CN107400179B - Double fat chain substituent phosphatidyl-ethanolamine chitosans and its preparation method and application - Google Patents
Double fat chain substituent phosphatidyl-ethanolamine chitosans and its preparation method and application Download PDFInfo
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- CN107400179B CN107400179B CN201710620704.2A CN201710620704A CN107400179B CN 107400179 B CN107400179 B CN 107400179B CN 201710620704 A CN201710620704 A CN 201710620704A CN 107400179 B CN107400179 B CN 107400179B
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- ethanolamine
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 115
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 title claims abstract description 50
- -1 substituent phosphatidyl-ethanolamine Chemical class 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 239000002502 liposome Substances 0.000 claims abstract description 53
- 239000003937 drug carrier Substances 0.000 claims abstract description 37
- 238000003780 insertion Methods 0.000 claims abstract description 23
- 230000037431 insertion Effects 0.000 claims abstract description 23
- 238000001338 self-assembly Methods 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 7
- 229940067605 phosphatidylethanolamines Drugs 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 46
- 238000000502 dialysis Methods 0.000 claims description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
- 239000008367 deionised water Substances 0.000 claims description 22
- 229910021641 deionized water Inorganic materials 0.000 claims description 22
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 22
- 239000012279 sodium borohydride Substances 0.000 claims description 22
- 238000003756 stirring Methods 0.000 claims description 22
- 238000002390 rotary evaporation Methods 0.000 claims description 18
- 239000003814 drug Substances 0.000 claims description 15
- 150000008104 phosphatidylethanolamines Chemical class 0.000 claims description 14
- HZAXFHJVJLSVMW-UHFFFAOYSA-N monoethanolamine hydrochloride Natural products NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 13
- 229940031098 ethanolamine Drugs 0.000 claims description 9
- NIYQLOFJJVVREI-UHFFFAOYSA-N acetic acid;[3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-dodecanoyloxypropyl] dodecanoate Chemical compound CC(O)=O.CCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCC NIYQLOFJJVVREI-UHFFFAOYSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 4
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Substances [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims description 4
- 230000001476 alcoholic effect Effects 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 2
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 claims description 2
- 230000006196 deacetylation Effects 0.000 claims description 2
- 238000003381 deacetylation reaction Methods 0.000 claims description 2
- 239000007800 oxidant agent Substances 0.000 claims description 2
- 230000001590 oxidative effect Effects 0.000 claims description 2
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 claims description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 2
- 235000010288 sodium nitrite Nutrition 0.000 claims description 2
- SLKDGVPOSSLUAI-PGUFJCEWSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine zwitterion Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCCCCCC SLKDGVPOSSLUAI-PGUFJCEWSA-N 0.000 claims 1
- 125000002252 acyl group Chemical group 0.000 claims 1
- 235000019441 ethanol Nutrition 0.000 claims 1
- 239000011261 inert gas Substances 0.000 claims 1
- 238000012377 drug delivery Methods 0.000 abstract description 5
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- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 238000010253 intravenous injection Methods 0.000 abstract 1
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 60
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- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical group [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 description 21
- 229910052757 nitrogen Inorganic materials 0.000 description 21
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- 239000007864 aqueous solution Substances 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 14
- ZZVUWRFHKOJYTH-UHFFFAOYSA-N diphenhydramine Chemical compound C=1C=CC=CC=1C(OCCN(C)C)C1=CC=CC=C1 ZZVUWRFHKOJYTH-UHFFFAOYSA-N 0.000 description 13
- 229940079593 drug Drugs 0.000 description 11
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 10
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- MWRBNPKJOOWZPW-NYVOMTAGSA-N 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine zwitterion Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-NYVOMTAGSA-N 0.000 description 5
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- OPOSNVQZZWJSOQ-UHFFFAOYSA-N C(CCCCCCCCCCCCC)(=O)[P]C(CCCCCCCCCCCCC)=O Chemical compound C(CCCCCCCCCCCCC)(=O)[P]C(CCCCCCCCCCCCC)=O OPOSNVQZZWJSOQ-UHFFFAOYSA-N 0.000 description 4
- JCABVIFDXFFRMT-DIPNUNPCSA-N [(2r)-1-[ethoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] octadec-9-enoate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OCC)OC(=O)CCCCCCCC=CCCCCCCCC JCABVIFDXFFRMT-DIPNUNPCSA-N 0.000 description 4
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- 125000001095 phosphatidyl group Chemical group 0.000 description 4
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- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
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- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 2
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- LVNGJLRDBYCPGB-UHFFFAOYSA-N 1,2-distearoylphosphatidylethanolamine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(COP([O-])(=O)OCC[NH3+])OC(=O)CCCCCCCCCCCCCCCCC LVNGJLRDBYCPGB-UHFFFAOYSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/12—Macromolecular compounds
- A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1806—Suspensions, emulsions, colloids, dispersions
- A61K49/1812—Suspensions, emulsions, colloids, dispersions liposomes, polymersomes, e.g. immunoliposomes
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Radiology & Medical Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Dispersion Chemistry (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention provides a kind of double fatty chain substituent phosphatidyl-ethanolamine chitosans and its preparation method and application, this method has synthesized double fatty chain substituent phosphatidyl-ethanolamine chitosans from chitosan and double fatty chain substituent phosphatidyl-ethanolamines.Liposome is modified by the method for rear insertion self assembly using double fatty chain substituent phosphatidyl-ethanolamine chitosans of synthesis, assembling forms double fatty chain substituent phosphatidyl-ethanolamine chitosan-lipidosome drug carriers.The pharmaceutical carrier that the present invention is assembled has strong cell adherence performance and antiserum ability, is suitable for intravenous injection.The present invention also provides above-mentioned chitosan-application of the liposome superparamagnetic Fe 3 O 4 nano-particles in drug delivery, have both high drug delivery efficiency and high biocompatibility, the target function and magnetic resonance imaging function of magnetic field guiding are provided simultaneously, had broad application prospects.
Description
Technical field
The present invention relates to a kind of double fatty chain substituent phosphatidyl-ethanolamine chitosans and its lipidosome drug carriers, specifically
Ground is said, is related to a kind of pair of fatty chain substituent phosphatidyl-ethanolamine chitosan and its is aoxidized with liposome building package superparamagnetic four
The pharmaceutical carrier of three Fe nanometer particles belongs to the preparation method of drug delivery field new drug carrier.
Background technique
Pharmaceutical carrier refers to that can change drug enters the mode of human body and the rate of release of distribution, control drug in vivo
And conduct drugs to the system of target organs.Pharmaceutical carrier effectively improves utilization rate, the safety of drug by control release
And validity.Chitosan and liposome are common pharmaceutical carriers, and chitosan biological compatibility and biodegradability are good, 2-
Amino and 6- hydroxyls are easy to carry out structural modification, have bioadhesion performance, and can improve drug by opening cell passage
Intercellular moment penetrating power；Liposome is to be dispersed in water the one kind formed by amphiphilic surfactant to have one layer
Or the ultra micro spherical particle of multilayer lipid vesicle structure, water-soluble or fat-soluble medicine can be loaded, medicine is widely used in
Object carrier.
Multifunctional nano-carrier is the nano-carrier of new generation to grow up on the basis of simple function nano-carrier, it
Simple function carrier some shortcomings present in tumor diagnosis and therapy are overcome, such as to the real-time prison of internal cellular activity
Control, for the effective transmitting of the special targeting of target area or drug in target cell.Multifunctional carrier is in single stable
Different functions is combined in structure.Tumor imaging agent or diagnostic reagent is such as combined to realize the early diagnosis of tumour, real-time monitoring
Oncotherapy effect etc..Multifunctional nano-carrier provides new opportunities for the early diagnosis of tumour and Individual drug treatment.
Magnetic resonance imaging (MRI) has good soft tissue resolution and spatial resolution, clear to show anatomic tissue knot
While structure, can the Features to soft tissue accurately positioned, quantitative analysis, be most having for early diagnosis of tumor
One of method of effect.In order to enhance the contrast between pathological tissues and the image of normal tissue to improve the clear of pathological tissues
Degree, needs that suitable contrast medium is selected to show anatomical features.T2 contrast medium has compared with the higher magnetic moment of paramagnet, right
The relaxation of proton has apparent acceleration effect in adjacent tissue, can significantly improve detection sensitivity.Common superparamagnetism comparison
Agent is mainly different size of microcrystalline metal particle (such as Fe3O4、Fe2O3)。
Malignant tumour is the first killer of human health.Although recently as the improvement of detection and treatment means, tumour
The survival rate of patient increases, but the death rate of tumor patient is still high.Currently, one of oncotherapy main means
It is chemotherapy, but the toxic reaction of drug and tumor cell drug resistance cause chemotherapy cure rate low.On the other hand, lack
Effective early diagnosis is also the main reason for causing cure rate low.Therefore, seek new effective early diagnosis of tumor and control
Treatment method is Clinical Oncology problem urgently to be resolved.Gene therapy is by the way that therapeutic gene to be imported into target cell core to repair
The dcc gene or inhibition for leading to disease lead to the deleterious gene of disease, to make body recovery normal function, reach treatment
The purpose of disease.Safe and efficient carrier is one of successful key of gene therapy.
The stability of pharmaceutical carrier in blood is very crucial to the effect for playing pharmaceutical carrier.Liposome structure is vulnerable to blood
The destruction of the ingredients such as clear middle-high density lipoprotein, leads to the leakage of entrapped drug.Chitosan has good antiserum performance, has
Help improve stability of the drug-loading nanoparticles in serum.Chitosan-modified, structure is carried out to liposome by rear inserted mode
The pharmaceutical carrier that surface has chitosan brush is built, and wraps up nano ferriferrous oxide nanoparticle (SPIO), forms set medicine
Object delivering and diagnostic imaging are in the multifunctional carrier of one, using gene as model drug, carry out gene and transfect performance evaluation, realize
Treatment and diagnosis integration, open up new way for oncotherapy.
Summary of the invention
Technical problem to be solved by the present invention lies in the defect for overcoming existing pharmaceutical carrier, a kind of double aliphatic chains are provided and are taken
It is carried for base phosphatidyl-ethanolamine chitosan, and by the liposome with chitosan brush and SPIO that rear inserted mode constructs
Body and preparation method thereof.
The first purpose of this invention is to provide a kind of double fatty chain substituent phosphatidyl-ethanolamine chitosans have formula
(I) structure:
Wherein, R and R' is identical or different CxHy, wherein x=11~17, y=21~35.
Preferably, R and R' is identical or not identical C11H23、C13H27、C17H35Or C17H33。
Double fatty chain substituent phosphatidyl-ethanolamine chitosans are achieved through the following technical solutions:
Oxygen is used as using one or more of sodium hypochlorite, sodium nitrite, periodate or hydrogen peroxide first
Agent, react 1 under conditions of pH=2~6,0~50 DEG C with chitosan~for 24 hours, reaction solution is dialysed, is freeze-dried, preparation
Formoxyl chitosan；Formoxyl chitosan is distributed in alcoholic solution, double fatty chain substituent phosphatidyl-ethanolamines, inertia is added
Under gas shield, 20~100 DEG C are stirred at reflux 2~48h, and sodium borohydride is added and continues stirring 3~for 24 hours, rotary evaporation removes second
After alcoholic solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtains double fatty chain substituent phosphatidyl-ethanolamines
Chitosan.
Preferably, the amino of chitosan glucose unit and oxidant molar ratio 1:3~10:1.
Preferably, the weight average molecular weight of the chitosan is 500-10000Da, deacetylation 65-95%.
Preferably, double fatty chain substituent phosphatidyl-ethanolamine is 1,2- dilauroyl phosphatidyl-ethanolamine, 1,
2- distearoylphosphatidylethanolamine, bis- myristoyl phosphatidyl-ethanolamine of 1,2-, bis- palmityl phosphatidyl second of 1,2-
One or more of hydramine, 1,2- dioleoylphosphatidylethanolamine, but above-mentioned raw materials are not limited to, dosage is formoxyl
0.1-1 times of chitosan repetitive unit molar equivalent, preferably 0.3-0.6 times, reaction condition is 20-100 DEG C of reflux 2h-48h,
Preferably 30-50 DEG C reflux 4-12h.
Another object of the present invention, which also resides in, provides a kind of pair of fatty chain substituent phosphatidyl-ethanolamine chitosan and lipid
The compound (polysome@SPIO) of body@SPIO.
Preferably, the cationic-liposome is DOTAP, Lipofectin, LipofectaminTMOne of 2000,
The partial size of the SPIO nanoparticle is 1~30nm.
The polysome@SPIO compound is achieved through the following technical solutions: preparing liposome@using film ultrasound
SPIO composite material, then double fatty chain substituent phosphatidyl-ethanolamine chitosans are inserted into liposome by way of rear insertion
Phospholipid bilayer obtains polysome@SPIO compound.
Application of the above-mentioned polysome@SPIO compound as pharmaceutical carrier is claimed simultaneously in the present invention, especially exists
Application in gene transfection.
The present invention modifies chitosan by double fatty chain substituent phosphatidyl-ethanolamines, then passes through rear inserted mode
Liposome SPIO is modified, the lipid composite material that surface has chitosan brush is obtained, improves liposome antiserum
While ability and biocompatibility, high potency drugs delivering is realized.
Compared with prior art, the invention has the following advantages that
1. the present invention modifies chitosan using double fatty chain substituent phosphatidyl-ethanolamines, it is prepared for double aliphatic chains
Substituent group phosphatidyl-ethanolamine chitosan.
2. the present invention using double fatty chain substituent phosphatidyl-ethanolamine chitosans by rear inserted mode to liposome into
Row modification, improves liposome biocompatibility and blood stability.
3. invented liposomes encapsulate SPIO simultaneously, polysome@SPIO complex carrier is obtained, realizes it in drug delivery
In application, especially gene transfection in application.The complex carrier has both high drug delivery efficiency and high biofacies
Capacitive, while the target function and magnetic resonance imaging function of magnetic field guiding being provided, it has broad application prospects.
Detailed description of the invention
Fig. 1 is the FTIR spectrogram of double fatty chain substituent phosphatidyl-ethanolamine chitosans prepared by embodiment 2；
Fig. 2 is double fatty chain substituent phosphatidyl-ethanolamine chitosans prepared by embodiment 21HNMR spectrogram；
Fig. 3 is double fatty chain substituent phosphatidyl-ethanolamine chitosan-DOTAP liposome-SPIO prepared by embodiment 2
The TEM photo of complex carrier；
Fig. 4 is that polysome@SPIO complex carrier prepared by embodiment 2 examines or check the retardation ability of DNA；
Fig. 5 is the efficiency gene transfection of polysome@SPIO complex carrier prepared by the present invention；
Fig. 6 is the cytotoxicity of polysome@SPIO complex carrier prepared by the present invention.
Specific embodiment
The present invention is described in detail below by the drawings and specific embodiments, but is not limited the scope of the invention.Such as without special
Illustrate, experimental method of the present invention is conventional method, and experiment equipment used, material, reagent etc. can be chemically public
Department's purchase.
Low-molecular weight chitoglycan (CSO) 0.8554g, is dissolved in the buffer solution of NaAc/HAc (pH=4.5) of 50mL, surpasses
Sound 30min, makes it dissolve.Sodium metaperiodate 0.1093g is dissolved in the buffer solution of NaAc/HAc (pH=4.5) of 50mL, ultrasound
30min.Both the above solution is respectively placed in ice bath, and is passed through high pure nitrogen degassing 30min, two kinds of solution is mixed, at 0-4 DEG C
Under be stirred to react for 24 hours, be added 10mL ethylene glycol stopped reaction.Reaction solution is transferred in bag filter (MWCO=3000Da), dialysis,
Freeze-drying, prepares formoxyl chitosan.
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2- distearoyl base phosphatidyl second is added
Hydramine 0.14g, nitrogen protection are stirred to react 42h at 60 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation
After removing alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2- distearoyl base phosphatidyl
Ethanol amine chitosan.
1, the 2- distearoyl base phosphatidyl-ethanolamine chitosan aqueous solution for preparing 1mg/mL, takes 100uL, and includes SPIO
DOTAP cationic-liposome 1mL ultrasound by way of mix, be then allowed to stand 1h, it is right by way of rear insertion self assembly
Liposome is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 2
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2- dioleoyl phosphatidyl ethanol is added
Amine 0.14g, nitrogen protection are stirred to react 42h at 60 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation is gone
After alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2- dioleoyl phosphatidyl ethanol
Amine chitosan.
1, the 2- dioleoylphosphatidylethanolamine aqueous solution for preparing 1mg/mL, takes 100uL, with the DOTAP comprising SPIO
Cationic-liposome 1mL is mixed by way of ultrasound, 1h is then allowed to stand, by way of rear insertion self assembly, to liposome
It is modified, obtains the lipidosome drug carrier that surface has chitosan brush.
Embodiment 3
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2- dilauroyl phosphatidyl second is added
Hydramine 0.14g, nitrogen protection are stirred to react 42h at 60 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation
After removing alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2- dilauroyl phosphatidyl
Ethanol amine chitosan.
1, the 2- dilauroyl phosphatidyl ethanol amine aqueous solution for preparing 1mg/mL, takes 100uL, and includes SPIO's
DOTAP cationic-liposome 1mL is mixed by way of ultrasound, 1h is then allowed to stand, by way of rear insertion self assembly, to rouge
Plastid is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 4
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2-, bis- myristoyl phosphatidyl is added
Ethanol amine 0.14g, nitrogen protection are stirred to react 42h at 60 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotation is steamed
After hair removal alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2-, bis- myristoyl phosphorus
Acyl ethanol amine chitosan.
The 1,2-dimristoyl phosphatidylethanolamine l aqueous solution for preparing 1mg/mL, takes 100uL, and includes SPIO's
DOTAP cationic-liposome 1mL is mixed by way of ultrasound, 1h is then allowed to stand, by way of rear insertion self assembly, to rouge
Plastid is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 5
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2-, bis- palmityl phosphatidyl second is added
Hydramine 0.14g, nitrogen protection are stirred to react 42h at 60 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation
After removing alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2-, bis- palmityl phosphatidyl
Ethanol amine chitosan.
1, the 2- dipalmitoylphosphatidylethanolamine chitosan aqueous solution for preparing 1mg/mL, takes 100uL, and includes SPIO
DOTAP cationic-liposome 1mL ultrasound by way of mix, be then allowed to stand 1h, it is right by way of rear insertion self assembly
Liposome is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 6
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2- distearoyl base phosphatidyl second is added
Hydramine 0.3g, nitrogen protection are stirred to react 42h at 60 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation
After removing alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2- distearoyl base phosphatidyl
Ethanol amine chitosan.
1, the 2- distearoyl base phosphatidyl-ethanolamine chitosan aqueous solution for preparing 1mg/mL, takes 100uL, and includes SPIO
DOTAP cationic-liposome 1mL ultrasound by way of mix, be then allowed to stand 1h, it is right by way of rear insertion self assembly
Liposome is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 7
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2- dioleoyl phosphatidyl ethanol is added
Amine 0.3g, nitrogen protection are stirred to react 42h at 60 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation is gone
After alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2- dioleoyl phosphatidyl ethanol
Amine chitosan.
1, the 2- dioleoylphosphatidylethanolamine chitosan aqueous solution for preparing 1mg/mL, takes 100uL, and includes SPIO's
DOTAP cationic-liposome 1mL is mixed by way of ultrasound, 1h is then allowed to stand, by way of rear insertion self assembly, to rouge
Plastid is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 8
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2- dilauroyl phosphatidyl second is added
Hydramine 0.3g, nitrogen protection are stirred to react 42h at 60 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation
After removing alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2- dilauroyl phosphatidyl
Ethanol amine chitosan.
1, the 2- dilauroyl phosphatidyl ethanol amine aqueous solution for preparing 1mg/mL, takes 100uL, and includes SPIO's
DOTAP cationic-liposome 1mL is mixed by way of ultrasound, 1h is then allowed to stand, by way of rear insertion self assembly, to rouge
Plastid is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 9
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2-, bis- myristoyl phosphatidyl is added
Ethanol amine 0.3g, nitrogen protection are stirred to react 42h at 60 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotation is steamed
After hair removal alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2-, bis- myristoyl phosphorus
Acyl ethanol amine chitosan.
The 1,2-dimristoyl phosphatidylethanolamine l aqueous solution for preparing 1mg/mL, takes 100uL, and includes SPIO's
DOTAP cationic-liposome 1mL is mixed by way of ultrasound, 1h is then allowed to stand, by way of rear insertion self assembly, to rouge
Plastid is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 10
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2-, bis- palmityl phosphatidyl second is added
Hydramine 0.3g, nitrogen protection are stirred to react 42h at 60 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation
After removing alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2-, bis- palmityl phosphatidyl
Ethanol amine chitosan.
1, the 2- dipalmitoylphosphatidylethanolamine chitosan aqueous solution for preparing 1mg/mL, takes 100uL, and includes SPIO
DOTAP cationic-liposome 1mL ultrasound by way of mix, be then allowed to stand 1h, it is right by way of rear insertion self assembly
Liposome is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 11
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2- distearoyl base phosphatidyl second is added
Hydramine 0.3g, nitrogen protection are stirred to react 42h at 50 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation
After removing alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2- distearoyl base phosphatidyl
Ethanol amine chitosan.
1, the 2- distearoyl base phosphatidyl-ethanolamine chitosan aqueous solution for preparing 1mg/mL, takes 500uL, and includes SPIO
DOTAP cationic-liposome 1mL ultrasound by way of mix, be then allowed to stand 1h, it is right by way of rear insertion self assembly
Liposome is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 12
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2- dioleoyl phosphatidyl ethanol is added
Amine 0.3g, nitrogen protection are stirred to react 42h at 50 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation is gone
After alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2- dioleoyl phosphatidyl ethanol
Amine chitosan.
1, the 2- dioleoylphosphatidylethanolamine aqueous solution for preparing 1mg/mL, takes 500uL, with the DOTAP comprising SPIO
Cationic-liposome 1mL is mixed by way of ultrasound, 1h is then allowed to stand, by way of rear insertion self assembly, to liposome
It is modified, obtains the lipidosome drug carrier that surface has chitosan brush.
Embodiment 13
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2- dilauroyl phosphatidyl second is added
Hydramine 0.3g, nitrogen protection are stirred to react 42h at 50 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation
After removing alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2- dilauroyl phosphatidyl
Ethanol amine chitosan.
1, the 2- dilauroyl phosphatidyl ethanol amine aqueous solution for preparing 1mg/mL, takes 500uL, and includes SPIO's
DOTAP cationic-liposome 1mL is mixed by way of ultrasound, 1h is then allowed to stand, by way of rear insertion self assembly, to rouge
Plastid is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 14
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2-, bis- myristoyl phosphatidyl is added
Ethanol amine 0.3g, nitrogen protection are stirred to react 42h at 50 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotation is steamed
After hair removal alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2-, bis- myristoyl phosphorus
Acyl ethanol amine chitosan.
The 1,2-dimristoyl phosphatidylethanolamine l aqueous solution for preparing 1mg/mL, takes 500uL, and includes SPIO's
DOTAP cationic-liposome 1mL is mixed by way of ultrasound, 1h is then allowed to stand, by way of rear insertion self assembly, to rouge
Plastid is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 15
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2-, bis- palmityl phosphatidyl second is added
Hydramine 0.3g, nitrogen protection are stirred to react 42h at 50 DEG C, and 0.1g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation
After removing alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2-, bis- palmityl phosphatidyl
Ethanol amine chitosan.
1, the 2- dipalmitoylphosphatidylethanolamine chitosan aqueous solution for preparing 1mg/mL, takes 500uL, and includes SPIO
DOTAP cationic-liposome 1mL ultrasound by way of mix, be then allowed to stand 1h, it is right by way of rear insertion self assembly
Liposome is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 16
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2- distearoyl base phosphatidyl second is added
Hydramine 0.3g, nitrogen protection are stirred to react 42h at 80 DEG C, and 0.2g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation
After removing alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2- distearoyl base phosphatidyl
Ethanol amine chitosan.
1, the 2- distearoyl base phosphatidyl-ethanolamine chitosan aqueous solution for preparing 1mg/mL, takes 500uL, and includes SPIO
DOTAP cationic-liposome 1mL ultrasound by way of mix, be then allowed to stand 2h, it is right by way of rear insertion self assembly
Liposome is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 17
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2- dioleoyl phosphatidyl ethanol is added
Amine 0.3g, nitrogen protection are stirred to react 42h at 80 DEG C, and 0.2g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation is gone
After alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2- dioleoyl phosphatidyl ethanol
Amine chitosan.
1, the 2- dioleoylphosphatidylethanolamine aqueous solution for preparing 1mg/mL, takes 500uL, with the DOTAP comprising SPIO
Cationic-liposome 1mL is mixed by way of ultrasound, 2h is then allowed to stand, by way of rear insertion self assembly, to liposome
It is modified, obtains the lipidosome drug carrier that surface has chitosan brush.
Embodiment 18
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2- dilauroyl phosphatidyl second is added
Hydramine 0.3g, nitrogen protection are stirred to react 42h at 80 DEG C, and 0.2g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation
After removing alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2- dilauroyl phosphatidyl
Ethanol amine chitosan.
1, the 2- dilauroyl phosphatidyl ethanol amine aqueous solution for preparing 1mg/mL, takes 500uL, and includes SPIO's
DOTAP cationic-liposome 1mL is mixed by way of ultrasound, 2h is then allowed to stand, by way of rear insertion self assembly, to rouge
Plastid is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 19
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2-, bis- myristoyl phosphatidyl is added
Ethanol amine 0.3g, nitrogen protection are stirred to react 42h at 80 DEG C, and 0.2g sodium borohydride room temperature is added and continues stirring for 24 hours, rotation is steamed
After hair removal alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2-, bis- myristoyl phosphorus
Acyl ethanol amine chitosan.
The 1,2-dimristoyl phosphatidylethanolamine l aqueous solution for preparing 1mg/mL, takes 500uL, and includes SPIO's
DOTAP cationic-liposome 1mL is mixed by way of ultrasound, 2h is then allowed to stand, by way of rear insertion self assembly, to rouge
Plastid is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Embodiment 20
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solution, 1,2-, bis- palmityl phosphatidyl second is added
Hydramine 0.3g, nitrogen protection are stirred to react 42h at 80 DEG C, and 0.2g sodium borohydride room temperature is added and continues stirring for 24 hours, rotary evaporation
After removing alcohol solvent, deionized water dialysis is used after being distributed in water, then be freeze-dried, obtain 1,2-, bis- palmityl phosphatidyl
Ethanol amine chitosan.
1, the 2- dipalmitoylphosphatidylethanolamine chitosan aqueous solution for preparing 1mg/mL, takes 500uL, and includes SPIO
DOTAP cationic-liposome 1mL ultrasound by way of mix, be then allowed to stand 2h, it is right by way of rear insertion self assembly
Liposome is modified, and the lipidosome drug carrier that surface has chitosan brush is obtained.
Liposome gene with chitosan brush transports measurement
It uses pGL3 plasmid for reporter gene, the gene delivery performance of the liposome vectors with chitosan brush is carried out
Evaluation, cell used are people's non-small cell lung cancer cell A549 cell line.By cultured plating cells, cultivate in the incubator
After reaching 80% to cell fusion degree, gene delivery is carried out, when transhipment, complete medium is sucked, is washed twice with PBS, blood
When transporting under the conditions of clear, the polysome@SPIO of culture medium and different N/P ratio (mass ratio) of the 400 μ L containing 10% serum is added
(embodiment 3) and DNA compound (every hole contains 1 μ g DNA), after cultivating 18h, are sucked out culture medium, change fresh containing 10% serum
Culture medium continue cultivate 32h after, it is multi-functional in BioTek Synergy2 according to specification provided by luciferase kit
The intensity that photon is detected in microplate reader, the concentration of total protein is detected with BCA, so that result, which is sought unity of standard, is melted into RLU/mg egg
White (opposite number of photons corresponding to every milligram of albumen).
The cytotoxicity of liposome with chitosan brush
The cytotoxicity of carrier is evaluated using mtt assay.The repopulating cell in 96 porocyte culture plates, parallel 3 hole, often
Hole plantation 5 × 104A cell, at 37 DEG C, 5%CO2Culture to cell fusion degree reaches 85% or more in cell incubator.It removes
After washing 2 times with PBS, fresh culture and carrier to be measured is added in culture medium, and after culture for 24 hours, every hole is added 20 μ L 5mg/mL's
MTT solution, 37 DEG C are continued to cultivate 4h, are removed culture medium, are terminated culture.Succinate dehydrogenase reduction in living cells mitochondria
MTT generates formazan, and 150 μ L DMSO, which are added, in every hole makes to dissolve, and continues to be incubated for 30min at 37 DEG C.In multi-function microplate reader
The absorption value that each hole of 570nm wavelength is measured on (Sunrise Tecan) shakes 96 orifice plate automatic mixing 600s before detecting, and adopts
It is returned to zero with cell-free culture medium to microplate reader.Cell survival rate is calculated by formula 1.1:
Cell survival rate (%)=A570SMP/A570CTL×100 (1.1)
Wherein A570SMPFor addition carrier to be measured or the light absorption value of the cell orifice plate of compound, A570CTLFor containing only culture medium
Cell orifice plate light absorption value.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (9)
1. pair fatty chain substituent phosphatidyl-ethanolamine chitosan, which is characterized in that have formula (I) structure:
Wherein, R and R' is identical or different CxHy, wherein x=11~17, y=21~35.
2. double fatty chain substituent phosphatidyl-ethanolamine chitosans according to claim 1, which is characterized in that R and R' is phase
With or not identical C11H23、C13H27、C17H35Or C17H33。
3. a kind of preparation method of fatty chain substituent phosphatidyl-ethanolamine chitosans double as described in claim 1, feature exist
In, oxidant is used as using one or more of sodium hypochlorite, sodium nitrite, periodate or hydrogen peroxide first,
React 1 under conditions of pH=2~6,0~50 DEG C with chitosan~for 24 hours, reaction solution is dialysed, is freeze-dried, and formoxyl is prepared
Chitosan；Formoxyl chitosan is distributed in alcoholic solution again, double fatty chain substituent phosphatidyl-ethanolamines, inert gas is added
Under protection, 20~100 DEG C are stirred at reflux 2~48h, and sodium borohydride is added and continues stirring 3~for 24 hours, it is molten that rotary evaporation removes ethyl alcohol
After agent, deionized water dialysis is used after being distributed in water, then be freeze-dried, it is poly- to obtain double fatty chain substituent phosphatidyl-ethanolamine shells
Sugar.
4. the preparation method of double fatty chain substituent phosphatidyl-ethanolamine chitosans according to claim 3, which is characterized in that
The weight average molecular weight of the chitosan is 500-10000Da, and deacetylation is 65~95%.
5. the preparation method of double fatty chain substituent phosphatidyl-ethanolamine chitosans according to claim 3, which is characterized in that
Double fatty chain substituent phosphatidyl-ethanolamines are 1,2- dilauroyl phosphatidyl-ethanolamine, 1,2- distearyl acyl group phosphatide
Acyl ethanol amine, bis- myristoyl phosphatidyl-ethanolamine of 1,2-, 1,2-dipalmitoylphosphatidylethanolamine, 1,2- dioleoyl
One or more of phosphatidyl-ethanolamine.
6. a kind of preparation method of double fatty chain substituent phosphatidyl-ethanolamine chitosan-lipidosome drug carriers, feature exist
In, using double fat chain substituent phosphatidyl-ethanolamine chitosan described in claim 1, by way of rear insertion self assembly,
Cationic-liposome is modified, double fatty chain substituent phosphatidyl-ethanolamine chitosan-lipidosome drug carriers are obtained.
7. the preparation of double fatty chain substituent phosphatidyl-ethanolamine chitosan-lipidosome drug carriers according to claim 6
Method, which is characterized in that cationic-liposome be 1,2-Dioleoyloxy-3-trimethylaminopropane, Lipofectin,
LipofectaminTMOne of 2000.
8. the preparation of double fatty chain substituent phosphatidyl-ethanolamine chitosan-lipidosome drug carriers according to claim 6
Method, which is characterized in that cationic-liposome hydrophilic core wraps up the super-paramagnetic ferriferrous oxide nano grain that partial size is 1~30nm
Son.
9. a kind of fatty chain substituent phosphatidyl-ethanolamine chitosan-lipidosome drug carriers double as claimed in claim 6 are in medicine
Application in object delivering.
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