CN107629142A - Cross-link dextran microballoon of modification and preparation method thereof and the medical composition containing this microballoon - Google Patents

Cross-link dextran microballoon of modification and preparation method thereof and the medical composition containing this microballoon Download PDF

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CN107629142A
CN107629142A CN201710852690.7A CN201710852690A CN107629142A CN 107629142 A CN107629142 A CN 107629142A CN 201710852690 A CN201710852690 A CN 201710852690A CN 107629142 A CN107629142 A CN 107629142A
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cross
microballoon
link dextran
modification
reaction
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何浩明
于帆
于一帆
唐小康
栾立标
管汉亮
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CHANGZHOU INSTITUTE OF MATERIA MEDICA Co Ltd
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CHANGZHOU INSTITUTE OF MATERIA MEDICA Co Ltd
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Abstract

The invention discloses a kind of cation modified cross-link dextran microballoon and preparation method thereof and the medical composition containing this microballoon, described cation modified cross-link dextran refer to that the H of the part OH in cross-link dextran chemical constitution is substituted by cation group.Described medical composition is composed of the following components：The cross-link dextran microballoon of modification, cross-linking hyaluronic acid sodium, PBS surplus.Composition is injected in vivo relevant position in the form of injection, cross-link dextran microballoon is delivered to injection site by cross-linking hyaluronic acid sodium as carrier, as cross-linking hyaluronic acid sodium is absorbed by tissue, cross-link dextran microballoon is gradually surrounded in target position by the connective tissue of body itself, and promote the generation of fibroblast and collagen between microballoon, so as to the volume of stabilisation implant, reach lasting filling effect.The medical composition of the present invention is a kind of three class medicine equipments for being preferably used to treat VUR, the urinary incontinence and incontinence of faces.

Description

Cross-link dextran microballoon of modification and preparation method thereof and medical group containing this microballoon Compound
Technical field
The present invention relates to a kind of degradable polymer microsphere, and in particular to a kind of cross-link dextran microballoon of modification and its Preparation method and the composition for treating VUR, the urinary incontinence and incontinence of faces containing this microballoon.
Background technology
Vesicoureteric reflux（VUR）Refer to that urine is from bladder reflux to ureter and renal plevis during urination.Reflux nephropathy （RN）It is due to VUR and intrarenal reflux（IRR）With urinary tract infections repeatedly, kidney is caused to form scar, atrophy, renal dysfunction Syndrome, chronic renal failure can be developed into by such as treating and correcting not in time.VUR not only occurs on children, and in UTI repeatedly （Urinary tract infections）On the basis of continue to adult, cause kidney function damage.
Incontinence of faces refers to that anal sphincter loses the control ability to excrement and gas discharge, belongs to bowel movement function disorder One kind.The incidence of general population's incontinence of faces is l%~2.2%.With the increase at age, the incidence of incontinence of faces Increase, the incidence of disease of over-65s incontinence of faces are 5 times of young people.Incontinence of faces easily causes multiple complications（Perineum, sacrum Afterbody dermatitis and pressure ulcer）, great pain not only is brought to patient, and many be stranded is brought to nursery work It is difficult.Treatment means include drug therapy and operative treatment, drug therapy at present mainly using Loperamide and diphenoxylate, But effect is not notable, and raised with larger side effect, such as blood pressure；For operative treatment, many gerontal patients are Operative treatment can not be born, surgical procedure and post operative recovery procedures can also bring great pain to patient.
Stress incontinence（Stress Urinary Incontinence, SUI）Refer to that the abdominal pressures such as sneeze or cough increase When there is involuntary urine from orificium urethrae externum seepage.At present, the whole world has 200,000,000 people to perplex by the different degrees of urinary incontinence, its Middle adult female urinary incontinence incidence is 25%~45%, and 7% or so has obvious incontinence, wherein about 50% is pressure The urinary incontinence.The multizone investigation of one, China shows that Chinese women urinary incontinence incidence is 30.9%, and the Chinese male urinary incontinence occurs Rate is 3%~10%.To the year two thousand thirty, the elderly population of China 60 years old and the above are up to 4.2 hundred million or so.Due in the elderly Urinary incontinence ratio is higher, therefore will be more and more by the number that the urinary incontinence perplexs.The serious urinary incontinence can use operative treatment, but one As light moderate the urinary incontinence there has been no effective treatment means, can only passively use adult's paper diaper, the painful topic of patient It is agonizing.
The composition gels of cross-link dextran microballoon are injected in the submucous layer of bladder of adjacent Ureteral orifice, and injection is formed Gel tissue distal ureter is attached in filling of bladder and contraction process, gradually by body itself connective tissue surround； Along with being injected in vivo as after implant, cross-link dextran microballoon can promote the life of fibroblast and collagen between microballoon Into so that the position of stabilisation implant and volume, reach lasting filling effect.Therefore can using this physiologic properties come Treat VUR, stress incontinence and incontinence of faces.
On the preparation method of cross-link dextran, Chinese patent literature CN 101182380（Application number 200610107398.4）A kind of synthetic method of anti-phase sephadex gel is disclosed, weighs glucan 100g, sodium hydroxide 10g, which is dissolved in 75ml distilled water, is configured to glucan alkali lye, weighs polyvinyl acetate 25-60g, molten under agitation Solution is configured to polyvinyl acetate epoxychloropropane solution in 500-900ml epoxychloropropane, is stirring speed as 500rpm's Under the conditions of, glucan alkali lye is poured into polyvinyl acetate with epoxychloropropane solution, carrying out cross-linking reaction, temperature 50 DEG C, react 5 hours；Reaction product with 3 times of amount ethanol and distills water washing 3 times successively, then is dried 2 hours through 50 DEG C, you can Product.Irregular sephadex can be made according to the method described above, but cross-link dextran microballoon can not be obtained, can not The cross-link dextran microballoon modified.
Applicant submitted entitled on April 14th, 2017《Treat VUR, stress incontinence and big Composition of fecal incontinence and preparation method thereof》With《The degree of cross linking detection method of cross-link dextran microballoon》Application for a patent for invention, Applicant is existing to declare present patent application by further developmental achievement.
The content of the invention
The technical problems to be solved by the invention be to provide a kind of biodegradable modification cross-link dextran microballoon and Its preparation method and the composition for treating VUR, the urinary incontinence and incontinence of faces containing this microballoon.Realize this hair The technical scheme of bright first purpose is a kind of cross-link dextran microballoon of modification, is microspheric, cation modified crosslinking Portugal Glycan, described cation modified cross-link dextran refer to the H of the part-OH in cross-link dextran chemical constitution by-CH2- CH2-N+H-(C2H5)2Substitute or by-CH2-CH2-N+H-(CH3)2Substitution.
The nitrogen content of the cross-link dextran of the modification is 0.5%~2.0%.
The particle diameter of the cross-link dextran dry microspheres of the modification is 80 μm~250 μm.
The technical scheme for realizing the second purpose of the invention is a kind of preparation method of the cross-link dextran microballoon of modification, described The cross-link dextran microballoon of modification is microspheric, cation modified cross-link dextran, described cation modified crosslinking Glucan refers to the H of the part-OH in cross-link dextran chemical constitution by-CH2-CH2-N+H-(C2H5)2Substitute or by-CH2- CH2-N+H-(CH3)2Substitution.
Cross-link dextran microballoon is first made when prepared by the first syntheti c route, then by cross-link dextran microballoon and cation Dressing agent is reacted, and the cross-link dextran microballoon modified, is comprised the following steps：
1. glucan and reducing agent are dissolved in sodium hydrate aqueous solution, glucan alkaline aqueous solution is uniformly mixed so as to obtain, as water Phase；The reducing agent is one kind in sodium borohydride, sodium sulfite, sodium pyrosulfite, VC.
2. surfactant is dissolved in organic solvent, obtain the organic phase containing surfactant, and will prepare Organic phase is transferred in the reaction vessel with agitating device.
3. opening stirring, 1. aqueous phase that step is obtained is added to step and 2. added in the reaction vessel of organic phase, enters Row emulsifies, and the w/o type emulsification systems of balance is formed after mechanical agitation, emulsion process temperature control is at 20 DEG C~50 DEG C.
4. crosslinking agent is added dropwise in the emulsification system 3. balanced to step, after crosslinking agent is added dropwise to complete, continue react 12h~ Reaction finishes after 36, and cross-linking reaction temperature is 30 DEG C~80 DEG C.
5. after cross-linking reaction terminates, the material in reaction vessel is all poured out, reaction solution is filtered out, collects what is obtained Microballoon then washs microballoon first with organic solvent washing to remove the surfactant and crosslinking agent that microsphere surface remains with ethanol To remove the organic solvent of microsphere surface residual, microballoon is washed followed by with distilled water, removes other water-solubility impurities； Obtain drying cross-link dextran microballoon after microballoon dehydration, drying after washing.
6. 5. drying cross-link dextran microballoon that step is obtained is added in sodium hydrate aqueous solution, mix；Continue to hydrogen Cation modified dose is added in sodium hydroxide solution, 6~10h of stirring reaction at 50 DEG C~90 DEG C is finished, obtains cation modified Cross-link dextran microballoon；After reaction terminates, the material in reaction vessel is all poured out, reaction solution is filtered out, collects The microballoon arrived is washed, dried.
The concentration of glucan is 0.11g/mL~0.67g/mL in the glucan alkaline aqueous solution of above-mentioned steps 1., glucan In glucose monomer and reducing agent material amount ratio be 9~25:1；2. the concentration of surfactant is step in organic phase 0.01~0.1g/mL；The crosslinking agent of step 4. is that epoxychloropropane or Isosorbide-5-Nitrae butanediol diglycidyl ether, crosslinking agent gather with Portugal The mass ratio of sugar is 1~8:10.
Above-mentioned steps 6. cation modified dose of middle addition be 2- lignocaine chloroethanes hydrochloride, 2- diethylamino bromide second Heptane hydrochloride salt, lignocaine chloromethanes hydrochloride, lignocaine bromomethane hydrochloride, the hydroxyethylamino chloroethanes hydrochlorides of 2- bis-, One kind in the hydroxyethylamino bromoethane hydrochlorides of 2- bis-.
Cation modified glucan is first made when preparing in second of syntheti c route, is then crosslinked balling-up and obtains microballoon, wraps Include following steps：
1. glucan is dissolved in sodium hydrate aqueous solution, mix；Continue to add into sodium hydroxide solution cation modified Agent, finish 6~10h of stirring reaction at 50 DEG C~90 DEG C, after completion of the reaction, to reaction after ethanol is added in solution, it is heavy to separate out Form sediment, what is be filtrated to get is precipitated as cation modified glucan.
2. 1. cation modified glucan, reducing agent that step is obtained are dissolved in sodium hydrate aqueous solution, mix Glucan alkaline aqueous solution is obtained, as aqueous phase；The reducing agent is in sodium borohydride, sodium sulfite, sodium pyrosulfite, VC It is a kind of.
3. surfactant is dissolved in organic solvent, obtain the organic phase containing surfactant, and will prepare Organic phase is transferred in the reaction vessel with agitating device；Surfactant is polyethylene glycol stearate diester, polyethylene glycol pair One kind in oleate, AEO.
4. opening stirring, 2. aqueous phase that step is obtained is added to step and 3. added in the reaction vessel of organic phase, enters Row emulsifies, and the w/o type emulsification systems of balance is formed after mechanical agitation, emulsion process temperature control is at 20 DEG C~50 DEG C.
5. crosslinking agent is added dropwise in the emulsification system 4. balanced to step, cross-linking reaction is now proceeded by, treats that crosslinking agent drips After the completion of adding, continue react and finish after reacting 12h~36h, cross-linking reaction temperature is 30 DEG C~80 DEG C；Crosslinking agent is epoxy chloropropionate The mass ratio of alkane or Isosorbide-5-Nitrae butanediol diglycidyl ether, crosslinking agent and glucan is 1~8:10.
6. step after 5. cross-linking reaction terminates, the material in reaction vessel is all poured out, filters out reaction solution, collect Obtained cation modified cross-link dextran microballoon is washed, dried.
Above-mentioned steps 1. cation modified dose of middle addition be 2- lignocaine chloroethanes hydrochloride, 2- diethylamino bromide second Heptane hydrochloride salt, lignocaine chloromethanes hydrochloride, lignocaine bromomethane hydrochloride, the hydroxyethylamino chloroethanes hydrochlorides of 2- bis-, One kind in the hydroxyethylamino bromoethane hydrochlorides of 2- bis-.
Use one pot reaction when prepared by the third syntheti c route when preparing, crosslinking balling-up and it is cation modified carry out simultaneously, Comprise the following steps：
1. glucan and reducing agent are dissolved in sodium hydrate aqueous solution, glucan alkaline aqueous solution is uniformly mixed so as to obtain, as water Phase；The reducing agent is one kind in sodium borohydride, sodium sulfite, sodium pyrosulfite, VC.
2. surfactant is dissolved in organic solvent, obtain the organic phase containing surfactant, and will prepare Organic phase is transferred in the reaction vessel with agitating device.
3. opening stirring, 1. aqueous phase that step is obtained is added to step and 2. added in the reaction vessel of organic phase, enters Row emulsifies, and the w/o type emulsification systems of balance is formed after mechanical agitation, emulsion process temperature control is at 20 DEG C~50 DEG C.
4. crosslinking agent is added dropwise in the emulsification system 3. balanced to step, after crosslinking agent is added dropwise to complete, at 30 DEG C~80 DEG C It is lower first to react 6~8 hours, cation modified dose is then added into reaction system, finishes the stirring reaction at 50 DEG C~90 DEG C The cross-link dextran microballoon that 15~20h is modified；Cation modified dose is 2- lignocaine chloroethanes hydrochloride, 2- diethylaminos Bromide ethane hydrochloride, lignocaine chloromethanes hydrochloride, lignocaine bromomethane hydrochloride, the hydroxyethylamino chloroethanes salt of 2- bis- One kind in hydrochlorate, the hydroxyethylamino bromoethane hydrochlorides of 2- bis-.
After 5. crosslinking, modification reaction terminate, the material in reaction vessel is all poured out, filters out reaction solution, collected Obtained microballoon is washed, dried.
The technical scheme for realizing the 3rd purpose of the invention is a kind of Combined medical of the cross-link dextran microballoon containing modification Thing, by weight percentage said composition are composed of the following components：The % of cross-link dextran microballoon 4%~6 of modification, cross-linked transparent matter The sour % of sodium 1%~2, PBS surplus；The cross-link dextran microballoon of modification therein is microspheric, cation modified friendship Join glucan, described cation modified cross-link dextran refer to the H of the part-OH in cross-link dextran chemical constitution by- CH2-CH2-N+H-(C2H5)2Substitute or by-CH2-CH2-N+H-(CH3)2Substitution.
The cross-link dextran microballoon of above-mentioned composition cationic modification is dispersed in cross-linking sodium hyaluronate gel； The nitrogen content of the cross-link dextran of composition cationic modification is 0.5%~2.0%.
Above-mentioned composition is to treat VUR, the composition of the urinary incontinence and incontinence of faces, the formulation of composition For injection.
The technical scheme for realizing the 4th purpose of the invention is a kind of detection of the nitrogen content of the cross-link dextran microballoon of modification Method, the cross-link dextran microballoon of the modification is microspheric, cation modified cross-link dextran, and described cation is repaiied The cross-link dextran of decorations refers to the H of the part-OH in cross-link dextran chemical constitution by-CH2-CH2-N+H-(C2H5)2Substitution or Person is by-CH2-CH2-N+H-(CH3)2Substitution；The detection method is applied to using cross-link dextran microballoon as raw material, is repaiied through cation Adorn the detection of nitrogen content in obtained microballoon；
The detection method of nitrogen content comprises the following steps：
The degree of cross linking X1 of cation modified cross-link dextran microballoon and the cross-link dextran microballoon as raw material are detected first Degree of cross linking X2；Then calculating formula is substituted into（MPortugal+MHand over*X2）*[100/（ X1- X2）]=A；A+MModification=B；Nitrogen content=14/B*100%； Wherein MPortugalIt is the relative atomic mass of glucan, MHand overIt is the relative atomic mass of the crosslinking agent used, MModificationBe modification group- CH2-CH2-N+H-(C2H5)2Or-CH2-CH2-N+H-(CH3)2Relative atomic mass.
The detection method of the wherein degree of cross linking of microballoon is as follows.
With electronic balance precision weigh quality be m, unit g tested microballoon, it is V to transfer them to capacityaML volumetric flasks In, measure sodium periodate solution with pipette and be transferred in above-mentioned volumetric flask, shake up, volumetric flask periphery is wrapped with tinfoil to be kept away Light, carries out redox reaction at 2 DEG C~10 DEG C, and the addition of sodium periodate solution guarantees complete oxidation cross-link dextran Microballoon.
Solution in the volumetric flask of course of reaction interval time absorption progress oxidation reaction is into volumetric flask, after being diluted with water Shake up, determine the light absorption value of solution.
Treat that light absorption value reaches stable, assert that reaction terminates, V is now drawn from volumetric flaskbML supernatants are to conical flask In, V is added into conical flaskb/ 10 mL ethylene glycol, are shaken up, and after being stood under room temperature, dark condition, 2 are added into conical flask ~3 drop phenolphthalein reagents, shake up, be then c with concentration, unit mol/L sodium hydroxide solution titrated, record consumption Volume V1。
With same volume VbML distilled water adds Vb/ 10 mL ethylene glycol solutions do blank, operate in the same way, carry out Titration, record the volume V of blank consumption0。
The substitution degree of cross linking calculation formula degree of cross linking=Calculated in X100% Obtain the degree of cross linking.
The present invention has positive effect：（1）The cross-link dextran microballoon of the modification of the present invention is cationic microspheres, due to Have cation modified group in chemical constitution, improve the bacteriostasis rate of microballoon, also improve microballoon and water compatibility and with The Percentage bound and adhesiveness of organism.
（2）The method that the present invention prepares the cross-link dextran microballoon of modification has three kinds, and first method is first obtained crosslinking Dextran microspheres, then cross-link dextran microballoon is modified to obtain cationic microspheres；Second method is first obtained modification Glucan, then be crosslinked balling-up obtain cationic microspheres；The third method is one pot reaction, and crosslinking balling-up and modification are entered simultaneously OK.The raw material of above-mentioned three kinds of preparation methods is controllable, solvent-free Residual Toluene in obtained product, product it is safe, can use In prescription；The cross-link dextran of microphoto display modification is smooth mellow and full spherical after being swelled with PBS cushioning liquid, and impurity contains Measure low, there is good biocompatibility.
（3）The composition of the present invention is by the hydrophilic cation modified cross-link dextran microballoon and work as filler For the non-immunogenicity of the cross-linking hyaluronic acid sodium composition of the non-animal source of carrier, without sensitization risk, no cytotoxicity, biology The sterile gel preparation of tolerability.
After the composition of the present invention is injected in vivo relevant position in the form of injection, returned available for treatment vesicoureteral Stream, the urinary incontinence and incontinence of faces.
The cross-link dextran microballoon modified in composition is dispersed in cross-linking sodium hyaluronate gel, cross-linked transparent matter Decentralized medium of the sour sodium as composition, and the cross-link dextran microballoon of modification is delivered to injection site as carrier；With Cross-linking hyaluronic acid sodium is absorbed by tissue, and the cross-link dextran microballoon of modification is in target position gradually by the connective tissue bag of body itself Enclose, and promote the generation of fibroblast and collagen between microballoon, so as to the volume of stabilisation implant, reach lasting filling effect Fruit.
By taking vesicoureteric reflux as an example, vesicoureteric reflux and its symptom cause of disease are that ureter prevents urine reflux Valve mechanical injuries, a kind of material is now needed to serve as filler, the increase tissue thickness in injection site so that in muscle just Urethra is helped to completely close prevention reflux when often shrinking.Filling effect can be played after the composition injection of the present invention, eliminates wing Guang ureter reflux symptom.
（4）Cross-linking sodium hyaluronate gel in the composition of the present invention as carrier is a kind of polymerization polysaccharide, and it is injected It can be absorbed by organisms after in body within some months to year, the cation modified cross-link dextran of filler is dried The size of microballoon is 80 μm~250 μm, will not be ruptured, it is thus eliminated that the risk of migration, slowly biodegradable by hydrolyzing.
Brief description of the drawings
Fig. 1 is that the chemical constitution of the cation modified cross-link dextran microballoon of the present invention illustrates formula；
Fig. 2 a are the microphoto of the cross-link dextran dry microspheres of modification prepared by embodiment 2, and Fig. 2 b are prepared by embodiment 2 Microphoto after the cross-link dextran dry microspheres swelling of modification；
Fig. 3 is the particle size distribution figure after the cross-link dextran microspheres swell of modification prepared by embodiment 2；
Cation modified dose of gas chromatogram is remained in the cross-link dextran microballoon for the modification that Fig. 4 is prepared for embodiment 2；
Fig. 5 a are the microphoto of the cross-link dextran dry microspheres of modification prepared by embodiment 3, and Fig. 5 b are prepared by embodiment 3 Microphoto after the cross-link dextran dry microspheres swelling of modification；
Fig. 6 is the particle size distribution figure after the cross-link dextran microspheres swell of modification prepared by embodiment 3；
Cation modified dose of gas chromatogram is remained in the cross-link dextran microballoon for the modification that Fig. 7 is prepared for embodiment 3；
Fig. 8 a are the microphoto of the cross-link dextran dry microspheres of modification prepared by embodiment 4, and Fig. 8 b are prepared by embodiment 4 Microphoto after the cross-link dextran dry microspheres swelling of modification；
Fig. 9 is the particle size distribution figure after the cross-link dextran microspheres swell of modification prepared by embodiment 4；
Cation modified dose of gas chromatogram is remained in the cross-link dextran microballoon for the modification that Figure 10 is prepared for embodiment 4；
Figure 11 a are the microphoto of the cross-link dextran dry microspheres of modification prepared by embodiment 5, and Figure 11 b are prepared by embodiment 5 Modification cross-link dextran dry microspheres swelling after microphoto；
Figure 12 is the particle size distribution figure after the cross-link dextran microspheres swell of modification prepared by embodiment 5；
Cation modified dose of gas chromatogram is remained in the cross-link dextran microballoon for the modification that Figure 13 is prepared for embodiment 5；
Figure 14 a are the microphoto of the cross-link dextran dry microspheres of modification prepared by embodiment 6, and Figure 14 b are prepared by embodiment 6 Modification cross-link dextran dry microspheres swelling after microphoto；
Figure 15 is the particle size distribution figure after the cross-link dextran microspheres swell of modification prepared by embodiment 6；
Cation modified dose of gas chromatogram is remained in the cross-link dextran microballoon for the modification that Figure 16 is prepared for embodiment 6.
Embodiment
（Embodiment 1, the cross-link dextran microballoon of modification）
The cross-link dextran microballoon of the modification of the present invention is dry, microspheric cation modified cross-link dextran, described The cross-link dextran of modification refer to the H of part-OH in cross-link dextran chemical constitution by-CH2-CH2-N+H-(C2H5)2Take Generation or by-CH2-CH2-N+H-(CH3)2Substitution, so as to obtain cation modified microballoon.
The chemical constitution signal formula of the cross-link dextran of modification is shown in Fig. 1, in Fig. 1 the part-OH of cross-link dextran H by- CH2-CH2-N+H-(C2H5)2Substitution, Fig. 1 are only to illustrate substituent, do not make specific modification point locator qualification.If instead of base It is-CH2-CH2-N+H-(CH3)2, by-the CH in Fig. 12-CH2-N+H-(C2H5)2It is changed to-CH2-CH2-N+H-(CH3)2.
The degree that how many-OH H is substituted with a substituent in cross-link dextran, with the nitrogen content numerical tabular of final products Existing, nitrogen content is higher, represents that few-OH substituted in cross-link dextran H is more；Otherwise nitrogen content is lower, crosslinking Portugal is represented The H for the few-OH being substituted in glycan is fewer.
Nitrogen content uses Kjeldahl nitrogen determination.
The nitrogen content of the cross-link dextran of the modification of the present invention is 0.5%~2.0%.
The particle diameter of the cross-link dextran dry microspheres of modification is 80 μm~250 μm.
The preparation method of the cross-link dextran microballoon of modification has three kinds, and first method is that first obtained cross-link dextran is micro- Ball, then cross-link dextran microballoon is modified to obtain cationic microspheres；Second method is the glucan of first obtained modification, Then crosslinking balling-up obtains cationic microspheres；The third method is one pot reaction, and crosslinking balling-up and modification are carried out simultaneously.For adopting The cross-link dextran microballoon modified made from first method, nitrogen content can also use degree of cross linking differential technique to determine.
Described degree of cross linking differential technique detection according to be compared to unmodified cross-link dextran microballoon, it is micro- after modification Substituted radical-CH is added in ball2-CH2-N+H-(C2H5)2Or-CH2-CH2-N+H-(CH3)2.For degree of cross linking detection method Speech, the definition of the degree of cross linking is that the quality of crosslinking agent modified part in microballoon accounts for the percentage of whole microspheres quality, wherein modificationt part Sub-prime amount includes glucan quality, the glucan quality of monosubstituted modification and the crosslinking agent fragment masses sum of crosslinking modification.And For the cross-link dextran microballoon of modification, when the degree of cross linking detects by cation modified group-CH2-CH2-N+H-(C2H5)2Or- CH2-CH2-N+H-(CH3)2Also monosubstituted modified part has been regarded, so as to also contribute to partial cross-linked degree.By the crosslinking Portugal of modification The degree of cross linking of glycan microballoon and the degree of cross linking of raw material cross-link dextran microballoon detect respectively, are obtained by series conversion nitrogenous Amount.
Specific detection method is as follows：First detect degree of cross linking X1 and the crosslinking Portugal of cation modified cross-link dextran microballoon The degree of cross linking X2 of glycan microballoon；（MPortugal+MHand over*X2）*[100/（ X1- X2）]=A；A+MModification=B；Nitrogen content=14/B*100%.Wherein MPortugalIt is the relative atomic mass of glucan, MHand overIt is the relative atomic mass of the crosslinking agent used, MModificationIt is modification group-CH2-CH2- N+H-(C2H5)2Or-CH2-CH2-N+H-(CH3)2Relative atomic mass.
The degree of cross linking detection method of wherein microballoon is as follows：
With electronic balance precision weigh quality be m, unit g tested cross-link dextran microballoon（It is modification or micro- without what is modified Ball）, it is V to transfer them to capacityaIn mL volumetric flasks, measure sodium periodate solution with pipette and be transferred to above-mentioned volumetric flask In, shake up, lucifuge is wrapped in volumetric flask periphery with tinfoil, and redox reaction is carried out at 2 DEG C~10 DEG C；Sodium periodate solution Addition guarantees complete oxidation cross-link dextran microballoon.The concentration of sodium periodate solution is the mol/L of 0.03mol/L~0.1.
Solution in the volumetric flask of course of reaction interval time absorption progress oxidation reaction is into volumetric flask, after being diluted with water Shake up, determine the light absorption value of solution.
Treat that light absorption value reaches stable, assert that reaction terminates, V is now drawn from volumetric flaskbML supernatants are to conical flask In, V is added into conical flaskb/ 10 mL ethylene glycol, are shaken up, and after being stood under room temperature, dark condition, 2 are added into conical flask ~3 drop phenolphthalein reagents, shake up, be then c with concentration, unit mol/L sodium hydroxide solution titrated, record consumption Volume V1.The concentration of the sodium hydroxide solution of titration is the mol/L of 0.005 mol/L~0.1.
With same volume VbML distilled water adds Vb/ 10 mL ethylene glycol solutions do blank, operate in the same way, carry out Titration, record the volume V of blank consumption0。
The substitution degree of cross linking calculation formula degree of cross linking=X100% falls into a trap Calculation obtains the degree of cross linking.
For the cross-link dextran microballoon modified made from first method, nitrogen content can use two methods to determine, If the nitrogen content error that two methods determine is within 20%, it is believed that the measured value of two methods is effective；For only with triumphant Microballoon prepared by second of the measurement of family name's nitriding and the third method, sets more than three parallel test groups during detection.
（The preparation method of embodiment 2, the cross-link dextran microballoon modified）
The H manufactured in the present embodiment for being glucan-OH is by-CH2-CH2-N+H-(C2H5)2Substituted cationic microspheres.
The process route of the present embodiment is first obtained cross-link dextran microballoon, then by cross-link dextran microballoon and cation Dressing agent is reacted, the cross-link dextran microballoon modified, i.e. cross-link dextran cationic microspheres.
The preparation method of the cross-link dextran microballoon of the modification of the present embodiment specifically includes following steps：
1. glucan, sodium borohydride are dissolved in 3mol/L sodium hydrate aqueous solution, it is water-soluble to be uniformly mixed so as to obtain glucan alkalescence Liquid, as aqueous phase.The concentration of glucan is 0.65g/mL in glucan alkaline aqueous solution, glucose monomer and boron in glucan The ratio between amount of material of sodium hydride is 15:1.When selecting VE as reducing agent, VE is dissolved in the organic solvent of step 2..
Reducing agent can also be one in sodium sulfite, sodium pyrosulfite, VC, VE except the above-mentioned sodium borohydride referred to Kind, the amount ratio of the material of glucose monomer and reducing agent in glucan is 9~25:1.
2. using as the double stearates of the polyethylene glycol 400 of surfactant（PEG400DS）It is dissolved in organic solvent toluene In, the organic phase containing surfactant is obtained, and the organic phase prepared is transferred in the reaction vessel with agitating device.Have The concentration of surfactant is 0.07g/mL in machine phase.
Except the above-mentioned polyethylene glycol stearate diester referred to, surfactant is also an option that polyethylene glycol dioleic acid ester （Such as PEG400DO）, AEO（Such as AEO MOA-3）In one kind.
Organic solvent is except toluene, it is also an option that the aromatic hydrocarbon of liquid（Apart from toluene）, aliphatic hydrocarbon, halogenated aliphatic One kind in hydrocarbon, halogenated aromatic；Specifically, for example can be normal octane, n -nonane, dichloromethane, 1,2- dichloros（Bromine）Second One kind in alkane, o-dichlorohenzene.
3. opening stirring, regulation rotating speed is 380r/min（300~2000rpm of agitator frequency control）, by step 1. To aqueous phase be added in 2. reaction vessel that step has added organic phase, the volume ratio of aqueous phase and organic phase is 1:2, carry out breast Change, through 30min~90min（It is 45min in the present embodiment）The w/o type emulsification systems of balance are formed after mechanical agitation, were emulsified Journey temperature control is at 20 DEG C~50 DEG C（It is 40 DEG C in the present embodiment）.
4. crosslinking agent epoxychloropropane is added dropwise in the emulsification system 3. balanced to step, it is 1~10s/ to control rate of addition It is added dropwise to, now proceeds by cross-linking reaction, after crosslinking agent is added dropwise to complete, continues to react 12h~36h（It is in the present embodiment 18h）Reaction finishes afterwards, and cross-linking reaction temperature is 30 DEG C~80 DEG C（It is 50 DEG C in the present embodiment）.Epoxychloropropane and glucan Mass ratio be 1~8:10（It is 4 in the present embodiment:10）.
5. step after 4. cross-linking reaction terminates, the material in reaction vessel is all poured out, filters out reaction solution, collect Obtained microballoon is first washed with organic solvent toluene to remove the surfactant and crosslinking agent of microsphere surface residual, repeats toluene Washing operation, until surfactant and cross-linked dosage through gas chromatographic detection microsphere surface residual reach below 10ug/g.
Then microballoon is washed with 95% ethanol to remove the toluene of microsphere surface residual, repeats ethanol washing operation, until warp Gas chromatographic detection microsphere surface Determination of Residual Toluene reaches below 10ug/g.
Microballoon is washed followed by with distilled water, and adjust the pH value of distilled water and mixture of microspheres material for 6~ 7.5, water washing operations are repeated, remove for example uncrosslinked glucan of other water-solubility impurities.
Microballoon after washing is first with 95% ethanol dehydration 3~5 times（The dosage of 95% ethanol guarantees to submerge microballoon）, then with nothing Water-ethanol continues dehydration 3~5 times（The dosage of absolute ethyl alcohol guarantees to submerge microballoon）；By the microballoon after dehydration 60 DEG C~100 8~24h is dried under the conditions of DEG C to obtain drying cross-link dextran microballoon.
6. 5. 5g that step is obtained dries the 4 mol/L sodium hydrate aqueous solutions that cross-link dextran microballoon adds 50ml In, mix；Continue to add cation modified dose of 2- lignocaine chloroethanes hydrochloride of 6.07g into sodium hydroxide solution（CAS NO.869-24-9）, the stirring reaction 8h at 65 DEG C is finished, obtains cation modified cross-link dextran microballoon.
Except the above-mentioned 2- lignocaine chloroethanes hydrochlorides used, cation modified dose it is also an option that 2- lignocaines Bromoethane hydrochloride, lignocaine chloromethanes hydrochloride, lignocaine bromomethane hydrochloride, the hydroxyethylamino chloroethanes hydrochloric acid of 2- bis- One kind in salt, the hydroxyethylamino bromoethane hydrochlorides of 2- bis-.
To be made by-CH2-CH2-N+H-(CH3)2Substituted cross-link dextran, 2- diformazans are selected by cation modified dose Amino chloroethanes hydrochloride, 2- dimethylamino bromoethanes hydrochloride, dimethylamino chloromethanes hydrochloride, dimethylamino bromomethane salt One kind in hydrochlorate, 2- dihydroxy methylamino chloroethanes hydrochloride, 2- dihydroxy methylamino bromoethane hydrochlorides.
After reaction terminates, the material in reaction vessel is all poured out, filters out reaction solution, the microballoon for collecting to obtain is first
With distillation water washing, and the pH value for adjusting distilled water and mixture of microspheres material is 6~7.5, repeats water washing operations, removes it His water-solubility impurity.
Microballoon after washing is first with 95% ethanol dehydration 3~5 times（The dosage of 95% ethanol guarantees to submerge microballoon）, then with nothing Water-ethanol continues dehydration 3~5 times（The dosage of absolute ethyl alcohol guarantees to submerge microballoon）；By the microballoon after dehydration 60 DEG C~100 The cross-link dextran microballoon that 8~24h is dried, modified is dried under the conditions of DEG C.
7. the cross-link dextran microballoon of 6. modification that step is obtained first passes through the screen cloth that aperture is 250 μm, take under screen cloth The microballoon of side, then sieved with the screen cloth that aperture is 80 μm, the microballoon above screen cloth is taken, obtains particle diameter as 80 μm~250 μm Modification cross-link dextran dry microspheres, be used to configure composition preferably through the microballoon of screening, dry microspheres is aobvious after screening Micro- photo is shown in Fig. 2 a.After the microballoon of the particle size range is swelled with PBS cushioning liquid, the microphoto of swollen microsphere is shown in Fig. 2 b, The particle size distribution figure of microballoon is shown in Fig. 3 after swelling, and the average grain diameter of swollen microsphere is 195.215 μm.
Further, with the residual of gas chromatographic detection dry microspheres cationic dressing agent, it is 6. step is located to see Fig. 4, Fig. 4 The gas chromatogram of obtained dry microspheres is managed, gas chromatogram shows that dry microspheres cationic dressing agent does not detect.
Detected using Kjeldahl's method, the nitrogen content of the cross-link dextran microballoon of the obtained modification of the present embodiment is 0.96%.
Again using degree of cross linking differential technique measure nitrogen content, comprise the following steps that：
First, the friendship of 6. modification that the degree of cross linking of 5. drying cross-link dextran microballoon that detecting step obtains and step obtain respectively Join the degree of cross linking of dextran microspheres.
The degree of cross linking detection method of step 5. cross-link dextran microballoon is as follows：
0.20006g tested cross-link dextran microballoon is weighed with electronic balance precision, is transferred them in 50mL volumetric flasks, is used Pipette measures 50mM sodium periodate solution 50.00mL and is transferred in above-mentioned 50mL volumetric flasks, shakes up, volumetric flask periphery Lucifuge is wrapped with tinfoil, carries out redox reaction at 4 DEG C, in course of reaction every 24h shake wells volumetric flask once.
In course of reaction quarter is diluted with water to every the solution in 24h absorption 0.1mL volumetric flasks into 100mL volumetric flasks Degree, is shaken up, and the light absorption value of solution is determined at 223nm.
After light absorption value reaches stable, 20.00mL supernatants are drawn from volumetric flask into conical flask, are added into conical flask 2mL ethylene glycol, shakes up, and after standing 20min under room temperature, dark condition, 2~3 drop phenolphthalein reagents is added into conical flask, are shaken It is even, then titrated with 0.01M sodium hydroxide solution, record the volume V of consumption1For 17.46mL.
Add 2mL ethylene glycol solutions to do blank with same volume 20mL distilled water, operate, titrated in the same way, Record the volume V of blank consumption0For 0.05ml.
Substitute into degree of cross linking calculation formula：The degree of cross linking= X100%=64.8%。
The degree of cross linking for measuring the cross-link dextran microballoon of 6. modification that step obtains according to the method described above is 85.5%.
According to the conversion method of embodiment 1, nitrogen content=1.06%.The error of testing result is within 15% twice.
The cross-link dextran microballoon purity of the obtained modification of the present embodiment is high, non-immunogenicity, without sensitization risk；Microballoon Spherical state does not well rupture.
The structure of the reaction vessel with agitating device used in above-mentioned preparation process referring to Application No. 2017200920968, Patent name is a kind of Chinese patent literature of multi-functional composite stirring reactor.
7. for two steps screening apparatus structure used referring to Application No. 2017200921496, patent name is one kind to step For the Chinese patent literature for the shaking screen for preparing microballoon, the screen cloth on concussion sieve selects according to screening situation.
（The preparation method of embodiment 3, the cross-link dextran microballoon modified）
Remaining is same as Example 2 for the preparation method of the cross-link dextran microballoon of the modification of the present embodiment, and difference is：
Step 6. in, 5. 5g that step is obtained dry cross-link dextran microballoon add 50ml 4 mol/L sodium hydroxides it is water-soluble In liquid, mix；Continue to add cation modified dose of 2- lignocaine chloroethanes hydrochloride of 10.6g into sodium hydroxide solution（CAS NO.869-24-9）, the stirring reaction 8h at 65 DEG C is finished, obtains cation modified cross-link dextran microballoon.
Detected using Kjeldahl's method, the nitrogen content of the cross-link dextran microballoon of the obtained modification of the present embodiment is 1.71%.
The microballoon of the present embodiment obtains the modification cross-link dextran dry microspheres that particle diameter is 80 μm~250 μm through sieving twice Afterwards, the microphoto of dry microspheres is shown in Fig. 5 a；After the microballoon of the particle size range is swelled with PBS cushioning liquid, swollen microsphere Microphoto is shown in Fig. 5 b, and the particle size distribution figure of microballoon is shown in Fig. 6 after swelling, and the average grain diameter of swollen microsphere is 348.465 μm.
Further, with the residual of gas chromatographic detection dry microspheres cationic dressing agent, it is 6. step is located to see Fig. 7, Fig. 7 The gas chromatogram of obtained dry microspheres is managed, gas chromatogram shows the μ g/ of dry microspheres cationic dressing agent residual quantity 6.6 g。
（The preparation method of embodiment 4, the cross-link dextran microballoon modified）
Remaining is same as Example 2 for the preparation method of the cross-link dextran microballoon of the modification of the present embodiment, and difference is：
Step 6. in, 5. 5g that step is obtained dry cross-link dextran microballoon add 50ml 4 mol/L sodium hydroxides it is water-soluble In liquid, mix；Continue to add cation modified dose of 2- dimethylamino chloroethanes hydrochloride of 5.03g into sodium hydroxide solution, add Finish the stirring reaction 8h at 65 DEG C, obtain cation modified cross-link dextran microballoon.
Detected using Kjeldahl's method, the nitrogen content of the cross-link dextran microballoon of the obtained modification of the present embodiment is 0.64%.
The microballoon of the present embodiment obtains the modification cross-link dextran dry microspheres that particle diameter is 80 μm~250 μm through sieving twice Afterwards, the microphoto of dry microspheres is shown in Fig. 8 a；After the microballoon of the particle size range is swelled with PBS cushioning liquid, swollen microsphere Microphoto is shown in Fig. 8 b, and the particle size distribution figure of microballoon is shown in Fig. 9 after swelling, and the average grain diameter of swollen microsphere is 112.501 μm.
Further, with the residual of gas chromatographic detection dry microspheres cationic dressing agent, see Figure 10, Figure 10 be step 6. The gas chromatogram of obtained dry microspheres is handled, gas chromatogram shows the μ of dry microspheres cationic dressing agent residual quantity 22 g/g。
（The preparation method of embodiment 5, the cross-link dextran microballoon modified）
The H manufactured in the present embodiment for being glucan-OH is by-CH2-CH2-N+H-(C2H5)2Substituted cationic microspheres.
The process route of the present embodiment is first obtained cation modified glucan, is then crosslinked balling-up and obtains microballoon.
The preparation method of the cross-link dextran microballoon of the modification of the present embodiment specifically includes following steps：
1. 20g is dried into glucan to add in 50ml 4 mol/L sodium hydrate aqueous solutions, mix；Continue molten to sodium hydroxide Cation modified dose of 2- lignocaine bromoethane hydrochloride of 17g is added in liquid, stirring reaction 8h, reaction at 65 DEG C is finished and finishes Afterwards, 500mL95% ethanol is added into solution after reaction, separates out precipitation, the cation modified Portugal that is precipitated as being filtrated to get gathers Sugar.
2. 1. cation modified glucan that step is obtained, as the sodium borohydride of reducing agent it is dissolved in 3mol/L's In sodium hydrate aqueous solution, glucan alkaline aqueous solution is uniformly mixed so as to obtain, as aqueous phase.Glucan in glucan alkaline aqueous solution Concentration is 0.4g/mL, and the ratio between amount of material of glucose monomer and sodium borohydride in glucan is 10:1.
3. using as the double stearates of the polyethylene glycol 400 of surfactant（PEG400DS）It is dissolved in organic solvent toluene In, the organic phase containing surfactant is obtained, and the organic phase prepared is transferred in the reaction vessel with agitating device.Have The concentration of surfactant is 0.07g/mL in machine phase.
Except the above-mentioned polyethylene glycol stearate diester referred to, surfactant is also an option that polyethylene glycol dioleic acid ester （Such as PEG400DO）, AEO（Such as AEO MOA-3）In one kind.
Organic solvent is except toluene, it is also an option that the aromatic hydrocarbon of liquid（Apart from toluene）, aliphatic hydrocarbon, halogenated aliphatic One kind in hydrocarbon, halogenated aromatic；Specifically, for example can be normal octane, n -nonane, dichloromethane, 1,2- dichloros（Bromine）Second One kind in alkane, o-dichlorohenzene.
4. opening stirring, regulation rotating speed is 380r/min（300~2000rpm of agitator frequency control）, by step 2. To aqueous phase be added in 3. reaction vessel that step has added organic phase, the volume ratio of aqueous phase and organic phase is 1:2, carry out breast Change, through 30min~90min（It is 45min in the present embodiment）The w/o type emulsification systems of balance are formed after mechanical agitation, were emulsified Journey temperature control is at 20 DEG C~50 DEG C（It is 40 DEG C in the present embodiment）.
5. crosslinking agent epoxychloropropane is added dropwise in the emulsification system 4. balanced to step, it is 1~10s/ to control rate of addition It is added dropwise to, now proceeds by cross-linking reaction, after crosslinking agent is added dropwise to complete, continues to react 12h~36h（It is in the present embodiment 18h）Reaction finishes afterwards, and cross-linking reaction temperature is 30 DEG C~80 DEG C（It is 50 DEG C in the present embodiment）.Epoxychloropropane and glucan Mass ratio be 1~8:10（It is 4 in the present embodiment:10）.
6. step after 5. cross-linking reaction terminates, the material in reaction vessel is all poured out, filters out reaction solution, collect Obtained microballoon is first washed with organic solvent toluene to remove the surfactant and crosslinking agent of microsphere surface residual, repeats toluene Washing operation, until surfactant and cross-linked dosage through gas chromatographic detection microsphere surface residual reach below 10ug/g.
Then microballoon is washed with 95% ethanol to remove the toluene of microsphere surface residual, repeats ethanol washing operation, until warp Gas chromatographic detection microsphere surface Determination of Residual Toluene reaches below 10ug/g.
Microballoon is washed followed by with distilled water, and adjust the pH value of distilled water and mixture of microspheres material for 6~ 7.5, water washing operations are repeated, remove for example uncrosslinked glucan of other water-solubility impurities.
Microballoon after washing is first with 95% ethanol dehydration 3~5 times（The dosage of 95% ethanol guarantees to submerge microballoon）, then with nothing Water-ethanol continues dehydration 3~5 times（The dosage of absolute ethyl alcohol guarantees to submerge microballoon）；By the microballoon after dehydration 60 DEG C~100 The cross-link dextran microballoon that 8~24h is dried, modified is dried under the conditions of DEG C.
7. the cross-link dextran microballoon screening of 6. modification that step is obtained, obtains the modification that particle diameter is 80 μm~250 μm Cross-link dextran dry microspheres, the microphoto of dry microspheres is shown in Figure 11 a after screening, and the microballoon of the particle size range is delayed with PBS After rushing solution swelling, the microphoto of swollen microsphere is shown in Figure 11 b, and the particle size distribution figure of microballoon is shown in Figure 12, swollen microsphere after swelling Average grain diameter be 601.424 μm.
Further, with the residual of gas chromatographic detection dry microspheres cationic dressing agent, see Figure 13, Figure 13 be step 6. The gas chromatogram of obtained dry microspheres is handled, gas chromatogram shows the μ of dry microspheres cationic dressing agent residual quantity 23 g/g。
According to Kjeldahl's method, the nitrogen content of the cross-link dextran microballoon of the obtained modification of the present embodiment is 1.84%.
（The preparation method of embodiment 6, the cross-link dextran microballoon modified）
The process route of the present embodiment is one pot reaction, is crosslinked balling-up and cation modified carries out simultaneously.
The preparation method of the cross-link dextran microballoon of the modification of the present embodiment specifically includes following steps：
1. being dissolved in using 20g glucans, as the sodium borohydride of reducing agent in 30mL 8mol/L sodium hydrate aqueous solution, mix It is even to obtain glucan alkaline aqueous solution, as aqueous phase.The concentration of glucan is 0.65g/mL in glucan alkaline aqueous solution, and Portugal gathers The ratio between amount of material of glucose monomer and sodium borohydride in sugar is 15:1.
Reducing agent can also be one in sodium sulfite, sodium pyrosulfite, VC, VE except the above-mentioned sodium borohydride referred to Kind, the amount ratio of the material of glucose monomer and reducing agent in glucan is 9~25:1.
2. using as the double stearates of the polyethylene glycol 400 of surfactant（PEG400DS）It is dissolved in organic solvent toluene In, the organic phase containing surfactant is obtained, and the organic phase prepared is transferred in the reaction vessel with agitating device.Have The concentration of surfactant is 0.07g/mL in machine phase.
Except the above-mentioned polyethylene glycol stearate diester referred to, surfactant is also an option that polyethylene glycol dioleic acid ester （Such as PEG400DO）, AEO（Such as AEO MOA-3）In one kind.
Organic solvent is except toluene, it is also an option that the aromatic hydrocarbon of liquid（Apart from toluene）, aliphatic hydrocarbon, halogenated aliphatic One kind in hydrocarbon, halogenated aromatic；Specifically, for example can be normal octane, n -nonane, dichloromethane, 1,2- dichloros（Bromine）Second One kind in alkane, o-dichlorohenzene.
3. opening stirring, regulation rotating speed is 380r/min（300~2000rpm of agitator frequency control）, by step 1. To aqueous phase be added in 2. reaction vessel that step has added organic phase, the volume ratio of aqueous phase and organic phase is 1:2, carry out breast Change, the w/o type emulsification systems of balance are formed after 45min mechanical agitations, emulsion process temperature control is at 40 DEG C.
4. crosslinking agent epoxychloropropane is added dropwise in the emulsification system 3. balanced to step, the matter of epoxychloropropane and glucan Amount is than being 1~8:10, control rate of addition to be added dropwise to for 1~10s/, now proceed by cross-linking reaction, treat that crosslinking agent drips Cheng Hou, at 30 DEG C~80 DEG C（It is 50 DEG C in the present embodiment）It is lower first to react 7h；Then 20g cations are added into reaction system to repair Adorn agent 2- lignocaine chloroethanes hydrochlorides（CAS NO.869-24-9）, the stirring reaction 17h at 65 DEG C is finished, is modified Cross-link dextran microballoon.
5. after 4. step is crosslinked, modification reaction terminates, the material in reaction vessel is all poured out, reaction is filtered out Liquid, the microballoon for collecting to obtain first washed with organic solvent toluene with remove microsphere surface residual surfactant, crosslinking agent and Dressing agent, toluene washing operation is repeated, up to the surfactant through gas chromatographic detection microsphere surface residual, crosslinking agent and repaiied Decorations dosage reaches below 10ug/g.
Then microballoon is washed with 95% ethanol to remove the toluene of microsphere surface residual, repeats ethanol washing operation, until warp Gas chromatographic detection microsphere surface Determination of Residual Toluene reaches below 10ug/g.
Microballoon is washed followed by with distilled water, and adjust the pH value of distilled water and mixture of microspheres material for 6~ 7.5, water washing operations are repeated, remove for example uncrosslinked glucan of other water-solubility impurities.
Microballoon after washing is first with 95% ethanol dehydration 3~5 times（The dosage of 95% ethanol guarantees to submerge microballoon）, then with nothing Water-ethanol continues dehydration 3~5 times（The dosage of absolute ethyl alcohol guarantees to submerge microballoon）；By the microballoon after dehydration 60 DEG C~100 The cross-link dextran microballoon that 8~24h is dried, modified is dried under the conditions of DEG C.
6. the cross-link dextran microballoon of 5. modification that step is obtained first passes through the screen cloth that aperture is 250 μm, take under screen cloth The microballoon of side, then sieved with the screen cloth that aperture is 80 μm, the microballoon above screen cloth is taken, obtains particle diameter as 80 μm~250 μm Modification cross-link dextran dry microspheres, the microphoto of dry microspheres is shown in Figure 14 a after screening, and the microballoon of the particle size range is used After the swelling of PBS cushioning liquid, the microphoto of swollen microsphere is shown in Figure 14 b, and the particle size distribution figure of microballoon is shown in Figure 15 after swelling, swelling The average grain diameter of microballoon is 383.489 μm.
Further, with the residual of gas chromatographic detection dry microspheres cationic dressing agent, see Figure 16, Figure 16 be step 6. The gas chromatogram of obtained dry microspheres is handled, gas chromatogram shows the μ of dry microspheres cationic dressing agent residual quantity 155 g/g。
According to Kjeldahl's method, the nitrogen content of the cross-link dextran microballoon of the obtained modification of the present embodiment is 1.96%.
（Embodiment 7, the composition for treating VUR, the urinary incontinence and incontinence of faces）
The composition for treating VUR, the urinary incontinence and incontinence of faces of the present embodiment is by weight percentage by with the following group It is grouped into：The % of cross-link dextran microballoon 4%~6 of modification（It is 4.5% in the present embodiment）, the % of cross-linking hyaluronic acid sodium 1%~2（This It is 1.5% in embodiment）, PBS surplus；PBS therein configures according to pharmacopeia.The crosslinking Portugal wherein modified gathers Sugared microballoon weight fraction in terms of dry microballoon, cross-linking hyaluronic acid sodium mass fraction in terms of cross-linking hyaluronic acid sodium dry powder.
Cross-linking hyaluronic acid sodium is swelled into gel in PBS, and the cross-link dextran microballoon of modification is in PBS In be swelled into gel micro-ball, therefore the actual form of composition is that sephadex microballoon is dispersed in cross-linked transparent matter In sour sodium gel.
The particle diameter of the cross-link dextran dry microspheres for the modification that composition configuration uses is 80 μm~250 μm.
The cross-linking hyaluronic acid sodium dry powder that configuration above-mentioned composition uses is first using entitled《High quality crosslinking is saturating Bright matter acid sodium gel and preparation method thereof》Chinese patent literature CN 103923328A（Application number 201410153564.9）Institute Cross-linking sodium hyaluronate gel is prepared in disclosed method, and then gel is dried in vacuo under conditions of less than 30 DEG C and is obtained Dry powder is standby.
The cross-link dextran microballoon optional embodiment 2 of the modification used in composition any road into embodiment 6 Prepared by line, the syntheti c route that preferred embodiment 2,3,4 uses；And the cross-link dextran microballoon of the modification used in composition contains Nitrogen quantity is in 0.5%~2.0%, preferably 0.8%~1.2%.
The preparation method of composition is as follows：
Weigh the cross-link dextran dry microspheres and cross-linked-hyaluronic acid of the modification of the method preparation according to one of embodiment 2 to 6 Sodium dry powder, PBS then is added in the cross-link dextran dry microspheres to modification and cross-linking hyaluronic acid sodium dry powder, modified Cross-link dextran dry microspheres and cross-linking hyaluronic acid sodium dry powder swelling completely after, be uniformly mixed so as to obtain treatment vesicoureteral return The combination of compositions thing of stream, the urinary incontinence and incontinence of faces.It is saturating to be dispersed in crosslinking for sephadex microballoon in composition In bright matter acid sodium gel.
The formulation of composition is injection agent, and it is micro- using the drying of 50mg cross-link dextrans that the present embodiment configures each injection Ball, 15mg cross-linking hyaluronic acid sodiums dry powder and 1mLPBS cushioning liquid.
Composition is used in a manner of injecting to treat VUR, the urinary incontinence and incontinence of faces.
When treating VUR and the urinary incontinence, composition is injected under the mucous membrane of urinary bladder of adjacent Ureteral orifice Layer, the gel tissue for injecting formation are attached to distal ureter, sephadex in filling of bladder and during shrinking Microballoon is gradually surrounded by the connective tissue of body itself, so as to reach final barrier effect.
When treating incontinence of faces, composite injection is injected in closest at the submucosa of anal tube high pressure part, every time note Penetrate need to apart from dentate line about at 5mm, respectively be spaced it is equal respectively squeeze into a pin everywhere, per combination of the pin containing 1mL or so Thing.By injectable composition, it can increase closest to the submucosa at anal tube, so as to strengthen intestines control ability.
（Test example 1）
Histologic reaction's experiment after in composition injection rat body
First, test material and method
The rat that 24 weight are 225g~275mg is divided into 2 groups, wherein control group 8, experimental group 16.
For 16 rats of experimental group, under inhalation anesthesia state, every rat is placed in dorsal position progress skin and disappeared Poison, then the composition described in the mark position hypodermic injection 0.35mL embodiments 1 in belly, every rat only receive once to note Penetrate.
For 8 rats of control group, under inhalation anesthesia state, every rat is placed in dorsal position progress skin and disappeared Poison, then belly mark position be subcutaneously injected 0.35mL PBS.
Overdose of anesthesia causes 2 rats deaths of 4 rats of experimental group and control group.
Reach 1 month, 6 months and 12 months after injection, in batches, respectively under magnifying glass by rats in test groups implantation Filler（The composition injected, calls implant in the following text）Carefully cut off, without any perienchyma during excision, once obtain Implant is taken, the tissue of 10mm around implant is just scaled off into carry out histopathological examination.
It is to probe into the histologic reaction in implant site and its peripheral part generation, planted in excision to choose edge tissues After entering thing and perienchyma, the abdominal incision of every rat is gone through abdominal cavity and abdominal organs by us（Such as liver, spleen） To have seen whether that any macroscopic view changes or whether occurred implant migration.
2nd, Histopathology assessment method
Obtained implant will be cut off and perienchyma is put into 10% buffered formaldehyde liquid and fixes 24 hours, then in embedded paraffin 4 microns of slabs are made, then use hematoxylin-eosin（hematoxyline-cosin）Coloring agent is to implant and peripheral group Knit and coloured, using mono blind method, that is, do not know about tested be injected in the case of which kind of implant by a urological pathology man Histopathological analysis is carried out to sample.Then entered by inflammatory reaction, fibrosis and fibroblast in microexamination sample Invade the degree of implant and adjacent tissue.Sxemiquantitative is carried out by following standard to fibrosis, fibroblast invasion and inflammatory reaction Analysis：0 grade=reactionless, 1 grade=mild reaction, the reaction of 2 grades=moderate, the reaction of 3 grades=moderate.
3rd, result
1st, there is not any complication, and none animal dead during research after experimental group injection（Exclude overdose of anesthesia）.
2nd, the filler of implantation can be touched easily in the injection site of experimental group rat, and can perceive it and distal end does not occur Migration.
3rd, experimental group does not occur the symptoms of allergic of such as erythema or swelling, each injection site and its surrounding at injection Tumour is not observed in tissue.
4th, the filler of experimental group rat injection is wrapped well, and easily is removed.
5th, for experimental group rat, first month after injection, the mean fibrosis degree of perienchyma is 1.21 ± 1.04, There is slight foreign body reaction；Six month after injection, the mean fibrosis degree of perienchyma is 1.15 ± 0.54, there is slight foreign matter Reaction；The 12nd month after injection, the mean fibrosis degree of perienchyma is 0.68 ± 0.41, granulation inflammatory reaction, inflammation It is most light, it has been fiberized substitution.The difference in statistical significance with control group be present.

Claims (13)

  1. A kind of 1. cross-link dextran microballoon of modification, it is characterised in that：It is microspheric, cation modified cross-link dextran, Described cation modified cross-link dextran refers to the H of the part-OH in cross-link dextran chemical constitution by-CH2-CH2-N+ H-(C2H5)2Substitute or by-CH2-CH2-N+H-(CH3)2Substitution.
  2. 2. the cross-link dextran microballoon of modification according to claim 1, it is characterised in that：The cross-link dextran of modification contains Nitrogen quantity is 0.5%~2.0%.
  3. 3. the cross-link dextran microballoon of modification according to claim 1 or 2, it is characterised in that：The cross-link dextran of modification The particle diameter of dry microspheres is 80 μm~250 μm.
  4. 4. the preparation method of the cross-link dextran microballoon of a kind of modification, it is characterised in that the cross-link dextran microballoon of the modification is Microspheric, cation modified cross-link dextran, described cation modified cross-link dextran refer to cross-link dextran The H of the part-OH in structure is learned by-CH2-CH2-N+H-(C2H5)2Substitute or by-CH2-CH2-N+H-(CH3)2Substitution；Prepare When cross-link dextran microballoon is first made, then cross-link dextran microballoon and cation modified dose are reacted, modified Cross-link dextran microballoon, comprises the following steps：
    1. glucan and reducing agent are dissolved in sodium hydrate aqueous solution, glucan alkaline aqueous solution is uniformly mixed so as to obtain, as water Phase；The reducing agent is one kind in sodium borohydride, sodium sulfite, sodium pyrosulfite, VC；
    2. surfactant is dissolved in organic solvent, the organic phase containing surfactant is obtained, and it is organic by what is prepared In phase transfer to the reaction vessel with agitating device；
    3. opening stirring, 1. aqueous phase that step is obtained is added to step and 2. added in the reaction vessel of organic phase, carries out breast Change, the w/o type emulsification systems of balance are formed after mechanical agitation, emulsion process temperature control is at 20 DEG C~50 DEG C；
    4. crosslinking agent is added dropwise in the emulsification system 3. balanced to step, after crosslinking agent is added dropwise to complete, continue after reacting 12h~36 Reaction finishes, and cross-linking reaction temperature is 30 DEG C~80 DEG C；
    5. after cross-linking reaction terminates, the material in reaction vessel is all poured out, reaction solution is filtered out, collects obtained microballoon First microballoon then is washed with ethanol to remove to remove the surfactant and crosslinking agent that microsphere surface remains with organic solvent washing The organic solvent for going microsphere surface to remain, washs to microballoon followed by with distilled water, removes other water-solubility impurities；Washing Microballoon afterwards is dehydrated, obtains drying cross-link dextran microballoon after drying；
    6. 5. drying cross-link dextran microballoon that step is obtained is added in sodium hydrate aqueous solution, mix；Continue to hydroxide Cation modified dose is added in sodium solution, 6~10h of stirring reaction at 50 DEG C~90 DEG C is finished, obtains cation modified friendship Join dextran microspheres；After reaction terminates, the material in reaction vessel is all poured out, reaction solution is filtered out, collects what is obtained Microballoon washing, dry.
  5. 5. the preparation method of the cross-link dextran microballoon of modification according to claim 4, it is characterised in that：The Portugal of step 1. The concentration of glucan is 0.11g/mL~0.67g/mL in glycan alkaline aqueous solution, glucose monomer and reducing agent in glucan Material amount ratio be 9~25:1；2. the concentration of surfactant is 0.01~0.1g/mL to step in organic phase；4. step Crosslinking agent is epoxychloropropane or Isosorbide-5-Nitrae butanediol diglycidyl ether, and the mass ratio of crosslinking agent and glucan is 1~8:10.
  6. 6. the preparation method of the cross-link dextran microballoon of modification according to claim 4, it is characterised in that：Step 6. in plus Cation modified dose entered is 2- lignocaine chloroethanes hydrochloride, 2- lignocaine bromoethanes hydrochloride, lignocaine chloromethane Heptane hydrochloride salt, lignocaine bromomethane hydrochloride, the hydroxyethylamino chloroethanes hydrochlorides of 2- bis-, the hydroxyethylamino bromoethane hydrochloric acid of 2- bis- One kind in salt.
  7. 7. the preparation method of the cross-link dextran microballoon of a kind of modification, it is characterised in that the cross-link dextran microballoon of the modification is Microspheric, cation modified cross-link dextran, described cation modified cross-link dextran refer to cross-link dextran The H of the part-OH in structure is learned by-CH2-CH2-N+H-(C2H5)2Substitute or by-CH2-CH2-N+H-(CH3)2Substitution；Prepare When cation modified glucan is first made, then be crosslinked balling-up obtain microballoon, comprise the following steps：
    1. glucan is dissolved in sodium hydrate aqueous solution, mix；Continue to add into sodium hydroxide solution cation modified Agent, finish 6~10h of stirring reaction at 50 DEG C~90 DEG C, after completion of the reaction, to reaction after ethanol is added in solution, it is heavy to separate out Form sediment, what is be filtrated to get is precipitated as cation modified glucan；
    2. 1. cation modified glucan, reducing agent that step is obtained are dissolved in sodium hydrate aqueous solution, it is uniformly mixed so as to obtain Glucan alkaline aqueous solution, as aqueous phase；The reducing agent is one in sodium borohydride, sodium sulfite, sodium pyrosulfite, VC Kind；
    3. surfactant is dissolved in organic solvent, the organic phase containing surfactant is obtained, and it is organic by what is prepared In phase transfer to the reaction vessel with agitating device；Surfactant is polyethylene glycol stearate diester, the double oleic acid of polyethylene glycol One kind in ester, AEO；
    4. opening stirring, 2. aqueous phase that step is obtained is added to step and 3. added in the reaction vessel of organic phase, carries out breast Change, the w/o type emulsification systems of balance are formed after mechanical agitation, emulsion process temperature control is at 20 DEG C~50 DEG C；
    5. crosslinking agent is added dropwise in the emulsification system 4. balanced to step, cross-linking reaction is now proceeded by, treats that crosslinking agent drips Cheng Hou, continue react and finish after reacting 12h~36h, cross-linking reaction temperature is 30 DEG C~80 DEG C；Crosslinking agent be epoxychloropropane or The mass ratio of Isosorbide-5-Nitrae butanediol diglycidyl ether, crosslinking agent and glucan is 1~8:10；
    6. step after 5. cross-linking reaction terminates, the material in reaction vessel is all poured out, filters out reaction solution, collection obtains Cation modified cross-link dextran microballoon washing, dry.
  8. 8. the preparation method of the cross-link dextran microballoon of modification according to claim 7, it is characterised in that：Step 1. in plus Cation modified dose entered is 2- lignocaine chloroethanes hydrochloride, 2- lignocaine bromoethanes hydrochloride, lignocaine chloromethane Heptane hydrochloride salt, lignocaine bromomethane hydrochloride, the hydroxyethylamino chloroethanes hydrochlorides of 2- bis-, the hydroxyethylamino bromoethane hydrochloric acid of 2- bis- One kind in salt.
  9. 9. the preparation method of the cross-link dextran microballoon of a kind of modification, it is characterised in that the cross-link dextran microballoon of the modification is Microspheric, cation modified cross-link dextran, described cation modified cross-link dextran refer to cross-link dextran The H of the part-OH in structure is learned by-CH2-CH2-N+H-(C2H5)2Substitute or by-CH2-CH2-N+H-(CH3)2Substitution；Prepare Shi Caiyong one pot reactions, be crosslinked balling-up and it is cation modified carry out simultaneously, comprise the following steps：
    1. glucan and reducing agent are dissolved in sodium hydrate aqueous solution, glucan alkaline aqueous solution is uniformly mixed so as to obtain, as water Phase；The reducing agent is one kind in sodium borohydride, sodium sulfite, sodium pyrosulfite, VC；
    2. surfactant is dissolved in organic solvent, the organic phase containing surfactant is obtained, and it is organic by what is prepared In phase transfer to the reaction vessel with agitating device；
    3. opening stirring, 1. aqueous phase that step is obtained is added to step and 2. added in the reaction vessel of organic phase, carries out breast Change, the w/o type emulsification systems of balance are formed after mechanical agitation, emulsion process temperature control is at 20 DEG C~50 DEG C；
    4. crosslinking agent is added dropwise in the emulsification system 3. balanced to step, after crosslinking agent is added dropwise to complete, at 30 DEG C~80 DEG C first Reaction 6~8 hours, then adds cation modified dose into reaction system, finish at 50 DEG C~90 DEG C stirring reaction 15~ The cross-link dextran microballoon that 20h is modified；Cation modified dose is 2- lignocaine chloroethanes hydrochloride, 2- diethylamino bromides Ethane hydrochloride, lignocaine chloromethanes hydrochloride, lignocaine bromomethane hydrochloride, the hydroxyethylamino chloroethanes hydrochloric acid of 2- bis- One kind in salt, the hydroxyethylamino bromoethane hydrochlorides of 2- bis-；
    After 5. crosslinking, modification reaction terminate, the material in reaction vessel is all poured out, filters out reaction solution, collection obtains Microballoon washing, dry.
  10. A kind of 10. medical composition of the cross-link dextran microballoon containing modification, it is characterised in that：The combination by weight percentage Thing is composed of the following components：The % of cross-link dextran microballoon 4%~6 of modification, the % of cross-linking hyaluronic acid sodium 1%~2, PBS Surplus；The cross-link dextran microballoon of modification therein is microspheric, cation modified cross-link dextran, described cation The cross-link dextran of modification refers to the H of the part-OH in cross-link dextran chemical constitution by-CH2-CH2-N+H-(C2H5)2Substitution Or by-CH2-CH2-N+H-(CH3)2Substitution.
  11. 11. the medical composition of the cross-link dextran microballoon according to claim 10 containing modification, it is characterised in that：Group The cross-link dextran microballoon of compound cationic modification is dispersed in cross-linking sodium hyaluronate gel；Composition cationic The nitrogen content of the cross-link dextran of modification is 0.5%~2.0%.
  12. 12. the medical composition of the cross-link dextran microballoon containing modification according to claim 10 or 11, its feature exist In：Composition is the composition for treating VUR, the urinary incontinence and incontinence of faces, and the formulation of composition is injection.
  13. A kind of 13. detection method of the nitrogen content of the cross-link dextran microballoon of modification, it is characterised in that the crosslinking Portugal of the modification Glycan microballoon is microspheric, cation modified cross-link dextran, and described cation modified cross-link dextran refers to hand over Join the H of the part-OH in glucan chemical constitution by-CH2-CH2-N+H-(C2H5)2Substitute or by-CH2-CH2-N+H-(CH3)2 Substitution；The detection method is applied to using cross-link dextran microballoon as raw material, through nitrogen content in cation modified obtained microballoon Detection；
    The detection method of nitrogen content comprises the following steps：
    The degree of cross linking X1 of cation modified cross-link dextran microballoon and the cross-link dextran microballoon as raw material are detected first Degree of cross linking X2；Then calculating formula is substituted into（MPortugal+MHand over*X2）*[100/（ X1- X2）]=A；A+MModification=B；Nitrogen content=14/B*100%； Wherein MPortugalIt is the relative atomic mass of glucan, MHand overIt is the relative atomic mass of the crosslinking agent used, MModificationBe modification group- CH2-CH2-N+H-(C2H5)2Or-CH2-CH2-N+H-(CH3)2Relative atomic mass；
    The detection method of the wherein degree of cross linking of microballoon is as follows：
    With electronic balance precision weigh quality be m, unit g tested microballoon, it is V to transfer them to capacityaIn mL volumetric flasks, use Pipette measures sodium periodate solution and is transferred in above-mentioned volumetric flask, shakes up, and lucifuge is wrapped in volumetric flask periphery with tinfoil, 2 DEG C~10 DEG C at carry out redox reaction, the addition of sodium periodate solution guarantees complete oxidation cross-link dextran microballoon；
    Solution in the volumetric flask of course of reaction interval time absorption progress oxidation reaction shakes into volumetric flask after being diluted with water It is even, determine the light absorption value of solution；
    Treat that light absorption value reaches stable, assert that reaction terminates, V is now drawn from volumetric flaskbML supernatants are into conical flask, to cone V is added in shape bottleb/ 10 mL ethylene glycol, are shaken up, and after being stood under room temperature, dark condition, 2~3 drop phenol are added into conical flask Phthalein reagent, shakes up, be then c with concentration, unit mol/L sodium hydroxide solution titrated, record the volume V of consumption1；
    With same volume VbML distilled water adds Vb/ 10 mL ethylene glycol solutions do blank, operate, are titrated in the same way, Record the volume V of blank consumption0；
    The substitution degree of cross linking calculation formula degree of cross linking=Calculated in X 100% To the degree of cross linking.
CN201710852690.7A 2017-09-20 2017-09-20 Cross-link dextran microballoon of modification and preparation method thereof and the medical composition containing this microballoon Pending CN107629142A (en)

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CN112075662A (en) * 2020-10-19 2020-12-15 甘肃烟草工业有限责任公司 Preparation method of natural ylang-rosea flavor-carrying gel filter stick and application of natural ylang-rosea flavor-carrying gel filter stick in cigarettes
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CN117229992A (en) * 2023-06-06 2023-12-15 广东龄值生物科技有限公司 Microcarrier for stem cell 3D culture and preparation method and application thereof

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CN117229992A (en) * 2023-06-06 2023-12-15 广东龄值生物科技有限公司 Microcarrier for stem cell 3D culture and preparation method and application thereof

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