CN100522145C - Preparation process of nano drug-loaded biological micro-capsule - Google Patents
Preparation process of nano drug-loaded biological micro-capsule Download PDFInfo
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- CN100522145C CN100522145C CNB2006100983817A CN200610098381A CN100522145C CN 100522145 C CN100522145 C CN 100522145C CN B2006100983817 A CNB2006100983817 A CN B2006100983817A CN 200610098381 A CN200610098381 A CN 200610098381A CN 100522145 C CN100522145 C CN 100522145C
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Abstract
The invention relates to a method for producing nanometer carrier biological micro capsule, wherein it comprises that: using general method to prepare oil-water reverse micro emulsion; degrading anion and cation polyelectrolyte polysaccharide, to obtain low molecular anion and cation polyelectrolyte polysaccharide; adding said polysaccharide into micro emulsion, to form capsule; separating solid and liquid, to obtain solid micro capsule. The invention has low cost and better slow-release property.
Description
One, technical field
The invention belongs to the pharmaceutical carrier preparing technical field, particularly a kind of method for preparing nano drug-loaded biological micro-capsule.
Two, background technology
For organizational project, the surface that has now found that lyophilizing decalcification bone is a nanostructured; Can some zest medicine directly be brought in the body by Transdermal absorption the nano level pharmaceutical carrier of pharmaceutical engineering, accomplish local application; For biotechnology, have an effect with extracellular mechanism so that the nano-particle passenger gene is easier.To not have the immunosuppressant natural product and make the nanoscale microcapsule, and can solve organizational project, a lot of problems that pharmaceutical engineering and biological engineering face have certain meaning to the development of three large-engineerings.As no immunosuppressant natural product, natural polysaccharide is because its superior performance is the object of educational circles's research always, reported the application of relevant natural polysaccharide aspect biomaterial over 10 years in the document in a large number, for example Lee etc. is a capsule material load insulin with the sodium alginate, people such as Lim transplant the microencapsulation technology and combine with histiocyte, preparation sodium alginate/polylysine (APA) microcapsule is as immune isolating tool, these achievements in research preferably resolve the immunologic rejection problem in the histiocyte migration process, avoid or reduced the use of expensive immunosuppressant, for histiocyte transplantation treatment nerve/endocrine system disease provides new approaches.But because the restriction of technology of preparing and material, the capsule of preparation can't reach ideal effect, often there are problems such as big or small heterogeneity or particle diameter be excessive, such capsule is limited in actual applications, research to such capsule medicine carrying in recent years reduces gradually, and turns to other aspects such as Risbud etc. to prepare support etc. with oligochitosan, gelatin.
For traditional material, prepare capsular technology and comprise physical method, chemical method and physico-chemical process, emulsion method and spray drying method are comparatively commonly used in each class methods, spray drying method encystation uniform particle diameter, size is controlled in micron arrives the millimeter scope, but need special instrument and equipment difficult universal, though the emulsion method special installation that need not simple to operate, the granule that forms often is difficult to control in many, the big or small heterogeneities of micron order and influence factor, encystation condition.Microemulsion method was because the system homogeneous in recent years, easy and simple to handle, the equal first-class advantage of capsule grain diameter of preparation is widely used, though the use of this method is by wide coverage, but only be confined to combining nano reactor and two kinds of technology of microemulsion polymerization such as the polymer substance of inorganic matter and some chemosynthesis such as Catherine, p-poly-phenyl amine coats barium sulfate nanoparticles and studies, and finds in microemulsion system two class material encystation big or small homogeneous satisfactory for result and is nanoscale.But in the report, the employed material of microemulsion system and nano-reactor does not often possess the good biocompatibility of natural product and can cause immune system response, is not ideal biomaterial, can't apply.Therefore, contradiction between current solution preparation method, material and the practical application will be the key subjects of technical field of biological material, will be to organizational project if can carry out the encystation reaction with ideal preparation method and desirable material, good impetus is played in field such as pharmacy and biomedicine.
Three, summary of the invention
Technical problem to be solved by this invention is at having preparation method in the above traditional handicraft, and the contradiction between material and the practical application proposes a kind of method for preparing nano drug-loaded biological micro-capsule.
Technical solution of the present invention is: a kind of method for preparing nano drug-loaded biological micro-capsule, and preparation process is: prepare water-in-oil type reverse microemulsion system by conventional method; Respectively yin, yang ion polyelectrolyte polysaccharide is carried out degradation treatment, obtain corresponding low-molecular-weight yin, yang ion polyelectrolyte polysaccharide; In microemulsion system, add the low-molecular-weight yin, yang ion polyelectrolyte polysaccharide after degradation treatment, by self-assembling reaction polymerization encystation; With gained contain capsule microemulsion system solid-liquid separation, obtain the solid microcapsule.
What the technical program proposed is the encystation environment with the microemulsion system, with sodium alginate and chitosan material is example, improved sodium alginate can form nano-micelle in microemulsion system, in nano-reactor, sodium alginate aggregates into outer surface for nuclear and oligochitosan by negative ions, such capsule having many uses aspect pharmaceutical preparation is for pharmaceutical science provides new dosage form.
The capsular synthetic route of such microemulsion as shown in Figure 1.From accompanying drawing 1 as can be seen: capsular synthetic preparation, the preparation of oligochitosan/sodium alginate microemulsion system, synthetic three steps of capsule nano-reactor that can be divided into the microemulsion system of sodium alginate of microemulsion.Described microemulsion system is that karyomorphism becomes nano-micelle by sodium alginate soln, and oligochitosan is that shell is combined into microsphere in skin.Wherein the single nanoparticle particle diameter is generally about 130nm.Such capsule has avoided some complex microsphere by macromolecular material (as polystyrene etc.) or the particulate complex steps of inorganic material (as silicon dioxide, carbon etc.) embedded nano, and particle diameter is even, good dispersion, can further control particulate size, encystation factors such as the thickness of film by conditions such as mixing speeds.
At microemulsion system may be in capsule the problem of residual surfactant and cosurfactant, this paper has carried out evaluation of its biocompatibility to capsule, by cell toxicity test and general toxicity test, researcher discovery cell degree of propagation relatively brought up to 95.39% from 80.30% in seven days, illustrative material leachable pair cell is injury not, and toxicity is less.Observe mice by the whole body acute toxicity test, find that the mouse movement diet is all normal, do not see the abdominal part irritation, blepharoptosis, etc. negative characteristics, the every index of mice all meets the specified in more detail of being done on the front page " medical apparatus and instruments supervision and management and evaluation " in June, 2000.Experimental result confirms that oligochitosan/sodium alginate micro gel capsule there is no acute toxicity effect, good biocompatibility.
The material price that the present invention adopts is cheap easily to be obtained, the new technique that relates to, route is simple, and is with high content of technology, and product drug loading height, slow-releasing is good, and pair cell and organism are nontoxic, is expected to replace present stage employed traditional material and preparation method, solved current same type of material big or small heterogeneity in capsular preparation process, oversize, exist the high-leakage rate in the use, the problem of low embedding rate.
Four, description of drawings
Fig. 1 is the preparation layout;
Fig. 2 is the capsule structure sketch map;
Fig. 3 is a BSA capsule slow-releasing result of study curve chart;
Fig. 4 is the result of study curve chart of lysophospholipase A1;
Fig. 5 is a capsular TEM photo in the microemulsion system;
Fig. 6 is a capsular TEM photo in the microemulsion system;
Fig. 7 is the photo of embodiment 11;
Fig. 8 is the photo of embodiment 12.
Five, the specific embodiment
One, natural polyelectrolyte polysaccharide such as sodium carboxymethyl cellulose, gelatin, transparent fat acid sodium, sodium alginate, oligo-glucosamine, methylol chitosan, chitosan and oligochitosan being carried out degradation treatment, is that example illustrates one by one with sodium alginate, methylol chitosan, chitosan:
Embodiment 1: with the research of Preparation of Chitosan low-molecular weight chitoglycan:
Be reflected in the heterogeneous system and carry out, chitosan at first is scattered in the deionized water, add hydrogen peroxide with mass ratio 1:1 then, in water bath with thermostatic control, carry out degradation reaction, control molecular weight of product by adjusting the response time, the step of concrete degradation reaction is as follows: after installing experimental apparatus according to requirement of experiment, add (3.000g) chitosan that takes by weighing, add deionized water with mass ratio 25:1 (W:W) subsequently, chitosan is fully disperseed, and heat temperature raising to 75 ℃ stirs between 85 ℃, make the chitosan isothermal reaction to solution colour by colourless transfer to yellowish green, stopped reaction.The elimination insoluble substance, transfer pH value of filtrate to 8, filter, filtrate is concentrated into about 1/3rd of original volume at 50 ℃ of reduction vaporizations on the rotary evaporator, dehydrated alcohol precipitation with 9 times of filtrate volumes, twice, 50 ℃ of 90% washing with alcohol obtains water miscible low-molecular weight chitoglycan at last with interior drying under reduced pressure.
Embodiment 2: study with methylol Preparation of Chitosan low-molecular-weight methylol chitosan
Be reflected in the heterogeneous system and carry out, the methylol chitosan at first is scattered in the deionized water, add hydrogen peroxide with mass ratio 1:1 then, in water bath with thermostatic control, carry out degradation reaction, control molecular weight of product by adjusting the response time, the step of concrete degradation reaction is as follows: after installing experimental apparatus according to requirement of experiment, add (3.000g) methylol chitosan that takes by weighing, add deionized water with mass ratio 25:1 subsequently, the methylol chitosan is fully disperseed, and heat temperature raising to 75 ℃ stirs between 85 ℃, make the isothermal reaction of methylol chitosan to solution colour by colourless transfer to yellowish green, stopped reaction.The elimination insoluble substance, transfer pH value of filtrate to 8, filter, filtrate is concentrated into about 1/3rd of original volume at 50 ℃ of reduction vaporizations on the rotary evaporator, dehydrated alcohol precipitation with 9 times of filtrate volumes, twice, 50 ℃ of 90% washing with alcohol obtains water miscible low-molecular-weight methylol chitosan at last with interior drying under reduced pressure.
Embodiment 3: the research for preparing low molecular weight seaweed acid sodium with sodium alginate is example:
Carry out the degraded of sodium alginate in homogeneous system, at first the sodium alginate that drying is crossed is dissolved in the deionized water, is configured to concentration and is 2% solution, and the parallel sodium alginate homogeneous phase solution 10mL that gets three part 2% is tiled in 18cm
2* 8cm
2Flat board on, in ultraviolet device, degrade, choose degradation model voluntarily, ultraviolet ray intensity is 48W, wavelength is 312nm, every square centimeter irradiation dose is 0.120J/cm
2
Embodiment 4: preparing the low-molecular-weight sodium carboxymethyl cellulose with sodium carboxymethyl cellulose is example
In homogeneous system, carry out the degraded of sodium carboxymethyl cellulose, at first the sodium carboxymethyl cellulose that drying is crossed is dissolved in the deionized water, is configured to concentration and is 2% solution, is heated to 100 ℃, be cooled to the parallel sodium carboxymethyl cellulose homogeneous phase solution 10mL that gets three part 2% after the room temperature, be tiled in 18cm
2* 8cm
2Flat board on, in ultraviolet device, degrade, choose degradation model voluntarily, ultraviolet ray intensity is 48W, wavelength is 312nm, every square centimeter irradiation dose is 0.120J/cm
2
Two, the design of microemulsion system: the fixing addition of oil phase (O), prepare the mixed liquor of a series of [surfactant (S)/oil phase (O)]=1:3, wherein oil phase can be paraffin oil, a kind of in cyclohexane extraction or the octane, surfactant can be in TX-100, polyoxyethylene laurel ether, sulfo-succinic acid two (2-ethyl oneself) ester sodium, polyoxyethylene (20), sorbitan mono-oleic acid ester, n-butyl alcohol or the n-amyl alcohol one or more, drips the polyelectrolyte polysaccharide solution and form microemulsion system in mixed liquor.
Embodiment 5: with paraffin oil, sorbitan mono-oleic acid ester, n-butyl alcohol and low-molecular weight chitoglycan solution is the example explanation
Paraffin oil is scattered in sorbitan mono-oleic acid ester and the n-butyl alcohol, disperse the polyelectrolyte polysaccharide in paraffin oil in the process of high-speed stirred (more than 2000 revolutions per seconds), sorbitan mono-oleic acid ester, in the n-butyl alcohol, with sorbitan mono-oleic acid ester/n-butyl alcohol is 3:2 (S component), the ratio of paraffin oil and S component mixed liquor is that 3:1 makes mixed system under the condition of high-speed stirred more than 2000 revolutions per seconds, under stirring condition, in system, drip low-molecular weight chitoglycan solution, concentration is according to conventional use amount, dripping quantity is about the 20-30% of above-mentioned system volume, continue to stir more than ten minutes, form microemulsion system.
Embodiment 6: with cyclohexane extraction, n-amyl alcohol and TX-100 and low-molecular-weight sodium alginate soln is the example explanation
N-amyl alcohol and TX-100 are thought that 3:2 mixes (S mixed liquor), the S mixed liquor is mixed with 1:3 with cyclohexane extraction, drip low-molecular-weight sodium alginate soln in (more than 500 revolutions per seconds) the dispersive process of stirring, concentration is according to conventional use amount, dripping quantity is about the 20-30% of above-mentioned system volume, continue to stir more than 10 minutes, form microemulsion system.
Three, the preparation of microcapsule
Embodiment 7: preparing capsule in conjunction with microemulsion system and nano-reactor, is the polyelectrolyte polysaccharide with sodium alginate and chitosan, and paraffin oil, sorbitan mono-oleic acid ester, n-butyl alcohol are that oil phase and surfactant are example:
The preparation of microcapsule is carried out in homogeneous system, at first with low molecular weight seaweed acid sodium, the low-molecular weight chitoglycan powder is mixed with concentration respectively and is lower than 7% solution, at first the low molecular weight seaweed acid sodium solution is scattered in volume ratio 1:1 and makes mixed liquor S ' in the emulsifying agent, the oil phase that adds 4 times of volume mixture liquid S ' in 15 minutes rapidly, continue to stir the water-in-oil microemulsion liquid system that forms sodium alginate aqueous solution/oil phase/emulsifying agent, to stir more than 30 minutes more than 500 revolutions per seconds, in solution, at the uniform velocity drip the chitosan solution that is no less than sodium alginate soln amount 1/3rd in the 30min, continuation is stirred with same speed, more than ten minutes, stop to stir, with mixed system with greater than 3000 revolutions per seconds speed centrifugalize more than 10 minutes, take out the washing with alcohol that precipitates with 90%, filter and obtain microcapsule.As shown in Figure 2, Fig. 5, Fig. 6 are capsular TEM photo in the microemulsion system.This preparation method technology is simple to operation, and the nano particle diameter of formation is evenly distributed, and it is ideal nano-particle that granule does not reunite that adhesion is easy to separate and has excellent biological compatibility.
Embodiment 8: preparing capsule in conjunction with microemulsion system and nano-reactor, is the polyelectrolyte polysaccharide with low-molecular-weight sodium carboxymethyl cellulose and low-molecular-weight carboxymethyl chitosan, and cyclohexane extraction, n-amyl alcohol, TX-100 are that oil phase and surfactant are example:
The preparation of microcapsule is carried out in homogeneous system, at first with the low-molecular-weight sodium carboxymethyl cellulose, low-molecular-weight carboxymethyl chitosan powder is mixed with concentration respectively less than 7% solution, the low-molecular-weight carboxymethylcellulose sodium solution is scattered in volume ratio 1:1 makes mixed liquor A in the emulsifying agent, add 4 times of volumes in 15 minutes rapidly in the oil phase of mixed liquor A, the water-in-oil microemulsion liquid system that forms low-molecular-weight sodium carboxymethyl cellulose solution/oil phase/emulsifying agent is stirred in continuation with the rotating speed more than 500 revolutions per seconds, disperse more than 30 minutes, at the uniform velocity in solution, drip and the low-molecular-weight carboxymethyl chitosan sugar juice that is no less than low-molecular-weight carboxymethylcellulose sodium solution 1/3rd in 30 minutes, continuation is stirred with same speed, more than ten minutes, stop to stir, with mixed system with mixed system with greater than 3000 revolutions per seconds speed centrifugalize more than 10 minutes, take out the washing with alcohol that precipitates with 90%, filter and obtain microcapsule.
Four, the research of counter-example.The polyelectrolyte polysaccharide is not carried out technical finesse, will directly add emulsification system less than the chitosan or the sodium alginate of degraded,
Can embodiment 9: investigate not degrade chitosan and make microemulsion system
Paraffin oil is scattered in sorbitan mono-oleic acid ester and the n-butyl alcohol, disperse chitosan in paraffin oil in the process of high-speed stirred (more than 2000 revolutions per seconds), sorbitan mono-oleic acid ester, in the n-butyl alcohol, with sorbitan mono-oleic acid ester/n-butyl alcohol is 3:2 (S component), the ratio of paraffin oil and S component mixed liquor is that 3:1 makes mixed system under the condition of high-speed stirred more than 2000 revolutions per seconds, under stirring condition, in system, drip low chitosan solution, concentration is according to conventional use amount, dripping quantity is about the 20-30% of above-mentioned system volume, continue to stir more than ten minutes, form flocculent deposit, can't form microemulsion system and also can't carry out the work that next step makes microcapsule.
Can embodiment 10: investigate the sodium alginate of not degrading and make microemulsion system
Paraffin oil is scattered in sorbitan mono-oleic acid ester and the n-butyl alcohol, disperse chitosan in paraffin oil in the process of high-speed stirred (more than 2000 revolutions per seconds), sorbitan mono-oleic acid ester, in the n-butyl alcohol, with sorbitan mono-oleic acid ester/n-butyl alcohol is 3:2 (S component), the ratio of paraffin oil and S component mixed liquor is that 3:1 makes mixed system under the condition of high-speed stirred more than 2000 revolutions per seconds, under stirring condition, in system, drip low chitosan solution, concentration is according to conventional use amount, dripping quantity is about the 20-30% of above-mentioned system volume, continue to stir more than ten minutes, form flocculent deposit, can't form microemulsion system and also can't carry out the work that next step makes microcapsule.
Embodiment 11: investigate similar system
Get sodium alginate aqueous solution and be emulsifiable in the paraffin oil with 1:10, fully stirring makes reaction system be emulsion, and keeps 20min.Slowly drip then and contain the following 3%Ca-Cl of 7% chitosan
2Aqueous solution.Behind the 30min, centrifugalize (rotating speed is not less than 2000 revolutions per seconds), filtration washing is used the kerosene thorough washing, and residual organic matter is removed in the extracting in apparatus,Soxhlet's of reuse dehydrated alcohol.Oven dry, vacuum drying, stand-by, show that by photo capsule is difficult to reach nanoscale as shown in Figure 7.
Embodiment 12: investigate similar system:
Get sodium alginate aqueous solution and be emulsifiable in the paraffin oil with 1:10, fully stirring makes reaction system be emulsion, and keeps 20min.Slowly drip then and contain the following aqueous solution of 7% chitosan.Behind the 30min, centrifugalize (rotating speed is not less than 2000 revolutions per seconds), filtration washing is used the kerosene thorough washing, and the extracting in apparatus,Soxhlet's of reuse dehydrated alcohol is removed residual organic matter, vacuum drying.Show the sticking connection of capsule phenomenon seriously as shown in Figure 8 by photo.
Five, in microemulsion system, can prepare embedding BSA, the capsule of protide such as BCA or enzyme material, and capsule is carried out the investigation of drug loading and sustained release performance.
Embodiment 11: investigate the capsular process of preparation BSA in the microemulsion system
Prepare the BSA capsule with microemulsion system
BSA and sodium alginate soln blend are prepared into the homogeneous system of 0.2-1.0mg/mL, note during preparation and can not stir rapidly, otherwise the activity that a large amount of foams influence BSA occurs in the system.The homogeneous system of embedding BSA is scattered in to make the volume ratio of BSA and S mixed liquor in the blended emulsifier (S mixed liquor) that n-amyl alcohol and TX-100 are 3:2 be that 1:1 makes the A mixed liquor, add 4 times in 15 minutes rapidly to the oil phase of A mixed liquor, continue to stir the water in oil translucent emulsion system that forms sodium alginate/BSA/ aqueous solution (W)/oil phase/emulsifying agent, high-speed stirred (more than 1000 revolutions per seconds), dripping concentration in 30min in solution is lower than 7% low-molecular weight chitoglycan solution and makes its addition that is no less than sodium alginate 1/3rd, continuation is stirred more than 30 minutes with same rotating speed, stop to stir, with mixed liquor centrifugalize (more than 3000 revolutions per seconds), taking out precipitation is 90% washing with alcohol with concentration, filters and obtains microcapsule.
Capsule is to the research of BSA drug loading
With microcapsule concentration is the liquefaction of 4% sodium citrate, and by the BSA envelop rate of microplate reader micrometer capsule, by BSA test kit production standard curve, the OD value of BSA is calculated the content of BSA in the broken capsule liquid of test, and calculates the envelop rate of microcapsule by following formula:
Envelop rate=(medicament contg/dosage) * 100%
Know that as calculated capsule can reach 88.4% to the drug loading of BSA.
The research of capsule sustained release performance
It is an amount of that precision takes by weighing microcapsule, with PBS (pH=7.4) is release medium, constant temperature (37 ± 0.5) ℃, rate oscillation with 100r/min was got the release liquid that the 5mL release medium is replenished the equivalent equality of temperature simultaneously to increase progressively trend in 48 hours, measure solution absorbance, draw cumulative release rate-time graph by drug level.See Fig. 3
Embodiment 12: investigate the capsular process of preparation lysophospholipase A1 in the microemulsion system
Prepare lysophospholipase A1 capsule with microemulsion system
Get 0.5ml enzyme liquid and the blend of 5ml3% sodium alginate soln is prepared into homogeneous system.The homogeneous system of embedding lysozyme A1 is scattered in the emulsifier tween, adds a certain amount of oil phase in 15 minutes rapidly, the ratio that makes emulsifying agent and oil phase is 1; 3, continuing to stir formation is the as clear as crystal emulsion system of anti-phase W/O that water comprises oil phase and emulsifying agent simultaneously with sodium alginate/lysophospholipase A1 aqueous solution, disperse more than 30 minutes, in 30min, in solution, slowly drip the low-molecular weight chitoglycan solution that is no less than sodium alginate soln 1/3rd, continue to react 30 minutes to stir more than 500 revolutions per seconds, stopped reaction, with the system centrifugalize, take out precipitation with the gradient washing with alcohol, filter and obtain microcapsule.
Capsule is to the research of lysophospholipase A vigor
Fresh egg yolk 5ml with 5% is a substrate, Tris-HCl with 10ml0.05mol/L is a buffer solution, the sodium deoxycholate that adds 5ml0.01mol/L, the CaCl2 of 5ml25mmol/L, the enzyme liquid that adds experiment institute consumption, 50 ℃ of constant temperature vibrations are in 10min NaOH titration with 0.1mol/L in the time, keep constant pH, measure the NaOH volume that is consumed.Enzyme activity unit is defined as, and per minute discharges the enzyme amount of 1 μ mol free fatty under these conditions, is a zymetology unit (μ).
It is an amount of that precision takes by weighing microcapsule, lives with the enzyme of said method mensuration in one week, draws enzyme half-life curve alive.See Fig. 4
Five, capsule is carried out evaluation of its biocompatibility
Embodiment 13: capsule is carried out mice whole body acute toxicity, cell toxicity test
The test of mice general toxicity
This experiment is a kind of non-specific acute toxicity test, with experiment material or material lixiviating solution by animal vein or lumbar injection in animal body, observe its biologically to judge the acute toxicity effect of material.
Select not pass through ten of the Kunming white mice of any other experiment, every heavily about 23g-25g under same environmental condition, uses same feedstuff cultivation 3 days before the experiment, and body weight does not alleviate during this period, no abnormal phenomenons such as spirit, appetite, drainage.With the negative contrast of 0.9% sodium chloride solution, investigate the biocompatibility of sodium alginate/chitosan oligosaccharide capsule lixiviating solution.The lixiviating solution preparation method is: before making lixiviating solution, use earlier and sterilization 0.9% sodium chloride solution of the same lot number of negative control flushing 3 times, lixiviating solution is according to " sample ": " 0.9% sodium chloride solution of sterilizing "=0.1 (g): 1 (ml) (W/V) lixiviate, external condition is: on the constant temperature shaking table in 37 ± 0.15 ℃, 50rmin
-1Jolting, extraction time are 48h.
Kunming mice is divided into two groups every group five, be that two groups of mices are injected sterilization 0.9% sodium chloride solution and material lixiviating solution respectively, injection volume is per kilogram 50mL (50mL/kg), two groups of kunming mices are weighed and under same environmental condition, use same feedstuff to cultivate 24h, 48h, the 72h body weight of weighing mice respectively.The record mice has or not bad reflection.The phenomenon that mice had the light exercise ability to go down at four hours in the viewing test group is recovered normal other mices again and abnormal conditions all do not occurred after 18 hours.The dependency rule that meets " pharmacopeia "
Cell toxicity test
Recovery L-929 cell is in the 25mL culture bottle, and 0.25% trypsinization is gone down to posterity two to three times, is mixed with 1 * 10
4Individual/mL cell suspension inoculation in 96 hole plastic culture dish, every hole 100u L, the hole of removing the orifice plate edge, every group of each issue parallel observations, 6 holes, plastic culture dish places to contain cultivates 24h in 5% (V/V) CO2 gas incubator.Discard former culture medium, with PBS liquid washed twice, test group adds above-mentioned sample lixiviating solution 0.1mL respectively on aseptic filter paper, adds 100 μ L/ hole fresh mediums in the lump together with matched group then, puts into above-mentioned culture environment and keeps 2d.MTT (2mg/mL) liquid that adds 20 μ L/ holes, continuation cultivation 6h abandons culture fluid and the sample in the hole, and remaining sample is two to three times in the usefulness PBS solution washing hole, adds the DMSO in 150 μ L/ holes, concussion 10min measures absorbance with the 490nm wavelength on immune microplate reader.Calculate the relative propagation degree (RGR) of cell according to following formula:
RGR=(experimental group mean light absorbency/negative control group mean light absorbency) * 100%
Claims (1)
1. method for preparing nano drug-loaded biological micro-capsule is characterized in that preparation process is:
A. with the hydrogen peroxide edman degradation Edman cationic polyelectrolyte polysaccharide is carried out degradation treatment, with light radiation degradation method the anionic polyelectrolyte polysaccharide is carried out degradation treatment, obtain the moon through degrading accordingly, the cationic polyelectrolyte polysaccharide, the cationic polyelectrolyte polysaccharide that described hydrogen peroxide edman degradation Edman is 1 mass parts is scattered in the deionized water of 25 mass parts, add hydrogen peroxide with mass ratio 1:1 then, heat temperature raising to 75 ℃ is between 85 ℃, stir, make the isothermal reaction of cationic polyelectrolyte polysaccharide to solution colour by colourless transfer to yellowish green, stopped reaction, the elimination insoluble substance, transfer pH value of filtrate to 8, filtrate is concentrated into about 1/3rd of original volume at 50 ℃ of reduction vaporizations on the rotary evaporator, dehydrated alcohol precipitation with 9 times of filtrate volumes, twice, 50 ℃ of 90% washing with alcohol obtains water miscible low molecular weight cationic polyelectrolyte polysaccharide at last with interior drying under reduced pressure; Described light radiation edman degradation Edman is for the anionic polyelectrolyte polysaccharide is dissolved in the deionized water, is configured to mass concentration and is 2% solution, degrades in ultraviolet device, and ultraviolet ray intensity is 48W, and wavelength is 312nm, and every square centimeter irradiation dose is 0.120J/cm
2, the son amount that makes low score anionic polyelectrolyte polysaccharide; Above-mentioned cationic polyelectrolyte polysaccharide is that chitosan, anionic polyelectrolyte polysaccharide are sodium alginate; Perhaps above-mentioned cationic polyelectrolyte polysaccharide is that methylol chitosan, anionic polyelectrolyte polysaccharide are sodium carboxymethyl cellulose;
B. the moon after will degrading, the cationic polyelectrolyte polysaccharide is mixed with mass concentration less than 7% solution, the anionic polyelectrolyte polysaccharide solution is scattered in volume ratio 1:1 makes mixed liquor S in the emulsifying agent, add 4 times of volumes in 15 minutes rapidly in the oil phase of mixed liquor S, stir the water-in-oil microemulsion liquid system that forms polyelectrolyte aqueous solution/oil phase/emulsifying agent, stir more than 30 minutes with the mixing speed more than 500 revolutions per seconds, in 30 minutes, in solution, at the uniform velocity drip the mass concentration be no less than anionic polyelectrolyte polysaccharide solution amount 1/3rd again less than 7% cationic polyelectrolyte polysaccharide solution, reaction encystation; Described oil phase and emulsifying agent are respectively: be oil phase with the paraffin oil, be emulsifying agent with the mixed liquor of sorbitan mono-oleic acid ester and n-butyl alcohol, perhaps be oil phase with the cyclohexane extraction, be emulsifying agent with the mixed liquor of n-amyl alcohol and triton x-100;
C. with step c gained contain capsule microemulsion system solid-liquid separation, obtain the solid microcapsule.
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| CN107281159B (en) * | 2017-06-29 | 2019-12-20 | 安徽大学 | Preparation method of sustained-release drug-loaded microcapsule with multilayer core-shell structure |
| CN108486879A (en) * | 2018-06-08 | 2018-09-04 | 天津工业大学 | A kind of preparation method of natural polysaccharide LBL self-assembly microcapsules |
| CN112370437B (en) * | 2020-10-20 | 2022-10-21 | 好医生药业集团有限公司 | Amoxicillin capsule and preparation method thereof |
| CN115212189A (en) * | 2022-03-30 | 2022-10-21 | 江南大学 | Nano-microsphere for improving oral bioavailability of chitosan oligosaccharide and preparation method thereof |
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| 壳聚糖微胶囊的制备. 张所信，刘丽荣.应用科技，第30卷第4期. 2003 |
| 壳聚糖微胶囊的制备. 张所信，刘丽荣.应用科技，第30卷第4期. 2003 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103769017A (en) * | 2012-10-23 | 2014-05-07 | 中国科学院理化技术研究所 | Microencapsulation method of water-soluble inorganic salt |
| CN103769017B (en) * | 2012-10-23 | 2015-08-12 | 中国科学院理化技术研究所 | Microencapsulation method of water-soluble inorganic salt |
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