CN104784120A - Preparation of chitosan-electronegative antibiotic nanoparticles through ion crosslinking method and bacteriostatic activity of nanoparticle - Google Patents
Preparation of chitosan-electronegative antibiotic nanoparticles through ion crosslinking method and bacteriostatic activity of nanoparticle Download PDFInfo
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- CN104784120A CN104784120A CN201410489772.6A CN201410489772A CN104784120A CN 104784120 A CN104784120 A CN 104784120A CN 201410489772 A CN201410489772 A CN 201410489772A CN 104784120 A CN104784120 A CN 104784120A
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Abstract
The invention provides a nano-preparation of electronegative antibiotic. The nano-preparation mainly comprises chitosan and the electronegative antibiotic. The electronegative antibiotic is prepared into chitosan-electronegative antibiotic nanoparticles through an ion crosslinking method. The method is simple in preparation process and operation; no organic solvent is used; nanoparticles with uniform diameters are obtained; and the method has good reproducibility. The pharmacological activity of the antibiotic nanoparticles is as good as raw materials. The antibiotic nanoparticles have advantages such as a broad antimicrobial spectrum and a low toxic and side effect. By preparing the nanoparticle preparation, the purpose of safe, effective, reliable treatment can be achieved.
Description
Technical field
The present invention relates to nanotechnology and antibiotic delivery field, particularly relate to the preparation of antibiotic nano-particle, come for antibiotic controlled release system and bacteriostatic activity.
Background technology
Elecrtonegativity antibiotic is the class antibiotic occurring negative charge group after ionizing in aqueous, and it mainly containing groups such as carboxylic acid, phosphoric acid, sulfonic acid, comprises daptomycin, fosfomycin, colistimethate sodium, amfomycin, cefonicid, bambermycin etc.At this, using daptomycin as main object of study.
Daptomycin is the Cyclic lipopeptide antibiotic extracting a kind of brand new obtained in streptomycete fermentation liquid, and it has significant effect to the clinical symptoms that high drug-resistance bacterial strain causes.The end of the year 2003, FDA (FDA) ratifies injection daptomycin and is used for the treatment of the concurrency skin and skin structure infection that some gram positive bacterias cause, as abscess, surgery cut infection and skin ulcer, and the bacteremia that staphylococcus aureus causes.2006, approval daptomycin was used for the treatment of infective endocarditis.Daptomycin, except acting on most of clinical related gram-positive bacterium, the more important thing is that it has strong active to the isolated strains presenting the drug resistance character such as methicillin (methicillin), vancomycin and linwzolid in vitro.The mechanism of action of daptomycin is different from other antibiotic, and it is by upsetting cell membrane to amino acid whose transhipment, thus hinders the biosynthesis of bacteria cell wall Peptidoglycan, changes the character of cytoplasma membrane; In addition, it also by destroying the cell membrane of antibacterial, makes its content leak and reach the object of sterilization, and therefore antibacterial may be more difficult to daptomycin generation drug resistance.Daptomycin has effective in vitro and in vivo bactericidal activity, causes seriously and the clinical related gram-positive antibacterial of life-threatening disease to resist.
On July 29th, 2010, FDA claims the problem with regard to using intravenous injection medicine daptomycin (daptomycin, Cubist drugmaker produces, trade name Cubicin) may cause eosinophilic pneumonia in diagnosis and treatment process.
Utilize biological macromolecule material to prepare nano-medicament carrier, it is desirable to both to meet the requirement to drug conveying, weaken again the toxic and side effects to organism.Protein and the large class of polysaccharide (as chitosan) two is mainly comprised for the biomacromolecule in nano-medicament carrier.Because these macromole can obtain from natural animals and plants, abundance, also be a kind of Renewable resource simultaneously, they have good organism affinity, rejection is little, and can by the enzymatic degradation in organism, product after degraded is also less to the toxic and side effects of organism, in addition on these macromolecular strands usually with the functional group that hydroxyl, amino, carboxyl etc. can react in a large number, can as the site of chemical modification, therefore they have boundless application prospect as the carrier material of medicine.
Chitosan is the polysaccharide that chitin is hydrolyzed by highly basic or after enzymolysis, deacetylate obtains, chemistry poly-(Isosorbide-5-Nitrae)-2-amino-2-deoxidation-β-D-Glucose by name.The good biocompatibility of chitosan, has short Absorption, and to human non-toxic, also can not produce secondary pollution to environment, therefore, is widely used in every respect.In addition, chitosan can multiple enzyme biodegradation in body, and catabolite is nontoxic and can be absorbed completely by organism, and therefore, be the ideal carrier of medicament slow release, in recent years, as drug carrier material, chitosan receives very large concern.
Summary of the invention
One is the object of the present invention is to provide effectively to send the antibiotic nanometer formulation of elecrtonegativity.The present invention prepares chitosan-elecrtonegativity antibiotic nano-particle by ionic cross-linking can controlled release drug and have suitable bacteriostatic activity effectively.
Further aim of the present invention is the polymer providing a kind of biocompatibility being used for effectively carrying out elecrtonegativity antibiotic delivery, thus reduces its side effect.
Above-mentioned and other objects are realized by a kind of natural cationic polysaccharide, and wherein said polymer is chitosan (molecular weight 1k-500kDa).In addition, this polymer shell polysaccharide can be dissolved in weakly acidic solution, and with a large amount of positive charge, therefore it is easy to act under aqueous phase condition with the molecule with negative charge and make nanoparticle.The present invention mainly uses ionic cross-linking to prepare chitosan-elecrtonegativity antibiotic nanoparticle.The method can solve the problems such as drug absorption difficulty, toxicity and instability, has slow-releasing and controlled-releasing action simultaneously and increases the advantages such as targeting.
The present invention is realized by following technical scheme, and concrete steps are as follows:
The preparation method of chitosan-elecrtonegativity antibiotic nanoparticle is as follows: take a certain amount of chitosan, be dissolved in Acetic acid-sodium acetate buffer, be made into certain density chitosan solution, regulates pH to 5.0 with 1mol/LNaOH solution.Separately take a certain amount of elecrtonegativity antibiotic to be dissolved in distilled water and to be made into certain density elecrtonegativity aqueous topical antibiotics.Under magnetic agitation condition, elecrtonegativity aqueous topical antibiotics is slowly added drop-wise in above-mentioned chitosan solution, continues to stir 30min, with 0.8 μm of filtering with microporous membrane, obtain chitosan-elecrtonegativity antibiotic nanoparticle.The medicine removing and do not wrap up of dialysing is carried out with MWCO=3500 bag filter.Last nanoparticle lyophilizing or for subsequent use at being kept at 4 DEG C.
The present invention has following beneficial effect: the chitosan based on ionic cross-linking prepared by the present invention-elecrtonegativity antibiotic nanoparticle can controlled release drug effectively, and has suitable bacteriostatic activity, has broad application prospects in antibiotic delivery field.
Accompanying drawing explanation
Fig. 1 is chitosan-daptomycin nanoparticle outward appearance photo of synthesizing according to embodiment 1 of the present invention and particle size distribution;
Fig. 2 is chitosan-daptomycin nanoparticle release profiles under condition of different pH that the present invention is prepared according to embodiment 1;
Fig. 3 is the bacteriostatic activity of chitosan-daptomycin nanoparticle of preparing according to embodiment 1 of the present invention and control sample thereof: 1-daptomycin nanoparticle; 2-daptomycin standard substance; 3-chitosan; Concrete dosage is as follows: (a) drug loading is 1.25 μ g/ sheets; (b) drug loading 2.5 μ g/ sheet; (c) drug loading 5 μ g/ sheet; (d) drug loading 10 μ g/ sheet
Fig. 4 is the chitosan-daptomycin nanoparticle of the present invention according to embodiment 1, the antibacterial circle diameter of daptomycin mark product and chitosan.
Detailed description of the invention
Embodiment 1
Take a certain amount of chitosan, be dissolved in the Acetic acid-sodium acetate solution of 50mM, be made into the chitosan solution of 0.5mg/ml, regulate pH to 5.0 with 1mol/LNaOH solution.Separately take a certain amount of daptomycin and be dissolved in purified water the daptomycin aqueous solution being made into 4mg/ml.Under magnetic agitation condition, 4ml daptomycin aqueous solution is slowly added drop-wise in above-mentioned chitosan solution, continues to stir 30min, with 0.8 μm of filtering with microporous membrane, obtain chitosan-daptomycin nanoparticle.The medicine removing and do not wrap up of dialysing is carried out with MWCO=3500 bag filter.Last nanoparticle lyophilizing or for subsequent use at being kept at 4 DEG C.
Embodiment 2
The sign of nanoparticle
The size of nanoparticle uses Dynamic Light Scattering Determination, and sample is 2ml.
Embodiment 3
The mensuration of chitosan-daptomycin nanoparticle drug loading
First drawing standard curve.The daptomycin standard solution of preparation variable concentrations, get the 1.0075mg/ml daptomycin aqueous solution of 5,10,20,40,60,80,120 μ l respectively, be diluted to 4ml, obtain the daptomycin aqueous solution of 1.260,2.519,5.038,10.076,15.113,20.150,30.225 μ g/ml, its absorbance A at 221nm place is measured with UV-Vis spectrophotometry photometry, matched curve obtains equation: y=0.0255x+0.0181, R
2=0.9959.
Get 2ml chitosan-daptomycin nanoparticle, lyophilizing in freezer dryer, obtain nanoparticle 1.30mg, drip the HCl of 0.1mol/L after adding distil water redissolves to clarification.Survey ultraviolet absorptivity after diluting 20 times, it is 0.3952mg/ml that substitution daptomycin absorbance standard curve obtains daptomycin concentration.Substitute into drug loading computing formula: drug loading=(Wt/Wn) × 100% (Wt: the quality of daptomycin in nanoparticle; Wn: the gross mass of nanoparticle), calculate drug loading.
Embodiment 4
The release in vitro of chitosan-daptomycin nanoparticle
Draw the tablets in vitro behavior curve of nanoparticle: precision measures 3ml nanoparticle 4 parts, be placed in bag filter (MWCO=3500) respectively, bag filter two ends are tightened, be placed in respectively and 40mL pH is housed is respectively 4.5, 5.0, in the conical flask of the phosphate buffered solution of 6.0 and 7.4, conical flask is positioned over temperature 37 DEG C, in the water bath chader of frequency of oscillation 120 times/min, respectively at 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h and 72h takes out 3mL solution in conical flask, its absorbance at λ=221nm place is surveyed with ultraviolet-visible spectrophotometer, in conical flask, add the phosphate buffered solution of 3mL same pH immediately simultaneously.Concentration and the preparation of daptomycin in solution is calculated by standard curve.Repeat above-mentioned steps twice.Get the meansigma methods of three lot samples preparation originally, draw the cumulative release curve chart of daptomycin in nanoparticle.
Embodiment 5
Daptomycin nano-particle staphylococcus activity research
The nutrient agar of 20ml sterilizing joined in sterilized plate, cool, prepare bottom culture medium.Get the staphylococcus aureus liquid culture 2ml cultivating 8h be added to 100ml sterilizing and be cooled in 1% nutrient agar of 55 DEG C, concussion mixing, draw 5ml and join in bottom culture medium, preparation is containing the double-layer plate of staphylococcus aureus.Be 10,5 with tweezers by drug loading, the daptomycin nanoparticle aseptic filter paper sheet of 2.5,1.25 μ g/ sheets is placed on the double-layer plate of staphylococcus aureus, sets adjuvant core oligosaccharide and daptomycin standard substance as contrast simultaneously, cultivates 24h for 37 DEG C.In the different drug loading group of observation and comparison daptomycin nanoparticle, daptomycin standard substance and core oligosaccharide inhibition zone size and measure antibacterial circle diameter.
Claims (7)
1., containing an antibiotic nanometer formulation, its feature is mainly that this nanometer formulation mainly comprises following composition: cationic polymer, elecrtonegativity antibiotic.Preparation method is ionic cross-linking.
2. the polymer described in claim 1 is a kind of cationic chitosan, molecular weight ranges 1k-500k;
3. the antibiotic described in claim 1 is elecrtonegativity antibiotic, specifically comprises daptomycin, fosfomycin, colistimethate sodium, amfomycin, cefonicid, bambermycin etc.
4. the preparation method of the nano-particle according to claim 1, is characterized in that described reactions steps is as follows:
Take a certain amount of chitosan, be dissolved in the Acetic acid-sodium acetate solution of 50mM, be made into the chitosan solution of 0.01-5.0mg/ml, regulate pH to 3.0-7.0 by 1mol/L NaOH solution.Separately take a certain amount of elecrtonegativity antibiotic and be dissolved in distilled water the aqueous solution being made into 0.5-20mg/ml.Under magnetic agitation condition, elecrtonegativity antibiotic solution is slowly added drop-wise in above-mentioned chitosan solution, the volume ratio of elecrtonegativity antibiotic solution and chitosan solution is 1: 1-10: 1, continue to stir 30min, with 0.8 μm of filtering with microporous membrane, obtain chitosan-elecrtonegativity antibiotic nanoparticle.The medicine removing and do not wrap up of dialysing is carried out with bag filter (MWCO=3.5k-14k).Last nanoparticle lyophilizing or for subsequent use at being kept at 4 DEG C.
5. the purification process of the nano-particle according to claim 4, is characterized by: the molecular weight of bag filter is within the scope of 3.5k-14k.
6. the nano-particle according to claim 1 or 4 is as antibacterial applications.
7., by an antibiotic transmission system for drug administration by injection, require described daptomycin and the antibiotic nanoparticle of elecrtonegativity thereof according to right 1, it is characterized in that, nanoparticle not only can be prepared into injection as preparation also can prepare liquid preparation.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1586488A (en) * | 2004-07-12 | 2005-03-02 | 复旦大学 | Chitosan glycyrrhizic acid nano particle and its preparing method |
| WO2009108407A1 (en) * | 2008-02-25 | 2009-09-03 | The University Of North Carolina At Charlotte Office Of Technology Transfer | Biodegradable therapeutic nanoparticles containing an antimicrobial agent |
| CN101838467A (en) * | 2010-05-21 | 2010-09-22 | 南京农业大学 | Novel chitosan nanoparticles and preparation method thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1586488A (en) * | 2004-07-12 | 2005-03-02 | 复旦大学 | Chitosan glycyrrhizic acid nano particle and its preparing method |
| WO2009108407A1 (en) * | 2008-02-25 | 2009-09-03 | The University Of North Carolina At Charlotte Office Of Technology Transfer | Biodegradable therapeutic nanoparticles containing an antimicrobial agent |
| CN101838467A (en) * | 2010-05-21 | 2010-09-22 | 南京农业大学 | Novel chitosan nanoparticles and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| NÁDIA C.SILVA，等: "Chitosan nanoparticles for daptomycin delivery in ocular treatment of bacterial endophthalmitis", 《DRUG DELIVERY》 * |
| 库马尔 主编，梁伟 等译: "《药用生物纳米材料》", 30 September 2009, 科学出版社 * |
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Application publication date: 20150722 |