CN107903341A - The application of double fat chain substituent phosphatidyl-ethanolamine chitosans - Google Patents

The application of double fat chain substituent phosphatidyl-ethanolamine chitosans Download PDF

Info

Publication number
CN107903341A
CN107903341A CN201711363440.3A CN201711363440A CN107903341A CN 107903341 A CN107903341 A CN 107903341A CN 201711363440 A CN201711363440 A CN 201711363440A CN 107903341 A CN107903341 A CN 107903341A
Authority
CN
China
Prior art keywords
chitosan
phosphatidyl
ethanolamine
chain substituent
fatty chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201711363440.3A
Other languages
Chinese (zh)
Other versions
CN107903341B (en
Inventor
张树彪
陈会英
马羽
秦晓利
蓝浩铭
崔韶晖
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dalian Minzu University
Original Assignee
Dalian Nationalities University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dalian Nationalities University filed Critical Dalian Nationalities University
Priority to CN201711363440.3A priority Critical patent/CN107903341B/en
Publication of CN107903341A publication Critical patent/CN107903341A/en
Application granted granted Critical
Publication of CN107903341B publication Critical patent/CN107903341B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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

Landscapes

  • 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

This divisional application provides a kind of application of double fatty chain substituent phosphatidyl-ethanolamine chitosans, from chitosan and double fatty chain substituent phosphatidyl-ethanolamines, has synthesized double fatty chain substituent phosphatidyl-ethanolamine chitosans.Using double fatty chain substituent phosphatidyl-ethanolamine chitosans of synthesis, by the method for rear insertion self assembly, liposome is modified, 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, suitable for intravenous injection.The present invention also provides application of the above-mentioned chitosan liposome superparamagnetic Fe 3 O 4 nano-particles in medicine delivery, have high drug delivery efficiency and high biocompatibility concurrently, the target function and magnetic resonance imaging function of magnetic field guiding are provided at the same time, had broad application prospects.

Description

The application of double fat chain substituent phosphatidyl-ethanolamine chitosans
The application is Application No. 2017106207042, the applying date is on July 27th, 2017, entitled " double fat The divisional application of chain substituent phosphatidyl-ethanolamine chitosan and its preparation method and application ".
Technical field
The present invention relates to a kind of double fatty chain substituent phosphatidyl-ethanolamine chitosans and its lipidosome drug carrier, specifically Ground is said, is related to a kind of double fatty chain substituent phosphatidyl-ethanolamine chitosans and its is aoxidized with liposome structure parcel superparamagnetic four The pharmaceutical carrier of three Fe nanometer particles, belongs to the preparation method of drug delivery field new drug carrier.
Background technology
Pharmaceutical carrier refers to change medicine into the mode of human body and distribution in vivo, the rate of release of control medicine And conduct drugs to the system of target organs.Pharmaceutical carrier is discharged by controlling, and effectively improves utilization rate, the security of medicine 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 medicine 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, is widely used in medicine Thing carrier.
Multifunctional nano-carrier is the nano-carrier of new generation to grow up on the basis of simple function nano-carrier, it Overcome simple function carrier some shortcomings present in tumor diagnosis and therapy, such as real-time prison to internal cellular activity Control, for the effective transmission of the special targeting of target site or medicine in target cell.Multifunctional carrier is in single stable Different functions is combined in structure.Such as combine tumor imaging agent or diagnostic reagent realizes the early diagnosis of tumour, monitoring in real time 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, the Features of soft tissue can be carried out with accurately positioning, quantitative analysis, be most having for early diagnosis of tumor One of method of effect.In order to strengthen the contrast between pathological tissues and the image of normal structure to improve the clear of pathological tissues Degree is, it is necessary to select suitable contrast medium to show anatomical features.T2 contrast medium has the magnetic moment compared with paramagnet higher, right The relaxation of proton has obvious acceleration effect in adjacent tissue, can significantly improve detection sensitivity.Common superparamagnetism contrast 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 detection and the improvement for the treatment of means, tumour The survival rate of patient increases, but the death rate of tumor patient is still high.At present, one of oncotherapy main means It is chemotherapy, but the toxic reaction of medicine 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 hurrily.Gene therapy is by the way that therapeutic gene is imported into target cell core to repair The defects of causing disease, or suppresses to cause the deleterious gene of disease gene, so that body recovery normal function, reaches treatment The purpose of disease.Safe and efficient carrier is one of successful key of gene therapy.
Effect of the stability of pharmaceutical carrier in blood to playing pharmaceutical carrier is very crucial.Liposome structure is easily by blood The destruction of the components such as clear middle-high density lipoprotein, causes 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 Building surface has the pharmaceutical carrier of chitosan brush, and wraps up nano ferriferrous oxide nano-particle (SPIO), forms set medicine Thing delivers and diagnostic imaging is in the multifunctional carrier of one, using gene as model drug, carries out gene transfection performance evaluation, realizes Treatment and diagnosis integration, new way is opened up for oncotherapy.
The content of the invention
The defects of technical problems to be solved by the invention are to overcome existing pharmaceutical carrier, there is provided a kind of double aliphatic chains take For base phosphatidyl-ethanolamine chitosan, and by what rear inserted mode was built there is the liposome of chitosan brush and SPIO to carry Body and preparation method thereof.
First purpose of the present 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 differ 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 both of sodium hypochlorite, sodium nitrite, periodate or hydrogen peroxide above first Agent, reacts 1~24h, reaction solution is prepared through dialysing, being freeze-dried with chitosan under conditions of pH=2~6,0~50 DEG C Formoxyl chitosan；Formoxyl chitosan is distributed in alcoholic solution, adds double fatty chain substituent phosphatidyl-ethanolamines, inertia Under gas shield, 20~100 DEG C are stirred at reflux 2~48h, add sodium borohydride and continue 3~24h of stirring, rotary evaporation removes second After alcoholic solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 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 be 1,2- dilauroyls phosphatidyl-ethanolamine, 1, 2- distearoylphosphatidylethanolamine, bis- myristoyl phosphatidyl-ethanolamines of 1,2-, bis- palmityl phosphatidyl second of 1,2- More than one or both 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, is preferably 0.3-0.6 times, and 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 also resides in a kind of double fatty chain substituent phosphatidyl-ethanolamine chitosans of offer and lipid The compound (polysome@SPIO) of body@SPIO.
Preferably, the cationic-liposome is DOTAP, Lipofectin, LipofectaminTMOne kind in 2000, The particle diameter of the SPIO nano-particles is 1~30nm.
The polysome@SPIO compounds are achieved through the following technical solutions：Liposome@is prepared using film ultrasound SPIO composite materials, then double fatty chain substituent phosphatidyl-ethanolamine chitosans are inserted into liposome by way of rear insertion Phospholipid bilayer, obtains polysome@SPIO compounds.
Application of the above-mentioned polysome@SPIO compounds as pharmaceutical carrier is claimed at the same time 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, obtaining surface has the lipid composite material of chitosan brush, improves liposome antiserum While ability and biocompatibility, realize that high potency drugs deliver.
Compared with prior art, the present invention has the following advantages：
1. the present invention modifies chitosan using double fatty chain substituent phosphatidyl-ethanolamines, double aliphatic chains are prepared for Substituent 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 at the same time, polysome@SPIO complex carriers are obtained, realize it in medicine delivery In application, especially gene transfection in application.The complex carrier has high drug delivery efficiency and high biofacies concurrently Capacitive, while the target function and magnetic resonance imaging function of magnetic field guiding are provided, have broad application prospects.
Brief description of the drawings
Fig. 1 is the FTIR spectrograms 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 spectrograms；
Fig. 3 is double fatty chain substituent phosphatidyl-ethanolamine chitosan-DOTAP liposomes-SPIO prepared by embodiment 2 The TEM photos of complex carrier；
Fig. 4 is that the polysome@SPIO complex carriers prepared by embodiment 2 examine or check the retardation ability of DNA；
Fig. 5 is the efficiency gene transfection of polysome@SPIO complex carriers prepared by the present invention；
Fig. 6 is the cytotoxicity of polysome@SPIO complex carriers prepared by the present invention.
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 its dissolving.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 Lower stirring reaction 24h, adds 10mL ethylene glycol stopped reactions.Reaction solution is transferred in bag filter (MWCO=3000Da), dialysis, It is lyophilized, prepare formoxyl chitosan.
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2- distearoyl base phosphatidyl second Hydramine 0.14g, nitrogen are protected, and stirring reaction 42h at 60 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation After removing alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2- distearoyl base phosphatidyls Monoethanolamine chitosan.
1, the 2- distearoyl base phosphatidyl-ethanolamine chitosan aqueous solutions of 1mg/mL are prepared, 100uL are taken, with including SPIO DOTAP cationic-liposomes 1mL ultrasound by way of mix, then stand 1h, it is rear insertion self assembly by way of, it is right Liposome is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 2
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2- dioleoyl phosphatidyl ethanols Amine 0.14g, nitrogen are protected, and stirring reaction 42h at 60 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation is gone After alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2- dioleoyl phosphatidyl ethanols Amine chitosan.
1, the 2- dioleoylphosphatidylethanolamine aqueous solutions of 1mg/mL are prepared, 100uL are taken, with the DOTAP comprising SPIO Cationic-liposome 1mL is mixed by way of ultrasound, then stands 1h, by way of rear insertion self assembly, to liposome Modified, obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 3
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2- dilauroyl phosphatidyl second Hydramine 0.14g, nitrogen are protected, and stirring reaction 42h at 60 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation After removing alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2- dilauroyl phosphatidyls Monoethanolamine chitosan.
1, the 2- dilauroyl phosphatidyl ethanol amine aqueous solutions of 1mg/mL are prepared, 100uL are taken, with including SPIO's DOTAP cationic-liposomes 1mL is mixed by way of ultrasound, then stands 1h, by way of rear insertion self assembly, to fat Plastid is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 4
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2-, bis- myristoyl phosphatidyls Monoethanolamine 0.14g, nitrogen are protected, and stirring reaction 42h at 60 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotate and steam After hair removes alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2-, bis- myristoyl phosphorus Acyl monoethanolamine chitosan.
The 1,2-dimristoyl phosphatidylethanolamine l aqueous solution of 1mg/mL is prepared, 100uL is taken, with including SPIO's DOTAP cationic-liposomes 1mL is mixed by way of ultrasound, then stands 1h, by way of rear insertion self assembly, to fat Plastid is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 5
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2-, bis- palmityl phosphatidyl second Hydramine 0.14g, nitrogen are protected, and stirring reaction 42h at 60 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation After removing alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2-, bis- palmityl phosphatidyls Monoethanolamine chitosan.
1, the 2- dipalmitoylphosphatidylethanolamine chitosan aqueous solutions of 1mg/mL are prepared, 100uL are taken, with including SPIO DOTAP cationic-liposomes 1mL ultrasound by way of mix, then stand 1h, it is rear insertion self assembly by way of, it is right Liposome is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 6
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2- distearoyl base phosphatidyl second Hydramine 0.3g, nitrogen are protected, and stirring reaction 42h at 60 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation After removing alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2- distearoyl base phosphatidyls Monoethanolamine chitosan.
1, the 2- distearoyl base phosphatidyl-ethanolamine chitosan aqueous solutions of 1mg/mL are prepared, 100uL are taken, with including SPIO DOTAP cationic-liposomes 1mL ultrasound by way of mix, then stand 1h, it is rear insertion self assembly by way of, it is right Liposome is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 7
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2- dioleoyl phosphatidyl ethanols Amine 0.3g, nitrogen are protected, and stirring reaction 42h at 60 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation is gone After alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2- dioleoyl phosphatidyl ethanols Amine chitosan.
1, the 2- dioleoylphosphatidylethanolamine chitosan aqueous solutions of 1mg/mL are prepared, 100uL are taken, with including SPIO's DOTAP cationic-liposomes 1mL is mixed by way of ultrasound, then stands 1h, by way of rear insertion self assembly, to fat Plastid is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 8
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2- dilauroyl phosphatidyl second Hydramine 0.3g, nitrogen are protected, and stirring reaction 42h at 60 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation After removing alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2- dilauroyl phosphatidyls Monoethanolamine chitosan.
1, the 2- dilauroyl phosphatidyl ethanol amine aqueous solutions of 1mg/mL are prepared, 100uL are taken, with including SPIO's DOTAP cationic-liposomes 1mL is mixed by way of ultrasound, then stands 1h, by way of rear insertion self assembly, to fat Plastid is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 9
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2-, bis- myristoyl phosphatidyls Monoethanolamine 0.3g, nitrogen are protected, and stirring reaction 42h at 60 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotate and steam After hair removes alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2-, bis- myristoyl phosphorus Acyl monoethanolamine chitosan.
The 1,2-dimristoyl phosphatidylethanolamine l aqueous solution of 1mg/mL is prepared, 100uL is taken, with including SPIO's DOTAP cationic-liposomes 1mL is mixed by way of ultrasound, then stands 1h, by way of rear insertion self assembly, to fat Plastid is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 10
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2-, bis- palmityl phosphatidyl second Hydramine 0.3g, nitrogen are protected, and stirring reaction 42h at 60 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation After removing alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2-, bis- palmityl phosphatidyls Monoethanolamine chitosan.
1, the 2- dipalmitoylphosphatidylethanolamine chitosan aqueous solutions of 1mg/mL are prepared, 100uL are taken, with including SPIO DOTAP cationic-liposomes 1mL ultrasound by way of mix, then stand 1h, it is rear insertion self assembly by way of, it is right Liposome is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 11
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2- distearoyl base phosphatidyl second Hydramine 0.3g, nitrogen are protected, and stirring reaction 42h at 50 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation After removing alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2- distearoyl base phosphatidyls Monoethanolamine chitosan.
1, the 2- distearoyl base phosphatidyl-ethanolamine chitosan aqueous solutions of 1mg/mL are prepared, 500uL are taken, with including SPIO DOTAP cationic-liposomes 1mL ultrasound by way of mix, then stand 1h, it is rear insertion self assembly by way of, it is right Liposome is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 12
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2- dioleoyl phosphatidyl ethanols Amine 0.3g, nitrogen are protected, and stirring reaction 42h at 50 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation is gone After alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2- dioleoyl phosphatidyl ethanols Amine chitosan.
1, the 2- dioleoylphosphatidylethanolamine aqueous solutions of 1mg/mL are prepared, 500uL are taken, with the DOTAP comprising SPIO Cationic-liposome 1mL is mixed by way of ultrasound, then stands 1h, by way of rear insertion self assembly, to liposome Modified, obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 13
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2- dilauroyl phosphatidyl second Hydramine 0.3g, nitrogen are protected, and stirring reaction 42h at 50 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation After removing alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2- dilauroyl phosphatidyls Monoethanolamine chitosan.
1, the 2- dilauroyl phosphatidyl ethanol amine aqueous solutions of 1mg/mL are prepared, 500uL are taken, with including SPIO's DOTAP cationic-liposomes 1mL is mixed by way of ultrasound, then stands 1h, by way of rear insertion self assembly, to fat Plastid is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 14
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2-, bis- myristoyl phosphatidyls Monoethanolamine 0.3g, nitrogen are protected, and stirring reaction 42h at 50 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotate and steam After hair removes alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2-, bis- myristoyl phosphorus Acyl monoethanolamine chitosan.
The 1,2-dimristoyl phosphatidylethanolamine l aqueous solution of 1mg/mL is prepared, 500uL is taken, with including SPIO's DOTAP cationic-liposomes 1mL is mixed by way of ultrasound, then stands 1h, by way of rear insertion self assembly, to fat Plastid is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 15
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2-, bis- palmityl phosphatidyl second Hydramine 0.3g, nitrogen are protected, and stirring reaction 42h at 50 DEG C, adds 0.1g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation After removing alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2-, bis- palmityl phosphatidyls Monoethanolamine chitosan.
1, the 2- dipalmitoylphosphatidylethanolamine chitosan aqueous solutions of 1mg/mL are prepared, 500uL are taken, with including SPIO DOTAP cationic-liposomes 1mL ultrasound by way of mix, then stand 1h, it is rear insertion self assembly by way of, it is right Liposome is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 16
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2- distearoyl base phosphatidyl second Hydramine 0.3g, nitrogen are protected, and stirring reaction 42h at 80 DEG C, adds 0.2g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation After removing alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2- distearoyl base phosphatidyls Monoethanolamine chitosan.
1, the 2- distearoyl base phosphatidyl-ethanolamine chitosan aqueous solutions of 1mg/mL are prepared, 500uL are taken, with including SPIO DOTAP cationic-liposomes 1mL ultrasound by way of mix, then stand 2h, it is rear insertion self assembly by way of, it is right Liposome is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 17
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2- dioleoyl phosphatidyl ethanols Amine 0.3g, nitrogen are protected, and stirring reaction 42h at 80 DEG C, adds 0.2g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation is gone After alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2- dioleoyl phosphatidyl ethanols Amine chitosan.
1, the 2- dioleoylphosphatidylethanolamine aqueous solutions of 1mg/mL are prepared, 500uL are taken, with the DOTAP comprising SPIO Cationic-liposome 1mL is mixed by way of ultrasound, then stands 2h, by way of rear insertion self assembly, to liposome Modified, obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 18
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2- dilauroyl phosphatidyl second Hydramine 0.3g, nitrogen are protected, and stirring reaction 42h at 80 DEG C, adds 0.2g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation After removing alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2- dilauroyl phosphatidyls Monoethanolamine chitosan.
1, the 2- dilauroyl phosphatidyl ethanol amine aqueous solutions of 1mg/mL are prepared, 500uL are taken, with including SPIO's DOTAP cationic-liposomes 1mL is mixed by way of ultrasound, then stands 2h, by way of rear insertion self assembly, to fat Plastid is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 19
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2-, bis- myristoyl phosphatidyls Monoethanolamine 0.3g, nitrogen are protected, and stirring reaction 42h at 80 DEG C, adds 0.2g sodium borohydride room temperatures and continue to stir 24h, rotate and steam After hair removes alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2-, bis- myristoyl phosphorus Acyl monoethanolamine chitosan.
The 1,2-dimristoyl phosphatidylethanolamine l aqueous solution of 1mg/mL is prepared, 500uL is taken, with including SPIO's DOTAP cationic-liposomes 1mL is mixed by way of ultrasound, then stands 2h, by way of rear insertion self assembly, to fat Plastid is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Embodiment 20
Formoxyl chitosan 0.16g is weighed, is distributed in 20mL methanol solutions, adds 1,2-, bis- palmityl phosphatidyl second Hydramine 0.3g, nitrogen are protected, and stirring reaction 42h at 80 DEG C, adds 0.2g sodium borohydride room temperatures and continue to stir 24h, rotary evaporation After removing alcohol solvent, dialysed, then be freeze-dried with deionized water after being distributed in water, obtain 1,2-, bis- palmityl phosphatidyls Monoethanolamine chitosan.
1, the 2- dipalmitoylphosphatidylethanolamine chitosan aqueous solutions of 1mg/mL are prepared, 500uL are taken, with including SPIO DOTAP cationic-liposomes 1mL ultrasound by way of mix, then stand 2h, it is rear insertion self assembly by way of, it is right Liposome is modified, and obtaining surface has the lipidosome drug carrier of chitosan brush.
Liposome gene transhipment measure with chitosan brush
PGL3 plasmids are used as reporter gene, the gene delivery performance of the liposome vectors with chitosan brush is carried out Evaluation, cell behaviour non-small cell lung cancer cell A549 cell lines used.By cultured plating cells, cultivate in the incubator After reaching 80% to cell fusion degree, gene delivery is carried out, during transhipment, complete medium is sucked, is washed twice with PBS, blood When being transported 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 are added (embodiment 3) and DNA compounds (contain 1 μ g DNA) per hole, and after cultivating 18h, sucking-off culture medium, changes and fresh contain 10% serum Culture medium continue cultivate 32h after, it is multi-functional in BioTek Synergy2 according to the specification that luciferase kit is provided The intensity of 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 eggs (the opposite number of photons corresponding to every milligram of albumen) in vain.
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 more than 85% in cell incubator.Remove Culture medium, after washing 2 times with PBS, adds fresh culture and carrier to be measured, after cultivating 24h, adds 20 μ L 5mg/mL's per hole 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 Jia Za, and 150 μ L DMSO are added per hole makes dissolving, continues to be incubated 30min at 37 DEG C.In multi-function microplate reader The absorption value in each hole of 570nm wavelength is measured on (Sunrise Tecan), 96 orifice plate automatic mixing 600s of vibrations before detecting, and adopt Returned to zero with acellular culture medium to microplate reader.Cell survival rate is calculated by formula 1.1：
Cell survival rate (%)=A570SMP/A570CTL×100 (1.1)
Wherein A570SMPTo add the light absorption value of the cell orifice plate of carrier or compound to be measured, 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 essences in the present invention All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.

Claims (1)

1. the application of pair fatty chain substituent phosphatidyl-ethanolamine chitosan, it is characterised in that by double fatty chain substituent phosphatide Acyl monoethanolamine chitosan prepares double fatty chain substituent phosphatidyl-ethanolamine chitosan-liposomes and is used for anti-human non-small cell lung cancer It is prepared by cell A549 pharmaceutical carriers：
Weighing formoxyl chitosan to be distributed in methanol solution, add 1,2- dilauroyl phosphatidyl-ethanolamines, nitrogen is protected, Stirring reaction 42h at 60 DEG C, adds sodium borohydride room temperature and continues to stir 24h, after rotary evaporation removes alcohol solvent, be distributed to water In after dialysed with deionized water, then be freeze-dried, obtain 1,2- dilauroyl phosphatidyl-ethanolamine chitosans；
Prepare 1,2- dilauroyl phosphatidyl ethanol amine aqueous solutions and pass through ultrasound with the DOTAP cationic-liposomes comprising SPIO Mode mix, stand, it is rear insertion self assembly by way of, liposome is modified, obtain surface there is chitosan brush The lipidosome drug carrier of son；
Non-small cell lung carcinoma cell A549 cell lines are cultivated in the incubator after reaching 80% to cell fusion degree, will cultivated Base sucks, and is washed with PBS, when being transported under serum condition, adds culture medium and lipidosome drug carrier and DNA containing 10% serum Compound, after cultivating 18h, suctions out culture medium, changes the fresh culture medium containing 10% serum and continues to cultivate 32h.
CN201711363440.3A 2017-07-27 2017-07-27 Application of double-aliphatic-chain substituent phosphatidyl ethanolamine chitosan Active CN107903341B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201711363440.3A CN107903341B (en) 2017-07-27 2017-07-27 Application of double-aliphatic-chain substituent phosphatidyl ethanolamine chitosan

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201711363440.3A CN107903341B (en) 2017-07-27 2017-07-27 Application of double-aliphatic-chain substituent phosphatidyl ethanolamine chitosan
CN201710620704.2A CN107400179B (en) 2017-07-27 2017-07-27 Double fat chain substituent phosphatidyl-ethanolamine chitosans and its preparation method and application

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN201710620704.2A Division CN107400179B (en) 2017-07-27 2017-07-27 Double fat chain substituent phosphatidyl-ethanolamine chitosans and its preparation method and application

Publications (2)

Publication Number Publication Date
CN107903341A true CN107903341A (en) 2018-04-13
CN107903341B CN107903341B (en) 2019-12-13

Family

ID=60401298

Family Applications (2)

Application Number Title Priority Date Filing Date
CN201711363440.3A Active CN107903341B (en) 2017-07-27 2017-07-27 Application of double-aliphatic-chain substituent phosphatidyl ethanolamine chitosan
CN201710620704.2A Active CN107400179B (en) 2017-07-27 2017-07-27 Double fat chain substituent phosphatidyl-ethanolamine chitosans and its preparation method and application

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN201710620704.2A Active CN107400179B (en) 2017-07-27 2017-07-27 Double fat chain substituent phosphatidyl-ethanolamine chitosans and its preparation method and application

Country Status (1)

Country Link
CN (2) CN107903341B (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1827096A (en) * 2006-04-12 2006-09-06 中国药科大学 Docetaxel liposome containing chitosan derivative, lyophiled preparation and preparation method thereof
CN101062417A (en) * 2007-05-17 2007-10-31 浙江大学 Method for preparing gene medication carrier by using chitosan modified liposome
CN101265304A (en) * 2008-04-18 2008-09-17 中国药科大学 Bionic chitosan derivatives, preparation method thereof and lipoid plastid preparation containing the same
CN102000351A (en) * 2010-12-02 2011-04-06 上海纳米技术及应用国家工程研究中心有限公司 Method for preparing chitosan-modified magnetic liposomes
CN102133405A (en) * 2011-03-08 2011-07-27 大连民族学院 Preparation method and application of liposome-chitosan compound gene carrier
CN103553970A (en) * 2013-11-22 2014-02-05 大连民族学院 Preparation method of carbamate cationic lipid and application of carbamate cationic lipid in medicine or gene delivery
CN103565744A (en) * 2012-07-26 2014-02-12 上海晟纳实业有限公司 Macromolecular phosphatidylglycerol nanoliposome, and preparation methods and applications thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2382994A1 (en) * 2010-04-26 2011-11-02 Maurizio Victor Cattaneo Ligand targeted nanocapsules for the delivery of RNAi and other agents
CN102952263B (en) * 2011-08-23 2014-05-07 国家纳米科学中心 Graft polymer, and preparation method and use thereof
WO2014054026A1 (en) * 2012-10-04 2014-04-10 University Of The Witwatersrand, Johannesburg Liposomal drug delivery system

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1827096A (en) * 2006-04-12 2006-09-06 中国药科大学 Docetaxel liposome containing chitosan derivative, lyophiled preparation and preparation method thereof
CN101062417A (en) * 2007-05-17 2007-10-31 浙江大学 Method for preparing gene medication carrier by using chitosan modified liposome
CN101265304A (en) * 2008-04-18 2008-09-17 中国药科大学 Bionic chitosan derivatives, preparation method thereof and lipoid plastid preparation containing the same
CN102000351A (en) * 2010-12-02 2011-04-06 上海纳米技术及应用国家工程研究中心有限公司 Method for preparing chitosan-modified magnetic liposomes
CN102133405A (en) * 2011-03-08 2011-07-27 大连民族学院 Preparation method and application of liposome-chitosan compound gene carrier
CN103565744A (en) * 2012-07-26 2014-02-12 上海晟纳实业有限公司 Macromolecular phosphatidylglycerol nanoliposome, and preparation methods and applications thereof
CN103553970A (en) * 2013-11-22 2014-02-05 大连民族学院 Preparation method of carbamate cationic lipid and application of carbamate cationic lipid in medicine or gene delivery

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BING WANG等: "Chitosan enhanced gene delivery of cationic liposome", 《BIOTECHNOL LETT》 *
M. PRABAHARAN等: "Carboxymethyl chitosan-graft-phosphatidylethanolamine: Amphiphilic matrices for controlled drug delivery", 《REACTIVE & FUNCTIONAL POLYMERS》 *

Also Published As

Publication number Publication date
CN107400179A (en) 2017-11-28
CN107400179B (en) 2019-06-28
CN107903341B (en) 2019-12-13

Similar Documents

Publication Publication Date Title
CN107982547B (en) Application of redox response chitosan-liposome
Lee et al. Platinum (II) drug-loaded gold nanoshells for chemo-photothermal therapy in colorectal cancer
Zhu et al. Bubble-generating polymersomes loaded with both indocyanine green and doxorubicin for effective chemotherapy combined with photothermal therapy
Yu et al. Triple-functional albumin-based nanoparticles for combined chemotherapy and photodynamic therapy of pancreatic cancer with lymphatic metastases
CN104398493B (en) A kind of tumor-targeting nanoscale medicine delivery system of reversible tumor drug resistance
CN102188382B (en) DSPE-PEG-FA-modified nanometer paclitaxel liposome and preparation method thereof
CN108653733B (en) Polymer vesicle of double-loaded anthracycline drug and photosensitizer with bubble generation function and preparation method thereof
CN106265514B (en) A kind of doxorubicin hydrochloride magnetic nano particle and preparation method thereof
Chen et al. Transferrin and folic acid co-modified bufalin-loaded nanoliposomes: Preparation, characterization, and application in anticancer activity
CN106344924A (en) Nano-formulation combined with metabolic block and drug resistant reverse application thereof
Lin et al. A phthalocyanine-based liposomal nanophotosensitizer with highly efficient tumor-targeting and photodynamic activity
CN106420664A (en) Application of adopting aspirin conjugate with antitumor activity as drug carrier or molecular probe carrier
Li et al. Self-assembled dual-targeted epirubicin-hybrid polydopamine nanoparticles for combined chemo-photothermal therapy of triple-negative breast cancer
Wang et al. Polyphenol-based nanoplatform for MRI/PET dual-modality imaging guided effective combination chemotherapy
CN101732724B (en) Composite micelle carrying anti-tumor medicine and preparation method thereof
CN100998563A (en) Magnetic pegylated liposomal doxorubicin and preparation method and application
Liu et al. Magnetic resonance imaging-guided multi-stimulus-responsive drug delivery strategy for personalized and precise cancer treatment
Wang et al. Recent progress in metal-organic cages for biomedical application: Highlighted research during 2018–2023
Zhao et al. Hepatocellular carcinoma targeting and pharmacodynamics of paclitaxel nanoliposomes modified by glycyrrhetinic acid and ferric tetroxide
CN105287612B (en) Salinomycin Sodium and adriamycin nano liposome and the preparation method and application thereof are carried altogether
CN107400179B (en) Double fat chain substituent phosphatidyl-ethanolamine chitosans and its preparation method and application
Liu et al. Precise delivery of multi-stimulus-responsive nanocarriers based on interchangeable visual guidance
CN113616790B (en) Engineered neutrophil sonosensitizer drug delivery system and application thereof
CN105056239A (en) Composite material of functional mesoporous silica loaded drug and siRNA, preparation and application thereof in preparation of anticancer drugs
CN113577277B (en) PEOz and polydopamine-gadolinium ion network modified degradable mesoporous silicon nano drug delivery system and preparation method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant