DE10157046A1 - Nano and microcapsules comprising reactive polymers - Google Patents

Abstract

The invention relates to nanocapsules and microcapsules comprising reactive polymers. The preferred polymers P1 are soluble in water and particularly comprise anhydride, imide, acid chloride and/or aldehyde functions. Polymers P1 together with other water-soluble polymers P2, which have a plurality of functional groups, form covalently bonded networks on the surface of colloidal carriers.

Description

State of the art

Colloidal carriers can be stabilized sterically and electrostatically by applying polymer layers. Suitable structures and processes are described in the prior art, which are briefly summarized below:
WO 00/28972 discloses the coating of liposomes with several layers of water-soluble polymers which are covalently crosslinked with one another. In addition to the use of low molecular weight crosslinkers, the use of reactive polymers, such as oxidized or hydrazylated dextrans or starches, is also described. The use of polymeric imines or anhydrides is not disclosed.

US 5837747 and subsequent documents of this family disclose the Production and use of biopolymers with ethylenically unsaturated Groups. However, the compounds listed are in their preparation comparatively complex and not readily available. The presence unreacted ethylenically unsaturated groups in biological Systems is not without problems since these groups use free thiols the proteins or glutathione can undergo further reactions.

Covalently cross-linked cladding layers on colloidal templates or Nano hollow bodies are also accessible according to WO 00/03797. According to this procedure water-soluble polyelectrolytes are initially in several layers on the Template deposited and then by adding bifunctional Cross-linked reagents. The use is disadvantageous low molecular weight crosslinkers such as glutaraldehyde, the ecological and brings health problems.

A first group of the polymers proposed here derives from Polysuccinimide. This connection has long been used in technical Made to scale and can lead to functionalized derivatives or Polyaspartic acid are implemented. Polysuccinimide is not water-soluble, polyaspartic acid is readily water-soluble, so that at Hydrolysis intermediates occur which are both water soluble as well have reactive imine functions.

A possibility for the targeted synthesis of a water-soluble copolymer from succinimide and aspartic acid units and their derivatives Connection is in Sikes et al. disclosed in US 5981681. Advantageous compared to the synthesis from polysuccinimide, the manufacturability of Polymers made from naturally occurring L-aspartic acid that lead to a very biocompatible product.

Microcapsules using polyaspartic acid are about from the US 5540927 known. They arise during the coacervation of gelatin and Polyaspartate and can subsequently be stabilized by glutaraldehyde become. The disadvantage is the use of the ecologically problematic Aldehyde and the comparatively high consumption of gelatin and Polyaspartate in the manufacture of the microcapsules.

The manufacture of vol microcapsules or the coating of colloids under Use of polysuccinimide or copolymers comprising succinimide, is not known.

The object of the present invention was to provide an encapsulation method of nano or micro particles to give the above disadvantages does not have. In particular, the use of low molecular weight crosslinkers be avoided in the manufacture of said capsules.

invention

The task is solved by using nano- or microparticles different polymers are coated, at least one of these Polymers has a variety of acid anhydride or imine functions and another of these polymers with the first polymer covalent bond like can form about amide bonds or esters.

invention description

The encapsulation of liposomes with water-soluble polymers is in the described above WO 00/28972 and leads to structures with very thin, often monomolecular layers are surrounded by polymers. By means of covalent bonds a firm connection between the Polymers and the particle surface achieved in further Reaction steps can be applied to multiple layers of polymer so that a stable network is created. After removing the inside Liposomes can also be produced in this way.

However, WO 00/28972 is disadvantageous in most cases, but also in the preparation according to US 5540827 the presence of low molecular weight Crosslinkers, which make the manufacturing process more expensive, difficult to separate are and pose health problems. So is for the production of Cosmetics the use of glutaraldehyde only to a limited extent authorized.

This disadvantage can be avoided by using reactive polymers which, due to their functional groups, are built into the Allow polymer layer.

It has been found to include the use of reactive polymers Imine functions or acid anhydride functions is particularly advantageous. On a significant advantage over other polymers described is their Reactivity to water, which leads to hydrolysis and thus to a The reactivity disappears within a short time. In the end product then no unwanted further reactions are possible.

In a preferred embodiment, a polymer P1. including succinimide and other functional groups. Here is polysuccinimide not a suitable polymer even because of its insolubility in water. Rather, other hydrophilic functions such as Sulfonic acids, hydroxyl groups, ethoxy groups, amides or carboxylic acids necessary to ensure the solubility of the polymer in water. The presence of carboxylic acid functions can be achieved by partial hydrolysis of polysuccinimide. To do this, the product is submerged with water Adding alkali is heated. The reaction can be stopped if one Most of the polysuccinimide has gone into solution.

Such a preparation can lead to the desired product. A The more elegant and controllable method is the thermal one Copolymerization of aspartic acid and its salts with suitable Conditions as described in US 5981691. Here you get immediately a water-soluble product that is well suited for further use is. A variation in the chain length can be achieved by installing additional comonomers, about bifunctional monoamines can be achieved.

The imino groups present in polymer P1 can be slightly alkaline React conditions with water, alcohols and primary amines. With further polymers P2, which comprise a large number of such groups, can first polymer P1 thus form networks. Another advantage is there in that the reactivity of the polymer P1 after the completion of here encapsulation reactions subsides and finally completely disappears. This creates products with very low toxicity. On Copolymer of aspartic acid and succinimide hydrolyzes completely Polyaspartic acid, a hardly toxic and ecologically harmless Polymer from amino acids (see Baypure ™ DS100, Bayer, Product description).

Suitable polymers P2 include proteins, peptides, gelatin, Albumins, globins, hemoglobins, casein, or protein mixtures such as whey, Soy protein, wheat glue, serum or herbal extracts, protamines, Polylysine, polyserine, polyornithine, carbohydrates such as Chitosan, amino functionalized dextrans, dextrans, starches, Polyethyleneimine, polyallylamine or acrylates, including primary Amino functions, polyvinyl alcohol or polythiols.

It is advantageous - but not absolutely necessary if both polymers P1 and P2 or polymer P1 or P2 and the particles to be encapsulated Develop electrostatic interactions. These can become one non-covalent pre-fixation can be used to stabilize the Structures then take place through the comparatively slow formation of the covalent bonds.

WO 01/64330 describes the rapid and continuous encapsulation of Colloids with polyelectrolytes described, this publication is here expressly included as a reference.

It is irrelevant for the implementation of the invention whether P1 or P2 first bound to the particle surface and whether this attachment is covalent or by hydrophobic parts of molecules or by ionic interactions is conveyed. In any case, P1 and P2 form a stable, covalently linked network that is a single colloidal particle completely encloses. A decrease in the interaction between P1 and P2, for example by changing the pH, is therefore the end of the Crosslinking reaction harmless because of the enclosed colloid its size cannot escape. In the case of liposomal templates, a Elimination of the interaction may be useful when, for example, by Clustering of lipids an undesirable permeabilization of the structure occurs. One observes an unwanted permeabilization of Liposomes, comprising phosphatidylethanolamine and other charged lipids, when coating with complementarily charged polyelectrolytes.

In another embodiment of the invention, polymers are comprised Acid anhydride functions or acid chlorides to encapsulate particles used.

Suitable polymers are made from monomers such as Acrylic anhydride, citraconic anhydride, methacrylic anhydride, Methacryloyl trimellitic anhydride, acryloyl chloride, methacryloyl chloride, or maleic anhydride accessible. They contain other functional ones Groups to improve their water solubility, such as Sulfonic acid groups, carboxyl groups or hydroxyl functions or Ethoxy groups or tertiary or quaternary amino functions or acid amides. Another group of polymers contains acid anhydride functions or Acid chlorides in the side chain in the sense of a graft polymer. polymers which are not water soluble, such as poly-maleic anhydride or Copolymers of maleic anhydride and olefins or polyacryloyl chloride can be partially hydrolyzed to increase water solubility improve. However, they are also available as copolymers of anhydride and carboxylic monomers and fall under those above described connections.

A preferred embodiment is the use of copolymers with hydrophilic substituents, such as sulfonyl, hydroxyl, tertiary amino, Ammonio, amide or carboxylic acid functions. Preferred comonomers are therefore, for example, acrylic acid, methacrylic acid, diethylaminoethyl acrylate, Dimethylaminoethyl acrylamide, acryloxyethyl trimethyl ammonium chloride, Dimethyldiallylammonium chloride, propenesulfonic acid, sulfoethyl methacrylate, Styrene sulfonic acid, acrylamide, methacrylamide, Acryloyltris-hydroxymethylmethylamine, glycerol acrylate, hydroxyethyl acrylate, glycol acrylate, Methacryloyloxyethyl glucoside, polyethylene glycol acrylates, vinyl pyrrolidone, Ethanol and other such compounds.

A preferred copolymer includes acrylic acid and maleic anhydride and optionally other hydrophilic monomers such as acrylamide.

The acid anhydride groups or acid chlorides present in polymer P1 can with water, primary and secondary alcohols and primary as well secondary amines react. With other polymers P2, which is a variety of such groups, the first polymer can thus form networks. Another advantage is that the reactivity of the polymer increases the completion of the encapsulation reactions undertaken here and finally disappears completely.

The suitable and preferred further polymers correspond to those above mentioned connections.

Process for the preparation of the micro or nanocapsules using the reactive polymers include those described in the prior art Procedures without being limited to them.

A preferred embodiment is the layer-by-layer deposition of polymers on liposomal templates according to WO 00/28972. Surprisingly found that other structures, such as cells, o / w Emulsions, w / o / w double emulsions, pigments, protein aggregates or Let latex particles be coated in this way. Minimum requirement for a coating of the type mentioned is only the presence functional groups such as amino, thiol, hydrazo, aldehyde, Carboxyl, active ester, hydroxyl or azide hydrogen groups on the Surface of the colloidally dissolved particles.

In any case, the actual build-up of the cladding layer takes place on one Phase boundary, such as that between a lipid layer and the medium, however not over this limit. All polymers P1 and P2 are water soluble and there is no separation of the polymers in two different phases such as in known polycondensation reactions between one water-soluble and an oil-soluble polymer.

It is irrelevant whether a reactive polymer or another polymer is used first is brought into contact with the templates. A preferred version is that a reactive polymer first comes into contact with the template is brought and before the complete hydrolysis of the imino or anhydride or acid chloride functions, another polymer is added.

A particularly preferred embodiment of the encapsulation is based on the Formation of electrostatic interactions between the template and the first Polymer or between an already single or multiple coated Particles and a next polymer. In the latter case, they are simple or multiple coated particles themselves again as a template for the Take a reaction. The number of layers applied is at least two, but can also be much larger.

An advantageous method for producing electrostatically stabilized Polymer layers on colloidal templates is in WO 01/64330 disclosed here as a reference in the disclosure content of the invention is expressly included. The reactive polymers described here can be electrically charged, such as when carboxyls or other charged Groups to increase the hydrophilicity of the molecule are present. Carboxyl groups also arise during the hydrolysis of the reactive groups Polymers.

With this procedure, too, it is irrelevant whether this is the first step Reactive polymer or an ordinary polyelectrolyte can be applied. A covalent bond between the template and the reactive polymer can be formed , for example if the template has free amino or hydroxy groups features. The formation of a covalent bond between the template and However, polymer layer is not absolutely necessary and not always desirable, for example if the conjugates formed are difficult to degrade or are toxic.

All electrostatic are suitable as templates for this process charged colloidal particles, such as cells, liposomes, o / w emulsions, w / o / w double emulsions, crystals, protein aggregates, Pigments, latex particles and others.

Are o / w emulsions or w / o / w emulsions or liposomes encapsulated a first polymer with amphipathic properties be used.

Further preferred processes for the production of nano- or microcapsules with reactive polymers are known to those skilled in the art as coacervate formation (see R. Voigt: Pharmaceutical Technology, Ullstein Mosby, 1995, pp. 289 ff.) Reactive polymers such as those described here can be used in the complex Coacervation, for example of gelatin, can be used. But you can also used for the subsequent consolidation of simple coacervates become.

The teaching according to the invention is used in the encapsulation of Colloids or in the production of nano and hollow micro spheres. A Encapsulation of colloids can increase mechanical or physiological stability of such particles can be exploited. Let it be about liposomes against mechanical and biochemical stressors protect by encapsulation. They can also be mechanically sensitive droplets of a w / o / w double emulsion in this way stabilize.

In another embodiment, the colloid stability, i.e. avoidance of flocculation or aggregation. For example Liposomes unstable in the presence of an emulsion and fuse with the Droplets of the emulsion. By individual encapsulation of the liposomes or the Emulsion droplets can avoid this process. Then it's one improved use of liposomes as stable drug containers or as depot forms or as transporters in cosmetics or pharmaceuticals. Other uses of the particles are in encapsulation of fragrances, flavors or other volatile substances that are slow should be released.

If the colloids are encapsulated with reactive polyaspartates, one can slow release in biological systems by hydrolysis or proteolytic cleavage can be achieved. That is about the case when the Particles are injected subcutaneously or intramuscularly. That is also the one Case when the particles are applied to the skin and by action be dissolved by bacteria. In this case, the capsules are suitable for example for the production of deodorants or to avoid bacterial infections.

example 1 Encapsulation of liposomes Preparation of the liposomes

60mol% distearoylphosphytidylglycerol and 40mol% cholesterol are in a mixture of isopropanol and chloroform (2/1) dissolved and under vacuum evaporated to dryness. The two lipid films are then in so much buffer (10 mM Na acetate, 100 mM NaCl, pH 4) that a Lipid concentration of 15 mg / ml is obtained. The suspensions are several times through isopore polycarbonate membranes with a pore size of 200 nm extruded.

Coating the liposomes with chitosan and polysuccinimide / aspartate

Chitosan (Pronova) and polyaspartate (Folia Inc., 30% imine groups) are dissolved in a concentration of 1 mg / ml in buffer (10 mM Na acetate, pH 4). Liposomes A are diluted in the same buffer to a lipid concentration of 0.15 mg / ml. The amounts of polymer given below are successively added to liposomes A:

Na borate buffer (100 mM pH 9.5) is then added and the Suspension is kept at 50 ° C for 4 h. After cooling, it becomes a pH adjusted from 7.5 with hydrochloric acid.

Evidence of the formation of envelope structures

Liposomes or part of the suspension is used after the reaction Triton X-100 (0.2% in water) treated. This will make the liposomes dissolved. Liposomes can no longer be detected using light scattering the encapsulated structures also scatter after the dissolution of the internal liposomes still the incident light.

Example 2 Stabilization of liposomes in o / w emulsions Preparation of the liposomes

Liposomes are prepared as in Example 1. The hydration takes place in a buffer containing 10 mM HEPES, 50 mM NaCl, 100 mM carboxyfluorescein, pH 7.5) so that a lipid concentration of 15 mg / ml is obtained. The Suspension is extruded as above.

Not included carboxyfluorescein is by gel filtration separated. A buffer (10 mM Na acetate pH 4) is used for this.

The liposomes are coated as in Example 1.

Structural stability analyzes

1 ml of the liposomes A and / ml of the nanocapsules produced therefrom mixed with 5 ml each of an o / w emulsion (Lipovenös,) and incubated for 24 h. The mixture of liposomes A and the emulsion then fluoresces with more than 30% of the maximum intensity. The mixture of the nanocapsules and the Emulsion fluoresces with less than 15% of the maximum intensity.

Claims (17)

1. Micro- or nanocapsule made of crosslinked polymers, characterized in that the capsule consists of at least one reactive polymer P1 comprising imine, anhydride or acid chloride functions and at least one different water-soluble polymer P2. 2. Micro or nanocapsule according to claim 1, characterized in that the reactive polymer is a copolymer containing succinimide monomers includes. 3. Micro or nanocapsule according to claim 2, characterized in that the copolymer comprises aspartic acid and succinimide, preferably L- Aspartic acid and succinimide. 4. Micro or nanocapsule according to claim 1, characterized in that the reactive polymer is a copolymer which Maleic anhydride monomers. 5. Micro or nanocapsule according to claim 4, characterized in that the copolymer comprises maleic acid and acrylic acid. 6. Micro- or nanocapsules according to one of claims 1 to 5, characterized characterized in that the water-soluble polymers P2 via a Variety of amino or hydroxyl functions. 7. micro- or nanocapsules according to claim 6, characterized in that the polymers P2 proteins, peptides, gelatin, albumins, globins, Hemoglobins, casein, or protein mixtures such as whey, soy protein, Wheat glue, serum or vegetable extracts, protamines, polylysine, Polyserine, polyornithine, also carbohydrates such as chitosan, amino-functionalized dextrans, dextrans, starches, polyethyleneimine, Polyallylamine or acrylates, comprising primary amino functions, Are polyvinyl alcohol or polythiols. 8. micro- or nanocapsules according to claim 1, characterized in that colloidally dissolved particles are inside the capsule. 9. micro- or nanocapsule according to claim 8, characterized in that the colloidal particles liposomes, cells, latex particles, crystals, Oil-in-water emulsions or water-in-oil-in-water emulsions or Are protein aggregates or pigments. 10. Process for the production of micro or nanocapsules, thereby characterized in that reactive polymers comprising a variety of Imide or anhydride or carboxylic acid chloride functions and others Polymers are used to make the capsule wall. 11. A method for producing micro- or nanocapsules according to claim 10, characterized in that the deposition of the polymers is carried out in layers. 12. A method for producing micro- or nanocapsules according to claim 10, characterized in that the deposition of the polymers by complex coacervation happens. 13. A method for producing micro or nanocapsules according to claim 10 or 13, characterized in that reactive polymers for subsequent hardening of preformed capsules can be used. 14. Use of the micro or nanocapsules according to claim 1 for Manufacture of cosmetic products. 15. Use of the micro or nanocapsules according to claim 1 for transport of active ingredients in pharmaceutical applications. 16. Use of the micro or nanocapsules according to claim 1 for delayed release of active ingredients in pharmaceutical Applications. 17. Use of the micro and nanocapsules according to claim 1 for Encapsulation of flavors or fragrances.