CN107137377A - A kind of tumor stem cell targeting lipids nanoparticle and preparation method and application - Google Patents

A kind of tumor stem cell targeting lipids nanoparticle and preparation method and application Download PDF

Info

Publication number
CN107137377A
CN107137377A CN201710198933.XA CN201710198933A CN107137377A CN 107137377 A CN107137377 A CN 107137377A CN 201710198933 A CN201710198933 A CN 201710198933A CN 107137377 A CN107137377 A CN 107137377A
Authority
CN
China
Prior art keywords
stem cell
polyethylene glycol
octadecyl alcolol
tumor stem
reaction
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
CN201710198933.XA
Other languages
Chinese (zh)
Other versions
CN107137377B (en
Inventor
胡富强
孟廷廷
袁弘
袁铭
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.)
Zhejiang University ZJU
Original Assignee
Zhejiang University ZJU
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 Zhejiang University ZJU filed Critical Zhejiang University ZJU
Priority to CN201710198933.XA priority Critical patent/CN107137377B/en
Publication of CN107137377A publication Critical patent/CN107137377A/en
Application granted granted Critical
Publication of CN107137377B publication Critical patent/CN107137377B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Biomedical Technology (AREA)
  • Nanotechnology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention provides a kind of tumor stem cell targeting lipids nanoparticle, it is made up of A15 polyethylene glycol octadecyl alcolols grafting, glycerin monostearate, salinomycin, the polyethylene glycol of the hydrophilic end carboxyl containing Amino End Group links octadecyl alcolol by esterification, the long fatty segment of octadecyl alcolol can be inserted into lipid nano particle, wherein A15 is RNA aptamers, it is strong with CD133 molecules affinity, the hepatic carcinoma stem cell of CD133 height expression can be targetted.The lipid nano particle encapsulates tumor stem cell specific drug salinomycin as carrier, by A15 targeting, is delivered to tumor stem cell and plays antineoplastic effect, targets and treat the hepatic carcinoma stem cell of CD133 molecules height expression.

Description

A kind of tumor stem cell targeting lipids nanoparticle and preparation method and application
Technical field
The invention belongs to targeting lipids nanoparticle and preparation method, a kind of tumor stem cell targeting lipids nanoparticle is related generally to And preparation method thereof.
Technical background
Solid lipid nano granule (Solid lipid nanoparticle, SLN) was developed in early 1990s The new colloidal delivery system of a kind of alternative emulsion, liposome and the polymer nanoparticle that come, belongs to submicron delivery system. SLN is easily prepared and available for intravenously administrable, it can also be used to oral or topical administration, is adapted to a variety of methods of administration.SLN is used as medicine Carrier can improve the bioavilability of medicine, can control insoluble drug release and play good targeting.
Aptamer (Aptamer) is short DNA or RNA fragments, is typically made up of 12~30 bases.Aptamers can The steric configuration of specific structure is formed, is specifically bound with target protein.Compared with antibody, aptamers possess molecular weight Small, low immunogenicity, the advantages of easily obtain.A15 is RNA aptamers, can recognize that tumor stem cell mark CD133 molecules, and Have compared with strong affinity and specificity.A15 is made up of 15 ribonucleotides, document report, compared with monoclonal antibody, the adaptation Body penetrating power in tumour cell ball is strong, retention time is long.
Salinomycin is a kind of polyethers ionic antibiotic, has stronger to most of gram-positive bacterias and various coccidias Suppression and killing action.Its preclinical and clinical data is less, a small number of document reports its there is serious muscle and god Through toxicity, limit it and be mainly used in poultry disease.Gupta in 2009 et al. passes through high flux screening method, finds salinomycin tool There is the ability of killing breast carcinoma stem cell, the effect for reducing breast carcinoma stem cell ratio is higher than conventional chemotherapy medicine taxol 100 times. In other tumours, such as chronic lymphocytic leukemia, also having been demonstrated can specific killing tumor stem cell.
The content of the invention
It is an object of the present invention to provide a kind of lipid nano particle of tumor stem cell targeting, the nanoparticle is gathered by A15- Ethylene glycol-octadecyl alcolol grafting, glycerin monostearate, monostearate macrogol ester, salinomycin composition, the poly- second of wherein A15- The percentage by weight of glycol-octadecyl alcolol grafting is that 9.1~17.9wt%, the percentage by weight of glycerin monostearate are 69.5 ~72.0wt%, the percentage by weight of monostearate macrogol ester is 3.6~7.6wt%, and the percentage by weight of salinomycin is 3.6~7.6wt%.
Second object of the present invention is to provide the preparation method of the tumor stem cell targeting lipids nanoparticle, specific logical Cross following steps realization：
(1) 20mg glycerin monostearates, 1.0~2.0mg monostearates macrogol ester and 1.0~2.0mg are taken respectively Amino End Group polyethylene glycol-octadecyl alcolol grafting, 1.0~2.0mg salinomycins are placed in 2.0mL absolute ethyl alcohols, and 50 DEG C of heating of water-bath are molten Solution, is organic phase, and under the conditions of ultra-pure water is dispersed phase, magnetic agitation, organic phase injection dispersed phase continues to stir 5min, must contained Lipid nano particle (the NH of free amine group2-PEG-SLN/SAL)；
(2) free amine group lipid nano particle (NH is taken2- PEG-SLN/SAL) dispersion liquid, pyrocarbonic acid diethyl ester processing water dilution Nanoparticle concentration is to 100 μ g/mL.The N of 1~2 times of amino mole of addition, the succinimidyl carbonates of N- bis- (DSC), room temperature is stirred Mix (400rpm) reaction 9h.The amino A15 of equimolar number is added, continues stirring reaction and stays overnight.After reaction terminates, reaction solution is put In the ultra-filtration centrifuge tube that molecular weight is 30kDa.Centrifugation, abandons filtrate, adds the cleaning of pyrocarbonic acid diethyl ester processing water twice, removes not The aptamers of reaction.Non- filtered solution is A15-PEG3400- SLN/SAL nano dispersion fluids.
Third object of the present invention is to provide the synthetic method of Amino End Group polyethylene glycol-octadecyl alcolol grafting, by with Lower step is realized：Polyethylene glycol (the NH containing Amino End Group end carboxyl is taken respectively2-PEG3400- COOH), be dissolved in anhydrous methylene chloride Concentration 10mg/mL solution is made into, the octadecyl alcolol (ODC) with mole is added.According to mol ratio octadecyl alcolol：Dicyclohexyl carbon two Imines：N-hydroxysuccinimide 1:1:1~1.5 ratio adds dicyclohexylcarbodiimide and n-hydroxysuccinimide, 24h is reacted at room temperature, after reaction terminates, reaction solution is added dropwise to the cold absolute ether of 8~10 times of volumes, 4 DEG C overnight, analyse product Go out, precipitation is collected by centrifugation, abandon supernatant, collect precipitation, 40 DEG C of drying obtain Amino End Group polyethylene glycol-octadecyl alcolol grafting (NH2- PEG3400-ODC)。
The lipid nano particle that fourth object of the present invention is to provide described tumor stem cell targeting is preparing administration system Application in system.The tumor stem cell is the liver-cancer stem cell of CD133 molecules height expression.Contained medicine is that tumor stem cell is special Specific agent salinomycin.
The A15 aptamers that the present invention chooses, the surface molecular label CD133 molecules with hepatic carcinoma stem cell, have Stronger affinity, can pass through the specific binding with CD133 molecules, liver cancer targeting tumor stem cell.Hydrophilic contains Amino End Group The polyethylene glycol of end carboxyl links octadecyl alcolol by esterification, and the long fatty segment of octadecyl alcolol can be inserted into lipid nano particle.Should The Amino End Group of polyethylene glycol is free in lipid nano particle periphery, and N, the succinimidos of N- bis- are passed through with the amino of A15 aptamers Carbonic ester is connected chemically, and realizes that A15 aptamers modify lipid nano particle.Liver-cancer stem cell targeting lipids nanoparticle is used as carrier bag Tumor stem cell specific drug salinomycin is sealed, tumor stem cell is delivered to by A15 targeting, and improve salinomycin The characteristics of slightly water-soluble and in vivo distribution difference, strengthen salinomycin drug effect.
Brief description of the drawings
Fig. 1 is infrared point of octadecyl alcolol, Amino End Group end carboxyl polyethylene glycol and Amino End Group polyethylene glycol-octadecyl alcolol grafting Analyse collection of illustrative plates.
Fig. 2 is that tumor spheres absorb the cell percentages of the nanoparticle and absorb the flow cytometer showed figure of fluorescence intensity, Blank control is not to be incubated nanoparticle group cell.
Embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
First, Amino End Group polyethylene glycol-octadecyl alcolol grafting (NH2-PEG3400- ODC) prepare
Embodiment 1：
Take 200mg NH2-PEG3400- COOH, 15.6mg octadecyl alcolols (ODC), are dissolved in 20mL anhydrous methylene chlorides, 11.9mg Add dicyclohexylcarbodiimide 6.6mgN- HOSu NHSs, room temperature magnetic agitation (300rpm) reaction 24h.Reaction knot Shu Hou, by the cold absolute ethers of the 200mL being added dropwise in reaction solution.4 DEG C overnight, make product fully separate out.Under the conditions of 4 DEG C 3000rpm centrifuges 5min, collects precipitation, and drying obtains Amino End Group polyethylene glycol-octadecyl alcolol grafting (NH2-PEG3400-ODC)。
Embodiment 2：
Take 200mg NH2-PEG3400- COOH, 7.8mg octadecyl alcolols (ODC), are dissolved in 20mL anhydrous methylene chlorides, add 11.9mg dicyclohexylcarbodiimide 10mg n-hydroxysuccinimides, room temperature magnetic agitation (300rpm) reaction 24h.Reaction After end, by the cold absolute ethers of the 200mL being added dropwise in reaction solution.4 DEG C overnight, make product fully separate out.Under the conditions of 4 DEG C 3000rpm centrifuges 5min, collects precipitation, and drying obtains Amino End Group polyethylene glycol-octadecyl alcolol grafting (NH2-PEG3400-ODC)。
Embodiment 3：
Take 200mg NH2-PEG3400- COOH, 7.8mg octadecyl alcolols (ODC), are dissolved in 20mL anhydrous methylene chlorides, add 11.9mg dicyclohexylcarbodiimide 7.9mg n-hydroxysuccinimides, room temperature magnetic agitation (300rpm) reaction 24h.Instead After should terminating, by the cold absolute ethers of the 160mL being added dropwise in reaction solution.4 DEG C overnight, make product fully separate out.Under the conditions of 4 DEG C 3000rpm centrifuges 5min, collects precipitation, and drying obtains Amino End Group polyethylene glycol-octadecyl alcolol grafting (NH2-PEG3400-ODC)。
Fourier transform infrared spectroscopy confirms grafting structure, such as accompanying drawing 1.At product infrared spectrogram, 720ppm positions There is the deformation vibration peak for continuous four methylene (- (CH2) 4-) for belonging to octadecyl alcolol, and the hydroxyl (- OH) at 3350ppm Stretching vibration peak disappears, and 1650ppm absworption peaks are attributed to the new stretching vibration peak for forming ester bond (C=O).
2nd, free amine group lipid nano particle (NH2- PEG-SLN/SAL) prepare
Embodiment 1
Take 20mg glycerin monostearates, 1.0mg salinomycins (SAL), 2mg monostearates macrogol ester and 2mg NH2- PEG3400- ODC graftings, it is accurately weighed, 2.0mL absolute ethyl alcohols are placed in, 50 DEG C of dissolvings of water-bath, are organic phase.20mL ultra-pure waters For dispersed phase, under mechanical agitation (400rpm), organic phase is rapidly injected dispersed phase, stirs 5min.Saturated nacl aqueous solution 0.5mL adjusts ionic strength, and flocculate nanoparticle, obtains (the NH of lipid nano particle containing free amine group2-PEG-SLN/SAL).After measured NH2- PEG-SLN/SAL particle diameter and surface potential be respectively
Embodiment 2
Take 20mg glycerin monostearates, 1.5mg salinomycins (SAL), 2.0mg monostearates macrogol ester and 2mg NH2-PEG3400- ODC graftings, it is accurately weighed, 2.0mL absolute ethyl alcohols are placed in, 50 DEG C of dissolvings of water-bath, are organic phase.20mL is ultrapure Water is that under dispersed phase, mechanical agitation (400rpm), organic phase is rapidly injected dispersed phase, stirs 5min.Saturated sodium-chloride is molten Liquid 0.5mL adjusts ionic strength, and flocculate nanoparticle, obtains (the NH of lipid nano particle containing free amine group2-PEG-SLN/SAL).After measured NH2- PEG-SLN/SAL particle diameter 90.2nm and surface potential is -9.5mV.
Embodiment 3
Take 20mg glycerin monostearates, 2.0mg salinomycins (SAL), 2.0mg monostearates macrogol ester and 2.0mg NH2-PEG3400- ODC graftings, it is accurately weighed, 2.0mL absolute ethyl alcohols are placed in, 50 DEG C of dissolvings of water-bath, are organic phase.20mL is ultrapure Water is that under dispersed phase, mechanical agitation (400rpm), organic phase is rapidly injected dispersed phase, stirs 5min.Saturated sodium-chloride is molten Liquid 0.5mL adjusts ionic strength, and flocculate nanoparticle, obtains (the NH of lipid nano particle containing free amine group2-PEG-SLN/SAL).After measured NH2- PEG-SLN/SAL particle diameter 93.2nm and surface potential is -10.2mV.
Embodiment 4
Take 20mg glycerin monostearates, 2.0mg salinomycins (SAL), 2.0mg monostearates macrogol ester and 1.5mg NH2-PEG3400- ODC graftings, it is accurately weighed, 2.0mL absolute ethyl alcohols are placed in, 50 DEG C of dissolvings of water-bath, are organic phase.20mL is ultrapure Water is that under dispersed phase, mechanical agitation (400rpm), organic phase is rapidly injected dispersed phase, stirs 5min.Saturated sodium-chloride is molten Liquid 0.5mL adjusts ionic strength, and flocculate nanoparticle, obtains (the NH of lipid nano particle containing free amine group2-PEG-SLN/SAL).After measured NH2- PEG-SLN/SAL particle diameter is 92.5nm and surface potential is -10.7mV.
Embodiment 5
Take 20mg glycerin monostearates, 2.0mg salinomycins (SAL), 2.0mg monostearates macrogol ester and 1.0mg NH2-PEG3400- ODC graftings, it is accurately weighed, 2.0mL absolute ethyl alcohols are placed in, 50 DEG C of dissolvings of water-bath, are organic phase.20mL is ultrapure Water is that under dispersed phase, mechanical agitation (400rpm), organic phase is rapidly injected dispersed phase, stirs 5min.Saturated sodium-chloride is molten Liquid 0.5mL adjusts ionic strength, and flocculate nanoparticle, obtains (the NH of lipid nano particle containing free amine group2-PEG-SLN/SAL).After measured NH2- PEG-SLN/SAL particle diameter 94.5nm and surface potential is -11.0mV.
Embodiment 6
Take 20mg glycerin monostearates, 2.0mg salinomycins (SAL), 1.5mg monostearates macrogol ester and 2.0mg NH2-PEG3400- ODC graftings, it is accurately weighed, 2.0mL absolute ethyl alcohols are placed in, 50 DEG C of dissolvings of water-bath, are organic phase.20mL is ultrapure Water is that under dispersed phase, mechanical agitation (400rpm), organic phase is rapidly injected dispersed phase, stirs 5min.Saturated sodium-chloride is molten Liquid 0.5mL adjusts ionic strength, and flocculate nanoparticle, obtains (the NH of lipid nano particle containing free amine group2-PEG-SLN/SAL).After measured NH2- PEG-SLN/SAL particle diameter 96.8nm and surface potential is -10.2mV.
Embodiment 7
Take 20mg glycerin monostearates, 2.0mg salinomycins (SAL), 1.0mg monostearates macrogol ester and 2.0mg NH2-PEG3400- ODC graftings, it is accurately weighed, 2.0mL absolute ethyl alcohols are placed in, 50 DEG C of dissolvings of water-bath, are organic phase.20mL is ultrapure Water is that under dispersed phase, mechanical agitation (400rpm), organic phase is rapidly injected dispersed phase, stirs 5min.Saturated sodium-chloride is molten Liquid 0.5mL adjusts ionic strength, and flocculate nanoparticle, obtains (the NH of lipid nano particle containing free amine group2-PEG-SLN/SAL).After measured NH2- PEG-SLN/SAL particle diameter 98.0nm and surface potential is -10.7mV.
Embodiment 8
Take 20mg glycerin monostearates, 1.0mg salinomycins (SAL), 1.0mg monostearates macrogol ester and 2.0mg NH2-PEG3400- ODC graftings, it is accurately weighed, 2.0mL absolute ethyl alcohols are placed in, 50 DEG C of dissolvings of water-bath, are organic phase.20mL is ultrapure Water is that under dispersed phase, mechanical agitation (400rpm), organic phase is rapidly injected dispersed phase, stirs 5min.Saturated sodium-chloride is molten Liquid 0.5mL adjusts ionic strength, and flocculate nanoparticle, obtains (the NH of lipid nano particle containing free amine group2-PEG-SLN/SAL).After measured NH2- PEG-SLN/SAL particle diameter 85.4nm and surface potential is -10.1mV.
3rd, prepared by liver-cancer stem cell targeting lipids nanoparticle
Embodiment 1
Take by 20mg glycerin monostearates, 2.0mg salinomycins (SAL), 2.0mg monostearates macrogol ester and 2.0mg NH2-PEG3400Free amine group lipid nano particle (NH prepared by-ODC graftings2- PEG-SLN/SAL) 200 μ of dispersion liquid L.Pyrocarbonic acid diethyl ester processing water dilutes nanoparticle concentration to 100 μ g/mL.Add 14 μ L 1.0mg/mL N, the succinyls of N- bis- Iminocarbonates (DSC) aqueous solution.Reaction 9h is stirred at room temperature.Add 54.9 μ L 1mM Amino End Group aptamers A15-NH2, after Continuous stirring reaction is stayed overnight.After reaction terminates, the ultra-filtration centrifuge tube that molecular weight is 30kDa is placed reaction liquid into.Under the conditions of 4 DEG C 20000rpm centrifuges 10min, abandons filtrate, adds the processing water cleaning of 0.5mL pyrocarbonic acid diethyl esters, repeats the step twice, remove Unreacted aptamers.Non- filtered solution is A15-PEG3400- SLN/SAL nanosuspensions.The particle diameter of nanoparticle after measured For 126nm, current potential is -14.6mV.
Embodiment 2
Take by 20mg glycerin monostearates, 2.0mg salinomycins (SAL), 2.0mg monostearates macrogol ester and 2.0mg NH2-PEG3400Free amine group lipid nano particle (NH prepared by-ODC graftings2- PEG-SLN/SAL) 200 μ of dispersion liquid L.Pyrocarbonic acid diethyl ester processing water dilutes nanoparticle concentration to 100 μ g/mL.Add 21 μ L 1.0mg/mL N, the succinyls of N- bis- Iminocarbonates (DSC) aqueous solution.Reaction 9h is stirred at room temperature.Add 54.9 μ L 1mM Amino End Group aptamers A15-NH2, after Continuous stirring reaction is stayed overnight.After reaction terminates, the ultra-filtration centrifuge tube that molecular weight is 30kDa is placed reaction liquid into.Under the conditions of 4 DEG C 20000rpm centrifuges 10min, abandons filtrate, adds the processing water cleaning of 0.5mL pyrocarbonic acid diethyl esters, repeats the step twice, remove Unreacted aptamers.Non- filtered solution is A15-PEG3400- SLN/SAL nanosuspensions.The particle diameter of nanoparticle after measured For 106nm, current potential is -16.2mV.
Embodiment 3
Take by 20mg glycerin monostearates, 2.0mg salinomycins (SAL), 2.0mg monostearates macrogol ester and 2.0mg NH2-PEG3400Free amine group lipid nano particle (NH prepared by-ODC graftings2- PEG-SLN/SAL) 200 μ of dispersion liquid L.Pyrocarbonic acid diethyl ester processing water dilutes nanoparticle concentration to 100 μ g/mL.Add 28 μ L 1.0mg/mL N, the succinyls of N- bis- Iminocarbonates (DSC) aqueous solution.Reaction 9h is stirred at room temperature.Add 54.9 μ L 1mM Amino End Group aptamers A15-NH2, after Continuous stirring reaction is stayed overnight.After reaction terminates, the ultra-filtration centrifuge tube that molecular weight is 30kDa is placed reaction liquid into.Under the conditions of 4 DEG C 20000rpm centrifuges 10min, abandons filtrate, adds the processing water cleaning of 0.5mL pyrocarbonic acid diethyl esters, repeats the step twice, remove Unreacted aptamers.Non- filtered solution is A15-PEG3400- SLN/SAL nanosuspensions.The particle diameter of nanoparticle after measured For 106nm, current potential is -14.4mV.
Connect after A15 aptamers, A15-PEG3400- SLN/SAL particle diameter is slightly increase；Amino End Group disappears, nanoparticle Current potential declines.
4th, the tumor spheres intake of tumor stem cell targeting lipids nanoparticle
Embodiment 1：
Take 20mg glycerin monostearates, 2mg monostearate macrogol esters, 2mg salinomycins and 2mg NH2-PEG3400- ODC, it is accurately weighed；100 μ g stearylamine-fluorescein isothiocynate is separately taken, 2.0mL absolute ethyl alcohols are placed in, 50 DEG C of water-bath is dissolved, For organic phase.20mL ultra-pure waters are organic phase injection dispersed phase under dispersed phase, mechanical agitation (400rpm), stir 5min. Saturated nacl aqueous solution adjusts the ionic strength of dispersion liquid, and flocculate nanoparticle, prepares the free amine group lipid nanometer of fluorescence labeling Grain
Take the fluorescence labeling NH of preparation2- PEG-SLN nanoparticles, pyrocarbonic acid diethyl ester processing water dilution nanoparticle concentration is extremely 100μg/mL.21 μ L 1.0mg/mL N, the succinimidyl carbonates of N- bis- (DSC) solution.Reaction 9h is stirred at room temperature.Add 54.9 μ L 1mM Amino End Group aptamers A15-NH2, continue stirring reaction and stay overnight.After reaction terminates, molecule is placed reaction liquid into Measure the ultra-filtration centrifuge tube for 30kDa.20000rpm centrifuges 10min under the conditions of 4 DEG C, abandons filtrate, adds appropriate pyrocarbonic acid diethyl ester Water cleaning is handled, the step is repeated twice, unreacted aptamers are removed.Non- filtered solution is the A15-PEG- of fluorescence labeling SLN/SAL nanosuspensions.
Immunological magnetic bead sorting method is taken to sort to obtain CD133+Hepatic carcinoma stem cell, and carry out serum free suspension ball culture, When cell ball growth in thickness is to about 200 μm, the A15-PEG-SLN/SAL nanosuspensions of fluorescence labeling are added, are incubated different Time (0.5h, 2h, 6h, 12h).
Referring to accompanying drawing 2, flow cytomery intake positive cell ratio and cell fluorescence intensity, 7.5% during 0.5h Cell is detectable higher than the fluorescent value for not being incubated cellular control unit, and 94.9% cell has green florescent signal during 6h, almost All cells of cell ball have intake.Incubation time continues to extend, and fluorescence intensity is moved to right, and cellular uptake amount is on the increase.A15 The liver-cancer stem cell targeting lipids nanoparticle intake of modification lipid nano particle formation is delivered to tumor stem cell.
5th, application of the tumor stem cell targeting lipids nanoparticle as pharmaceutical carrier in antineoplastic is prepared
Embodiment 1
Using Acid Phosphatase Test method method, free salinomycin and liver-cancer stem cell targeting lipids nanoparticle A15- are determined PEG-SLN/SAL tumor stem cell antiproliferative activity.Take and sort to obtain CD133+ tumor stem cells, serum free suspension ball culture, When cell ball growth in thickness is to about 200 μm, A15-PEG-SLN/SAL nanoparticles and salinomycin dimethyl sulfoxide solution are added.37 Under the conditions of DEG C 48h is incubated with cell ball.Determine the absorbance at 405nm and calculate cell survival rate.
The tumor stem cell IC of the hepatic carcinoma stem cell target lipid grain of rice50It is worth for 0.69 ± 0.015 μ g/mL.Salinomycin Group cell survival rate declines with the increase of concentration, when concentration is 0.8 μ g/mL, when survival rate is down to 55%, continues increase salt mould Plain concentration, because being limited by salinomycin dissolubility, medicine is separated out, and cell survival rate changes unobvious.Result of study shows that liver cancer swells Knurl stem cell target lipid nano particle, can improve the dissolubility of salinomycin, and salinomycin drug effect can be strengthened as pharmaceutical carrier.

Claims (6)

1. a kind of tumor stem cell targeting lipids nanoparticle, it is characterised in that by A15- polyethylene glycol-octadecyl alcolol grafting, single Tristerin is constituted, and the percentage by weight of wherein A15- polyethylene glycol-octadecyl alcolol grafting is 9.1~17.9wt%, list The percentage by weight of tristerin is 69.5~72.0wt%, and the percentage by weight of monostearate macrogol ester is 3.6 ~7.6wt%, the percentage by weight of salinomycin is 3.6~7.6wt%.
2. a kind of preparation method of tumor stem cell targeting lipids nanoparticle described in claim 1, it is characterised in that by with Lower step is realized：
(1) polyethylene glycol containing Amino End Group and end carboxyl, the octadecyl alcolol with mole are taken respectively, are dissolved in anhydrous methylene chloride. According to mol ratio octadecyl alcolol：Dicyclohexylcarbodiimide：N-hydroxysuccinimide 1:1:1~1.5 ratio adds two hexamethylenes Base carbodiimide and n-hydroxysuccinimide, after room temperature reaction, reaction solution are added dropwise to the cold anhydrous second of 8~10 times of volumes Ether, 4 DEG C overnight, separate out product, precipitation are collected by centrifugation, dry to obtain free terminal amino group polyethylene glycol-octadecyl alcolol grafting；
(2) 20mg glycerin monostearates, 1.0~2.0mg monostearates macrogol ester and 1.0~2.0mg ends ammonia are taken respectively Base polyethylene glycol-octadecyl alcolol grafting, 1.0~2.0mg salinomycins are placed in 2.0mL absolute ethyl alcohols, and 50 DEG C of heating for dissolving of water-bath are Organic phase, under the conditions of ultra-pure water is dispersed phase, magnetic agitation, organic phase injection dispersed phase continues to stir 5min, obtained containing free ammonia The lipid nano particle of base；
(3) take the lipid nano particle of free amine group, add the N of 1~2 times of amino molal quantity, the succinimidyl carbonates of N- bis-, 9h is reacted at room temperature, the Amino End Group aptamers A15 of amino molal quantity such as adds, reaction stays overnight, and after reaction terminates, places reaction liquid into Molecular weight is 30kDa ultra-filtration centrifuge tube, and high speed centrifugation abandons filtrate, plus the cleaning of pyrocarbonic acid diethyl ester processing water is twice, removes not The A15 of reaction, non-filtered solution is hepatic carcinoma stem cell target lipid nano particle dispersion liquid.
3. a kind of tumor stem cell targeting lipids nanoparticle according to claim 1, it is characterised in that Amino End Group therein Step realization is synthesized by the following way in polyethylene glycol-octadecyl alcolol grafting：Take the polyethylene glycol containing Amino End Group end carboxyl, be dissolved in nothing Water dichloromethane is made into concentration 10mg/mL solution, the octadecyl alcolol with mole is added, according to mol ratio octadecyl alcolol：Two hexamethylenes Base carbodiimide：N-hydroxysuccinimide 1:1:1~1.5 ratio adds dicyclohexylcarbodiimide and N- hydroxysuccinimidyl acyls Imines, reacts at room temperature 24h, after reaction terminates, and reaction solution is added dropwise to the cold absolute ether of 8~10 times of volumes, and 4 DEG C overnight, make Product is separated out, and precipitation is collected by centrifugation, and abandons supernatant, collects precipitation, and 40 DEG C of drying obtain Amino End Group polyethylene glycol-octadecyl alcolol grafting Thing.
4. tumor stem cell targeting lipids nanoparticle according to claim 1 is preparing antineoplastic as drug-loading system In application.
5. application according to claim 4, it is characterised in that the tumor stem cell is the liver of CD133 molecules height expression Cancer stem cell.
6. application according to claim 4, it is characterised in that contained medicine is salinomycin.
CN201710198933.XA 2017-03-29 2017-03-29 A kind of tumor stem cell targeting lipids nanoparticle and preparation method and application Active CN107137377B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710198933.XA CN107137377B (en) 2017-03-29 2017-03-29 A kind of tumor stem cell targeting lipids nanoparticle and preparation method and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710198933.XA CN107137377B (en) 2017-03-29 2017-03-29 A kind of tumor stem cell targeting lipids nanoparticle and preparation method and application

Publications (2)

Publication Number Publication Date
CN107137377A true CN107137377A (en) 2017-09-08
CN107137377B CN107137377B (en) 2019-10-11

Family

ID=59783673

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710198933.XA Active CN107137377B (en) 2017-03-29 2017-03-29 A kind of tumor stem cell targeting lipids nanoparticle and preparation method and application

Country Status (1)

Country Link
CN (1) CN107137377B (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108866061A (en) * 2018-06-21 2018-11-23 中山大学附属第五医院 A kind of aptamer identifying liver cancer cells and its screening technique and purposes
CN112472686A (en) * 2020-12-07 2021-03-12 中国药科大学 Lipid nanoparticle of PEG-PLA-SN38 linker and preparation method thereof
CN116426532A (en) * 2023-06-08 2023-07-14 时夕(广州)生物科技有限公司 Targeting aptamer and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103145968A (en) * 2012-11-23 2013-06-12 杭州师范大学 Folate-conjugated polyethylene glycol monostearate, and preparation method and application thereof
CN105287612A (en) * 2015-10-30 2016-02-03 中国人民解放军第二军医大学 Salinomycin sodium and adriamycin co-loaded nano-liposome as well as preparation method and application thereof
CN105708803A (en) * 2014-12-04 2016-06-29 佳木斯大学 Folic acid targeting silymarin solid lipid nanosphere preparation method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103145968A (en) * 2012-11-23 2013-06-12 杭州师范大学 Folate-conjugated polyethylene glycol monostearate, and preparation method and application thereof
CN105708803A (en) * 2014-12-04 2016-06-29 佳木斯大学 Folic acid targeting silymarin solid lipid nanosphere preparation method
CN105287612A (en) * 2015-10-30 2016-02-03 中国人民解放军第二军医大学 Salinomycin sodium and adriamycin co-loaded nano-liposome as well as preparation method and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JIANG J. X.,ET AL.: "The promotion of salinomycin delivery to hepatocellular carcinoma cells through EGFR and CD133 aptamers conjugation by PLGA nanoparticles", 《NANOMEDICINE》 *
孟廷廷: "纳米给药系统的肿瘤干细胞治疗及机制研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 *
朱冰 等: "盐霉素纳米制剂的研究进展", 《药学实践杂志》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108866061A (en) * 2018-06-21 2018-11-23 中山大学附属第五医院 A kind of aptamer identifying liver cancer cells and its screening technique and purposes
CN112472686A (en) * 2020-12-07 2021-03-12 中国药科大学 Lipid nanoparticle of PEG-PLA-SN38 linker and preparation method thereof
CN116426532A (en) * 2023-06-08 2023-07-14 时夕(广州)生物科技有限公司 Targeting aptamer and application thereof
CN116426532B (en) * 2023-06-08 2023-09-12 时夕(广州)生物科技有限公司 Targeting aptamer and application thereof

Also Published As

Publication number Publication date
CN107137377B (en) 2019-10-11

Similar Documents

Publication Publication Date Title
US10343903B2 (en) Cationic polymer coated mesoporous silica nanoparticles and uses thereof
Song et al. Lactoferrin modified graphene oxide iron oxide nanocomposite for glioma-targeted drug delivery
Barani et al. Nanotechnology in ovarian cancer: Diagnosis and treatment
Babaei et al. Synthesis of theranostic epithelial cell adhesion molecule targeted mesoporous silica nanoparticle with gold gatekeeper for hepatocellular carcinoma
Du et al. Regulating the surface poly (ethylene glycol) density of polymeric nanoparticles and evaluating its role in drug delivery in vivo
Yu et al. In vivo biodistribution and pharmacokinetics of silica nanoparticles as a function of geometry, porosity and surface characteristics
Babic et al. Poly (L-lysine)-modified iron oxide nanoparticles for stem cell labeling
Sarparanta et al. Intravenous delivery of hydrophobin-functionalized porous silicon nanoparticles: stability, plasma protein adsorption and biodistribution
Zhou et al. Synthesis and characterization of PEGylated polyethylenimine-entrapped gold nanoparticles for blood pool and tumor CT imaging
Yan et al. Transferrin-conjugated, fluorescein-loaded magnetic nanoparticles for targeted delivery across the blood–brain barrier
Nguyen et al. Hierarchical self-assembly of magnetic nanoclusters for theranostics: Tunable size, enhanced magnetic resonance imagability, and controlled and targeted drug delivery
Emilian Leucuta Nanotechnology for delivery of drugs and biomedical applications
Huang et al. Suppression of breast cancer cell migration by small interfering RNA delivered by polyethylenimine-functionalized graphene oxide
Parsian et al. Half generations magnetic PAMAM dendrimers as an effective system for targeted gemcitabine delivery
JP6251294B2 (en) Targeted nanoparticles
Bober et al. Application of dendrimers in anticancer diagnostics and therapy
CN102639153B (en) Treating water insoluble nanoparticles with hydrophilic alpha-hydroxyphosphonic acid conjugates, the so modified nanoparticles and their use as contrast agents
Krishna et al. An efficient targeted drug delivery through apotransferrin loaded nanoparticles
CN107137377B (en) A kind of tumor stem cell targeting lipids nanoparticle and preparation method and application
Wen et al. Brain-targeted delivery of trans-activating transcriptor-conjugated magnetic PLGA/lipid nanoparticles
US10624976B2 (en) Dendronized metallic oxide nanoparticles, a process for preparing the same and their uses
Chen et al. Acidity and Glutathione Dual‐Responsive Polydopamine‐Coated Organic‐Inorganic Hybrid Hollow Mesoporous Silica Nanoparticles for Controlled Drug Delivery
Khodadust et al. Development of poly (I: C) modified doxorubicin loaded magnetic dendrimer nanoparticles for targeted combination therapy
KR20140022904A (en) A method for the treatment of a solid tumour
CN104491868A (en) Novel chemotherapeutic drug nano ADC based on antibody conjugation and preparation method and application thereof

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