CA2097801C - Method of producing collagen particles, and the use of such particles as substrates for active substances - Google Patents
Method of producing collagen particles, and the use of such particles as substrates for active substances Download PDFInfo
- Publication number
- CA2097801C CA2097801C CA002097801A CA2097801A CA2097801C CA 2097801 C CA2097801 C CA 2097801C CA 002097801 A CA002097801 A CA 002097801A CA 2097801 A CA2097801 A CA 2097801A CA 2097801 C CA2097801 C CA 2097801C
- Authority
- CA
- Canada
- Prior art keywords
- collagen
- microparticles
- process according
- active substance
- collagen microparticles
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5169—Proteins, e.g. albumin, gelatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1658—Proteins, e.g. albumin, gelatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
Landscapes
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Optics & Photonics (AREA)
- Biomedical Technology (AREA)
- Nanotechnology (AREA)
- Physics & Mathematics (AREA)
- Peptides Or Proteins (AREA)
- Cosmetics (AREA)
- Medicinal Preparation (AREA)
- Manufacturing Of Micro-Capsules (AREA)
- Processes Of Treating Macromolecular Substances (AREA)
- Materials For Medical Uses (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention concerns a process for the production of cross-linked collagen particles whose size lies in the micro and nanometer range, in which a solution or disper-sion of collagen in water is distributed finely in discrete droplets in a waterimmiscible organic phase, whereby emul-sifying agents are added, a water-in-oil emulsion thereby being formed, the collagen being subsequently cross-linked in the interface of the droplets, using a cross-linking agent, and the collagen particles produced in this manner being purified and isolated by separating the organic phase and washing.
Description
~o~~$o~
Process for the roduction of collaaen barticles and their Use as Carriers for ~rctive Substances DR$CRIPTIUN
The invention concerns a process for the production of collagen particles whose size lies in the micro and manome-ter range, and their use as carriers for active substances in medicine, the food processing industry and cosmetics.
The inclusion of a great many active substances and other components such as dyestuffs, catalysts, enzymes, radioac-tive material, flavours, volatile compounds and the like in just as many kinds of casing materials in capsules of a size lying in the micro and manometer range is well-known.
In the pharmaceutical sphere distilled oils in gelatine cases, for example, are used as aroma carriers, which only release the aroma when the case has dissolved.
The purpose of the encapsulation is - in addition to effec-ting the release of the contents after the case has dis-solved - for example the protection of active substances against oxygen, moisture or other. chemical agents; the transformation of a fluid into a "dry" powder; the produc-tion of depot forms of active substances by means of the appropriate treatment or the choice of relevant casing material; or the production of complexes of active substan-ces and adjuvants by loading the surface of the carrier particles with active substances.
Particle sizes in the manometer range offer, in addition to this, the possibility - as a result of the extremely small dimensions of the capsules - of dissolving the latter colloidally in water together with the enclosed or absorbed active material. It is thus possible to administer such solutions intravenously as well.
_ 2 _ Collagen is the main component of the connective tissue in vertebrates and is almost ubiquitous.
Particulate carriers of drugs made of collagen possess the advantage, compared with other carrier systems, that. owing to the biocompatibility and the complete biodegradation and metabolic conversion of the particles, toxicological problems are not to be expected when they are applied locally and systemically.
The use of collagen as a capsule encasing material offers the additional advantage that, in the case of substances possessing a high protein affinity, which bind very well with protein particles, a high loading of the carrier system is possible.
The capacity of the carrier system for adsorbent loading is, in these cases, a great deal higher than that of, fox example, carrier particles on an acrylate basis, taking the same particle surface area into account.
Processes for the production of collagen microparticles are described, for example, in gS Patent Specification 4 565 580 and in TJS Patent Specification 4 107 288. The expres-sion ''microparticle'° is used in the sense that hereby particle sizes in the micrometer range and smaller are described.
A common feature of the processes described there is that , the microparticles are produced according to the principle of coacervation.
In this process, desolvating agents, such as, for example, alcohols or electrolytes, are added to a molecular disper-sed solution of collagens. The solubility of the protein is reduced by means of the admixture, which results in a cloudiness of the solution, indicating the formation of light-scattering protein aggregates.
~~~~80~
- 3 _ Tf one separates the coacervate and dries it, in the end one obtains microcapsules, which can be hardened by a subsequent treatment. The particle diameters are in the range of 10 - 1000 ~tm (OS Patent Specification 4 565 580) and 10 - 1000 nm (US Patent Specification 4 107 288), respectively.
In US Patent Specification 4 837 285 a process is described in which an aqueous collagen dispersion is forced through a vibrating, hollow tube whose diameter is defined. Droplets form at the end of the tube, which drip into a cooling bath comprised of liquid nitrogen. After the nitrogen has evaporated, the frozen droplets are dry frozen and then cross-linked in an aqueous dispersion, so that they are aftercured. The particle diameter is between 100 and 400 N.m .
The above-mentioned collagen microparticle size distri-butions according to processes of the prier art already show the serious disadvantages. Por one thing, the par-ticle size distribution is spread over a very wide range.
Homogeneous particle fractions with a very narrow particle distribution cannot be obtained according to this process, as the agglomeration of the collagen molecules and the particle agglomeration happen at random and can scarcely be controlled.
Defined collagen particle fractions with a diameter in the low micrometer and in the manometer range cannot be ob-tained, according to this process, in particular. so that the microparticles produced according to this process are unsuitable for preparations for intravenous injection.
The present invention takes as its basis the task of fin-ding a process for the production of homogeneous callagen particles in the micrometer and manometer range, with a narrow particle size distribution to be controlled by the process.
The task is solved surprisingly by a process in which a solution or dispersion of collagen in water is distributed finely in discrete droplets in a waterimmiscible organic phase, whereby emulsifying agents are added, a water-in-oil emulsion thereby being formed, t:he collagen being subse-quently cross-linked in the interface of the droplets, using a cross-linking agent, and the collagen particles produced in this manner being purified and isolated by separating the organic phase and washing.
To produce the particles, an emulsion is prepared in a manner known to a person skilled in the art, from an a-queous collagen dispersion, which can contain native col-lagen or collagen which has been denatured by means of chemical agents or physical influences from sources of various origins known to the man of the art, from an emul-sifying agent and from an organic phase. The determination of the emulsion type can take place by means of conduc-timetry.
Emulsifying agents which can be employed to advantage in the processes according to the inventions are polyethylene-glycol fatty acid esters, such as, for example. polyethy-leneglycol-400-stearate: polyethyleneglycol fatty alcohol ethers, such as, for example, polythyleneglycol-200-lauryl ether; polyethyleneglycol sorbitane fatty acid esters, such as, for example, polyethyleneglycol-~~0)-sorbitane mono-oleate; partial fatty acids of polyhydric alcohols, such as, for example, glycerol monostearate or sorbitane tri-oleate; or partial fatty acid esters of sugars, such as, for example, saccharose monolauryl acid ester.
The waterimmiscible organic phase can contain natural, halfsynthetical or synthetical liquid fats, oils or waxes, such as, for example, olive oil, castor oil, cotton seed oil, soya bean oil, hydrated peanut oil, triglyceride mixtures (Miglyol~, Softisan~), silicone oil, oleic acid oleyl ester, isopropyl myristate or ethyl oleate.
The production of the emulsion is the decisive step influencing the size and homogeneity of the microparticles which ultimately result. The size of the dispersed water droplets in the organic phase is determined and controlled by both the kind of emulsifying agent or emulsifying complex and the kind and intensity of the emulsifying process.
The collagen molecules are then polymerized by adding a cross-linking agent in the interface of the droplets.
Mono- or bifunctional aldehydes such as, for example, formaldehyde, glutaric dialdehyde or dialdehyde starch; or bifunctional isocyanates, such as, for example, hexa-methylene diisocyanate, can be used as cross-linking agents.
The organic phase is subsequently separated and the par-ticles purified by washing, i.e. the remainders of the organic phase, the emulsifying agent and the cross-linking agent are got rid of.
It may be necessary to redisperse the collagen micropar-ticles in the ultrasonic bath, to break up the aggregates.
The determination of the particle size and the size distri-bution take place after freeze drying and redispersion by means of photon correlation spectrometry. At the same time, a laser beam is sent at 20 impulses per second through a vessel filled with a dilute, colloidal solution of collagen particles. The laser light, being scattered by the particles, is registered via a photoelectric cell at an angle of 90° to the irradiating laser.
The scattering is dependent on the size of the particles;
by conversion of the electric signal obtained, it is pos-sible to infer the particle size by means of a mathematical equation.
Because of the Brownian movement, at every impuls another "particle population'° is hit. In this manner the infor-mation gained from the large number of impulses (r 2500) provides an exact picture of the average size and size distribution of the particles.
Active substances can be added to the emulsion prior to the cross-linking of the particles.
The active substances axe enclosed in the particles during production of the particles by interface polymerisation, or are adsorbed at the surface of the particles after particle formation.
If the loading with active substances is to take place after the production of the particles, the collagen par-ticles are, after their production, separation and purifi-cation, subjected to any desired drying process known to the man skilled in the art, preferably using the freeze drying process.
At a high temperature reduction rate the particles are shock-freezed in a freeze-dryer to temperatures <_ -50°C, so as to form ice crystalls which are as small as possible, and are subsequently freeze-dried under vacuum.
The drying process increases the stability of the particles during storage until further processing.
For subsequent loading of the dried particles with active substance, the particles are first redispersed in water;
then the active substance. an active substance solution or active substance dispersion is added to the dispersion.
The particles, loaded with active substance through ad-sorption, are subsequently separated and purified.
According to the process of the present invention, it is possibl~, by means of emulsion polymerisation, to produce collagen particles with very small diametres which can be used in preparations for intravenous injections or for application on the eye; the comparatively simple process allows accurate process control and thus improved control of the production of homogenous amounts of particles with the respective, desired narrow size distribution and at high yields.
The invention is illustrated by means of the following examples:
Example 1:
Production of collagen microparticles from a water-in-oil emulsion:
By means of a pestle, 15 g of sorbitane monolaureate are homogenously incorporated into 100 g of cotton seed oil, which has been put into a mortar. After 10 minutes, 25 g of a 1 p. c. dispersion of native calf skin collagen is added in five portions and is carefully incorporated. The emulsion is homogenised for 3 x 10 minutes in the ultraso-nic bath, while being cooled.
In a further process step the emulsion is treated with a high-speed homoginizing mixer for 3 x 15 seconds, with intermediate cooling. The emulsion is stirred at 400 rpm onto a magnetic stirrer; to effect the cross-linking of the _ g _ particles, 5.0 m1 of glutaric aldehyde solution (25°'°) are added. After 10 minutes, the reaction is stopped by adding 7.0 ml of Na-disulfite solution (25°~), and the mixture is thereafter worked up.
An excess of ether is added three times to the preparation and the latter is centrifuged for 10 minutes each time, at 2500 rpm; this process is repeated twice with an excess of water. The particles are either freeze-dried or are im-mediately processed further.
In the present example, collagen particles with an average diametre of 2.15 ~tm were obtained; 90 °~ of the particles were smaller than 9,2 Nm.
The collagen particle yield was ca. 95%, relative to the amount of pure collagen used.
Example 2:
The experiment was carried out as described in example 1, whereby instead of a 1 p, c. dispersion of native calf skin collagen, a 1 p. c. dispersion of denatured calf skin collagen was used.
The average particle diametre obtained was 320 nm.
Yield: 90 %.
Example 3:
Production of collagen microcapsules from a water-in-oil emulsion g of polyoxyethylene-(20)-sorbitane monooleate are dispersed in 25 m1 of a 0.75 p. c. dispersion of native calf skin collagen. After 10 minutes 100 g of cotton seed oil are added in five portions and stirred. Each time. the ~:8'~3 _ g _ emulsion is subsecguently worked for a period of 5 minutes with the high-speed homogenizing mixer and the slit homoge-nizer.
1.0 g of glutaric aldehyde solution (25%) is then added and the dispersion is stirred for 10 minutes. To recover the excess of glutaric aldehyde, 1.4 ml of Na-disulfite solution (25%) are added. The removal of the organic phase is carried out as described in example 1. Yield: 92 °%.
The particles obtained exhibited an average diametre of 1.23 Vim. 90 % of the particles were smaller than 2.2 ~tm.
Exam~l~ 4:
Loading of the collagen particles with ethacridine lactate 50 mg of particles are dispersed in 50 ml of water, in the ultrasonic bath. 25 mg of ethacridine lactate are added to the particle dispersion and stirred for 24 hours at 200 rpm.
To determine the load, the suspension is ultrafiltrated;
the content of medicinal agent in the supernatant is deter-mined, and subsequently the particle load is calculated.
The particle load is 12 mg on 50 mg of collagen particles.
The absolute load is thus 24 °~-wt, relative to the medi-cinal agent used.
Example 5:
Loading of the collagen particles with chlorophyll 250 mg of collagen particles are dispersed in 22,5 ml of physiological NaCl solution. The dispersion is exposed to sonic waves for 10 minutes. Thereafter, 2.5 ml of satu-rated ethanolic chlorophyll solution are added and stirred for about 2~4 hours at 300 rpm. The particles are purified in a GPC column, which is filled with Sephadex~ G-50 (arose-linked polysaccharide).
A second band does not appear, which indicates that the loading was carried out quantitatively. The particles obtained are freeze-dried.
Process for the roduction of collaaen barticles and their Use as Carriers for ~rctive Substances DR$CRIPTIUN
The invention concerns a process for the production of collagen particles whose size lies in the micro and manome-ter range, and their use as carriers for active substances in medicine, the food processing industry and cosmetics.
The inclusion of a great many active substances and other components such as dyestuffs, catalysts, enzymes, radioac-tive material, flavours, volatile compounds and the like in just as many kinds of casing materials in capsules of a size lying in the micro and manometer range is well-known.
In the pharmaceutical sphere distilled oils in gelatine cases, for example, are used as aroma carriers, which only release the aroma when the case has dissolved.
The purpose of the encapsulation is - in addition to effec-ting the release of the contents after the case has dis-solved - for example the protection of active substances against oxygen, moisture or other. chemical agents; the transformation of a fluid into a "dry" powder; the produc-tion of depot forms of active substances by means of the appropriate treatment or the choice of relevant casing material; or the production of complexes of active substan-ces and adjuvants by loading the surface of the carrier particles with active substances.
Particle sizes in the manometer range offer, in addition to this, the possibility - as a result of the extremely small dimensions of the capsules - of dissolving the latter colloidally in water together with the enclosed or absorbed active material. It is thus possible to administer such solutions intravenously as well.
_ 2 _ Collagen is the main component of the connective tissue in vertebrates and is almost ubiquitous.
Particulate carriers of drugs made of collagen possess the advantage, compared with other carrier systems, that. owing to the biocompatibility and the complete biodegradation and metabolic conversion of the particles, toxicological problems are not to be expected when they are applied locally and systemically.
The use of collagen as a capsule encasing material offers the additional advantage that, in the case of substances possessing a high protein affinity, which bind very well with protein particles, a high loading of the carrier system is possible.
The capacity of the carrier system for adsorbent loading is, in these cases, a great deal higher than that of, fox example, carrier particles on an acrylate basis, taking the same particle surface area into account.
Processes for the production of collagen microparticles are described, for example, in gS Patent Specification 4 565 580 and in TJS Patent Specification 4 107 288. The expres-sion ''microparticle'° is used in the sense that hereby particle sizes in the micrometer range and smaller are described.
A common feature of the processes described there is that , the microparticles are produced according to the principle of coacervation.
In this process, desolvating agents, such as, for example, alcohols or electrolytes, are added to a molecular disper-sed solution of collagens. The solubility of the protein is reduced by means of the admixture, which results in a cloudiness of the solution, indicating the formation of light-scattering protein aggregates.
~~~~80~
- 3 _ Tf one separates the coacervate and dries it, in the end one obtains microcapsules, which can be hardened by a subsequent treatment. The particle diameters are in the range of 10 - 1000 ~tm (OS Patent Specification 4 565 580) and 10 - 1000 nm (US Patent Specification 4 107 288), respectively.
In US Patent Specification 4 837 285 a process is described in which an aqueous collagen dispersion is forced through a vibrating, hollow tube whose diameter is defined. Droplets form at the end of the tube, which drip into a cooling bath comprised of liquid nitrogen. After the nitrogen has evaporated, the frozen droplets are dry frozen and then cross-linked in an aqueous dispersion, so that they are aftercured. The particle diameter is between 100 and 400 N.m .
The above-mentioned collagen microparticle size distri-butions according to processes of the prier art already show the serious disadvantages. Por one thing, the par-ticle size distribution is spread over a very wide range.
Homogeneous particle fractions with a very narrow particle distribution cannot be obtained according to this process, as the agglomeration of the collagen molecules and the particle agglomeration happen at random and can scarcely be controlled.
Defined collagen particle fractions with a diameter in the low micrometer and in the manometer range cannot be ob-tained, according to this process, in particular. so that the microparticles produced according to this process are unsuitable for preparations for intravenous injection.
The present invention takes as its basis the task of fin-ding a process for the production of homogeneous callagen particles in the micrometer and manometer range, with a narrow particle size distribution to be controlled by the process.
The task is solved surprisingly by a process in which a solution or dispersion of collagen in water is distributed finely in discrete droplets in a waterimmiscible organic phase, whereby emulsifying agents are added, a water-in-oil emulsion thereby being formed, t:he collagen being subse-quently cross-linked in the interface of the droplets, using a cross-linking agent, and the collagen particles produced in this manner being purified and isolated by separating the organic phase and washing.
To produce the particles, an emulsion is prepared in a manner known to a person skilled in the art, from an a-queous collagen dispersion, which can contain native col-lagen or collagen which has been denatured by means of chemical agents or physical influences from sources of various origins known to the man of the art, from an emul-sifying agent and from an organic phase. The determination of the emulsion type can take place by means of conduc-timetry.
Emulsifying agents which can be employed to advantage in the processes according to the inventions are polyethylene-glycol fatty acid esters, such as, for example. polyethy-leneglycol-400-stearate: polyethyleneglycol fatty alcohol ethers, such as, for example, polythyleneglycol-200-lauryl ether; polyethyleneglycol sorbitane fatty acid esters, such as, for example, polyethyleneglycol-~~0)-sorbitane mono-oleate; partial fatty acids of polyhydric alcohols, such as, for example, glycerol monostearate or sorbitane tri-oleate; or partial fatty acid esters of sugars, such as, for example, saccharose monolauryl acid ester.
The waterimmiscible organic phase can contain natural, halfsynthetical or synthetical liquid fats, oils or waxes, such as, for example, olive oil, castor oil, cotton seed oil, soya bean oil, hydrated peanut oil, triglyceride mixtures (Miglyol~, Softisan~), silicone oil, oleic acid oleyl ester, isopropyl myristate or ethyl oleate.
The production of the emulsion is the decisive step influencing the size and homogeneity of the microparticles which ultimately result. The size of the dispersed water droplets in the organic phase is determined and controlled by both the kind of emulsifying agent or emulsifying complex and the kind and intensity of the emulsifying process.
The collagen molecules are then polymerized by adding a cross-linking agent in the interface of the droplets.
Mono- or bifunctional aldehydes such as, for example, formaldehyde, glutaric dialdehyde or dialdehyde starch; or bifunctional isocyanates, such as, for example, hexa-methylene diisocyanate, can be used as cross-linking agents.
The organic phase is subsequently separated and the par-ticles purified by washing, i.e. the remainders of the organic phase, the emulsifying agent and the cross-linking agent are got rid of.
It may be necessary to redisperse the collagen micropar-ticles in the ultrasonic bath, to break up the aggregates.
The determination of the particle size and the size distri-bution take place after freeze drying and redispersion by means of photon correlation spectrometry. At the same time, a laser beam is sent at 20 impulses per second through a vessel filled with a dilute, colloidal solution of collagen particles. The laser light, being scattered by the particles, is registered via a photoelectric cell at an angle of 90° to the irradiating laser.
The scattering is dependent on the size of the particles;
by conversion of the electric signal obtained, it is pos-sible to infer the particle size by means of a mathematical equation.
Because of the Brownian movement, at every impuls another "particle population'° is hit. In this manner the infor-mation gained from the large number of impulses (r 2500) provides an exact picture of the average size and size distribution of the particles.
Active substances can be added to the emulsion prior to the cross-linking of the particles.
The active substances axe enclosed in the particles during production of the particles by interface polymerisation, or are adsorbed at the surface of the particles after particle formation.
If the loading with active substances is to take place after the production of the particles, the collagen par-ticles are, after their production, separation and purifi-cation, subjected to any desired drying process known to the man skilled in the art, preferably using the freeze drying process.
At a high temperature reduction rate the particles are shock-freezed in a freeze-dryer to temperatures <_ -50°C, so as to form ice crystalls which are as small as possible, and are subsequently freeze-dried under vacuum.
The drying process increases the stability of the particles during storage until further processing.
For subsequent loading of the dried particles with active substance, the particles are first redispersed in water;
then the active substance. an active substance solution or active substance dispersion is added to the dispersion.
The particles, loaded with active substance through ad-sorption, are subsequently separated and purified.
According to the process of the present invention, it is possibl~, by means of emulsion polymerisation, to produce collagen particles with very small diametres which can be used in preparations for intravenous injections or for application on the eye; the comparatively simple process allows accurate process control and thus improved control of the production of homogenous amounts of particles with the respective, desired narrow size distribution and at high yields.
The invention is illustrated by means of the following examples:
Example 1:
Production of collagen microparticles from a water-in-oil emulsion:
By means of a pestle, 15 g of sorbitane monolaureate are homogenously incorporated into 100 g of cotton seed oil, which has been put into a mortar. After 10 minutes, 25 g of a 1 p. c. dispersion of native calf skin collagen is added in five portions and is carefully incorporated. The emulsion is homogenised for 3 x 10 minutes in the ultraso-nic bath, while being cooled.
In a further process step the emulsion is treated with a high-speed homoginizing mixer for 3 x 15 seconds, with intermediate cooling. The emulsion is stirred at 400 rpm onto a magnetic stirrer; to effect the cross-linking of the _ g _ particles, 5.0 m1 of glutaric aldehyde solution (25°'°) are added. After 10 minutes, the reaction is stopped by adding 7.0 ml of Na-disulfite solution (25°~), and the mixture is thereafter worked up.
An excess of ether is added three times to the preparation and the latter is centrifuged for 10 minutes each time, at 2500 rpm; this process is repeated twice with an excess of water. The particles are either freeze-dried or are im-mediately processed further.
In the present example, collagen particles with an average diametre of 2.15 ~tm were obtained; 90 °~ of the particles were smaller than 9,2 Nm.
The collagen particle yield was ca. 95%, relative to the amount of pure collagen used.
Example 2:
The experiment was carried out as described in example 1, whereby instead of a 1 p, c. dispersion of native calf skin collagen, a 1 p. c. dispersion of denatured calf skin collagen was used.
The average particle diametre obtained was 320 nm.
Yield: 90 %.
Example 3:
Production of collagen microcapsules from a water-in-oil emulsion g of polyoxyethylene-(20)-sorbitane monooleate are dispersed in 25 m1 of a 0.75 p. c. dispersion of native calf skin collagen. After 10 minutes 100 g of cotton seed oil are added in five portions and stirred. Each time. the ~:8'~3 _ g _ emulsion is subsecguently worked for a period of 5 minutes with the high-speed homogenizing mixer and the slit homoge-nizer.
1.0 g of glutaric aldehyde solution (25%) is then added and the dispersion is stirred for 10 minutes. To recover the excess of glutaric aldehyde, 1.4 ml of Na-disulfite solution (25%) are added. The removal of the organic phase is carried out as described in example 1. Yield: 92 °%.
The particles obtained exhibited an average diametre of 1.23 Vim. 90 % of the particles were smaller than 2.2 ~tm.
Exam~l~ 4:
Loading of the collagen particles with ethacridine lactate 50 mg of particles are dispersed in 50 ml of water, in the ultrasonic bath. 25 mg of ethacridine lactate are added to the particle dispersion and stirred for 24 hours at 200 rpm.
To determine the load, the suspension is ultrafiltrated;
the content of medicinal agent in the supernatant is deter-mined, and subsequently the particle load is calculated.
The particle load is 12 mg on 50 mg of collagen particles.
The absolute load is thus 24 °~-wt, relative to the medi-cinal agent used.
Example 5:
Loading of the collagen particles with chlorophyll 250 mg of collagen particles are dispersed in 22,5 ml of physiological NaCl solution. The dispersion is exposed to sonic waves for 10 minutes. Thereafter, 2.5 ml of satu-rated ethanolic chlorophyll solution are added and stirred for about 2~4 hours at 300 rpm. The particles are purified in a GPC column, which is filled with Sephadex~ G-50 (arose-linked polysaccharide).
A second band does not appear, which indicates that the loading was carried out quantitatively. The particles obtained are freeze-dried.
Claims (12)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for producing cross-linked collagen microparticles having a size in the micrometer or nanometer range, which process comprises:
finely distributing discrete droplets of a dispersion or solution of collagen in water into a water-immiscible organic phase together with an emulsifying agent, thereby forming a water-in-oil emulsion;
adding a cross-linking agent to the water-in-oil emulsion, thereby cross-linking the collagen at an interface of the droplets;
isolating the so-prepared collagen microparticles from the organic phase and washing the collagen microparticles, thereby purifying the collagen microparticles; and loading the collagen microparticles with an active substance for medicine or cosmetics or for use in food processing, either while preparing the collagen microparticles or after the purification of the collagen microparticles.
finely distributing discrete droplets of a dispersion or solution of collagen in water into a water-immiscible organic phase together with an emulsifying agent, thereby forming a water-in-oil emulsion;
adding a cross-linking agent to the water-in-oil emulsion, thereby cross-linking the collagen at an interface of the droplets;
isolating the so-prepared collagen microparticles from the organic phase and washing the collagen microparticles, thereby purifying the collagen microparticles; and loading the collagen microparticles with an active substance for medicine or cosmetics or for use in food processing, either while preparing the collagen microparticles or after the purification of the collagen microparticles.
2. The process according to claim 1, wherein the active substance is added to the emulsion during preparation of the collagen microparticles.
3. The process according to claim 1, wherein the active substance is loaded to the collagen microparticles after the purification of the prepared collagen microparticles.
4. The process according to claim 3, wherein the step of loading the collagen microparticles with the active substance comprises:
shock-freezing the collagen microparticles at a high temperature reduction rate (gradient) to a temperature of <= -50°C, thereby forming small ice crystals;
freeze-drying the so-frozen collagen microparticles under vacuum;
re-dispersing the freeze-dried collagen microparticles in water, thereby forming a dispersion of the collagen microparticles in water;
adding the active substance or a solution or dispersion of the active substance to the dispersion of the collagen microparticles, thereby loading the collagen microparticles with the active substance by adsorption; and separating the collagen microparticles loaded with the active substance from the dispersion and purifying the collagen microparticles.
shock-freezing the collagen microparticles at a high temperature reduction rate (gradient) to a temperature of <= -50°C, thereby forming small ice crystals;
freeze-drying the so-frozen collagen microparticles under vacuum;
re-dispersing the freeze-dried collagen microparticles in water, thereby forming a dispersion of the collagen microparticles in water;
adding the active substance or a solution or dispersion of the active substance to the dispersion of the collagen microparticles, thereby loading the collagen microparticles with the active substance by adsorption; and separating the collagen microparticles loaded with the active substance from the dispersion and purifying the collagen microparticles.
5. The process according to any one of claims 1 to 4, wherein the collagen is native collagen.
6. The process according to any one of claims 1 to 4, wherein the collagen is denatured collagen.
7. The process according to any one of claims 1 to 6, wherein the emulsifying agent is selected from the group consisting of polyethyleneglycol fatty acid esters, polyethyleneglycol fatty alcohol ethers, polyethyleneglycol-sorbitane-fatty acid esters, partial fatty acid esters of polyhydric alcohols and partial fatty acid esters of sugar.
8. The process according to any one of claims 1 to 7, wherein the cross-linking agent is mono- or bifunctional aldehyde or bifunctional isocyanate.
9. The process according to claim 8, wherein the cross-linking agent is a bifunctional aldehyde.
10. The process according to any one of one of claims 1 to 9, wherein the water-immiscible organic phase is a natural, halfsynthetic or synthetic liquid fat, oil or wax.
11. The process according to any one of claims 1 to 10, wherein the active substance is medicine suitable for intravenous injection.
12. A use of the collagen microparticles according to claim 11 for preparing intravenous injection.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4038887.5 | 1990-12-06 | ||
| DE4038887A DE4038887A1 (en) | 1990-12-06 | 1990-12-06 | METHOD FOR THE PRODUCTION OF COLLAGEN PARTICLES AND THEIR USE AS AN ACTIVE SUBSTANCE |
| PCT/EP1991/002195 WO1992010287A1 (en) | 1990-12-06 | 1991-11-21 | Method of producing collagen particles, and the use of such particles as substrates for active substances |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2097801A1 CA2097801A1 (en) | 1992-06-07 |
| CA2097801C true CA2097801C (en) | 2002-01-22 |
Family
ID=6419700
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002097801A Expired - Lifetime CA2097801C (en) | 1990-12-06 | 1991-11-21 | Method of producing collagen particles, and the use of such particles as substrates for active substances |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0561821B1 (en) |
| JP (1) | JP3233631B2 (en) |
| AT (1) | ATE116158T1 (en) |
| CA (1) | CA2097801C (en) |
| DE (2) | DE4038887A1 (en) |
| DK (1) | DK0561821T3 (en) |
| ES (1) | ES2069316T3 (en) |
| GR (1) | GR3015518T3 (en) |
| WO (1) | WO1992010287A1 (en) |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4414755C2 (en) * | 1994-04-27 | 2000-11-16 | Lohmann Therapie Syst Lts | Collagen preparation for the controlled delivery of active ingredients, processes and use |
| DE19624332B4 (en) * | 1996-06-19 | 2009-12-24 | Namos Gmbh | Metallic nanostructure based on highly ordered proteins and methods for their production |
| DE19739031A1 (en) * | 1997-09-05 | 1999-03-11 | Suwelack Nachf Dr Otto | Oral administration agent, its preparation and use |
| DE19839515B4 (en) * | 1998-08-29 | 2012-02-02 | Nanohale Gmbh | New pharmaceutical preparation containing colloidal polymer-drug associations, especially for mucosal drug administration |
| DE19934894A1 (en) * | 1999-07-24 | 2001-02-01 | Pharma Concepts Gmbh & Co Kg | Collagen particles for use as drug carriers, e.g. for administration of proteins or peptides by injection, prepared from emulsion containing collagen solution or dispersion without using crosslinking agents |
| DE10010113B4 (en) * | 2000-03-03 | 2009-05-07 | Wolfgang Dr. Schatton | Native sponge collagen, process for its isolation and its use, as well as native nanoparticulate sponge collagen, process for its preparation and its use |
| WO2002060486A2 (en) * | 2001-01-29 | 2002-08-08 | Fresco Bernard B | Controlled release microsphere system for administration of drug to the eye |
| EP1951762B1 (en) * | 2005-10-03 | 2018-02-07 | PINSKY, Mark A. | Compositions and methods for improved skin care |
| US20080175807A1 (en) | 2007-01-24 | 2008-07-24 | Fujifilm Corporation | Composition for hair |
| JP2008201767A (en) * | 2007-01-24 | 2008-09-04 | Fujifilm Corp | Composition for hair |
| JP2008255020A (en) * | 2007-04-02 | 2008-10-23 | Fujifilm Corp | Antiaging skin care preparation |
| DE102008036576A1 (en) * | 2008-07-31 | 2010-02-04 | Gelita Ag | Particles of collagen material and method of manufacture |
| CA2904143A1 (en) * | 2013-03-12 | 2014-10-09 | Cephalon, Inc. | Nanoparticulate and macroparticulate formulations |
| CN115350329A (en) * | 2022-08-19 | 2022-11-18 | 江苏西宏生物医药有限公司 | Long-acting particle type I and type III collagen composite implant |
| CN115350328A (en) * | 2022-08-19 | 2022-11-18 | 江苏西宏生物医药有限公司 | Long-acting particle III type collagen implant |
| CN115252898B (en) * | 2022-08-19 | 2023-08-18 | 江苏西宏生物医药有限公司 | Long-acting microparticle type I and type V collagen composite implant |
| CN115317668B (en) * | 2022-08-19 | 2023-12-29 | 北京西宏润美医药科技有限公司 | Long-acting microparticle type I collagen implant |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4798786A (en) * | 1982-05-06 | 1989-01-17 | Stolle Research And Development Corporation | Living cells encapsulated in crosslinked protein |
| JPS60174725A (en) * | 1984-02-22 | 1985-09-09 | Koken:Kk | Substrate containing regenerated collagen fibril and its preparation |
| SE459005B (en) * | 1985-07-12 | 1989-05-29 | Aake Rikard Lindahl | SET TO MANUFACTURE SPHERICAL POLYMER PARTICLES |
-
1990
- 1990-12-06 DE DE4038887A patent/DE4038887A1/en active Granted
-
1991
- 1991-11-21 AT AT91920426T patent/ATE116158T1/en not_active IP Right Cessation
- 1991-11-21 WO PCT/EP1991/002195 patent/WO1992010287A1/en active IP Right Grant
- 1991-11-21 ES ES91920426T patent/ES2069316T3/en not_active Expired - Lifetime
- 1991-11-21 EP EP91920426A patent/EP0561821B1/en not_active Expired - Lifetime
- 1991-11-21 JP JP51866791A patent/JP3233631B2/en not_active Expired - Lifetime
- 1991-11-21 CA CA002097801A patent/CA2097801C/en not_active Expired - Lifetime
- 1991-11-21 DK DK91920426.3T patent/DK0561821T3/en active
- 1991-11-21 DE DE59104102T patent/DE59104102D1/en not_active Expired - Lifetime
-
1995
- 1995-03-22 GR GR950400651T patent/GR3015518T3/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP0561821A1 (en) | 1993-09-29 |
| DK0561821T3 (en) | 1995-06-12 |
| ATE116158T1 (en) | 1995-01-15 |
| ES2069316T3 (en) | 1995-05-01 |
| JPH06503259A (en) | 1994-04-14 |
| JP3233631B2 (en) | 2001-11-26 |
| DE59104102D1 (en) | 1995-02-09 |
| GR3015518T3 (en) | 1995-06-30 |
| CA2097801A1 (en) | 1992-06-07 |
| DE4038887A1 (en) | 1992-07-02 |
| WO1992010287A1 (en) | 1992-06-25 |
| DE4038887C2 (en) | 1992-10-08 |
| EP0561821B1 (en) | 1994-12-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2097801C (en) | Method of producing collagen particles, and the use of such particles as substrates for active substances | |
| US5043161A (en) | Small, oily, free-flowing, silky-smooth, talc-like, dry microcapsules and aqueous formulations containing them | |
| RU2606854C1 (en) | Method of producing nanocapsules of dry spinach extract | |
| RU2590693C1 (en) | Method of producing nano capsules of adaptogens in pectin | |
| Chen et al. | Preparation of human serum albumin microspheres by a novel acetone-heat denaturation method | |
| RU2646482C2 (en) | Method for producing nanocapsules of metronidazole in carrageenan | |
| RU2555824C1 (en) | Method for production of microcapsules of dry girasol extract in pectin | |
| JP2003526503A (en) | Method for preparing colloidal particles in the form of nanocapsules | |
| Carvalho da Silva et al. | Methods of microencapsulation of vegetable oil: principles, stability and applications-A Minireview | |
| RU2631883C2 (en) | Method for production of nanocapules of penicillin group medicine preparations in konjac gum | |
| RU2613108C1 (en) | Production method of metronidazole nanocapsules in konjac gum | |
| RU2578403C2 (en) | Method of producing nanocapsules of cytokinins | |
| RU2605614C1 (en) | Method of producing nanocapsules of dry girasol extract | |
| RU2599007C1 (en) | Method of producing nanocapsules of ciprofloxacin hydrochloride in sodium alginate | |
| RU2609824C1 (en) | Method for obtaining nanocapsules of medications of penicillin goup in sodium alginate | |
| RU2547560C2 (en) | Method for producing drug preparations of penicillin in sodium alginate possessing supramolecular properties | |
| RU2634256C2 (en) | Method for producing nanocapules of dry extract of topinambur | |
| RU2555782C1 (en) | Method of producing glucosamine sulphate nanocapsules in konjac gum in hexane | |
| RU2557975C1 (en) | Method for producing glucosamine sulphate nanocapsules in carrageenan | |
| RU2555055C1 (en) | Method of obtaining nanocapsules of glucoamine sulphate in xanthan gum | |
| LU500375B1 (en) | Nano Microcapsule Made Of Modified Chitosan-Coated Macadamia Oil And Preparation Method And Application Thereof | |
| RU2554738C1 (en) | Method of producing chondroitin sulphate nanocapsules in carrageenan | |
| RU2573979C1 (en) | Method of production of nanocapsules of antibiotics in agar-agar | |
| RU2580613C1 (en) | Method of producing antibiotic nanocapsules in agar-agar | |
| RU2609742C1 (en) | Method for producing ciprofloxacin hydrochloride nanocapsules |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKEX | Expiry |