WO1999047130A1

WO1999047130A1 - Nanoparticles, method for producing nanoparticles and use of the same

Info

Publication number: WO1999047130A1
Application number: PCT/EP1999/001452
Authority: WO
Inventors: Javier Manero; Jennifer Filbey; Peter Boderke
Original assignee: Aventis Research & Technologies Gmbh & Co. Kg
Priority date: 1998-03-13
Filing date: 1999-03-06
Publication date: 1999-09-23
Also published as: EP1061904A1; DE19810965A1; AU3031899A; JP2002506814A; CN1292687A

Abstract

The invention relates to nanoparticles with a biocompatible, biodegradable polyelectrolyte complex consisting of polycations and polyanions, and at least one bioactive agent. The nanoparticles are obtained by additionally treating the polyelectrolyte complex with at least one cross-linking agent after it has been formed. The invention also relates to a method for producing said nanoparticles. An active agent in combined or non-combined form and an aqueous solution of an acidic polymer substance and a basic polymer substance are brought together and the polyelectrolyte is then produced in a nanoparticulate form or optionally, is converted into a nanoparticulate form. The invention is characterised in that the nanoparticulate polyelectrolyte complex is then treated with at least one cross-linking agent. The inventive nanoparticles can be used for applying bioactive agents. They have the advantages of being very stable, enabling the controlled release of active agents and not producing the burst effect. Due to the method by which they are produced, the inventive nanoparticles also only have very few residues of organic solvents.

Description

description

Nanoparticles, processes for their production and their use

The invention relates to nanoparticles, methods for producing nanoparticles and the use of nanoparticles.

In modern pharmaceutical technology, formulations and combinations of active substances are becoming increasingly interesting, the form of use of which can not only be applied in a gentle manner, but also have a targeted influence on the distribution, bioavailability or absorption of the drug.

In particular, particulate systems, so-called micro- or nanoparticles, which have a particle size in the range of less than 100 μm, have proven to be promising forms of application for delivering a wide variety of pharmaceuticals to the body.

A document that describes the advantages of the above-mentioned nanoparticles is WO 96/20698. It explains in particular that the surface of this

Particles can be modified in a variety of ways, for example to increase the retention time. It is also mentioned, for example, that the particles can be provided with antigens in order to release the pharmaceuticals which are in the particles in a very targeted manner at their intended site of action. However, it is problematic that the depot effect of the particles is relatively limited, since the release takes place very quickly. Furthermore, the stability of the particles described is relatively low.

Another document that describes the prior art is WO 96/05810. This affects microparticles that contain chitosan and are positively charged. Partial crosslinking of the particles reduces the residence time of the 2

Particles on the mucous membranes increased so that they are increasingly absorbed into the body. The problem here is that the particles are produced in emulsions. This makes it possible for residues of the solvent to remain on or in the particles. This is caused in particular by adsorption of the hydrophobic solvent onto hydrophobic polymers or bioactive substances, such as proteins. These solvent residues are of particular concern for pharmaceutical applications. The positive charge of the particles, which is measured by a positive zeta potential, can furthermore be disadvantageous for the surface modifications described in WO 96/20698.

In addition, the microparticles described are generally too large (1-100 μm) to ensure efficient absorption through biological membranes. Larger particles (> 5 μm) can also have a toxic effect because they can get stuck in the fine blood capillaries of the lungs.

The patents US 5,449,720 and WO 92/17167 of BIOTECH AUSTRALIA disclose similar systems with which the absorption of the microparticles can be increased significantly. These systems use vitamin B12 or analogues to increase the absorption of the particles in the digestive tract. The above mentioned

Problems of the rapid release of the active ingredients, low stability and loading are not solved in these documents either.

Patent specification EP 0 454 044 B1 describes the production of pharmaceutical preparations in microparticulate form, which are made up of a polyelectrolyte complex

Have polycations and polyanions and at least one active ingredient. This solves the organic solvent problems described above. However, despite their ionic crosslinking, these particles often have insufficient stability, so that the active ingredients could be released suddenly shortly after the particles have been administered. 3

In view of the prior art specified and discussed herein, it is an object of the present invention to provide particles which help to avoid or reduce the disadvantages of the known particles. In particular, it is an object of the present invention to provide nanoparticles which are stable and an initial sudden release

Avoid (so-called "burst effect") the active ingredient and release it in a controlled manner

Furthermore, it is an object of the present invention to provide nanoparticles which are largely free of residues of organic solvents

Another task is to provide the simplest and most advantageous method for producing nanoparticles

Finally, the specification of the use of nanoparticles is also the task of

invention

These tasks, as well as other tasks not explicitly mentioned, which can be derived or inferred from the contexts discussed here, are solved by the measures described in claim 1. Appropriate modifications of the nanoparticles according to the invention, processes for their production and their use are described in possibly subordinate and in the Claim 1 jerk-related subclaims under protection

Because the biocompatible and biodegradable polyelectrolyte complex of polycations and polyanions is treated with a crosslinking agent during or after its formation, active ingredients that are present in the nanoparticles are released in a controlled manner

The following advantages are additionally achieved by the measure according to the invention 4

Avoidance of a quick release of the active ingredient shortly after administration of the particles ("burst effect")

Sensitive active substances can be superbly protected against degradation by the nanoparticles according to the invention, since they are well embedded in the nanoparticles

Highly effective active ingredients can be controlled and released in a very targeted manner at their destination, so that a lower dose of the drug is required and the undesirable side effects are minimized

The nanoparticles are much more stable than conventional ones without losing their biodegradability

Due to their small size, the particles according to the invention are particularly easily absorbed

The nanoparticles are essentially free of residues of organic solvents, since they can be formed in an aqueous solution

The charge of the polyelectrolyte complex can be varied by the crosslinking in order to change the hydrophilicity of the particles. This causes a modification of the binding of plasma proteins to the nanoparticles. Furthermore, the retention time of the particles on the mucous membranes and the residence time of the particles in the body can also be influenced

Other substances that can be bound to the surface of the nanoparticles are more firmly bound to the particles through the crosslinking. These substances can, for example, increase the absorption of the particles by causing the particles to be actively transported 5

covalent bonding of these substances to the polyelectrolyte complex can be avoided. This improves the biodegradability.

In addition, the swelling capacity or the water absorption of the particle can be influenced by this measure.

For the purposes of the invention, nanoparticles refer to particles with an average size of 10 to 1000 nm, preferably 10 to 500 and particularly preferably 50 to 250 nm, which have a biocompatible and biodegradable polyelectrolyte complex 10.

The shape of the particles can be regular or irregular.

The term biocompatible here means that the compounds which are preferably used for the preparation of the polyelectrolyte complex are compatible with application, that is to say, for example, are not or only to a reasonable extent toxic and / or have only a very slight allergenic effect. So that there is no accumulation of the polymers in the body, they should be biodegradable or excreted. Depending on their use, the polymers are preferably also biocompatible.

The polyelectrolyte complex can result from the bringing together of polyanions and polycations, as is described in the patent specification EP 0 454 044 B1. Depending on the ratio of the polymeric ions, the surface of the 25 nanoparticles formed can be positively or negatively charged (a positive or negative zeta

Possess potential) or be uncharged.

All negatively charged, biodegradable or separable polymers are suitable as polyanions for this purpose. For this purpose, the polyacids can in particular have 30 charged phosphonate, phosphate, sulfonate, sulfate and / or carboxy groups. Preferred, without being restricted thereby, 6

Hepaπn, sumarin, protamine sulfate, polyvinyle, polyallylene, polystyrene and polyacrylates, derivatives of polyzuckerπ, such as starch hydrolysates, inulin, hydroxyethyl starch, dextrans, cellulose derivatives, alginates and xylan, which have sulfate groups or carbonate groups, such as pectinate and xylanolysulfate, which have carbonates , in particular polyamino acids, such as polyaspartic acid and polyglutamic acid. These charged polymers can also be partially substituted or in the form of a salt. The polyanions can also be used in copolymer form. These include copolymers of various monomers which can be used to prepare the polyanions listed above, as well as copolymers thereof Monomers with other biodegradable monomers, as can be used to produce the biodegradable polymers listed below, are to be understood. Mixtures of these polymers / copolymers are within the scope of the present invention Also suitable are xylan polysulfate and partially substituted xylan polysulfate

The weight average molecular weight of the polyanions is preferably 1,000 to 2,000,000 daltons, particularly preferably 40,000 to 600,000 daltons. They are generally commercially available. However, the polyanions can also be prepared in any manner known to those skilled in the art

For the production of the nanoparticles, the polyanions are preferably used in a concentration of 0.1 to 40 g / l, particularly preferably 1 to 20 g / l

Suitable biodegradable or excretable polycations in the context of the invention include, without limitation, collagen and collagen derivatives, gelatin, poly-N-alkylvinylpyπdine, polyethyleneimine, polyvinylamine, polyallylamine and polyacrylate, poly-L-Lysiπ, poly -α, ß- (dιmethylamιnoethyl) -D, L-aspartamιd (PDAA), copolymers of PDAA and hydrophobically esterified poly-α, ß- (2-hydroxyethyl) -D, L-aspartamιd (PHEA), chitosan and derivatives, lysinoctadecyl ester , aminated dextrans, aminated cyclodextins, 7

aminated cellulose ethers, aminated pectins and their partly substituted derivatives and salts. The polycations can also be used in copolymer form. These include copolymers of various monomers that can be used to produce the above-mentioned polycations, as well as copolymers of these monomers with other biodegradable monomers. How they can be used to produce the biodegradable polymers listed below, to understand. Mixtures of these polymers / copolymers are likewise suitable in the context of the present inventions. Chitosan is preferred here, since its particularly high compatibility is recognized

The weight average molecular weight of the polycations is preferably 1,000 to 2,000,000 daltons, particularly preferably 40,000 to 600,000 daltons. Their preparation is known to the person skilled in the art. They are generally commercially available

To produce the nanoparticles, the polycations are preferably used in a concentration of 0.1 to 40 g / l, particularly preferably 1 to 20 g / l

The nanoparticles can be further biocompatible and / or biodegradable

Containing polymers which are known to the person skilled in the art, for example, without any limitation, the following polymers may be mentioned, such as polysaccharides, such as dextran and its derivatives, polyalkyl cyanoacrylates, polyalcohols, polymethylidene malonates, polyesters, such as PLGA (polylactic-polyglycolic acid copolymer) and polycaprolactone , Polyether, like

Polyethylene glycol, polyanhydrides, polyalkylcyanoacrylates, polyacrylamides, polyphosphazenes and biodegradable polyamides and polyurethanes. Derivatives of the polymers which have additional functional groups are particularly preferred. These functional groups are not intended to restrict the hydroxyl, amino, the thiol, the

Carbonyl, the thiocarbonyl, the imino, the carboxyl, the alkoxycarbonyl, the 8th

Carboxyamide, the phosphonate, the phosphate, the sulfonate, the sulfate and the epoxy group

These biodegradable polymers can also be used in copolymer form. These include copolymers of various monomers, which can be used to produce the biodegradable polymers listed above. Mixtures of these polymers / copolymers are also suitable in the context of the present invention

These polymers can serve to vary the stability of the nanoparticles

Furthermore, the polymers can serve the surface-modifying agents mentioned below, to bind the bioactive substances to the nanoparticles and / or to stabilize the substances

The weight average molecular weight of the polymers is more than 1,000, preferably 30,000 to 2,000,000, particularly preferably 50,000 to 300,000 daltons

For the production of the nanoparticles, the biodegradable polymers are preferably used in a concentration of 0 to 100 g / l, particularly preferably 0 to 40 g / l

The production of these polymers and of the polyacids or polybases is known from the literature. A large part of these polymers is also commercially available. In addition, other additives can be incorporated into the nanoparticles if desired. For example, absorption improvers such as phospholipids can be incorporated

Bioactive substances are substances that influence the properties or the behavior of living systems

Restriction should apply to therapeutics, diagnostics, cosmetics as well 9

prophylactic substances. It should be pointed out here that in special cases the substance itself does not have to become active. In the case of diagnostics, these include, for example, contrast agents such as oxygen or noble gases, etc. be understood.

Particularly interesting bioactive substances are, for example, active peptides and proteins, such as insulin, interferons, enzymes, somatropin, erythropoietin, G-CSF, human growth hormone, calcionin, LHRH, factor VIII, tPA, enkephaline, glucagon, TRH, thymopoietin, thymopentin, thymocartin as well as analogs and fragments;

Enzyme inhibitors such as HIV protease inhibitors;

Antigens and immunogens, for example influenza viruses or subunits of antigens;

Gangliosides;

Antibiotics, such as ß-lactam antibiotics (penicillins, cephalosporins, monobactams, carbapenems, etc.), aminoglycosides (e.g. streptomycin), tetracyclines,

Chloramphenicol, macrolide antibiotics (e.g. erythromycin), lincomycine, fosfomycin, fusidic acid, polymyxine, vancomycine and the like. Teicoplanin;

Local anesthetics;

Contraceptives;

Analgesics, such as hypnoanalgesics, in particular opium alkaloids, 4-phenylpiperidine derivatives (pethidine), 3,3-diphenylpropylamine derivatives (methadone), fentanyl derivatives, tramadol and nefopam and non-opioid analgesics, antipyretics and

Anti-inflammatory drugs, especially derivatives of salicylic acid (e.g. acetylsalicylic acid), 10

the aniline (e.g. paracetamol), the anthranilic acid (mefenammic acid), the pyrazole (metamizole, phenazone, propyphenazone) u from (hetero) arylessιg- u -propionsaureπ (indomethacm, diclofenac, ibuprofen, phenonobutene), sterucocoblene, steruco -Deπvate,

Anti-rheumatic drugs, such as oxyphenbutazone, arylacetic acid and propionic acid derivatives, in particular indomethacm, diclofenac, ibuprofen, ketoprofen, oxicams such as piroxicam, gold (l) preparations, D-penicillamine, chloroqum and immunosuppressants,

Hormones and antagonists, such as peptide hormones, in particular

Adrenocortiocotropin, vasopresin, desmopressm, parathyroid hormone, somatostatin and insulin, steroid hormones, in particular progesterones, estrogens and androgens, prostaglandms and adrenal hormones, such as adrenaline,

Cytostatics, such as alkylating agents, especially mechloroethamm,

Cyclophosphamide, Ifosfamide, Mephalan, Chlorambucil, Hexamethylmelamm, Thitepa, Busulfan, Carmustm, lomustm, Semustm, Steptozocm and Dacarbazin,

Antimetabohten, especially methotrexate, fluorouracil, Floxuπdm, Cytarabm, Mercaptopurm, Thioguanm, Pentostatin,

Alkaloids,

RNA, DNA, such as nucleotides, oligonucleotides, polynucleotides, genes or gene segments, plasmids and / or vectors and their derivatives, which are particularly active in HIV, rheumatoid arthritis, cancer, hormone deficiency diseases, hypertension, atherosclerosis, vascular diseases, viral infections and a lack of endogenous synthesis Peptides and proteins are used 1 1

Toxme or Vaccme, such as bacterial vaccines, such as tetanus and cholera toxin, such as viral vaccines, such as AIDS antigens or viral hepatitis components,

Carbohydrates, such as mono- or polysacchand, dextran, agar, agarose-denvate,

Protooglycans, such as Heparm, Heparan, dermatan sulfates,

Lipids, such as phosphopides, cholesteπn, Tngylceπde and Lipoproteme u

Mixtures of these bioactive substances can also be used

The nanoparticles according to the invention are particularly suitable dosage forms, particularly for unstable preparations, since the particles are particularly stable and thus protect the active ingredients, for example proteins, against decomposition by, for example, gastric acid. The active ingredient can therefore be released in a particularly targeted manner, so that, for example, after oral administration of the Active ingredient is not released in the stomach or intestine where it was broken down, but only when it has been taken ms blood

The active ingredient can be in at least four different ways

Nanoparticles arrive

1 inclusion of the active ingredient or mixture of active ingredients contained in the solution in the case of complex precipitation (“capture” from the solution)

2 Adsorption or absorption of an active ingredient or the active ingredient mixture from a solution with which the nanoparticles already produced come into contact (in the case of porous particles or gels with a “sponge effect”)

3 Failure of the polyelectrolyte complex, the active substance chemically attached to one

Complex partner is bound 12

4 Inclusion by using the active ingredient / active ingredient mixture as a polyelectrolyte complex formation partner

For the production of the nanoparticles, the bioactive agent / the bioactive agent mixture is preferably used in a concentration of 0.1 to 40 g / l, particularly preferably 1 to 20 g / i

According to the invention, the polyelectrolyte complex after its formation is additionally treated with at least one crosslinking agent. These compounds link the polymers of the nanoparticles so that they become more stable and pharmaceutical

Active substances contained in the nanoparticles are released more slowly

These crosslinking agents include, but are not intended to restrict them,

Aldehydes and ketones such as formaldehyde, glyoxal and glutaraldehyde, benzoquinone

halogenated triazine pvates, such as 2,4,6-trichloro-1, 3,5-tricazione, 2,4-dichloro-6-methoxy-1, 3,5-tricazione,

2,4-Dιchlor-6-ethoxy-1, 3,5-trιazιn, 2,4-dιchlor-6-phenoxy-1, 3,5-trιazιn, 2-chloro-4,6-dιmethoxy-1, 3, 5-trιazιn, 2-choir-4,6-dιethoxy-1, 3,5-trιazιn, 2-choir-4,6-dιphenoxy-1, 3,5-trιazιn,

Phosphomum salts, such as

O- (1, 2-dιhydro-2 -oxo-1 -pyπdyl) -trιpyrrolιdιnophosphonιumchlorιd,

O- (1, 2-dιhydro-2-oxo-1 -pyπdyl) -tπpyrrolιdιnophosphonιumhexafluorophosphat,

O- (1, 2-Dιhydro-2 -oxo-1 -pyπdyl) -trιpyrrolιdιnophosphonιumperchlorat, O- (1, 2-Dιhydro-2-oxo-1 -pyπdyl) -tπpyrrolιdιnophosphonιumtetrafluoroborat,

O- (1,2-dihydro-2-oxo-1-pyridyl) tris (dimethylamino) phosphonic chloride, 1 3

O- (1,2-dihydro-2-oxo-1-pyridyl) -tπs (dimethylamino) phosphonium hexafluorophosphate,

O- (1,2-dihydro-2-oxo-1-pyridyl) -tπs (dimethylamino) phosphonium chlorate,

O- (1,2-dihydro-2-oxo-1-pyridyl) tris (dimethylamino) phosphonium tetrafluoroborate, O- (3,4-dihydro-4-oxo-1, 2,3-benzotrιazιn-3-yl) - trιpyrrolιdιnophosphonιumchloπd,

O- (3,4-dihydro-4-oxo-1, 2,3-benzotrιazιn-3-yl) -trιpyrrolιdιophosphonιumhexa- fluorophosphate,

O- (3,4-dιhydro-4-oxo-1, 2,3-benzotπazιn-3-yl) -trιpyrrolιdιnophosphonιumperchlorat,

O- (3,4-dιhydro-4-oxo-1, 2,3-benzotrιazιn-3-yl) -trιpyrrolιdιnophosphonιumtetra fluoroborate,

O- (3,4-dιhydro-4-oxo-1, 2,3-benzotrιazιn-3-yl) -trιs (dιmethylamιno) phosphonium chloπd,

O- (3,4-dihydro-4-oxo-1, 2,3-benzotrιazιn-3-yl) trιs (dimethylamino) phosphonium hexafluorophosphate, O- (3,4-dihydro-4-oxo-1,2 , 3-benzotrιazιn-3-yl) -tπs (dιmethylamιno) phosphonιum perchlorate,

O- (3,4-dιhydro-4-oxo-1, 2,3-benzotrιazιn-3-yl) -tπs (dιmethylamιno) phosphonium tetrafluoroborate,

O- (5-norbomene-2,3-dicarboxamido) -tπpyrrolιdιnophosphonumumchloπd, O- (5-norbomene-2,3-dicarboxamido) -trιpyrrolidicnophosphonum hexafluorophosphate,

O- (5-norbomen-2,3-dicarboxamido) -tripyrroleidophosphonium perchlorate,

O- (5-norbornene-2,3-dιcarboxamιdo) -trιpyrrolιdιnophosphonιum tetrafluoroborate,

O- (5-norbomen-2,3-dιcarboxamιdo) -trιs (dιmethylamιno) phosphonιumchlorιd,

O- (5-norbornene-2,3-dιcarboxamιdo) -trιs (dιmethylamιno) phosphonium hexafluorophosphate,

O- (5-norbornene-2,3-dιcarboxamιdo) -trιs (dιmethylamιno) phosphonιumperchlorate,

O- (5-norbornene-2,3-dιcarboxamιdo) -trιs (dιmethylamιno) phosphonιumtetra-fluoroborate,

O- (benzotrιazol-1-yl) -trιpyrrolιdιnophosphonιumchlorιd, O- (benzotπazol-1-yl) -tπpyrrolιdιnophosphonιumhexafluorophosphate,

O- (benzotrιazol-1-yl) -trιpyrrolιdιnophosphonιumperchlorat, 14

0- benzotrιazol-1 yl) -tπpyrrolιdιnophosphonιumtetrafluoroborat,

O- benzotrιazol-1 yl) trιs (dimethylamιno) phosphonium chlorid,

O-benzotrιazol-1 yl) trιs (dimethylamino) phosphonium hexafluorophosphate,

O-benzotrιazol-1 yl) -tπs (dimethylamino) phosphonium jumpchlorate,

O-Benzotπazol-1 yl) trιs (dimethylamino) phosphonium tetrafluoroborate,

O-N-Malemimidy l) -trιpyrrolιdιnophosphonιumchloπd,

O-N-Maleinimidy l) -tπpyrrolιdιnophosphonιumhexafluorophosphat,

O- N-Malemimidy l) -tπpyrrolιdιnophosphonιumperchlorat,

O-ι N-Malemimidy l) -trιpyrrolιdιnophosphonιumtetrafluoroborat,

O-N-Malemimidy l) -tπs (dimethylamino) phosphonium chloride,

O-N-Malemimidy l) -tπs (dιmethyiamιno) phosphonιumhexafluorophosphat,

O- N-Malemimidy l) -trιs (dimimethylamino) phosphonium jumpchlorate,

O N-Maleinimidy l) trιs (dimethylamιno) phosρhonιumtetrafluoroborat,

O-ι N-Succmimidy l) -tπpyrrolιdιnophosphonιumchlorιd,

O N-succinimidy l) -tπpyrrolιdιnophosphonιumhexafluorophosphat,

O- N-succinimidy l) -tπpyrrolιdιnophosphonιumperchlorat,

O N-Succmimidy l) -trιpyrrolιdιnophosphonιumtetrafluoroborat,

O-N-succinimidyl) trιs (dimethylamine) phosphonium chloride,

O-N-Succmimidy l) trιs (dimethylamino) phosphonium hexafluorophosphate,

O-N-Succmimidy l) -trιs (dimimethylamno) phosphonium jumpchlorate,

O-ι N-Succinimidy l) -trιs (dιmethylamιno) phosphonιumtetrafluoroborat,

O N-Phtha midy l) -tπpyrrolιdιnophosphonιumchlorιd,

O N-phthahmidy l) -trιpyrrolιdιnophosphonιumhexafluorophosphat,

OH N-phthahmidy l) -trιpyrrolιdιnophosphonιumperchlorat,

O N-phthahmidy l) -trιpyrrolιdιnophosphonιumtetrafluoroborat,

O N-phthalimidyl) trιs (dimethylamine) phosphonium chloride,

O N-phthahmidyl) trιs (dimethylamino) phosphonium hexafluorophosphate,

O N-Phthahmidy l) trιs (dimethylamιno) phosphonium jumpchlorate,

O-N-phthalimidyl) trιs (dimethylamino) phosphonium tetrafluoroborate,

O-N-perhydroph 'thalιmιdyl) -tπpyrrolιdιnophosphonιumchloπd,

O-N-perhydrophthalic) tripyrrolidophosphonum hexafluorophosphate, 15

O- (N-perhydrophthalimidyl) tripyrrolidinophosphoπium perchlorate, O- (N-perhydrophthalimidyl) thpyrrolidinophosphonium tetrafluoroborate, O- (N-perhydrophthalimidyl) tris (dimethylamino) phosphonium chloride, O- (N-perhexamophosphonium), (N-perhydrophthalimidyl) tris (dimethylamino) phosphonium perchlorate,

O- (N-perhydrophthalimidyl) tris (dimethylamino) phosphonium tetrafluoroborate, tris-di-methyl-amino-chlorophosphonium hexafluorophosphate, tris-di-methyl-amino-bromo-phosphonium hexafluorophosphate, tris-di-methyl-amino- cyano-phosphonium hexafluorophosphate, tris-di-methyl-amino-isothiocyanoto-phosphonium hexafluorophosphate,

Tris-di-methyl-amino-azido-phosphonium hexafluorophosphate, tris-di-methyl-amino-trichloromethyl-phosphonium hexafluorophosphate, tris-di-methylamino-trifluoromethyl-phosphonium hexafluorophosphate, tris-di-methyl-amino-phenoxy- phosphonium hexafluorophosphate, tris-dimethylamino-p-nitrophenoxyphosphonium hexafluorophosphate,

Tris-pyrrolidino-chloro-phosphonium-hexafluorophosphate, tris-pyrrolidino-bromo-phosphonium-hexafluorophosphate, tris-pyrrolidino-cyano-phosphonium-hexafluorophosphate, tris-pyrrolidino-isothiocyanoto-phosphonium-hexafluorophosphoronophosphate, tris

Tris-pyrrolidino-trichloromethyl-phosphonium hexafluorophosphate, tris-pyrrolidino-trifluoromethyl-phosphonium-hexafluorophosphate, tris-pyrrolidino-phenoxy-phosphonium-hexafluorophosphate, tris-pyrrolidino-p-nitrophenoxy-phosphonium-hexafluorophosphate;

Uronium salts such as

1,2-dihydro-2-oxo-1-pyridyl-oxy-biscyclohexylidenuronium chloride, 1, 2-dihydro-2-oxo-1-pyridyl-oxy-biscyclohexylidenuronium hexafluorophosphate, 1,2-dihydro-2-oxo-1-pyridyl oxy-biscyclohexylidenuronium perchlorate, 1, 2-dihydro-2-oxo-1-pyridyl-oxy-biscyclohexylidenuronium tetrafluoroborate,

1, 2-dihydro-2-oxo-1-pyridyl-oxy-biscyclopentylidenuronium chloride, 1 6

1, 2-dihydro-2-oxo-1-pyhdyl-oxy-biscyclopentylidenuronium hexafluorophosphate, 1, 2-dihydro-2-oxo-1-pyridyl-oxy-biscyclopentylidenuronium perchlorate, 1, 2-dihydro-2-oxo-1-pyridyl- oxy-biscyclopentylidenuronium tetrafluoroborate, 1, 2-dihydro-2-oxo-1-pyridyl-oxy-N, N, N ', N'-tetramethyluronium chloride, 1, 2-dihydro-2-oxo-1-pyridyl-oxy-N, N, N ', N'-tetramethyluronium hexafluorophosphate,

1, 2-dihydro-2-oxo-1-pyridyl-oxy-N, N, N ', N'-tetramethyl-uronium perchlorate, 1, 2-dihydro-2-oxo-1-pyridyl-oxy-N, N, N ', N'-tetramethyl uronium tetrafluoroborate, 3,4-dihydro-4-oxo-1, 2,3-benzothazin-3-yl-oxy-biscyclohexylidenuronium chloride, 3,4-dihydro-4-oxo-1, 2, 3-benzotriazin-3-yl-oxy-biscyclo-hexylidenuronium hexafluorophosphate,

3,4-dihydro-4-oxo-1, 2,3-benzotriazin-3-yl-oxy-biscyclo-hexylidenuronium perchlorate, 3,4-dihydro-4-oxo-1, 2,3-benzotriazin-3-yl- oxy-biscyclo-hexylidenuronium tetrafluoroborate, 3,4-dihydro-4-oxo-1, 2,3-benzotriazin-3-yl-oxy-biscyclo-pentylidenuronium chloride, 3,4-dihydro-4-oxo-1, 2, 3-benzotriazin-3-yl-oxy-biscyclopentylidenuronium hexafluorophosphate,

3,4-dihydro-4-oxo-1, 2,3-benzotriazin-3-yl-oxy-biscyclopentylidenuronium perchlorate, 3,4-dihydro-4-oxo-1, 2,3-benzotriazin-3-yl- oxy-biscyclopentylidenuronium tetrafluoroborate, 3,4-dihydro-4-oxo-1, 2,3-benzotriazin-3-yl-oxy-N, N, N ', N'-tetramethyluronium chloride,

3,4-dihydro-4-oxo-1, 2,3-benzotriazin-3-yl-oxy-N, N, N \ N'-tetramethyluronium hexafluorophosphate,

S ^ -Dihydro ^ -oxo-I ^ .S-benzotriazin-S-yl-oxy-N ^ .N'.N'- tetramethyluronium perchlorate, 3,4-dihydro-4-oxo-1, 2,3- benzotriazin-3-yl-oxy-N, N, N ', N'-tetramethyluronium tetrafluoroborate,

5-norbornene-2,3-dicarboxamido-oxy-biscyclohexylidenuronium chloride, 5-norbomene-2,3-dicarboxamido-oxy-biscyclohexylidenuronium hexafluorophosphate, 5-norbornene-2,3-dicarboxamido-oxy-biscyclohexylidenuronium perchlororene, 5 dicarboxamido-oxy-biscyclohexylidenuronium tetrafluoroborate,

5-norbornene-2,3-dicarboxamido-oxy-biscyclopentylidenuronium chloride, 17

5-norbomene-2,3-carboxamido-oxy-bicyclopentyl-uronic acid hexafluorophosphate,

5-norbornene-2,3-carboxamido-oxy-bicyclopentyl-denuronone chlorate,

5-norbornene-2,3-carboxamido-oxy-bicyclopentyl-denuron-umtetrafluoroborate,

5-norbornene-2,3-carboxamido-oxy-N, N, N ', N'-tetramethyluronium chloride,

5-norbomen-2,3-carboxamido-oxy-N, N, N ', N'-tetramethyluronum hexafluorophosphate,

5-norbornene-2,3-carboxamido-oxy-N, N, N ', N'-tetramethyluronium chlorate,

5-norbornene-2,3-carboxamido-oxy-N, N, N ', N'-tetramethyluronium tetrafluoroborate

Benzotπazol- '-y / 1l-oxy-bιscyclohexylιdenuronιumchlorιd, B B Beeennnzzzoootttrrπιιaaazzzooolll --- 11' - -yyylll-oxy-biscyclohexylidenuronium hexafluorophosphate,

Benzotπazol- '-y tιl \ l-oxy-biscyclohexylidenuronium perchlorate,

Benzotπazol- 'l-y tιll-oxy-biscyclohexyhdenuroniumtetrafluoroborat,

Benzotπazol- '-ytl \ l-oxy-biscyclopentylidenuroniumchlorid,

Benzotπazol- 'l-y tfl \ l-oxy-biscyclopentylidenuroniumhexafluorophosphat,

B Beennzzoottππaazzooll - 1 'l - yylll-oxy-biscyclopentylidenuronium perchlorate,

Benzotπazol- 'l-y tιl \ l-oxy-biscyclopentyhdenuronium tetrafluoroborate,

Benzotπazol- 'l-y ^ ιl \ l-oxy-N, N, N', N'-tetramethyluronιumchlorιd,

Benzotπazol- '1-y ttl \ l-oxy-N, N, N \ N'-tetramethyluronum hexafluorophosphate,

Benzotnazole- ly tιl \ l-- oxy-N, N, N ', N'-tetramethyluronιumperchlorat,

BB Beeennnzzzoootttrrπιιaaazzzooolll --- 11 '1 --- yyylll-oxy-N, N, N \ N'-tetramethyluronιumtetrafluoroborat,

N-maleinimidyl-oxy-biscyclohexylidenuronium chloride, N-malemimidyl-oxy-biscyclohexylidenuronium hexafluorophosphate, N-maleinimidyl-oxy-biscyclohexyhdenuronium perchlorate, N-maleinimidyl-oxy-biscyclohexy-denoronidylonatylonuronium xurobluronium xyluronium oxychloride

N-Maleinimidyl-oxy-biscyclopenty denuronium hexafluorophosphate, N-Maleinimidyl-oxy-biscyclopentylidenuronium perchlorate, N-Maleinimidyl-oxy-biscyclopentyhdenurιiumtetrafluoroborat, N-Maleιnιmιdyl-oxy-N-N, N, Male N, N ', N'-tetramethyluronum hexafluorophosphate,

N-maleimidyl-oxy-NNN'.N'-tetramethyluronium perchlorate, N-maleimidyl-oxy-N, N, N ', N'-tetramethyluronium tetrafluoroborate, N-succinimidyl-oxy-biscyclohexylidenuronium chloride, N-succinimidyl-oxy-biscyclohexyiidenuronium hexafluorophosphate, N-succyinidyl-oxychloride-bis-succinimidyl-oxy-cyclinimyl-bis-succinidyl-bis-succinidyl-bis-cyclinimidyl-oxy-succinidyl-bis-succinidyl-oxy-cyclinidyl-oxy-succinidyl-bis-succinidyl-oxy-oxychloride-N-succinyl-oxy-bis-cyclinidyl-N-succinidyl-oxy-oxychloride-N-succinidyl-bis-oxychloride-N-succinidyl-bis-oxychloride-N-succinidyl-bis-oxychloride biscyclohexylidenuronium tetrafluoroborate,

N-succinimidyl-oxy-biscyclopentylidenuronium chloride, N-succinimidyl-oxy-biscyclopentylidenuronium hexafluorophosphate, N-succinimidyl-oxy-biscyclopentylidenuronium perchlorate, N-succinimidyl-oxy-biscyclopentyluronium-ninoxin-n-oxy-n-oxy-n-oxy-n-oxy-n-oxy-n-oxy-n-cyclo

N-succinimidyl-oxy-N, N, N ', N'-tetramethyluronium hexafluorophosphate, N-succinimidyl-oxy-N, N, N', N'-tetramethyluronium perchlorate, N-succinimidyl-oxy-N, N, N ', N 'tetramethyluronium tetrafluoroborate, N-phthalimidyl-oxy-biscyclohexylidenuronium chloride, N-phthalimidyl-oxy-biscyclohexylidenuronium hexafluorophosphate,

N-phthalimidyl-oxy-biscyclohexylidenuronium perchlorate, N-phthalimidyl-oxy-biscyclohexylidπuronium tetrafluoroborate, N-phthalimidyl-oxy-biscyclopentylidenuronium chloride, N-phthalimidyl-oxy-biscyclopentylorylidononylylurylidylonylurylidyluronium

N-phthalimidyl-oxy-biscyclopentylidenuronium tetrafluoroborate, N-phthalimidyl-oxy-N, N, N ', N'-tetramethyluronium chloride, N-phthalimidyl-oxy-N, N, N', N'-tetramethyluronium hexafluorophimidate, N-phthalate N, N, N ', N'-tetramethyluronium perchlorate, N-phthalimidyl-oxy-N, N, N', N'-tetramethyluronium tetrafluoroborate,

N-perhydrophthalimidyl-oxy-biscyclohexylidenuronium chloride, N-perhydrophthalimidyl-oxy-biscyclohexylidenuronium hexafluorophosphate, N-perhydrophthalimidyl-oxy-biscyclohexylidenuronium perchlorate, N-perhydrophthalimidyl-oxy-biscyclo-oxychloride-oxychloride

N-perhydrophthalimidyl-oxy-biscyclopentylidenuronium hexafluorophosphate, 1 9

N-perhydrophthalimidyl-oxy-biscyclopentylidenuronium perchlorate, N-perhydrophthalimidyl-oxy-biscyclopentylidenuronium tetrafluoroborate, N-perhydrophthalimidyl-oxy-N, N, N ', N'-tetramethyluronium chloride, N-perhydrophthalimide, tetramethyluronium hexafluorophosphate, N-perhydrophthalimidyl-oxy-N, N, N ', N'-tetramethyluronium perchlorate,

N-perhydrophthalimidyl-oxy-N, N, N ', N'-tetramethyluronium tetrafluoroborate;

Derivatives of hydroxylamine, such as

Carbonic acid bis (1, 2-dihydro-2-oxo-1-pyridyl) ester, carbonic acid bis (3,4-dihydro-4-oxo-1, 2,3-benzotriazin-3-yl) ester,

Carbonic acid bis (5-norbomen-2,3-dicarboxamido) ester,

Carbonic acid bis (benzotriazol-1-yl) ester,

Carbonic acid bis (N-maleimidyl) ester,

Carbonic acid bis (N-succinimidyl) ester, carbonic acid bis (N-phthalimidyl) ester,

Carbonic acid bis (N-perhydrophthalimidyl) ester,

Oxalic acid bis (1,2-dihydro-2-oxo-1-pyridyl) ester,

Oxalic acid bis (3,4-dihydro-4-oxo-1, 2,3-benzotriazin-3-yl) ester,

Oxalic acid bis (5-norbornene-2,3-dicarboxamido) ester, oxalic acid bis (benzotriazol-1-yl) ester,

Oxalic acid bis (N-maleimidyl) ester,

Oxalic acid bis (N-succinimidyl) ester,

Oxalic acid bis (N-phthalimidyl) ester,

Bis (N-perhydrophthalimidyl) oxalic acid, bis (1,2-dihydro-2-oxo-1-pyridyl) pyrocarbonate,

Pyrocarbonate bis (3,4-dihydro-4-oxo-1, 2,3-benzotriazin-3-yl) ester,

Pyrocarbonate bis (5-norbomen-2,3-dicarboxamido) ester,

Pyrocarbonate bis (benzotriazol-1-yl) ester,

Pyrocarbonate bis (N-maleimidyl) ester, pyrocarbonate bis (N-succinimidyl) ester, 20th

Pyrocarbonate bis (N-phthalimidyl) ester, pyrocarbonate bis (N-perhydrophthalimidyl) ester; and

reactive carbonic acid derivatives, such as carbodiimides, in particular N- (3-dimethylaminopropyl) -N-ethylcarbodiimide.

Agents that form ester or amide groups are preferred because the resulting ester or amide groups can be biodegraded particularly well. These include all halogenated triazine derivatives, all phosphonium salts, all uronium salts and reactive carbonic acid derivatives. N- (3-Dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (EDAP) and O- (N-succinimidyl) -N, N, N ^' , N ^' - tetramethyluronium tetrafluoroborate (TSTU) are particularly preferred.

The degree of crosslinking can be influenced by the concentration of the polyelectrolyte complex and the crosslinking agent and the reaction time.

The reaction time depends, among other things, on the type, reactivity and concentration of the selected crosslinking agent and the polyelectrolyte complex, as well as the reaction temperature and the pH of the solution. Under certain circumstances, it can be influenced by catalysts. At room temperature, it is preferably 1 minute to 24 hours, particularly preferably 5 to 120 minutes.

The surface of the nanoparticles can be modified. This modification is described in the above-mentioned patent applications WO 96/20698, US 5,449,720 and WO 92/17167, which are intended to be included in the disclosure.

The properties of the nanoparticles can be influenced in a targeted manner by the modification. For example, antithrombocytic properties can be generated, the absorption of the particles via the intestine can be improved or substances can be transferred to the

Particles are bound so that the particles in very defined areas in the 21

Body will be enriched. Antigens against cancer cells, which can be linked to the particles, are mentioned as an example, so that the drugs are released directly from the drug saturated drug depots in the cancer cells.

This modification can be achieved in that at least one of the charged polymers of the polyelectrolyte complex is additionally treated with an agent which modifies the surface before, during or after the formation of the complex. These means include, without being limited by this, various synthetic polymers, biopolymers, low molecular weight

Oligomers, natural substances and surface-active substances.

The synthetic polymers with which the surface of the nanoparticles can be modified include carboxymethyl cellulose, cellulose, cellulose acetate, cellulose phthalate, polyethylene glycol (carbowax), polyvinyl alcohol

(PVA), hydroxypropyl methyl cellulose phthalate, hydroxypropyl cellulose, sodium or potassium salts of carboxymethyl cellulose, polyvinyl pyrolidone, polystyrene and silicates such as bentonite.

The biopolymers with which the surface of the nanoparticles can be modified include, in particular, proteins and peptides, such as gelatin, casein, albumins (ovalbumin), myoglobin, hemoglobin, monoclonal and polyclonal antibodies, cytokines, such as growth factors, interferons, lymphokines, monokines, Interleukins and chemokines; as well as polysaccharides and pectins.

The natural substances with which the surface of the nanoparticles can be modified include, in particular, cofactors, such as coenzymes, such as vitamins, in particular vitamin B12, and prosthetic groups, such as the heme group; Lipids, especially phospholipids such as lecithin and cholesterol; and prostaglandins. 22

The surface-active substances with which the surface of the nanoparticles can be modified include non-mastic surfactants, in particular sorbitan fatty acid esters, in particular polyoxyethylene sorbitan fatty acid esters, fatty alcohols, such as cetylaic alcohols or stearyl alcohols, and polyether sulfonates, anionic surfactants, in particular sodium, for example, fatty dodecyl sulfates Palmitic acid, stearic acid and oleic acid), Gylceπnester of fatty acids (for example Glyceπnmonostearat) and sodium and potassium salts of fatty acids (Natnumoleat, sodium palmitate, Natπumstearat, among others), Polyoxylstearat, Polyoxylethylenlaurylether, Sorbitansesanolamine, Methyldimbromidom, Ethylammonyl, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium Ethyl, Methylammonium Ethyl, Methyl Brodyl Hexadecyltπmethylammonumchlorid, Dimethyldodecylaminoprpan, N-Cetyl-N-ethylmorpholmiumethosulfat

These agents, which can be used to modify the surface of the nanoparticles, preferably include agents which enable active transport (for example absorption) of the particles. These agents are known as carπers

These carners include bile acids, adhesins, invasme,

Toxins, such as plant or bacterial toxins, cobalamins, viral Hamaglutimne, Lectme, Transferπn, Riboflavm and peptides that are transported intestinally (which use Carπersysteme for mtestinal peptide transport) derivatives of these substances, which also use the respective Camer systems , can also be used

Cobolamines, which are suitable as carriers, include, for example, substances such as vitamin B12 or analogues that bind to the intπnsic factor (IF), a glycoprotem of gastric juice. Through this binding, the nanoparticles are actively absorbed by the mucous membranes from the digestive tract. To the analogues belong, for example, without being restricted thereby, 23

Aquocobalamin, adenosylcobalamin, methylcobalamin, hydroxycobalamm, cyanocobalamin, carbana d and 5-methoxybenzalcyanocobalamin as well as the desdimethyl, monoethylamide and methylamide derivatives of the aforementioned compounds. Furthermore, these analogues include chlorocobalamin, sulfitocobalamin, nitrocobalamin, nitrocobalamin

Benzimidazolecyanocobalamm derivatives, such as 5,6-dichlorobenzimidazole, 5-hydroxybenzimidazole, trimethylbenzimidazole, as well as adenosylcyanocobalamm [(Ade) CN-Cbl], cobalt amalgamation, cobalt ammonium lactam and the anilide, ethyl or deoxy amide and dicarboxylate, monocarboxylate, monocarboxyl Corresponding analogues Further analogues of vitamin B12 result from the substitution of the cobalt atom by zinc or nickel

In the context of the present invention, these surface-modifying agents can also be used as mixtures in order to combine the various properties of the surface-modifying agents or to achieve synergistic effects

To produce the nanoparticles, the surface-modifying agents are preferably used in a concentration of 0 to 200 g / l, particularly preferably 0 to 20 g / l

These surface-modifying agents can be added directly to the aqueous solution. These agents can preferably be bound covalently or ionically to at least one of the hydrophilic polymers, to the crosslinking agent or to the other above-mentioned biocompatible and biodegradable polymers and to the bioactive agent, in order to do so to connect as firmly as possible with the polyelectrolyte complex

For this purpose, the surface-modifying agents can be activated with other substances. However, it is also possible to use the hydrophilic polymers, the crosslinking agents or to the other above-mentioned biocompatible and biodegradable 24

To activate polymers and the bioactive agent in order to then bring these substances together with the surface-modifying agents. These activators include, for example, without limitation, disuccinimidyl suberate, Bιs (sulfoauccιnιmιdyl) suberate, ethylene glycol bιs (succιnιmdylsucccolnate), ethylene glycol (sulfosuccιnιmdylsuccιnat), p-Aminophenylessigsaure, Dιthιo-bιs (succιnιmιdylpropιonat), 3 3'Dιthιo- bιs (sulfosuccιnιmιdylpropιonat) Disuccmimidyltartrat, Disulfosuccmimidyltartrat, -ethylene bιs [2- (Succιnιmιdooxycarbonyloxy) -ethylene] sulfone, bιs [2- (Sulfosuccιnιmιdooxycarbonyloxy) ] sulfone, N, N'-Dιmethyladιpιnsauredιamιd ^* 2HCI, N, N'-Dιmethylpιmelιnsauredιamιd ^* 2HCI,

N, N'-Dιmethylsubeπnsauredιamιd * 2HCI Furthermore, epoxides can be used as activators. These epoxides include, for example, ethylene oxide, 1, 2-propylene oxide, glycidyl ethers, such as diglycidylbutane diol ether, diglycidylethane diol ether, and erytholic acid anhydride

However, it is also possible to use activators which have a thiol group and are particularly readily biodegradable. These activators include, for example, N-succinomidyl-3- (2-pyridyldιthιo) propionate, iminothiolane, sulfosuccinidimidyl-6- [3- (2-pyridyldιthιo ) propιonamιdo] hexanoate, succinomidyl-6- [3- (2- pyπdyldιthιo) propιonamιdo] hexanoate, sulfosuccιnιmιdyl-6- [-methyl- - (2-pyrιdyl- dιthιo) toluamιdo] hexanoate, 1'-4-dι (2'pyπdyldιthιo) propιonamιdo] butane, 4- -Succιnιmιdyloxycarbonyl-α-methyl- - (2-pyπdyldιthιo) toluene, dimethyl-3,3'Dιthιobιspropιonιmιdat ^* 2HCI

These activators can be used all or as a mixture

To produce the nanoparticles, the activators are preferably used in a concentration of 0 to 40 g / l, particularly preferably 0 to 2 g / l

This is described in patent applications WO 96/20698, US 5,449,720 and WO 92/17167 25th

The nanoparticles can be produced, for example, by polyelectrolyte complexation, emulsion techniques, spray drying, solvent evaporation, solvent extraction, coacervation, extrusion, precipitation and filtration or other processes known to the person skilled in the art.

The nanoparticles are preferably produced by polyelectrolyte complexation. The nanoparticles can be brought together by bringing together an aqueous solution of polycations, an aqueous solution of polyanions and at least one bioactive agent and, if appropriate, further substances (further polymers, auxiliaries, etc.) which may be bound to one of the two ionic polymers or which are present in free form can be obtained, and subsequent treatment with a crosslinking agent.

The at least two aqueous solutions of the hydrophilic polymers are brought together in such a way that nanoparticles of the desired size and

Form size distribution. This can be done, for example, by controlled dropping of one of the two solutions into the other of the two solutions. The complex that forms during mixing precipitates as a result of neutralization. It may be necessary to adjust the pH in order to dissolve the substances, such as the polymers, bioactive substances, etc. These pH values depend, among other things, on the particular polyelectrolyte and are known to the person skilled in the art. In preferred embodiments, the person skilled in the art can orientate himself, for example, at the isoelectric point. The particle size can be controlled by the manner in which they are brought together, for example when adding drops, the dilution of the at least two solutions, the speed of the stirrer, the pH and the diameter of the nozzles used for the drops and the dropping speed. The particle size can also be influenced by ultrasound.

In particularly preferred embodiments of the invention

Manufacturing process can be dispensed with further auxiliary substances. This 2 6

However, depending on the bioactive active ingredient, for example as auxiliaries, auxiliaries may be necessary. Excipients may also be indispensable in the treatment with the crosslinking agent

However, depending on the bioactive active substance and the polymer used, different emulsion processes can be used. This may be necessary, for example, if particularly hydrophobic active substances or additionally hydrophobic polymers are to be incorporated into the nanoparticles. These emulsion processes are described in WO 96/05810

Here, for example, one of the hydrophilic polymers is dissolved in water. This solution is added to a non-polar solvent with vigorous stirring by dissolving the hydrophobic active ingredient. Then, for example, the second of the hydrophilic polymers can be added to the resulting emulsion, so that the polyelectrolyte complex is formed. This complex can be crosslinked in situ by adding one of the crosslinking agents mentioned above. It is preferred that this emulsion is stabilized by suitable means, for example dioctylsulphosuccmate

A non-polar polymer can also be dissolved in a hydrophobic solvent in order to introduce it into the polyelectrolyte complex. If both a hydrophobic bioactive agent and a non-polar polymer are to be introduced into the polyelectrolyte complex, it is possible to vary the process explained above slightly so that a multiple emulsion (oil-in-water-oil-oil emulsion) is formed

The particles can also be formed by spray drying. A suitable solution of at least one polyanion, at least one polycation and at least one bioactive active ingredient and optionally further substances is sprayed through a suitable nozzle, so that particles of the desired size are formed. These particles are then dried 27

The resulting particles can be crosslinked in situ by adding crosslinking agents in order to obtain the nanoparticles according to the invention. For this purpose, for example, one of the above-mentioned crosslinking agents can be added and preferably stirred at room temperature for a further 10 minutes to 24 hours, depending on the desired degree of crosslinking and crosslinking agent. The exact one

The regulation for the implementation of the particles depends on the crosslinking agent and can be optimized by a person skilled in the art with a few routine tests.

Crosslinking can be determined using methods known from the literature, such as NMR, NIR or exclusion chromatography.

The resulting particles, which contain a polyelectrolyte complex and at least one bioactive agent, can also be crosslinked later. For this purpose, these particles can be in a suitable solvent, for example water or a dipolar aprotic solvent, such as DMF (dimethylformamide) or

DMSO (Dimethylsoulfoxid), are added. One of the above-mentioned crosslinking agents can then be added to this solution and reacted with the polyelectrolyte complex in such a way that it is additionally crosslinked. This can be done, for example, by stirring for 10 minutes to 24 hours at room temperature.

The particles can then be isolated. This separation can take place, for example, by filtration or centrifugation. The particles are then preferably washed with water and dried, for example by lyophilization.

The nanoparticles thus obtained can be sterilized by radiation, as is well known in the art. However, the nanoparticles can also be produced under sterile conditions. 28

The particles can be administered in any manner known to the person skilled in the art. This includes, in particular, without being restricted by this, the oral form of administration. However, they can also be administered parenterally, for example by injection intravenously, intraarterially, intramuscularly, subcutaneously, intrathecally or intralumbally. The nanoparticles can also be administered nasally, occularly, rectally, vaginally, buccally, orally, transdermally and by inhalation.

The production is to be explained in more detail by the following examples.

Example 1a:

18 mg of insulin and 2 mg of FITC (fluorescein isothiocyanate) labeled insulin were stirred in a 50 ml Erlenmeyer flask with 12 ml of distilled water (from a water treatment system from Millipore) for 1 min at room temperature on a magnetic stirrer (from Ikamag RCT, level 5). A 0.1 N aqueous HCl solution was then added dropwise until a clear solution was obtained and the insulin had completely dissolved.

20 mg of xylan polysulfate (from the Ben-

Drug), which was dissolved in 4 ml of distilled water. Cloudiness occurred when added dropwise. Then 0.1 N NaOH solution was slowly added dropwise until the solution was completely clear.

To this solution, one was added with stirring at medium speed

Add a solution of 4 mg chitosan (from Fluka) in 2 ml Millipore water. The solution was now slightly clouded.

The crosslinking took place with Glyoxal (Riedel). 400 μl of an aqueous 2% glyoxal solution (corresponding to 8 mg of pure glyoxal) were added. The resulting suspension was stirred at room temperature for 10 minutes. 2 9

The suspension was then ultrafiltered over a 100 kD membrane (PLHK membrane from Millipore) (Amicon 8050 ultrafiltration cell, nitrogen pressure 0.2 bar, purity> 99.9%), the residue being washed with 5 ml of water. The retentate was then transferred to a 100 ml round bottom flask, frozen (with a mixture of isopropanol / dry ice) and freeze-dried overnight (model LDC-1, Christ).

The release of the insulin was tested by suspending 5 mg of the dried particles in 10 ml of pH 7.4 phosphate buffer (produced with Sigma phosphate buffer tablets) and heating them at 37 ° C. in a drying cabinet. After 30 minutes, a sample is taken, ultrafiltered (Filter Millipore PLHK) and examined by means of fluorescence spectroscopy for FITC insulin content according to the method known from the literature (excitation wavelength: 494 nm, emission wavelength: 518 nm).

It was found that only 30.1% of the insulin was released after 30 minutes.

Comparative Example 1:

Example 1 was repeated. However, the resulting particles were not cross-linked. This means that the resulting suspension was not mixed with glyoxal, but washed, ultrafiltered and dried directly as described above.

The release of the FITC insulin was tested as in Example 1. It was found that 59.4% of the FITC insulin was released into the solution.

Example 1 b:

The reaction was carried out similarly to that described in Example 1: 20 mg of insulin were stirred in a 50 ml Erlenmeyer flask with 12 ml of distilled water for 1 min and a 0.1 N HCl solution was added dropwise until a clear solution was obtained. 20 mg were added to this solution with stirring 30th

Xylan polysulfate, dissolved in 4 ml of water, was added dropwise and then a 0.1 N NaOH solution was added until a clear solution was formed. A solution of 4 mg of chitosan in 2 ml of water was added to this solution with stirring at medium speed Hydrolyzates of poly- (L-lysine methyl ester fumaramide) (LMF), (the preparation can, for example, according to the in the patent

The processes described in EP 0 245 840 B1) are dissolved in 4 ml of water and added dropwise instead of glyoxal, this time for crosslinking, first 8 μl of N-ethyldnsopropylamine (from Fluka) and then 13 mg (N-succinomidyl) -N, N, N ^' , N ^' - tetramethyluronium tetrafluoroborate (TSTU, Fluka) added with stirring and stirred for 15 mm at room temperature

Release was carried out analogously to Example 1a, this time the released amount of insulin was determined by means of the HPLC method known from the literature. After 4 hours, only 30% of the insulin was released in PBS buffer. The comparison test under exactly the same conditions but without crosslinking resulted in a release of 73% insulin 4 hours

Example 1 c

The reaction was again carried out as described in Example 1b, this time instead of TSTU as crosslinker, 16.4 mg of O- (1H benzotπazol-1-yl-N, N, N ^' , N ^' , tetramethyluronium hexafluorophosphate (HBTU, company Fluka ) used The release of insulin in buffer is now only 14% after 4 hours

Example 2a Inclusion of albumin

18 mg of bovine serum albumin (BSA) and 2 mg of BSA-FITC (from Sigma) were stirred in 4 ml of distilled water for 1 mm at room temperature on a magnetic stirrer (from IKA - combimag RCT, level 5). This solution was dripped in Ruhren 20 mg xylan polysulfate (from the company Bene Arzneimittel), which was previously dissolved in 4 ml of distilled water 31

A solution of 4 mg chitosan (from Fluka) in 2 ml Millipore water is added to this solution with stirring at medium speed. The solution was now slightly cloudy. 20 mg LMF (lysomethyl ester fumaramide hydrolyzate), dissolved in, were added to the entire solution 4 ml of water, added dropwise The crosslinking was carried out with 200 μl of a 40% aqueous glyoxal solution (Riedel).

The suspension was stirred for 15 mm at room temperature

The suspension was cleaned with the aid of an ultrafiltration (Amicon 8050 ultrafiltration cell, nitrogen pressure 1 bar) over a 300 kD membrane (PLMK from Millipore). The residue was washed 3 times with 30 ml of water, 100 ml of permeate being formed

The retentate was then transferred to a 250 ml round bottom flask, frozen and freeze-dried overnight

The release of the BSA-FITC was tested by suspending 5 mg of the dried particles in 10 ml of phosphate buffer pH 7.4 and incubating at 37 ° C in the drying cabinet. After 4 hours a sample was taken, ultrafiltered (filter

Millipore PLHK) and examined by means of a spectrophotometer for FITC content at 494 nm and after 4 hours was 54% of the FITC-BSA. For comparison, particles were produced without addition of crosslinking agent under the same conditions. The release of the FITC-BSA was 80.5 for these particles %

Example 2 b

The reaction was carried out as described in Example 1. Instead of glyoxal, 8 μl of N-ethyldnsopropylamine (the

Fluka) and then 13 mg (N-succinimidyl) -N, N, N ^' , N ^' -tetramethyluronum tetrafluoroborate (TSTU, Fluka) were added with stirring and stirred for 15 mm at room temperature. The work-up and release were carried out analogously to Example 2a performed After 4 hours, 67% of the BSA was released in PBS buffer 32

Example 2 c

The reaction was carried out again as described in Example 2b. This time, instead of TSTU, 16.4 mg of O- (1H benzotriazol-1-yl-N, N, N ^' , N ^' tetramethyluronium hexafluorophosphate (HBTU, Fluka) was used as the crosslinker

Only 45% of the BSA was approved using this crosslinker.

Example 3a: Inclusion of tetracycline

Instead of the model drug BSA, 20 mg of tetracycline (Sigma) were used this time and processed according to Example 2a. The release of the tetracycline was measured according to the method known from the literature using UV spectroscopy at 356 nm. Without crosslinking, the release of the tetracycline was 70% after 4 hours. If, as mentioned in Example 2a, the particles were crosslinked with 200 μl of a 40% glyoxal solution, the release of tetracycline was only 10%

Example 3b

Instead of glyoxal, 13 mg of TSTU and again 8 μl of N-ethyldiisopropylamine were used as crosslinkers. The release of the tetracycline was in this

Trap only 34%.

Claims

33 Patent claims

1. Nanoparticles comprising a biocompatible, biodegradable polyelectrolyte complex composed of at least one polycation and at least one polyanion and at least one bioactive agent, the nanoparticles being obtainable by treating the polyelectrolyte complex additionally with at least one crosslinking agent during or after its formation.

2. Nanoparticles according to claim 1, characterized in that the particles have an average size of 50 to 250 nm.

3. Nanoparticles according to at least one of the preceding claims, characterized in that the crosslinking agent glyoxal, TSTU or

EDAP is.

4. Nanoparticles, according to at least one of the preceding claims, characterized in that the polycation is chitosan.

5. Nanoparticles, according to at least one of the preceding claims, characterized in that the polyanion is xylan polysulfate

6. Nanoparticles according to at least one of the preceding claims, wherein the nanoparticles are obtainable in that at least one of the two charged polymers of the polyelectrolyte complex is additionally treated with at least one agent which modifies the surface of the nanoparticles before, during or after the formation of the complex.

7. Nanoparticle according to claim 6, characterized in that the surface-modifying substance contains a carrier. 34

8. A process for the production of particles according to one of the preceding claims, wherein an active ingredient in bound or unbound form, an aqueous solution of at least one polycation and an aqueous solution of at least one polyanion are brought together and then the polyelectrolyte is formed in nanoparticulate form or, if appropriate, in a nanoparticulate form Form is converted, characterized in that the nanoparticulate polyelectrolyte complex is treated with a crosslinking agent.

9. Use of the particles according to claims 1 to 8 or obtained according to claim 9 as an oral application form.