CN114652856B - Double-protein delivery carrier procedural drug delivery system and preparation method thereof - Google Patents
Double-protein delivery carrier procedural drug delivery system and preparation method thereof Download PDFInfo
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Abstract
The application discloses a double-protein delivery carrier procedural drug delivery system and a preparation method thereof. The dual protein delivery vehicle procedural drug delivery system of the present application comprises: a nanoparticle of a first protein, a first drug, and a hydrogel of a second protein; wherein the nanoparticles of the first protein are dispersed in the hydrogel of the second protein and are covalently linked to the second protein, and the first drug is loaded on the nanoparticles of the first protein. The double-protein delivery carrier procedural drug delivery system is a multi-drug co-drug delivery system, can realize the co-system procedural release of hydrophilic drugs and hydrophobic drugs and the ultra-long-acting release of the hydrophobic drugs, and is particularly suitable for the situations that a first drug is a hydrophobic drug and slow release drug delivery is carried out, and a second drug is a hydrophilic drug and quick release drug delivery is carried out.
Description
Technical Field
The technology relates to the technical field of biomedical materials, in particular to a double-protein delivery carrier procedural drug delivery system and a preparation method thereof. The double-protein delivery carrier procedural drug delivery system can realize procedural drug delivery of two drugs in different release forms, and can realize ultra-long-acting release drug delivery of hydrophobic drugs.
Background
Kartogenin (KGN) is a heterocyclic compound screened by Nohua corporation in 2012 and capable of promoting the selective differentiation of multifunctional mesenchymal stem cells into chondrocytes, and is currently developed into one of the main drugs for repairing osteoarthritis cartilage defects.
KGN is a hydrophobic drug, insoluble in water, and such drugs typically require administration using a drug delivery vehicle. In addition, repair of an osteoarthritic cartilage defect requires a long period (e.g., about 60 days), and in order for KGN to function for this long period, administration of KGN needs to be delayed as much as possible.
Currently, drug delivery vehicles for administration of hydrophobic drugs such as KGN are mainly high molecular polymer nano-encapsulation systems, for example, PLGA (poly (lactic-co-glycolic acid)) which can achieve slow release administration of KGN. Although slow release administration is already performed, the KGN administration mode in the prior art is basically 15-30 days, and KGN is not released any more, which is obviously unsuitable compared with the repair period of the cartilage defect of osteoarthritis of about 2 months. Therefore, there is a need to develop drug delivery vehicle systems capable of releasing hydrophobic drugs such as KGN more long-term.
Disclosure of Invention
In view of the above-mentioned problems in the prior art, an object of the present invention is to provide a drug delivery vehicle system capable of releasing hydrophobic drugs such as KGN with very long duration.
The invention comprises the following steps:
1. A dual protein delivery vehicle procedural drug delivery system comprising: a nanoparticle of a first protein, a first drug, and a hydrogel of a second protein; wherein,
The nanoparticles of the first protein are dispersed in the hydrogel of the second protein and are covalently linked to the second protein,
The first drug is supported on the nanoparticle of the first protein.
The first protein is not particularly limited, and is preferably a natural protein which is degradable from the viewpoint of reducing side effects of the administration carrier; from the viewpoint of facilitating the preparation of nanoparticles, preferred are those proteins capable of undergoing conformational changes such as β -sheet formation, nanoparticle formation, etc., in organic solvents such as acetone, ethanol, etc. Proteins are typically rich in amino groups that are capable of forming covalent linkages with carboxyl groups contained in the first drug. In combination, the first protein is preferably fibroin, bovine serum albumin, human serum albumin, or the like.
The second protein is not particularly limited, and is preferably a natural protein which is degradable from the viewpoint of reducing side effects of the administration carrier; from the viewpoint of enhancing the effect of repairing an osteoarthritic cartilage defect, it is preferable that the protein has a function of promoting cell growth and proliferation, and/or that the hydrogel formed by the protein has good pore penetration and can function as a scaffold. In combination, the second protein is preferably collagen, gelatin, or the like.
The first protein and the second protein may be covalently linked (cross-linked) using prior art techniques. For example, glutamine transaminase (TGase) is capable of catalyzing a transamidation reaction between the γ -amide group of a glutamine residue and the e-amino group of a lysine residue in a protein, thereby covalently linking a first protein and a second protein. In the present invention, there is no particular limitation on the method of covalently linking the first protein and the second protein, for example, covalent linking (crosslinking) may be performed by catalysis of the first protein and the second protein by glutamine transaminase (TGase).
There is no particular limitation on the weight ratio of the first drug to the first protein, for example, the lower limit thereof may be, for example, 1:100000, 1:10000, 1:1000, 1:100, 1:50, 1:25, 1:20; the upper limit may be, for example, 1:2, 1:5, 1:10, 1:20, 1:25, 1:30, 1:50.
There is no particular limitation on the weight ratio of the hydrogel of the second protein to the first protein as long as the nanoparticles of the first protein can be dispersed in the hydrogel of the second protein, for example, the lower limit thereof may be, for example, 1: 100. 1: 50. 1: 20. 1: 15. 1: 10. 1:5, a step of; the upper limit may be, for example, 1:10, 1:5, 1:3, 1:2, 1:1, 1:0.5, 1:0.1, 1:0.01.
The content of the second protein in the hydrogel of the second protein is not particularly limited as long as the hydrogel can be formed, and for example, the lower limit thereof may be, for example, 10 wt%, 5 wt%, 2 wt%, 1 wt%; the upper limit may be, for example, 30 wt%, 20 wt%, 15 wt%, 10 wt%.
2. The dual protein delivery vehicle procedural drug delivery system of item 1 wherein the first protein is selected from the group consisting of fibroin, bovine serum albumin, or human serum albumin.
3. The dual protein delivery vehicle procedural drug delivery system of item 1 wherein the second protein is selected from collagen or gelatin.
4. The dual protein delivery vehicle procedural drug delivery system of item 1 wherein the first drug comprises a hydrophobic drug.
5. The dual protein delivery vehicle procedural drug delivery system of item 1 wherein the first drug comprises KGN (Kartogenin).
6. The dual protein delivery vehicle procedural drug delivery system of item 1 further comprising a second drug dispersed in a hydrogel of the second protein.
7. The dual protein delivery vehicle procedural drug delivery system of item 6 wherein the second drug comprises a hydrophilic drug.
8. The dual protein delivery vehicle procedural drug delivery system of item 6 wherein the second drug comprises dexamethasone.
9. Use of the dual protein delivery vehicle procedural drug delivery system of item 1 in the manufacture of a medicament for the repair of an osteoarthritic cartilage defect wherein the first medicament comprises Kartogenin and the second medicament comprises dexamethasone.
10. A method of preparing a dual protein delivery vehicle procedural drug delivery system of any one of items 1 to 8 comprising:
A preparation step of a nanoparticle of a first protein loaded with a first drug: dripping the first protein aqueous solution into the first medicine solution or dripping the first medicine solution into the first protein aqueous solution, preferably stirring and mixing (stirring time is 2-24 h for example), centrifuging, and taking a precipitate, preferably washing (re-suspending and re-centrifuging) the precipitate with water for 1 or more times to obtain the first protein nanoparticle loaded with the first medicine; wherein the first aqueous protein solution is prepared by dissolving the first protein in water, preferably at a concentration of, for example, 1 to 10w/v%; the first drug solution is prepared by dissolving the first drug and a cross-linking agent, preferably acetone and ethanol, in an organic solvent, preferably EDC/NHS, the cross-linking agent acting to covalently link the first drug to the first protein; and
Preparation of the dual protein delivery vehicle procedural drug delivery system: adding a second protein (such that the concentration thereof is, for example, 6-12 w/v%) to the aqueous solution of the first protein-loaded nanoparticle (concentration thereof is, for example, 1-5 mg/mL), and adding a catalyst for crosslinking (the catalyst functions to covalently link the first protein-loaded nanoparticle to the second protein, for example, glutamine transaminase, in an amount of, for example, 20-100U, for example, 60U, per gram of the second protein) such that a hydrogel of the second protein is formed, and the first protein-loaded nanoparticle is dispersed in the hydrogel of the second protein and covalently linked to the second protein, thereby obtaining the dual protein delivery vehicle procedural drug delivery system.
11. The method of preparation of item 10, wherein, in the step of preparing the dual protein delivery vehicle procedural drug delivery system, a second drug is added to the aqueous solution of the first drug loaded nanoparticle of the first protein.
The invention has the following beneficial technical effects:
1. The dual protein delivery vehicle programming delivery system of the present invention is capable of achieving ultra-long-acting release of hydrophobic drugs. Taking KGN as an example, the main polymer nano-package drug delivery system in the prior art generally reaches the plateau (QiaFeng et al, adv. Hetherline Mater.1, 2102395) at maximum 28 days in 7-15 days (Arezou Baharlou Houreh et al, international Journal of Biological Macromolecules (2021) 589-600; wenshuai Fan et al, MATERIALS SCIENCE & ENGINEERING C110 (2020) 110705;Payam Baei et al, CHEMICAL ENGINEERING Journal 418 (2021) 129277;Seon Sook Lee et al, ACS appl. Mater. Interfaces 2017,9,42668-42675;Lixia Zheng et al, ACS biomater. Sci. Eng.2019,5,4564-4573; mi Lan Kang et al, biomaterials 35 (2014) 9984e9994; mi-Lan Kang et al, acta Biomaterialia 39 (2016) 65-78; jianbin Xu et al, biomaterials 210 (2019) 51-61) and the maximum 28 days. In contrast, the dual protein delivery vehicle programming delivery system of the present invention still continuously released KGN at 60 days and the amount released did not reach 50%, indicating that there was a considerable time for release to reach plateau. To the inventors' knowledge, this is the best effect achieved so far in the long-acting release administration of KGN.
2. In the case of the inclusion of a second drug, the dual protein delivery vehicle programming delivery system of the present invention is in fact a multi-drug co-delivery system, enabling the co-system programming release of hydrophilic and hydrophobic drugs, which is particularly suitable for situations where the first drug is a hydrophobic drug and slow release drug and the second drug is a hydrophilic drug and fast release drug.
3. As a typical example of application, where the first drug is KGN and the second drug is dexamethasone, the dual protein delivery vehicle procedural drug delivery system may be used as an osteoarthritis cartilage defect repair drug. Osteoarthritis and cartilage defect repair require rapid inflammation at the early stage and slow promotion of cell proliferation and differentiation at the later stage, whereas a dual protein delivery vehicle procedural drug delivery system is capable of perfectly achieving rapid release of dexamethasone (inflammation) and long-acting slow release of KGN (pro-cell proliferation and differentiation) in the same drug delivery system. Moreover, the double-protein delivery carrier procedural drug delivery system can be entirely composed of natural protein materials which can be degraded in vivo, is safe and nontoxic, and has small toxic and side effects on human bodies. In addition, collagen is the most abundant structural protein in ECM (extracellular matrix ), and collagen hydrogel can support growth/adhesion of Mesenchymal Stem Cells (MSCs) and cartilage differentiation.
Drawings
FIG. 1 is a conceptual diagram of the design of a dual protein delivery vehicle programming delivery system according to an embodiment of the present invention.
Fig. 2 is a scanning electron microscope image of the drug-loaded silk nanoparticle.
FIG. 3 is a transmission electron microscope image of the drug-loaded human serum albumin nanoparticle.
Fig. 4 is a view of the appearance of the hydrogel and a sem image of the hydrogel.
Fig. 5 is a graph showing the in vitro drug release profile of hydrogels.
FIG. 6 is a graph showing the cytotoxicity test results of hydrogels.
Fig. 7 is a photograph showing the cartilage repair promoting ability of the hydrogel in animal experiments.
Detailed Description
The present invention will be described in detail with reference to specific examples. It should be particularly pointed out that these descriptions are merely exemplary descriptions and do not constitute limitations on the scope of the invention.
Example 1:
as an embodiment of the invention, the collagen used in the hydrogel is recombinant collagen described in Chinese patent application publication CN1371919A, has good water solubility, the molecular weight is 97,000Da, the fibroin is extracted from silkworm cocoons, and the activity unit of glutamine transaminase is 200U/g. The preparation method of the hydrogel of the embodiment comprises the following steps:
1) Preparation of soluble fibroin: removing silkworm chrysalis from silkworm cocoons, cleaning, putting into 5g/L sodium carbonate solution, boiling for 30min for degumming, washing degummed fibroin with deionized water, putting into an oven for drying, dissolving a certain amount of fibroin in three-element solution of calcium chloride/water/ethanol (molar ratio of 1:8:2) at 80 ℃ for 1h, then pouring the mixed solution into a dialysis bag (molecular weight cut-off 8000-12000), dialyzing with distilled water for 3d at room temperature, removing salt, ethanol and other small molecular impurities in the solution, pouring the dialyzed fibroin solution into a funnel, removing insoluble impurities, preparing a fibroin aqueous solution, and performing vacuum freeze drying to obtain the soluble fibroin.
2) Preparation of drug-loaded fibroin nanoparticles: weighing soluble fibroin, dissolving in deionized water to prepare a 1% -5% solution, preparing fibroin nanoparticles by a desolvation method, firstly dissolving KGN in an acetone solvent to a concentration of 0.4mg/mL, adding EDC/NHS (molar ratio: 1:1), stirring for 2-4 h, and then adding the soluble fibroin dropwise (150 mu L/drop) into acetone. The mixed solution was then centrifuged for 30 minutes at 100000 Xg, the supernatant was aspirated, the pellet was then resuspended in distilled water, vortexed, centrifuged again, washed, and after at least 2 more final rotations of the suspension, to produce a uniform suspension of drug-loaded silk nanoparticles and stored at 4 ℃.
3) Preparation of double-protein-carrier hydrogel: dissolving drug-loaded natural protein nano particles (1-5 mg/mL) in deionized water, then adding dexamethasone injection (DXMS) to ensure that the concentration is 0.1-5mg/mL, then adding collagen to ensure that the collagen is completely dissolved in a water bath at 37 ℃, and ensuring that the concentration of the collagen is 6% -12%. After thorough mixing, TGase (60U/g protein) was added and dissolved completely in the solution by stirring. The mixture was then crosslinked at 4 ℃ for 10 hours to obtain two drug-loaded hydrogels.
Example 2:
As an example of the present invention, the collagen used in the hydrogel is recombinant collagen described in Chinese patent application publication CN1371919A, which has good water solubility, molecular weight of 97,000Da, bovine serum albumin is reagent grade (manufactured by Amresco company, U.S.) and activity unit of glutamine transaminase is 200U/g. The preparation method of the hydrogel of the embodiment comprises the following steps:
1) Preparation of drug-loaded bovine serum albumin nanoparticles: KGN was dissolved in ethanol at a concentration of 0.4mg/mL and EDC/NHS (molar ratio: 1:1) was added and stirred for 2 hours. Bovine serum albumin solution was prepared at a concentration of 2%, pH was adjusted to 8 with 0.2M NaOH, and stirred at room temperature for 30 minutes. The KGN-ethanol solution was added dropwise to the bovine serum albumin solution and stirred for a further 12h. The mixed solution was centrifuged at 10000 revolutions for 30 minutes, the supernatant was aspirated, and the pellet was resuspended in distilled water, vortexed, then centrifuged again, washed, and after at least 2 more repetitions of final rotations of the suspension, to produce a homogeneous drug loaded bovine serum albumin nanoparticle suspension and stored at 4 ℃.
2) Preparation of double-protein-carrier hydrogel: drug-loaded bovine serum albumin nanoparticles (1 mg/mL) were dissolved in deionized water, then dexamethasone was added to a concentration of 1mg/mL, then human-like collagen was added to a concentration of 10% in a 37℃water bath to completely dissolve the collagen. After thorough mixing, TGase (60U/g protein) was added and dissolved completely in the solution by stirring. The mixture was then crosslinked at 4 ℃ for 10 hours to obtain two drug-loaded hydrogels.
Example 3:
As an example of the present invention, the collagen used in the hydrogel is recombinant collagen described in Chinese patent application publication CN1371919A, which has good water solubility, molecular weight of 97,000Da, and human serum albumin is pharmaceutical grade (manufactured by Boya Biochemical Co., ltd.), and the activity unit of glutamine transaminase is 200U/g. The preparation method of the hydrogel of the embodiment comprises the following steps:
1) Preparation of drug-loaded human serum albumin nanoparticles: KGN was dissolved in ethanol at a concentration of 0.4mg/mL and EDC/NHS (molar ratio: 1:1) was added and stirred for 2 hours. A human serum albumin solution was prepared at a concentration of 2%, pH was adjusted to 8 with 0.2M NaOH, and the mixture was stirred at room temperature for 30 minutes. The KGN-ethanol solution was added dropwise to the human serum albumin solution and stirred for a further 12h. The mixed solution was centrifuged at 10000 rpm for 30 minutes, the supernatant was sucked off, and then the precipitate was resuspended in distilled water, vortexed, then centrifuged again, washed, and finally spun at least 2 more times after suspension was repeated to produce a uniform suspension of drug-loaded human serum albumin nanoparticles and stored at 4 ℃.
2) Preparation of double-protein-carrier hydrogel: drug-loaded human serum albumin nanoparticles (1 mg/mL) were dissolved in deionized water, then dexamethasone was added to a concentration of 1mg/mL, then human-like collagen was added to a concentration of 10% in a 37℃water bath to completely dissolve the collagen. After thorough mixing, TGase (60U/g protein) was added and dissolved completely in the solution by stirring. The mixture was then crosslinked at 4 ℃ for 10 hours to obtain two drug-loaded hydrogels.
Example 4:
As an embodiment of the invention, the collagen used in the hydrogel is recombinant collagen described in Chinese patent application publication CN1371919A, has good water solubility, has a molecular weight of 97,000Da, is extracted from silkworm cocoons, and has a glutamine transaminase activity unit of 200U/g. The preparation method of the hydrogel of the embodiment comprises the following steps:
1) Preparation of soluble fibroin: removing silkworm chrysalis from silkworm cocoons, cleaning, putting into 5g/L sodium carbonate solution, boiling for 30min for degumming, washing degummed fibroin with deionized water, putting into an oven for drying, dissolving a certain amount of fibroin in three-element solution of calcium chloride/water/ethanol (molar ratio of 1:8:2) at 80 ℃ for 1h, then pouring the mixed solution into a dialysis bag (molecular weight cut-off 8000-12000), dialyzing with distilled water for 3d at room temperature, removing salt, ethanol and other small molecular impurities in the solution, pouring the dialyzed fibroin solution into a funnel, removing insoluble impurities, preparing a fibroin aqueous solution, and performing vacuum freeze drying to obtain the soluble fibroin.
2) Preparation of fibroin nanoparticles: weighing soluble fibroin, dissolving in deionized water, preparing into 1% -5% solution, preparing fibroin nanoparticles by desolvation method, and adding the soluble fibroin dropwise (150 μl/drop) into acetone. The mixed solution was then centrifuged for 30 minutes at 100000 Xg, the supernatant was aspirated, the pellet was then resuspended in distilled water, vortexed, centrifuged again, washed, and after at least 2 more final rotations of the suspension, to produce a uniform suspension of drug-loaded silk nanoparticles and stored at 4 ℃.
3) Preparation of double-protein hydrogels: silk fibroin nano particles (1-5 mg/mL) are dissolved in deionized water, then collagen is added into the deionized water to be completely dissolved in a water bath at 37 ℃, and the concentration of the collagen is 6% -12%. After thorough mixing, TGase (60U/g protein) was added and dissolved completely in the solution by stirring. The mixture was then crosslinked at 4 ℃ for 10 hours to obtain a double protein hydrogel.
Performance testing was performed on the dual protein delivery vehicle procedural drug delivery system prepared in the examples of the present invention:
(1) The design concept is as follows: in this study, as shown in fig. 1, we designed a double-protein hydrogel drug sustained-release system with different release trajectories of double drugs, loaded with hydrophilic and hydrophobic drugs, for bone defect repair. The natural protein nano-particles are loaded with a hydrophobic drug KGN and then are mixed with collagen and a hydrophilic anti-inflammatory drug dexamethasone. A tissue engineering hydrogel scaffold useful for osteoarthritis and cartilage defects is prepared by enzymatic crosslinking. The hydrogel is characterized in that the natural protein double-protein drug can be completely degraded and nontoxic, and can realize the programmed release of the hydrophilic drug and the hydrophobic drug in a co-system.
(2) The encapsulation efficiency, drug loading and nano-average particle size of the KGN-loaded natural protein nanoparticle of example 1 were measured by drug loading (%) = (total drug amount-drug amount in supernatant)/total formulation amount of 100%, encapsulation efficiency (%) = (total drug amount-drug amount in supernatant)/total drug amount of 100%
Detecting the medicine amount in the supernatant: the method comprises the steps of detecting the medicine amount in supernatant liquid through a high performance liquid chromatography, determining the qualitative medicine at the peak position through chromatography, wherein the peak area is in direct proportion to the medicine concentration, detecting the medicine amount through taking a standard curve of the peak area and the medicine concentration, adding 0.1% formic acid into a KGN chromatographic condition C18 reverse phase column with the mobile phase of acetonitrile and water (the ratio is 70:30), the flow rate is 1mL/min, the detection wavelength is 281, the sample injection amount is 10uL, and the peak outlet time is 11min.
Finally, the encapsulation efficiency, drug loading capacity and nanometer average particle size of the silk fibroin nanoparticle loaded KGN, the human serum albumin nanoparticle loaded KGN and the bovine serum albumin nanoparticle loaded KGN are shown in the following table.
(2) The morphology of the drug-loaded fibroin nanoparticle of example 1 and the drug-loaded human serum albumin nanoparticle of example 3 was evaluated: and carrying out morphological evaluation on the drug-loaded fibroin nanoparticles by using a scanning electron microscope, and carrying out morphological evaluation on the drug-loaded human serum albumin nanoparticles by using a transmission electron microscope. Fig. 2 is a scanning electron microscope image of the drug-loaded silk nanoparticle of example 1. FIG. 3 is a transmission electron microscope image of the drug-loaded human serum albumin nanoparticle of example 3. As shown in the electron microscope image, the nano particles all show spherical shapes.
(3) FIG. 4 is a diagram of the appearance and scanning electron microscope of a double protein loaded drug hydrogel of example 1: the gel is milky white, and the internal structure is detected by SEM, so that the hydrogel is porous and has high porosity and pore interoperability.
(4) In vitro drug release: detecting the quantity of the drug released in the PBS solution by high performance liquid chromatography, and carrying out chromatography to obtain qualitative drug at peak position, wherein the peak area is in direct proportion to the concentration of the drug, and the peak area and the concentration of the drug are used as standard curves to detect the quantity of the drug, and the chromatographic conditions of KGN and dexamethasone are as follows:
KGN chromatographic conditions: c18 reverse phase column, mobile phase is acetonitrile and water (ratio is 70:30), 0.1% formic acid is added, flow rate is 1mL/min, detection wavelength 281, sample injection amount is 10uL, and peak time is 11min.
Dexamethasone chromatographic conditions: c18 reverse phase column, mobile phase is acetonitrile and water (ratio is 70:30), flow rate is 1mL/min, detection wavelength is 240, sample injection amount is 10-uL, and peak time is 3.5min.
FIG. 5 is a graph showing the in vitro drug release profile of a dual protein loaded hydrogel. "hydrogel-KGN" in FIG. 5 shows the KGN-releasing course of a double-protein-carrying hydrogel prepared according to example 1; "hydrogel-dexamethasone" represents the release profile of dexamethasone for the double protein loaded hydrogel prepared as described in example 1; "Silk fibroin nanoparticle-KGN" means the KGN-releasing course of KGN-loaded silk fibroin nanoparticles prepared according to step 2) of example 1.
Experimental results show that the double-protein loaded medicine hydrogel has different release tracks for KGN and dexamethasone, and the dexamethasone is released in a large amount in the early stage, and more than 90% of the dexamethasone is released in 40 days; the dual protein drug-loaded hydrogel has a small amount of slow release for KGN within 60 days, and the release amount is less than 50% at 60 days, which indicates that the release time is quite long after the slow release time, and the KGN is stopped to be released. Moreover, the release of KGN by the double protein-loaded drug hydrogel was slower compared to single silk fibroin nanoparticle loaded KGN (the time to plateau was about 40 days).
For KGN administration, the main-stream polymer nano-encapsulated administration systems in the prior art are generally used for 7-15 days (see, for example, arezou Baharlou Houreh et al, international Journal of Biological Macromolecules 177 (2021) 589-600; wenshuai Fan et al, MATERIALS SCIENCE & ENGINEERING C (2020) 110705;Payam Baei et al, CHEMICAL ENGINEERING Journal 418 (2021) 129277;Seon Sook Lee et al, ACS appl. Materials 2017,9,42668-42675;Lixia Zheng et al, ACS biomatter. Sci. Eng.2019,5,4564-4573; mi Lan Kang et al, biomaterials 35 (2014) 9984e9994; mi-Lan kan et al, acta Biomaterialia (2016) 65-78; jiaanbin Xu et al, biomaterials 210 (2019) 51-61), and the maximum reaching of the plateau (Qi Feng et al, adv. Health materials 2021, 21095) in 28 days. In contrast, the dual protein delivery vehicle programming delivery system of the present invention still continuously released KGN at 60 days and the amount released did not reach 50%, indicating that there was a considerable time for release to reach plateau. To the inventors' knowledge, this is the best effect achieved so far in the long-acting release administration of KGN.
(5) Cell experiment: hydrogel cytotoxicity test:
the effect of the leach liquor of the hydrogels of example 1, example 2, example 3 on cell viability was evaluated using the MTT method and its safety was determined by cytotoxicity analysis. The stent extract was prepared with reference to GB/T16886.5-2003/ISO 10993-5:199 standard. Cells grown well and diluted were plated in 96-well plates (plating density 1X 10 4 cells/well). The extract solutions of example 1, example 2 and example 3 were added to the well plate (100. Mu.L/well) respectively as experimental groups, and the other group was cultured using the prepared DMEM high-sugar culture solution as a control group. Placing the experimental group and the control group into an incubator for constant temperature culture, respectively culturing for 24 hours, adding 50 mu L of MTT (methyl thiazolyl tetrazolium) into each hole, incubating for 3 hours at 37 ℃, removing the culture solution, adding 150 mu L of DMSO into each hole, and continuously shaking to dissolve purple formazan sediment at the bottom of the hole plate. Then, absorbance (OD) values of the respective wells were measured at a wavelength of 490nm using an enzyme-labeled instrument. Cell relative viability (RELATIVE CELL Growth) was determined using equation RELATIVE CELL And (5) calculating. The results show that: as shown in FIG. 6, the relative proliferation rate of the cells was 110% or more for 24 hours of culture, and the hydrogel of example 1, example 2, and example 3 was rated as 0 in the cytotoxicity rating according to the national standard. Thus, it can be seen that the hydrogel of example 1, example 2, example 3 was not only free of cytotoxicity in terms of in vitro cell compatibility, but also promoted the growth and proliferation of cells to some extent, exhibiting good cell compatibility.
(6) Animal experiment: cartilage repair promoting ability of hydrogels:
Animals were divided into control defect groups, drug-free hydrogel groups and drug-loaded hydrogel groups prepared as in example 1, and were anesthetized by intravenous injection using 3% sodium pentobarbital. The animals were cut along a 2.0mm longitudinal row of unilateral knees, revealing joint furrows and then surgically drilled to create cartilage defects 3mm in diameter and 5mm deep in the knee pulley groove of the right leg of the animal. The defect is left blank without any treatment, and the drug-free hydrogel and the drug-loaded hydrogel are filled with the drug-free hydrogel group and the drug-loaded hydrogel group, respectively, and are flush with the surface of the natural cartilage after the stent is implanted. The ligament is reset, joint capsules are sewn layer by layer, and the skin is wrapped. 20 units of penicillin sodium are injected every day within three days after the operation, so that the postoperative infection is prevented. The results show that: all knee samples showed no inflammatory response and the hydrogel and host tissues integrated well. The repair state of the bone cartilage defect after the operation of 12 weeks is shown in fig. 7, the blank control group has larger defect recess, the defect part of the hydrogel group without medicine is filled with fibrous tissue or is not completely filled with repair tissue, the boundary between the implant of the hydrogel group with medicine and surrounding tissue disappears, the newly-born granulation tissue is filled, and the joint surface is basically smooth. In contrast, the drug-loaded hydrogel group exhibited relatively good repairability.
Finally, it should be noted that while the above describes embodiments of the invention in terms of drawings, the invention is not limited to the particular embodiments and fields of application described above, which are illustrative, instructive, and not limiting. Those skilled in the art, having the benefit of this disclosure, may effect numerous forms of the invention without departing from the scope of the invention as claimed.
Claims (2)
1. A method of manufacturing a dual protein delivery vehicle programming delivery system comprising: a nanoparticle of a first protein, a first drug, and a hydrogel of a second protein; wherein,
The nanoparticles of the first protein are dispersed in the hydrogel of the second protein and are covalently linked to the second protein,
The first drug is supported on the nanoparticle of the first protein;
wherein the first protein is fibroin;
the second protein is collagen;
The first drug is Kartogenin (KGN);
The dual protein delivery vehicle procedural drug delivery system further comprises a second drug dispersed in the hydrogel of the second protein, the second drug being dexamethasone;
The manufacturing method comprises the following steps:
1) Preparation of soluble fibroin: the silkworm cocoons are cleaned after silkworm pupas are removed, are put into 5g/L sodium carbonate solution to be boiled for 30min for degumming, the degummed fibroin is cleaned by deionized water, and is put into an oven for drying, and a certain amount of fibroin is subjected to a mole ratio of 1 at 80:: 8:2, dissolving in a ternary solution of calcium chloride/water/ethanol for 1 hour, pouring the mixed solution into a dialysis bag with molecular weight cut-off of 8000-12000, dialyzing with distilled water at room temperature for 3 days, removing salt, ethanol and other small molecular impurities in the solution, pouring the dialyzed silk fibroin solution into a funnel, removing insoluble impurities, preparing a silk fibroin aqueous solution, and performing vacuum freeze drying to obtain soluble silk fibroin;
2) Preparation of drug-loaded fibroin nanoparticles: weighing soluble fibroin and dissolving in deionized water
In the method, 1% -5% of solution is prepared, fibroin nano particles are prepared by a desolvation method, KGN is firstly dissolved in an acetone solvent, the concentration is 0.4mg/mL, and then the fibroin nano particles are added with the molar ratio of 1:1 EDC/NHS, stirring for 2-4 hr, and adding soluble fibroin
Drop wise addition to acetone at 150 μl/drop; then the mixed solution was centrifuged at 100000 Xg for 30 minutes, the supernatant was aspirated, and the precipitate was resuspended in distilled water and vortexed
Oscillating, then re-centrifuging, washing, repeating the final rotation for at least 2 times after suspending,
To produce a uniform suspension of drug-loaded silk nanoparticles and stored at 4 ℃;
3) Preparation of double-protein-carrier hydrogel: dissolving 1-5mg/mL of drug-loaded natural protein nano particles in deionized water, then adding dexamethasone injection to ensure that the concentration is 0.1-5mg/mL, then adding collagen to completely dissolve in a water bath at 37 ℃, wherein the concentration of the collagen is 6% -12%; after thorough mixing, 60U/g of TGase of protein was added and stirred
After being completely dissolved in the solution, the mixture was crosslinked at 4℃for 10 hours to obtain a hydrogel loaded with both drugs.
2. Use of a dual protein delivery vehicle procedural drug delivery system made according to the manufacturing method of claim 1 in the manufacture of an osteoarthritic cartilage defect repair drug.
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