CN114099707B - Collagenase modified PLGA microsphere and preparation method thereof - Google Patents
Collagenase modified PLGA microsphere and preparation method thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- 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
- A61K9/1682—Processes
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- 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
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to improvement of drug delivery efficiency in pathological fibrosis tissues, in particular to a collagenase modified PLGA microsphere and a preparation method thereof. The invention aims to provide a new choice for the collagen barrier of the drug permeable fibrotic tissue. The technical scheme of the invention is a preparation method of collagenase modified PLGA microspheres, which comprises the following steps: co-dissolving the medicine micro powder and polylactic acid-glycolic acid copolymer (PLGA) to prepare microspheres; then modified with collagenase. Collagenase is fixed on the surface of PLGA microspheres, so that the PLGA microspheres loaded with drugs can pass through a collagen barrier, the penetration of the drugs loaded in the microspheres in tissues is improved, and the treatment efficiency is greatly improved.
Description
Technical Field
The invention relates to improvement of drug delivery efficiency in pathological fibrosis tissues, in particular to a collagenase modified PLGA microsphere and a preparation method thereof.
Background
The pathologic altered process of fibrosis is often accompanied by abnormal increase and excessive deposition of extracellular matrix within the tissue, not only increasing tissue stiffness, but also acting as a barrier to drug delivery. Breaking collagen barrier in tissue can help the medicine to penetrate in fibrosis tissue, raise bioavailability of medicine and raise treatment efficiency. Current strategies to overcome the collagen barrier are primarily based on injection or mechanical action of microneedles to provide a physical pathway for drug delivery. However, the drug entering the tissue is still affected by the surrounding collagen barrier, impeding its diffusion within the tissue.
Disclosure of Invention
The invention aims to provide a new choice for the collagen barrier of the drug permeable fibrotic tissue.
The technical scheme of the invention is a preparation method of collagenase modified PLGA microspheres, which comprises the following steps:
s1, co-dissolving medicine micro powder and polylactic acid-glycolic acid copolymer (PLGA) in an organic solvent to obtain a raw material solution; adding the raw material solution into a 2% PVA solution, and emulsifying to form a suspension; adding the suspension into 0.5% PVA solution, stirring at room temperature, completely evaporating the organic solvent, sieving, collecting the hardened microspheres, and thoroughly cleaning with deionized water; the organic solvent is dichloromethane and/or acetone
s2, adding Traut's reagent into collagenase solution with the concentration of 10mg/mL, and reacting for 1 h under stirring at room temperature;
s3, adding the microspheres obtained in the step s1 into the reaction solution in the step s2 to obtain PLGA microsphere suspension; reacting for 1-12 h at room temperature; and (3) centrifuging, washing with PBS solution for three times, dialyzing, and freeze-drying to obtain PLGA microspheres with collagenase immobilized on the surfaces.
In step s2, the collagenase is collagenase type I (collagenase I).
Further, in step s1, the volume of the 2% PVA solution is 5 to 10 times that of the raw material solution.
In the step s1, the drug micro powder accounts for 1-20% of the weight of the PLGA.
Specifically, in the step s1, the sieved screen mesh is 60-100 meshes.
Wherein, in step s2, the volume of Traut's reagent is 5 times that of collagenase solution.
Further, in step s3, the concentration of the PLGA microsphere suspension is 1-10 mg/mL.
Wherein, the PLGA microsphere obtained in the step s3 is preserved at the temperature of minus 20 ℃.
Specifically, the drug in step s1 is triamcinolone acetonide (Triamcinolone acetonide, TA), 5-fluorouracil, doxorubicin or botulinum toxin.
The invention also provides the collagenase modified PLGA microsphere loaded with the drug, which is prepared by the method.
The invention has the beneficial effects that: collagenase is specific hydrolase of collagen, which can effectively reduce collagen deposition in tissues, and reconstruct the tissue and mechanical environment of extracellular matrix. Therefore, based on the thought of immobilized enzyme, collagenase is immobilized on the surface of the PLGA microsphere, so that the PLGA microsphere loaded with the drug can pass through the collagen barrier, the permeation of the drug loaded in the microsphere in tissues is improved, and the treatment efficiency is greatly improved. The invention utilizes the degradation effect of collagenase on collagen, so that the barrier of the collagen barrier in the tissue is broken more forcefully and less, and more space is provided for the diffusion of the medicine in the tissue.
Drawings
FIG. 1 is a schematic diagram of the structure of a collagenase modified PLGA microsphere of type I; wherein PLGA represents polylactic acid-glycolic acid copolymer, collageenase I represents type I collagenase, triamcinolone acetonide, TA represents triamcinolone acetonide.
FIG. 2 preparation route of collagenase modified PLGA microspheres, type I.
FIG. 3 diffusion behavior of non-collagenase modified PLGA microspheres (left) and collagenase modified PLGA microspheres (right) in three-dimensional collagen gels.
Detailed Description
EXAMPLE 1 preparation of triamcinolone acetonide-loaded collagenase modified PLGA microspheres
The preparation flow is shown in figure 2:
triamcinolone Acetonide (TA) drug micropowder is co-dissolved with polylactic-glycolic acid (PLGA) (TA/PLGA, 5% w/w) in methylene chloride and completely dissolved under ultrasound. Next, the mixture was added to 5 volumes of 2% PVA solution and emulsified under ultrasound for 1 minute to form an o/w suspension. The o/w emulsion was quickly transferred to 10 volumes of 0.5% PVA solution, the trichloroethylene was evaporated to completion with stirring at room temperature, the hardened microspheres were collected with a screen (100 mesh) and thoroughly washed with deionized water.
The collagenase was thiolated by the Traut's reaction: 5 times Traut's reagent was added to 10mg/mL collagenase PBS solution and reacted at room temperature with stirring for 1 h.
PLGA microsphere suspension was then added at a final concentration of 1mg/mL and reacted at room temperature for 12h. Centrifugation, PBS washing three times, dialysis, and freeze-drying to obtain PLGA microsphere with collagenase immobilized on the surface (its structure schematic diagram is shown in figure 1). The microspheres were lyophilized and stored at-20 ℃ prior to use.
Example 2 microsphere diffusion experiments in three-dimensional collagen
Referring to the above experimental procedure, the collagen enzyme modified PLGA microspheres with fluorescence properties were prepared by substituting nile red dye for triamcinolone acetonide. Preparing microsphere suspension with concentration of 1mg/mL by using PBS solution, adding the microsphere suspension to the upper surface of collagen gel, placing the mixture in 37 ℃ for reaction for 24 hours, taking out the mixture, and observing the diffusion result of the microsphere in the three-dimensional collagen by using laser confocal. Under the same conditions, PLGA microspheres without modified collagenase were used as a control group.
As a result, as shown in fig. 3, the collagenase-modified PLGA microspheres diffuse significantly longitudinally inside the three-dimensional collagen, while the non-modified collagenase PLGA microspheres remain on the surface of the collagen gel. The result shows that the collagen gel is degraded under the action of collagenase, the internal three-dimensional structure is collapsed, and the inhibition for the diffusion of the microspheres is reduced.
Claims (10)
1. The preparation method of the collagenase modified PLGA microsphere is characterized by comprising the following steps:
s1, co-dissolving medicine micro powder and polylactic acid-glycolic acid copolymer (PLGA) in an organic solvent to obtain a raw material solution; adding the raw material solution into a 2% PVA solution, and emulsifying to form a suspension; adding the suspension into 0.5% PVA solution, stirring at room temperature, completely evaporating the organic solvent, sieving, collecting the hardened microspheres, and thoroughly cleaning with deionized water; the organic solvent is dichloromethane or/and acetone;
s2, adding Traut's reagent into collagenase solution with the concentration of 10mg/mL, and reacting for 1 h under stirring at room temperature;
s3, adding the microspheres obtained in the step s1 into the reaction solution in the step s2 to obtain PLGA microsphere suspension; reacting for 1-12 h at room temperature; and (3) centrifuging, washing with PBS solution for three times, dialyzing, and freeze-drying to obtain PLGA microspheres with collagenase immobilized on the surfaces.
2. The method of claim 1, wherein in step s2, the collagenase is type I collagenase.
3. The method according to claim 1, wherein in step s1, the volume of the 2% PVA solution is 5 to 10 times that of the starting material solution.
4. The method according to any one of claims 1 to 3, wherein in step s1, the drug fine powder accounts for 1 to 20% by weight of the PLGA.
5. A method according to any one of claims 1 to 3, wherein in step s1, the screened mesh is 60 to 100 mesh.
6. A method according to any one of claims 1 to 3, wherein in step s2 the volume of the Traut's reagent is 5 times the volume of the collagenase solution.
7. The method according to any one of claims 1 to 3, wherein in step s3, the concentration of the suspension of PLGA microspheres is 1 to 10mg/mL.
8. A method according to any one of claims 1 to 3, wherein the PLGA microspheres obtained in step s3 are stored at-20 ℃.
9. A method according to any one of claims 1 to 3, wherein the drug in step s1 is triamcinolone acetonide, 5-fluorouracil, doxorubicin or botulinum toxin.
10. A drug-loaded collagenase-modified PLGA microsphere obtained by the method of any one of claims 1 to 3.
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| CN202111458056.8A CN114099707B (en) | 2021-12-01 | 2021-12-01 | Collagenase modified PLGA microsphere and preparation method thereof |
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| CN114099707B true CN114099707B (en) | 2023-08-01 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103768027A (en) * | 2014-01-15 | 2014-05-07 | 上海交通大学 | Collagenase sustained-release microsphere as well as preparation method and application thereof |
| CN108273061A (en) * | 2018-03-02 | 2018-07-13 | 中国药科大学 | A kind of anti-fibrosis medicine nanometer formulation and preparation method thereof |
-
2021
- 2021-12-01 CN CN202111458056.8A patent/CN114099707B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103768027A (en) * | 2014-01-15 | 2014-05-07 | 上海交通大学 | Collagenase sustained-release microsphere as well as preparation method and application thereof |
| CN108273061A (en) * | 2018-03-02 | 2018-07-13 | 中国药科大学 | A kind of anti-fibrosis medicine nanometer formulation and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Facile fabrication of PEG-coated PLGA microspheres via SPG membrane emulsification for the treatment of scleroderma by ECM degrading enzymes;Seiichi Ohta 等;《Colloids and Surfaces B: Biointerfaces》;第179卷;第453-461页 * |
| Nanoparticles functionalized with collagenase exhibit improved tumor accumulation in a murine xenograft model;Surya Murty 等;《Part Part Syst Charact》;第31卷(第12期);第1307-1312页 * |
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