymersome. It was also proven that the thiophene groups are not oxidized so that no subsequent crosslinking of the membrane occurs. The possibility to encapsulate enzymes that routinely serve in diagnostic processes such as HRP illustrate the possibility that the PS-PIAT biohybrid may be used as a

"microsensor" or "nanosensor".

The PS-PIAT polymersome may encapsulate even large proteins having the mass of

150 kDa and more. For instance, the enzyme glucose oxidase (GOX) 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.

Thus, 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, ρH7). The not encapsulated enzyme was removed from the PS-PIAT/GOX aggregates by a Sephadex column.

Microscopic observations showed the vesicular nature of the aggregates, and the testing in presence of the suitable color-forming substrate (ABTS) has proven the enzymatic activity of the aggregates.

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. In combination with the known electron-producing capacity of GOX a "nanobattery" system might be developed, at least in principle, which is capable to generate electricity. When a system can be designed in which glucose is able to migrate through their membrane continuously, a working fuel cell might be possible.

The invention will be further described with reference to the following non-limiting examples.

Example 1 : General Synthesis of PS-PIAT

Step l

Initiator Complex

While stirring , 1.02 g (0.242 mmol) of amino-functionalized polystyrene(40) [van Hest et al. ,Chem.Eur.J. 1996, 12,1616 ] dissolved in 10 ml of dichloromethane was added to a solution of 0.143 g (0.242 mmol) of (t-BuNC)₄Ni(C10₄)₂ [ Stephany, Drenth, Recl.Trav.Chim.Pays-Bas 1972,91,1453] in 25 ml of dichloromethane under a nitrogen atmosphere. After one hour of stirring, the solvent was evaporated, yielding the Initiator Complex as a yellow solid (1.16 g). Step II

Polymerization

In a typical polymerization reaction, the desired amount of the Initiator Complex dissolved in 5.0 ml of dichloromethane was added to a stirred solution of IAT in 10 ml of dichloromethane. Complete consumption of the isocyanide, as observed by IR spectroscopy, was obtained after 2 days. The product was evaporated to dryness and redissolved in minimal amount of dichloromethane. The polymer was then precipitated by dropping this solution into well-stirred mixture of methanol/water (1 :1 v/v). The solid was filtered off and washed with methanol/water mixture (1 : 1 v/v)

Example 2 Encapsulation of CAL-B by injection

A 0.50 g.l^"1 solution of PS-PIAT dissolved in tetrahydrofuran was injected into a 30 mg.r¹ solution of CAL B enzyme. The final ratio of water to tetrahydrofuran was 12: 1 v/v. After two days of equilibrating, the mixture was filtrated to dispose all non-included enzymes with the help of an eppendorf equipped with a 100 kDa cutoff filter. After centrifugation until the filter was dry the same volume of water was added to the filter and centrifugation was continued until dryness. This step was repeated one more time. The PS-PIAT/CAL B biohybrid was redispersed in the same volume of water. Example 3

Crosslinking of PS-PIAT membrane Into an aqueous solution prepared by mixing 0.20 ml of 30 mg.l^"1 CAL B and 1.0 ml of 1.3 μM BRP was injected 0.10 ml of a solution containing 0.50 g.l^"1 PS-PIAT solution in tetrahydrofuran, resulting in final water/THF ratio 12:1 (v/v). Subsequently, the dispersion was placed in a water bath of 6OC for 30 minutes. After cooling to room temperature, 0.50 ml of the dispersion was transferred to an eppendorf having a filter unit with a cutoff of 100 kDa. The dispersion was centrifuged to dryness after which 0.50 ml of pure water was added and the dispersion was centrifuged again to dryness. After repeating this step for the second time, 0.50 ml of water was added to re-disperse the crosslinked aggregates. \-/ I / I >.L. IU — — w vr w •«•

Example 4

Preparation of the PS-PIAT biohybrids by lyophilization

A solution of 1.0 ml of 1.0 g.1^"1 PS-PIAT in THF was injected into 0.20 ml of a 0.10 g.l^"1 CAL B solution in water, resulting in a dispersion with a PS-PIAT/CAL B molar ratio of 50: 1. To this solution a tenfold excess of pure water was added after which the mixture was lyophilized. The resulting fluffy off-white powder was redissolved in THF and injected into water to obtain a final PS-PIAT/CAL B concentration of 0.50 g.l^'1 in water/THF (8:5 v/v).

Example 5

Encapsulation of two enzymes inside PS-PIAT aggregates Un the same manner as in Example 4, CAL B labeled with an Alexa-488 dye was lyophilized with PS-PIAT and the resulting powder was redissolved in THF and injected into water containing 30 mg.l^"1 of CAL B labeled with an Alexa -633 dye. Not encapsulated enzymes were removed by column chromatography using Sephadex G-50.

Example 6 Encapsulation of HRP enzyme

A solution of HRP, labeled with rhodamine isothiocyanate, was prepared by dissolving 0.90 mg of the enzyme in 10 ml aqueous phosphate buffer solution (20 mM, pH 7.5). Into this solution a 1.0 mg.ml^"1 solution of PS-PIAT in THF was injected. The dispersion was left to equilibrate for one day. In order to remove not encapsulated enzyme, 0.50 ml of the dispersion was transferred to an eppendorf fitted with a 100 IcDa cutoff filter unit. The eppendorf was centrifuged until all buffer had passed the filter. Fresh 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). Example 7

Encapsulation of GOX enzyme

A solution of 48 mg.l^"1 GOX dissolved in phosphate buffer (20 niM, pH 7.0) was prepared. Into this solution a 1.0 mg.ml^"1 solution of PS-PIAT in THF was injected resulting a final buffer/THF ratio of 6:1 (v/v). The free enzyme was removed by size exclusion chromatography using Sephadex G-50 and an aqueous phosphate buffer (pH 7.5) as eluent.

Each of the articles and publications mentioned above is incorporated herein by reference in its entirety. The invention having been thus described, it will be obvious to the worker skilled in the art that the same may be varied in many ways without departing from the spirit of the invention and all such modifications are included within the scope of the present invention as set forth in the following claims.

Claims

I Claim:

1. A polymersome comprising a vesicular polymer membrane of a PS-PIAT polymer represented by the formula:

(i) wherein 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.

2. The polymersome according to claim 1 , wherein n represents a number between 10 and 45, preferably about 15 to about 40, and more preferably 20 to 35 such as about 31.

3. The polymersome according to claims 1-2, wherein said polymersome has a diameter within the range of about 30 to about 150 nanometers.

4. The polymersome according to claims 1 -3, which further comprises a catalyst inside said vesicular membrane.

5. The polymersome according to claims 1-4, which further comprises a catalyst in said membrane.

6. The polymersome according to claim 4, which further comprises a catalyst in said membrane.

7. The polymersome according to claims 5-6, wherein said catalyst inside said vesicular membrane is different from said catalyst in said membrane.

8. The polymersome according to claims 4-7, wherein said catalyst, or catalysts if more than one, is an enzyme.

9. The polymersome according to claims 1-8, wherein said PS-PIAT polymer was formed by reacting an IC of formula (4) with an IAT monomer of formula (5) in a ratio of IAT:IC of about 50:1.

10. A process which comprises adding an organic solution of a PS-PIAT polymer of formula (1):

(1) wherein 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.

11. The process according to claim 10, wherein said catalyst is a water-soluble enzyme.

12. The process according to claims 10-11, which further comprises forming said organic solution by dissolving solid PS-PIAT polymer into an organic solvent, such as tetrahydrofuran.

13. The process according to claim 12, which further comprises pre-treating said PS-PIAT prior to said formation of said organic solution of PS-PIAT, said pre-treating comprising dissolving said PS-PIAT polymer and a catalyst in a solvent to form a solution and lyophilizing said solution to form a catalyst coated PS-PIAT polymer.

14. A process which comprises: dissolving a PS-PIAT polymer of formula (1)

W wherein 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.

15. The process according to claim 14, wherein said aqueous solution or water is an aqueous solution.

16. The process according to claim 15, wherein said aqueous solution further contains a second catalyst and wherein said PS-PIAT polymersomes contain said second catalyst inside the vesicular membrane thereof.

17. The process according to claim 14-16 wherein said catalyst and said second catalyst are the same or different enzymes.