CN107970453A - A kind of double targeted delivery methods of the pectin nano-particle of modified with folic acid - Google Patents
A kind of double targeted delivery methods of the pectin nano-particle of modified with folic acid Download PDFInfo
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- CN107970453A CN107970453A CN201711272031.2A CN201711272031A CN107970453A CN 107970453 A CN107970453 A CN 107970453A CN 201711272031 A CN201711272031 A CN 201711272031A CN 107970453 A CN107970453 A CN 107970453A
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- pectin
- polyethylene glycol
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- 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
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
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- 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
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
Abstract
The invention discloses a kind of double targeted delivery methods of the pectin nano-particle of modified with folic acid.The pectin nano-particle is by pectin and eight arm polyethylene glycol conjugateds, pectin passes through acid amides key connection with folic acid, eight arm polyethylene glycol are combined with cancer therapy drug ursolic acid by ester bond, obtain folic acid (pectin multi-arm polyethylene glycol) ursolic acid prodrug, mixed with cancer therapy drug hydroxycamptothecin with certain proportion, double targeted nano-particles of core shell structure are prepared by the method for self assembly.Double targeting pectin nano-particle good biocompatibilities prepared by this method, pectin can be degraded by the pectase of colon, and eight arm polyethylene glycol are easily non-toxic carrier, and for transportational process without phenomenon of burst release, extracorporeal releasing experiment shows good pH value response to medicine in vivo.Nano medication carrying drug ratio produced herein is high, and embedding rate is controllable, and yield is high, and as a kind of new pharmaceutical carrier, pectin has good potential applicability in clinical practice.
Description
Technical field
A kind of method for preparing new type anticancer medicament as pharmaceutical carrier the present invention relates to natural pectin, a kind of specific folic acid
Double targeted delivery methods of the pectin nano-particle of modification, belong to high molecular material application, advanced nanometer technology and bio-pharmaceuticals
Field.
Background technology
Pectin is widely present in fruit, root, stem, the Ye Zhong of plant, is the constituent of cell membrane, forms in flanking cell
Interbed adhesive, makes plant tissue cell tightly be bonded together.Pectin is linear polysaccharide polymer, mainly by galacturonic
Acid composition, containing having hundreds to about 1000 Anhydrogalactose aldehydic acid residues, its corresponding average molecular mass for 50000~
150000, it is mainly used in food-processing industry, such as gelling agent, emulsifying agent and thickener.The good bioactivity of pectin and
Biocompatibility, becomes the new lover of pharmaceutical carrier, and researcher had found the galectin-3 (Gal-3) of pectin in recent years
Ligand is acted on recognition interference tumour growth, and cannot be digested in intestines and stomach, and the pectase of colon secretion can degrade
Pectin, realize it is segmented intestine targeted, while study find pectin can liver cancer targeting, inducing cell apoptosis, suppresses transcellular work
With, it is and existing seldom as the report of pharmaceutical carrier on pectin, it is based particularly on targeting pectin and carries the report of medicine there has been no report
Road, therefore, establishes it is particularly important that a kind of brand-new pectin drug-loading system becomes, and provide a kind of new way for research from now on
Footpath.
The content of the invention
The object of the present invention is to provide a kind of double targeted delivery methods of the pectin nano-particle of modified with folic acid, solve hydrophobic
The solubility problem of medicine, it is the carrier material good biocompatibility of this method, nontoxic, biodegradable, pectin is designed in pairs
The effect of targeting, double pectin nano-particles for carrying medicine realize joint anticancer.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：Pectin nano-particle is by pectin and the poly- second two of eight arms
Alcohol conjugated, pectin are combined by acid amides key connection, eight arm polyethylene glycol with folic acid with cancer therapy drug ursolic acid by ester bond,
Folic acid-(pectin-multi-arm polyethylene glycol)-ursolic acid prodrug is obtained, is mixed with cancer therapy drug hydroxycamptothecin with certain proportion, is led to
The method for crossing self assembly prepares double targeted nano-particles of core shell structure.Comprise the following steps that：
(1) appropriate folic acid is dissolved in dimethyl sulfoxide (DMSO), adds 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide salt
Hydrochlorate (EDC) activates the terminal carboxyl group of folic acid, and reaction adds ethylenediamine and pyridine after 30 minutes, stirs evenly, lucifuge reaction 24
Hour, amination folic acid (FA-NH2) is obtained, reaction solution is transferred in dialysis membrane, phosphate buffer solution (pH=7.4) conduct
Extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect dialysis membrane in solution, freeze-drying, obtain yellow
Powder；
(2) take appropriate pectin to be dissolved in deionized water, stir evenly, add EDC and react 30 minutes, activate the carboxyl of pectin,
FA-NH2 and 4-dimethylaminopyridine (DMAP) are added, lucifuge reaction 24h, reaction solution is transferred in dialysis membrane, phosphoric acid delays
Rush solution (pH=7.4) and be used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect molten in dialysis membrane
Liquid, freeze-drying, obtains folic acid-pectin (FA-Pectin) yellow powder；
(3) take appropriate eight arms polyethylene glycol to be dissolved in pyridine, stir evenly, add EDC and react 30 minutes, activate poly- second two
The carboxyl of alcohol, adds cancer therapy drug ursolic acid (UA) and catalyst DMAP, continuously logical nitrogen, when reaction 48 is small at 35 DEG C, takes
Go out mixed liquor, add three times ether (v/v, relative to mixeding liquid volume), stir evenly, there is white precipitate precipitation, 4000 leave
The heart collects precipitation, and centrifugation is repeatedly washed with ether, obtains eight arm polyethylene glycol-ursolic acid couplings crude product, is transferred into
In dialysis membrane, phosphate buffer solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis,
Solution in dialysis membrane is collected, freeze-drying, obtains eight arm polyethylene glycol-ursolic acid couplings (8armPEG-UA) white powder；
(4) eight arm polyethylene glycol-ursolic acid couplings are taken, EDC is dissolved in dimethyl sulfoxide (DMSO), is reacted 30 minutes, is added
Folic acid-pectin and DMAP, continuously lead to nitrogen, when 35 DEG C of lucifuge reactions 48 are small, take out mixed liquor, add three times ether (v/v, phase
For mixeding liquid volume) stir evenly, have yellow mercury oxide precipitation, 4000 turns are collected by centrifugation precipitation, and repeatedly washed with ether from
The heart, obtains the folic acid-arm of pectin-eight polyethylene glycol-ursolic acid (FA-Pectin-8armPEG-UA) crude product, is transferred into dialysis
In film, phosphate buffer solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect
Solution in dialysis membrane, freeze-drying, the folic acid the purified-arm of pectin-eight polyethylene glycol-ursolic acid yellow powder；
(5) the dmso solution folic acid-arm of pectin-eight polyethylene glycol-ursolic acid couplings, liquid-transfering gun separate out, dropwise
Instill in phosphate buffer solution (PBS, pH=7.4), stirring a period of time, obtain the folic acid-arm of pectin-eight polyethylene glycol-black bearberry
Sour nano-particle solution, dialysis, extracellular fluid dialysis are replaced when 6 is small, overnight, collect liquid in dialysis membrane, freeze-drying, obtains
Folic acid-the arm of pectin-eight polyethylene glycol-ursolic acid nano-particle yellow powder；
(6) the appropriate folic acid-arm of pectin-eight polyethylene glycol-ursolic acid couplings and dewatering medicament hydroxycamptothecin is taken to be dissolved in
Dimethyl sulfoxide (DMSO), stirs evenly, and instills dropwise in phosphate buffer solution (PBS, pH=7.4), stirring a period of time, obtains leaf
Acid-the arm of pectin-eight polyethylene glycol-ursolic acid/hydroxycamptothecin (FA-Pectin-8armPEG-UA/HCPT) nano-particle is molten
Liquid, dialysis, extracellular fluid dialysis are replaced when 6 is small, overnight, collect liquid in dialysis membrane, freeze-drying, obtains folic acid-pectin-eight
Arm polyethylene glycol-ursolic acid/hydroxycamptothecin nano particle yellow powder.
Preferably, cancer therapy drug ursolic acid can be replaced by other cancer therapy drugs, such as taxol, oleanolic acid, tripterygium wilfordii
A prime etc..Preferably, hydroxycamptothecin can be replaced by the strong medicine of other hydrophobicitys or have the Jenner of optical property
Rice corpuscles, or the ferriferrous oxide nano-particle of magnetic targeted function.
Preferably, hydroxycamptothecin can be replaced by the strong medicine of other hydrophobicitys or have the gold of optical property
Nano-particle, or the ferriferrous oxide nano-particle of magnetic targeted function.
Preferably, double 100 rans of targeting pectin nano particle diameter scope.
The present invention has the following advantages：
(1) present invention selects pectin to substantially increase the biocompatibility of medicine as pharmaceutical carrier, and no phenomenon of burst release, keeps away
Exempt from existing burst release, poor biocompatibility after general carrier dispenser, cannot degrade, toxic side effect caused by accumulation in vivo
Deng.
(2) present invention utilizes the amphipathic nature of eight arm polyethylene glycol, improves pectin and is coupled as with eight arm polyethylene glycol
The stability and yield for the nano-particle that carrier is self-assembly of altogether, improves the water-soluble and dispersed of nano-particle.
(3) double targeting pectin nano-particles of the invention are the single-minded medicines for being directed to frequently-occurring colon cancer and developing, and are expected to
Clinical practice.
(4) two kinds of hydrophobic anticancer drugs of present invention delivery, according to the obstructed of the mechanism of action of two kinds of medicines kill cancer cells
Realize two kinds of medicine synergistic treatment cancers.
Figure of description
The Technology Roadmap of Figure 1A synthesizing amino folic acid, B synthesize the Technology Roadmap of eight arm polyethylene glycol-ursolic acid, C
Synthesize the Technology Roadmap of the folic acid-arm of pectin-eight polyethylene glycol-ursolic acid；
The conceptual design of Fig. 2 self assemblies folic acid-arm of pectin-eight polyethylene glycol-ursolic acid/hydroxycamptothecin nano particle
Figure；
The hydrogen spectrum nuclear-magnetism figure of Fig. 3 folic acid-arm of pectin-eight polyethylene glycol-ursolic acid couplings；
Fig. 4 folic acid-the arm of pectin-eight polyethylene glycol-ursolic acid/hydroxycamptothecin nano particle diameter distribution map；
Fig. 5 folic acid-the arm of pectin-eight polyethylene glycol-ursolic acid/hydroxycamptothecin nano particle stability experiment；
The hemolytic experiment figure of Fig. 6 folic acid-arm of pectin-eight polyethylene glycol-ursolic acid/hydroxycamptothecin nano particle；
The external release profile of ursolic acid and hydroxycamptothecin in Fig. 7 nano-particles；
The cytotoxicity figure of Fig. 8 folic acid-arm of pectin-eight polyethylene glycol-ursolic acid/hydroxycamptothecin nano particle；
The co-focusing imaging figure of Fig. 9 folic acid-arm of pectin-eight polyethylene glycol-ursolic acid nano-particle, A- hydroxycamptothecin,
B- folic acid-the arm of pectin-eight polyethylene glycol-ursolic acid nano-particle, the C- folic acid-arm of pectin-eight polyethylene glycol-ursolic acid/hydroxyl
Camptothecine nano-particle；
Ursolic acid and hydroxycamptothecin content in blood and the graph of a relation of time in Figure 10 nano-particles；
Influence of the concentration of Figure 11 folic acid to colon cancer cell activity；
Figure 12 applies the mouse tumor volume change figure of pure medicine and Nano medication；
The relative tumour volume figure of mouse after Figure 13 dispensers, mouse survival rate figure, mouse weight variation diagram, medicine it is super quick
React, WBC cell numbers in blood.
Embodiment
Embodiment one
(1) 1.0g folic acid is dissolved in dimethyl sulfoxide (DMSO), adds 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide salt
Hydrochlorate (EDC) activates the terminal carboxyl group of folic acid, and reaction adds ethylenediamine and pyridine after 30 minutes, stirs evenly, lucifuge reaction 24
Hour, obtain amination folic acid (FA-NH2), reaction solution is transferred in dialysis membrane, phosphate buffer solution (pH=7.4) conduct
Extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect dialysis membrane in solution, freeze-drying, obtain yellow
Powder；
(2) take 1.0g pectin to be dissolved in deionized water, stir evenly, add EDC and react 30 minutes, activate the carboxyl of pectin,
Add 0.1g FA-NH2With 4-dimethylaminopyridine (DMAP), lucifuge reaction 24h, reaction solution is transferred in dialysis membrane, phosphorus
Acid buffering solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect in dialysis membrane
Solution, freeze-drying, obtains folic acid-pectin (FA-Pectin) yellow powder；
(3) take eight arm polyethylene glycol of 2.0g to be dissolved in pyridine, stir evenly, add EDC and react 30 minutes, activate poly- second two
The carboxyl of alcohol, adds 0.1g cancer therapy drugs ursolic acid (UA) and catalyst DMAP, continuously leads to nitrogen, and reaction 48 is small at 35 DEG C
When, mixed liquor is taken out, three times ether (v/v, relative to mixeding liquid volume) is added, stirs evenly, there is a white precipitate precipitation, 4000
Turn that precipitation is collected by centrifugation, and centrifugation is repeatedly washed with ether, eight arm polyethylene glycol-ursolic acid couplings crude product is obtained, by its turn
Move on in dialysis membrane, phosphate buffer solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when to replace once dialysis outer
Liquid, collects solution in dialysis membrane, freeze-drying, obtains the white powder of eight arm polyethylene glycol-ursolic acid couplings (8armPEG-UA)
End；
(4) eight arm polyethylene glycol of 1.0g-ursolic acid couplings are taken, EDC is dissolved in dimethyl sulfoxide (DMSO), is reacted 30 minutes,
0.5g folic acid-pectin and DMAP are added, continuously leads to nitrogen, when 35 DEG C of lucifuge reactions 48 are small, take out mixed liquor, add three times ether
(v/v, relative to mixeding liquid volume) is stirred evenly, and has yellow mercury oxide precipitation, and 4000 turns are collected by centrifugation precipitation, and multiple with ether
Washing centrifugation, obtains the folic acid-arm of pectin-eight polyethylene glycol-ursolic acid (FA-Pectin-8armPEG-UA) crude product, is shifted
Into dialysis membrane, phosphate buffer solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when to replace once dialysis outer
Liquid, collects solution in dialysis membrane, freeze-drying, the folic acid the purified-arm of pectin-eight polyethylene glycol-ursolic acid yellow powder；
(5) the dmso solution 10mg folic acid-arm of pectin-eight polyethylene glycol-ursolic acid couplings, liquid-transfering gun separate out,
Dropwise instill phosphate buffer solution in (PBS, pH=7.4), stirring a period of time, obtain the folic acid-arm of pectin-eight polyethylene glycol-
Ursolic acid nano-particle solution, dialysis, extracellular fluid dialysis are replaced when 6 is small, overnight, are collected liquid in dialysis membrane, are freeze-dried,
Obtain the folic acid-arm of pectin-eight polyethylene glycol-ursolic acid nano-particle yellow powder；
(6) the 12mg folic acid-arm of pectin-eight polyethylene glycol-ursolic acid couplings and dewatering medicament hydroxycamptothecin is taken to be dissolved in
Dimethyl sulfoxide (DMSO), stirs evenly, and instills dropwise in phosphate buffer solution (PBS, pH=7.4), stirring a period of time, obtains leaf
Acid-the arm of pectin-eight polyethylene glycol-ursolic acid/hydroxycamptothecin (FA-Pectin-8armPEG-UA/HCPT) nano-particle is molten
Liquid, dialysis, extracellular fluid dialysis are replaced when 6 is small, overnight, collect liquid in dialysis membrane, freeze-drying, obtains folic acid-pectin-eight
Arm polyethylene glycol-ursolic acid/hydroxycamptothecin nano particle yellow powder.
Embodiment two
(1) 1.0g folic acid is dissolved in dimethyl sulfoxide (DMSO), adds 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide salt
Hydrochlorate (EDC) activates the terminal carboxyl group of folic acid, and reaction adds ethylenediamine and pyridine after 30 minutes, stirs evenly, lucifuge reaction 24
Hour, amination folic acid (FA-NH2) is obtained, reaction solution is transferred in dialysis membrane, phosphate buffer solution (pH=7.4) conduct
Extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect dialysis membrane in solution, freeze-drying, obtain yellow
Powder；
(2) take 1.0g pectin to be dissolved in deionized water, stir evenly, add EDC and react 30 minutes, activate the carboxyl of pectin,
0.2g FA-NH2 and 4-dimethylaminopyridine (DMAP) are added, lucifuge reaction 24h, reaction solution is transferred in dialysis membrane, phosphorus
Acid buffering solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect in dialysis membrane
Solution, freeze-drying, obtains folic acid-pectin (FA-Pectin) yellow powder；
(3) take eight arm polyethylene glycol of 2.0g to be dissolved in pyridine, stir evenly, add EDC and react 30 minutes, activate poly- second two
The carboxyl of alcohol, adds 1.0g cancer therapy drugs ursolic acid (UA) and catalyst DMAP, continuously leads to nitrogen, and reaction 48 is small at 35 DEG C
When, mixed liquor is taken out, three times ether (v/v, relative to mixeding liquid volume) is added, stirs evenly, there is a white precipitate precipitation, 4000
Turn that precipitation is collected by centrifugation, and centrifugation is repeatedly washed with ether, eight arm polyethylene glycol-ursolic acid couplings crude product is obtained, by its turn
Move on in dialysis membrane, phosphate buffer solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when to replace once dialysis outer
Liquid, collects solution in dialysis membrane, freeze-drying, obtains the white powder of eight arm polyethylene glycol-ursolic acid couplings (8armPEG-UA)
End；
(4) eight arm polyethylene glycol of 1.0g-ursolic acid couplings are taken, EDC is dissolved in dimethyl sulfoxide (DMSO), is reacted 30 minutes,
0.5g folic acid-pectin and DMAP are added, continuously leads to nitrogen, when 35 DEG C of lucifuge reactions 48 are small, take out mixed liquor, add three times ether
(v/v, relative to mixeding liquid volume) is stirred evenly, and has yellow mercury oxide precipitation, and 4000 turns are collected by centrifugation precipitation, and multiple with ether
Washing centrifugation, obtains the folic acid-arm of pectin-eight polyethylene glycol-ursolic acid (FA-Pectin-8armPEG-UA) crude product, is shifted
Into dialysis membrane, phosphate buffer solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when to replace once dialysis outer
Liquid, collects solution in dialysis membrane, freeze-drying, the folic acid the purified-arm of pectin-eight polyethylene glycol-ursolic acid yellow powder；
(5) the dmso solution 10mg folic acid-arm of pectin-eight polyethylene glycol-ursolic acid couplings, liquid-transfering gun separate out,
Dropwise instill phosphate buffer solution in (PBS, pH=7.4), stirring a period of time, obtain the folic acid-arm of pectin-eight polyethylene glycol-
Ursolic acid nano-particle solution, dialysis, extracellular fluid dialysis are replaced when 6 is small, overnight, are collected liquid in dialysis membrane, are freeze-dried,
Obtain the folic acid-arm of pectin-eight polyethylene glycol-ursolic acid nano-particle yellow powder；
(6) the 12mg folic acid-arm of pectin-eight polyethylene glycol-ursolic acid couplings and dewatering medicament hydroxycamptothecin is taken to be dissolved in
Dimethyl sulfoxide (DMSO), stirs evenly, and instills dropwise in phosphate buffer solution (PBS, pH=7.4), stirring a period of time, obtains leaf
Acid-the arm of pectin-eight polyethylene glycol-ursolic acid/hydroxycamptothecin (FA-Pectin-8armPEG-UA/HCPT) nano-particle is molten
Liquid, dialysis, extracellular fluid dialysis are replaced when 6 is small, overnight, collect liquid in dialysis membrane, freeze-drying, obtains folic acid-pectin-eight
Arm polyethylene glycol-ursolic acid/hydroxycamptothecin nano particle yellow powder.
Embodiment three
(1) 1.0g folic acid is dissolved in dimethyl sulfoxide (DMSO), adds 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide salt
Hydrochlorate (EDC) activates the terminal carboxyl group of folic acid, and reaction adds ethylenediamine and pyridine after 30 minutes, stirs evenly, lucifuge reaction 24
Hour, amination folic acid (FA-NH2) is obtained, reaction solution is transferred in dialysis membrane, phosphate buffer solution (pH=7.4) conduct
Extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect dialysis membrane in solution, freeze-drying, obtain yellow
Powder；
(2) take 1.0g pectin to be dissolved in deionized water, stir evenly, add EDC and react 30 minutes, activate the carboxyl of pectin,
0.1g FA-NH2 and 4-dimethylaminopyridine (DMAP) are added, lucifuge reaction 24h, reaction solution is transferred in dialysis membrane, phosphorus
Acid buffering solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect in dialysis membrane
Solution, freeze-drying, obtains folic acid-pectin (FA-Pectin) yellow powder；
(3) take eight arm polyethylene glycol of 1.0g to be dissolved in pyridine, stir evenly, add EDC and react 30 minutes, activate poly- second two
The carboxyl of alcohol, adds 0.5g cancer therapy drugs ursolic acid (UA) and catalyst DMAP, continuously leads to nitrogen, and reaction 48 is small at 35 DEG C
When, mixed liquor is taken out, three times ether (v/v, relative to mixeding liquid volume) is added, stirs evenly, there is a white precipitate precipitation, 4000
Turn that precipitation is collected by centrifugation, and centrifugation is repeatedly washed with ether, eight arm polyethylene glycol-ursolic acid couplings crude product is obtained, by its turn
Move on in dialysis membrane, phosphate buffer solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when to replace once dialysis outer
Liquid, collects solution in dialysis membrane, freeze-drying, obtains the white powder of eight arm polyethylene glycol-ursolic acid couplings (8armPEG-UA)
End；
(4) eight arm polyethylene glycol of 1.0g-ursolic acid couplings are taken, EDC is dissolved in dimethyl sulfoxide (DMSO), is reacted 30 minutes,
0.1g folic acid-pectin and DMAP are added, continuously leads to nitrogen, when 35 DEG C of lucifuge reactions 48 are small, take out mixed liquor, add three times ether
(v/v, relative to mixeding liquid volume) is stirred evenly, and has yellow mercury oxide precipitation, and 4000 turns are collected by centrifugation precipitation, and multiple with ether
Washing centrifugation, obtains the folic acid-arm of pectin-eight polyethylene glycol-ursolic acid (FA-Pectin-8armPEG-UA) crude product, is shifted
Into dialysis membrane, phosphate buffer solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when to replace once dialysis outer
Liquid, collects solution in dialysis membrane, freeze-drying, the folic acid the purified-arm of pectin-eight polyethylene glycol-ursolic acid yellow powder；
(5) the dmso solution 10mg folic acid-arm of pectin-eight polyethylene glycol-ursolic acid couplings, liquid-transfering gun separate out,
Dropwise instill phosphate buffer solution in (PBS, pH=7.4), stirring a period of time, obtain the folic acid-arm of pectin-eight polyethylene glycol-
Ursolic acid nano-particle solution, dialysis, extracellular fluid dialysis are replaced when 6 is small, overnight, are collected liquid in dialysis membrane, are freeze-dried,
Obtain the folic acid-arm of pectin-eight polyethylene glycol-ursolic acid nano-particle yellow powder；
(6) the 12mg folic acid-arm of pectin-eight polyethylene glycol-ursolic acid couplings and dewatering medicament hydroxycamptothecin is taken to be dissolved in
Dimethyl sulfoxide (DMSO), stirs evenly, and instills dropwise in phosphate buffer solution (PBS, pH=7.4), stirring a period of time, obtains leaf
Acid-the arm of pectin-eight polyethylene glycol-ursolic acid/hydroxycamptothecin (FA-Pectin-8armPEG-UA/HCPT) nano-particle is molten
Liquid, dialysis, extracellular fluid dialysis are replaced when 6 is small, overnight, collect liquid in dialysis membrane, freeze-drying, obtains folic acid-pectin-eight
Arm polyethylene glycol-ursolic acid/hydroxycamptothecin nano particle yellow powder.
Example IV
(1) 1.0g folic acid is dissolved in dimethyl sulfoxide (DMSO), adds 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide salt
Hydrochlorate (EDC) activates the terminal carboxyl group of folic acid, and reaction adds ethylenediamine and pyridine after 30 minutes, stirs evenly, lucifuge reaction 24
Hour, amination folic acid (FA-NH2) is obtained, reaction solution is transferred in dialysis membrane, phosphate buffer solution (pH=7.4) conduct
Extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect dialysis membrane in solution, freeze-drying, obtain yellow
Powder；
(2) take 1.0g pectin to be dissolved in deionized water, stir evenly, add EDC and react 30 minutes, activate the carboxyl of pectin,
0.1g FA-NH2 and 4-dimethylaminopyridine (DMAP) are added, lucifuge reaction 24h, reaction solution is transferred in dialysis membrane, phosphorus
Acid buffering solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect in dialysis membrane
Solution, freeze-drying, obtains folic acid-pectin (FA-Pectin) yellow powder；
(3) take eight arm polyethylene glycol of 2.0g to be dissolved in pyridine, stir evenly, add EDC and react 30 minutes, activate poly- second two
The carboxyl of alcohol, adds 1.0g cancer therapy drugs ursolic acid (UA) and catalyst DMAP, continuously leads to nitrogen, and reaction 48 is small at 35 DEG C
When, mixed liquor is taken out, three times ether (v/v, relative to mixeding liquid volume) is added, stirs evenly, there is a white precipitate precipitation, 4000
Turn that precipitation is collected by centrifugation, and centrifugation is repeatedly washed with ether, eight arm polyethylene glycol-ursolic acid couplings crude product is obtained, by its turn
Move on in dialysis membrane, phosphate buffer solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when to replace once dialysis outer
Liquid, collects solution in dialysis membrane, freeze-drying, obtains the white powder of eight arm polyethylene glycol-ursolic acid couplings (8armPEG-UA)
End；
(4) eight arm polyethylene glycol of 1.0g-ursolic acid couplings are taken, EDC is dissolved in dimethyl sulfoxide (DMSO), is reacted 30 minutes,
0.2g folic acid-pectin and DMAP are added, continuously leads to nitrogen, when 35 DEG C of lucifuge reactions 48 are small, take out mixed liquor, add three times ether
(v/v, relative to mixeding liquid volume) is stirred evenly, and has yellow mercury oxide precipitation, and 4000 turns are collected by centrifugation precipitation, and multiple with ether
Washing centrifugation, obtains the folic acid-arm of pectin-eight polyethylene glycol-ursolic acid (FA-Pectin-8armPEG-UA) crude product, is shifted
Into dialysis membrane, phosphate buffer solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when to replace once dialysis outer
Liquid, collects solution in dialysis membrane, freeze-drying, the folic acid the purified-arm of pectin-eight polyethylene glycol-ursolic acid yellow powder；
(5) the dmso solution 10mg folic acid-arm of pectin-eight polyethylene glycol-ursolic acid couplings, liquid-transfering gun separate out,
Dropwise instill phosphate buffer solution in (PBS, pH=7.4), stirring a period of time, obtain the folic acid-arm of pectin-eight polyethylene glycol-
Ursolic acid nano-particle solution, dialysis, extracellular fluid dialysis are replaced when 6 is small, overnight, are collected liquid in dialysis membrane, are freeze-dried,
Obtain the folic acid-arm of pectin-eight polyethylene glycol-ursolic acid nano-particle yellow powder；
(6) the 12mg folic acid-arm of pectin-eight polyethylene glycol-ursolic acid couplings and dewatering medicament hydroxycamptothecin is taken to be dissolved in
Dimethyl sulfoxide (DMSO), stirs evenly, and instills dropwise in phosphate buffer solution (PBS, pH=7.4), stirring a period of time, obtains leaf
Acid-the arm of pectin-eight polyethylene glycol-ursolic acid/hydroxycamptothecin (FA-Pectin-8armPEG-UA/HCPT) nano-particle is molten
Liquid, dialysis, extracellular fluid dialysis are replaced when 6 is small, overnight, collect liquid in dialysis membrane, freeze-drying, obtains folic acid-pectin-eight
Arm polyethylene glycol-ursolic acid/hydroxycamptothecin nano particle yellow powder.
Claims (5)
- A kind of 1. double targeted delivery methods of the pectin nano-particle of modified with folic acid, it is characterised in that：The pectin nanoparticle Son is to pass through acid amides key connection, eight arm polyethylene glycol and anticancer with folic acid by pectin and eight arm polyethylene glycol conjugateds, pectin Medicine ursolic acid is combined by ester bond, folic acid-(pectin-multi-arm polyethylene glycol)-ursolic acid prodrug is obtained, with cancer therapy drug hydroxyl Camptothecine is mixed with certain proportion, and double targeted nano-particles of core shell structure are prepared by the method for self assembly.Specific steps are such as Under：(1) appropriate folic acid is dissolved in dimethyl sulfoxide (DMSO), adds 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) terminal carboxyl group of folic acid is activated, reaction adds ethylenediamine and pyridine after 30 minutes, stirs evenly, when lucifuge reaction 24 is small, Obtain amination folic acid (FA-NH2), reaction solution is transferred in dialysis membrane, phosphate buffer solution (pH=7.4) is outer as dialysis Liquid, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect dialysis membrane in solution, freeze-drying, obtain yellow powder；(2) take appropriate pectin to be dissolved in deionized water, stir evenly, add EDC and react 30 minutes, activate the carboxyl of pectin, then add Enter FA-NH2With 4-dimethylaminopyridine (DMAP), lucifuge reaction 24h, reaction solution is transferred in dialysis membrane, phosphoric acid buffer is molten Liquid (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect solution in dialysis membrane, it is cold It is lyophilized dry, obtain folic acid-pectin (FA-Pectin) yellow powder；(3) take appropriate eight arms polyethylene glycol to be dissolved in pyridine, stir evenly, add EDC and react 30 minutes, activate polyethylene glycol Carboxyl, adds cancer therapy drug ursolic acid (UA) and catalyst DMAP, continuously logical nitrogen, when reaction 48 is small at 35 DEG C, takes out mixed Liquid is closed, three times ether (v/v, relative to mixeding liquid volume) is added, stirs evenly, there is white precipitate precipitation, 4000 leave heart receipts Collection precipitation, and centrifugation is repeatedly washed with ether, eight arm polyethylene glycol-ursolic acid couplings crude product is obtained, is transferred into dialysis In film, phosphate buffer solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect Solution in dialysis membrane, freeze-drying, obtains eight arm polyethylene glycol-ursolic acid couplings (8armPEG-UA) white powder；(4) eight arm polyethylene glycol-ursolic acid couplings are taken, EDC is dissolved in dimethyl sulfoxide (DMSO), is reacted 30 minutes, addition folic acid- Pectin and DMAP, continuously lead to nitrogen, when 35 DEG C of lucifuge reactions 48 are small, take out mixed liquor, add three times ether (v/v, relative to mixed Close liquid product) stir evenly, there is yellow mercury oxide precipitation, 4000 turns are collected by centrifugation precipitation, and centrifugation is repeatedly washed with ether, obtain Folic acid-the arm of pectin-eight polyethylene glycol-ursolic acid (FA-Pectin-8armPEG-UA) crude product, is transferred into dialysis membrane, phosphorus Acid buffering solution (pH=7.4) is used as extracellular fluid dialysis, dialysed overnight, it is every 4 it is small when replace an extracellular fluid dialysis, collect in dialysis membrane Solution, freeze-drying, the folic acid the purified-arm of pectin-eight polyethylene glycol-ursolic acid yellow powder；(5) the dmso solution folic acid-arm of pectin-eight polyethylene glycol-ursolic acid couplings, liquid-transfering gun are separated out, instilled dropwise In phosphate buffer solution (PBS, pH=7.4), stirring a period of time, obtain the folic acid-arm of pectin-eight polyethylene glycol-ursolic acid and receive Rice corpuscles solution, dialysis, extracellular fluid dialysis are replaced when 6 is small, overnight, collect liquid in dialysis membrane, freeze-drying, obtains leaf Acid-the arm of pectin-eight polyethylene glycol-ursolic acid nano-particle yellow powder；(6) the appropriate folic acid-arm of pectin-eight polyethylene glycol-ursolic acid couplings and dewatering medicament hydroxycamptothecin is taken to be dissolved in diformazan Base sulfoxide, stirs evenly, and instills dropwise in phosphate buffer solution (PBS, pH=7.4), stirring a period of time, obtains folic acid-fruit The arm of glue-eight polyethylene glycol-ursolic acid/hydroxycamptothecin (FA-Pectin-8armPEG-UA/HCPT) nano-particle solution, thoroughly Analysis, extracellular fluid dialysis are replaced when 6 is small, overnight, collect liquid in dialysis membrane, freeze-drying, obtains folic acid-arm of pectin-eight and gather Ethylene glycol-ursolic acid/hydroxycamptothecin nano particle yellow powder.
- 2. a kind of double targeted delivery methods of the pectin nano-particle of modified with folic acid according to claim 1, its feature exist In：The cancer therapy drug ursolic acid can be replaced by other cancer therapy drugs, such as taxol, oleanolic acid, triptolide.
- 3. a kind of double targeted delivery methods of the pectin nano-particle of modified with folic acid according to claim 1, its feature exist In：The hydroxycamptothecin can be replaced by the strong medicine of other hydrophobicitys or Jenner's grain of rice with optical property Son, or the ferriferrous oxide nano-particle of magnetic targeted function.
- 4. a kind of double targeted delivery methods of the pectin nano-particle of modified with folic acid according to claim 1, its feature exist In：The eight arm polyethylene glycol can be replaced by single-stranded polyethylene glycol, four arm polyethylene glycol, can be selected according to obstructed medicine Different polyethylene glycol terminal groups.
- 5. a kind of double targeted delivery methods of the pectin nano-particle of modified with folic acid according to claim 1, its feature exist In：Double 100 rans of targeting pectin nano particle diameter scope.
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| WO2020156513A1 (en) * | 2019-01-30 | 2020-08-06 | 同宜医药（苏州）有限公司 | Bi-ligand drug conjugate and use thereof |
| CN112641762A (en) * | 2021-01-05 | 2021-04-13 | 吉林大学 | Nanoparticles of eight-arm polyethylene glycol oleanolic acid drug carrier and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109044978A (en) * | 2018-09-28 | 2018-12-21 | 佳木斯大学 | A kind of preparation method and applications of oleanolic acid nano particle |
| CN109044978B (en) * | 2018-09-28 | 2020-12-29 | 佳木斯大学 | Preparation method and application of oleanolic acid nanoparticles |
| WO2020156513A1 (en) * | 2019-01-30 | 2020-08-06 | 同宜医药（苏州）有限公司 | Bi-ligand drug conjugate and use thereof |
| CN110975842A (en) * | 2019-12-16 | 2020-04-10 | 健帆生物科技集团股份有限公司 | Immunoadsorbent, preparation method thereof and adsorber for hemoperfusion |
| CN110974973A (en) * | 2019-12-25 | 2020-04-10 | 中国科学院过程工程研究所 | Cationic poly-prodrug polymer, preparation method and application thereof |
| CN112641762A (en) * | 2021-01-05 | 2021-04-13 | 吉林大学 | Nanoparticles of eight-arm polyethylene glycol oleanolic acid drug carrier and preparation method thereof |
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