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CN118415979A - Preparation and application of phentolamine nanometer slow release injection - Google Patents

Preparation and application of phentolamine nanometer slow release injection Download PDF

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Publication number
CN118415979A
CN118415979A CN202410874586.8A CN202410874586A CN118415979A CN 118415979 A CN118415979 A CN 118415979A CN 202410874586 A CN202410874586 A CN 202410874586A CN 118415979 A CN118415979 A CN 118415979A
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phentolamine
cartilage
oil phase
release injection
water
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CN118415979B (en
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白晓春
魏小翠
丘菁杨
赖锐军
黄仕江
江媛君
温嘉欣
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Southern Medical University
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Southern Medical University
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Abstract

The application relates to a preparation method and application of a phentolamine nanometer slow-release injection, which takes phentolamine as a medicine active ingredient and adopts an improved emulsifying solvent volatilization method to prepare the phentolamine nanometer slow-release injection. Meanwhile, the application constructs a phentolamine-loaded nanometer slow release injection system, which can effectively promote the regeneration of hyaline cartilage after the local cartilage defect of the mice by injecting into the joint cavity, inhibit the progress of osteoarthritis and resist the cartilage degradation under the stimulation of inflammation. A plurality of in-vivo and in-vitro models comprehensively verify that the phentolamine-loaded nano sustained-release injection can promote cartilage formation and inhibit cartilage degeneration, and is expected to be popularized and applied in joint cartilage diseases such as osteoarthritis, cartilage injury, joint hose degradation and the like.

Description

Preparation and application of phentolamine nanometer slow release injection
Technical Field
The application relates to the technical field of medicines, in particular to a preparation method and application of phentolamine nanometer slow-release injection.
Background
Cartilage damage due to trauma, infection, tumor, joint degenerative disease, etc. often results in or exacerbates the development of Osteoarthritis (OA), and in severe cases, total joint destruction. Cartilage tissue is composed of single chondrocytes, lacks vascular nerve distribution, and has poor proliferation capacity of chondrocytes, so that repair after cartilage injury is extremely difficult.
Aiming at the articular cartilage diseases, the patients are easy to have drug-resistant reaction and digestive tract adverse reaction when taking the drugs orally. The joint cavity injection can directly inject the medicine into a lesion site, so that the consumption of the medicine by the metabolism of the body in the in-vivo transportation and adverse reactions possibly caused are reduced, and the medicine plays the maximum role in minimum dosage. In addition, the intra-articular environment can be improved and maintained by adopting the intra-articular cavity injection mode, so that the joint is well lubricated, the inflammatory substance level is inhibited or reduced, the inflammation and the pain are relieved, the cartilage can be protected, the cartilage cell repair is promoted, and the patient acceptability is high. Conventional intra-articular injection drugs, however, are rapidly cleared from the joint and require frequent injections to maintain effective drug concentrations. Therefore, the study of long-acting drug delivery systems is necessary to reduce the frequency of intra-articular injection and facilitate clinical conversion applications of drugs.
Phentolamine (Phentolamine, PM) is an alpha adrenergic receptor blocker, which has been widely used clinically, mainly for: diagnosing pheochromocytoma and treating hypertension attacks caused by the pheochromocytoma; treating left ventricular failure; the medicine is used for treating the overflow of norepinephrine intravenous administration and preventing skin necrosis, and the safety and the curative effect of the medicine are subjected to long-term clinical examination, so that the medicine can further develop the clinical application potential of the medicine.
Disclosure of Invention
Based on the above, it is necessary to provide a phentolamine nanometer sustained-release injection which can effectively promote hyaline cartilage regeneration after local cartilage defect of mice, inhibit osteoarthritis progression, resist cartilage degradation under inflammatory stimulus, and can be used for treating articular cartilage diseases.
In a first aspect of the present invention, a method for preparing phentolamine nanometer sustained release injection is provided, comprising the following steps:
dissolving phentolamine in a first oil phase solvent, dissolving an oil phase matrix in a second oil phase solvent, and mixing to prepare an oil phase;
adding an inner water phase to the oil phase to prepare water-in-oil colostrum;
adding the water-in-oil colostrum into an external water phase to prepare water-in-oil-in-water compound emulsion; removing the first oil phase solvent and the second oil phase solvent, centrifuging, and collecting phentolamine-loaded nanoparticles;
the nanoparticles were washed with water and resuspended in buffer solution, stirred and filtered.
In one embodiment, the oil phase matrix is an amphiphilic graft copolymer; and/or
The external water phase is a nonionic emulsifier; and/or
The oil phase also includes an emulsifier.
In one embodiment, the oil phase matrix is a poly (lactic-co-glycolic acid); and/or
The aqueous phase matrix is polyvinyl alcohol; and/or
The oil phase also includes the fatty acid sorbitan 80.
In one embodiment, the first oil phase solvent is dimethyl sulfoxide; and/or
The second oil phase solvent is dichloromethane; and/or
The internal aqueous phase is water.
In one embodiment, the volume ratio of the inner aqueous phase, the oil phase and the outer aqueous phase is (0.5-1.5): (5-15): (80-120).
In one embodiment, the buffer solution is a mixture of a viscoelastic lubricant and an acetic acid buffer solution.
In one embodiment, the viscoelastic lubricant is hyaluronic acid.
In a second aspect of the present invention, there is provided a phentolamine nanometer sustained release injection prepared according to the preparation method of the phentolamine nanometer sustained release injection as described above.
In a third aspect, the invention provides an application of the phentolamine nanometer slow release injection in preparing a medicament for treating articular cartilage diseases.
In one embodiment, the drug is administered by a joint cavity injection.
Compared with the prior art, the application has the following beneficial effects:
The application takes phentolamine as the active ingredient of the medicine, and adopts an improved emulsifying solvent volatilization method to prepare the phentolamine nanometer slow-release injection. Meanwhile, the application constructs a phentolamine-loaded nanometer slow release injection system, which can effectively promote the regeneration of hyaline cartilage after the local cartilage defect of the mice by injecting into the joint cavity, inhibit the progress of osteoarthritis and resist the cartilage degradation under the stimulation of inflammation. A plurality of in-vivo and in-vitro models comprehensively verify that the phentolamine-loaded nano sustained-release injection can promote cartilage formation and inhibit cartilage degeneration, and is expected to be popularized and applied in joint cartilage diseases such as osteoarthritis, cartilage injury, joint hose degradation and the like.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following description will briefly explain the drawings needed in the embodiments or the prior art, and it is obvious that the drawings in the following description are some embodiments of the present application and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of drug release profile of HA-PLGA-PM-NPs provided in example 1, where the abscissa indicates time and the ordinate indicates drug release ratio.
FIG. 2 is a graph of results of promoting repair of articular cartilage defects by HA-PLGA-PM-NPs provided in example 1, wherein A in FIG. 2 is a rough photograph of cartilage of each group (upper scale, 500 μm) and H & E staining (lower scale, 200 μm), and B in FIG. 2 is a macroscopic ICRS scoring result.
FIG. 3 is a graph showing the results of inhibition of osteoarthritis progression in mice by HA-PLGA-PM-NPs provided in example 1, wherein Sham refers to Sham surgery group, HA-PLGA-NPs refers to HA-PLGA-NPs injection control group, and HA-PLGA-PM-NPs refers to HA-PLGA-PM-NPs injection group.
FIG. 4 is a graph showing the results of inhibition of inflammatory degeneration of human cartilage explants by HA-PLGA-PM-NPs as provided in example 1, wherein A in FIG. 4 is the result of staining with Saf-O and toluidine blue (scale bar, 500 μm); b in fig. 4 is immunohistochemical staining of ACAN in cartilage tissue (scale bar, 100 μm); c in fig. 4 is a semi-quantitative analysis of the Saf-O stained proteoglycan content (n=3) in a in fig. 4, data expressed as mean ± standard deviation. * P <0.05, < P <0.01, < P <0.001; d in fig. 4 is a quantitative statistical analysis (n=3) of ACAN positive cells in B in fig. 4, data are expressed as mean ± standard deviation. * P <0.05, < P <0.01, < P <0.001.
Detailed Description
The present application will be described in further detail with reference to specific examples. The present application may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:
herein, "one or more" refers to any one, any two, or any two or more of the listed items.
As used herein, the term "and/or," and/or, "and/or" includes any one of the two or more of the associated listed items and also includes any and all combinations of the associated listed items, including any two or more of the associated listed items, or all combinations of the associated listed items.
Herein, "further," "still further," "particularly," and the like are used for descriptive purposes and are not to be construed as limiting the scope of the application.
Herein, "first aspect," "second aspect," "third aspect," "fourth aspect," etc. are for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature being indicated. Furthermore, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list of descriptive purposes only and are not to be construed as limiting the number of closed forms. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present application, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise. In the description of the present application, the meaning of "several" means at least one, such as one, two, etc., unless specifically defined otherwise.
In the present application, the numerical ranges are referred to as continuous, and include the minimum and maximum values of the ranges, and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
The percentage content referred to in the present application refers to mass percentage for both solid-liquid mixing and solid-solid mixing and volume percentage for liquid-liquid mixing unless otherwise specified.
The percentage concentrations referred to in the present application refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system after the component is added.
The temperature parameter in the present application is not particularly limited, and may be a constant temperature treatment or a treatment within a predetermined temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations in a range such as + -5 deg.C, + -2 deg.C, + -1 deg.C, + -0.5 deg.C, + -0.4 deg.C, + -0.3 deg.C, + -0.2 deg.C, + -0.1 deg.C. The normal temperature or room temperature in the present application means that no temperature control operation is applied, and generally means 4 ℃ to 35 ℃, preferably means 20±5 ℃.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present application, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.
Phentolamine (Phentolamine, PM) is an alpha adrenergic receptor blocker, which has been widely used clinically, mainly for: diagnosing pheochromocytoma and treating hypertension attacks caused by the pheochromocytoma; treating left ventricular failure; the medicine is used for treating the overflow of norepinephrine intravenous administration and preventing skin necrosis, and the safety and the curative effect of the medicine have been subjected to long-term clinical examination. In order to further develop clinical application potential of PM, the application adopts an improved emulsifying solvent volatilization method to construct a sustained-release injection for loading PM, and performs joint cavity injection in a knee joint cartilage defect model and an arthritis model of mice, so as to investigate whether PM can treat arthritis and cartilage defect in vivo. In addition, in order to further verify the therapeutic effect of PM on human-derived tissue samples, human cartilage explants were used for in vitro culture, and IL-1α stimulation was used to investigate whether PM treatment could protect cartilage tissue and prevent cartilage degeneration under inflammatory stimulation.
Based on the above, the first aspect of the invention provides a preparation method of phentolamine nanometer slow release injection, which comprises the following steps:
dissolving phentolamine in a first oil phase solvent, dissolving an oil phase matrix in a second oil phase solvent, and mixing to prepare an oil phase;
adding an inner water phase to the oil phase to prepare water-in-oil colostrum;
adding the water-in-oil colostrum into an external water phase to prepare water-in-oil-in-water compound emulsion; removing the first oil phase solvent and the second oil phase solvent, centrifuging, and collecting phentolamine-loaded nanoparticles;
the nanoparticles were washed with water and resuspended in buffer solution, stirred and filtered.
In some of these examples, the oil phase matrix is an amphiphilic graft copolymer.
In some of these examples, the aqueous phase matrix is a nonionic emulsifier.
In some of these examples, the oil phase further comprises an emulsifier.
In one specific example, the oil phase matrix is a polylactic acid glycolic acid copolymer.
In one specific example, the aqueous phase matrix is polyvinyl alcohol.
In one specific example, the oil phase further comprises the fatty acid sorbitan 80.
In some examples, the first oil phase solvent is dimethyl sulfoxide.
In some examples, the second oil phase solvent is methylene chloride.
In some examples, the internal aqueous phase is water.
In some examples, the buffer solution is a mixture of a viscoelastic lubricant and an acetic acid buffer solution.
In some examples, the viscoelastic lubricant is hyaluronic acid.
In particular, polylactic-co-glycolic acid (PLGA) is a biocompatible and biodegradable polymer, a commonly used FDA approved supporting matrix for the development of active drug delivery systems with selective targeting and release. By utilizing the amphipathy of the grafted polymer, hydrophilic HA can be coated on the surface of PLGA Nano Particles (NPs) with hydrophobic characteristics. HA is a viscoelastic lubricant that reduces friction in the knee. And has the capability of specifically binding with CD44, and CD44 is a type I transmembrane receptor protein which is abundant in human articular chondrocytes. Therefore, the drug delivery system of the present application grafts Hyaluronic Acid (HA) onto PLGA nanoparticles and is used for intra-articular cavity injection, which can target chondrocytes, improve tissue specificity of drug delivery, and is a safe drug delivery system with better receptor specificity, which may represent an advantageous alternative to current nanotherapeutics.
In some examples, the volume ratio of the inner aqueous phase, the oil phase, and the outer aqueous phase is (0.5-1.5): (5-15): (80-120).
In one specific example, the volume ratio of the inner aqueous phase, the oil phase and the outer aqueous phase is 1:10:100.
In some examples, the mass to volume ratio of the lubricant to the solution is (6-9) mg: (20-40) mL. It is understood that the mass to volume ratio of the lubricant to the solution includes, but is not limited to, 6mg:30mL, 7mg:30mL, 7.5mg:30mL, 8mg:30mL, 9mg:30mL.
In some examples, the conditions of centrifugation include: the rotation speed is 10000rpm-15000rpm, and the time is 8min-15min. In one specific example, the conditions of centrifugation include: the rotation speed was 12000rpm and the time was 10min.
In some examples, the stirring time is 0.5h-2h. It is understood that the time of agitation includes, but is not limited to, 0.5h, 1h, 1.5h, 2h. Further, the stirring time was 1h.
In a second aspect of the present invention, there is provided a phentolamine nanometer sustained release injection prepared according to the preparation method of the phentolamine nanometer sustained release injection as described above.
In a third aspect, the invention provides an application of the phentolamine nanometer slow release injection in preparing a medicament for treating articular cartilage diseases.
In some examples, the articular cartilage disease includes one or more of osteoarthritis, articular cartilage damage, articular cartilage degradation.
In some examples, the drug is administered by a route of administration that is by intra-articular injection.
In a fourth aspect of the application there is provided the use of phentolamine in the manufacture of a medicament for the treatment of articular cartilage disease. The application discovers the brand-new application of the phentolamine which is a clinical common medicine in the repair of arthritis and cartilage injury, constructs a phentolamine-loaded nanometer slow release injection system, and can effectively promote the regeneration of hyaline cartilage after the local cartilage defect of a mouse and inhibit the progress of osteoarthritis through joint cavity injection. Experiments on human bone cartilage explants prove that the presence of phentolamine can resist the degradation of cartilage surface matrix induced by IL-1 beta, and further prove that the phentolamine has a protective effect on cartilage tissues in an inflammatory state. The comprehensive verification of various in-vivo and in-vitro models proves that the phentolamine not only can promote cartilage formation, but also can inhibit cartilage degeneration, and the PM-loaded nanometer slow-release injection is expected to be popularized and applied in osteoarthritis and cartilage injury.
In some examples, the medicament can be prepared into a proper dosage form according to clinical requirements, wherein the dosage form of the medicament comprises injection, oral liquid, pill, powder, paste, tablet, granule, powder or capsule.
The following examples are further illustrative, and the raw materials used in the following examples, unless otherwise specified, are commercially available; the instruments used, unless otherwise specified, may be commercially available; the processes involved, unless otherwise specified, are routinely chosen by those skilled in the art.
Example 1
1. Preparation and characterization of phentolamine nanometer slow release injection
And preparing PM-loaded polylactic-co-glycolic acid (PLGA) nanoparticles by adopting an improved emulsion solvent volatilization method. Firstly, the following solutions are weighed and prepared: 5mg PM (TargetMol, T1275) was dissolved in 100. Mu.L DMSO, 100mg PLGA (molecular weight 100kDa, jinan Daida Di) was dissolved in 4mL dichloromethane, and the two phases were mixed as an oil phase; purified water is used as an inner water phase; 40mL of emulsifier polyvinyl alcohol (PVA) was used as the external aqueous phase. The ultrasonic cell pulverizer probe reaches the position below the liquid level, purified water is injected into an oil phase containing fatty acid sorbitan 80 (Span-80) at a constant speed, and ultrasonic power is 200W and 80s, so that W/O colostrum is formed. And injecting the primary emulsion into the PVA outer water phase at a constant speed, and stirring strongly to form the W/O/W compound emulsion. The organic phase dichloromethane was evaporated by low-speed magnetic stirring overnight. The next day, the mixed emulsion was centrifuged at 12000rpm for 10min to collect the nanoparticles, and washed three times with ddH 2 O for resuspension. PLGA concentration was 25mg/mL, colostrum emulsifier Span-80 concentration was 2wt%, PVA concentration was 1wt%, and the volume ratio of inner aqueous phase/oil phase/outer aqueous phase was 1:10:100.
The resulting nanoparticles (Nanoparticles, NPs) were immediately resuspended in 30mL of acetic acid buffer (4 ℃, ph=6.8) containing 0.25mg/mL Hyaluronic Acid (HA) (molecular weight 50-80 kda, merck). The mixture was then stirred for 1h and filtered through a 0.45 μm polycarbonate membrane to obtain HA-PLGA-PM-NPs. By the same method, HA-PLGA-NPs were prepared without adding PM as a control.
After ultrafiltration centrifugation, particle size distribution and zeta potential of HA-PLGA-NPs were measured by room temperature dynamic light scattering (Malven Zetasizer Nano ZS, MALVERN PANALYTICAL, malvern, UK); measuring the drug loading rate and encapsulation efficiency of the HA-PLGA-PM-NPs by adopting a high performance liquid chromatography; the drug release experiment was continued for one month and a drug release profile was drawn.
2. Construction of a model of local cartilage defect in mice
The therapeutic effect of PM on cartilage defects was studied using 9 week old, skeletally mature C57BL/6 male mice. The mice are anesthetized by an isoflurane anesthesia system, the flow is regulated to 300-500 mL/min, after the anesthetic is filled in the induction box for about 1min, the mice are placed in the induction box, the induction box is closed immediately, and the mice are waited for complete anesthesia (the process takes about 2-3 min). The maintenance concentration is regulated, the mice are generally maintained at 1% -1.5%, the mice are taken out from the induction box, the heads/noses of the mice are placed in the anaesthetic mask for fixation, the skin is prepared on the two lower limbs, and the mice are sterilized and prepared for operation.
The inner side of the knee joint of the mouse is provided with a incision which is about 0.5cm in size beside the patella, the skin is separated, the joint capsule is exposed, the patella is gently separated, and the knee joint is flexed to expose the femoral condyle. A blunt needle (0.5 mm diameter) was used to drill holes in the femoral articular surface until the subchondral bone (1 mm depth) was accessed, and blood flow was seen. The right femur was resected exposing the patella but without drilling as a control (Sham, sham surgery group). After flushing the joint with sterile saline to clear the fragments, the patella is repositioned and the joint capsule is closed with absorbable suture. The skin was sutured with 6-0 propylene suture and antibiotics were applied topically. After 1 week of implantation, mice were intra-articular injected with HA-PLGA-PM-NPs, and mice injected with HA-PLGA-NPs served as a control group. Mice were sacrificed 28 days post-surgery and the materials were harvested for histological analysis.
3. Construction of a mouse arthritis model
An OA model was established by the medial meniscus (DMM) instability method using 9 week old, skeletally mature C57BL/6 male mice, anesthetized as above. A near patellar incision was made in the medial knee of the mice about 0.5cm in size, the skin was separated, and after opening the knee capsule, the fat pad was blunt stripped in the intercondylar area, exposing the meniscal tibial ligament of the medial meniscus. The medial meniscal tibial ligament is then severed with a scalpel blade, destabilizing the joint. The joint capsule was closed with 6-0 absorbable suture. The Sham group only revealed ligaments and not severed. After 1 week of molding, mice were intra-articular injected with HA-PLGA-PM-NPs, mice injected with HA-PLGA-NPs served as a control group, and after 12 weeks of surgery, mice were sacrificed and harvested for histological analysis.
4. Human articular cartilage explant in vitro culture model
The experiment was approved by ethics committee of southern medical university, and all tissue acquisitions obtained patient informed consent and adhered to hospital guidelines. Cartilage samples were obtained in unaffected cartilage areas of Osteoarthritis (OA) patients receiving total knee arthroplasty at a third affiliated hospital of the university of south medical science, immediately placed in sterile medium (dmem+10% fbs). Samples were immediately processed in an ultra clean bench, the articular cartilage with the intact surface was selected, cut into blocks of 0.5X0.5 cm 2 size, placed in an orifice plate, and full broth (DMEM+10% FBS+1% P/S) was added to submerge the cartilage pieces. Cartilage explants were divided into three groups, each with complete broth (Control group), complete broth+100 ng/mL IL-1β (IL-1β group), complete broth+100 ng/mL IL-1β+5. Mu.M PM culture (IL-1β/PM injection group), every two days, tissue samples were collected for histological analysis after 7 days, including detection of proteoglycan content in human cartilage explants by Saf-O and toluidine blue staining after 7 days of culture under 3 conditions, and immunohistochemical staining of ACAN in cartilage tissue.
The histological analysis method is as follows:
1. General observational analysis
After drawing materials, the patella of the mouse is separated, the modeling part is exposed, the cartilage defect repair condition is recorded by photographing under a split type microscope, and the cartilage repair score is evaluated through the International Cartilage Repair Society (ICRS). The ICRS score is a quantitative index for evaluating the cartilage repair degree specified by the International cartilage repair institute, and is mainly quantitatively evaluated according to the defect repair degree, the edge integration degree of normal cartilage tissues and three dimensions of general macroscopic appearance, wherein the total score range is 1-12 scores, and the score is graded: grade I (complete normal): 12 minutes; grade ii (substantially normal): 8-11 minutes; grade iii (anomaly): 4-7 minutes; grade IV (severe anomaly): 1-3 minutes. Specific scoring criteria are shown in table 1. Three orthopedics not participating in the experiment in the group were asked to score blindly and to take the mean value for analysis.
TABLE 1 ICRS scoring criteria
2. Histological staining
The samples of the mouse femur and human explant were placed in 4wt% paraformaldehyde for 24h, transferred to 10wt% ethylenediamine tetraacetic acid (EDTA) buffer, placed on a room temperature shaker, decalcified for one month, fresh decalcified liquid was changed daily until the tissue was soft and can be pierced by a sharp needle without resistance, the tissue was transferred to an automatic dehydrator for dehydration, paraffin embedding, and sliced with a lycra automatic microtome at a thickness of 4 μm. Dewaxing and hydrating paraffin sections, specifically, placing the sections in a 65 ℃ oven for baking the sections 1h, melting the tissues and surrounding paraffin, preventing the subsequent experiment from flaking, and then transferring the sections to the following liquids in sequence and soaking for corresponding time: xylene I10 min, xylene II 10min, absolute ethanol I10 min, absolute ethanol II 10min,95% ethanol 5min,90% ethanol 5min,80% ethanol 5min,70% ethanol 5min,50% ethanol 5min, distilled water 5min. Sections were subjected to the following histological staining analysis:
(1) H & E staining
The slices after conventional dewaxing and hydration are immersed in hematoxylin for 3min-8min, washed with running water, differentiated for a few seconds by 1% hydrochloric acid alcohol, washed with running water, returned to blue by 0.6% ammonia water, and washed with running water. The sections were stained in eosin staining solution for 1min-3min to stain the cytoplasm. Sequentially placing the slices into 95% alcohol I5 min-95% alcohol II 5 min-absolute alcohol I5 min-absolute alcohol II 5 min-xylene I5 min-xylene II 5min for dehydration and transparency, taking out the slices from the xylene, slightly airing, and sealing the slices with neutral resin. And observing under a microscope, and performing image acquisition analysis.
(2) Safranin (Saf-O) fast green staining
The slices after conventional dewaxing and hydration are immersed in PBS for 5min, dyed in 1% fixed green solution for 1min, and quickly soaked in deionized water for 1s, and redundant dye liquor is removed. 3% acetic acid solution is fixed and differentiated for 3s-5s, and superfluous liquid on the surface is thrown away. 0.5% safranin O dye liquor is used for dyeing for 3min, and the solution is quickly soaked in deionized water for 1s, so that redundant dye liquor is removed. Differentiation is carried out for 3s-5s in 3% acetic acid solution, and proper color is observed under a mirror. Sequentially placing the slices into 95% alcohol I5 min-95% alcohol II 5 min-absolute alcohol I5 min-absolute alcohol II 5 min-xylene I5 min-xylene II 5min, dehydrating and transparency, air drying in a fume hood, and sealing with neutral resin.
(3) Toluidine blue dyeing
Immersing the slice subjected to conventional dewaxing and hydration in PBS for 5min, sucking toluidine blue by a pipette, dripping or dip-dyeing for 5-30 min, controlling the dyeing process under a mirror, and adjusting specific time according to factors such as the concentration of the dyeing liquid, the room temperature and the like; washing with flowing water for 2min, adding 1% hydrochloric acid alcohol dropwise for differentiation, and removing excessive staining solution in cell nucleus and excessive staining solution in cell plasma until the cells are in purplish blue color and clear visible; washing with running water for 5min after differentiation is completed, washing with distilled water for 1s-2s, and air-drying the washed slices in a fume hood and sealing with neutral resin.
(4) Immunohistochemical staining
Taking a slice after conventional dewaxing and hydration, immersing the slice into a sodium citrate antigen retrieval liquid, and placing the slice in a water bath kettle at 65 ℃ for heating overnight to retrieve the antigen. Sections were washed 3 times with PBS for 5min each. The mixture was treated with a permeation solution (IF buffer:1% BSA+0.1% triton X-100 in PBS) for 30min.3% H 2O2 inactivated endogenous peroxidase, and treated at room temperature in the dark for 15min. The combined pen was circled, and a blocking solution (5% goat serum in IF buffer) was added dropwise and blocked at room temperature for 60min. Primary antibody was instilled and placed in a wet box and incubated overnight at 4 degrees. The sections were removed and rewarmed for 30min, washed 3 times with PBS for 10min each. Dripping HRP-labeled secondary antibody, and treating at room temperature for 60min. PBS was washed 3 times for 10min each. DAB color development, observation under a mirror and timely flushing termination by tap water. Hematoxylin counterstains the nuclei 30 s and water washes back to blue for 15min. Sequentially placing the slices into 95% alcohol I5 min-95% alcohol II 5 min-absolute alcohol I5 min-absolute alcohol II 5 min-xylene I5 min-xylene II 5min for dehydration and transparency, taking out the slices from the xylene, slightly airing, and sealing the slices with neutral resin. And observing under a microscope, and performing image acquisition analysis.
The experimental results are as follows:
1. construction of long-lasting PM-sustained-release articular cavity delivery system
The PLGA nanoparticle loaded with PM is successfully prepared by an improved emulsion solvent evaporation method, HA is further coated to form HA-PLGA-PM-NPs, the result is shown in table 2, the average particle size is 245nm, zeta potential (zeta potential) is-31.5 mV, encapsulation rate (Encapsulation effciency) is 90.3%, and drug loading rate (Drug Loading) is 4.5%. The drug release curve is shown in figure 1, and shows that the HA-PLGA-PM-NPs can release about 25% in the initial 12h, can meet the high-concentration drug environment in the initial administration period, release PM slowly and continuously from the nanoparticles in 7 days later, and release about 60% in a cumulative way after 7 days, and the release curve is stable.
TABLE 2 HA general characterization of PLGA-PM-NPs
2. HA-PLGA-PM-NPs injection can promote repair of local articular cartilage defect of mice
As shown in fig. 2, the mice were sampled to expose the molding area and observed under a split microscope, and the defects of the HA-PLGA-NPs injection control group failed to heal completely, the cartilage pits were visible in the center, and the edges of the pits were white clustered fibrous tissue, which was poorly integrated with the surrounding tissues. Most of the sample cartilage defects in the HA-PLGA-PM-NPs injection group are well recovered, the defect area is highly consistent with the surrounding cartilage or slightly concave, the new tissue is hyaline cartilage tissue, and the new tissue is well integrated with the surrounding tissue. The ICRS score was generally significantly higher than the control, and the differences were statistically significant.
Histological analysis showed that the HA-PLGA-NPs injection control group showed a visual fibrous tissue repair after surgery compared to normal mouse cartilage, with the new tissue being lower than the surrounding cartilage and poorly integrated with the surrounding tissue. The HA-PLGA-PM-NPs injection group can be used for ensuring that the new tissue is highly consistent with the surrounding cartilage and well integrated with the surrounding tissue.
3. HA-PLGA-PM-NPs can inhibit the progression of osteoarthritis
As shown in fig. 3, the detection of the materials obtained after 12 weeks of the model formation of arthritis shows that the cartilage damage of the mice injected with the HA-PLGA-NPs is obvious, the safranin O staining of the cartilage tissue is obviously weakened, obvious cartilage matrix degradation occurs, the cartilage matrix of the mice injected with the HA-PLGA-PM-NPs is not obviously degraded, and the safranin O staining is similar to that of the mice injected with the artificial operation group, so that the cartilage tissue is not obviously damaged, which indicates that the HA-PLGA-PM-NPs can inhibit the progress of osteoarthritis.
4. HA-PLGA-PM-NPs can resist cartilage degradation under inflammatory stimulus
As shown in fig. 4, in the human cartilage explant in vitro culture model, after stimulation with IL-1 β, it was seen that the edge cartilage matrix of the explant was partially degraded, showing that the coloration of the side of the explant near the edge became light when stained with safranin and toluidine blue, while the cartilage matrix remained uniformly and deeply colored from edge to inside when PM was present.
Immunohistochemical detection showed that the number of positive cells of explant ACAN treated with IL-1β was significantly reduced, and that when IL-1β and PM were present simultaneously, the number of positive cells of ACAN was similar to that of the control group. Experiments on human bone cartilage explants prove that PM can resist IL-1 beta-induced degradation of cartilage surface matrix, and further prove that PM has a protective effect on cartilage tissues in an inflammatory state.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. The scope of the application is, therefore, indicated by the appended claims, and the description may be intended to interpret the contents of the claims.

Claims (10)

1. The preparation method of the phentolamine nanometer slow-release injection is characterized by comprising the following steps:
dissolving phentolamine in a first oil phase solvent, dissolving an oil phase matrix in a second oil phase solvent, and mixing to prepare an oil phase;
adding an inner water phase into the oil phase to prepare water-in-oil colostrum;
adding the water-in-oil colostrum into an external water phase to prepare water-in-oil-in-water compound emulsion; removing the first oil phase solvent and the second oil phase solvent, centrifuging, and collecting phentolamine-loaded nanoparticles;
the nanoparticles were washed with water and resuspended in buffer solution, stirred and filtered.
2. The method for preparing phentolamine nanometer slow release injection according to claim 1, wherein the oil phase matrix is an amphiphilic graft copolymer; and/or
The external water phase is a nonionic emulsifier; and/or
The oil phase also includes an emulsifier.
3. The method for preparing phentolamine nanometer slow release injection according to claim 2, wherein the oil phase matrix is polylactic acid-glycolic acid copolymer; and/or
The outer water phase is polyvinyl alcohol; and/or
The oil phase also includes the fatty acid sorbitan 80.
4. The method for preparing phentolamine nanometer slow release injection according to claim 1, wherein the first oil phase solvent is dimethyl sulfoxide; and/or
The second oil phase solvent is dichloromethane; and/or
The internal aqueous phase is water.
5. The method for preparing phentolamine nanometer slow release injection according to claim 1, wherein the volume ratio of the inner water phase, the oil phase and the outer water phase is (0.5-1.5): (5-15): (80-120).
6. The method for preparing phentolamine nanometer slow release injection according to claim 1, wherein the buffer solution is a mixture of a viscoelastic lubricant and an acetic acid buffer solution.
7. The method for preparing phentolamine nanometer slow release injection according to claim 6, wherein the viscoelastic lubricant is hyaluronic acid.
8. The phentolamine nanometer slow release injection prepared by the preparation method of the phentolamine nanometer slow release injection according to any one of claims 1-7.
9. The use of phentolamine nanometer slow release injection as defined in claim 8 in the preparation of medicament for treating articular cartilage diseases.
10. The use according to claim 9, wherein the route of administration of the medicament is by intra-articular injection.
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