CN108379582A - A kind of preparation method of dexamethasone magnetic microsphere - Google Patents
A kind of preparation method of dexamethasone magnetic microsphere Download PDFInfo
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Abstract
The present invention provides a kind of preparation methods of dexamethasone magnetic microsphere, belong to dexamethasone application technology field, and for this method using PLGA as carrier shell, dexamethasone is drug core, Fe3O4Nanometer powder is magnetic kernel, and dichloromethane is organic phase, and polyvinyl alcohol (polyvinyl alcohol, PVA) is used as water phase, is made by emulsion-solvent evaporation method (S/O/W).By microsphere injections to affected part in clinical application, and apply moving field in vitro, gradually anti-inflammatory effect is played in release to dexamethasone, magnetic microsphere shakes under the action of outer moving field, play the role of improving local circulation, inflammatory products is promoted to absorb, in addition part can play the role of physical therapy, by above-mentioned principle, treatment chronic soft tissue pain can be reached.
Description
Technical field
The invention belongs to dexamethasone application technology fields, and in particular to a kind of preparation side of dexamethasone magnetic microsphere
Method.
Background technology
Dexamethasone also known as dexamethasone, fluorine methylprednisolone, Dexamethasone, are glucocorticosteroid hormones.Its derivative has hydrogen
Change can ground pine, prednisone etc., pharmacological action is mainly anti-inflammatory, antitoxin, antiallergy, antirheumatic, and Clinical practice is wide.Easily
Self-digestion road absorbs, and blood plasma T1/2 is 190 minutes, and tissue T 1/2 is 3, intramuscular injection dexamethasone sodium phosphate or dexamethasone
After acetate peak plasma concentrations were reached respectively at l hours and 8 hours.The product plasma protein binding rate is compared with other corticoid medicines
Object is low, and the anti-inflammatory activity of product 0.75mg is equivalent to 5mg prednisolones.Aeroseb-Dex, anti-inflammatory, antiallergy
It is stronger compared with prednisone with antitoxic action, water-sodium retention and promote row's potassium effect very light, can intramuscular injection or intravenous infusion Pituitary Adrenal is pressed down
It is stronger to make use.
Dexamethasone has following effect：(1) anti-inflammatory effect：The reaction that can mitigate and prevent tissue to inflammation, to mitigate
The performance of inflammation.Hormone inhibits inflammatory cell, including macrophage and leucocyte gathering in inflammation part, and phagocytosis is inhibited to make
With, the release of lysosomal enzyme and the synthesis and release of inflammation chemistry intermediary；(2) immunosuppressive action：Including preventing or pressing down
Cell-mediated immune response is made, the allergic reaction of retardance reduces the number of T lymphocytes, monocyte, acidophic cell
Mesh, reduces the binding ability of immunoglobulin and cell surface receptor, and inhibits the synthesis and release of interleukin, to reduce T
Lymphocyte mitigates the extension of primary immune response to lymphoblastic transformation.Immune complex can be reduced by basilar memebrane,
And the concentration of Complement and immunoglobulin can be reduced.
Poly(D,L-lactide-co-glycolide (poly (lactic-co-glycolic acid), PLGA) is by two kinds of lists
Body --- lactic acid (PLA) and hydroxyacetic acid are polymerized at random, are a kind of degradable functional polymer organic compounds, have
The performance of good biocompatibility, nontoxic, good encystation and film forming, be widely used in pharmacy, medical engineering material and
Modernization industry field.
Chronic soft tissue pain is a kind of most common, most multiple antalgesic, refers to muscle, ligament, fascia, flesh
The disease of pain caused by the soft tissue damage of the human bodies such as tendon, synovial membrane, fat, capsular ligament and related sign, mostly by acute injury
Or caused soft tissue aseptic inflammation, proliferation of fibrous tissue, inflammatory tissue adhesion, denaturation and contracture after chronic strain, to
Pain is generated, clinic is most common with neck-shoulder pain, lumbago and leg pain.Gently then pain is hard to bear for harm of the soft tissue pain to human body, heavy then can lose
Lose labour capacity and self care ability.It not only influences the labour capacity of patient, diet, sleep, mood even major depression.
It is indicated in the research of Crofford, the present whole world has the crowd of 20%-25% to be perplexed by chronic ache, wherein about 10%
Crowd pain be chronic generalized plain.These patients are more likely to occur another pain more concentrated, for example, inflammatory
The patient of pain/degenerative arthritis, the incidence of fibromyalgia are 4 times of normal person.Therefore, it is with cancer, coronary heart disease
Etc. diseases it is the same, be listed in global great research topic.
Currently, opioid drug, nonsteroidal anti-inflammatory agent and glucocorticoid are the mainstreams of pain therapy instantly.And inflammatory
Reaction is often an Important cause of disease and influence factor for pain, and tolerable pain and additive strong spy are easy with opioid drug
Property, glucocorticoid becomes the drug class of this project first choice because of its anti-inflammatory, antitoxin, Hemorrhagic shock and immunosuppressive effect feature
Not, wherein artificial synthesized hormone --- dexamethasone is using cost performance higher as research target.
Clinically common scheme is dexamethasone and the united single injection of other drugs, this is filled in resulting in regard to invisible
The multiple injection of meter Song.It is well known that if glucocorticoid is long-term, a large amount of uses easy to produce adverse reaction.For example, high blood
Pressure, hyperglycemia, caput femoris necrosis, fungal infection, digestive tract ulcer, more severe patient may occur in which mental symptom.And single-dose
The shortcomings that be that cannot maintain stable drug concentration in diseased region, concentration rises sharply rapid drawdown, drug cannot be utilized effective well
Treatment disease.
How dexamethasone is used in chronic soft tissue pain, reach preferable therapeutic effect, be people in the art
The hot spot of member's research.
Invention content
More adverse reaction is brought to patient in order to overcome to treat in chronic soft tissue Pain Process in existing technology, and
The deficiency that cannot effectively treat, the present invention provide a kind of preparation method of dexamethasone magnetic microsphere, and this method is with PLGA
Carrier shell, dexamethasone are drug core, Fe3O4Nanometer powder is magnetic kernel, and dichloromethane is organic phase, polyvinyl alcohol
(polyvinyl alcohol, PVA) is used as water phase, is made by emulsification-evaporation method (S/O/W).In clinical application
By microsphere injections to affected part, and apply moving field in vitro, gradually anti-inflammatory effect is played in release to dexamethasone, and magnetic microsphere exists
It is shaken under the action of outer moving field, plays the role of improving local circulation, inflammatory products is promoted to absorb, in addition part can play reason
The effect for the treatment of can reach treatment chronic soft tissue pain by above-mentioned principle.
The technical solution adopted by the present invention to solve the technical problems is：
A kind of preparation method of dexamethasone magnetic microsphere, the method are：
(1) PLGA, Fe are weighed according to rate of charge3O4Nanometer powder, dexamethasone pulvis；
(2) PLGA is added in dichloromethane, oscillation adds Fe up to being completely dissolved3O4Nanometer powder vibrates mixing；
(3) dexamethasone pulvis is added in step (2), oscillation is until be completely dissolved；
(4) solution that step (3) obtains is slowly dropped into dropper in the PVA solution of ice bath, is stirred, volatilization, until
Dichloromethane volatilizees completely；
(5) solution for obtaining step (4) filters, and collects microballoon, is lyophilized after deionized water washing, you can be prepared ground
Sai meter Song magnetic microspheres.
Wherein, PLGA is carrier shell, and dexamethasone is drug core, Fe3O4Nanometer powder is magnetic kernel, dichloromethane
Alkane is organic phase, and polyvinyl alcohol (polyvinyl alcohol, PVA) is used as water phase, passes through emulsification-evaporation method (S/O/
W it) is made.
PLGA is artificial-synthetic copolymer, selects it as important auxiliary material because it has the advantage that：It, can be with 1. moderate
The making and application of big batch and clinic；2. easy to use, the injection of microballoon can be with muscle and subcutaneous administration；3. stability
Height is not readily dissolved in water and hydrochloric acid in gastric juice, reduces the loss of first pass effect, and is not easy to be degraded by enzymes, and controllability is strong；4. safe and nontoxic, can
Metabolism, in vivo without rejection, and its metabolite be lactic acid and hydroxyacetic acid, the above product after tricarboxylic acid cycle most
Final product is that carbon dioxide and water are expelled directly out in vitro.In order to increase the targeting of PLGA, the present invention adds certain hereby
Magnetism (Fe3O4Nanometer powder), therefore the microballoon can more have diseased region under the intervention in magnetic field the treatment being directed to,
The utilization rate and therapeutic effect of drug can be enhanced, and because reducing the intake of drug to reduce the incidence of side effect.
Clinically our most common pain are inflammatory pain, and in its therapeutic process, steroids are anti-inflammatory because of its
The effect of as common drug clinically, but hormone is if long-term widely apply, and can cause numerous heavier bad
Reaction or complication, this was once a pain spot for being difficult to balance in therapeutic process.And the appearance of PLGA magnetic microspheres
It can preferably solve the problems, such as this --- the microballoon is suitble to make the protein or peptide medicament for needing long-term multiple dosing, especially
It is steroids.
Preferably, in step (1), the rate of charge is (1:3)~(1:5)；Rate of charge is equal to (dexamethasone matter
Amount)/(dexamethasone quality+PLGA quality+Fe3O4Nanometer powder quality).
Preferably, in step (2), the PLGA is by lactic acid and hydroxyacetic acid in mass ratio 50:50 preparations obtain；PLGA
Its mass concentration is 1 after dichloromethane is added:20, later using interruption vortex oscillation 10min, until being completely dissolved.
It is furthermore preferred that in step (2), Fe3O4The addition of nanometer powder and the mass ratio of PLGA are 4:1；Fe is added3O4
Sonic oscillation 1-2min is carried out after nanometer powder.
Preferably, in step (3), after dexamethasone pulvis is added, sonic oscillation is until be completely dissolved, in the process of ultrasound
Middle homogeneous stirring instrument 10000r/min stirrings.
Preferably, in step (4), solution is slowly instilled into ice bath 1h and matter by the speed of 30drops/min with dropper
It measures in the PVA solution that score is 2%, while it is the 4h that volatilizees under conditions of 1800r/min to control PVA solution in rotating speed, until two
Chloromethanes volatilizees completely.
In order to improve therapeutic effect, administering mode is also the problem of being worthy of consideration.It is well known that local administration is given than vein
The bioavilability of medicine is high, and effect is good.So the subsequent zoopery of the present invention has then selected local administration.The administering mode pair
The grain size of microballoon has certain requirement --- because to there is good syringeability, ideal microsphere diameter to should be less than 125um, pass through
The prepared microballoon of the present invention, diameter in 30um or so, it is far be respectively less than more than requirement.And pass through inventor
A large amount of clinical trials, the drug of all of above lot number can by 5# (a diameter of 0.5mm) syringe needle of standard, in other words,
It is exactly the syringe needle of the syringe of 2.5ml or more can well pass through.
Because dexamethasone is not readily dissolved in water, that the present invention selects then is solvent evaporation method (S/O/W), wherein this experiment S
(solid phase) refers to dexamethasone powder；O (oil phase) refers to dichloromethane；And W (water phase) then refers to
For polyvinyl alcohol (Polyvinyl Alcohol, PVA).And during preparation, the main of microsphere diameter and appearance is influenced
There are four factors：First, microsphere diameter is related to rotating speed when emulsification and volatilization.Such as initially dichloromethane volatilization in the present invention
When the rotating speed that selects be 1000r/min, the diameter for making microballoon is mostly 100-150um, or even also part microsphere diameter is more than
200um, after after the adjustment of 1500r/min and 1800r/min, the diameter of microballoon is gradually decreased to present 30um；Second,
The diameter and appearance of microballoon are related with the mass concentration of PLGA in oil phase；The mass concentration of PLGA can not only influence colostric fluid
Stabilization, and influence microballoon homogeneity；If PLGA mass concentrations too low (1:30-1:60), colostric fluid unstability,
The microballoon produced it is in irregular shape, size is inhomogenous；And PLGA mass concentrations excessively high (1:10-1:15) that, produces is micro-
Ball is smaller, but can and lumps product cotton-shaped there are many irregular PLGA.Only PLGA mass concentrations moderate (1:20),
Uniform and smooth sphere can be obtained, diameter range is between both excessively high/low microballoons of mass concentration diameter range；Third,
Water phase PVA mass fractions also have an impact the diameter of microballoon with appearance.The viscosity of PVA solution being associated between colostrum drop
Certain influence.As PVA mass fractions low (0.1%-0.5%), the smaller stability for being not enough to keep colostric fluid of viscosity,
The microsphere diameter produced is larger, and inhomogenous；And when PVA mass fractions high (4%), viscosity is larger and prevents to a certain degree
The mobility of colostric fluid is easy to form the microballoon of diameter bigger compared with when mass fraction is relatively low；Only when PVA mass fractions
When moderate (1%-2%), the diameter of microballoon is fine, and more uniform, meets requirement of experiment.Fourth, the volume ratio of oil phase and water phase
Also the diameter and shape of microballoon can be influenced.Such as when its ratio too low (1:1-1:2) when, though not balling-up or energy balling-up substantially,
Microballoon shell is loose, broken；And work as ratio excessively high (6:1) when, lead to the reduction of colostric fluid surface tension, be easy to be formed larger micro-
Ball；When ratio moderate (2:1-4:1) when, it is 4 that can just form appearance rounding, uniform microballoon, and ratio:It is made when 1
Microsphere diameter is less than 2:Made microballoon when 1, but ratio is 2:When 1, made microballoon size is more uniform.But this hair
It is bright it is middle use the emulsion-solvent evaporation method for being S/O/W to prepare the microballoon, so this factor has no influence, and first three factor exists
It is corrected in time after being had been found to during the preparation method of the present invention.The parameter used respectively in the last present invention is distinguished successively
Its mass concentration is 1 after dichloromethane is added for volatilization revolution 1800r/min, PLGA:20, PVA mass fractions are 2%, above
Microsphere features smooth surface rounding made by condition, size is uniform, good dispersion, and cleansing pin rate is high.
In addition, different from other microballoons is exactly the targeting type of magnetic microsphere.The microballoon for carrying medicine can be in the guiding in magnetic field
It is lower gradually to degrade in lesions position and discharge drug to lesion swimming, stable treatment can be maintained dense for a long time in affected area
Degree makes the still inmature gradual atrophy of cancer cell, the chance do not grown up and recovered lead to cancer cell slow-decay, until disappearing
It dies.If applied to the treatment of local inflammation, under the guiding in magnetic field, the microballoon quantity magnetic microsphere into lesion is more micro- than common
Ball increases 23% within 48 hours, and increases to 60% quickly, and the smaller increase of microsphere diameter is more apparent.Therefore, with micro-
Ball manufacture craft progress, the case in later stage magnetic field guiding magnetic microballoon in lesion increased quantity and rate it is more common
Microballoon is increased more obvious, to there is clinical efficacy more precisely.
Finally, the purpose of the invention is to develop a safety for being suitable for local treatment for inflammatory pain, have
The dexamethasone magnetic microsphere of effect.This microballoon has its slow release and targeting as other magnetic microspheres, after local injection,
Under the guiding in magnetic field, concentrate on lesions position, as PLGA slowly degrades, dexamethasone will gradually slow release, Zhi Houke
Reach a relatively stable drug concentration.
Description of the drawings
Fig. 1 is the aspect graph under the micro- light microscopic of dexamethasone magnetic microsphere that the embodiment of the present invention makes, wherein (a) (b)
(c) it is 1 to correspond to rate of charge respectively:3、1:4、1:5.
Specific implementation mode
Below in conjunction with attached drawing, the present invention is further illustrated：
1, the preparation of dexamethasone magnetic microsphere：
It is that S/O/W methods are made that microballoon, which uses,.According to PLGA/Fe3O4It is 4:1 ratio, by 1:3,1:4,1:5
Rate of charge【Rate of charge=(dexamethasone quality)：(dexamethasone quality+PLGA quality+Fe3O4Nanometer powder)】, calculate
It after specific quality, weighs respectively, load weighted PLGA is put into 3ml dichloromethane (O), interruption vortex oscillation instrument is with 3000r/
Min vibrates 10min, until being completely dissolved；The Fe that will have been weighed afterwards3O4Nanometer powder is dissolved in the above solution, ultrasonic vibration
After 1min mixings, then 100mg dexamethasone pulvis (S) is dissolved in dichloromethane, sonic oscillation be used in combination equal until be completely dissolved
Matter stirs instrument 10000r/min, then, the above dichloromethane mixed solution is slow by the speed of 30drops/min with dropper
It instills in the 2%PVA solution 30ml of ice bath 1h, rotating speed 1800r/min, volatilize 4h, until dichloromethane volatilizees completely.Microballoon
It after solidification, is filtered through filter membrane negative pressure of vacuum, collects microballoon, be lyophilized after deionized water washing, you can dexamethasone magnetic is prepared
Property microballoon.
2, dexamethasone magnetic microsphere form and characteristic
2.1 morphological observation
Dexamethasone magnetic microsphere is applied on glass slide, after being disperseed with appropriate distilled water, it is set and is seen under 400 times of light microscopics
It examines and takes pictures, measure the grain size of at least 500 microballoons, calculate average diameter, microspherulite diameter=(stage micrometer and eyepiece survey
Length (um)/stage micrometer that micro- ruler overlaps overlapped with eyepiece micrometer shared by lattice number) x microballoons occupy the micro- ruler of eyepiece
Lattice number, summation/microballoon sum of average diameter=microsphere diameter, span=(D90-D10)/D50, D90, D50 and D10 points
Do not indicate that the grain size of the microballoon of 90%, 50% and 10% quantity is respectively less than the value.
2.2 magnetic responsivenesses measure
Dexamethasone magnetic microsphere 5mg is taken, 100ml distilled waters is added, standard solution is made, is seen in 40x10 power microscopes
Examine distribution and movement of the microballoon under the magnetic field of 4000GS.After drying, absorption and unadsorbed microballoon are weighed respectively, and according to public affairs
Formula --- magnetic suck rate (%)=adsorption microspheres/(adsorption microspheres+unadsorbed microballoon) x100% --- calculates adsorption rate.
2.3 microspheres qualities are assessed
The assessment of microspheres quality mainly passes through the following aspects：The span (S1) of microballoon, yield (S2), encapsulation rate
(S3), the summing value of drugloading rate (S4) is assessed, the summing value S=-S1+S2 of the microspheres quality prepared by volatility process used herein
+S3+S4。
The computational methods of its central span (S1) see above 2.1.1 morphological observations part, and yield (S2)=wrap up medicine
Drug total amount × 100% of total amount/input coefficient of object；Drug total amount/input all the components for encapsulation rate (S3)=wrapped up
Quality × 100%.
2.4 statistical method
Average data indicates that statistical software is calculated using 17.0 versions of SPSS using x ± s, compares between metering sample group
Compare using one-way analysis of variance compared with multisample in using paired t-test, organizing, counting sample, sample compares using card side two-by-two
It examines.
2.5 result
2.5.1 the form of microballoon and quality evaluation
The dexamethasone magnetic microsphere that this experiment makes is black spherical shape molecule, more under micro- light microscopic (40x10)
Rounded, rate of charge is respectively 1:3,1:4,1:5 microballoon average diameter (span, S1) is respectively 27.47 ± 8.78um,
29.66 ± 10.02um, 30.14 ± 11.2um, form refers to Fig. 1 under light microscopic.Rate of charge is respectively 1:3,1:4,1:5 microballoon
Yield (S2) is respectively (87.02 ± 2.25) %, (87.25 ± 3.14) %, (84.00 ± 5.53) %；Encapsulation rate (S3) is respectively
For (76.44 ± 3.11) %, (78.89 ± 2.42) %, (71.33 ± 5.60) %；Drugloading rate (S4) be respectively (33.09 ±
1.89) %, (30.33 ± 2.01) %, (24.29 ± 1.78) %；Summing value (S) is respectively 168.55 ± 3.62,167.47 ±
4.15,149.48 ± 4.21.Dispensing is than being 1:5 groups of other S values have apparent significant difference, and P compared with remaining is two groups<
0.01.Details are shown in Table 1.
The Property comparison (x ± s, n=500) of 1 dexamethasone magnetic microsphere of table
* P compared with other groups<0.01
The quality of microballoon is generally related with span (S1), yield (S2), encapsulation rate (S3) and drugloading rate (S4), wherein with
S1 is inversely proportional, directly proportional to S2, S3, S4.Last rate of charge is 1:3,1:4 and 1:The summing value (S) of 5 microspheres quality is respectively
It is 168.55 ± 3.62,167.47 ± 4.15 and 149.48 ± 4.21.Rate of charge is 1:The summing value S of 5 microspheres quality and its
He two groups relatively have apparent significant difference, that is to say, that rate of charge 1:5 microballoon is relatively inferior.And rate of charge is 1:3 Hes
1:4 two groups of microspheres quality summing value S are compared, no difference of science of statistics, that is to say, that the quality of the two is suitable.
2.5.2 magnetic field responsiveness
Under an optical microscope, when magnetic field intensity is 4000GS, dexamethasone magnetic microsphere is in diethyl ether solution in not
Regular motion, direction are that move distance is about (80.0 ± 5.0) mm until bottle wall, illustrates magnetic responsiveness and shape towards magnet
State is preferable.
3, the release in vitro and estimation of stability of dexamethasone magnetic microsphere
The release in vitro of 3.1 dexamethasone magnetic microspheres
It is 1 that precision, which weighs rate of charge, respectively:3,1:4,1:5 each 10mg of dexamethasone magnetic microsphere is respectively placed in tool plug
In flask, wherein being put into the PBS solution (phosphate-buffered salt, PH=7.4) of 50ml.Flask is put into water bath with thermostatic control concussion instrument,
Water level is more than PBS solution plane, shakes condition：36-37 DEG C, 100-120 beats/min.In 0.5h, 1.0h, 1.5h, 2.0h, 2.5h,
At interval of 24 hours after 3.0, supernatant is taken, until the 14th day.Sample 2ml is pipetted every time, equivalent is filled after sampling every time,
Institute's collecting sample is stored in 4 DEG C of refrigerators and preserves.Its drug concentration is detected, and calculates the release percentage of the drug.It calculates public
Formula is Q=CtV/m x100%, wherein Q are the Cumulative release amount (%) of the microballoon, CtFor t when PBS solution in dexamethasone
Drug concentration (ug/ml), V are drug dilution volume (ml), and m is the gross mass of dexamethasone in every bottle.
Rate of charge is 1:3,1:4 and 1:When the cumulative in vitro release rate of 5 dexamethasone magnetic microsphere is respectively the 2nd day
79.12%, 32.91% and 40.33%；82.6%, 62.74% and 76.04% at the 7th day；The 86.34% of 14th day,
66.99% and 97.85%, it is shown in Table 2 in detail.
The cumulative in vitro release rate (%) of the different rate of charge microballoons of table 2
The stability test of 3.2 dexamethasone magnetic microspheres
The microballoon (n >=500) of different rate of charges is respectively placed in EP pipes, and puts it into 4-8 DEG C of refrigerator 3
Month, respectively the 0th, 1,2, March observe within 1st whether its physical features (grain size and appearance under light microscopic) has change, and simultaneously
Detect its drugloading rate.
Although microballoon its rate of charge difference being placed in 4-8 DEG C of refrigerator, its physical property and its drugloading rate nothing within March
Significant change, that is to say, that the microballoon system has preferable stability, is shown in Table 3 in detail.
The different rate of charge microballoon stability tests (n >=500) of table 3
4, the Study on biocompatibility of dexamethasone magnetic microsphere
Experimental method
4.1 blank magnetic microsphere production methods
Similar to dexamethasone magnetic microsphere production method is emulsification -- solvent evaporation method (S/O/W).With unique difference
Be whether to be put into dexamethasone in microballoon manufacturing process.Cobalt ray, radiation sterilization are used after completing and being lyophilized.
4.2 hemolysis in vitro are tested
4.2.1 the preparation of test solution
The sterile blank microballoon and sterile saline are configured to the mother liquor of 4mg/ml in proportion, physiology salt is used in combination
Water is diluted to the microballoon lotion of a concentration of 1mg/ml, 2mg/ml and 4mg/ml by multiple respectively.
4.2.2 the preparation of red cell suspension
It takes SD rat whole bloods 2ml to be placed in teat glass, fibrin is removed in a manner of continuously stirring with glass paddle
Original is used in combination physiological saline by 10 times of defibrinated hemodilution, is centrifuged after mixing, rotating speed 3000r/min, centrifugation
5min pipettes supernatant, abandons it.The red blood cell of lower sediment is washed repeatedly to supernatant in colourless, clarification with physiological saline
Until transparent.Finally, by the above Washed Red Blood Cells normal saline at 2% red cell suspension.
4.2.3 hemolysis rate is tested
Precision pipettes each 3 parts of the test sample of various concentration, every part of 0.3ml, referred to as X solution.
A given the test agent：Red cell suspension 2.5ml+ physiological saline 2.2ml+X solution 0.3ml
B negative controls：Red cell suspension 2.5ml+ physiological saline 2.5ml
C positive controls：Red cell suspension 2.5ml+ distilled water 2.5ml
It by the above A+B+C sample blendings, is immediately placed in water bath with thermostatic control concussion instrument and is incubated 3h, water temperature is 36-37 DEG C.It will be upper
The given the test agent centrifugation of processing is stated, 3000r/min, 5min pipette supernatant, stay it, are stored at room temperature 30min.With negative control
Supernatant is blank, according to the light absorption value that spectral scan at 540nm obtains, calculates hemolysis rate (%), formula HL%=
(the At given the test agent light absorption values of At/Apc * 100%；Apc positive controls light absorption value), the above light absorption value is sky with negative control
White standard.Negative control absorbance≤0.03, positive control range of absorbency 0.8 ± 0.3.Hemolysis rate (%)<5%, illustrate this
Given the test agent is without haemocylolysis.
Blood compatibility is tested in 4.3 bodies
3 SD rats are taken, are weighed in respectively, by above-mentioned test sample mother liquor, 0.8ml is injected per 100g weight according to rat
(high dose), 0.5ml (middle dosage) and 0.2ml (low dosage) calculate injection dosage, respectively through in tail vein injection rat body.
0.5h, 6h, 12h before injection and after injection, take blood 0.5ml, are placed in anticoagulant tube (purple) for 24 hours.Carry out blood routine inspection
It looks into.
4.4 Experiment of Histocompatibility
After rat sevoflurane inhalation anesthesia, the fine hair on birds or animals hair at left side back is shaved, test sample mother liquor is subcutaneously injected in experimental group
1ml physiological saline is subcutaneously injected in 1ml, control group.10th day and injection site skin and subcutaneous tissue is taken to do HE dyeing after 30 days.
The above experimental data of 4.5 statistical analysis is analyzed using 17.0 softwares of SPSS, and paired t-test is used between group,
And it is front and back relatively using one-way analysis of variance, P in organizing<0.05 is statistically significant.
4.6 result：
4.6.1 hemolysis in vitro is tested
Above-mentioned sample to be tested is after hemolysis in vitro is tested, a concentration of 1mg/ml, 2mg/ml, the sample extinction of 4mg/ml
Degree is respectively 0.006,0.011,0.032；Hemolysis rate is respectively 0.5%, 1.2%, 3.3% (negative control and positive control
As a result absorbance is respectively 0.003 and 0.802；Hemolysis rate is respectively 0% and 99.9%), equal less than 5% by hemolysis rate
Regard standard of the given the test agent without haemocylolysis as to consider, sample above is without cause haemocylolysis.It is shown in Table 4 in detail.
The light absorption value and hemolysis rate (%, n=3) of the different samples to be tested of table 4
4.6.2 blood routine result
It is as follows that rat injects influence of the microballoon test sample of various dose to blood routine：
(1) compare with administration proleukocyte, red blood cell and enumeration of thrombocytes, the note of the high, medium and low other rat of dosage group
Rear 0.5h, 6h, 12h are penetrated, leucocyte for 24 hours, the variation of red blood cell and enumeration of thrombocytes changes nothing in normal range (NR)
Statistical significance, details are shown in Table 5.
(2) compared with the volume of administration proerythrocyte and blood platelet, after the injection of the high, medium and low other rat of dosage group
0.5h, 6h, 12h, the variation of red blood cell and blood platelet for 24 hours is in normal range (NR), and change is not statistically significant, and details are shown in
Table 6.
(3) compared with administration proleukocyte classification (%), 0.5h after the injection of the high, medium and low other rat of dosage group is neutral
Granulocyte (%) variation is not statistically significant, but 6h, 12h, neutrophil leucocyte (%) for 24 hours are substantially reduced；And lymphocyte
The variation of (%) is then on the contrary --- and 0.5h after the injection of the high, medium and low other rat of dosage group, variation is not statistically significant, but
6h, 12h, it is significantly raised for 24 hours；For monocyte (%) then in 12h, variation for 24 hours is statistically significant；Eosinophil
(%) 0.5h, 6h, 12h after injection, significantly reduces, and P for 24 hours<0.01；The variation of basophilic granulocyte is anticipated without statistics
Justice；The above details are shown in Table 7.
Influence of the 5 various dose microballoon of table to blood count
Influence of the 6 various dose microballoon of table to blood cell volume
Influence of the 7 various dose microballoon of table to leukocyte differential count (%)
*P<0.05,**P<0.01
4.6.3 histocompatbility result
(1) skin after rat skin lower injection the skin injuries such as festers and infects the 10th day and the 30th day without redness
Sign.
(2) rat injection site pathological section (HE dyeing) result
Rat injection site pathological section (HE dyeing) has a small amount of inflammatory cell on the 10th day in 40x10 times of the visual field, the
Inflammatory cell increases earlier above within 30 days, and has fibroblast proliferation and package.But compared with the control group and no significant difference.
5, dexamethasone magnetic microsphere pharmacodynamic study
Experimental method
5.1 experimental animals and grouping
The male SD rat 36 for choosing the SPF ranks that quality is 220-250g has only succeeded modeling rat.It is randomly divided into 6
Group, every group 6.Respectively microballoon group, microballoon+magnetic therapy group, rice group, rice+magnetic therapy group, positive controls, magnetic therapy group.Model
Day alternates with night in 12 hours for rat feeding environment, room temperature, quiet environment raising.It usually drinks water, feed and arbitrarily ask for as needed.
Fasting water 12h before anesthesia.
5.2 experimental method
5.2.1 method for preparation of drug
Dexamethasone microspheres solution：Weighing 100ug PVA and 10ml waters for injection respectively, 60 DEG C of water-bath 30min after mixing,
It is allowed to be completely dissolved.It weighs 10mg and makes dexamethasone microballoon by oneself, launch at twice in above-mentioned PVA solution, launch microballoon every time
Ultrasonic vibration 2min afterwards is allowed to evenly dispersed.
Dexamethasone sodium phosphate solution：4ml waters for injection dilution 5 is added in dexamethasone sodium phosphate injection 5mg/ml
Again to 1mg/ml.
5.2.2 pentobarbitol
After rat is placed in inhalation anesthesia case, the Sevoflurane (permanent auspicious) of 3%-5% is sucked, oxygen flow 2L/min, when sucking
Between 5-10min, until rat be in appropriateness narcosis --- pain disappear, righting reflex loss, skeletal muscle relaxation, exhale
It inhales uniformly, stablizes and gentle.
5.2.3 drug injection method
Microballoon：1ml syringes draw 0.3ml dexamethasone microspheres solution (about 0.1mg dexamethasone), will be postanesthetic big
Mouse places operating table, is all slowly subcutaneously injected in the left back whole center portion position of rat.Rat takes other mouse cages individually to put after injection
It sets.
Dexamethasone sodium phosphate：1ml syringes draw 0.1ml dexamethasone microspheres solution (about 0.1mg dexamethasone),
Postanesthetic rat is placed into operating table, is all slowly subcutaneously injected in the left back whole center portion position of rat.Rat takes it after injection
His mouse cage is individually placed.
5.2.4 magnetotherapy method
Rat is placed in above magnetic field, and apart from In The Rat Sole 10cm, magnetic field is away from metal object 30cm or more.Daily treatment 2 times, often
Secondary 30min, two treatments interval 6 hours, specific treatment time are 10:00-10:30,16:00-16:30.
5.2.5 the measurement method of the mechanical threshold of pain
With the 2nd after injection, 4,6,8,10 days, using Von Frey filaments measure the left back whole heart of rat.It, will before measurement
Rat shifts to an earlier date 30min and is put into the flip organic glass fixator that bottom is metal strip grid, is allowed to adapt to environment.It is surveyed after peace and quiet
Amount, time 15:00-17:Between 00.
5.2.6Von Frey filament measurement methods
The measurement of the mechanical threshold of pain uses classics Von Frey filaments, measure filament sequence be 2.0g, 4.0g, 6.0g,
8.0g、10.0g、15.0g、26g.It is measured since 2.0g first, each model filament measures 5 times, is if measured 3 times
1.4g is then down in positive reaction, if be still it is positive anti-if should continue to be down to 1.0g and start to measure., whereas if measuring 3 times
For feminine gender, then 4.0g is risen to, and so on, until 26g.Measuring point is the left back whole heart of rat, every time lasting 5s, twice
The stimulus intervals time is 10s, and measurement dynamics is that bent filaments to nearly right angle are advisable.Positive performance is rat to filament
Reflection is hidden in appearance, for example paw withdrawal and is licked and licked tested vola etc..It is reactionless to filament that feminine gender shows as rat.Recording method is
Positive findings " X ", negative findings are "○".
5.2.7 rat part pathology
It is injected the 10th day in microballoon/sodium phosphate injection/CFA, 2 modeling rats is randomly selected, after Sevoflurane
(the same 2.2.1 of method), after the left back whole heart takes skin 0.3cm-0.5cm, neutral formalin to fix, row HE dyeing.
5.2.8 rat Ipsilateral foot ocular estimate method
It is divided into following three ranks by degree of inflammation different manifestations：It is red and swollen, red, without redness, and in the light of actual conditions register
Number of elements.Evaluation personnel does not know about rat grouping situation, is from start to finish same people's evaluation.
The treatment of 5.3 observation index before and after suffer from during different group rat models treatments on the the 2nd, 4,6,8,10 sufficient appearance,
The mechanical threshold of pain and pathological section on the 10th and between comparison.
5.4 statistical method
Using SPSS17.0 software row statistical analysis, the rat machinery threshold of pain is indicated with (x ± s).Enumeration data is relatively adopted
Use Chi-square Test.Front and back comparison uses one-way analysis of variance in measurement data group.Comparison among groups use paired-samples T-test.P<
0.05 is to have apparent significant difference.
5.5 result
5.5.1 suffer from sufficient appearance during different group rat model treatments to compare
It is red and swollen sample inflammation appearance that rat model, which suffers from foot, before treatment.2nd to the 10th rat suffers from the comparison of sufficient appearance,
Microballoon group and ground rice group no significant difference；Microballoon+magnetic therapy group is compared with ground rice+magnetic therapy group without significant difference；Magnetic therapy group and the positive
Control group compares also without significant difference.Refer to table 8-12.
8. rat of table, which is treated, suffers from sufficient appearance (n=36) on the 2nd
9. rat of table, which is treated, suffers from sufficient appearance (n=36) on the 4th
10. rat of table, which is treated, suffers from sufficient appearance (n=36) on the 6th
11. rat of table, which is treated, suffers from sufficient appearance (n=36) on the 8th
12. rat of table, which is treated, suffers from sufficient appearance (n=36) on the 10th
5.5.2. rat suffers from the variation of the mechanical threshold of pain of foot before and after treatment
Compared with before each group treatment, other than positive controls and magnetic therapy group, other groups are on day 4 and the 6th day when is equal
It has clear improvement；And it is significantly raised compared with other groups in the threshold of pain of the 4th, 6 day microballoon+magnetic therapy group.And microballoon group and microballoon+magnetic therapy group
, the P also significantly raised compared with other groups in the 8th day pain threshold<0.05.Details see the table below.
* compared with other groups, P<0.05
5.5.3. rat suffers from sufficient pathological change (HE) before and after treatment
Compared with positive control other than magnetic therapy group, the inflammatory cell of remaining each group significantly reduces.Improvement degree is in micro-
Ball group and microballoon+magnetic therapy group are the most apparent.
On end, a kind of preparation method of dexamethasone magnetic microsphere provided by the invention, the microballoon production method is emulsification
Solvent evaporation method (S/O/W), manufacturing conditions are PLGA (50/50), and PLGA mass concentrations are 1:20, PVA mass fractions are
2%, revolution 1800r/min.The dexamethasone magnetic microsphere that the present invention makes is black spherical shape molecule, in micro- light microscopic
How rounded under (40X10), dexamethasone magnetic microsphere, rate of charge is respectively 1:3,1:4,1:5 microballoon average diameter (across
Away from S1) it is 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) %；Encapsulation rate (S3) be respectively (76.44 ±
3.11) %, (78.89 ± 2.42) %, (71.33 ± 5.60) %；Drugloading rate (S4) is respectively (33.09 ± 1.89) %,
(30.33 ± 2.01) %, (24.29 ± 1.78) %；Summing value (S) be respectively (168.55 ± 3.62), (167.47 ± 4.15),
(149.48±4.21).The above microsphere features smooth surface rounding, size is uniform, good dispersion, and cleansing pin rate is high.
Meanwhile rate of charge 1:3,1:4,1:5 dexamethasone magnetic microsphere discharges in vitro, accumulative release point on the 2nd
Not Wei 79.12%, 32.91%, 40.33%.Rate of charge is 1:3 microballoon burst release is obvious, and rate of charge is 1:5 it is micro-
Balloon borne dose is above minimum, so rate of charge is 1:4 microballoon is more satisfactory microballoon.
Again, rate of charge 1:4 microballoon is in the biocompatibility of Organism of Rats：Hemolysis in vitro tests the micro- of various concentration
Ball test sample hemolysis rate is below 5%, meets the standard that country formulates implantation material；Internal blood compatibility and histocompatbility
Meet the requirements.
Finally, dexamethasone sodium phosphate injection is injected at the whole bottom of rat model trouble respectively and dexamethasone PLGA magnetism is micro-
Ball (1:4) and after magnetic therapy, be divided into microballoon group, microballoon+magnetic therapy group, rice group, rice+magnetic therapy group, positive controls, magnetic therapy group.
Compared with the effect that microballoon (+magnetic therapy) is organized with ground rice (+magnetic therapy) is organized, first 2 days mechanical threshold of pain no difference of science of statistics, from the 4th day,
The mechanical threshold of pain of microballoon+magnetic therapy group is apparently higher than other groups.At this time microballoon group, compare between rice group and ground rice+magnetic therapy group
No difference of science of statistics.And the mechanical threshold of pain advantage of microballoon+magnetic therapy group is continued for the 10th day treatment end, pathological examination and
Physical appearance variation tendency is also same.Therefore, the dexamethasone magnetic microsphere that the present invention makes combined with magnetic therapy after control
Therapeutic effect is the most apparent, there is definite curative effect in terms of pathological examination.
Finally it should be noted that：Obviously, the above embodiment is merely an example for clearly illustrating the present invention, and simultaneously
The non-restriction to embodiment.For those of ordinary skill in the art, it can also do on the basis of the above description
Go out other various forms of variations or variation.There is no necessity and possibility to exhaust all the enbodiments.And thus drawn
The obvious changes or variations that Shen goes out are still in the protection scope of this invention.
Claims (6)
1. a kind of preparation method of dexamethasone magnetic microsphere, which is characterized in that the method is：
(1) PLGA, Fe are weighed according to rate of charge3O4Nanometer powder, dexamethasone pulvis；
(2) PLGA is added in dichloromethane, oscillation adds Fe up to being completely dissolved3O4Nanometer powder vibrates mixing；
(3) dexamethasone pulvis is added in step (2), oscillation is until be completely dissolved；
(4) solution that step (3) obtains is slowly dropped into dropper in the PVA solution of ice bath, is stirred, volatilization, until dichloro
Methane volatilizees completely；
(5) solution for obtaining step (4) filters, and collects microballoon, is lyophilized after deionized water washing, you can fill in rice with being prepared
Loose magnetic microsphere.
2. a kind of preparation method of dexamethasone magnetic microsphere as described in claim 1, which is characterized in that in step (1),
The rate of charge is (1:3)~(1:5)；
Rate of charge is equal to (dexamethasone quality)/(dexamethasone quality+PLGA quality+Fe3O4Nanometer powder quality).
3. a kind of preparation method of dexamethasone magnetic microsphere as claimed in claim 2, which is characterized in that in step (2),
The PLGA is by lactic acid and hydroxyacetic acid in mass ratio 50:50 preparations obtain；
Its mass concentration is 1 after dichloromethane is added in PLGA:20, later using interruption vortex oscillation 10min, until completely molten
Solution.
4. a kind of preparation method of dexamethasone magnetic microsphere as claimed in claim 3, which is characterized in that in step (2),
Fe3O4The addition of nanometer powder and the mass ratio of PLGA are 4:1；
Fe is added3O4Sonic oscillation 1-2min is carried out after nanometer powder.
5. a kind of preparation method of dexamethasone magnetic microsphere as claimed in claim 4, which is characterized in that in step (3),
After dexamethasone pulvis is added, sonic oscillation is until be completely dissolved, with homogeneous stirring instrument 10000r/ during ultrasound
Min is stirred.
6. a kind of preparation method of dexamethasone magnetic microsphere as claimed in claim 5, which is characterized in that in step (4),
Solution is slowly instilled into the PVA solution that ice bath 1h and mass fraction are 2% by the speed of 30drops/min with dropper
In, while it is the 4h that volatilizees under conditions of 1800r/min to control PVA solution in rotating speed, until dichloromethane volatilizees completely.
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| IZABELA GALESKA ET AL.: "Controlled Release of Dexamethasone from PLGA Microspheres Embedded Within Polyacid-Containing PVA Hydrogels", 《THE AAPS JOURNAL》 * |
| 陈晓慧等: "微球制剂的研究进展", 《临床医药实践》 * |
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