CN108727597A - Polyphosphate-polycaprolactone nano-medicament carrier and its application - Google Patents
Polyphosphate-polycaprolactone nano-medicament carrier and its application Download PDFInfo
- Publication number
- CN108727597A CN108727597A CN201810587964.9A CN201810587964A CN108727597A CN 108727597 A CN108727597 A CN 108727597A CN 201810587964 A CN201810587964 A CN 201810587964A CN 108727597 A CN108727597 A CN 108727597A
- Authority
- CN
- China
- Prior art keywords
- nano particle
- drug
- caprolactone
- copolymer
- polyphosphate
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G79/00—Macromolecular compounds obtained by reactions forming a linkage containing atoms other than silicon, sulfur, nitrogen, oxygen, and carbon with or without the latter elements in the main chain of the macromolecule
- C08G79/02—Macromolecular compounds obtained by reactions forming a linkage containing atoms other than silicon, sulfur, nitrogen, oxygen, and carbon with or without the latter elements in the main chain of the macromolecule a linkage containing phosphorus
- C08G79/04—Phosphorus linked to oxygen or to oxygen and carbon
-
- 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
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
-
- 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
- A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
- C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
- C08G63/08—Lactones or lactides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Nanotechnology (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
The present invention provides a kind of diblock copolymer being made of polyphosphate and poly-epsilon-caprolactone, and the diblock copolymer is expressed from the next, and the wherein degree of polymerization x of poly-epsilon-caprolactone block is 15-186, and the degree of polymerization y of polyphosphate block is 7-200.The present invention also provides assemble the nano particle formed, purposes of the nano particle as pharmaceutical carrier, including the preparation method of the drug delivery system of the nano particle and the copolymer and nano particle by the copolymer.
Description
Technical field
The invention belongs to the drug delivery fields based on biological medical polymer material, relate generally to one kind by aliphatic poly
Ester, polyphosphate synthesis block copolymer, and nano particle that thus based block copolymer is formed in aqueous solution, and
Nano-medicament carrier based on the polymer and nano particle.The nano-carrier has good biocompatibility and degradable
Property, is primarily adapted for use in vivo medicine delivery, it can also be used to Gene transfer vector, biological radiography agent other fields such as exploitation.
Background technology
The morbidity and mortality of malignant tumour rise year by year, have become the major reason of China's death, to me
State's social and economic development harm is huge.Classic chemotherapy is due to the problems such as lacking tumour-specific, drug resistance, for common solid tumors
Generally it is only capable of reaching the effective percentage of 20-30%；And to human normal tissue toxic side effect.High molecular nanometer drug delivery system
System can protect drug molecule, increase stability in drug body, extend drug blood half-life period, can be efficiently defeated by chemotherapeutics
It send to tumor focus position, improves drug in the enrichment of tumor locus, reduce the drug concentration of normal portions, improve drug
The effect of.These advantages make nano-drug transporter highlight great application potential in oncotherapy.
Biodegradable high-molecular has good application prospect in drug delivery, the reason is that Biodegradable high-molecular
It can degrade, catabolite or be excreted in vivo, or participate in life entity metabolism, so as to reduce secondary work
With.A large amount of degradable macromolecules of the synthesising biological degradable macromolecule including polycaprolactone, polyanhydride etc., these macromolecules
It is had been widely used in bio-medical fields such as drug delivery, gene delivery, cell culture and organizational projects.Polyphosphate
It is a kind of degradable high polymer material for connecting backbone structure unit by phosphoric acid ester bond, is used as fire proofing, polyphosphate earliest
With good hydrophily, the hydrophilic shell of nano particle is can be used as, extends the body-internal-circulation of particle.Such as Shanghai drug institute Li Ya
Flat researcher etc., as drug conveying carrier, it is independent to disclose polyphosphate using polyphosphate-polycaprolactone block polymer
To the body-internal-circulation of nano particle, there are facilitations when formation hydrophilic shell.The abundant controllable synthesis method of polyphosphoric acid esters material
It disclosure satisfy that the demand of nano-carrier hydrophilic shell characteristic regulation and control with changeable structure, there is apparent advantage.
Invention content
The present invention provides a kind of polyphosphate-pla-pcl block copolymer, the nanometer formed by the copolymer
Grain and preparation method thereof, the internal destiny and drug-loading nanoparticles of the system research nano particle are in oncotherapy
With.
The present invention provides the method for preparing the block copolymer of the present invention comprising makees solvent with toluene, synthesis gathers in oneself
Ester (PCL)；In solvent benzol, dichloromethane, tetrahydrofuran, MEP is prepared；Tetrahydrofuran is solvent, using PCL-OH as macromolecular
Initiator, stannous iso caprylate are catalyst, polymerize to obtain PCL-b-PMEP by the ring-opening polymerization of cyclic phosphate ester monomer
Block copolymer.
Polyphosphate provided by the invention-polycaprolactone di-block copolymer can be self-assembly of nano particle, this
The grain size of nano particle is in 40nm or so, and surface charge is in -14mV or so.The nano particle can effectively contain drug (such as
Docetaxel) to form stable Nano medication, drug is efficiently delivered to tumor locus, and achieve good tumour and control
Therapeutic effect.
The present invention has obtained a kind of polyphosphate-pla-pcl block copolymer, and the polymeric reaction condition used is mild, former
Material is easy to get, and purification process is easy after reaction；Such polymer assembles to form nano particle after, can realize internal long circulating and swollen
Tumor tissue is enriched with, and can be used for containing and its conveying in vivo for chemotherapeutics, and effectively inhibits the growth of tumour.The nano particle
It has a good application prospect in conveying chemotherapeutics is for oncotherapy.
The advantage of the synthetic method of the present invention is that good control is obtained by the reaction, and dispersion degree is lower, can be very good to control
The length of hydrophobic block processed and hydrophilic block reaches the last stabilization of particle.
The advantage of the particle of the present invention prepared by the method for the present invention is：Compared to the PCL-b-PEEP reported
Nano-medicament carrier, the nano-medicament carriers that are self-assembly of of PCL-b-PMEP have more hydrophilic polyphosphate in the present invention
Shell, thus there is better stability.Have studies have shown that the surface hydrophilicity of nano-medicament carrier significantly affects its surface
Albumen hat formation, and then influence carrier in vivo with the interaction of macrophage.The higher carrier surface of surface hydrophilicity
Less albumen can be adsorbed, to less be absorbed by macrophage.PCL-b-PMEP in the present invention is compared to PCL-b-
PEEP protein adsorption quantities are less, thus it is more difficult removed by macrophage, to show longer blood circulation time
With higher tumour enriching quantity.PCL-b-PMEP shows as nano-medicament carrier for tumour medicine delivering more excellent
Performance, therapeutic field of tumor have better application prospect.
More specifically, the present invention provides the following terms：
1. a kind of diblock copolymer being made of polyphosphate and poly-epsilon-caprolactone, the diblock copolymer is by following formula
It indicates, the degree of polymerization x of wherein poly-epsilon-caprolactone block is 15-186 (corresponding number-average molecular weight be 1710-21,200), poly- phosphorus
The degree of polymerization y of acid esters block is 7-200 (corresponding number-average molecular weight is 1,060-30,285)
2. the copolymer according to 1, wherein x are 22 and y is 62.
3. a kind of nano particle is assembled by the copolymer according to 1 or 2.
4. the nano particle according to 3 is used to prepare the purposes of pharmaceutical carrier.
5. a kind of drug delivery system, it includes according to 3 nano particle and loaded by the nano particle
Active constituent.
6. the system according to 5, wherein the active constituent is the drug for treating cancer.
7. the system according to 6, wherein the cancer is selected from breast cancer and lung cancer.
8. the system according to 6, wherein the drug is Docetaxel.
9. the method for being used to prepare the copolymer according to 1, the described method comprises the following steps：
A) 6-caprolactone, initiator benzyl alcohol and catalyst stannous iso caprylate is utilized to prepare the poly-epsilon-caprolactone of one-ended hydroxy
Macromole evocating agent；
B) the chloro- 2- oxygen -1,3 of 2- is prepared by phosphorus trichloride and glycol reaction, 2- dioxaphospholane (COP), so
COP is reacted with methanol afterwards and prepares 2- methoxyl group -2- oxygen -1,3,2- dioxaphospholane (MEP)；And
C) in the presence of solvents tetrahydrofurane and catalyst stannous iso caprylate, keep the poly-epsilon-caprolactone of the one-ended hydroxy big
Initiator molecule carries out block copolymerization with MEP and reacts.
10. the method for being used to prepare the nano particle according to 3, the method includes making the copolymerization according to 1 or 2
Object is self-assembled into nano particle.
Description of the drawings
Aforementioned aspect of the present invention can be more easily understood and permitted by reading following detailed description when read in conjunction with the accompanying drawings
Mostly adjoint advantage, wherein：
Fig. 1 provides the synthetic route chart of PCL-b-PMEP in embodiment 1；
Fig. 2 provides PCL-b-PMEP's in embodiment 11H H NMR spectroscopies；
Fig. 3 provides the GPC spectrums of PCL-b-PMEP in embodiment 1；
Fig. 4 (A) provides the grain size of PCL-b-PMEP nano-carriers in example 2；
Fig. 4 (B) provides the surface potential of PCL-b-PMEP nano-carriers in example 2；
Fig. 4 (C) provides the freezing transmission electron microscope photo of PCL-b-PMEP nano-carriers in example 2；
Fig. 4 (D) provides stability of the PCL-b-PMEP nano-carriers in serum in example 2；
Fig. 5 provides the PCL-b-PMEP nano-micelles for containing Docetaxel in example 2 in phosphate buffer
In, add up the curve of release Docetaxel；
Fig. 6 provides the micellar nanoparticles with DiO fluorescent markers in embodiment 3 and is entering MDA-MB-231 cells
The laser confocal microscope photo being distributed afterwards；
Fig. 7 provides the cytotoxicity of various concentration PCL-b-PMEP nano particles in embodiment 3；
Fig. 8 (A) is provided in example 4 through tail vein injection micellar nanoparticles PCL-b-PMEP to ICR mouse
In after, living small animal imaging in different time points mouse blood nir dye DiR fluorescent markers nano-carrier carry out
It is quantitative；
Fig. 8 (B) is provided and is based on the Drug-time curve that Fig. 8 (A) is obtained in example 4；
Fig. 9 (A) is provided in example 4 through tail vein injection micellar nanoparticles PCL-b-PMEP to MDA-MB-
After in 231 breast cancer mouses, the nano-carrier different time of living small animal imaging representation DiR labels is enriched with and removes in tumour
Situation；
Fig. 9 (B) is provided in example 4 through tail vein injection micellar nanoparticles PCL-b-PMEP to MDA-MB-
After in 231 breast cancer mouses, the quantitative analysis of each internal organs DiR fluorescence intensities；
Figure 10 (A) is provided in the MDA-MB-231 breast cancer solid tumor models of embodiment 5, and tail vein injection contains
After the micellar nanoparticles of Docetaxel enter in Mice Body, to the inhibition curve of tumour growth；
Figure 10 (B) is provided in the MDA-MB-231 breast cancer solid tumor models of embodiment 5, and tail vein injection contains
After the micellar nanoparticles of Docetaxel enter in Mice Body, after treatment in 22 days, the tumor weight of each group compares；
Figure 11 (A) is provided in the B16 melanoma metastasis models in embodiment 5, and it is purple that tail vein injection contains more west
After the micellar nanoparticles of China fir alcohol enter in Mice Body, after treatment in 20 days, the growth curve chart of mouse；
Figure 11 (B) is provided in the B16 melanoma metastasis models in embodiment 5, and it is purple that tail vein injection contains more west
After the micellar nanoparticles of China fir alcohol enter in Mice Body, after treatment in 20 days, mouse lung shifts photo；
Figure 11 (C) is provided in the B16 melanoma metastasis models in embodiment 5, and it is purple that tail vein injection contains more west
After the micellar nanoparticles of China fir alcohol enter in Mice Body, the photo of mouse lung H&E dyeing in the 20th day.
Specific implementation mode
The present invention is better understood from by means of following experiments example, they illustrate the synthesis of the block copolymer of the present invention
The preparation method of method and nano-carrier, and its property as pharmaceutical carrier.These embodiments are merely illustrative, and are not
The entire reflection of the present invention.
The synthesis of embodiment 1, PCL-b-PMEP block copolymers
The present invention provides AB types block polyphosphate-polycaprolactone co-polymer, the B block PMEP in the present invention, for parent
Aqueous polyphosphate, relative molecular weight are 1,060-3,0285.A block PCL in the present invention are hydrophobic fat adoption
Ester, relative molecular weight 3,080-2,1200.Its advantage is that：1. relative hydrophobicity, therefore between polymer chains
Hydrophobic-hydrophobic interaction promotes copolymer self assembly；2. biodegradable；3. nontoxic and bio-compatible；4. synthesis letter
It is single and controllable.
Terminal hydroxy group PCL macromole evocating agents with various molecular weight are cyclic annular single by caprolactone under THF solvent conditions
Body is benzyl alcohol causes, the ring-opening polymerization under the catalytic condition of stannous iso caprylate obtains.With the polycaprolactone of one-ended hydroxy
It is that catalyst has synthesized a series of PCL-b-PMEP block copolymers for macromole evocating agent, stannous iso caprylate.Yin Qigao is catalyzed
Efficiency and nontoxicity, stannous iso caprylate are by most widely used urging for lactone and lactide cyclic monomer ring-opening polymerization
Agent has been approved by the FDA in the United States as food additives.Fig. 1 is the conjunction object route of PCL-b-PMEP block copolymers.
6-caprolactone (CL) is under room temperature with calcium hydride drying vacuum distillation in 48 hours.Benzyl alcohol is steamed using preceding decompression
It evaporates.It is evaporated under reduced pressure again twice with solvent toluene azeotropic after stannous iso caprylate vacuum distillation.After tetrahydrofuran is handled with sodium-potassium-sodium alloy
It steams in a nitrogen atmosphere.
The preparation of MEP：Synthesize the chloro- 2- oxygen -1,3,2- dioxy phosphorus heterocycles of 2- with glycol reaction by phosphorus trichloride first
Pentane (2-chloro-2-oxo-1,3,2-dioxaphospholane, COP), then COP is reacted with methanol synthesizes 2- methoxies
Base -2- oxygen -1,3,2- dioxaphospholane (MEP), specific synthetic method are as follows：
In the three neck round bottom flask of 1000mL, phosphorus trichloride (3.0mol) is dissolved in solvent anhydrous methylene chloride
In (500mL), ethylene glycol (3.0mol) is slowly dropped by constant pressure funnel, after all dripping off, it is 0.5 small that the reaction was continued
When, solvent is boiled off under decompression, twice by product 2- chloro- 1,3,2- dioxaphospholane (CDP) steam (50 for continuous vacuum distillation
℃,200Pa).Product is dissolved in solvent benzol, O is passed through2Reaction 48 hours, steams solvent benzol under decompression, connects under decompression
Product COP is steamed (88-89 DEG C, 20Pa) by continuous distillation twice, in N2It is sealed up for safekeeping in 1 DEG C of refrigerator under atmosphere.
In a dry 1000mL three-necked flask, 0.3mol methanol, three second of 0.3mol are sequentially added with syringe
The solvents tetrahydrofurane (THF) of amine (HCL for being generated except dereaction) and 500mL, after system is cooled to -5 DEG C, side stirring
While the tetrahydrofuran solution (0.154mol COP are dissolved in 100mL tetrahydrofurans) of COP is slowly added to constant pressure funnel, about 1
Hour drips off, and system is reacted 24 hours again at -5 DEG C.The triethylamine hydrochloride sediment of white is filtered, is taken out under vacuum molten
Agent, under reduced pressure distill (92-94 DEG C, 20Pa) purify twice.
The preparation of the polycaprolactone macromole evocating agent of one-ended hydroxy in glove box by CL (17.35g, 0.152mol),
Initiator benzyl alcohol (0.0498g, 0.604mmol) and catalyst stannous iso caprylate Sn (Oct)2(0.145g, 0.302mmol) adds
Enter into dry round-bottomed flask.It is dissolved in chloroform after being heated 5 hours in 130 DEG C of oil bath, is deposited in cold n-hexane.
Filtering, sediment are dried under vacuum to constant weight, yield 88%.It is fed intake by reaction and different amounts of caprolactone CL is added and extends
Reaction time can obtain the PCL of different molecular weight.
Block copolymerization is at 35 DEG C, and tetrahydrofuran is solvent, and using PCL-OH as macromole evocating agent, stannous iso caprylate is
Catalyst is polymerize by the ring-opening polymerization of cyclic phosphate ester monomer.In the tetrahydrochysene furan of the drying of MEP and PCL-OH
It mutters and stannous iso caprylate is added in solution, concentration reprecipitation is in cold ether after reaction 3 hours.Sediment is dried extremely under vacuum
Constant weight, yield 80%.
The composition of block copolymer passes through1H NMR are analyzed, its molecular structure and number-average molecular weight are measured, it is confirmed
The block copolymer contains PCL and PMEP blocks, is characterized to the molecular weight and molecular weight distribution of polymer with GPC.
Fig. 3 gives the GPC superposition objects of a typical macromole evocating agent and block copolymer.As shown, the GPC figures of copolymer
To be unimodal, without the peak of PCL macromole evocating agents, show that PCL macromole evocating agents have completely consumed and obtained PCL-b-
PMEP block copolymer structures.
Embodiment 2, the preparation of nano particle (hereinafter referred to as NPs) and performance evaluation
One, the preparation of nano particle
Amphipathic nature block polymer can be self-assembly of nanometer due to the hydrophobic interaction of hydrophobic section in aqueous solution
Grain.There are many ways to preparing nano particle, ours is dialysis.Nano particle is prepared by dialysis：By 10mg systems
Standby PCL-b-PMEP block copolymers are dissolved in 1mL n,N-Dimethylformamide, then under fast stirring with 60mL/h's
Speed instills 5mL ultra-pure waters, and after being stirred for 10min, dialysis removes organic solvent, and last constant volume to 5mL obtains a concentration of 2mg/
The nanoparticles solution of mL.The preparation of drug-loading nanoparticles is the input 1mg Docetaxels before adding water, and being sufficiently stirred makes system
Mixing, remaining operation are same as above；The preparation of the nano particle of fluorescent marker is that 25 μ g DiO or DiR fluorescence dye is put into before adding water
Material, being sufficiently stirred makes system mixing, remaining operation is same as above.
Two, the form of nano particle
The form of nano particle is formed by using transmission electron microscope observation PCL-b-PMEP copolymers.Such as Fig. 4 (C)
Shown, as seen from the figure, PCL-b-PMEP copolymers are formed by nano particle and regular spherical structure are presented, and grain size is about 40nm
And size distribution is uniform.
Three, the grain size and surface potential of nano particle
The nanoparticles solution of a concentration of 2mg/mL is diluted 10 times, Malvern Zetasizer Nanao ZS90 dynamics
The grain size and particle diameter distribution of solution nano particle when light scattering apparatus detects under the conditions of 25 DEG C.Setting repeats to test three times, as a result
It is analyzed and is averaged using 4.2 softwares of Malvern Dispersion Technology Software.
Fig. 4 (A) is the grain size of nano particle, and the average diameter under granule strength statistics is 40nm or so；Fig. 4 (B) is to receive
The surface potential of rice grain, surface potential are -14mV.
Four, the serum stability of nano particle
0.05mL fetal calf serums (FBS) are added in the nanoparticles solution of 0.45mL 1mg/mL, are incubated jointly at 37 DEG C
Different time is educated, the flat grain size of nano particle is monitored using dynamic light scattering.Existed by the size of the visible nano particles of Fig. 4 (D)
It has almost no change within 50h.Illustrate the simulation it is internal under the conditions of it is highly stable.
Five, releasability of the nano particle to drug
Drug release in vitro experiment is carried out at 37 DEG C in phosphate buffer (PBS, 0.02M, pH=7.4), concrete operations
It is as follows：The drug-loading nanoparticles for containing chemotherapeutic Docetaxel are resuspended with phosphate buffer, nano particle it is final concentration of
1.0mg/mL；Take the solution of 3 parts of 3mL be placed directly in dialysis tubing (Float-A-Lyzer, load stay molecular weight
14,000Da) it, is placed in 22mL phosphate buffer solutions at 37 DEG C and rocks, in different times point, by the external solution of dialysis tubing
(22mL PBS) all takes out, and supplements with the phosphate buffer solution of same volume.The phosphate buffer solution of taking-up is lyophilized
The accumulation burst size of drug is measured by HPLC afterwards, result is the average value of three repetition experiments.Release kinetics profile result
See Fig. 5, in starting 10 hours, Docetaxel there are one faster release, this be due to be located at polymer shell compared with
Close Docetaxel diffusion.And in next 40 hours, the release of Docetaxel is slower and gentle, only discharges 8%,
The above percentage refers to that the chemotherapeutic of release accounts for the mass percentage of load chemotherapeutic.Illustrate that the drug-loading nanoparticles can be with
Realization slowly releases drug in vivo, this medicine stability in blood by when applying in vivo with positive meaning
Justice.
Embodiment 3, nano particle as pharmaceutical carrier cellular level performance evaluation
One, laser scanning co-focusing microscope observation nano particle enters the ability of MDA-MB-231 cells
The case where nano particle by observing DiO labels enters MDA-MB-231 cells.The specific method is as follows：
1) MDA-MB-231 cells are planted in 24 holes containing round coverslip with the density of 8 × 104 cells in every hole
In plate, using 500 μ L DMEM culture mediums, it is placed in 37 DEG C, 5%CO2Incubator culture is for 24 hours；
2) culture medium is removed, the DMEM complete mediums of the new nano particle marked containing DiO are changed to, in 37 DEG C of conditions
Lower and cell co-cultures 4h；
3) culture solution is removed, is cleaned twice using PBS, 4% paraformaldehydes of 1mL (pH 7.4, be dissolved in PBS) are added,
Room temperature fixes 15min；
4) paraformaldehyde solution is removed, is cleaned twice using PBS, 200 μ L 1%Triton X-100 are added and wear film 5min；
5) it uses PBS to clean twice, Alexa is added568phalloidin room temperatures dye 20min；
6) it uses PBS to clean twice, DAPI dye liquor room temperatures is added and dye 2min；
7) PBS is used to clean twice, by coverslip tipped upside down on one side containing an anti-fluorescent quenching mounting of drop with cell
Mounting on the glass slide of agent；
8) laser scanning co-focusing microscope is used to observe intracellular Fluorescence, microscopical model Carl Zeiss LSM
710, Germany.
As shown in fig. 6, blue-fluorescence is the nucleus of DAPI dyes, red fluorescence is the cytoskeleton that Alexa 568 contaminates, and
Green fluorescence is then the nano particle of DiO labels.It can be seen that after cultivating 4h, can into the cell be observed in MDA-MB-231
The nano particle of green fluorescent label shows that nano particle can be absorbed by tumour cell.
Two, the biological effect evaluation of drug-loading nanoparticles
DTXL is loaded using MTT (3- (4,5- dimethylthiazoles -2) -2,5- diphenyltetrazolium bromide bromides) colorimetric determination
Nano particle to the killing ability of breast cancer cell MDA-MB-231 (be purchased from ATCC).It is as follows：Cell is with 10000
The density kind in a/hole is added 100 μ L DMEM culture mediums (10% fetal calf serum) in every hole, is placed in 96 porocyte culture plates
CO2In incubator (37 DEG C, CO2After a concentration of 5%) culture 48 hours, drug-loading nanoparticles are diluted to not with complete medium
(100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.12 μM, 1.56 μM, 0.78 μM, 0.39 μM and 0.19 μM total for same concentration
Count 10 concentration gradients), and 25 μ L MTT solution are added in every hole after 48 hours with MDA-MB-231 cell co-cultures
(5mg/mL is dissolved in aqueous solution).Continue after cultivating 4h, culture solution is sucked out, 100 μ L dimethyl sulfoxides are added in every hole, 37 DEG C put
Set 10min；The light absorption value of solution in hole is measured at 490nm with Bio-Rad microplate reader.Cell viability is calculated with following formula：
Wherein Abs is light absorption value of the solution in 490nm.IC50When being expressed as the cell death half detected by MTT methods
Drug concentration.
As a contrast with the free Docetaxels (DTXL) of same concentration.As a result see Fig. 7.As seen from the figure, when nanometer
It is more than 98% cell death when the DTXL that grain carries is 100 μM a concentration of, and free drug group still has about 40% or so cell dead
It dies, it was demonstrated that the nanometer formulation for carrying medicine has stronger tumor cell killing potential.
The case where embodiment 4, nano particle blood circulation, tumour enrichment
One, Pharmacokinetic Evaluation
27 male ICR mouses (6-8 weeks) are randomly assigned to be 9 groups, every group 3.It is received what is marked with nir dye DiR
Rice grain passes through in tail vein injection to Mice Body.Cycle 0.083,0.5,1,2,4,8,12,24,48 hour in vivo, each
Every group of time point taking-up 3 mouse is collected in blood to heparin sodium anticoagulant tube by the method that eye socket takes blood, and passed through immediately
5000rpm, 4 DEG C of centrifugation 10min, are collected in upper plasma sample to clean Ep pipes.100 μ L plasma samples are taken, with live body petty action
Object imaging quantifies the nano-carriers marked of nir dye DiR in different time points mouse blood.As a result such as Fig. 8 (A)
Shown, nano particle is not removed quickly in blood；Fig. 8 (B) pharmacokinetic curve is shown, after injection 48 hours,
It still has more than 1% nano particle to recycle in blood, this is because highly hydrophilic the playing of nano grain surface PMEP molecules
The effect of stable nanoparticles, and hydrated sheath is formed in nano grain surface, it is effective against the absorption of haemocyanin, escapes machine
The removing of body immune system, to extend the circulation time of nano particle in blood.
Two, the case where tumour is enriched with
It is that nano particle is carried by EPR effects in tumor locus enrichment that nano particle has longer circulation time in blood
Chance is supplied.Therefore next we assess the enrichment feelings at MDA-MB-231 tumor tissues after nano particle intravenous administration
Condition.
The nano particle that nir dye DiR is marked by tail vein injection to 3 nude mices use small animal living body at
As instrumentSpectrum monitors 0.5,2,6,10,24,48,72 and 96 hour time point nano particle in intravital mouse body surface
The distribution situation of tumour.96 hour time point put to death mouse, collected each internal organs and tumor tissues.Using PBS cleansing tissues, with filter
Paper suck dry moisture carries out quantitative analysis with small animal imaging instrument to the fluorescence intensity of each internal organs and tumour.
From Fig. 9 (A) result, nano particle is gradually accumulative in tumor locus as time went on, and is reached at 24 hours
To maximum enriching quantity, then since scavenging effect slowly reduces.The nano particle of time point tumor locus after 24 hours
Average fluorescent strength is all higher than other positions, illustrates that the nano particle has good tumour enrichment.It is each from Fig. 9 (B)
The fluorescence imaging result of internal organs and tumor locus, which can be seen that, goes back nano particle and is only second in the average fluorescent strength of tumor locus
Liver with powerful Scavenging activity further demonstrates the good tumour enrichment of the nano particle.This is attributed to the fact that nanometer
The circulation time for the length that particle has in blood reaches tumor tissues by " EPR " effect for nano particle and provides more
Chance.
The inhibition of embodiment 5, drug-loading nanoparticles to breast cancer in situ mice tumors grew
Breast cancer in situ mouse tumor model is established：
The large-scale culture for carrying out MDA-MB-231 cells first, uses serum-free DMEM before preparing to be inoculated with
(Hyclone) culture cell 6 hours, cell is collected by centrifugation in 1000rpm after pancreatin digestion, and cell, which is resuspended, with PBS makes its density reach
To 2.5 × 107/ mL, will be under the fat pad of second mammary gland on the right side of 200 μ L pallium cell injections to BALB/c nu nude mices.It is in situ
The nude mice of inoculation breast cancer cell is raised about 10 days or so in SPF grades of animal houses can form visual tumors.The volume of tumour is pressed
According to formula：V=0.5 × a × b2It calculates, wherein a refers to the longer diameter of tumour, and b refers to the shorter diameter of tumour.
The tenth day after tumor cell inoculation, the gross tumor volume of nude mice reaches about 50mm3, it is randomly divided into six groups and is started
It is treated.Every group of 5 nude mices, are administered according to following processing method：
PBS：Every nude mice is by 200 μ L PBS of tail vein injection, as a contrast.
Empty NP：Every nude mice does not carry medicine particle by 200 μ L of tail vein injection, as a contrast.
Free DTXL(1mg/kg)：Every nude mice passes through 200 μ L DTXL solution of tail vein injection.The dosage of DTXL is
1mg/kg mouse weights.
NP@DTXL(1mg/kg)：Every nude mice carries the nano particle of DTXL, dosage by 150 μ L of tail vein injection
The dosage for being scaled DTXL is 1mg/kg mouse weights.
Free DTXL(2mg/kg)：Every nude mice passes through 200 μ L DTXL solution of tail vein injection.The dosage of DTXL is
2mg/kg mouse weights.
NP@DTXL(1mg/kg)：Every nude mice carries the nano particle of DTXL, dosage by 150 μ L of tail vein injection
The dosage for being scaled DTXL is 2mg/kg mouse weights.
Phase before the treatment starts every other day carries out one-shot measurement to gross tumor volume, and Vertical Square is detected using vernier caliper
Two upward diameters test the later stage since tumor volume change is larger, daily to tumour body respectively as the length and width of tumour
Product measures primary, and shown in embodiment result such as Figure 10 (A), Figure 10 (B) is to put to death and dissect mouse after treatment end, by tumour from
The tumor weight comparison diagram weighed after being taken out in Mice Body, PBS and blank granules are to the growth of tumour without inhibiting to make
Use, DTXL has certain Tumor growth inhibition effect as present widely used cancer chemotherapeutic drug, but only in 2mg/kg
Significant therapeutic effect is shown under dosage.And no matter NP@DTXL show significantly under 2mg/kg or 1mg/kg dosage
Therapeutic effect, and under 2mg/kg dosage, final tumour almost disappears.It can be seen that nano particle delivering DTXL has
Good tumor growth inhibitory effect.
The inhibition of embodiment 6, drug-loading nanoparticles to B16 Melanoma Growths
2 × 10 are injected using tail vein5Melanoma cells B16 establishes melanoma metastasis model to C57/BL6 mouse.
After four days, the C57/BL6 mouse of lotus knurl are randomly divided into 3 groups, every group 10.At the 10th, 13,16,19,22,25 day, by as follows
Method is administered：
1) PBS groups：Specified time passes through 200 μ L PBS buffer solutions of tail vein injection；
2) DTXL groups：The PBS solution that specified time passes through 200 μ L DTXL of tail vein injection, wherein DTXL's gives medicament
Amount is 3mg/kg mouse weights.
3) NP@DTXL groups：Specified time carries the NP solution of DTXL, dosage 3mg/ by 200 μ L of tail vein injection
Kg mouse weights.
Mouse state is observed after inoculation daily, 3 mouse is taken within 20 days after administration to put to death from every group at random, dissects mouse, it will
Tumour first carries out taking pictures and then tissue is fixed with 4% paraformaldehyde after taking out out of Mice Body, is contaminated with H&E after slice
Color, observation lung transfer case, remaining mouse are put to death when being in moribund condition.
Figure 11 (A) indicates the growth curve of the mouse during entire treatment；PBS groups are all dead after treating 30 days, DTXL
All death after group treatment 33 days, drug-loading nanoparticles group also have 25% mouse survival when treating 90 days；Figure 11 (B) expressions are controlled
The transfer photo of the entire lung of mouse after treating 20 days；Figure 11 (C) indicates that each processing group tumor tissue section is through H&E after treating 20 days
The result of dyeing.It can be clearly seen that in the tumor tissues handled through PBS that shifting tumor tissues accounts for normal structure about 50%,
DTXL groups lung metastatic tumour tissue ratio is reduced to 25% or so, and the nano particle group lung for carrying DTXL only finds that ratio is less than
5% neoplasm metastasis illustrates that drug-loading nanoparticles also have good therapeutic effect to melanoma metastasis tumour.
Claims (10)
1. a kind of diblock copolymer being made of polyphosphate and poly-epsilon-caprolactone, the diblock copolymer is by following formula table
Show, the wherein degree of polymerization x of poly-epsilon-caprolactone block is 15-186, and the degree of polymerization y of polyphosphate block is 7-200
2. copolymer according to claim 1, wherein x are 22 and y is 62.
3. a kind of nano particle is assembled by copolymer according to claim 1 or 2.
4. nano particle according to claim 3 is used to prepare the purposes of pharmaceutical carrier.
5. a kind of drug delivery system is born it includes nano particle according to claim 3 and by the nano particle
The active constituent of load.
6. system according to claim 5, wherein the active constituent is the drug for treating cancer.
7. system according to claim 6, wherein the cancer is selected from breast cancer and lung cancer.
8. system according to claim 6, wherein the drug is Docetaxel.
9. the method for being used to prepare copolymer according to claim 1, the described method comprises the following steps：
A) poly-epsilon-caprolactone for utilizing 6-caprolactone, initiator benzyl alcohol and catalyst stannous iso caprylate to prepare one-ended hydroxy divides greatly
Sub- initiator；
B) the chloro- 2- oxygen -1,3 of 2- is prepared by phosphorus trichloride and glycol reaction, 2- dioxaphospholane (COP) then will
COP is reacted with methanol prepares 2- methoxyl group -2- oxygen -1,3,2- dioxaphospholane (MEP)；And
C) in the presence of solvents tetrahydrofurane and catalyst stannous iso caprylate, make the poly-epsilon-caprolactone macromolecular of the one-ended hydroxy
Initiator carries out block copolymerization with MEP and reacts.
10. the method for being used to prepare nano particle according to claim 3, the method includes making according to claim 1
Or the copolymer described in 2 is self-assembled into nano particle.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810587964.9A CN108727597A (en) | 2018-06-08 | 2018-06-08 | Polyphosphate-polycaprolactone nano-medicament carrier and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810587964.9A CN108727597A (en) | 2018-06-08 | 2018-06-08 | Polyphosphate-polycaprolactone nano-medicament carrier and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108727597A true CN108727597A (en) | 2018-11-02 |
Family
ID=63932969
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810587964.9A Pending CN108727597A (en) | 2018-06-08 | 2018-06-08 | Polyphosphate-polycaprolactone nano-medicament carrier and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108727597A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090238858A1 (en) * | 2008-02-21 | 2009-09-24 | Rutgers, The State University Of New Jersey | Compositions and Methods for Treating Ophthalmic Diseases |
| CN102335432A (en) * | 2010-07-26 | 2012-02-01 | 中国科学院上海药物研究所 | Phosphate-based drug delivery system for intracerebral drug delivery |
| CN106810689A (en) * | 2016-12-28 | 2017-06-09 | 四川国纳科技有限公司 | Bioabsorbable polyphosphate amino acid copolymer material |
-
2018
- 2018-06-08 CN CN201810587964.9A patent/CN108727597A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090238858A1 (en) * | 2008-02-21 | 2009-09-24 | Rutgers, The State University Of New Jersey | Compositions and Methods for Treating Ophthalmic Diseases |
| CN102335432A (en) * | 2010-07-26 | 2012-02-01 | 中国科学院上海药物研究所 | Phosphate-based drug delivery system for intracerebral drug delivery |
| CN106810689A (en) * | 2016-12-28 | 2017-06-09 | 四川国纳科技有限公司 | Bioabsorbable polyphosphate amino acid copolymer material |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2950458C (en) | Carbonate polymer containing a functional group of disulfide five-membered ring in the side chain and application thereof | |
| CN102085177B (en) | Reducible and degradable nano medicine-carrying micelle and preparation method thereof | |
| CN105213348B (en) | A kind of drug-loading nanoparticles and its preparation method and application of reduction response | |
| CN102875818B (en) | Polyamino acid grafted copolymer and preparation method thereof | |
| CN101787119A (en) | Polymer with tumor organization pH responsiveness and micelle thereof | |
| CN102218027A (en) | Polymer micelle lyophilized agent encapsulating insoluble antitumor drug | |
| CN106905519B (en) | Biodegradable amphiphilic polymers, polymer vesicle prepared therefrom and preparing the application in targeted therapy of lung cancer drug | |
| Han et al. | Redox-sensitive micelles for targeted intracellular delivery and combination chemotherapy of paclitaxel and all-trans-retinoid acid | |
| CN104548109B (en) | biomedical compositions | |
| Huang et al. | Fabrication and reduction-sensitive behavior of polyion complex nano-micelles based on PEG-conjugated polymer containing disulfide bonds as a potential carrier of anti-tumor paclitaxel | |
| CN101812227A (en) | Micelle based on non-linear polyethylene glycol-polylactic acid block copolymer and preparation method thereof | |
| Yi et al. | Synthesis, characterization, and formulation of poly-puerarin as a biodegradable and biosafe drug delivery platform for anti-cancer therapy | |
| CN105963706A (en) | Branched polyHPMA copolymer-DOX conjugate, and preparation method and application thereof | |
| CN101205302B (en) | Polyphosphate ester-polycaprolactone tri-block copolymer and uses thereof | |
| CN108727597A (en) | Polyphosphate-polycaprolactone nano-medicament carrier and its application | |
| CN106581691B (en) | Restore targeting polyethylene glycol carbonic ester maytansine prodrug micelle, preparation method and the application of response | |
| CN106188550B (en) | Dendritic block copolymer p AM-PGlu-b-TPGS and the preparation method and application thereof | |
| CN103263671B (en) | Preparation and application of anti-tumor activator nanostructure lipid carrier | |
| CN102552930B (en) | Water-soluble paclitaxel derivative with cell targeting effect and preparation thereof | |
| CN106474486A (en) | A kind of polymer micelle and its application | |
| CN105770900A (en) | Application of carbonate polymer containing bi-sulfur five-member ring functional gene in side chain | |
| CN101628970B (en) | Hyperbranched polymer and preparation method and application thereof | |
| CN110105562A (en) | The bi-block copolymer and its synthetic method of the amine ligand containing DOPA and application | |
| CN109734892B (en) | Poly-diselenocarbonate polymer, preparation method and application thereof | |
| CN105997867B (en) | The preparation method of functional drug composition |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181102 |
|
| RJ01 | Rejection of invention patent application after publication |