CN110452314A - Anoxic sensitivity response type chitosan nitroimidazole grafting and preparation and application - Google Patents
Anoxic sensitivity response type chitosan nitroimidazole grafting and preparation and application Download PDFInfo
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- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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Abstract
The present invention provides a kind of anoxic sensitivity response type chitosan nitroimidazole grafting and preparation and application, and the weight average molecular weight of chitosan is 2000Da-20000Da, and the grafting rate of nitroimidazole is 1~20%.The present invention uses the chitosan with good biocompatibility for hydrophilic material, using N-Boc- bromo alkanamine as bridge linkage group, selects nitroimidazole for anoxic sensitive group, synthesizes the chitosan nitroimidazole grafting with anoxic sensitivity responsiveness.Self aggregation forms polymer micelle to the grafting material in water, with lower critical micelle concentration, the chitosan nitroimidazole grafting can be used as pharmaceutical carrier, with pharmaceutical composition, the drug-carrying nanometer particle of building, can be in hypoxic cell under highly expressed nitroreductase effect, rapid delivery of pharmaceuticals, it realizes Targeting delivery of the drug in the anoxics lesion environment such as tumour, cerebral apoplexy, improves drug effect.The structural formula of chitosan nitroimidazole grafting of the invention are as follows:
Description
Technical field
The invention belongs to compound synthesis, are related to the synthetic method of anoxic sensitivity response type chitosan nitroimidazole grafting,
And chitosan nitroimidazole grafting is as the application in anoxic sensitivity response medicine release carrier.
Background technique
Nano medication can realize lesions position Targeting distribution, and drug targeting delivering can be improved drug effectiveness, it is secondary to reduce poison
Effect.But the release of Nano medication lesion target area still has problem.According to the pathologic feature of disease, design environment response medicine
Carrier is the effective means for realizing drug targeting release.
Under anoxic conditions, series of physiological and pathological variation will occur for body.Oxygen is that cell carries out normal physiological activity
Institute's necessary material.Glycolytic cycle gradually replaces aerobic respiration when anoxic, becomes the main path of cellular energy metabolism, therefrom
Intracellular products type is caused to change, lactic acid accumulation, pH value intracellular changes.Cell energy supply is insufficient when due to anoxic,
The energy consumption organelle physiological function such as mitochondria, golgiosome, endoplasmic reticulum is abnormal.Meanwhile endocellular enzyme express spectra changes, cell
Physiological function associated signal paths change.Anoxic can also cause cellular stress, lead to gene damage.
The reason of anoxic is a variety of disease developments.Tumour is the second largest reason of human diseases death.Tumor development
In the process, tumour cell fast breeding, aerobic respiration is vigorous, increases the consumption of oxygen, leads to ischaemia.Tumor blood vessels
It is formed not exclusively, vascular system missing, blood conveying and oxygen conveying capacity are weak.Tumor tissues volume constantly increases simultaneously, makes
The extruding of pairs of peripheral vessels, further decreases blood supply.The pathological characteristics and structure feature of above-mentioned tumor tissues, cause to swell jointly
Tumor anoxic.Anoxic causes tumour cell metabolic pathway to change, and glycolysis increases, and intracellular products change, make
It is accumulated at lactic acid, tumor microenvironment pH value reduces.Meanwhile anoxic destroys the homeostasis of cellular redox balance, into the cell
Nitroreductase (Nitroreductase, NTRs), hypoxia inducible factor (Hypoxia induced factor-1 α, HIF-1
α) etc. protein expressions increase, and malignancy and tumor stem cell sample property is caused to increase.Therefore, anoxic is that tumour is caused to be disliked
One of the important environmental factor changed.In addition, angiemphraxis causes local blood in the cardiovascular and cerebrovascular diseases such as cerebral ischemia, coronary heart disease
Circulatory stasis, lesions position oxygen supply are obstructed, and cause nerve cell or Myocytes Anoxia impaired, generate inflammation, initiation office
Portion's necrosis, causes the organic damage of body.
The treatment method of anoxic related disease predominantly improves local oxygen and supplies and protect lesions position hypoxic cell.Early period
Result of study is shown, is that skeleton structure links fatty acid, building polymerization with chitosan (weight average molecular weight 2000Da-20000Da)
Object Micellar drug passs release system, can effectively encapsulate therapeutic agent, delivers the medicament to the anoxics lesion such as tumour.Chitosan has life
The advantages that object compatibility is good, toxicity is low, good water solubility, chitosan link the pharmaceutical carrier of fatty acid construct, can be by various kinds of cell
Efficiently intake.But it with existing chitosan aliphatic acid grafting, constructs drug and passs release system, be difficult to realize in anoxic lesions position
Quickly and effectively drug release.
The characteristics of for anoxic related disease, is changed using the physiological parameter of cell when anoxic, design anoxic sensitivity response
Property drug passs release system, it is possible to significantly improve drug and quickly and effectively discharge in anoxic lesion.Nitroreductase (NTRs) is thin
A kind of enzyme that nitroaromatic catalysis is reduced to aromatic amine intracellular.Under anaerobic condition, NTRs is with reduced form nicotinoyl amine gland
Purine dinucleotides (Reduced form of nicotinamide-adenine dinucleotide, NADH) is electronics confession
Body can be catalyzed a variety of exogenous nitro-aromatic compounds and single electron transfers occur, generate Free radicals, go forward side by side one
Free radicals are reduced into azanol or amino by step.2- nitroimidazole is a kind of hydrophobic nitro-aromatic compound,
After NTRs is acted on, hydrophilic 2- aminooimidazole can be reduced to.Select 2- nitroimidazole for hydrophobic grouping, with hydrophilic material shell
Glycan link, constructs amphipathic grafting pharmaceutical carrier, can dredge carrier material under the action of hypoxic cell height expresses NTRs
Water end (W.E.) is changed into hydrophilic radical, promotes grafting micellar structure Anaerobic response depolymerization, realizes the release of drug anoxic sensitivity.
Summary of the invention
It is quickly and effectively discharged to improve drug in the targeting of anoxic lesions position, the object of the present invention is to provide a kind of shells
Glycan nitroimidazole grafting has structural unit represented by following formula:
Wherein, the bromine atom of N-Boc- bromo alkanamine is replaced by nitroimidazole, and the free amino in the part on chitosan chain is logical
Superamide key is connected with the amino for the N-BOC- alkanamine for having sloughed N-terminal BOC protection, and the weight average molecular weight of chitosan is 2000Da-
20000Da, the grafting rate of nitroimidazole are 1~20%, and the critical micell of chitosan nitroimidazole grafting in aqueous solution is dense
Degree is 15.63~103.5 μ g/mL.
It is a further object to provide the preparation methods of chitosan nitroimidazole grafting, pass through following scheme reality
It is existing:
Weigh quantitative 2- nitroimidazole, the potassium carbonate and 2- nitroimidazole 0.8~3 of 0.5~5 times of mole of 2- nitroimidazole
The N-Boc- bromo alkanamine (alkyl carbon atoms number is 6-12) of times mole is placed in the dry round-bottomed flask of 50mL, it is added 10~
20mL is dehydrated n,N-Dimethylformamide (N, N-dimethylformamide, DMF), 80 in constant-temperature heating magnetic stirring apparatus
DEG C reaction 4h~8h.After reaction, centrifugation extracts reaction solution supernatant, puts into the deionized water of 5 times of volumes of reaction solution and anti-
The ethyl acetate of 1 times of volume of liquid is answered to extract, gas bath shaker at room temperature vibrates 5min, stands 30min and takes to liquid layered
Upper layer ethyl acetate is placed in 25mL round-bottomed flask, 40 DEG C of revolvings on Rotary Evaporators, until solution completely removes in flask.Flask
It is middle to be added appropriate n,N-Dimethylformamide (DMF), two succinimide esters of 0.5~5 times of mole of 2- nitroimidazole (N,
N'-Disuccinimidyl Carbonate, DSC), the chitosans of 2~10 times of moles of 2- nitroimidazole (weight average molecular weight=
2000-20000Da) with the deionized water of DMF equimultiple volume, 40 DEG C of reactions for 24 hours, are obtained instead in constant-temperature heating magnetic stirring apparatus
Answer product.Reaction product is placed in bag filter (1000~3500Da of MWCO, Beijing is through hongda Bioisystech Co., Ltd, section) thoroughly
48h is analysed, to remove DMF and aqueous by-product.Reaction product is freeze-dried after dialysis, obtains chitosan nitroimidazole grafting.
It is also another object of the present invention to provide the chitosan nitroimidazole graftings antitumor as cell toxicant class
Application in pharmaceutical carrier.The drug can be cell toxicant series antineoplastic medicament, MAPK signal pathway inhibitor, PI3K signal
Pathway inhibitor, cMYC inhibitor and fat-soluble fluorescent dye etc., for example, adriamycin, taxol, SB203580, LY294002,
Axitinib, JQ1, cumarin, Nile red etc..
Experiment in vitro proves that chitosan nitroimidazole grafting of the present invention is containing nitroreductase as carrier
37 DEG C of 10-100 μm of ol, in pH7.4 phosphate buffered saline solution, or in 37 DEG C, 5%CO2Under+95% air conditions, containing
In the tumour cell of the DMEM culture solution culture of 10~500 μm of ol cobalt chlorides, or in 37 DEG C, 5%CO2+ 95%N2Under the conditions of, training
Nutrient solution is rapid delivery of pharmaceuticals in the tumour cell of the DMEM culture solution culture containing 10% fetal calf serum.
The present invention uses the chitosan with good biocompatibility for hydrophilic material, using N-Boc- bromo alkanamine as bridge
Symbasis group selects nitroimidazole for anoxic sensitive group, and synthesizing, there is the chitosan nitroimidazole of anoxic sensitivity responsiveness to graft
Object.Self aggregation forms polymer micelle to the grafting material in water, has lower critical micelle concentration, the chitosan nitro
Imidazoles grafting can be used as pharmaceutical carrier, and pharmaceutical composition, the drug-carrying nanometer particle of building, can in hypoxic cell highly expressed nitre
Base restores under enzyme effect, rapid delivery of pharmaceuticals, realizes Targeting delivery of the drug in anoxic lesion environment, improves drug effect.
The invention has the characteristics that (1) present invention constructs the chitosan grafting nitroimidazole polymerization that can be used for entrapped drug for the first time
Object pharmaceutical carrier.(2) the chitosan nitroimidazole polymer support that the present invention constructs is remarkably improved the grafting of chitosan fatty acid
Object drug passs drug release efficiency of the release system in hypoxic cell.(3) the chitosan nitroimidazole polymer that the present invention constructs
Carrier, can be with a variety of drugs such as adriamycin, taxol, SB203580, LY294002, Axitinib, JQ1, cumarin, Nile red
Deng, building drug pass release system.The drug of building is passed release system property and is stablized, and may be implemented in containing nitroreductase 10-100
37 DEG C of μm ol, be released effectively drug in pH7.4 phosphate buffered saline solution.
Detailed description of the invention
Fig. 1 is 2- nitroimidazole, N-Boc- bromo hexylamine, intermediate product, chitosan and chitosan nitro miaow in embodiment 5
The nuclear magnetic resonance spectroscopy of azoles grafting.
Fig. 2 is the chitosan nitroimidazole drug-carrying nanometer particle In-vitro release curves for encapsulating adriamycin.
Fig. 3 is the chitosan nitroimidazole drug-carrying nanometer particle In-vitro release curves of envelope paclitaxel.
Fig. 4 is the chitosan nitroimidazole drug-carrying nanometer particle In-vitro release curves for encapsulating SB203580.
Fig. 5 is the chitosan nitroimidazole drug-carrying nanometer particle In-vitro release curves for encapsulating LY294002.
Fig. 6 is the chitosan nitroimidazole drug-carrying nanometer particle In-vitro release curves for encapsulating Axitinib.
Fig. 7 is the In-vitro release curves for encapsulating the chitosan nitroimidazole drug-carrying nanometer particle of JQ1.
Fig. 8 is drug release percentage of the drug-carrying nanometer particle in cobalt chloride incubated cell for encapsulating adriamycin.
Fig. 9 is drug release percentage of the drug-carrying nanometer particle in cobalt chloride cell for encapsulating cumarin.
Figure 10 is drug release percentage of the drug-carrying nanometer particle of encapsulating Nile red in hypoxic cell.
Specific embodiment
The mode of present invention combination attached drawing and following embodiment is described in detail the present invention, but the invention is not limited to this
A little embodiments.
One, the synthesis of chitosan nitroimidazole grafting
Embodiment 1
Precision weighs 64.8mg 2- nitroimidazole, and 39.6mg potassium carbonate and 128.5mg N-Boc-6- bromo hexylamine are placed in 50mL
In dry round-bottomed flask, 10mL dehydration n,N-Dimethylformamide (N, N-dimethylformamide, DMF) is added, in constant temperature
Heat 80 DEG C of reaction 4h in magnetic stirring apparatus.After reaction, centrifugation extracts reaction solution supernatant, investment 100mL deionized water and
20mL ethyl acetate, gas bath shaker at room temperature vibrate 5min, stand 30min and take upper layer ethyl acetate to liquid layered, set
In 40 DEG C of water-bath revolvings on 100mL round-bottomed flask, Rotary Evaporators, until solution completely removes in flask.30mL is added in flask
Bis- succinimide ester of DMF, 73.4mg (N, N'-Disuccinimidyl Carbonate, DSC), 186.8mg chitosan (Mw
=2000Da) and 30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product
The 48h that dialyses is placed in bag filter (MWCO 1000Da), to remove DMF and aqueous by-product.Reaction product freezing is dry after dialysis
It is dry, obtain chitosan nitroimidazole grafting.
Embodiment 2
Precision weighs 64.8mg 2- nitroimidazole, and 79.2mg potassium carbonate and 160.6mg N-Boc-6- bromo hexylamine are placed in 50mL
In dry round-bottomed flask, 20mL is added and is dehydrated DMF, 80 DEG C of reaction 4h in constant-temperature heating magnetic stirring apparatus.After reaction, from
The heart extracts reaction solution supernatant, puts into 100mL deionized water and 20mL ethyl acetate, gas bath shaker at room temperature vibrates 5min, quiet
30min to be set, to liquid layered, takes upper layer ethyl acetate, is placed in 100mL round-bottomed flask, 40 DEG C of water-baths rotate on Rotary Evaporators,
Into flask, solution is completely removed.30mL DMF, 146.8mg DSC, 373.6mg chitosan (Mw=5000Da) are added in flask
With 30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product is placed in dialysis
Dialyse 48h in bag (MWCO 3500Da), to remove DMF and aqueous by-product.Reaction product is freeze-dried after dialysis, obtains shell
Glycan nitroimidazole grafting.
Embodiment 3
Precision weighs 64.8mg 2- nitroimidazole, and 158.2mg potassium carbonate and 240.9mg N-Boc-6- bromo hexylamine are placed in 50mL
In dry round-bottomed flask, 20mL is added and is dehydrated DMF, 80 DEG C of reaction 4h in constant-temperature heating magnetic stirring apparatus.After reaction, from
The heart extracts reaction solution supernatant, puts into 100mL deionized water and 20mL ethyl acetate, gas bath shaker at room temperature vibrates 5min, quiet
30min to be set, to liquid layered, takes upper layer ethyl acetate, is placed in 100mL round-bottomed flask, 40 DEG C of water-baths rotate on Rotary Evaporators,
Into flask, solution is completely removed.30mL DMF, 220.2mg DSC, 934.1mg chitosan (Mw=are added in flask
20000Da) with 30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product
The 48h that dialyses is placed in bag filter (MWCO 3500Da), to remove DMF and aqueous by-product.Reaction product freezing is dry after dialysis
It is dry, obtain chitosan nitroimidazole grafting.
Embodiment 4
Precision weighs 64.8mg 2- nitroimidazole, and 79.2mg potassium carbonate and 240.9mg N-Boc-6- bromo hexylamine are placed in 50mL
In dry round-bottomed flask, 10mL is added and is dehydrated DMF, 80 DEG C of reaction 4h in constant-temperature heating magnetic stirring apparatus.After reaction, from
The heart extracts reaction solution supernatant, puts into 100mL deionized water and 20mL ethyl acetate, gas bath shaker at room temperature vibrates 5min, quiet
30min to be set, to liquid layered, takes upper layer ethyl acetate, is placed in 100mL round-bottomed flask, 40 DEG C of water-baths rotate on Rotary Evaporators,
Into flask, solution is completely removed.30mL DMF, 293.6mg DSC, 311.3mg chitosan (Mw=2000Da) are added in flask
With 30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product is placed in dialysis
Dialyse 48h in bag (MWCO 1000Da), to remove DMF and aqueous by-product.Reaction product is freeze-dried after dialysis, obtains shell
Glycan nitroimidazole grafting.
Embodiment 5
Precision weighs 64.8mg 2- nitroimidazole, and 237.6mg potassium carbonate and 401.5mg N-Boc-6- bromo hexylamine are placed in 50mL
In dry round-bottomed flask, 15mL is added and is dehydrated DMF, 80 DEG C of reaction 4h in constant-temperature heating magnetic stirring apparatus.After reaction, from
The heart extracts reaction solution supernatant, puts into 100mL deionized water and 20mL ethyl acetate, gas bath shaker at room temperature vibrates 5min, quiet
30min to be set, to liquid layered, takes upper layer ethyl acetate, is placed in 100mL round-bottomed flask, 40 DEG C of water-baths rotate on Rotary Evaporators,
Into flask, solution is completely removed.30mL DMF, 513.8mg DSC, 747.3mg chitosan (Mw=5000Da) are added in flask
With 30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product is placed in dialysis
Dialyse 48h in bag (MWCO 3500Da), to remove DMF and aqueous by-product.Reaction product is freeze-dried after dialysis, obtains shell
Glycan nitroimidazole grafting.
Embodiment 6
Precision weighs 64.8mg 2- nitroimidazole, and 158.2mg potassium carbonate and 240.9mg N-Boc-6- bromo hexylamine are placed in 50mL
In dry round-bottomed flask, 20mL is added and is dehydrated DMF, 80 DEG C of reaction 4h in constant-temperature heating magnetic stirring apparatus.After reaction, from
The heart extracts reaction solution supernatant, puts into 100mL deionized water and 20mL ethyl acetate, gas bath shaker at room temperature vibrates 5min, quiet
30min to be set, to liquid layered, takes upper layer ethyl acetate, is placed in 100mL round-bottomed flask, 40 DEG C of water-baths rotate on Rotary Evaporators,
Into flask, solution is completely removed.30mL DMF, 220.2mg DSC, 311.3mg chitosan (Mw=are added in flask
20000Da) with 30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product
The 48h that dialyses is placed in bag filter (MWCO 3500Da), to remove DMF and aqueous by-product.Reaction product freezing is dry after dialysis
It is dry, obtain chitosan nitroimidazole grafting.
Embodiment 7
Precision weighs 64.8mg 2- nitroimidazole, and 158.2mg potassium carbonate and 160.6mg N-Boc-6- bromo hexylamine are placed in 50mL
In dry round-bottomed flask, 10mL is added and is dehydrated DMF, 80 DEG C of reaction 4h in constant-temperature heating magnetic stirring apparatus.After reaction, from
The heart extracts reaction solution supernatant, puts into 100mL deionized water and 20mL ethyl acetate, gas bath shaker at room temperature vibrates 5min, quiet
30min to be set, to liquid layered, takes upper layer ethyl acetate, is placed in 100mL round-bottomed flask, 40 DEG C of water-baths rotate on Rotary Evaporators,
Into flask, solution is completely removed.In flask be added 30mL DMF, 734mg DSC, 207.6mg chitosan (Mw=2000Da) and
30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product is placed in bag filter
Dialyse 48h in (MWCO 1000Da), to remove DMF and aqueous by-product.Reaction product is freeze-dried after dialysis, and it is poly- to obtain shell
Sugared nitroimidazole grafting.
Embodiment 8
Precision weighs 64.8mg 2- nitroimidazole, and 396.0mg potassium carbonate and 401.5mg N-Boc-6- bromo hexylamine are placed in 50mL
In dry round-bottomed flask, 20mL is added and is dehydrated DMF, 80 DEG C of reaction 4h in constant-temperature heating magnetic stirring apparatus.After reaction, from
The heart extracts reaction solution supernatant, puts into 100mL deionized water and 20mL ethyl acetate, gas bath shaker at room temperature vibrates 5min, quiet
30min to be set, to liquid layered, takes upper layer ethyl acetate, is placed in 100mL round-bottomed flask, 40 DEG C of water-baths rotate on Rotary Evaporators,
Into flask, solution is completely removed.In flask be added 30mL DMF, 734mg DSC, 207.6mg chitosan (Mw=5000Da) and
30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product is placed in bag filter
Dialyse 48h in (MWCO 3500Da), to remove DMF and aqueous by-product.Reaction product is freeze-dried after dialysis, and it is poly- to obtain shell
Sugared nitroimidazole grafting.
Embodiment 9
Precision weighs 64.8mg 2- nitroimidazole, and 396.0mg potassium carbonate and 481.7mg N-Boc-6- bromo hexylamine are placed in 50mL
In dry round-bottomed flask, 20mL is added and is dehydrated DMF, 80 DEG C of reaction 4h in constant-temperature heating magnetic stirring apparatus.After reaction, from
The heart extracts reaction solution supernatant, puts into 100mL deionized water and 20mL ethyl acetate, gas bath shaker at room temperature vibrates 5min, quiet
30min to be set, to liquid layered, takes upper layer ethyl acetate, is placed in 100mL round-bottomed flask, 40 DEG C of water-baths rotate on Rotary Evaporators,
Into flask, solution is completely removed.30mL DMF, 220.2mg DSC, 207.6mg chitosan (Mw=are added in flask
20000Da) with 30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product
The 48h that dialyses is placed in bag filter (MWCO 3500Da), to remove DMF and aqueous by-product.Reaction product freezing is dry after dialysis
It is dry, obtain chitosan nitroimidazole grafting.
Embodiment 10
Precision weighs 64.8mg 2- nitroimidazole, and 316.8mg potassium carbonate and 240.9mg N-Boc-6- bromo hexylamine are placed in 50mL
In dry round-bottomed flask, 15mL is added and is dehydrated DMF, 80 DEG C of reaction 4h in constant-temperature heating magnetic stirring apparatus.After reaction, from
The heart extracts reaction solution supernatant, puts into 100mL deionized water and 20mL ethyl acetate, gas bath shaker at room temperature vibrates 5min, quiet
30min to be set, to liquid layered, takes upper layer ethyl acetate, is placed in 100mL round-bottomed flask, 40 DEG C of water-baths rotate on Rotary Evaporators,
Into flask, solution is completely removed.30mL DMF, 293.6mg DSC, 186.8mg chitosan (Mw=2000Da) are added in flask
With 30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product is placed in dialysis
Dialyse 48h in bag (MWCO 1000Da), to remove DMF and aqueous by-product.Reaction product is freeze-dried after dialysis, obtains shell
Glycan nitroimidazole grafting.
Embodiment 11
Precision weighs 64.8mg 2- nitroimidazole, and 396.0mg potassium carbonate and 240.9mg N-Boc-6- bromo hexylamine are placed in 50mL
In dry round-bottomed flask, 15mL is added and is dehydrated DMF, 80 DEG C of reaction 4h in constant-temperature heating magnetic stirring apparatus.After reaction, from
The heart extracts reaction solution supernatant, puts into 100mL deionized water and 20mL ethyl acetate, gas bath shaker at room temperature vibrates 5min, quiet
30min to be set, to liquid layered, takes upper layer ethyl acetate, is placed in 100mL round-bottomed flask, 40 DEG C of water-baths rotate on Rotary Evaporators,
Into flask, solution is completely removed.30mL DMF, 220.2mg DSC, 186.8mg chitosan (Mw=5000Da) are added in flask
With 30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product is placed in dialysis
Dialyse 48h in bag (MWCO 3500Da), to remove DMF and aqueous by-product.Reaction product is freeze-dried after dialysis, obtains shell
Glycan nitroimidazole grafting.
Embodiment 12
Precision weighs 64.8mg 2- nitroimidazole, and 158.2mg potassium carbonate and 481.7mg N-Boc-6- bromo hexylamine are placed in 50mL
In dry round-bottomed flask, 20mL is added and is dehydrated DMF, 80 DEG C of reaction 4h in constant-temperature heating magnetic stirring apparatus.After reaction, from
The heart extracts reaction solution supernatant, puts into 100mL deionized water and 20mL ethyl acetate, gas bath shaker at room temperature vibrates 5min, quiet
30min to be set, to liquid layered, takes upper layer ethyl acetate, is placed in 100mL round-bottomed flask, 40 DEG C of water-baths rotate on Rotary Evaporators,
Into flask, solution is completely removed.30mL DMF, 220.2mg DSC, 186.8mg chitosan (Mw=are added in flask
20000Da) with 30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product
The 48h that dialyses is placed in bag filter (MWCO 3500Da), to remove DMF and aqueous by-product.Reaction product freezing is dry after dialysis
It is dry, obtain chitosan nitroimidazole grafting.
Embodiment 13
Precision weighs 64.8mg 2- nitroimidazole, and 316.8mg potassium carbonate and 289.1mg N-Boc-10- bromo decyl amine are placed in
In the dry round-bottomed flask of 50mL, 20mL is added and is dehydrated DMF, 80 DEG C of reaction 4h in constant-temperature heating magnetic stirring apparatus.Reaction terminates
Afterwards, centrifugation extracts reaction solution supernatant, puts into 100mL deionized water and 20mL ethyl acetate, the oscillation of gas bath shaker at room temperature
5min stands 30min to liquid layered and takes upper layer ethyl acetate, is placed in 100mL round-bottomed flask, 40 DEG C of water on Rotary Evaporators
Bath revolving, until solution completely removes in flask.30mL DMF, 220.2mg DSC, 311.3mg chitosan (Mw=are added in flask
5000Da) with 30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product is set
Dialyse 48h in bag filter (MWCO 3500Da), to remove DMF and aqueous by-product.Reaction product freezing is dry after dialysis
It is dry, obtain chitosan nitroimidazole grafting.
Embodiment 14
Precision weighs 64.8mg 2- nitroimidazole, and 237.6mg potassium carbonate and 313.3mg N-Boc-12- bromododecane amine are set
In the dry round-bottomed flask of 50mL, 20mL is added and is dehydrated DMF, 80 DEG C of reaction 4h in constant-temperature heating magnetic stirring apparatus.Reaction knot
Shu Hou, centrifugation extract reaction solution supernatant, put into 100mL deionized water and 20mL ethyl acetate, the oscillation of gas bath shaker at room temperature
5min stands 30min to liquid layered and takes upper layer ethyl acetate, is placed in 100mL round-bottomed flask, 40 DEG C of water on Rotary Evaporators
Bath revolving, until solution completely removes in flask.30mL DMF, 220.2mg DSC, 311.3mg chitosan (Mw=are added in flask
5000Da) with 30mL deionized water, 40 DEG C of reactions for 24 hours, obtain reaction product in constant-temperature heating magnetic stirring apparatus.Reaction product is set
Dialyse 48h in bag filter (MWCO 3500Da), to remove DMF and aqueous by-product.Reaction product freezing is dry after dialysis
It is dry, obtain chitosan nitroimidazole grafting.
Two, the characteristic of chitosan nitroimidazole grafting
The nuclear magnetic resonance spectroscopy of 5 gained chitosan nitroimidazole grafting of embodiment and its synthesis material and intermediate product is such as
Fig. 1 shows.There is figure as it can be seen that the bromine atom of N-Boc- bromo amine is replaced to form intermediate product by 2- nitroimidazole, chitosan is in
Between product amino be connected, realize the chemical grafting of chitosan and nitroimidazole.
The chitosan nitroimidazole grafting that above-described embodiment 1-14 is obtained is weighed respectively to match using deionized water as solvent
Concentration processed is the grafting solution of 10mg/mL.Using particle size and surface potential instrument, grafting micella is flat in measurement solution
Equal partial size.By well known pyrene fluorescence method, it is dense that the critical micell of chitosan nitroimidazole grafting micella in water is measured respectively
Degree.NMR spectrum is composed by known hydrogen, measures nitroimidazole grafting rate.The above-mentioned characteristic of embodiment 1-14 is listed in the table below
1。
Seen from table 1, chitosan nitroimidazole grafting of the invention has self aggregation formation polymerization in an aqueous medium
The characteristic of object micella.Chitosan nitroimidazole of the invention has lower critical micelle concentration, and substantially less than general surface is living
The critical micelle concentration of property agent.By adjusting the molecular weight and 2- nitroimidazole grafting rate of chitosan, chitosan nitro can be changed
The average grain diameter and surface potential of imidazoles grafting micella adapt to various disease treatment to be applied to the encapsulating of a variety of drugs
Needs.
1 chitosan nitroimidazole grafting physicochemical property of table
Application in three, chitosan nitroimidazole grafting micellar pharmaceutical compositions
1. adriamycin
The accurate chitosan nitroimidazole grafting prepared in 100mg embodiment 2,3,5,6,8,9 that weighs (sets 100mL burning respectively
In cup, it is separately added into 45mL deionized water, water bath sonicator 1min), it is transferred in volumetric flask, is settled to respectively with deionized water
50mL obtains the grafting solution of 2mg/mL.Chitosan nitroimidazole grafting solution 10mL is pipetted respectively, and it is dense that 1.5mL is added
Degree is the adriamycin dimethyl sulfoxide solution of 2mg/mL, and after stirring 4h under the conditions of 35 DEG C, being placed in molecular cut off is the saturating of 1000Da
Analyse bag in, deionized water dialysis for 24 hours after, in bag filter liquid 8000rpm be centrifuged 10min, supernatant is through 0.22 μm of miillpore filter
Filtering, must encapsulate the chitosan nitroimidazole grafting drug-carrying nanometer particle solution of adriamycin.
Using meagre profit granularity and surface potential analyzer, the partial size and surface potential of drug-carrying nanometer particle are measured；Efficient liquid phase
Chromatography determination doxorubicin content simultaneously calculates entrapment efficiency, the results are shown in Table 2.
The chitosan nitroimidazole drug-carrying nanometer particle physicochemical property of the encapsulating adriamycin of table 2
| Carrier | Average grain diameter (nm) | Drugloading rate (w/w) | Encapsulation rate (%) |
| Embodiment 2 | 186.6±3.7 | 13.53% | 90.2% |
| Embodiment 3 | 222.2±2.9 | 13.82% | 92.1% |
| Embodiment 5 | 159.7±2.1 | 14.25% | 95.0% |
| Embodiment 6 | 171.9±1.5 | 14.52% | 96.8% |
| Embodiment 8 | 137.9±7.9 | 13.91% | 92.7% |
| Embodiment 9 | 165.4±5.8 | 14.01% | 93.4% |
The release in vitro of six kinds of drug-carrying nanometer particles is referring to fig. 2, it is seen that passes through chitosan in chitosan nitroimidazole grafting
The control of molecular weight and nitroimidazole grafting rate can effectively realize encapsulating and the sensitive release of anoxic to drug.
2. taxol
Precision weighs the chitosan nitroimidazole grafting prepared in 100mg embodiment 1,4,7,10 and (sets 100mL beaker respectively
In, it is separately added into 45mL deionized water, water bath sonicator 1min), it is transferred in volumetric flask, is settled to respectively with deionized water
50mL obtains the grafting solution of 2mg/mL.Chitosan nitroimidazole grafting solution 10mL is pipetted respectively, and 2mL concentration is added
For the taxol methanol solution of 2mg/mL, after stirring 4h under the conditions of 40 DEG C, it is placed in the bag filter that molecular cut off is 1000Da,
Deionized water dialysis for 24 hours after, liquid 8000rpm is centrifuged 10min in bag filter, and supernatant obtains through 0.22 μm of filtering with microporous membrane
The chitosan nitroimidazole grafting drug-carrying nanometer particle solution of envelope paclitaxel.
Using meagre profit granularity and surface potential analyzer, the partial size and surface potential of drug-carrying nanometer particle are measured；Efficient liquid phase
Chromatography determination content of taxol simultaneously calculates entrapment efficiency, the results are shown in Table 3.The release in vitro of four kinds of drug-carrying nanometer particles is referring to figure
3。
The chitosan nitroimidazole drug-carrying nanometer particle physicochemical property of 3 envelope paclitaxel of table
| Carrier | Average grain diameter (nm) | Drugloading rate (w/w) | Encapsulation rate (%) |
| Embodiment 1 | 602.3±5.5 | 10.70% | 53.5% |
| Embodiment 4 | 295.4±5.3 | 9.06% | 45.3% |
| Embodiment 7 | 62.6±3.1 | 5.68% | 28.4% |
| Embodiment 10 | 105.0±1.7 | 10.06% | 50.3% |
3.SB203580
Precision weighs the chitosan nitroimidazole grafting prepared in 100mg embodiment 5,6,11,12,13,14 and (sets respectively
In 100mL beaker, it is separately added into 45mL deionized water, water bath sonicator 1min), it is transferred in volumetric flask, is distinguished with deionized water
It is settled to 50mL, obtains the grafting solution of 2mg/mL.Chitosan nitroimidazole grafting solution 10mL is pipetted respectively, is slowly dripped
The drug SB203580 chloroformic solution that 0.6mL concentration is 5mg/mL is added, after stirring 12h under room temperature,
4000rpm is centrifuged 10min, and supernatant must encapsulate the chitosan nitro miaow of SB203580 through 0.22 μm of filtering with microporous membrane
Azoles grafting drug-carrying nanometer particle solution.
Using meagre profit granularity and surface potential analyzer, the partial size and surface potential of drug-carrying nanometer particle are measured；Efficient liquid phase
Chromatography determination SB203580 content simultaneously calculates entrapment efficiency, the results are shown in Table 4.The release in vitro of six kinds of drug-carrying nanometer particles referring to
Fig. 4.
The chitosan nitroimidazole drug-carrying nanometer particle physicochemical property of the encapsulating of table 4 SB203580
| Carrier | Average grain diameter (nm) | Drugloading rate (w/w) | Encapsulation rate (%) |
| Embodiment 5 | 169.5±3.2 | 14.75% | 98.3% |
| Embodiment 6 | 185.9±2.5 | 14.63% | 97.5% |
| Embodiment 11 | 155.8±2.9 | 14.76% | 98.4% |
| Embodiment 12 | 157.9±5.1 | 14.69% | 97.9% |
| Embodiment 13 | 169.2±2.1 | 14.81% | 98.7% |
| Embodiment 14 | 175.7±2.3 | 14.48% | 96.5% |
4.LY294002
Precision weighs the chitosan nitroimidazole grafting prepared in 100mg embodiment 5,12,13,14 and (sets 100mL beaker respectively
In, it is separately added into 45mL deionized water, water bath sonicator 1min), it is transferred in volumetric flask, is settled to respectively with deionized water
50mL obtains the grafting solution of 2mg/mL.Pipette chitosan nitroimidazole grafting solution 10mL respectively, be slowly added dropwise into
0.5mL concentration is the drug LY294002 chloroformic solution of 10mg/mL, and after stirring 9h under room temperature, 4000rpm is centrifuged 10min,
For supernatant through 0.22 μm of filtering with microporous membrane, the chitosan nitroimidazole grafting drug-carrying nanometer particle that must encapsulate LY294002 is molten
Liquid.
Using meagre profit granularity and surface potential analyzer, the partial size and surface potential of drug-carrying nanometer particle are measured；Efficient liquid phase
Chromatography determination LY294002 content simultaneously calculates entrapment efficiency, the results are shown in Table 5.The release in vitro of four kinds of drug-carrying nanometer particles referring to
Fig. 5.
The chitosan nitroimidazole drug-carrying nanometer particle physicochemical property of the encapsulating of table 5 LY294002
| Carrier | Average grain diameter (nm) | Drugloading rate (w/w) | Encapsulation rate (%) |
| Embodiment 5 | 157.3±2.2 | 12.30% | 49.2% |
| Embodiment 12 | 163.8±3.1 | 13.71% | 54.8% |
| Embodiment 13 | 151.3±1.9 | 16.84% | 67.4% |
| Embodiment 14 | 167.5±1.9 | 17.61% | 70.4% |
5. Axitinib
Precision weighs the chitosan nitroimidazole grafting prepared in 100mg embodiment 5,8,13,14 and (sets 100mL beaker respectively
In, it is separately added into 45mL deionized water, water bath sonicator 1min), it is transferred in volumetric flask, is settled to respectively with deionized water
50mL obtains the grafting solution of 2mg/mL.Pipette chitosan nitroimidazole grafting solution 10mL respectively, be slowly added dropwise into
1.0mL concentration is the drug Axitinib chloroformic solution of 5mg/mL, and after stirring 12h under room temperature, 4000rpm is centrifuged 10min,
For supernatant through 0.22 μm of filtering with microporous membrane, the chitosan nitroimidazole grafting drug-carrying nanometer particle that must encapsulate Axitinib is molten
Liquid.
Using meagre profit granularity and surface potential analyzer, the partial size and surface potential of drug-carrying nanometer particle are measured；Efficient liquid phase
Chromatography determination Axitinib content simultaneously calculates entrapment efficiency, the results are shown in Table 6.The release in vitro of five kinds of drug-carrying nanometer particles referring to
Fig. 6.
The chitosan nitroimidazole drug-carrying nanometer particle physicochemical property of the encapsulating Axitinib of table 6
| Carrier | Average grain diameter (nm) | Drugloading rate (w/w) | Encapsulation rate (%) |
| Embodiment 5 | 165.3±2.1 | 7.12% | 28.48% |
| Embodiment 8 | 151.7±3.9 | 6.31% | 25.24% |
| Embodiment 13 | 165.1±2.8 | 7.06% | 28.24% |
| Embodiment 14 | 173.5±1.9 | 8.01% | 32.04% |
6.JQ1
Precision weighs the chitosan nitroimidazole grafting prepared in 100mg embodiment 5,6,11,12,13,14 and (sets respectively
In 100mL beaker, it is separately added into 45mL deionized water, water bath sonicator 1min), it is transferred in volumetric flask, is distinguished with deionized water
It is settled to 50mL, obtains the grafting solution of 2mg/mL.Chitosan nitroimidazole grafting solution 10mL is pipetted respectively, is slowly dripped
The drug JQ1 chloroformic solution that 0.5mL concentration is 8mg/mL is added, after stirring 12h under room temperature, 4000rpm is centrifuged 10min,
Supernatant must encapsulate the chitosan nitroimidazole grafting drug-carrying nanometer particle solution of JQ1 through 0.22 μm of filtering with microporous membrane.
Using meagre profit granularity and surface potential analyzer, the partial size and surface potential of drug-carrying nanometer particle are measured；Efficient liquid phase
Chromatography determination JQ1 content simultaneously calculates entrapment efficiency, the results are shown in Table 7.The release in vitro of six kinds of drug-carrying nanometer particles is referring to Fig. 7.
The chitosan nitroimidazole drug-carrying nanometer particle physicochemical property of the encapsulating of table 7 JQ1
| Carrier | Average grain diameter (nm) | Drugloading rate (w/w) | Encapsulation rate (%) |
| Embodiment 5 | 178.7±4.2 | 13.40% | 67.0% |
| Embodiment 6 | 191.9±3.5 | 14.16% | 70.8% |
| Embodiment 11 | 163.8±3.7 | 15.26% | 76.3% |
| Embodiment 12 | 175.9±4.3 | 15.35% | 76.8% |
| Embodiment 13 | 180.1±4.1 | 16.71% | 83.6% |
| Embodiment 14 | 191.7±5.3 | 17.18% | 85.9% |
7. cumarin
Precision weighs the chitosan nitroimidazole grafting prepared in 100mg embodiment 5,6,8,9,13,14 and (sets 100mL respectively
In beaker, it is separately added into 45mL deionized water, water bath sonicator 1min), it is transferred in volumetric flask, is settled to respectively with deionized water
50mL obtains the grafting solution of 2mg/mL.Chitosan nitroimidazole grafting solution 10mL is pipetted respectively, and 2mL concentration is added
For the cumarin ethanol solution of 2mg/mL, after stirring 2h under the conditions of 35 DEG C, it is placed in the bag filter that molecular cut off is 3500Da,
Deionized water dialysis for 24 hours after, liquid 8000rpm is centrifuged 10min in bag filter, and supernatant obtains through 0.22 μm of filtering with microporous membrane
Encapsulate the chitosan nitroimidazole grafting drug-carrying nanometer particle solution of cumarin.
Using meagre profit granularity and surface potential analyzer, the partial size and surface potential of drug-carrying nanometer particle are measured；Fluorescence spectrophotometer
Photometry measurement tonka-bean cellulose content simultaneously calculates entrapment efficiency, the results are shown in Table 8.
The chitosan nitroimidazole drug-carrying nanometer particle physicochemical property of the encapsulating cumarin of table 8
| Carrier | Average grain diameter (nm) | Drugloading rate (w/w) | Encapsulation rate (%) |
| Embodiment 5 | 150.4±1.2 | 16.81% | 84.1% |
| Embodiment 6 | 161.7±0.9 | 17.23% | 86.2% |
| Embodiment 8 | 134.5±1.2 | 14.31% | 71.6% |
| Embodiment 9 | 158.3±1.3 | 13.25% | 66.3% |
| Embodiment 13 | 143.2±0.7 | 18.91% | 94.6% |
| Embodiment 14 | 135.3±1.4 | 17.55% | 87.9% |
8. Nile red
Precision weighs the chitosan nitroimidazole grafting prepared in 100mg embodiment 1,2,4,7,11,12 and (sets 100mL respectively
In beaker, it is separately added into 45mL deionized water, water bath sonicator 1min), it is transferred in volumetric flask, is settled to respectively with deionized water
50mL obtains the grafting solution of 2mg/mL.Chitosan nitroimidazole grafting solution 10mL is pipetted respectively, and it is dense that 0.8mL is added
Degree is the Nile red ethanol solution of 2mg/mL, after stirring 2h under the conditions of 35 DEG C, is placed in the bag filter that molecular cut off is 1000Da
In, deionized water dialysis for 24 hours after, liquid 8000rpm is centrifuged 10min in bag filter, supernatant through 0.22 μm of filtering with microporous membrane,
The chitosan nitroimidazole grafting drug-carrying nanometer particle solution of Nile red must be encapsulated.
Using meagre profit granularity and surface potential analyzer, the partial size and surface potential of drug-carrying nanometer particle are measured；Fluorescence spectrophotometer
Photometry measurement Nile red content simultaneously calculates entrapment efficiency, the results are shown in Table 9.
The chitosan nitroimidazole drug-carrying nanometer particle physicochemical property of the encapsulating Nile red of table 9
| Carrier | Average grain diameter (nm) | Drugloading rate (w/w) | Encapsulation rate (%) |
| Embodiment 1 | 552.3±5.5 | 1.02% | 12.8% |
| Embodiment 2 | 187.6±3.1 | 2.35% | 29.4% |
| Embodiment 4 | 254.1±3.3 | 1.21% | 15.1% |
| Embodiment 7 | 76.6±2.7 | 1.31% | 16.4% |
| Embodiment 11 | 171.3±1.9 | 3.16% | 39.5% |
| Embodiment 12 | 121.1±2.7 | 2.97% | 37.1% |
Four, chitosan nitroimidazole grafting micella answering in the release of antineoplastic pharmaceutical compositions anaerobic environment specificity
With
1. encapsulating drug release of the nitroimidazole grafting drug-carrying nanometer particle of adriamycin in hypoxic cell
Precision weighs the chitosan nitroimidazole grafting prepared in 100mg embodiment 5 and (sets in 100mL beaker, 45mL is added and goes
Ionized water, water bath sonicator 1min), it is transferred in volumetric flask, 50mL is settled to deionized water, obtain 2mg/mL grafting solution.
Chitosan nitroimidazole grafting solution 10mL is pipetted, the adriamycin dimethyl sulfoxide solution that 1.5mL concentration is 2mg/mL is added,
Stir 4h under the conditions of 35 DEG C, be placed in molecular cut off be 3500Da bag filter in, deionized water dialysis for 24 hours after, in bag filter
Liquid 8000rpm is centrifuged 10min, and supernatant must encapsulate the chitosan nitroimidazole of adriamycin through 0.22 μm of filtering with microporous membrane
Grafting drug-carrying nanometer particle solution.
Take 4T1 cell inoculation in 24 porocyte culture plates, 37 DEG C, 5%CO2Under+95% air conditions, respectively containing 0 μM,
10 μM, 50 μM or 100 μM cobalt chlorides are trained containing 10% fetal calf serum (v/v), containing the DMEM of penicillin and each 100U/mL of streptomysin
It supports and is cultivated in base.It is separately added into the chitosan nitroimidazole grafting drug-carrying nanometer particle of the encapsulating adriamycin of above method preparation,
Make the final concentration of 20 μ g/ml of grafting carrier.The chitosan nitroimidazole grafting that adriamycin is encapsulated in cell is detected after being incubated for 12h
Object drug-carrying nanometer particle drug release percentage.As a result see Fig. 8.
2. encapsulating drug release of the nitroimidazole grafting drug-carrying nanometer particle of cumarin in hypoxic cell
Precision weighs the chitosan nitroimidazole grafting prepared in 100mg embodiment 5 and (sets in 100mL beaker, 45mL is added and goes
Ionized water, water bath sonicator 1min), it is transferred in volumetric flask, 50mL is settled to deionized water, the grafting for obtaining 2mg/mL is molten
Liquid.It pipettes chitosan nitroimidazole grafting solution 10mL, is added the cumarin ethanol solution that 2mL concentration is 2mg/mL, 35
Under the conditions of DEG C stir 4h after, be placed in molecular cut off be 3500Da bag filter in, deionized water dialysis for 24 hours after, in bag filter
Liquid 8000rpm is centrifuged 10min, and supernatant must encapsulate the chitosan nitroimidazole of cumarin through 0.22 μm of filtering with microporous membrane
Grafting drug-carrying nanometer particle solution.
Take 4T1 cell inoculation in 24 porocyte culture plates, 37 DEG C, 5%CO2Under+95% air conditions, containing respectively
It is cultivated in 100 μM of cobalt chlorides and 10% fetal calf serum (v/v), the DMEM culture medium containing penicillin and each 100U/mL of streptomysin.Point
Not Jia Ru above method preparation encapsulating cumarin chitosan nitroimidazole grafting drug-carrying nanometer particle, keep grafting carrier whole
Concentration is 20 μ g/ml.Respectively detection be incubated for 0h, 4h, 8h, 12h, for 24 hours after, in cell encapsulate cumarin chitosan nitroimidazole
Grafting drug-carrying nanometer particle drug release percentage.As a result see Fig. 9.
3. encapsulating drug release of the nitroimidazole grafting drug-carrying nanometer particle of Nile red in hypoxic cell
Precision weighs the chitosan nitroimidazole grafting prepared in 100mg embodiment 11 and (sets in 100mL beaker, 45mL is added
Deionized water, water bath sonicator 1min), it is transferred in volumetric flask, with deionized water respectively to 50mL, the grafting for obtaining 2mg/mL is molten
Liquid.Chitosan nitroimidazole grafting solution 10mL is pipetted, the Nile red ethanol solution that 0.8mL concentration is 2mg/mL is added,
Under the conditions of 35 DEG C stir 2h after, be placed in molecular cut off be 1000Da bag filter in, deionized water dialysis for 24 hours after, bag filter
Middle liquid 8000rpm is centrifuged 10min, and supernatant must encapsulate the chitosan nitro miaow of Nile red through 0.22 μm of filtering with microporous membrane
Azoles grafting drug-carrying nanometer particle solution.
Take 4T1 cell inoculation in 24 porocyte culture plates, in 37 DEG C, 5%CO2+ 95%N2Under the conditions of, containing 10% tire ox
It is cultivated in serum (v/v), the DMEM culture medium containing penicillin and each 100U/mL of streptomysin.Nostoc commune Vanch liquid processing group is control.
It is separately added into the chitosan nitroimidazole grafting drug-carrying nanometer particle of the encapsulating cumarin of above method preparation, makes grafting carrier
Final concentration of 20 μ g/ml.It is incubated for the chitosan nitroimidazole grafting drug-carrying nanometer particle for detecting afterwards encapsulate adriamycin in cell for 24 hours
Drug release percentage.The result is shown in Figure 10.
Claims (8)
1. a kind of anoxic sensitivity response type chitosan nitroimidazole grafting, which is characterized in that have structure represented by following formula
Unit:
Wherein, the bromine atom of N-Boc- bromo alkanamine is replaced by nitroimidazole, and the free amino in the part on chitosan chain passes through acyl
Amine key is connected with the amino for the N-BOC- alkanamine for having sloughed N-terminal BOC protection, and the weight average molecular weight of chitosan is 2000Da-
20000Da, the grafting rate of nitroimidazole are 1~20%, and the critical micell of chitosan nitroimidazole grafting in aqueous solution is dense
Degree is 15.63~103.5 μ g/mL.
2. the preparation method of anoxic sensitivity response type chitosan nitroimidazole grafting described in claim 1, which is characterized in that
It is realized by following steps:
2- nitroimidazole is weighed respectively, and the potassium carbonate of 0.5~5 times of mole of 2- nitroimidazole and 0.8~3 times of 2- nitroimidazole rub
The N-Boc- bromo alkanamine of your amount is placed in the dry round-bottomed flask of 50mL, and 20~50mL is added and is dehydrated n,N-Dimethylformamide,
80 DEG C of 4~8h of reaction in constant-temperature heating magnetic stirring apparatus.After reaction, appropriate ethyl acetate extracts reaction product, is placed in
40 DEG C of revolvings of round-bottomed flask remove solvent, appropriate n,N-Dimethylformamide are added in flask, 0.5~5 times of 2- nitroimidazole is rubbed
Two succinimide esters of your amount, the chitosan and n,N-Dimethylformamide isoploid of 2~10 times of moles of 2- nitroimidazole
The deionized water of accumulated amount, in constant-temperature heating magnetic stirring apparatus 40 DEG C reaction 4~for 24 hours, obtain reaction product.
3. preparation method according to claim 2, which is characterized in that reaction product is placed in 8~72h of dialysis in bag filter,
To remove N,N-dimethylformamide and aqueous by-product.
4. preparation method according to claim 2, which is characterized in that reaction product is freeze-dried after dialysis, obtains chitosan
Nitroimidazole grafting.
5. preparation method according to claim 1, which is characterized in that the weight average molecular weight of chitosan is 2000Da-
20000Da。
6. preparation method according to claim 1, which is characterized in that in 37 containing 10-100 μm of ol of nitroreductase
DEG C, in pH7.4 phosphate buffered saline solution, micella depolymerization occurs.
7. the anoxic sensitivity response type chitosan nitroimidazole grafting of claim 1 the method preparation is as cell toxicant class
Application in antineoplastic drug carrier.
8. application according to claim 7, which is characterized in that the drug is MAPK signal pathway inhibitor, PI3K letter
Number pathway inhibitor, cMYC inhibitor and fat-soluble fluorescent dye, select adriamycin, taxol, SB203580, LY294002,
Axitinib, JQ1, cumarin, Nile red.
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