CN103191066A - Dimethyl-sulfone-contained biodegradation microsphere and preparation method thereof - Google Patents
Dimethyl-sulfone-contained biodegradation microsphere and preparation method thereof Download PDFInfo
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Abstract
The invention provides a dimethyl-sulfone-contained biodegradation microsphere and a preparation method thereof. The method comprises the following steps of dissolving dimethyl sulfone into a first organic solvent to obtain a first mixed solution; dissolving biodegradable macromolecules into a second organic solvent to obtain a second mixed solution; mixing the first mixed solution and the second mixed solution, pressing the mixed solution into a continuous phase through a film, and continuously stirring the mixed solution to obtain the dimethyl-sulfone-contained biodegradation microsphere. Compared with the oral dimethyl sulfone, by utilizing the biodegradable effect of the biological degradable macromolecules in a human body, the action time of the dimethyl sulfone in the body can be prolonged, the burst phenomenon of the dimethyl sulfone can be solved, a slow release effect can be realized, the action time of a drug can be increased, the time of taking medicine can be reduced, and the utilization effect of the dimethyl sulfone also can be improved; and by adopting a film emulsification technology, and the biological microsphere containing the dimethyl sulfone is enabled to have good consistency and drug loading capacity.
Description
Technical field
The invention belongs to the slow releasing carrier of medication technical field, relate in particular to a kind of dimethyl sulfone biodegradable microspheres and preparation method thereof that contains.
Background technology
Osteoarthritis (osteoarthritis OA) is a kind of commonly encountered diseases, frequently-occurring disease that has a strong impact on patients ' life quality and work, a kind of chronic osteoarthritis of the regression of ankle cartilage and Secondary cases caused by hyperosteogeny, and knee joint OA sickness rate is the highest.Because its etiology and pathology still imperfectly understands, therefore, the OA treatment becomes one of difficult problem of clinical treatment.Domestic preliminary survey shows that the prevalence that the total prevalence rate of osteoarthritis is about 15%, 40 years old crowd is 10～17%, then reach 50% more than 60 years old, and in crowd more than 75 years old, 80% suffers from osteoarthritis, and the final disability rate of this disease is 53%.The incidence of China's osteoarthritis accounts for 10% of total population, be about 100,000,000 people, nineteen ninety, China has only more than 4,000 ten thousand osteoarthritis patients, and reaching 8,000 ten thousand in 2000, patient's number has reached people more than 100,000,000 at present, predicts according to WHO, to reach 1.5 hundred million to Chinese osteopathia patient in 2015, will become one of maximum country of world's osteoarthritis number of patients.
Because division potential and the repair ability thereof of chondrocyte are limited, therefore use exogenous medicine and promote that cartilage injury's reparation is important research direction.At present, at different patients, different joint position and different pathological stages many Therapeutic Method are arranged, but the whole bag of tricks all has its limitation, success rate is extremely low, can not fundamentally cure arthritis.Though and the nonsteroidal anti-inflammatory drug of extensive use can ease the pain in the osteoarthritis treatment, has very strong untoward reaction; The hormone medicine joint cavity injection has good relief of symptoms, improves the effect of function, but can not promote to damage the recovery of cartilage.The repairing of skeleton, cartilage and connective tissue will be raw material with sulfur all with rebuilding, and sulfur also helps the absorption of calcium simultaneously, therefore has the scholar to advise the food of polyphagia sulfur-bearing.
Dimethyl sulfone is a kind of organic sulfur compound, it is the synthetic necessary material of human collagen albumen, be external health product commonly used, be mainly used in auxiliary treatment such as skin, hair, bone, cartilage, its metabolism to saccharide plays a driving role, and also can promote wound healing, and dimethyl sulfone also is the most effective external chondrocyte mitogen simultaneously, have the metabolism of the articular cartilage of acceleration, promote the effect of regenerating bone or cartilage.In recent years, the report that adopts dimethyl sulfone oral medication knee osteoarthritis is arranged successively, and many basic research show that also it has the effect of good control articular cartilage degeneration, are expected to one of effective ways that become the treatment osteoarthritis abroad.
The external oral result of dimethyl sulfone that uses shows, dimethyl sulfone and other treatment medicine for treating arthritis have synergism, but its single oral dose is bigger, and need repeatedly take, the prominent phenomenon of releasing is arranged, increase burden to the patient, and medicine stability is poor, its molecular weight is less, half-life in vivo is shorter, discharged by organism metabolism easily, the effect of whole body or topical application all can not satisfy treats requirement clinically, therefore studies the dimethyl sulfone slow releasing agent the local active drug concentration of long term maintenance damage is had great importance.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of dimethyl sulfone biodegradable microspheres and preparation method thereof that contains, and this biodegradable microspheres has solved the prominent problem of releasing of dimethyl sulfone.
The invention provides a kind of dimethyl sulfone biodegradable microspheres that contains, formed by dimethyl sulfone and biodegradability macromolecule.
Preferably, described biodegradability macromolecule is selected from a kind of in polylactic acid, polyglycolic acid, polylactic acid-glycolic guanidine-acetic acid copolymer and the polycaprolactone.
The present invention also provides a kind of preparation method that contains the dimethyl sulfone biodegradable microspheres, may further comprise the steps:
A) dimethyl sulfone is dissolved in first organic solvent, the mixed solution of winning; The biodegradability macromolecule is dissolved in second organic solvent, gets second mixed solution;
B) described first mixed solution is mixed with described second mixed solution, obtain the 3rd mixed solution, go in the continuous phase by mould then, obtain O/W type emulsion;
C) described O/W type emulsion is stirred, obtain containing the dimethyl sulfone biodegradable microspheres.
Preferably, described film is microporous membrane.
Preferably, described continuous phase is the aqueous solution that comprises surfactant and dispersant.
Preferably, the speed of described stirring is 300～700rpm/min, and the time of stirring is 6～8h.
Preferably, described first organic solvent is selected from a kind of in ethanol, benzene, methanol and the acetone.
Preferably, described second organic solvent is selected from chloroform, dichloromethane and N, a kind of in the dinethylformamide.
Preferably, the high molecular concentration of biodegradability is 1～7wt% in described the 3rd mixed solution.Preferably, the concentration of dimethyl sulfone is 1～15wt% in described the 3rd mixed solution.
The invention provides a kind of dimethyl sulfone biodegradable microspheres and preparation method thereof that contains, this method is dissolved in first organic solvent with dimethyl sulfone, the mixed solution of winning; The biodegradability macromolecule is dissolved in second organic solvent, gets second mixed solution; Described first mixed solution is mixed with described second mixed solution, obtain the 3rd mixed solution, go in the continuous phase by mould then, stir then, obtain containing the dimethyl sulfone biodegradable microspheres.Compare with the prior art oral administration of dimethyl sulfone, the present invention utilizes the film emulsifying technology that dimethyl sulfone and biodegradable molecule are prepared into to contain the dimethyl sulfone biodegradable microspheres, at first, the present invention utilizes the effect of biodegradability macromolecule biodegradable in vivo, prolong dimethyl sulfone action time in vivo, solved the prominent phenomenon of releasing of dimethyl sulfone, reach the effect of slow release, improve drug treating time and reduced medicining times, also improved the effect of utilizing of dimethyl sulfone simultaneously; Secondly, adopt the film emulsifying technology, make to contain the dimethyl sulfone biological microsphere and have homogeneity and drug loading preferably.
Description of drawings
The stereoscan photograph that contains the dimethyl sulfone biodegradable microspheres that Fig. 1 prepares for the embodiment of the invention 1;
The stereoscan photograph that contains the dimethyl sulfone biodegradable microspheres that Fig. 2 prepares for the embodiment of the invention 2;
The stereoscan photograph that contains the dimethyl sulfone biodegradable microspheres that Fig. 3 prepares for the embodiment of the invention 3;
The stereoscan photograph that contains the dimethyl sulfone biodegradable microspheres that Fig. 4 prepares for the embodiment of the invention 4;
The stereoscan photograph that contains the dimethyl sulfone biodegradable microspheres that Fig. 5 prepares for the embodiment of the invention 2;
The stereoscan photograph that contains the dimethyl sulfone biodegradable microspheres that Fig. 6 prepares for the embodiment of the invention 5;
The stereoscan photograph that contains the dimethyl sulfone biodegradable microspheres that Fig. 7 prepares for the embodiment of the invention 6;
The stereoscan photograph that contains the dimethyl sulfone biodegradable microspheres that Fig. 8 prepares for the embodiment of the invention 2;
The stereoscan photograph that contains the dimethyl sulfone biodegradable microspheres that Fig. 9 prepares for the embodiment of the invention 7;
Figure 10 is the curve chart of the release cumulative percentage that contains the dimethyl sulfone biodegradable microspheres for preparing of the embodiment of the invention 1,2 and 3 to the time;
Figure 11 is the curve chart of the release cumulative percentage that contains the dimethyl sulfone biodegradable microspheres for preparing of the embodiment of the invention 4,2 and 5 to the time;
The laser co-focusing photo that contains the dimethyl sulfone biodegradable microspheres that Figure 12 prepares for the embodiment of the invention 4;
The laser co-focusing photo that contains the dimethyl sulfone biodegradable microspheres that Figure 13 prepares for the embodiment of the invention 2;
The laser co-focusing photo that contains the dimethyl sulfone biodegradable microspheres that Figure 14 prepares for the embodiment of the invention 5;
Figure 15 is that the dimethyl sulfone biodegradable microspheres that contains that MC3T3-E1 osteoblast and the embodiment of the invention 1,2 and 3 prepare is cultivated the bar diagram that obtains altogether;
Figure 16 is that the dimethyl sulfone biodegradable microspheres that contains that MC3T3-E1 osteoblast and the embodiment of the invention 4,2 and 5 prepare is cultivated the bar diagram that obtains altogether;
Figure 17 is that the diameter that contains the dimethyl sulfone biodegradable microspheres for preparing of the embodiment of the invention 6,2 and 7 is to the bar diagram of stir speed (S.S.).
The specific embodiment
The invention provides a kind of dimethyl sulfone biodegradable microspheres that contains, formed by dimethyl sulfone and biodegradability macromolecule.
Wherein, described dimethyl sulfone is that the dimethyl sulfone that can be used as medicine well known to those skilled in the art gets final product, and there is no special restriction.
Described biodegradability macromolecule is that biodegradability macromolecule well known to those skilled in the art gets final product, there is no special restriction, the macromolecule of biodegradability described in the present invention is preferably polylactic acid, polyglycolic acid, polylactic acid-glycolic guanidine-acetic acid copolymer or poly-second lactone.The biodegradability macromolecule has excellent biological compatibility, degradability and characteristic such as can absorb, and field such as fixing and medicine controlled releasing has a wide range of applications in environmental conservation, organizational project, fracture.The biodegradability macromolecule is applied to pharmaceutical carrier, can utilizes the characteristic of its biodegradable in vivo, prolong drug action time in vivo improve curative effect of medication, solve the prominent phenomenon of releasing of dimethyl sulfone.
The present invention also provides a kind of above-mentioned preparation method that contains the dimethyl sulfone biodegradable microspheres, may further comprise the steps: a) dimethyl sulfone is dissolved in first organic solvent, the mixed solution of winning; The biodegradability macromolecule is dissolved in second organic solvent, gets second mixed solution; B) described first mixed solution is mixed with described second mixed solution, obtain the 3rd mixed solution, go in the continuous phase by mould then, obtain O/W type emulsion; C) described O/W type emulsion is stirred, obtain containing the dimethyl sulfone biodegradable microspheres.Dimethyl sulfone and biodegradation high molecular are dissolved in respectively in first organic solvent and second organic solvent, obtain first mixed solution and second mixed solution.Wherein, the organic solvent that described first organic solvent is solubilized dimethyl sulfone well known to those skilled in the art gets final product, and there is no special restriction, is preferably ethanol, benzene, methanol or acetone among the present invention, more preferably acetone; Described second organic solvent is that the high molecular organic solvent of solubilized biodegradability well known to those skilled in the art gets final product, and there is no special restriction, is preferably chloroform, dichloromethane or N among the present invention, dinethylformamide, more preferably acetone.
Obtain after first mixed solution and second mixed solution, both are mixed, obtain the 3rd mixed solution and be decentralized photo.Wherein, the high molecular concentration of biodegradability is 1～7wt% in the 3rd mixed solution that obtains, and is preferably 3～5wt%; The concentration of dimethyl sulfone is 1～15wt% in the 3rd mixed solution, is preferably 5～10wt%.
After obtaining the 3rd mixed solution, the 3rd mixed solution is gone in the continuous phase by mould, obtain O/W type emulsion.Wherein, described film is that the film for film emulsifying well known to those skilled in the art gets final product, and there is no special restriction, is preferably microporous membrane among the present invention, more preferably the SPG film.
Among the present invention, described continuous phase is that used continuous phase gets final product in the film emulsion process well known to those skilled in the art, there is no special restriction, is preferably the aqueous solution that comprises surfactant and dispersant.Wherein, described surfactant is that surfactant well known to those skilled in the art gets final product, and there is no special restriction, is preferably sodium lauryl sulphate or the fatty acid Pyrusussuriensis is smooth; The content of surfactant is preferably 0.5～6g/L in the described continuous phase, more preferably 2～4g/L; Described dispersant is that dispersant well known to those skilled in the art gets final product, and there is no special restriction, is preferably polyvinyl alcohol or Polysorbate; The content of dispersant is preferably 0.5～4g/L in the described continuous phase, more preferably 1～2g/L.Both all can reduce surface tension and surface free energy.
The present invention utilizes the film emulsifying technology that dimethyl sulfone and biodegradable molecule are prepared into to contain the dimethyl sulfone biodegradable microspheres, at first, the present invention utilizes the effect of biodegradability macromolecule biodegradable in vivo, prolong dimethyl sulfone action time in vivo, solved the prominent phenomenon of releasing of dimethyl sulfone, reach the effect of slow release, improved drug treating time and reduced medicining times, also improved the effect of utilizing of dimethyl sulfone simultaneously; Secondly, adopt the film emulsifying technology, make to contain the dimethyl sulfone biological microsphere and have homogeneity and drug loading preferably.
After obtaining O/W type emulsion, its constant temperature is stirred, in this process, remove along with the organic solvent in the decentralized photo constantly also volatilizees gradually to the continuous phase diffusion, obtain containing the dimethyl sulfone biodegradable microspheres.Wherein, the temperature of described constant temperature is preferably 12 ℃～20 ℃, more preferably 12 ℃～15 ℃; The speed of described stirring is preferably 300～700rpm/min, more preferably 400～600rpm/min; The time of described stirring is preferably 6～8h, more preferably 7～8h.
In the process that stirs, organic facies is constantly also volatilized gradually to the continuous phase diffusion and is removed, there is solid to separate out gradually, be solidified into then and contain the dimethyl sulfone biodegradable microspheres, the final suspension that contains the dimethyl sulfone biodegradable microspheres that forms, the present invention is preferably centrifugal with this suspension, and washing obtains containing the dimethyl sulfone biodegradable microspheres after the drying.Described centrifugal rotation speed is preferably 6000～9000r/min, and the centrifugal time is preferably 20～40min; In order to prevent the dimethyl sulfone distillation, described drying is preferably lyophilization.
In order to further specify the present invention, below in conjunction with embodiment a kind of dimethyl sulfone biodegradable microspheres and preparation method thereof that contains provided by the invention is described in detail.
Used reagent is commercially available in following examples.
1.1 polylactic acid and dimethyl sulfone are dissolved in respectively in chloroform and the acetone, and fully stir, ultrasonic even then with both blend, obtain decentralized photo, the content of polylactic acid is 3wt% in the decentralized photo, the content of dimethyl sulfone is 4.5wt%.
1.2 the decentralized photo that obtains in 1.1 is added in the bin, under the impressed pressure effect, decentralized photo is pressed in the continuous phase by cellular glass film (SPG microporous membrane), thereby prepare monodispersed O/W type emulsion, continuous phase is that 300ml contains sodium lauryl sulphate (SDS) 2g/L and polyvinyl alcohol 1g/L; The SPG membrane aperture is 0.6 μ m.
1.3 the O/W type emulsion that obtains in 1.2 is transferred to rapidly in 15 ℃ of temperature chambers, 500rpm/min stirs 7h, along with constantly also volatilizing gradually to the continuous phase diffusion, decentralized photo solvent chloroform and acetone removes, be solidified into and contain the dimethyl sulfone biodegradable microspheres, the final suspension that contains the dimethyl sulfone biodegradable microspheres that forms, the centrifugal 30min of 8000r/min, deionized water wash 3 times obtains containing the dimethyl sulfone biodegradable microspheres after the lyophilization.
The dimethyl sulfone biodegradable microspheres that contains that obtains in 1.3 that takes a morsel is suspended in the distilled water, drop on the silicon chip on a small quantity with the absorption of 1ml pipettor, it is evenly spread out, naturally dry, be fixed on the sample platform with double faced adhesive tape, after metal spraying is handled under the vacuum condition, utilize scanning electron microscope that it is analyzed, obtain its stereoscan photograph, as shown in Figure 1, its scale is 100 μ m.
Place distilled water to carry out ultra-sonic dispersion the dimethyl sulfone biodegradable microspheres that contains that obtains in 1.3, adopt scanning electron microscope to observe, calculate by formula by the particle diameter of 200 microspheres on the statistics stereoscan photograph and to contain dimethyl sulfone biodegradable microspheres mean diameter and particle size distribution, mean diameter (D
n) formula: D
n=(∑ d
i)/n, d
iBe microsphere diameter, n is the microsphere quantity of statistics, and the computing formula of dispersibility (CV) is: CV=SD/D
n, SD is standard deviation.1.3 in the D that contains the dimethyl sulfone biodegradable microspheres that obtains
n=4.78 μ m, CV=26.35%.
What obtain among the weighing 20.0mg1.3 contains the dimethyl sulfone biodegradable microspheres, be dissolved in the 10ml nitric acid, utilize inductive coupling plasma emission spectrograph (ICP-AES) to analyze, measure the wherein content of element sulphur, calculate dimethyl sulfone content by the shared molecular weight of element sulphur in the dimethyl sulfone, get 3 parallel samples.The medicine drug loading and the embedding rate that contain the dimethyl sulfone biodegradable microspheres calculate as follows: drug loading (DL)=(quality of the content/microsphere of dimethyl sulfone in the microsphere) * 100%; Medicine embedding rate (EE)=(quality of dimethyl sulfone in drug loading * microsphere gross mass/theoretical microsphere) * 100% obtains the results are shown in Table 1.
The dimethyl sulfone biodegradable microspheres that contains that obtains among the 10.0mg1.3 is mixed with 1.5ml PBS buffer (pH=7.4), placing 37 ℃ of constant temperature oscillators to carry out jolting with the speed of 120r/min hatches, vitro drug release under the simulation physiological condition, in 12h, 24h, 48h, 72h, 96h, 120h, 168h, 196h, 244h and 504h centrifugalize, take out the 1.0ml supernatant, mend the fresh PBS buffer of 1.0ml simultaneously, utilize ICP-AES to measure dimethyl sulfone concentration in the supernatant, each time point of sample is established 3 parallel samples, results averaged, the release cumulative percentage is done curve to the time, the result as among Figure 10-■-shown in.
Utilize tetrazolium blue chromogenic reaction drug sensitive experiment (MTT experiment) to detect the MC3T3-E1 osteoblast of growing up under the dimethyl sulfone biodegradable microspheres condition of culture that contains for preparing in 1.3 containing, obtain bar diagram, shown in a among Figure 15.
Embodiment 2
2.1 polylactic acid and dimethyl sulfone are dissolved in respectively in chloroform and the acetone, and fully stir, ultrasonic even then with both blend, obtain decentralized photo, the content of polylactic acid is 3wt% in the decentralized photo, the content of dimethyl sulfone is 4.5wt%.
2.2 the decentralized photo that obtains in 2.1 is added in the bin, under the impressed pressure effect, decentralized photo is pressed in the continuous phase by cellular glass film (SPG microporous membrane), thereby prepare monodispersed O/W type emulsion, continuous phase is that 300ml contains sodium lauryl sulphate (SDS) 2g/L and polyvinyl alcohol 1g/L; The SPG membrane aperture is 5.1 μ m.
2.3 the O/W type emulsion that obtains in 2.2 is transferred to rapidly in 15 ℃ of temperature chambers, 500rpm/min stirs 7h, along with constantly also volatilizing gradually to the continuous phase diffusion, decentralized photo solvent chloroform and acetone removes, be solidified into and contain the dimethyl sulfone biodegradable microspheres, the final suspension that contains the dimethyl sulfone biodegradable microspheres that forms, the centrifugal 30min of 8000r/min, deionized water wash 3 times obtains containing the dimethyl sulfone biodegradable microspheres after the lyophilization.
The dimethyl sulfone biodegradable microspheres that contains that obtains in 2.3 that takes a morsel is suspended in the distilled water, drop on a small quantity on the silicon chip with the absorption of 1ml pipettor, it is evenly spread out, dry naturally, be fixed on the sample platform with double faced adhesive tape, after metal spraying is handled under the vacuum condition, utilize scanning electron microscope that it is analyzed, obtain its stereoscan photograph, as Fig. 2, Fig. 5 and shown in Figure 8, Fig. 2 scale is 100 μ m, and the scale of Fig. 5 is 20 μ m, and the scale of Fig. 8 is 10 μ m.
Place distilled water to carry out ultra-sonic dispersion the dimethyl sulfone biodegradable microspheres that contains that obtains in 2.3, adopt scanning electron microscope to observe, calculate by formula by the particle diameter of 200 microspheres on the statistics stereoscan photograph and to contain dimethyl sulfone biodegradable microspheres mean diameter and particle size distribution, mean diameter (D
n) formula: D
n=(∑ d
i)/n, d
iBe microsphere diameter, n is the microsphere quantity of statistics, and the computing formula of dispersibility (CV) is: CV=SD/D
n, SD is standard deviation.2.3 in the D that contains the dimethyl sulfone biodegradable microspheres that obtains
n=26.24 μ m, CV=8.65%.
What obtain among the weighing 20.0mg2.3 contains the dimethyl sulfone biodegradable microspheres, be dissolved in the 10ml nitric acid, utilize inductive coupling plasma emission spectrograph (ICP-AES) to analyze, measure the wherein content of element sulphur, calculate dimethyl sulfone content by the shared molecular weight of element sulphur in the dimethyl sulfone, get 3 parallel samples.Calculating contains medicine drug loading (DL) and the embedding rate (EE) of dimethyl sulfone biodegradable microspheres, obtains the results are shown in Table 1.
The dimethyl sulfone biodegradable microspheres that contains that obtains among the 10.0mg2.3 is mixed with 1.5ml PBS buffer (pH=7.4), placing 37 ℃ of constant temperature oscillators to carry out jolting with the speed of 120r/min hatches, vitro drug release under the simulation physiological condition, in 12h, 24h, 48h, 72h, 96h, 120h, 168h, 196h, 244h and 504h centrifugalize, take out the 1.0ml supernatant, mend the fresh PBS buffer of 1.0ml simultaneously, utilize ICP-AES to measure dimethyl sulfone concentration in the supernatant, each time point of sample is established 3 parallel samples, results averaged, the release cumulative percentage is done curve to the time, among result such as Figure 10-●-shown in Figure 11 in-▲-shown in.By Figure 10 and Figure 11 as can be known, to arrive the required time of plateau be 7～10 days to the dimethyl sulfone biodegradable microspheres that contains of preparation, biodegradable microspheres reaches the drug release time of 50% release rate more than 3 days, therefore can think and reach the good slow release effect, significantly do not dash forward and release phenomenon, its rate of releasing drug increases and accelerates with dimethyl sulfone mass ratio in the reduction of membrane aperture and the polylactic acid, because the mechanism of drug release is to be controlled simultaneously by the degraded of drug diffusion and carrier material, rate of releasing drug may be and the microsphere size, specific surface area and internal structure are relevant, if but played a decisive role by the drug diffusion control of hydrophobic polylactic acid mediation preceding 10 God.
What preparation contained a small amount of Fluorescein isothiocyanate (FITC) as stated above contains the dimethyl sulfone biodegradable microspheres, microsphere is placed the distilled water ultra-sonic dispersion, on tiling and the microscope slide, adopt laser confocal microscope to observe the microsphere internal structure after the drying at room temperature, obtain the laser co-focusing photo, as shown in figure 13.
Utilize tetrazolium blue chromogenic reaction drug sensitive experiment (MTT experiment) to detect containing the MC3T3-E1 osteoblast of growing up under the dimethyl sulfone biodegradable microspheres condition of culture that contains of preparation in 2.3, obtain bar diagram, shown in b among Figure 15 and among Figure 16 shown in the c '.
3.1 polylactic acid and dimethyl sulfone are dissolved in respectively in chloroform and the acetone, and fully stir, ultrasonic even then with both blend, obtain decentralized photo, the content of polylactic acid is 3wt% in the decentralized photo, the content of dimethyl sulfone is 4.5wt%.
3.2 the decentralized photo that obtains in 3.1 is added in the bin, under the impressed pressure effect, decentralized photo is pressed in the continuous phase by cellular glass film (SPG microporous membrane), thereby prepare monodispersed O/W type emulsion, continuous phase is that 300ml contains sodium lauryl sulphate (SDS) 2g/L and polyvinyl alcohol 1g/L; The SPG membrane aperture is 10.1 μ m.
3.3 the O/W type emulsion that obtains in 3.2 is transferred to rapidly in 15 ℃ of temperature chambers, 500rpm/min stirs 7h, along with constantly also volatilizing gradually to the continuous phase diffusion, decentralized photo solvent chloroform and acetone removes, be solidified into and contain the dimethyl sulfone biodegradable microspheres, the final suspension that contains the dimethyl sulfone biodegradable microspheres that forms, the centrifugal 30min of 8000r/min, deionized water wash 3 times obtains containing the dimethyl sulfone biodegradable microspheres after the lyophilization.
The dimethyl sulfone biodegradable microspheres that contains that obtains in 3.3 that takes a morsel is suspended in the distilled water, drop on the silicon chip on a small quantity with the absorption of 1ml pipettor, it is evenly spread out, naturally dry, be fixed on the sample platform with double faced adhesive tape, after metal spraying is handled under the vacuum condition, utilize scanning electron microscope that it is analyzed, obtain its stereoscan photograph, as shown in Figure 3, its scale is 100 μ m.
Place distilled water to carry out ultra-sonic dispersion the dimethyl sulfone biodegradable microspheres that contains that obtains in 3.3, adopt scanning electron microscope to observe, calculate by formula by the particle diameter of 200 microspheres on the statistics stereoscan photograph and to contain dimethyl sulfone biodegradable microspheres mean diameter and particle size distribution, mean diameter (D
n) formula: D
n=(∑ d
i)/n, d
iBe microsphere diameter, n is the microsphere quantity of statistics, and the computing formula of dispersibility (CV) is: CV=SD/D
n, SD is standard deviation.3.3 in the D that contains the dimethyl sulfone biodegradable microspheres that obtains
n=35.18 μ m, CV=3.24%.
What obtain among the weighing 20.0mg3.3 contains the dimethyl sulfone biodegradable microspheres, be dissolved in the 10ml nitric acid, utilize inductive coupling plasma emission spectrograph (ICP-AES) to analyze, measure the wherein content of element sulphur, calculate dimethyl sulfone content by the shared molecular weight of element sulphur in the dimethyl sulfone, get 3 parallel samples.Calculating contains medicine drug loading (DL) and the embedding rate (EE) of dimethyl sulfone biodegradable microspheres, obtains the results are shown in Table 1.
The dimethyl sulfone biodegradable microspheres that contains that obtains among the 10.0mg3.3 is mixed with 1.5ml PBS buffer (pH=7.4), placing 37 ℃ of constant temperature oscillators to carry out jolting with the speed of 120r/min hatches, vitro drug release under the simulation physiological condition, in 12h, 24h, 48h, 72h, 96h, 120h, 168h, 196h, 244h and 504h centrifugalize, take out the 1.0ml supernatant, mend the fresh PBS buffer of 1.0ml simultaneously, utilize ICP-AES to measure dimethyl sulfone concentration in the supernatant, each time point of sample is established 3 parallel samples, results averaged, the release cumulative percentage is done curve to the time, the result as among Figure 10-▲-shown in.
Utilize tetrazolium blue chromogenic reaction drug sensitive experiment (MTT experiment) to detect the MC3T3-E1 osteoblast of growing up under the dimethyl sulfone biodegradable microspheres condition of culture that contains for preparing in 3.3 containing, obtain bar diagram, shown in c among Figure 15.As shown in Figure 15, growth along with incubation time, the number of cells of each group increases gradually, membrane aperture is that cultured cells propagation situation is better than other two groups under the condition that contains the dimethyl sulfone biodegradable microspheres for preparing of 5.1 μ m in containing embodiment 2, when cultivating 1 day, a has significant difference (P＜0.05) with the b ratio, has compared propagation trend with c, but has not had significant difference; When cultivating 3 days, b has compared propagation trend with other two groups, but does not have significant difference; When cultivating 7 days, b with other two groups to compare propagation trend more obvious, and significant difference (P＜0.05) is arranged.
The medicine drug loading and the embedding rate that contain the dimethyl sulfone biodegradable microspheres of the different membrane aperture preparations of table 1
Embodiment 4
4.1 polylactic acid and dimethyl sulfone are dissolved in respectively in chloroform and the acetone, and fully stir, ultrasonic even then with both blend, obtain decentralized photo, the content of polylactic acid is 3wt% in the decentralized photo, the content of dimethyl sulfone is 0.9wt%.
4.2 the decentralized photo that obtains in 4.1 is added in the bin, under the impressed pressure effect, decentralized photo is pressed in the continuous phase by cellular glass film (SPG microporous membrane), thereby prepare monodispersed O/W type emulsion, continuous phase is that 300ml contains sodium lauryl sulphate (SDS) 2g/L and polyvinyl alcohol 1g/L; The SPG membrane aperture is 5.1 μ m.
4.3 the O/W type emulsion that obtains in 4.2 is transferred to rapidly in 15 ℃ of temperature chambers, 500rpm/min stirs 7h, along with constantly also volatilizing gradually to the continuous phase diffusion, decentralized photo solvent chloroform and acetone removes, be solidified into and contain the dimethyl sulfone biodegradable microspheres, the final suspension that contains the dimethyl sulfone biodegradable microspheres that forms, the centrifugal 30min of 8000r/min, deionized water wash 3 times obtains containing the dimethyl sulfone biodegradable microspheres after the lyophilization.
The dimethyl sulfone biodegradable microspheres that contains that obtains in 4.3 that takes a morsel is suspended in the distilled water, drop on the silicon chip on a small quantity with the absorption of 1ml pipettor, it is evenly spread out, naturally dry, be fixed on the sample platform with double faced adhesive tape, after metal spraying is handled under the vacuum condition, utilize scanning electron microscope that it is analyzed, obtain its stereoscan photograph, as shown in Figure 4, its scale is 20 μ m.
Place distilled water to carry out ultra-sonic dispersion the dimethyl sulfone biodegradable microspheres that contains that obtains in 4.3, adopt scanning electron microscope to observe, calculate by formula by the particle diameter of 200 microspheres on the statistics stereoscan photograph and to contain dimethyl sulfone biodegradable microspheres mean diameter, obtaining the result is 28.64 μ m.
What obtain among the weighing 20.0mg4.3 contains the dimethyl sulfone biodegradable microspheres, be dissolved in the 10ml nitric acid, utilize inductive coupling plasma emission spectrograph (ICP-AES) to analyze, measure the wherein content of element sulphur, calculate dimethyl sulfone content by the shared molecular weight of element sulphur in the dimethyl sulfone, get 3 parallel samples.Calculating contains medicine drug loading (DL) and the embedding rate (EE) of dimethyl sulfone biodegradable microspheres, obtains the results are shown in Table 2.
The dimethyl sulfone biodegradable microspheres that contains that obtains among the 10.0mg4.3 is mixed with 1.5ml PBS buffer (pH=7.4), placing 37 ℃ of constant temperature oscillators to carry out jolting with the speed of 120r/min hatches, vitro drug release under the simulation physiological condition, in 12h, 24h, 48h, 72h, 96h, 120h, 168h, 196h, 244h and 504h centrifugalize, take out the 1.0ml supernatant, mend the fresh PBS buffer of 1.0ml simultaneously, utilize ICP-AES to measure dimethyl sulfone concentration in the supernatant, each time point of sample is established 3 parallel samples, results averaged, the release cumulative percentage is done curve to the time, among result such as Figure 11-●-shown in.
What preparation contained a small amount of Fluorescein isothiocyanate (FITC) as stated above contains the dimethyl sulfone biodegradable microspheres, microsphere is placed the distilled water ultra-sonic dispersion, on tiling and the microscope slide, adopt laser confocal microscope to observe the microsphere internal structure after the drying at room temperature, obtain the laser co-focusing photo, as shown in figure 12.
Utilize tetrazolium blue chromogenic reaction drug sensitive experiment (MTT experiment) to detect the MC3T3-E1 osteoblast of growing up under the dimethyl sulfone biodegradable microspheres condition of culture that contains for preparing in 4.3 containing, obtain bar diagram, shown in b ' among Figure 16, a ' grows up only containing under the condition of culture of biodegradable microspheres that polylactic acid do not contain dimethyl sulfone for blank assay contrast is osteoblast.
5.1 polylactic acid and dimethyl sulfone are dissolved in respectively in chloroform and the acetone, and fully stir, ultrasonic even then with both blend, obtain decentralized photo, the content of polylactic acid is 3wt% in the decentralized photo, the content of dimethyl sulfone is 8.6wt%.
5.2 the decentralized photo that obtains in 5.1 is added in the bin, under the impressed pressure effect, decentralized photo is pressed in the continuous phase by cellular glass film (SPG microporous membrane), thereby prepare monodispersed O/W type emulsion, continuous phase is that 300ml contains sodium lauryl sulphate (SDS) 2g/L and polyvinyl alcohol 1g/L; The SPG membrane aperture is 5.1 μ m.
5.3 the O/W type emulsion that obtains in 5.2 is transferred to rapidly in 15 ℃ of temperature chambers, 500rpm/min stirs 7h, along with constantly also volatilizing gradually to the continuous phase diffusion, decentralized photo solvent chloroform and acetone removes, be solidified into and contain the dimethyl sulfone biodegradable microspheres, the final suspension that contains the dimethyl sulfone biodegradable microspheres that forms, the centrifugal 30min of 8000r/min, deionized water wash 3 times obtains containing the dimethyl sulfone biodegradable microspheres after the lyophilization.
The dimethyl sulfone biodegradable microspheres that contains that obtains in 5.3 that takes a morsel is suspended in the distilled water, drop on the silicon chip on a small quantity with the absorption of 1ml pipettor, it is evenly spread out, naturally dry, be fixed on the sample platform with double faced adhesive tape, after metal spraying is handled under the vacuum condition, utilize scanning electron microscope that it is analyzed, obtain its stereoscan photograph, as shown in Figure 6, its scale is 20 μ m.
Place distilled water to carry out ultra-sonic dispersion the dimethyl sulfone biodegradable microspheres that contains that obtains in 5.3, adopt scanning electron microscope to observe, calculate by formula by the particle diameter of 200 microspheres on the statistics stereoscan photograph and to contain dimethyl sulfone biodegradable microspheres mean diameter, obtaining the result is 20.58 μ m.
What obtain among the weighing 20.0mg5.3 contains the dimethyl sulfone biodegradable microspheres, be dissolved in the 10ml nitric acid, utilize inductive coupling plasma emission spectrograph (ICP-AES) to analyze, measure the wherein content of element sulphur, calculate dimethyl sulfone content by the shared molecular weight of element sulphur in the dimethyl sulfone, get 3 parallel samples.Calculating contains medicine drug loading (DL) and the embedding rate (EE) of dimethyl sulfone biodegradable microspheres, obtains the results are shown in Table 2.
The dimethyl sulfone biodegradable microspheres that contains that obtains among the 10.0mg5.3 is mixed with 1.5ml PBS buffer (pH=7.4), placing 37 ℃ of constant temperature oscillators to carry out jolting with the speed of 120r/min hatches, vitro drug release under the simulation physiological condition, in 12h, 24h, 48h, 72h, 96h, 120h, 168h, 196h, 244h and 504h centrifugalize, take out the 1.0ml supernatant, mend the fresh PBS buffer of 1.0ml simultaneously, utilize ICP-AES to measure dimethyl sulfone concentration in the supernatant, each time point of sample is established 3 parallel samples, results averaged, the release cumulative percentage is done curve to the time, the result as among Figure 11-■-shown in.
What preparation contained a small amount of Fluorescein isothiocyanate (FITC) as stated above contains the dimethyl sulfone biodegradable microspheres, microsphere is placed the distilled water ultra-sonic dispersion, on tiling and the microscope slide, adopt laser confocal microscope to observe the microsphere internal structure after the drying at room temperature, obtain the laser co-focusing photo, as shown in figure 14.By Figure 12～Figure 14 as can be known, it is loose structure that the present invention contains dimethyl sulfone biodegradable microspheres internal structure, and along with increasing of dimethyl sulfone concentration wherein, pore structure is network-like gradually, when dimethyl sulfone concentration is 8.6wt%, be typical radial loose structure, this structure more is conducive to supporting of medicine and discharges.
Utilize tetrazolium blue chromogenic reaction drug sensitive experiment (MTT experiment) to detect the MC3T3-E1 osteoblast of growing up under the dimethyl sulfone biodegradable microspheres condition of culture that contains for preparing in 5.3 containing, obtain bar diagram, shown in d ' among Figure 16.As shown in Figure 16, d ' group cell proliferation situation is better than other and contains two groups of dimethyl sulfone biodegradable microspheres; When cultivating 1 day, c ' and b ', d ' have significant difference (P＜0.05) respectively, illustrate in the time of 1 day, among the embodiment 2 preparation to contain the dimethyl sulfone biodegradable microspheres obvious to the MC3T3-E1 cel l proliferation, but and blank assay a ' do not have significant difference; When cultivating 3 days, the cell proliferation situation is along with the increase of drug level has propagation trend, but do not have significant difference; But after cultivating 7 days, cell has tangible propagation trend, and d ' has significant difference respectively with a ', b ', c '.The result shows that the MC3T3-E1 cell is containing during the dimethyl sulfone biodegradable microspheres cultivates altogether, its multiplication capacity and cytoactive all are better than simple polylactic acid biodegradable microspheres, therefore as can be known, dimethyl sulfone can promote the growth and breeding of MC3T3-E1 cell, also can strengthen cell viability, show good biological activity.
The medicine drug loading and the embedding rate that contain the dimethyl sulfone biodegradable microspheres of table 2 different MS M prepared at concentrations
Embodiment 6
6.1 polylactic acid and dimethyl sulfone are dissolved in respectively in chloroform and the acetone, and fully stir, ultrasonic even then with both blend, obtain decentralized photo, the content of polylactic acid is 3wt% in the decentralized photo, the content of dimethyl sulfone is 4.5wt%.
6.2 the decentralized photo that obtains in 6.1 is added in the bin, under the impressed pressure effect, decentralized photo is pressed in the continuous phase by cellular glass film (SPG microporous membrane), thereby prepare monodispersed O/W type emulsion, continuous phase is that 300ml contains sodium lauryl sulphate (SDS) 2g/L and polyvinyl alcohol 1g/L; The SPG membrane aperture is 5.1 μ m.
6.3 the O/W type emulsion that obtains in 6.2 is transferred to rapidly in 15 ℃ of temperature chambers, 300rpm/min stirs 7h, along with constantly also volatilizing gradually to the continuous phase diffusion, decentralized photo solvent chloroform and acetone removes, be solidified into and contain the dimethyl sulfone biodegradable microspheres, the final suspension that contains the dimethyl sulfone biodegradable microspheres that forms, the centrifugal 30min of 8000r/min, deionized water wash 3 times obtains containing the dimethyl sulfone biodegradable microspheres after the lyophilization.
The dimethyl sulfone biodegradable microspheres that contains that obtains in 6.3 that takes a morsel is suspended in the distilled water, drop on the silicon chip on a small quantity with the absorption of 1ml pipettor, it is evenly spread out, naturally dry, be fixed on the sample platform with double faced adhesive tape, after metal spraying is handled under the vacuum condition, utilize scanning electron microscope that it is analyzed, obtain its stereoscan photograph, as shown in Figure 7, its scale is 10 μ m.
7.1 polylactic acid and dimethyl sulfone are dissolved in respectively in chloroform and the acetone, and fully stir, ultrasonic even then with both blend, obtain decentralized photo, the content of polylactic acid is 3wt% in the decentralized photo, the content of dimethyl sulfone is 4.5wt%.
7.2 the decentralized photo that obtains in 7.1 is added in the bin, under the impressed pressure effect, decentralized photo is pressed in the continuous phase by cellular glass film (SPG microporous membrane), thereby prepare monodispersed O/W type emulsion, continuous phase is that 300ml contains sodium lauryl sulphate (SDS) 2g/L and polyvinyl alcohol 1g/L; The SPG membrane aperture is 5.1 μ m.
7.3 the O/W type emulsion that obtains in 7.2 is transferred to rapidly in 15 ℃ of temperature chambers, 700rpm/min stirs 7h, along with constantly also volatilizing gradually to the continuous phase diffusion, decentralized photo solvent chloroform and acetone removes, be solidified into and contain the dimethyl sulfone biodegradable microspheres, the final suspension that contains the dimethyl sulfone biodegradable microspheres that forms, the centrifugal 30min of 8000r/min, deionized water wash 3 times obtains containing the dimethyl sulfone biodegradable microspheres after the lyophilization.
The dimethyl sulfone biodegradable microspheres that contains that obtains in 7.3 that takes a morsel is suspended in the distilled water, drop on the silicon chip on a small quantity with the absorption of 1ml pipettor, it is evenly spread out, naturally dry, be fixed on the sample platform with double faced adhesive tape, after metal spraying is handled under the vacuum condition, utilize scanning electron microscope that it is analyzed, obtain its stereoscan photograph, as shown in Figure 9, its scale is 10 μ m.
The average diameter that contains the dimethyl sulfone biodegradable microspheres that embodiment 6, embodiment 2, embodiment 7 obtain is mapped to stir speed (S.S.), obtain bar diagram as shown in figure 17.As shown in Figure 17, increase along with speed of agitator, the size that contains the dimethyl sulfone biodegradable microspheres can diminish gradually, and has statistical significance (P＜0.05) between 300rpm/min and 500rpm/min, 300rpm/min and 700rpm/min, 500rpm/min and 700rpm/min.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. one kind contains the dimethyl sulfone biodegradable microspheres, it is characterized in that, is made up of dimethyl sulfone and biodegradability macromolecule.
2. the dimethyl sulfone biodegradable microspheres that contains according to claim 1 is characterized in that, described biodegradability macromolecule is selected from a kind of in polylactic acid, polyglycolic acid, polylactic acid-glycolic guanidine-acetic acid copolymer and the polycaprolactone.
3. a preparation method that contains the dimethyl sulfone biodegradable microspheres is characterized in that, may further comprise the steps:
A) dimethyl sulfone is dissolved in first organic solvent, the mixed solution of winning; The biodegradability macromolecule is dissolved in second organic solvent, gets second mixed solution;
B) described first mixed solution is mixed with described second mixed solution, obtain the 3rd mixed solution, go in the continuous phase by mould then, obtain O/W type emulsion;
C) described O/W type emulsion is stirred, obtain containing the dimethyl sulfone biodegradable microspheres.
4. preparation method according to claim 3 is characterized in that, described film is microporous membrane.
5. preparation method according to claim 3 is characterized in that, described continuous phase is the aqueous solution that comprises surfactant and dispersant.
6. preparation method according to claim 3 is characterized in that, the speed of described stirring is 300～700rpm/min, and the time of stirring is 6～8h.
7. preparation method according to claim 3 is characterized in that, described first organic solvent is selected from a kind of in ethanol, benzene, methanol and the acetone.
8. preparation method according to claim 3 is characterized in that, described second organic solvent is selected from chloroform, dichloromethane and N, a kind of in the dinethylformamide.
9. preparation method according to claim 3 is characterized in that, the high molecular concentration of biodegradability is 1～7wt% in described the 3rd mixed solution.
10. preparation method according to claim 3 is characterized in that, the concentration of dimethyl sulfone is 1～15wt% in described the 3rd mixed solution.
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