CN105218699A - The chitosan of oligomerization arginine covalent modification, preparation method, screening and application - Google Patents

The chitosan of oligomerization arginine covalent modification, preparation method, screening and application Download PDF

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CN105218699A
CN105218699A CN201410320869.4A CN201410320869A CN105218699A CN 105218699 A CN105218699 A CN 105218699A CN 201410320869 A CN201410320869 A CN 201410320869A CN 105218699 A CN105218699 A CN 105218699A
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arginine
chitosan
oligomerization
sirna
nanoparticle
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高钟镐
黄伟
杨飞飞
柳珊
金明姬
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Institute of Materia Medica of CAMS
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Abstract

(Arginine, is abbreviated as R, R to the invention provides a series of oligomerization arginine 2-R 7) the novel carriers CS-g-R of covalent modification chitosan n, and apply efficient carrier and load DNA and function siRNA and prepare nanoparticle and carry out inside and outside applied research.The present invention adopts one-step synthesis to be covalently bonded on the free amine group of chitosan molecule by oligomerization arginine, while the novel vector prepared maintains chitosan security, nanoparticle can be prepared with the nucleic acid of negative charge by complex coacervation with surface, effectively improve delivery of nucleic acids efficiency.CS-g-R nnanoparticle prepared by value-added tax function siRNA has good therapeutic action in vivo and in vitro, has good application prospect.

Description

The chitosan of oligomerization arginine covalent modification, preparation method, screening and application
Technical field
The invention belongs to field of biomedical materials, relate to a kind of polymer materials, be specifically related to the chitosan derivatives of a series of oligomerization arginine covalent modification, its preparation method, screening and purposes.
Background technology
Gene therapy (GeneTherapy), as a kind of emerging treatment means, has become the study frontier of current medicine.Wherein, exonuclease treatment is provide a kind of brand-new methods for the treatment of to the many difficult and complicated cases comprising cancer.But the shortage of safe and efficient nucleic acid delivery vector is the bottleneck problem limiting its application all the time, at present, nucleic acid delivery vector is mainly divided into virus vector and the large class of non-virus carrier two.Virus vector transfection efficiency is high, but there is the safety issues such as immune response, cell pathology change, teratogenesis, mutagenesis in the application.Based on above reason, non-virus carrier becomes comparatively ideal nucleic acid delivery vector.
Non-virus carrier uses chemical material to protect nucleic acid molecule and be delivered to target cell to play therapeutic action, can be divided into natural macromolecular material and synthesized polymer material.Chitosan (Chitosan, CS) as a kind of natural macromolecule amylose, 2 bit aminos with positive charge, can with electronegative interaction of molecules, and easily carrying out chemical reaction and carry out modification to its structure, is study nucleic acid non-viral delivery vector more widely at present.As a kind of excellent macromolecular material, chitosan has the advantages such as bioadhesive, biodegradable, safety non-toxic.
Within 2006, report application chitosan the earliest and send siRNA, (see KatasH, AlparHO, DevelopmentandcharacterizationofnanoparticlesforsiRNAdel ivery [J], JControlRelease, 2006,115 (2): 216-225.) but lower transfection efficiency limits field sent by chitosan application at siRNA.In order to improve transfection efficiency, have studied the impact on nanoparticle physico-chemical property and outer-gene silence efficiency of chitosan molecule amount and deacetylation (see LiuX, HowardKA, DongM, Theinfluenceofpolymericpropertiesonchitosan/siRNAnanopar ticlesformulationandgenesilencing [J], Biomaterials, 2007,28 (6): 1280-1288); Load siRNA and prepare the method for nanoparticle to the impact of nanoparticle physico-chemical property, (see MaoS, SunW, KisseiT, Chitosan-basedformulationsfordeliveryofDNAandsiRNA [J], AdvDrugDelivRev, 2010,62 (1): 12-27); On this basis, also by having carried out various chemically modified to CS molecule: modify (GhosnB with imidazoleacetic acid to CS, Singh, A, Roy, K, EfficientsiRNAdeliverybysecondaryandtertiaryaminemodifie dpolysaccharides [J], NSTI-Nanotech2008a, 2,338 – 341); With mannose-modified CS (gorgeous see stone, Zhang Huibin, Zong Li. the complete synthesis and Cytotoxic evaluation of the carrier mannose glycosylation chitosan of target mannose receptor.Pharmaceutical Biotechnology, 2009,16 (3): 207-211), with Surface-modified by Transferrin chitosan (MaoHQ, RoyK, Troung-LeVL, etal.Chitosan-DNAnanoparticlesasgenecarriers:synthesis, characterizationandtransfectionefficiency.J.JControlRele ase, 2001, 70 (3): 399), containing the guanidino group of positively charged in arginine molecule, can with the molecular dna of bear electric charge, the electrostatical bindings such as siRNA, thus effective permeate through cell membranes, existing research confirms that arginic number has impact (see FutakiS to the arginic born of the same parents' of the entering efficiency of oligomerization, SuzukiT, OhashiW, etal.Arginine-richpeptides.Anabundantsourceofmembrane-pe rmeablepeptideshavingpotentialascarriersforintracellular proteindelivery [J] .JBiolChem, 2001, 276 (8): 5836-5840).The present invention utilizes chitosan and the arginic feature of oligomerization, synthesizes a series of oligomerization arginine-chitosan derivative, and evaluates the performance that siRNA is sent in its inside and outside.The patent No. is the patent that CN1519035A discloses name and is called " preparation method of chitosan-arginine conjugate anticoagulant material ", using chitosan-arginine conjugate as blood compatibility material, for improving the anticoagulation function of chitosan; China Patent No. is the patent that CN101780281A discloses name and is called " N-arginine chitosan is as the application of Percutaneous absorption enhancer ", for promoting the Transdermal absorption of macromole and insoluble drug.The present invention, through patent consulting and literature search, not yet finds the report that discussion 2-7 oligomerization arginine affects chitosan delivery of nucleic acids efficiency at present.
Summary of the invention
The technical problem to be solved in the present invention be provide a kind of oligomerization arginine to modify by screening process chitosan derivatives as non-viral nucleic acid delivery vector, this carrier cell toxicity is low, and transfection efficiency is high, and inside and outside effect is good.
The present invention additionally provides the preparation method of the chitosan derivatives that this oligomerization arginine is modified on the other hand.
The present invention additionally provides the application of chitosan derivatives as nucleic acid carrier of this oligomerization arginine modification on the other hand.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide the chitosan non-viral delivery vector that a series of oligomerization arginine is modified, its molecular formula is as follows:
The deacetylation of raw material is expressed as: free amine group number/(the repeating unit number of free amine group number+non-deacetylate) i.e. (y+m)/(x+y+m), deacetylation scope is 60%-95%.M is the repeating unit number that Rn replaces that free amine group generates product.
N represents arginic number, n=2-7, and namely oligomerization arginine is two poly arginine (R 2), three poly arginine (R 3), four poly arginine (R 4), five poly arginine (R 5), six poly arginine (R 6), seven poly arginine (R 7).
Preferably five poly arginine (R 5), six poly arginine (R 6), seven poly arginine (R 7);
Most preferably six poly arginine (R 6).
Another aspect of the present invention, provides the preparation method of the chitosan that this poly arginine is modified, comprises the steps:
(1) be dissolved in acetic acid/sodium acetate buffer solution by oligomerization arginine, stirring at room temperature is dissolved, and obtains oligomerization arginine solution; Add EDC/NHS as coupling agent stirring at room temperature, activated carboxyl;
(2) added by chitosan in above-mentioned (1) solution, lucifuge is reacted;
(3) gained mixing solutions is transferred to dialysis tubing, deionized water is dialysed, the chitosan that after lyophilize, obtained oligomerization arginine is modified.
Macroscopic single crystal route as shown in Figure 1.
Wherein in step (1)
The pH scope of preferred acetic acid/sodium acetate buffer solution is: pH4.0-6.0, and preferably pH5.0-6.0, is most preferably pH5.5;
The consumption of preferred EDC, and the molar ratio of Rn is EDC:Rn=1 ~ 2:1, preferably 1.3 ~ 1.7:1; Most preferably 1.5:1;
The consumption of preferred NHS, and the molar ratio of Rn is NHS:Rn=1 ~ 2:1, preferably 1.3 ~ 1.7:1; Most preferably 1.5:1;
Preferred temperature of reaction is 10-40 DEG C; Preferably 20-30 DEG C; Most preferably 24-26 DEG C;
The preferred reaction times is 1.5-6h; Preferably 3-5h; Most preferably 4h.
Wherein in step (2)
The molecular weight ranges of chitosan: 5000Da ~ 50kDa; Preferably 10kDa-20kDa; Most preferably 11kDa-15kDa;
The consumption of chitosan, and the ratio of Rn is chitosan: Rn=4.5 ~ 5.5:1, preferably 4.3 ~ 5.2:1; Most preferably 5:1;
Preferred temperature of reaction is 10-40 DEG C; Preferably 20-30 DEG C; Most preferably 24-26 DEG C;
The preferred reaction times is 10-30h; Preferably 15-24h; Most preferably 19-21h.
In the preparation method of oligomerization arginine beautify chitosan, the ratio of most preferred chitosan and damping fluid solvent is that every 55mg chitosan is dissolved in 10ml solvent;
Wherein in step (3)
Dialysis tubing molecular weight cut-off scope: 3000Da ~ 5kDa, preferred scope is 3500-12000Da
Preferred dialyzate: 5% aqueous ethanolic solution, water, DMSO
The time of dialysis: 24-72h
In product, preferred chitosan free ammonia base (y) is 10:1-15:1 with the mol ratio of oligomerization arginine (m).Oligomerization arginine molecule replaces substitution value level and the m/ (x+y+m) ~ 10% of chitosan free amine group.
The substitution value warp of reaction product 1hNMR measuring and calculation: substitution value is 9-20%, preferably 10-17%; Most preferably 11-15%
Another aspect of the invention, provide chitosan that above-mentioned poly arginine modifies as in body, the application of external nucleic acid delivery vector.The invention provides chitosan that above-mentioned serial oligomerization arginine modifies as the screening process of non-viral delivery vector, obtain the efficient delivery vector of low toxicity, and be applied in body, external delivery of nucleic acids.Preparation-obtained chitosan derivatives all can increase cellular uptake amount to some extent, improves transfection efficiency.
Under room temperature condition, CS-g-Rn provided by the invention can be compounded to form nanoparticle according to certain mass ratio with nucleic acid molecule.In one embodiment, when above-mentioned CS-g-Rn and siRNA formation nanoparticle sends siRNA, the arginic length of oligomerization (i.e. arginic number) has remarkably influenced for siRNA transfection efficiency, when preferably few arginic length is 6, namely six poly arginines are the highest to target gene silence efficiency.Within provided by the invention year, function siRNA nanoparticle has significant lethal effect to tumour cell in vitro, illustrates that siRNA can send into born of the same parents by carrier, and nanoparticle can by method administration Tumor suppression growths such as local intratumor injections; When CS-g-Rn sends plasmid DNA, the expression that plasmid DNA is stronger can be observed, illustrate that plasmid delivery also effectively can be entered born of the same parents by this carrier.
Advantageous Effects
Adopt one-step synthesis to be covalently bonded on chitosan molecule free amine group by oligomerization arginine, preparation method is simple; CS-g-Rn and nucleic acid molecule form nanoparticle and transfectional cell, and the cellular uptake efficiency of nucleic acid molecule significantly improves; The support C S-g-R screened 6load siRNA lucthe nanoparticle prepared is suitable with commercially available transfection reagent Lipofectamin2000 to the silence efficiency of expression of target gene; CS-g-R 6value-added tax function siRNA is applied in body, and antitumous effect is good.
Accompanying drawing explanation
Fig. 1 is the synthetic route of embodiment 1.
Fig. 2 is a series of CS-g-R prepared in embodiment 1 6the cytotoxicity histogram of carrier.
Fig. 3 is CS-g-R prepared in test example 2 nthe transmission electron microscope figure of nanoparticle is formed with siRNA.A: the GFP carrying plasmid DNA nanometer particle expresses; The GFP of b:Lipofectamine2000 transfection expresses.
Fig. 4 is the expression of DNA after year plasmid DNA nanometer particle transfectional cell prepared by test example 3.
Fig. 5 is CS-g-R prepared in test example 2 nwith siRNA lucprepare nanoparticle, the nanoparticle that different carriers is formed lowers the effect of expression of target gene.
Fig. 6 is according to the cellular uptake situation of carrying fluorescently-labeled siRNA nanoparticle prepared by test example 2.In figure, a is the picked-up curve of blanc cell, and b is the picked-up curve of naked siRNA group cell, and c is the picked-up curve carrying siRNA nanoparticle group cell.
Fig. 7 is according to year function siRNA prepared by test example 2 bcl-2nanoparticle vitro inhibition cell proliferation effect.
Fig. 8 ~ 9 are according to year function siRNA prepared by test example 2 bcl-2nanoparticle anti-tumor in vivo effect.
Term and abbreviation
EDC:1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride
NHS:N-hydroxysuccinimide
CS: chitosan
Embodiment
In order to describe the present invention and feature thereof in detail, below in conjunction with the drawings and specific embodiments, the present invention will be further described.It should be pointed out that these embodiments only can not be used for limiting the scope of the invention for illustrating the present invention.
Embodiment 1: substitution value level is the preparation of the Rn beautify chitosan of 10%
(1) certain 5 × 10 are taken -4mole Rn, be placed in 25ml round-bottomed flask, add the acetic acid/sodium acetate buffer solution 10ml of pH5.5, be that 1:1.5 adds EDC, NHS by molar weight, stirring at room temperature activates 4 hours;
(2) by molar weight Rn:CS (Mw:11.3kDa) for 1:5 adds chitosan in above-mentioned mixed solution, stirring at room temperature is dissolved, and continues reaction 20 hours;
(3), after reaction terminates, be transferred to by reaction solution in the dialysis tubing of 3500-12000, deionized water is dialysed 48 hours, by the product lyophilize 36h after dialysis, and obtained oligomerization arginine beautify chitosan.
Macroscopic single crystal route as shown in Figure 1, the substitution value warp of product 1hNMR calculates, and the results are shown in Table 1 (the substitution value level of oligomerization arginine to chitosan is 10%).
Table 1CS-g-R nsubstitution value
Biological test:
Test example 1: the chitosan Cytotoxic evaluation that oligomerization arginine is modified
By 4T1 cell with 4 × 10 3/ hole density is inoculated in 96 orifice plates, after cultivating 24h, inhale and abandon old substratum, adding concentration is respectively after the CS-g-Rn cultivation 24h of 100 μ g/ml, old substratum is abandoned in suction, every hole adds the MTT solution 20 μ L containing 5mg/ml respectively, after continuing to cultivate 4h, adds 150 μ lDMSO solution, shaking table 37 DEG C, the speed jolting of 60rpm/min 10 minutes, then measure the OD value of each hole at 570nm place by microplate reader.Calculate the survival rate of cell, often organize Setup Experiments 6 repeating holes.As can be seen from accompanying drawing 2, CS-g-Rn when detectable level 100 μ g/ml, the equal >93% of cell survival rate, illustrates that the support C S-g-Rn safety of synthesis is good.
Test example 2: the preparation of carrying siRNA nanoparticle
Complex coacervation preparation is adopted to carry siRNA nanoparticle.Certain density oligomerization arginine beautify chitosan and certain density siRNA solution are placed in 55 DEG C of water bath with thermostatic control preheating 20min respectively.The chitosan solution drawing the modification of oligomerization arginine with pipettor is added in equal-volume siRNA solution, mixes 45s rapidly, must carry the chitosan nano of siRNA on vortex mixer.As seen from Figure 3, prepared nanoparticle particle diameter is about 200nm, and size of particles is homogeneous.
Test example 3: the expression of carrying plasmid DNA nanometer particle
By 4T1 cell with 4 × 10 3the density in/hole is inoculated in 24 holes, and after cultivating 24h, cytogamy degree about reaches 85-90%, starts transfection.The nanoparticle that green fluorescent protein plasmid gene GFP is carried in the method preparation of carrying siRNA nanoparticle prepared by embodiment 3.Inhale the old substratum abandoned in 24 orifice plates, PBS washs 2 times, and every hole adds the RPMI1640 substratum of 100 μ l serum-frees, and then every hole adds the transfection composite particles solution 50 μ l containing 400ng green fluorescence protein gene GFP.Using Lipofectamine2000/GFP mixture as positive control, after cultivating 4h, every hole adds 150 μ l and cultivates 20h containing the RPMI1640 substratum continuation of 10% serum, observes egfp expression situation with inverted fluorescence microscope.The results are shown in Figure 4, can find out from result and carry the transfection of GFP nanoparticle after 24 hours, plasmid has stronger expression, suitable with the expression intensity of commercially available transfection reagent group.Carrier synthesized by explanation can effectively be sent plasmid DNA and enter born of the same parents.
The screening of test example 4:CS-g-Rn carrier
To the MCF-7-Luc cell of logarithmic phase be in respectively according to 5 × 10 4the density in/hole is inoculated in 24 orifice plates, 5%CO 2, 37 DEG C cultivate 24 hours, prepare according to embodiment 3 and carry a siRNA nanoparticle transfectional cell.First, suck substratum, wash 2 times with PBS and change not containing the substratum of FBS.Nanoparticle suspension is added in respective aperture and cultivate 4 hours.3 multiple holes established by each sample, and the cell not adding process is set to negative control.Change the substratum containing 10%FBS, continue cultivation 20,44,68 hours.Result display transfection after 48 hours, the silence efficiency of nanoparticle to target gene is the highest, separately can find out CS-g-R from Fig. 5 result 6the highest to the silence efficiency of target gene, reach 64.9%.CS-g-R is described 6the highest to the delivery efficiency of siRNA.By measuring the target gene silence efficiency of different degree of substitution carrier, when substitution value horizontal position 10%, target gene silence efficiency is the highest.
Table 2CS-g-R ncarrier is to the silence efficiency of target gene
Table 3 different degree of substitution CS-g-R 6on the impact of target gene silence efficiency
Test example 5: carry siRNA nanoparticle cellular uptake
To the 4T1 cell of logarithmic phase be in, with 8 × 10 4the density in/hole is laid in 12 orifice plates, 37 DEG C, 5%CO 2cultivate 24 hours under condition.With the siRNA of FAM mark for model drug, prepare nanoparticle and cultivate 0.5 hour for 100pmol/ hole is transfected in respective aperture with siRNA dosage, naked for transfection same dose siRNA group being set to control group simultaneously.By concentration be 0.25% tryptic digestion collection hole in cell, 0.5mLPBS Solution Dispersion, keeps in Dark Place, and goes up machine testing as early as possible.
From picked-up laboratory test results (accompanying drawing 5), compared with naked siRNA group, the picked-up curve of nanoparticle group cell significantly moves to right, and illustrates that siRNA can more effectively send into born of the same parents by the chitosan modified through oligomerization arginine.
Test example 6: carry function siRNA bcl-2nanoparticle extracorporeal anti-tumor cell proliferation effect
To the 4T1 cell of logarithmic phase be in 8 × 10 3the density in/hole is laid in 96 orifice plates, 37 DEG C, 5%CO 2cultivate 24 hours, by CS-g-R n/ siRNA bcl-2nanoparticle in respective aperture with siRNA dosage 5pmo/ hole transfection, continues cultivation 24 hours, establishes blank and naked siRNA control group simultaneously, often organizes 6 multiple holes.Every hole adds CCK-8 reagent 10 μ L, continues cultivation 2 hours.Microplate reader 570nm place measures absorption value (A), is calculated as follows survival rate:
Cell survival rate %=A s/ A b× 100%
Wherein, A s, A bbe respectively administration group and blank group absorption value.By calculating cell survival rate, investigate nanoparticle to the kill capability (see Fig. 7) of cell.Result shows CS-g-R n/ siRNA bcl-2nanoparticle all has the effect of antiproliferative effect, and CS-g-R is described nsiRNA can be sent and play a role into born of the same parents.Wherein CS-g-R 6/ siRNA bcl-2the suppression efficiency of nanoparticle on cell proliferation is up to 71.9%, prompting CS-g-R 6delivery efficiency the highest.
Test example 7: carry function siRNA survivinand siRNA bcl-2nanoparticle anti-tumor in vivo effect
Get 15 female BAl BIc/c mouse, select the 4T1 mouse mastopathy cell being in logarithmic phase, with 1 × 10 5/ quantity only (suspending with the RPMI-1640 substratum 0.1mL not containing FBS) in-situ inoculating the 4th mammary gland pad on the right side of BALB/c mouse, preparation lotus mammary cancer bearing mouse model.Treat that tumor growth is to 150mm 3time (inoculation after the 13rd day), by mice with tumor be at random 3 groups of (often organizing 5) control groups (physiological saline group), naked siRNA treatment group, with CS-g-R 6for carrier prepares the nanoparticle treatment group of target Survivin and Bcl-2.Respectively at the administrations in the 1st day, 3 days, 5 days, 7 days, 9 days after grouping, the administering mode of nanoparticle is intratumor injection, and naked siRNA and physiological saline are also by intratumor injection administration.The 6th day after administration terminates, mouse dislocation is put to death, dissects and take out lung tissue and tumor tissues.From accompanying drawing 8,9, the knurl of naked siRNA group mouse weighs and physiological saline group indifference, illustrates that naked siRNA is without antitumor action; CS-g-R 6/ siRNA survivinand CS-g-R 6/ siRNA bcl-2nanoparticle group knurl representation work is less than physiological saline group and naked siRNA group; Illustrate and carry siRNA survivinand siRNA bcl-2nanoparticle antitumous effect is remarkable, has extraordinary Prospect of R & D.

Claims (10)

1. the chitin carrier CS-g-R of an oligomerization arginine modification n, it is characterized in that, CS-g-R nmolecular structural formula is as follows:
Wherein, the molecular weight of chitosan is 5000Da ~ 50kDa, R nfor oligomerization arginine, n represents arginic number and n=2-7 integer, and m is R nreplace the repeating unit number that free amine group generates product, x and y represents the number of repeating unit, and m/ (x+y+m) is 10%-20%.
2. the chitosan of oligomerization arginine modification according to claim 1, is characterized in that, R nbe selected from two poly arginine (R 2), three poly arginine (R 3), four poly arginine (R 4), five poly arginine (R 5), six poly arginine (R 6), seven poly arginine (R 7).
3. the preparation method of oligomerization arginine beautify chitosan according to claim 1 and 2, is characterized in that, comprise the steps:
(1) be dissolved in the acetic acid/sodium acetate buffer solution of pH4.0-6.0 by the oligomerization arginine containing 2-7 monomer, stirring at room temperature is dissolved, and adds coupling agent, activation 1.5-4h;
(2) be that 60-95% chitosan adds in above-mentioned arginine solution by substitution value, stirring at room temperature, lucifuge reaction 24-72h;
(3) by gained mixing solutions transfer dialysis tubing, deionized water is dialysed, the chitosan that after lyophilize, obtained oligomerization arginine is modified.
4. preparation method according to claim 3, is characterized in that, the coupling agent used is selected from 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxysuccinimide.
5. preparation method according to claim 3, is characterized in that, substitution value level and the m/ (x+y+m) of oligomerization arginine molecule replacement chitosan free amine group are 10%-20%.
6. CS-g-Rn according to claim 1 and 2 is as in body or the application of external nucleic acid delivery vector.
7. application according to claim 6, is characterized in that, described nucleic acid is selected from plasmid DNA or siRNA.
8. application according to claim 6, is characterized in that, the size of described plasmid DNA is 1-30Kb.
9. application according to claim 6, is characterized in that, the size of described siRNA is 15-30bp.
10. application according to claim 6, is characterized in that, described siRNA with Survivin and Bcl-2 for target spot.
CN201410320869.4A 2014-07-04 2014-07-04 The chitosan of oligomerization arginine covalent modification, preparation method, screening and application Pending CN105218699A (en)

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CN107344971A (en) * 2016-05-05 2017-11-14 中国科学院理化技术研究所 A kind of poly- ε-lysine modified chitosan and preparation method thereof
CN108239181A (en) * 2016-12-23 2018-07-03 中国医学科学院药物研究所 A kind of chitosan derivatives R6H6-CS, preparation method and application
CN109142560B (en) * 2018-07-06 2021-03-02 浙江工商大学 PEG-ACS/luxR-siRNA nano-composite, application thereof and method for reducing biogenic amine in litopenaeus vannamei
CN109142560A (en) * 2018-07-06 2019-01-04 浙江工商大学 PEG-ACS/luxR-siRNA nano-complex and its application and the method for reducing biogenic amine in litopenaeus vannamei
CN112569366A (en) * 2019-09-27 2021-03-30 中国医学科学院药物研究所 Oral nano polymer targeted delivery system for entrapped biomacromolecule medicine
CN112569366B (en) * 2019-09-27 2023-08-01 中国医学科学院药物研究所 Oral nano polymer targeted delivery system for encapsulating biological macromolecule medicine
CN113402630A (en) * 2021-06-11 2021-09-17 盐城师范学院 Chitosan derivative drug delivery carrier and preparation method and application thereof
CN113402630B (en) * 2021-06-11 2023-01-10 盐城师范学院 Chitosan derivative drug delivery carrier and preparation method and application thereof
CN113461834A (en) * 2021-07-09 2021-10-01 中科解码(北京)生物技术有限公司 Nano material and preparation method and application thereof
CN113461834B (en) * 2021-07-09 2022-03-11 中科解码(北京)生物技术有限公司 Nano material and preparation method and application thereof

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