CN108379582B - Preparation method of dexamethasone magnetic microspheres - Google Patents
Preparation method of dexamethasone magnetic microspheres Download PDFInfo
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
- A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
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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
- A61K47/30—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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Abstract
The invention provides a preparation method of dexamethasone magnetic microspheres, belonging to the technical field of dexamethasone application, wherein P L GA is taken as a carrier shell, dexamethasone is taken as a drug core, and Fe is taken as3O4The nano-powder is a magnetic core, dichloromethane is an organic phase, polyvinyl alcohol (PVA) is an aqueous phase, and the nano-powder is prepared by an emulsification-solvent evaporation method (S/O/W). In clinical application, the microspheres are injected to an affected part, a dynamic magnetic field is applied in vitro, dexamethasone is gradually released to play an anti-inflammatory role, the magnetic microspheres vibrate under the action of the external dynamic magnetic field to play a role in improving local circulation and promoting absorption of inflammatory products, in addition, the local part can play a physiotherapy role, and chronic soft tissue pain can be treated by the principle.
Description
Technical Field
The invention belongs to the technical field of dexamethasone application, and particularly relates to a preparation method of dexamethasone magnetic microspheres.
Background
Dexamethasone, also known as fluorometholone, dexamethone, is a glucocorticoid. The derivatives of the compound have hydrocortisone, prednisone and the like, have the pharmacological actions of anti-inflammation, antitoxic, antiallergic and antirheumatic, and are widely used clinically. Is easy to be absorbed by digestive tract, the plasma T1/2 is 190 minutes, the tissue T1/2 is 3 days, and the peak values of blood concentration are reached at l hours and 8 hours respectively after dexamethasone sodium phosphate or dexamethasone acetate is injected into muscle. The product has lower plasma protein binding rate than other corticoid drugs, and 0.75mg of the product has anti-inflammatory activity equivalent to 5mg of prednisolone. The adrenocortical hormone medicine has stronger anti-inflammatory, antiallergic and antitoxic effects than prednisone, has light water-sodium retention and potassium discharge promoting effects, and can be injected into muscle or dripped for inhibiting pituitary-adrenal gland.
Dexamethasone has the following effects: (1) anti-inflammatory action: can reduce and prevent the tissue reaction to inflammation, thereby reducing the inflammation expression. Hormones inhibit the accumulation of inflammatory cells, including macrophages and leukocytes, at sites of inflammation and inhibit phagocytosis, the release of lysosomal enzymes, and the synthesis and release of chemical mediators of inflammation; (2) immunosuppressive action: including preventing or inhibiting cell-mediated immune responses, delaying allergic responses, reducing the number of T lymphocytes, monocytes, eosinophils, reducing the binding capacity of immunoglobulins to cell surface receptors, and inhibiting the synthesis and release of interleukins, thereby reducing the conversion of T lymphocytes into lymphoblasts and reducing the spread of the primary immune response. Can reduce the passage of immune complex through the basement membrane and reduce the concentration of complement components and immunoglobulin.
Polylactic-co-glycolic acid (poly-lactic-co-glycolic acid), P L GA, is formed by random polymerization of two monomers, namely lactic acid (P L A) and glycolic acid, is a degradable functional polymer organic compound, has good biocompatibility, no toxicity and good encapsulation and film forming properties, and is widely applied to the fields of pharmacy, medical engineering materials and modern industry.
Chronic soft tissue pain is the most common and most common pain symptom, and refers to pain and related symptoms caused by injury of human body soft tissue such as muscle, ligament, fascia, tendon, synovium, fat, joint capsule and the like, and the pain is caused by aseptic inflammation, fibrous tissue hyperplasia, inflammatory tissue adhesion, degeneration and contracture of the soft tissue caused by acute injury or chronic strain, and the pain is most common in clinic. The pain of soft tissue is hard to be decocted if the pain is slight, and the labor capacity and the self-care ability of life can be lost if the pain is serious. It not only affects the patient's ability to work, diet, sleep, mood, and even major depression. Crofford's study showed that now 20% -25% of the population worldwide suffered from chronic pain, with about 10% of the population suffering from chronic widespread pain. These patients are more likely to develop another more concentrated pain, e.g., inflammatory pain/degenerative arthritis, with a 4-fold higher incidence of fibromyalgia than normal. Therefore, it is a serious research topic worldwide, like cancer and coronary heart disease.
At present, opioids, nonsteroidal anti-inflammatory drugs and glucocorticoids are the mainstream of current pain treatment. The inflammatory reaction is often an important cause and influencing factor of pain, and the characteristic that opioid drugs are easy to have pain tolerance and strong addiction is achieved, and glucocorticoid becomes the preferred drug category of the subject due to the characteristics of anti-inflammation, antitoxic, antishock and immunosuppression, wherein the synthetic hormone dexamethasone has higher cost performance as the research target.
A clinically common protocol is a single injection of dexamethasone in combination with other drugs, which results in virtually multiple injections of dexamethasone. It is well known that glucocorticoids, if used in large quantities for a long period of time, are prone to adverse reactions. For example, hypertension, hyperglycemia, femoral head necrosis, fungal infection, and peptic ulcer, and more serious patients may have mental symptoms. And the single administration has the defects that the stable drug concentration can not be maintained at the pathological change part, the concentration suddenly rises and falls, and the drugs can not be well utilized to effectively treat diseases.
How to use dexamethasone in chronic soft tissue pain to achieve better treatment effect is a hot research focus of technicians in the field.
Disclosure of Invention
In order to overcome the defects that more adverse reactions are brought to patients in the process of treating chronic soft tissue pain in the prior art and the patients cannot be effectively treated, the invention provides a preparation method of dexamethasone magnetic microspheres, which takes P L GA as a carrier shell, dexamethasone as an inner drug core and Fe3O4The nano-powder is a magnetic core, dichloromethane is an organic phase, polyvinyl alcohol (PVA) is an aqueous phase, and the nano-powder is prepared by an emulsification-solvent evaporation method (S/O/W). In clinical application, the microspheres are injected to an affected part, a dynamic magnetic field is applied in vitro, dexamethasone is gradually released to play an anti-inflammatory role, the magnetic microspheres vibrate under the action of the external dynamic magnetic field to play a role in improving local circulation and promoting absorption of inflammatory products, in addition, the local part can play a physiotherapy role, and chronic soft tissue pain can be treated by the principle.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a preparation method of dexamethasone magnetic microspheres comprises the following steps:
(1) weighing P L GA and Fe according to the feeding ratio3O4Nanopowder, dexamethasone powder;
(2) adding P L GA into dichloromethane, shaking to dissolve completely, and adding Fe3O4Oscillating and mixing the nano powder;
(3) adding dexamethasone powder into the step (2), and oscillating until the dexamethasone powder is completely dissolved;
(4) slowly dripping the solution obtained in the step (3) into the PVA solution which is subjected to ice bath by using a dropper, stirring and volatilizing until dichloromethane is completely volatilized;
(5) and (4) filtering the solution obtained in the step (4), collecting microspheres, washing with deionized water, and freeze-drying to obtain the dexamethasone magnetic microspheres.
Wherein P L GA is carrier shell, dexamethasone is drug core, and Fe3O4The nano-powder is a magnetic core, dichloromethane is an organic phase, polyvinyl alcohol (PVA) is an aqueous phase, and the nano-powder is prepared by an emulsification-solvent evaporation method (S/O/W).
P L GA is artificially synthesized polymer and is selected as important auxiliary material, because of its advantages of 1, moderate price, large batch production and application and clinic, 2, convenient use, intramuscular and subcutaneous administration of microsphere injection, 3, high stability, difficult dissolution in water and gastric acid, reduction of loss of first pass effect, difficult enzyme degradation, strong controllability, 4, safety, no toxicity, metabolizability, no rejection reaction in vivo, lactic acid and glycolic acid as metabolites, direct discharge of carbon dioxide and water as final products after tricarboxylic acid cycle of the above products, and in order to increase the targeting property of P L GA, the invention adds certain magnetism (Fe is specially adapted to the invention3O4Nanopowder), so the microspheres can be used for more targeted treatment of diseased parts under intervention of magnetic field, enhance utilization rate and treatment effect of medicine, and reduce side effect caused by reduced medicine intakeThe rate of generation.
The most common pain in clinic is inflammatory pain, in the treatment process, steroid hormones become common clinical medicines due to the anti-inflammatory effect of the steroid hormones, but if the hormones are applied in large quantities for a long time, a large number of heavy adverse reactions or complications can be caused, which is a pain point which is difficult to balance in the treatment process, and the appearance of the P L GA magnetic microspheres can better solve the problem that the microspheres are suitable for manufacturing protein or peptide medicines, particularly hormones, which need to be taken for a plurality of times for a long time.
Preferably, in the step (1), the feeding ratio is (1:3) - (1:5), and is equal to (dexamethasone mass)/(dexamethasone mass + P L GA mass + Fe)3O4Nanopowder mass).
Preferably, in the step (2), the P L GA is prepared from lactic acid and glycolic acid according to the mass ratio of 50:50, the mass concentration of the P L GA after dichloromethane is added is 1:20, and then intermittent vortex oscillation is adopted for 10min until complete dissolution.
More preferably, in step (2), Fe3O4The mass ratio of the added amount of the nano powder to the P L GA is 4:1, and Fe is added3O4And performing ultrasonic oscillation for 1-2min after the nano powder is prepared.
Preferably, in the step (3), after dexamethasone powder is added, ultrasonic oscillation is carried out until the dexamethasone powder is completely dissolved, and stirring is carried out by a homogenizing stirrer at 10000r/min in the ultrasonic process.
Preferably, in the step (4), the solution is slowly dropped into the PVA solution which is ice-cooled for 1 hour and has the mass fraction of 2% by using a dropper at the speed of 30drops/min, and the PVA solution is controlled to volatilize for 4 hours at the rotation speed of 1800r/min until the dichloromethane is completely volatilized.
Administration is also a considerable problem in order to improve the therapeutic effect. It is well known that topical administration has a higher bioavailability and better efficacy than intravenous administration. Therefore, the latter animal experiments of the present invention have selected topical administration. The administration mode has certain requirements on the particle size of the microspheres, because good needle penetration is needed, the diameter of the microspheres is ideally less than 125um, and the diameters of the microspheres prepared by the method are all about 30um and far less than the requirements. And through a large amount of clinical experiments of the inventor, the medicines of all the batches can pass through a standard 5# (the diameter is 0.5mm) needle, in other words, the needle of a syringe with the volume of more than 2.5ml can pass through the needle well.
The method is characterized in that dexamethasone is not easily dissolved in water, a solvent evaporation method (S/O/W) is selected, wherein S (solid phase) refers to dexamethasone powder, O (oil phase) refers to dichloromethane, W (water phase) refers to Polyvinyl Alcohol (PVA), the primary factors influencing the diameter and appearance of the microspheres in the preparation process are four, wherein the primary factors influencing the diameter and appearance of the microspheres in the emulsification and evaporation process are one, the diameter of the microspheres is related to the rotation speed in the emulsification and evaporation process, for example, the rotation speed selected in the initial dichloromethane evaporation process is 1000r/min, the diameter of the prepared microspheres is mostly 100-150um, even a part of the microspheres exceeds 200um, the diameter of the microspheres gradually decreases to the current 30um after the adjustment of 1500r/min and 1800r/min, the diameter and appearance of the microspheres are related to the mass concentration of P L GA in the oil phase, the mass concentration of P L GA is not only capable of influencing the stability of the primary emulsion, but also influencing the uniformity of the primary emulsion in the primary emulsion prepared by the initial emulsion preparation process, and the initial emulsion prepared by the initial emulsion preparation process, the initial emulsion preparation process is not only when the initial viscosity of PVA is not smaller than the initial emulsion prepared by the initial emulsion viscosity of PVA 2, the initial emulsion prepared by the initial emulsion, the initial emulsion viscosity of PVA 1-10 percent, the initial emulsion is not smaller than the initial emulsion, the initial emulsion viscosity of the initial emulsion prepared by the initial emulsion, the initial emulsion prepared by the initial emulsion of the initial emulsion, the initial emulsion prepared by the initial emulsion of PVA-10-60 percent of the initial emulsion, the initial emulsion of the initial emulsion, the initial emulsion prepared by the initial emulsion, the initial emulsion of the initial emulsion prepared by the initial emulsion of the initial emulsion, the initial emulsion of the initial emulsion is not only when the initial emulsion, the initial emulsion prepared by the initial emulsion of the initial emulsion prepared by the initial emulsion is not only when the initial emulsion of the initial emulsion is not only when the initial emulsion of the initial emulsion is not only when the initial emulsion is not more easily formed by the initial emulsion, the initial emulsion of the initial emulsion, the initial emulsion prepared by the initial emulsion of the initial emulsion prepared by the initial emulsion of the initial emulsion, the initial emulsion of the initial emulsion is not more easily formed by the initial emulsion prepared by the initial emulsion of the initial emulsion prepared by the initial emulsion of the initial emulsion.
In addition, unlike other microspheres, it is the targeted type of magnetic microsphere. The drug-loaded microspheres can migrate to the focus under the guidance of a magnetic field, gradually degrade and release drugs at the focus part, and maintain stable treatment concentration at the focus part for a long time, so that the cancer cells which are not young are gradually atrophied, and have no chance of growing and recovering, so that the cancer cells are slowly attenuated until being died. When the magnetic microsphere is applied to the treatment of local inflammation, the number of the magnetic microspheres entering a focus is increased by 23 percent within 48 hours and is quickly increased to 60 percent compared with the common microspheres under the guidance of a magnetic field, and the smaller the diameter of the microspheres is, the more obvious the increase is. Therefore, with the progress of the microsphere preparation process, the increase quantity and the increase speed of the magnetic microspheres in the focus of later-stage cases under the guidance of a magnetic field are more obvious than those of the common microspheres, so that the microsphere has more definite clinical curative effect.
The microspheres have the same slow release property and targeting property as other magnetic microspheres, are concentrated at a focus part under the guidance of a magnetic field after local injection, and gradually and slowly release dexamethasone along with the slow degradation of P L GA, so that a relatively stable drug concentration can be achieved.
Drawings
FIG. 1 is a morphology of dexamethasone magnetic microspheres prepared in the example of the invention under a microscope, wherein (a), (b) and (c) correspond to the respective dosage ratios of 1:3,1:4 and 1: 5.
Detailed Description
The invention will be further described with reference to the accompanying drawings in which:
1. preparation of dexamethasone magnetic microspheres:
the microspheres are prepared by an S/O/W method according to P L GA/Fe3O4The ratio of the raw materials is 4:1, and the raw materials are mixed according to the feed ratio of 1:3,1:4 and 1:5 (feed ratio is dexamethasone mass): dexamethasone mass + P L GA mass + Fe3O4Nanopowder), calculating the specific mass, weighing, adding weighed P L GA into 3ml dichloromethane (O), oscillating for 10min at 3000r/min with intermittent vortex oscillator, and dissolving weighed Fe3O4Dissolving the nano powder in the solution, uniformly mixing by ultrasonic oscillation for 1min, dissolving 100mg of dexamethasone powder (S) in dichloromethane, performing ultrasonic oscillation until the dexamethasone powder is completely dissolved, using a homogenizing stirrer for 10000r/min, then slowly dropping the dichloromethane mixed solution into 30ml of 2% PVA solution which is ice-bathed for 1h by using a dropper according to the speed of 30drops/min, rotating speed of 1800r/min, and volatilizing for 4h until dichloromethane is completely volatilized. After the microspheres are solidified, filtering the microspheres through a filter membrane under vacuum and negative pressure, collecting the microspheres, washing the microspheres with deionized water, and freeze-drying the microspheres to obtain the dexamethasone magnetic microspheres.
2. Dexamethasone magnetic microsphere morphology and characteristics
2.1 morphological Observation
Dexamethasone magnetic microspheres are coated on a glass slide, dispersed by using a proper amount of double distilled water, placed under a 400-fold optical microscope for observation and photographing, the particle size of at least 500 microspheres is measured, and the arithmetic mean diameter is calculated, wherein the particle size of the microspheres is (the length (um) of a microscope stage micrometer coinciding with an eyepiece micrometer/the number of lattices of the microscope stage micrometer coinciding with the eyepiece micrometer) x the number of the lattices of the eyepiece micrometer, the arithmetic mean diameter is the total sum of the diameters of the microspheres/the total number of the microspheres, the span is (D90-D10)/D50, and D90, D50 and D10 respectively represent that the particle sizes of 90%, 50% and 10% of the microspheres are smaller than the arithmetic mean diameter.
2.2 measurement of magnetic responsivity
5mg of dexamethasone magnetic microspheres are taken and added with 100ml of double distilled water to prepare a standard solution, and the distribution and the movement of the microspheres under the magnetic field of 4000GS are observed by a microscope at 40x10 times. After drying, the adsorbed and unadsorbed microspheres were weighed respectively, and the adsorption rate was calculated according to the formula-magnetic adsorption rate (%) -adsorbed microsphere/(adsorbed microsphere + unadsorbed microsphere) x 100%.
2.3 microsphere quality assessment
The quality of the microspheres is evaluated mainly by the following aspects: the sum of the span of the microspheres (S1), yield (S2), encapsulation efficiency (S3), drug loading (S4) was evaluated, and the sum of the mass of the microspheres prepared by the volatilization method used herein, S1+ S2+ S3+ S4.
The calculation of span (S1) was carried out as described in the previous 2.1.1 morphological observation section, and the yield (S2) was × 100% of the total amount of encapsulated drug/total amount of drug delivered to the system and the encapsulation yield (S3) was × 100% of the total amount of encapsulated drug/total amount of all components delivered.
2.4 statistical methods
The average data is represented by x +/-s, statistical software adopts SPSS17.0 version for calculation, pairing t test is adopted for comparison among metering sample groups, one-factor variance analysis is adopted for comparison among multiple samples in the groups, and chi-square test is adopted for comparison between every two counting samples.
2.5 results
2.5.1 morphology and quality assessment of microspheres
The dexamethasone magnetic microspheres prepared in the experiment are black spherical microparticles, are mostly circular under a microscope (40x10), the average diameters (span, S1) of the microspheres with the feeding ratios of 1:3,1:4 and 1:5 are respectively 27.47 +/-8.78 um, 29.66 +/-10.02 um and 30.14 +/-11.2 um, and the shape under the microscope is shown in figure 1. The microsphere yield (S2) of the feed ratio of 1:3,1:4 and 1:5 is respectively (87.02 +/-2.25)%, (87.25 +/-3.14)%, and (84.00 +/-5.53)%; the encapsulation efficiencies (S3) were (76.44. + -. 3.11)%, (78.89. + -. 2.42)%, and (71.33. + -. 5.60)%, respectively; the drug loading rate (S4) is (33.09 +/-1.89)%, (30.33 +/-2.01)%, and (24.29 +/-1.78)%; the summation values (S) are 168.55 + -3.62, 167.47 + -4.15, 149.48 + -4.21, respectively. The administration ratio was 1:5 with a significant statistical difference with P <0.01 compared to the other two groups. See table 1 for details.
Table 1 comparison of the properties of dexamethasone magnetic microspheres (x ± s, n ═ 500)
P <0.01 in comparison with other groups
The mass of microspheres is generally related to the span (S1), yield (S2), encapsulation efficiency (S3), and drug loading (S4), where S1 is inversely proportional and S2, S3, S4 are all proportional. The final batch ratios were 1:3, and the sum of the microsphere masses (S) at 1:4 and 1:5 was 168.55 + -3.62, 167.47 + -4.15 and 149.48 + -4.21, respectively. The sum S of the mass of the microspheres at a feed ratio of 1:5 was statistically significantly different from the other two groups, i.e.the feed ratio of 1:5 was relatively inferior. Compared with the mass summation S of the two groups of microspheres with the feeding ratio of 1:3 and 1:4, the two groups of microspheres have no statistical difference, namely the two groups of microspheres have equivalent mass.
2.5.2 magnetic field responsiveness
Under an optical microscope, when the magnetic field intensity is 4000GS, the dexamethasone magnetic microspheres move irregularly in an ether solution in a direction towards a magnet, and the movement distance is about (80.0 +/-5.0) mm till the bottle wall, which indicates that the magnetic responsiveness and the shape are better.
3. In-vitro release and stability evaluation of dexamethasone magnetic microspheres
3.1 in vitro Release of dexamethasone magnetic microspheres
10mg of each dexamethasone magnetic microsphere with the feeding ratio of 1:3,1:4 and 1:5 are precisely weighed respectively and respectively placed in a flask with a plug, and 50ml of PBS (phosphate buffer salt, PH 7.4) is placed in the flask. Putting the flask into a constant-temperature water bath oscillator, wherein the water level exceeds the plane of the PBS solution, and the oscillation condition is as follows: at 36-37 ℃ and 100-. At 0.5h,1.0h,1.5h,2.0h,2.5h,3.0 hEvery 24 hours thereafter, the supernatant was collected until day 14. 2ml of sample is removed each time, the sample is filled in equal amount after each sampling, and the collected sample is stored in a refrigerator at 4 ℃. The drug concentration was measured and the percent release of the drug was calculated. The formula is Q ═ CtV/m x 100% wherein Q is the cumulative release (%) of the microspheres, CtThe drug concentration of dexamethasone in the PBS solution at t (ug/ml), V is the drug dilution volume (ml), and m is the total mass of dexamethasone in each bottle.
The in vitro accumulation release rates of the dexamethasone magnetic microspheres with the feed ratio of 1:3,1:4 and 1:5 are 79.12%, 32.91% and 40.33% respectively on the 2 nd day; 82.6%, 62.74% and 76.04% on day 7; 86.34%, 66.99% and 97.85% on day 14, see Table 2 for details.
TABLE 2 in vitro cumulative release (%)
3.2 stability testing of dexamethasone magnetic microspheres
Placing microspheres (n is more than or equal to 500) with different feeding ratios in an EP tube respectively, placing the microspheres in a refrigerator with the temperature of 4-8 ℃ for 3 months, observing whether the physical characteristics (the particle size and the appearance under a light lens) are changed on days 1 of 0, 1, 2 and 3 months respectively, and simultaneously detecting the drug loading capacity.
Although the charging ratio of the microspheres put into a refrigerator at 4-8 ℃ is different, the physical properties and the drug loading capacity of the microspheres are not obviously changed within 3 months, namely the microsphere system has better stability, and the details are shown in Table 3.
TABLE 3 stability testing of microspheres at different feed ratios (n.gtoreq.500)
4. Biocompatibility research of dexamethasone magnetic microspheres
Experimental methods
4.1 method for preparing blank magnetic microsphere
Similar to the preparation method of the dexamethasone magnetic microspheres, the preparation method is an emulsification-solvent evaporation method (S/O/W). The only difference is whether dexamethasone was added during the microsphere preparation. After the preparation is finished and freeze-dried, cobalt rays are adopted for irradiation sterilization.
4.2 in vitro hemolysis assay
4.2.1 preparation of test solutions
The sterile blank microspheres and sterile physiological saline are prepared into 4mg/ml mother solution according to a proportion, and the mother solution is respectively diluted into microsphere emulsions with the concentrations of 1mg/ml,2mg/ml and 4mg/ml by using the physiological saline according to multiple times.
4.2.2 preparation of erythrocyte suspensions
Putting 2ml of SD rat whole blood into a glass test tube, removing fibrinogen by a glass stirring rod in a continuous stirring manner, diluting the fibrin-removed blood by 10 times by using physiological saline, uniformly mixing, centrifuging at the rotating speed of 3000r/min for 5min, removing supernatant, and discarding. The lower layer of red blood cells is washed repeatedly with normal saline until the supernatant is colorless, clear and transparent. Finally, the washed red blood cells were prepared into a 2% red blood cell suspension with physiological saline.
4.2.3 hemolysis rate test
Precisely transferring 3 parts of samples with different concentrations, each 0.3ml, and calling the sample as X solution.
A, test sample: 2.5ml of erythrocyte suspension, 2.2ml of physiological saline and 0.3ml of X solution
B, negative control: erythrocyte suspension 2.5ml + physiological saline 2.5ml
C positive control: 2.5ml of erythrocyte suspension and 2.5ml of distilled water
And (2) uniformly mixing the A + B + C samples, immediately placing the samples in a constant-temperature water bath oscillator for incubation for 3H, wherein the water temperature is 36-37 ℃, centrifuging the processed test samples, 3000r/min for 5min, removing supernatant, standing the supernatant At room temperature for 30min, taking the negative control supernatant as a blank, and calculating the hemolysis rate (%) according to the absorbance obtained by spectral scanning At 540nm, wherein the formula is H L ═ At/Apc: (At test sample absorbance; Apc positive control absorbance), the absorbance takes the negative control as the blank standard, the negative control absorbance is less than or equal to 0.03, the positive control absorbance range is 0.8 +/-0.3, and the hemolysis rate (%) < 5%, thus the test samples have no hemolysis.
4.3 in vivo blood compatibility test
The body weight of 3 SD rats was weighed, and the above test mother solutions were injected into the body of each rat via tail vein at a dose of 0.8ml (high dose), 0.5ml (medium dose) and 0.2ml (low dose) per 100g of body weight. 0.5ml of blood is taken before injection and 0.5h, 6h,12h and 24h after injection respectively and is placed in an anticoagulation tube (purple). Routine blood examination was performed.
4.4 histocompatibility test
After the rats were anesthetized by inhalation, vellus hairs on the left back were removed, and 1ml of stock solution of the test article was injected subcutaneously into the experimental group, and 1ml of physiological saline was injected subcutaneously into the control group. And (3) taking the skin and the subcutaneous tissues of the injection part for HE staining after 10 days and 30 days.
4.5 statistical analysis the experimental data were analyzed using SPSS17.0 software, using paired t-tests between groups, and single factor analysis of variance for comparisons before and after groups, with P <0.05 being statistically significant.
4.6 results:
4.6.1 in vitro hemolysis assay
After the in vitro hemolysis experiment is carried out on the sample to be detected, the absorbance of the sample with the concentration of 1mg/ml,2mg/ml and 4mg/ml is respectively 0.006,0.011 and 0.032; the hemolysis rates were 0.5%, 1.2%, and 3.3%, respectively (the absorbance of the results for the negative and positive controls was 0.003 and 0.802, respectively, and the hemolysis rates were 0% and 99.9%, respectively), and the above samples were considered as the standard considerations that the test samples had no hemolysis when the hemolysis rate was less than 5%. See table 4 for details.
TABLE 4 Absorbance and hemolysis (%, n ═ 3) for different samples tested
4.6.2 blood routine results
The effect of injecting different doses of microsphere test sample into rats on blood routine is as follows:
(1) the change in the white blood cell, red blood cell and platelet counts at 0.5h, 6h,12h,24h after injection in the high, medium and low dose groups of rats was within the normal range compared to the pre-dose white blood cell, red blood cell and platelet counts and the changes were not statistically significant, as detailed in table 5.
(2) Compared with the volume of the red blood cells and the platelets before administration, the change of the red blood cells and the platelets at 0.5h, 6h,12h and 24h after injection of the rats in the high, middle and low dose groups is within a normal range, and the change is not statistically significant, and the details are shown in a table 6.
(3) Compared with the classification (%) of the leukocytes before administration, the change of the neutrophils (%) at 0.5h after the injection of the rats in the high, medium and low dose groups has no statistical significance, but the neutrophils (%) at 6h,12h and 24h are obviously reduced; the change of lymphocyte (%) is on the contrary-0.5 h after the injection of rats of high, medium and low dose groups, the change has no statistical significance, but obviously increases in 6h,12h and 24 h; the change of the monocyte (%) in 12h and 24h has statistical significance; eosinophils (%) were significantly reduced at 0.5h, 6h,12h, and 24h post-injection, and P < 0.01; the change in basophils is not statistically significant; see table 7 for details above.
TABLE 5 Effect of different dosages of microspheres on blood cell counts
TABLE 6 Effect of different dosages of microspheres on blood cell volume
TABLE 7 Effect of different doses of microspheres on leukocyte Classification (%)
*P<0.05,**P<0.01
4.6.3 histocompatibility results
(1) The skin of the rat after subcutaneous injection has no signs of skin injury such as red swelling, ulceration, infection and the like on the 10 th day and the 30 th day.
(2) Results of pathological section (HE staining) of rat injection site
Pathological sections of rat injection sites (HE staining) had a small amount of inflammatory cells in a 40x 10-fold visual field at day 10, increased inflammatory cells earlier at day 30, and had fibrocyte proliferation and encapsulation. But no significant difference was observed compared to the control group.
5. Pharmacodynamic study of dexamethasone magnetic microsphere
Experimental methods
5.1 Experimental animals and groups
Male SD rats 36 of SPF rating of 220-250g mass were selected for successful modeling only. The groups were randomized into 6 groups of 6 individuals. Respectively microsphere group, microsphere + magnetotherapy group, rice + magnetotherapy group, positive control group, and magnetotherapy group. The model rats are raised in a 12-hour day-night alternating environment at room temperature in a quiet environment. The drinking water and the eating can be taken at will according to the needs. Water is prohibited for 12h before anesthesia.
5.2 Experimental methods
5.2.1 Process for preparing medicine
Dexamethasone microsphere solution: 100ug PVA and 10ml water for injection were weighed separately, mixed and dissolved completely in a water bath at 60 ℃ for 30 min. Weighing 10mg of self-made dexamethasone microspheres, putting the self-made dexamethasone microspheres into the PVA solution twice, and carrying out ultrasonic oscillation for 2min after each microsphere is put, so that the self-made dexamethasone microspheres are uniformly dispersed.
Dexamethasone sodium phosphate solution: dexamethasone sodium phosphate injection 5mg/ml is added with 4ml water for injection to dilute 5 times to 1 mg/ml.
5.2.2 rat anesthesia method
After the rat is placed in an inhalation anesthesia box, 3% -5% of sevoflurane (Henry) is inhaled, the oxygen flow is 2L/min, and the inhalation time is 5-10min until the rat is in a moderate anesthesia state, namely pain disappears, righting reflex disappears, skeletal muscle relaxes, and the breath is uniform, stable and gentle.
5.2.3 methods of drug injection
Microsphere preparation: a1 ml syringe sucks 0.3ml of dexamethasone microsphere solution (about 0.1mg of dexamethasone), and the anesthetized rat is placed on an operating table and is injected into the foot sole center part of the left hind paw of the rat slowly and subcutaneously. After injection, rats are independently placed in other mouse cages.
Dexamethasone sodium phosphate: a1 ml syringe sucks 0.1ml of dexamethasone microsphere solution (about 0.1mg of dexamethasone), and the anesthetized rat is placed on an operating table and is injected into the foot sole center part of the left hind paw of the rat slowly and subcutaneously. After injection, rats are independently placed in other mouse cages.
5.2.4 magnetotherapy method
The rat is placed above the magnetic field, 10cm away from the sole of the rat, and the distance between the magnetic field and the metal object is more than 30 cm. The treatment is carried out 2 times a day, each time is 30min, the interval between the two treatments is 6 hours, and the specific treatment time is 10:00-10:30 and 16:00-16: 30.
5.2.5 method for measuring mechanical pain threshold
And 2, 4, 6, 8 and 10 days after injection, the left hind paw arch of the rat was measured using Von Frey cellosilk. Before measurement, the rat is put into a flip organic glass fixer with a metal bar grid at the bottom for 30min in advance, so that the rat adapts to the environment. Measured after quiet, the time is between 15:00 and 17: 00.
5.2.6Von Frey fiber measuring method
The mechanical pain threshold is measured by using classical Von Frey fiber filaments, the order of the fiber filaments is measured from 2.0g, 4.0g, 6.0g, 8.0g, 10.0g, 15.0g and 26g, each type of filament is measured 5 times, if 3 times of measurement are positive reaction, the fiber filament is reduced to 1.4g, if positive reaction is still generated, the measurement is continued to be reduced to 1.0g, on the contrary, if 3 times of measurement are negative, the fiber filament is increased to 4.0g, and the like, 26g is used, the measurement part is the left and rear plantar arch of a rat, each time lasts for 5s, the interval time between two times of stimulation is 10s, the measurement strength is that the fiber filament is bent to be approximately right-angled, positive results are that the rat generates avoidance reflex on the fiber filament, such as foot contraction and sole licking, and the like, negative results are recorded as positive results ' X ' and negative results are ○ '.
5.2.7 local pathology in rat
On the 10 th day of microsphere/sodium phosphate injection/CFA injection, 2 molding rats were randomly selected, and after sevoflurane anesthesia (method 2.2.1), 0.3cm to 0.5cm of skin was taken from the left hind paw arch, and after neutral formalin fixation, HE staining was performed.
5.2.8 rat affected foot appearance evaluation method
The expression of the inflammation is divided into the following three grades: red, no red and swollen, and the number of them was registered according to the actual condition. The panelists were not aware of the rat group and were all evaluated by the same person throughout.
5.3 Observation of indices before and after treatment, pathological sections of rats with different groups of models on days 2, 4, 6, 8 and 10, appearance of affected feet, mechanical pain threshold and day 10, and comparison between the pathological sections.
5.4 statistical methods
Statistical analysis was performed using the SPSS17.0 software, and the mechanical pain threshold of rats was expressed as (x. + -. s). The counting data is compared by chi-square test. Single factor analysis of variance was used for the pre-and post-comparisons within the metrology data sets. The paired T test was used for group comparisons. P <0.05 is a significant statistical difference.
5.5 results
5.5.1 comparison of appearance of affected feet during treatment of rats in different groups of model rats
The affected feet of the model rats before treatment are all red and swollen-like inflammatory appearances. Comparing the appearance of the affected feet of the rats from the 2 nd to the 10 th days, the microsphere group and the rice group have no obvious difference; the microsphere and magnet therapy group has no obvious difference compared with the rice and magnet therapy group; the magnetic therapy group and the positive control group have no obvious difference. See tables 8-12 for details.
TABLE 8 appearance of feet on day 2 of treatment of rats (n ═ 36)
TABLE 9 treatment of rats with the appearance of a 4-day foot (n ═ 36)
TABLE 10 appearance of feet on day 6 of treatment of rats (n ═ 36)
TABLE 11 appearance of feet on day 8 of treatment of rats (n ═ 36)
TABLE 12 appearance of feet on day 10 of treatment of rats (n ═ 36)
5.5.2. Change of mechanical pain threshold of affected foot before and after rat treatment
Compared with the groups before treatment, the groups except the positive control group and the magnetic therapy group are obviously improved on the 4 th day and the 6 th day; on the 4 th and 6 th days, the pain threshold of the microsphere and magnet therapy group is obviously increased compared with that of other groups. The pain threshold of the microsphere group and the microsphere and magnet therapy group on the 8 th day is obviously increased compared with other groups, and P is less than 0.05. The details are given in the table below.
P <0.05 compared to other groups
5.5.3. Pathological changes of affected foot (HE) before and after treatment of rat
Compared with the positive control, the inflammatory cells of other groups except the magnetic therapy group are obviously reduced. The improvement degree is most obvious in microsphere group and microsphere and magnetic therapy group.
Finally, the preparation method of the dexamethasone magnetic microspheres is an emulsion solvent evaporation method (S/O/W), the preparation conditions are P L GA (50/50), the mass concentration of P L GA is 1:20, the mass fraction of PVA is 2%, the rotation speed is 1800r/min, the dexamethasone magnetic microspheres prepared by the invention are black spherical micro particles, the dexamethasone magnetic microspheres are mostly circular under a microscope (40X10), the average diameters (span, S1) of the microspheres with the feeding ratios of 1:3,1:4 and 1:5 are respectively (27.47 +/-8.78) um, (29.66 +/-10.02) um, (30.14 +/-11.2) um. yield (S2) is respectively (87.02 +/-2.25)%, (87.25 +/-3.14)%, (84.00 +/-5.53)%, the encapsulation ratios (S3) are respectively (76.44 +/-3.11), the dispersion rate is respectively, (89.42)%, (80)%, the dispersion rate is respectively) (80.42)%, (S62) is equal to 80.42)%, (80)%, (80.42)%, and the dispersion rate is preferably (33.42)%, (S42)%, (33.42)% of the dispersion rate is larger than 25) and the dispersion rate is larger than 25.42.42.42)% of the dispersion rate is equal to 80).
Meanwhile, dexamethasone magnetic microspheres with the feeding ratio of 1:3,1:4 and 1:5 are released in vitro, and the cumulative release on the 2 nd day is 79.12%, 32.91% and 40.33% respectively. The burst release of the microspheres with the feeding ratio of 1:3 is obvious, and the drug loading of the microspheres with the feeding ratio of 1:5 is the lowest, so the microspheres with the feeding ratio of 1:4 are more ideal microspheres.
Thirdly, the biocompatibility of the microspheres in the rat body is that the feeding ratio is 1: 4: the hemolysis rates of microsphere test products with different concentrations in an in vitro hemolysis experiment are all lower than 5 percent and meet the national standards made for implants; the in vivo blood compatibility and the tissue compatibility both meet the requirements.
Finally, after dexamethasone sodium phosphate injection and dexamethasone P L GA magnetic microspheres (1:4) are respectively injected on the soles of the affected feet of the model rats and are subjected to magnetic therapy, the affected feet of the model rats are divided into microsphere groups, microsphere + magnetic therapy groups, floor rice + magnetic therapy groups, positive control groups and magnetic therapy groups, compared with the effects of the floor rice (+ magnetic therapy) groups, the mechanical pain threshold of the previous 2 days has no statistical difference, from the 4 th day, the mechanical pain threshold of the microsphere + magnetic therapy groups is obviously higher than that of the other groups, at the moment, no statistical difference exists among the microsphere groups, the floor rice groups and the floor rice + magnetic therapy groups, the advantages of the mechanical pain threshold of the microsphere + magnetic therapy groups are continued until the 10 th day of treatment is finished, and the pathological results and the trend of physical appearance change are the same.
Finally, it should be noted that: it should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.
Claims (1)
1. A preparation method of dexamethasone magnetic microspheres is characterized by comprising the following steps:
(1) weighing P L GA and Fe according to the feeding ratio3O4Nanopowder, dexamethasone powder;
(2) adding P L GA into dichloromethane, shaking to dissolve completely, and adding Fe3O4Oscillating and mixing the nano powder;
(3) adding dexamethasone powder into the step (2), and oscillating until the dexamethasone powder is completely dissolved;
(4) slowly dripping the solution obtained in the step (3) into the PVA solution which is subjected to ice bath by using a dropper, stirring and volatilizing until dichloromethane is completely volatilized;
(5) filtering the solution obtained in the step (4), collecting microspheres, washing with deionized water, and freeze-drying to obtain dexamethasone magnetic microspheres;
wherein in the step (1), the feeding ratio is 1: 4;
the feed ratio is equal to (dexamethasone mass)/(dexamethasone mass + P L GA mass + Fe3O4Nanopowder mass);
in the step (2),
the P L GA is prepared from lactic acid and glycolic acid according to the mass ratio of 50:50, the mass concentration of the P L GA is 1:20 after dichloromethane is added, and then intermittent vortex oscillation is adopted for 10min until the P L GA is completely dissolved;
Fe3O4the mass ratio of the added amount of the nano powder to the P L GA is 4:1, and Fe is added3O4Performing ultrasonic oscillation for 1-2min after the nanometer powder;
in the step (3), the step (c),
after dexamethasone powder is added, ultrasonic oscillation is carried out until the dexamethasone powder is completely dissolved, and stirring is carried out by a homogenizing stirrer at 10000r/min in the ultrasonic process;
in the step (4), the step (c),
slowly dropping the solution into the PVA solution which is ice-cooled for 1h and has the mass fraction of 2% by a dropper at the speed of 30drops/min, and controlling the PVA solution to volatilize for 4h under the condition that the rotating speed is 1800r/min until dichloromethane is completely volatilized.
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| 微球制剂的研究进展;陈晓慧等;《临床医药实践》;20120731;第21卷(第7期);第534-538页 * |
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