CN105860057B - Biodegradable polymer based on the hydrophilic polyaminoacid of hydrophobic function small molecule and its preparation method and application - Google Patents
Biodegradable polymer based on the hydrophilic polyaminoacid of hydrophobic function small molecule and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of biodegradable polymer based on the hydrophilic polyaminoacid of hydrophobic function small molecule and its preparation method and application.Hydrophobic function initiator is prepared first, then triggers NCA monomer ring-opening polymerisations, obtains the amphiphilic surfactant based on functional molecular and poly- polypeptide, and its is emulsifiable poly-（Lactic-glycolic acid）（PLGA）, PLA（PLA）, it is poly-（ε caprolactones）（PCL）Material, polymer nano-particle is obtained, and emulsifying agent also serves as composition structure in nanoparticle surface, the active amino of its end, can further with the targeted molecular with carboxyl by chemical bond and, further to improve the stability of nano-particle, and introduce targeted molecular.Obtained load medicine targeted nano-particle has very high stability, and it can be enriched to tumor locus well, and have therapeutic action and low toxic side effect well to many kinds of solids tumour including human breast carcinoma.
Description
Technical field
The present invention relates to a kind of amphipathic nature polyalcohol, and in particular to one kind is by hydrophobic function small molecule and hydrophilic poly- amino
Amphipathic Biodegradable material of acid construct and preparation method and application.
Background technology
It is poly-（Lactic-co-glycolic acid）（PLGA）It is a kind of biodegradable polymer of FDA approvals, is widely used in
The bio-medical fields such as operation suture thread, tissue engineering bracket, drug controlled release carrier.Biodegradable based on PLGA is received
Rice corpuscles and micro particles turn into realizes one of most important carrier of drug targeting long-acting treatment.For example, a variety of contain albumen
The PLGA microballoons such as Depot of medicine and polypeptide drugs®, Decapeptyl®, Somatulline®, Nutropin®, Depot®,
It is clinically used for treating prostate cancer, acromegalia, growth hormone deficiency.PLGA nano-particles and micro particles are typically
Prepared with emulsification-evaporation method or nanoprecipitation method, this is generally with surfactant come the droplet of stable dispersion, reduction
Surface tension and prevent wadding accumulate.Polyvinyl alcohol（PVA）, poloxamer（poloxamer）And polyvinyl pyrilodone（PVP））Deng
Surfactant is because having in aqueous the advantages that viscosity is high, can be particularly well adsorbed in the surface of dispersed droplets and as system
The most frequently used surfactant of standby PLGA nano-particles and micro particles.But these surfactants exist can not it is biodegradable,
Potential toxicity in vivo be present, lack the shortcomings of functional group.For example, PVA is not only potentially carcinogenic, and zoopery finds skin
The problems such as lower or intravenous injection PVA can cause hypertension, organ damage, anaemia, nervous centralis to suppress（J. Biomed.
Mater. Res. Part A: 100A; 1998-2005, 2012）.
Recently, polyethylene glycol 1000 vitamin E succinic acid ester（TPGS）As a kind of biocompatible surfactants' quilt
It is widely used in nano-particle preparation（Biomaterials 33; 4889-4906, 2012）.For example, it is prepared with TPGS
PLGA, the PLA of a series of medicines such as parcel taxols（PLA）, polyethylene glycol-polylactic acid（PEG-PLA）Nano-particle；This
The size tunable system of a little nano-particles is between 200-800nm.Compared with traditional PVA emulsifying agents, TPGS has been shown preferably
Emulsifying effectiveness and contain efficiency.Research is it has also been found that TPGS emulsifying agents can be hindered by suppressing tumor cell surface P- glycoprotein function
Only anticancer chemotherapeutic agent is pumped out by cell, so as to greatly enhance cancer therapy drug（Adriamycin, taxol, Docetaxel etc.）To resistance to
The toxicity of medicine tumour cell.Moreover, TPGS is also found inherently have active anticancer, can suppress to be implanted in people's lung with nude mice
The growth of cancer cell.But the usual stability of nanoparticle such as the PLGA with TPGS emulsifications are relatively low, and surface is difficult to carry out function
Change.
The content of the invention
It is an object of the invention to provide a kind of biodegradable polymer of hydrophobic function small molecule-hydrophilic polyaminoacid
And preparation method thereof.The polymer can be used to solubilized hydrophobic drug and make as functionalization biodegradable surfactant
Standby multi-functional Nano medication.
To reach above-mentioned purpose, the technical solution adopted by the present invention is：It is a kind of with following structural formula based on hydrophobic work(
The biodegradable polymer of energy property small molecule-hydrophilic polyaminoacid：
,
,。
The polymer of the present invention includes hydrophobic function small molecule R1As the head of hydrophobic, water-soluble is poly-（γ-oligomerization second two
Alcohol-Pidolidone）With it is poly-（β-oligomeric ethylene glycol-L-Aspartic acid）Polyaminoacid segment be as hydrophilic tail, wherein x
2~5, n are 3~20, and the segment that wave represents comes from diamine compound；R1Molecular weight is more than 240 Da, and its structural formula is such as
Under：
1）Come from the substituent R of hydrophobic amine based compound1：
,,
,Deng；
2）Come from the substituent R of hydrophobicity hydroxy compounds1：
,,,,,Deng.
The invention also discloses the above-mentioned biodegradable polymer based on hydrophobic function small molecule-hydrophilic polyaminoacid
Preparation method, comprise the following steps：Using hydrophobic amine based compound as initiator, ring-opening polymerisation a-amino acid-N- carboxyls
Inner-acid anhydride compound obtains the biodegradable polymer based on hydrophobic function small molecule-hydrophilic polyaminoacid；α-the ammonia
Base acid-carboxyl inner-acid anhydride compound is γ-oligomeric ethylene glycol-Pidolidone-N- carboxyl inner-acid anhydrides（EGx-Glu-NCA）Or β-widow
Polyethylene glycol-L-Aspartic acid-N- carboxyl inner-acid anhydrides（EGx-Asp-NCA）.Hydrophobic amine based compound includes distearyl acyl group
Phosphatidyl-ethanolamine, DOPE, hexadecylamine or eribulin etc.；Or hydrophobicity amido chemical combination
Thing is prepared by hydrophobicity hydroxy compounds；Hydrophobic amine based compound, the mol ratio of carboxyl inner-acid anhydride compound are 1:3~
25；The molecular weight of hydrophobic amine based compound is more than 240 Da.
In above-mentioned technical proposal, hydrophobic amine based compound is prepared by hydrophobicity hydroxy compounds（Also known as terminal amino group
Hydrophobic small molecules）For using hydrophobicity hydroxy compounds, p-nitrophenyl chloro-formate as raw material, in anhydrous methylene chloride and pyrrole
In pyridine, reaction obtains the feature Hydrophobic small molecules of p-nitrophenyl chloroformate activation；Then with p-nitrophenyl chloro-carbonic acid
The feature Hydrophobic small molecules and diamines of ester activation are reactant, and in anhydrous methylene chloride and pyridine, reaction prepares hydrophobicity
Amine compound.Hydrophobicity hydroxy compounds includes vitamin e, cholesterol, cumarin, cholic acid, camptothecine, Irinotecan etc..
Diamine compound structure is, one kind in following compound：It is ethylenediamine, butanediamine, pungent
Diamines, lysine methyl ester, ethyl ester of lysine, omithine methyl ester, ornithine ethyl ester, cystine methyl esters, cystine ethyl ester, cystamine
Deng its concrete structure is as follows：
,,,,,,,；
Wherein y is 2,4,8.
In above-mentioned technical proposal, the mol ratio of hydrophobicity hydroxy compounds, p-nitrophenyl chloro-formate and pyridine is 1: 2
∶5；The mol ratio of the feature Hydrophobic small molecules of p-nitrophenyl chloroformate activation, diamines and pyridine is 1: 20: 20；By R1-
4-NC is added drop-wise in excessive diamines, it is ensured that R1- 4-NC only reacts with the single-ended amino of diamines.
In above-mentioned technical proposal, with Hydrophobic small molecules, p-nitrophenyl chloro-formate（4-NC）For raw material, in anhydrous dichloro
In methane and pyridine, reaction obtains the Hydrophobic small molecules of p-nitrophenyl chloroformate activation；Again with the activation of above-mentioned preparation
Hydrophobic small molecules and diamines are reactant, are reacted in anhydrous methylene chloride and pyridine, and the hydrophobic small of terminal amino group is prepared
Molecule（R1-NH2）；Finally with the R of above-mentioned preparation1-NH2Or the hydrophobic function small molecule of amino-contained itself（R1-NH2）To trigger
Agent, ring-opening polymerisation γ-oligomeric ethylene glycol-Pidolidone-N- carboxyl inner-acid anhydrides（EGx-Glu-NCA）Or β-oligomeric ethylene glycol-L-
Aspartic acid-N- carboxyl inner-acid anhydrides（EGx-Asp-NCA）It is prepared based on hydrophobic function small molecule-hydrophilic polyaminoacid
Biodegradable polymer；
The Hydrophobic small molecules of the terminal amino group（R1-NH2）Molecular formula such as formula III（The hydrophobic function of amino-contained itself is small
Molecule）And IV（Hydrophobic amine based compound is prepared by hydrophobicity hydroxy compounds）It is shown；
Formula III
Formula IV.
In above-mentioned technical proposal, when preparing the feature Hydrophobic small molecules of p-nitrophenyl chloroformate activation, in 0 DEG C of degree
Under the conditions of be added dropwise p-nitrophenyl chloro-formate, then 30 DEG C react 24 hours；When preparing hydrophobic amine based compound, reaction temperature
It is 24 hours to spend for room temperature, time；Prepare the amphipathic biodegradable polymerization based on feature Hydrophobic small molecules-polyaminoacid
During thing, reaction temperature is 25-50 DEG C, and the reaction time is 12-72 hours.
Above-mentioned preparation process can represent as follows：
（1）The Hydrophobic small molecules of intermediate p-nitrophenyl chloroformate activation（R1-4-NC）Preparation：In 0 DEG C of condition
Under, the dichloromethane that the dichloromethane solution of p-nitrophenyl chloro-formate is added drop-wise to hydrophobicity hydroxy compounds and pyridine is molten
In liquid.After being added dropwise, reaction solution is transferred to filtering after being reacted 24 hours in 30 DEG C of oil baths will remove pyridiniujm, and revolving removes
Dichloromethane obtains crude product.Crude product is dissolved in petroleum ether, is then centrifuged off insoluble matter in -5 DEG C of degree, then rotate and remove
Solvent is gone to obtain the intermediate R of yellow oily1-4-NC；
（2）The Hydrophobic small molecules of terminal amino group（R1-NH2）Preparation：By step（1）The p-nitrophenyl chloro-carbonic acid of preparation
The Hydrophobic small molecules of ester activation（R1-4-NC）It is dissolved in anhydrous methylene chloride solution, is often dripped within 10 seconds with constant pressure funnel
It is added in the solution of diamines and pyridine, after reacting at room temperature 24 hours, is washed with deionized, organic phase is dried with anhydrous magnesium sulfate
Overnight, filtering, rotary evaporation remove dichloromethane, are dried in vacuo 24 hours, obtain product.After drying, filter and remove magnesium sulfate,
Organic phase filtrate is collected, rotary evaporation removes dichloromethane, and is dried in vacuo 24 hours, obtains the Hydrophobic small molecules of terminal amino group
（R1-NH2）；
（3）The preparation of biodegradable polymer based on hydrophobic function small molecule-hydrophilic polyaminoacid：By step
（2）The Hydrophobic small molecules of the terminal amino group of preparation（R1-NH2）Or the hydrophobic function small molecule of amino-contained itself is dissolved in anhydrous two
It is placed in chloromethanes or dimethylformamide in closed reactor, then by γ-oligomeric ethylene glycol-Pidolidone-N- carboxyls
Acid anhydrides（EGx-Glu-NCA）Or β-oligomeric ethylene glycol-L-Aspartic acid-N- carboxyl inner-acid anhydrides（EGx-Asp-NCA）Dichloromethane
Or dimethyl formamide solution is added rapidly in initiator solution in a nitrogen environment, then 12~72 are reacted at 25~50 DEG C
Hour.Reaction terminate after, by reaction solution with ice ether precipitate, centrifuge, vacuum drying, obtain based on hydrophobic function small molecule-
The biodegradable polymer of hydrophilic polyaminoacid.
In above-mentioned technical proposal, the hydrophobic function small molecule of the Hydrophobic small molecules of terminal amino group or amino-contained itself（R1-
NH2）With γ-oligomeric ethylene glycol-Pidolidone-N- carboxyl inner-acid anhydrides（EGx-Glu-NCA）Or β-oligomeric ethylene glycol-L- asparagus fern ammonia
Acid-N- carboxyl inner-acid anhydrides（EGx-Asp-NCA）Mol ratio as needed control 1:3~25.Organic solvent used can be
Dichloromethane, chloroform, DMF, dimethyl sulfoxide (DMSO), METHYLPYRROLIDONE, the present invention preferably two
Chloromethanes.
The invention also discloses a kind of hydrophobic amine based compound, it is prepared by hydrophobicity hydroxy compounds, is specially
Using hydrophobicity hydroxy compounds, p-nitrophenyl chloro-formate as raw material, in anhydrous methylene chloride and pyridine, reaction obtains pair
The feature Hydrophobic small molecules of chloroformate nitrophenyl ester activation；Then dredged with the feature of p-nitrophenyl chloroformate activation
Water small molecule and diamines are reactant, and in anhydrous methylene chloride and pyridine, reaction prepares hydrophobic amine based compound.Hydrophobicity
Hydroxy compounds includes vitamin e, cholesterol, cumarin, cholic acid, camptothecine, Irinotecan etc.；Diamine compound is selected from following
One kind in compound：Ethylenediamine, butanediamine, octamethylenediamine, lysine methyl ester, ethyl ester of lysine, omithine methyl ester, ornithine second
Ester, cystine methyl esters, cystine ethyl ester, cystamine etc..
In above-mentioned technical proposal, the mol ratio of hydrophobicity hydroxy compounds, p-nitrophenyl chloro-formate and pyridine is 1: 2
∶5；The mol ratio of the feature Hydrophobic small molecules of p-nitrophenyl chloroformate activation, diamine compound and pyridine is 1: 20:
20；By R1- 4-NC is added drop-wise in excessive diamines, it is ensured that R1- 4-NC only reacts with the single-ended amino of diamines.
In above-mentioned technical proposal, when preparing the hydrophobic function small molecule of p-nitrophenyl chloroformate activation, in 0 DEG C of degree
Under the conditions of be added dropwise p-nitrophenyl chloro-formate, then 30 DEG C react 24 hours；When preparing hydrophobic amine based compound, reaction temperature
It is 24 hours to spend for room temperature, time.
Above-mentioned preparation method can represent as follows：
Hydrophobic small molecules disclosed by the invention by above-mentioned end amino（R1-NH2）Trigger γ-oligomeric ethylene glycol-L- paddy ammonia
Acid-N- carboxyl inner-acid anhydrides（EGx-Glu-NCA）Or β-oligomeric ethylene glycol-L-Aspartic acid-N- carboxyl inner-acid anhydrides（EGx-Asp-
NCA）It polymerize the biodegradable polymer based on hydrophobic function small molecule-hydrophilic polyaminoacid prepared, its molecular weight is
0.5~6.5 kDa, wherein polyaminoacid percetage by weight are 20~95%.Its terminal amido can be used to connect bioactivity point
Son, these bioactive molecules include but are not limited to targeted molecular：The small peptides such as cRGD, iRGD, AP, Octreotide, ACUPA point
The protein molecular such as son, antibody and its fragment, iron transfer protein, galactolipin（Gal）With monose or the polysaccharide molecule such as hyaluronic acid, leaf
Acid, biotin etc.；With wear membrane molecule：TAT, Angiopep-2, iRGD, T7, Cilengitide etc..
The present invention further discloses the above-mentioned biodegradable poly based on hydrophobic function small molecule-hydrophilic polyaminoacid
Compound is used as application of the high molecular surfactant in multi-functional nano-medicament carrier is prepared.
Because above-mentioned polymer is made up of feature Hydrophobic small molecules and hydrophilic polyaminoacid, therefore there is good biology
Compatibility and biological degradability.Hydrophobic feature Hydrophobic small molecules head and hydrophilic polyaminoacid tail have macromolecular structure simultaneously
And high surface area, possess as the essential characteristic of good surface activating agent.Moreover, polymeric hydrophilic chain length can pass through regulation
The degree of polymerization of polyaminoacid controls, and so as to obtain the polymer of different hydrophobe ratios, can conveniently prepare have a different emulsifications
The polymer of performance.In addition, amido is contained in the end of polyaminoacid hydrophilic segment, can be used to introduce the bioactivity such as targeted molecular
Molecule, so as to which multifunctional nano pharmaceutical carrier be made, carrying medicament is used for the safe and efficient treatment of tumour.
Specifically preparation method is：
First hydrophobic function small molecule-hydrophilic polyaminoacid terminal amido connection on targeted molecular for example small peptide, monose,
Folic acid, biotin, antibody etc., then again by the use of the polymer containing targeted molecular as emulsifying agent, by emulsification-evaporation method or
The precipitation method prepare the nano-medicament carrier that targeted molecular is contained on surface；Or first use hydrophobic function small molecule-hydrophilic poly- amino
Acid is made surface for emulsifying agent and contains the nano-medicament carrier of amido, then again with the bioactive molecule containing multifunctional group such as
Hyaluronic acid, transferrins etc. react, and nano-medicament carrier surface-crosslinked and containing targeted molecular is prepared.
The first strategy is first by the amphipathic biodegradable polymer based on feature Hydrophobic small molecules-polyaminoacid
It is dissolved in water equal solvent, then under EDC/NHS catalytic conditions, is lived with biologies such as small peptide, monose, folic acid, biotin, antibody
Property molecule reaction, be made the surfactant containing bioactive molecules such as targeted moleculars；Then to the water-soluble of the surfactant
The acetone soln of the biodegradable polymers such as PLGA, PLA, PCL or PEG-PLA is added dropwise in liquid, then is stirred at room temperature
Volatilization removes organic solvent, and the multi-functional nano-particle for containing the bioactive molecules such as targeted molecular to surface is collected by centrifugation and carries
Body.
Second of strategy is that the biodegradable polymers such as PLGA, PLA, PCL or PEG-PLA first are dissolved in into acetone etc. is organic
In solvent, the biodegradable polymer dissolved with hydrophobic function small molecule-hydrophilic polyaminoacid is then added drop-wise to dropwise
In the aqueous solution, then 6 h volatilizations are stirred at room temperature and remove acetone；The nano-particle for containing amido to surface is collected by centrifugation.Then,
Such as hyaluronic acid of the bioactive molecule containing multiple carboxyls after being activated with EDC/NHS（HA）Or transferrins etc. is added to table
Face is contained in the aaerosol solution of the nano-particle of amido, is reacted overnight under the conditions of pH 8-9, is prepared surface-crosslinked and contains
The multi-functional nanoparticulate carriers of targeted molecular.
Above-mentioned multi-functional nanoparticulate carriers can load multi-medicament, for being treated inside a variety of diseases.
The present invention further discloses the above-mentioned biodegradable poly based on hydrophobic function small molecule-hydrophilic polyaminoacid
Application of the compound in terms of the solubilising of hydrophobic medicine.Above-mentioned polymer has good biocompatibility, biological degradability and Yi Gong
The advantages that energy property.Hydrophobic feature small molecule head and hydrophilic polyaminoacid tail have macromolecular structure and high surface area simultaneously,
Possess excellent emulsifiability, there is preferable application prospect in terms of solubilized hydrophobic drug.In addition, the poly- ammonia of polymeric hydrophilic
Amido is contained in the end of base acid segment, available for the bioactive molecule such as targeted molecular and cell-penetrating peptide is introduced, is advantageous to improve medicine
The bioavilability of thing and the enrichment in affected area, so as to which the therapeutic effect of medicine be greatly improved.Specific preparation method：
First configure a series of various concentrations（0.5-20 mg/mL）Two based on feature Hydrophobic small molecules-polyaminoacid
The aqueous solution of parent's property biodegradable polymer, then again by the hydrophobic drug of excess（Taxol, Docetaxel, camplotheca acuminata
Alkali, Irinotecan, vincristine, TPT, Belotecan, vinorelbine etc.）Above-mentioned polymer solution is added, is then placed
Onto 37 DEG C of shaking tables.After 48 h, then centrifugation removes undissolved medicine, obtains using the hydrophobic drug after polymer solubilising
Solution.
Because above-mentioned technical proposal is used, the present invention has following advantages compared with prior art:
1. in the biodegradable polymer disclosed by the invention based on hydrophobic function small molecule-hydrophilic polyaminoacid,
Feature Hydrophobic small molecules and polyaminoacid all have good biocompatibility；Polyaminoacid material has good biology can
Degradability, and its catabolite is the natural amino acid having no toxic side effect；The hydrophilic chain end of Inventive polymers contains amine
Base functional group, it can be easy to introduce the bioactive molecule such as targeted molecular and cell-penetrating peptide by coupling reaction, so as to be prepared into
Contain targeted molecular or multifunctional nano pharmaceutical carrier surface-crosslinked and containing targeted molecular to surface；In addition, should preparing
During polymer, the diamines small molecules such as the diamines containing ketal, cystine ester or cystamine can be facilitated the introduction of, it is quick so as to which pH or reduction be made
Sense type nano-carrier, realize that nano-particle triggers release medicine in the cell.
2. the present invention is used as macromolecule by the use of the biodegradable polymer of hydrophobic function small molecule-hydrophilic polyaminoacid
The surface of multifunctional nano pharmaceutical carrier prepared by surfactant is made up of polyaminoacid segment, is overcome very well existing most
Nanoparticle surface can not biodegradable, easily trigger allergic reaction caused by polyethylene glycol is formed and be difficult to carry out nanometer
The problems such as particle surface is modified；Hydrophilic polyaminoacid chain not only has good biological degradability, and the amido energy of its end
It is convenient to introduce targeted molecular in nanoparticle surface and wear the bioactive molecules such as mould peptide, so as to strengthen Nano medication in affected area
Enriching quantity.
3. the biodegradable polymer conduct disclosed by the invention based on hydrophobic function small molecule-hydrophilic polyaminoacid
Macromolecule solubilizer can overcome existing most solubilizer（Poloxamer, Tween 80, castor oil etc.）Without biodegradable
Property, biocompatibility is bad, is difficult the shortcomings of being functionalized modification；Build the feature Hydrophobic small molecules of Inventive polymers
All there is good biocompatibility with polyaminoacid；Polyaminoacid material has good biodegradability, and it drops
It is the natural amino acid having no toxic side effect to solve product；The hydrophilic chain end of the polymer contains amino functional group, can be easy to
Introduced by coupling reaction and targeted molecular and wear the bioactive molecules such as mould molecule, can preferably improve drug bioavailability with
And targeting is enriched to affected area, improves the therapeutic effect of medicine.
4. the biodegradable polymer disclosed by the invention based on hydrophobic function small molecule-hydrophilic polyaminoacid is made
Existing high molecular surfactant can be overcome very well for high molecular surfactant（PVA, poloxamer, PVP etc.）Without life
Biodegradable, biocompatibility are bad, are difficult the shortcomings of being functionalized modification；Prepared using it as surfactant
The biodegradable polymer nano-medicament carrier such as PLGA, PLA, PCL or PEG-PLA, is successfully solved existing for existing carrier
Surfactant can not it is biodegradable, potential toxicity in vivo be present, lack the shortcomings of functional group, achieve unexpected
Technique effect.
Therefore, hydrophobic function small molecule-hydrophilic polyamino acid polymers that prepared by the present invention have good biofacies
Capacitive, biological degradability and contain functional group, have in terms of the preparation of the solubilising and functional nano medicine of hydrophobic medicine
Huge application potential.
Brief description of the drawings
Fig. 1 is the hydrogen nuclear magnetic spectrogram of initiator vitamin e amino in embodiment one；
Fig. 2 is initiator cholesterol amino hydrogen nuclear magnetic spectrogram in embodiment two；
Fig. 3 is polymer vitamin e in embodiment three-poly-（γ-diethylene glycol monomethyl ether-Pidolidone ester）Proton magnetic
Spectrogram；
Fig. 4 is polymer vitamin e in embodiment three-poly-（γ-diethylene glycol monomethyl ether-Pidolidone ester）MALDI-
TOF phenograms；
Fig. 5 is polymer cholesterol in example IV-poly-（γ-diethylene glycol monomethyl ether-L-Aspartic acid ester）Proton
Magnetic spectrum figure；
Fig. 6 is vitamin e in embodiment five-poly-（γ-diethylene glycol monomethyl ether-Pidolidone ester）To dewatering medicament Japanese yew
The solubilising figure of alcohol；
Fig. 7 is PLGA in embodiment seven, VE-poly (EG2- Glu, nano-particle PLGA NPs and surface key and hyalomitome
The x-ray photoelectron energy spectrum diagram of the nano-particle HA-PLGA NPs of acid analysis for surface composition；
Fig. 8 is nano-particle in embodiment seven（PLGA NPs）The hydrogen nuclear magnetic spectrogram of surface-active contents；
Fig. 9 be in embodiment 11 nano-particle to dewatering medicament taxol（PTX）Cumulative in vitro burst size and time
Graph of a relation；
Figure 10 is nano-particle in embodiment 12（HA-PLGA NPs）To normal cell（L929）And tumour cell
（MCF-7, MG U87）Toxicity data figure；
Figure 11 is medicine-carried nano particles in embodiment 13（PTX-HA-PLGA NPs）To human breast cancer cell（MCF-7）
Toxicity data figure；
Figure 12 is medicine-carried nano particles in embodiment 14（PTX-HA-PLGA NPs）In lotus MCF-7 human breast carcinoma mouse
In tumor killing effect, oncotherapy picture, changes of weight and survival rate figure；
Figure 13 is medicine-carried nano particles in embodiment 14（PTX-HA-PLGA NPs）In lotus MCF-7 human breast carcinoma mouse
Histomorphometric analysis figure during treatment end 21 days.
Embodiment
Below in conjunction with the accompanying drawings and embodiment the invention will be further described：
The initiator vitamin e amino of embodiment one（VE-NH2）Synthesis
（1）Intermediate VE-4-NC preparation：Under nitrogen environment, by p-nitrophenyl chloro-formate（4-NC, 1.98 g,
9.8 mmol）Dichloromethane（30 mL）Solution is added drop-wise to vitamin e under the conditions of 0 DEG C with the speed of 5 seconds one drops（VE, 2.12
g, 4.9 mmol）And pyridine（1.98 mL, 24.5 mmol）Dichloromethane（10 mL）In solution.After being added dropwise, transfer
Reacted 24 hours into 30 DEG C of oil baths.After reaction, accessory substance pyridiniujm is filtered to remove, then filtrate is spin-dried for Rotary Evaporators, is obtained
To faint yellow sticky VE-4-NC crude products.Then petroleum ether is used（b.p: 60-90 ℃）Crude product is dissolved, is centrifuged off miscellaneous
Matter, revolving, finally vacuum drying obtains clear yellow viscous oily product VE-4-NC, yield 93.4%；
（2）Initiator vitamin e amino（VE-NH2）Preparation：Under nitrogen environment, by step（1）The p-nitrophenyl of preparation
The VE-4-NC of base chloroformate activation（0.6 g, 1.0 mmol）Dichloromethane（14 mL）Solution constant pressure funnel 10
Second is often added drop-wise to ethylenediamine（1.35 mL, 20.0 mmol）And pyridine（1.6 mL, 20 mmol）The dichloromethane of mixing（4
mL）In solution, room temperature magnetic agitation is reacted 24 hours.Then the dichloromethane of certain volume is added, and is extracted with deionized water,
Until aqueous phase becomes no color.The dichloromethane being collected into mutually is dried 24 hours with anhydrous magnesium sulfate under the conditions of -24 DEG C,
Filter and remove magnesium sulfate, rotary evaporation removes dichloromethane, is finally dried in vacuo 24 hours, obtains clear yellow viscous oily product
VE-NH2, yield 78.1%.
VE-NH2Nuclear-magnetism, which characterizes, sees accompanying drawing 1,1HNMR (400MHz, CDCl3): δ 3.33 (t, -NHCH2CH2NH2);
2.90 (t, -NHCH2CH2NH2); 2.58 (t, -Ph(CH3)3CH2CH2-); 2.08, 2.02 (s, -Ph(CH3)3-);
1.77 (t, -Ph(CH3)3CH2CH2-); 1.52 (m, CH3(CH(CH3)CH2CH2CH2)3-); 1.37-1.07 (m, CH3
(CH(CH3)CH2CH2CH2)3-; s, -C(CH3)O-); 0.87-0.83 (d, CH3(CH(CH3)CH2CH2CH2)3-)。
The initiator cholesterol amino of embodiment two（Chol-NH2）Synthesis
（1）Intermediate Chol-4-NC preparation：Nitrogen protection under, by p-nitrophenyl chloro-formate (4-NC, 1.61 g,
8.0 mmol）Dichloromethane（About 30 mL）Solution is slowly added dropwise under condition of ice bath（Drip within about 5 seconds one）To cholesterol（Chol,
1.55 g, 4.0 mmol）And pyridine（1.56 mL, 20.0 mmol）Dichloromethane（10 mL）In.Then, in 30 DEG C of oil baths
Middle reaction one day, is filtered to remove pyridiniujm, then rotary evaporation obtains white solid crude product.Finally crude product is washed with ice acetone
Purifying, obtains Chol-4-NC products.Yield is：50.4%；
（2）Initiator cholesterol amino（Chol-NH2）Preparation：By Chol-4-NC（1.05 g, 1.9 mmol）It is dissolved in
Dichloromethane（30 mL）In, the ethylenediamines of 20 times of excess is then slowly dropped under conditions of 30 DEG C of oil baths（2.57
mL, 38.0 mmol）Dichloromethane（7 mL）In solution, reacted one day after dripping.After reaction terminates, by solution core
Funnel filters, and is then spin-dried for clear liquid, adds absolute ether to dissolve to obtain yellow solution.Yellow solution buck（pH=12）Washing two
It is secondary to colourless to remove remaining pyridine and ethylenediamine, then with secondary water washing three times, finally dried with anhydrous magnesium sulfate.Then use
Sand core funnel filters, rotary evaporation, then is dried in vacuo 24 hours, produces white product.Yield is about：67.7%.
Chol-NH2Nuclear-magnetism, which characterizes, sees accompanying drawing 2,1H NMR (400 MHz, CDCl3): δ 5.37 (t, -CH=C-);
4.49 (m, -(-CH2)2CHOCONH-); 3.21 (t, -NHCH2CH2NH2); 2.81(t, -NHCH2CH2NH2); 2.34,
2.27(d, -CH=CCH2CH(-CH2) O-)。
The vitamin E- of embodiment three gathers（γ-diethylene glycol monomethyl ether-Pidolidone）（VE-poly(EG2-Glu)）Conjunction
Into
So that n is 5 as an example：By initiator vitamin e amino VE-NH2（1.16 g, 2.25 mmol）It is dissolved in 37 mL dichloros
It is placed in methane solvent in closed reactor.Under nitrogen environment, by γ-diethylene glycol monomethyl ether-Pidolidone-N- carboxyls
Acid anhydrides（EG2-Glu-NCA）（3.71 g, 13.50 mmol）The dichloromethane solution of monomer（37 mL）Quickly it is added to initiator
In, 25 DEG C are reacted 12 hours.Course of reaction is monitored with Fourier infrared spectrograph.After reaction terminates, by reaction solution rotary evaporation
About 18 milliliters are concentrated into, is precipitated with ice ether, low-temperature centrifugation（-5℃, 5000 rpm）Collect precipitation.Finally washed with ice ether
Three times, it is dried in vacuo 48 hours, obtains light yellow product, yield 55.8%.
VE-poly(EG2- Glu) nuclear-magnetism characterize see accompanying drawing 3,1H NMR (600 MHz, CDCl3): δ 4.24 (m, -
NHCOCH-; t, CH3OCH2CH2OCH2CH2-); 3.68-3.54 (m, CH3OCH2CH2OCH2CH2-); 3.40-3.36
(t, -NHCH2CH2NH-; s, CH3OCH2CH2 OCH2CH2-); 2.66-2.34 (t, -Ph(CH3)3CH2CH2-, t,-
COCH(NH)CH2CH2-); 2.07, 2.03 (s, -Ph(CH3)3-); 1.99-1.88 (m, -COCH(NH)CH2CH2-);
1.78 (t, -Ph(CH3)3CH2CH2-); 1.53 (m, CH3(CH(CH3)CH2CH2CH2)3-); 1.37-1.07 (m, CH3
(CH(CH3)CH2CH2CH2)3-; s,-C(CH3)(CH2)-); 0.87-0.83 (d, CH3(CH(CH3)CH2CH2CH2)3-)。
VE-poly(EG2- Glu) MALDI-TOF characterize see accompanying drawing 4, molecular weight 1695.1, unit number is 5.
With similar method, by from different monomers（EGx- Glu-NCA or EGx-Asp-NCA）With control monomer/initiation
The rate of charge of agent, a series of different VE-poly (EG of degree of polymerization can be preparedx-Glu)nWith VE-poly (EGx-Asp)n
Amphipathic nature polyalcohol, its sign are shown in Table 1.
Polymer VE-poly (the EG of table 1x-Glu)nWith VE-poly (EGx-Asp)nPreparation and sign.
Example IV cholesterol-poly-（γ-oligomeric ethylene glycol-L-Aspartic acid）（Chol-poly(EGx-Asp)）Conjunction
Into
To synthesize Chol-poly (EG2-Asp)19（x=2, n=19）Exemplified by：By initiator cholesterol amino Chol-NH2
(0.18 g, 0.38 mmol), which is dissolved in 19.8 mL DMF solutions, to be placed in closed reactor.Under nitrogen environment, by EG2-
Asp-NCA monomers (1.98 g, 7.6 mmol）DMF solution (19.8 ml) be quickly added in initiator, 40 DEG C of reactions are 48 small
When.Course of reaction is monitored with Fourier infrared spectrograph.After reaction terminates, concentrated solvent is sunk to about 4 mL of residue with ice ether
Form sediment, low-temperature centrifugation（-5℃, 5000 rpm）Collect precipitation.Finally washed three times with ice ether, be dried in vacuo 48 hours, obtain
Faint yellow solid, yield 62.1%.Its sign is shown in Table 2.
Chol-poly(EG2-Asp)19Nuclear-magnetism, which characterizes, sees accompanying drawing 5, 1H NMR (400 MHz, CDCl3): δ 5.35
(t, -CH=C-); 4.46 (m, -(-CH2)2CHOCONH-); 4.24 (m, -NHCOCH-; t,
CH3OCH2CH2OCH2CH2-) 3.65-3.54 (m, CH3OCH2CH2OCH2CH2-); 3.42-3.34 (t, -
NHCH2CH2NH-; s, CH3OCH2CH2OCH2CH2-)。
With similar method, from different initiators such as cumarin amine compound（Cou-NH2）, cholic acid amido chemical combination
Thing（CA-NH2）, camptothecine amine compound（CPT-NH2）, Irinotecan amine compound（INT-NH2）, the courage containing double sulphur consolidates
Alcohol amine compound（Chol-SS-NH2）, DSPE（DSPE）, DOPE
（DOPE）, eribulin（Eri）And hexadecylamine（THDA）, a series of hydrophobic functions can be made in ring-opening polymerisation NCA monomers
Small molecule-hydrophilic polyamino acid polymers, its sign are shown in Table 2.
The preparation of the hydrophobic function small molecule of table 2-hydrophilic polyamino acid polymers and sign.
VE-poly (the EG of embodiment fivex-Glu)nPolymer solubilizing hydrophobic drugs taxol（PTX）
With with VE-poly (EG2-Glu)5Solubilisation of hydrophobic anti-cancer medicine paclitaxel（PTX）Exemplified by, the PTX of excess is added to
Various concentrations（0.5、1.0、2.5、5.0、10.0、15.0、20.0 mg/mL）VE-poly (EG2-Glu)5In the aqueous solution（2
mL）, then seal, be placed on 37 DEG C of shaking table（200 rpm）In.Two days later, centrifugation removes undissolved medicine, obtains
With the PTX solution after polymer solubilising.The PTX amounts of solubilising can be measured with HPLC.As a result show PTX solubilising amount with
VE-poly(EG2-Glu)5The increase of polymer concentration and dramatically increase, as VE-poly (EG2-Glu)5Concentration is 20 mg/mL
When, PTX solubility may be up to 381.9 μ g/mL, the solubilizing effect of other concentration, refer to accompanying drawing 6.Its solubilizing effect is better than existing
Solubilizing effect in widely used TPGS to PTX（Eur. J. Pharm. Sci. 25; 445-453, 2005）.It is more important
, the particle surface after solubilising contains amino-functional group, can be used to introducing targeted molecular and wear the bioactivity such as mould molecule
Molecule, improves drug bioavailability and targeting is enriched to affected area.
Chol-SS-poly (EG of the embodiment six containing double sulphurx-Asp)nPolymer solubilizing hydrophobic drugs Docetaxel
（DTX）
With with Chol-SS-poly (EG3-Asp)6Exemplified by solubilisation of hydrophobic cancer therapy drug DTX, the DTX of excess is added to not
Same concentration（1.0、2.0、5.0、10.0、15.0、20.0 mg/mL）Chol-SS-poly (EG3-Asp)6In the aqueous solution（2
mL）, then seal, be placed on 37 degrees Celsius of shaking table（200 rpm）In.Two days later, centrifugation removes undissolved medicine,
Obtain using the DTX solution after polymer solubilising.The DTX amounts of solubilising can be measured with HPLC.As a result show DTX solubilising amount with
Chol-SS-poly (EG3-Asp)6The increase of polymer concentration and dramatically increase.For example, work as Chol-SS-poly (EG3-
Asp)6When concentration is respectively 1 mg/mL and 2 mg/mL, DTX solubility may be up to 28.0 μ g/mL and 57.5 μ g/mL.And
And nano-particle has been self-assembly of after polymer solubilising DTX, its particle diameter is about 60 nm, and particle diameter distribution is about 0.25.
VE-poly (the EG of embodiment sevenx-Glu)nPLGA nano-particles are prepared for high molecular surfactant
PLGA nano-particles（PLGA NPs）Preparation：With VE-poly (EG2-Glu)5It is for high molecular surfactant
Example, by 0.9 mL PLGA acetone soln（10 mg/mL）9 mL VE-poly (EG are added drop-wise to dropwise2-Glu)5The aqueous solution
（0.45 mg/mL）In, 6 h are stirred at room temperature, acetone is volatilized, are then centrifuged for（12000 rpm, 10 min, 4 ℃;
Sorvall Biofuge Stratos, Thermo Scientific）Nano-particle is collected, and with secondary washing once, is removed
Free emulsifying agent, surface (EG containing VE-poly is prepared2-Glu)5PLGA nano-particles.The particle diameter and grain of nano-particle
Footpath distribution can be respectively 135 nm and 0.06 with dynamic light scattering measurement.Nanoparticle surface contains VE-poly (EG2-Glu)5It is poly-
Amino acid segment can observe being confirmed for N peaks by x-ray photoelectron power spectrum（Fig. 7）.Moreover, nanoparticle surface
Amount containing surfactant can compose nuclear-magnetism with hydrogen and specifically measure, and pass through EG on comparison surface activating agent2In d 3.54-3.68
（At Fig. 8 B *）Methylene and PLGA in d 5.21（Fig. 8 A）The peak area of the methine at place, PLGA surfaces can be calculated and contained
7.3 wt.% VE-poly (EG2-Glu)5.By adjusting VE-poly (EG2-Glu)5For the concentration of high molecular surfactant
For 0.15,030 mg/mL, nano-particle of the particle diameter in 150 nm can be prepared（Characterization result is shown in Table 3）.
Contain the preparation of the PLA nano-particles of targeted molecular in the surface of embodiment eight
Targeted molecular is first connected to the end of the hydrophilic polyaminoacid segment of high molecular surfactant.With connection
ACUPA small peptides targeted molecular is to VE-poly (EG3-Glu)14.4Exemplified by polymer, polymer ends amido is converted into carboxylic first
Base, specific method are as follows：By VE-poly (EG3-Glu)14.4（900 mg, 0.20 mmol）, succinic anhydride（24.0 mg,
0.24 mmol）, DMAP（24.4 mg, 0.20 mmol）And triethylamine（20.4 mg, 0.20 mmol）It is dissolved in 9 milliliters of nothing
In water Isosorbide-5-Nitrae-dioxane, it is stirred at room temperature 24 hours；Reaction terminates, and revolving removes solvent, then is dissolved with a small amount of dichloromethane, so
After filtrate is collected by filtration, precipitated with absolute ether, centrifuge, be finally dried in vacuo, obtain VE-poly (EG3-Glu)14.4- COOH,
Yield 78.1%.Then, VE-poly (EG are activated with EDC/NHS3-Glu)14.4- COOH terminal carboxyl groups, by VE-poly (EG3-
Glu)14.4-COOH（680mg, 0.15 mmol）, NHS（51.8 mg, 0.45 mmol）And EDC（57.5 mg, 0.30 mmol）
It is dissolved in dichloromethane（6.8 mL）In, after reacting at room temperature 24 hours, precipitated, centrifuged with absolute ether, vacuum drying, obtained
VE-poly(EG3-Glu)14.4- NHS, yield 87.1%.Finally, by obtained VE-poly (EG3-Glu)14.4- NHS is with containing amino
ACUPA targeted moleculars reaction, specifically, first by ACUPA（44.7 mg, 0.14 mmol）With VE-poly (EG3-Glu)14.4-
NHS（585mg, 0.13 mmol）It is dissolved in DMF（6 mL）In, then 30 DEG C react 2 days.After reaction, precipitated with absolute ether,
Centrifugation, vacuum drying, obtains the VE-poly (EG with targeted molecular3-Glu)14.4- ACUPA polymer, yield 77.9%.Then,
According to the similar method of embodiment seven VE-poly (EG3-Glu)14.4- ACUPA is that table is prepared in high molecular surfactant
The PLA nano-particles of ACUPA targeted moleculars are contained in face, and the targeted molecular density that the nanoparticle surface contains can pass through regulation
The concentration of high molecular surfactant controls.
With similarity method can by other targeted moleculars such as cRGD, iRGD, AP, Octreotide, TAT, Angiopep-2,
IRGD, T7, Cilengitide, antibody and its fragment, galactolipin（Gal）, folic acid, biotin etc. be connected to nanoparticle surface,
So as to realize the surface-functionalized of nano-particle and the targeting conveying of Nano medication after medicine is contained as pharmaceutical carrier.
Embodiment nine is surface-crosslinked and the preparation of PLGA nano-particles containing hyaluronic acid targeted molecular
The PLGA nano-particles prepared with embodiment seven are surface-crosslinked and containing hyaluronic acid targeted molecular to prepare
PLGA nano-particles.First by hyaluronic acid（HA, 45.5 mg, 1.3 μm of ol, 120.0 μm of ol carboxyls）It is dissolved in 4.0 mL vinegar
Acid-sodium acetate buffer（0.1 M, pH 5.0）In, then EDC is added at room temperature（6.9 mg, 36.0 μm of ol）And NHS
（2.1 mg, 18.0 μm of ol）Activate 0.5 h.Then, the HA of activated carboxylic is added to PLGA nano-particles prepared by embodiment seven
Aaerosol solution（NaCO3/NaHCO3Buffer medium, pH 9.0）In, 37 DEG C of reactions are overnight.After reaction, molecular cut off is used
（MWCO）Unreacted HA and other small molecule by-products are removed for the dialysis of 350000 Da bag filters, freeze-drying obtains surface
Crosslinking and the PLGA nano-particles containing hyaluronic acid targeted molecular.The amount that nanoparticle surface contains HA molecules is by first will
HA is marked with nir dye Cy5, the Cy5 HA marked then is connected into nanoparticle surface with above-mentioned same procedure, then
The fluorescence intensity of nano-particle is measured with fluophotometer again, the HA contained so as to calculate nanoparticle surface is 5.15
wt.%（Characterization result is shown in Table 3）.
The VE-poly (EG2-Glu) of table 35Surfactant prepares PLGA nano-particles（PLGA NPs）And on its surface
Introduce hyaluronic acid（HA）, surface-crosslinked and containing targeted molecular nano-particle is made（HA-PLGA NPs）Characterize
The surface reversible crosslink of embodiment ten and contain transferrins（Tf）The preparation of the PCL nano-particles of targeted molecular
The N- glycosidic linkage oxidations of transferrins are prepared into the transferrins of aldehyde radical functionalization first（Tf-CHO）, iron egg will be turned
In vain（Tf, 25 mg）It is dissolved in 900 μ L sodium acetate（0.1 M, pH 5.5）In solution, then to its add 225 μ L sodium metaperiodates
Solution（50 mM）, after reacting 1 h at room temperature.After reaction, Sephadex G-25 chromatographic columns are used under the conditions of 4 DEG C（1.8 ×
25 cm）Purification（Mobile phase is sodium acetate（0.1 M, pH 5.5）Solution）, Tf-CHO is prepared.Then, with containing cystine linkage
VE-SS-poly(EG2-Glu)6Polymer is that high molecular surfactant prepares PCL nano-particles, by 0.9 mL PCL acetone
And tetrahydrofuran solution（10 mg/mL）9 mL VE-SS-poly (EG are added drop-wise to dropwise2-Glu)6The aqueous solution（0.45 mg/mL）
In, 6 h are stirred at room temperature, acetone and tetrahydrofuran is volatilized, are then centrifuged for collecting nano-particle, and with secondary washing once, remove
Free emulsifying agent is removed, surface (EG containing VE-SS-poly is prepared2-Glu)6PCL nano-particles.Finally, by Tf-CHO plus
To the aaerosol solution of PCL nano-particles（NaCO3/NaHCO3Buffer medium, pH 9.0）In, reacted overnight under room temperature condition.Reaction
Afterwards, molecular cut off is used（MWCO）Unreacted Tf and other small molecule by-products are removed for the dialysis of 350000 Da bag filters,
Freeze-drying obtains surface reversible crosslink and the PCL nano-particles containing Tf targeted moleculars.Due in high molecular surfactant
In introduce cystine linkage, the nano-particle has reduction-sensitive.Dynamic optical dissipates color result and shows the average straight of the nano-particle
Footpath is about 150 nm.
Loading and release in vitro of the nano-particle of embodiment 11 to dewatering medicament
Dewatering medicament taxol is contained with nano-particle（PTX）Exemplified by, first by PTX and PLGA（Theoretical drugloading rate 8
wt.%）It is dissolved into acetone soln, adds dropwise the solution to VE-poly (EG2-Glu)5The water of high molecular surfactant
Solution（0.45 mg/mL）In, 6 h are stirred at room temperature, acetone is volatilized, are then centrifuged for collecting nano-particle, and with secondary water washing
Free emulsifying agent is removed, the PLGA nano-particles for containing PTX are prepared（PTX-PLGA NPs）.Then by targeting point
Son（Such as HA）Surface is bonded in, obtains targeted nano-particle（PTX-HA-PLGA NPs）, the method for reference implementation example nine.
The particle diameter and particle diameter distribution of nano-particle can be respectively 164 nm and 0.16 with dynamic light scattering measurement.Nano-particle
Drugloading rate usable highly effective liquid chromatogram（HPLC）Measure.0.2 mL PTX-PLGA NPs solution is first taken, it is lyophilized to weigh.Again
With water and acetonitrile（50:50, v/v）Again dissolve, after 0.45 μm of membrane filtration, drugloading rate is determined with HPLC（DLC）And load
Drug effect rate（DLE）Respectively 6.4 wt.% and 80.1%.VE-poly (EG can be used with similar method2-Glu)5Macromolecule surface is lived
Property agent prepare nano-particle realize to other hydrophobic drugs such as Docetaxel（DTX）, camptothecine（CPT）, Irinotecan
（INT）, vincristine（VCR）, TPT（TPT）, Belotecan（BLT）, vinorelbine（NVB）, adriamycin（DOX）Deng
Contain, it the results are shown in Table 4.
VE-poly (the EG of table 42-Glu)5The nano-particle for containing dewatering medicament prepared by high molecular surfactant
PTX extracorporeal releasing experiment is shaken in 37 DEG C of constant-temperature tables（200 rpm）Carry out, there are three Duplicate Samples.Will
0.5 mL PTX-HA-PLGA NPs medicine-carried nano particles solution is placed in bag filter（MWCO 12000-14000 Da,
Spectrum Laboratories, USA）In, then the bag filter is put into containing Tween 80（0.1%, v/v）PB（10
mM, pH 7.4）Cushioning liquid（25 mL）Middle carry out extracorporeal releasing experiment.At interval of certain time, taken out from dissolution medium
5.0 mL solution are used as test, while 5.0 mL respective medias are added into test tube.The dissolution medium solution of taking-up is freezed, then
Add 0.5 mL acetonitrile/waters（1:4, v/v）Dissolving, then with HPLC determine drug concentration in solution, finally observe PTX from
The behavior that PTX-HA-PLGA NPs Cumulative release amounts change over time（Accompanying drawing 9）.As a result show in PB（10 mM, pH 7.4）
In medium, about 54.0% PTX discharges from HA-PLGA NPs after 7 days.Importantly, the nano-particle is big
The big medicine that reduces was released in initial two days prominent, realized long-acting release of the medicine from PLGA nano-particles.
The mtt assay of embodiment 12 analyzes empty nano-particle（HA-PLGA NPs）Cytotoxicity
From normal cell（Mouse is into fiber L929 cells）And tumour cell（Human breast carcinoma MCF-7, human glioma
MG U87）With mtt assay test HA-PLGA NPs cytotoxicity.First by cell kind on 96 orifice plates（1×104Individual cell/
Hole）, through culture in 24 hours to cell attachment about 70%.Then, various concentrations are added（10-350 µg/mL）HA-PLGA skies receive
Grain of rice subsample.After culture 48 hours, 10 μ L MTT is added to every hole（5.0 mg/mL）Solution, followed by culture 4 hours.
Then, crystallization of 150 μ L DMSO dissolving generations is added to every hole, and absorbance is surveyed at 492 nm with ELIASA（A）,
Calculate cell survival rate.It is reference with cell blank control wells and culture medium blank well.Test result shows that sky nano-particle is led
Causing the survival rate of tumour cell substantially reduces.Such as after the effect of MCF-7 tumour cells and 350 μ g/mL empty nano-particle, its
Survival rate drops to about 60%（Figure 10）.The polymer of this explanation present invention is as PLGA made from high molecular surfactant
NPs has certain lethality to tumour cell.On the contrary, even if L929 normal cells are received with the sky at concentrations up to 350 μ g/mL
Rice corpuscles acts on, and its survival rate is still above 90%（Figure 10）.The polymer of this explanation present invention is as high molecular surfactant
Obtained PLGA NPs have good biocompatibility, the defects of overcoming poisonous existing emulsifying agent, poor biocompatibility.
The medicine-carried nano particles of embodiment 13（PTX-HA-PLGA NPs）Vitro cytotoxicity
PTX-HA-PLGA NPs are analyzed to MCF-7 with mtt assay（Excessive surface expresses CD44 acceptors）The poison of tumour cell
Property.First by cell kind on 96 orifice plates（1×104Individual cells/well）, through culture in 24 hours to cell attachment about 70%.Then,
Add PTX-HA-PLGA NPs, PTX concentration ranges be chosen to be 0.0005,0.005,0.01,0.05,0.1,0.25,0.5,1,
2.5th, 5 and 10 μ g/mL.After culture 4 hours, the nutrient solution containing medicine-carried nano particles is suctioned out, fresh culture is then added and continues
It is incubated 44 h.10 μ L MTT is added to every hole again（5.0 mg/mL）Solution, it is then incubated for 4 hours.Then, added to every hole
Crystallization of 150 μ L DMSO dissolving generations, and absorbance is surveyed at 492 nm with ELIASA（A）, calculate cell survival rate.
Experiment is compared with PTX clinical preparations Taxol.In addition to confirm that nano-particle of the surface containing HA has targeting, we add
One group of cell surface receptor enclosed experiment, specific method are：First by MCF-7 cells and excessive HA macromoleculars（5 mg/
mL）4 h are incubated, HA macromoleculars is combined and occupies the CD44 acceptors of cell surface, then add PTX-HA-PLGA
NPs, analyze its cytotoxicity.Experimental result shows that PTX-HA-PLGA NPs are thin to the MCF-7 tumours of overexpression CD44 acceptors
Born of the same parents have compared with high toxicity, its half lethal concentration（IC50 = 0.37 μg/mL）Less than Taxol（IC50 = 0.82 μg/mL）（Figure 11）,
This explanation medicine-carried nano particles has higher antitumor activity in vitro.In addition, after medicine-carried nano particles are closed to surface receptor
The toxicity of MCF-7 cells substantially reduce, its IC50For 2.16 μ g/mL, this explanation PTX-HA-PLGA NPs mainly by by
Body Guidance means enter tumour cell, have good tumor-targeting.
The medicine-carried nano particles of embodiment 14（PTX-HA-PLGA NPs）Inside antitumor activity
Internal antitumor activity experiment is to use lotus MCF-7 human breast carcinoma Balb/c nude mices（18 ~ 20 grams, 4 ~ 6 week old）Enter
OK.First by mouse bare subcutaneous injection MCF-7 human breast cancer cells（5×106Individual/only）Establish subcutaneous breast cancer tumour mould
Type, tumor growth is waited to about 50-80 mm3When（About 1 week）Proceed by treatment.Treatment is divided into two groups, i.e. medicine-carried nano particles
（PTX-HA-PLGA NPs）With PTX clinical preparations（Taxol）, with empty nano-particle（HA-PLGA NPs）And physiological saline
（PBS）As a control group.Administration starting date is defined the 0th day, passed through mouse tail vein injection at the 0th, 3,6,9 and 12 day respectively
Administration（PTX doses are 5 mg/kg）.During treatment, gross tumor volume, changes of weight and the survival rate of tumor-bearing mice are observed.It is small
The body weight of mouse is to weigh once for every three days.The gross tumor volume vernier caliper measurement of mouse, computational methods are：V=（L×W×H）/
2,（Wherein L is the length of tumour, and W is the width of tumour, and H is the thickness of tumour）.The existence of continuous observation mouse was by 38 days.It is real
Mouse natural death or gross tumor volume are more than 1000 mm during testing3When, it is determined as death.As a result PTX-HA-PLGA is found
NPs and Taxol can suppress the growth of tumour to a certain extent, and PTX-HA-PLGA NPs show it is significantly superior
Suppression tumor growth effect more, after intravenous injection medicament-carried nano 21 days, tumour remains to be suppressed well, increases without obvious
It is long（Figure 12 A and B）.Body weight test result shows that PTX-HA-PLGA NPs will not cause mouse weight significant change, which demonstrate
PTX-HA-PLGA NPs will not produce obvious toxic side effect（Figure 12 C）.Kaplan-Meier survivorship curves observation result is shown
After lotus knurl mouse intravenous administration PTX-HA-PLGA NPs, there is not death in whole treatment cycle（Figure 12 D）；And PBS groups
With blank nanoparticle subgroup animal after treatment 38 days it is all dead, Taxol groups also have 60% animal after Ureteral Calculus terminates
It is dead.Histologic analysis after H＆E dyeing shows PTX-HA-PLGA NPs energy induced tumor cell large area necrosis, and to liver
The normal structures such as dirty, kidney are without overt toxicity；And although Taxol can kill tumour cell to a certain extent, also can simultaneously
Cause normal structure（Liver and kidney）Damage（Figure 13）.Therefore, the load prepared with the high molecular surfactant of the present invention
Targeted drug delivery to lesions position can be realized the effect of being remarkably reinforced by medicine nano-particle；Also substantially reduce small point simultaneously
The toxic side effect of sub- hydrophobic medicine.
In summary, it is of the invention from amido Hydrophobic small molecules（Molecular weight is more than 240）Set out, react and prepare with polyaminoacid
A series of polymer hydrophilic, hydrophobic units are controllable are obtained, possess good biodegradability；It can be used as dewatering medicament
Solubilizer, be added significantly to the meltage of dewatering medicament, improve the bioavilability of medicine, and be applicable various hydrophobic medicine
Thing, be advantageous to improve the application performance of dewatering medicament；It is also used as surfactant, prepares PLGA, PLA, PCL or PEG-
A variety of Biodegradable nano particles such as PLA, the biocompatibility and feature of polymer nanoparticle drug carriers are added, further
, targeted molecular can also be added, obtains targeted nano-particle, for the carrier of medicine, increases drug-rich rate, improves medicine
Therapeutic effect.
Claims (9)
- A kind of 1. biodegradable polymer based on hydrophobic function small molecule-hydrophilic polyaminoacid, it is characterised in that：It is described The chemical structural formula of biodegradable polymer based on hydrophobic function small molecule-hydrophilic polyaminoacid is following chemical constitution One kind in formula：,,Wherein x is that 2~5, n is 3~20；R1Structural formula it is as follows：1）Hydrophobic amine based compound substituent R1：,,,；2）Hydrophobicity hydroxy compounds substituent R1：。
- 2. the preparation of the biodegradable polymer based on hydrophobic function small molecule-hydrophilic polyaminoacid described in claim 1 Method, it is characterised in that comprise the following steps：Using hydrophobic amine based compound as initiator, ring-opening polymerisation a-amino acid-N- Carboxyl inner-acid anhydride compound obtains the biodegradable polymer based on feature Hydrophobic small molecules-hydrophilic polyaminoacid；It is described A-amino acid-N- carboxyl inner-acid anhydrides compound is γ-oligomeric ethylene glycol-Pidolidone-N- carboxyls inner-acid anhydride or β-oligomerization second two Alcohol-L-Aspartic acid-N- carboxyl inner-acid anhydrides.
- 3. biodegradable polymer according to claim 2 based on hydrophobic function small molecule-hydrophilic polyaminoacid Preparation method, it is characterised in that：Hydrophobic amine based compound includes DSPE, dioleoyl phosphatidyl Monoethanolamine, hexadecylamine or eribulin；Or the hydrophobic amine based compound is prepared by hydrophobicity hydroxy compounds Obtain；Hydrophobic amine based compound, the mol ratio of a-amino acid-N- carboxyl inner-acid anhydride compounds are 1: 3~25.
- 4. biodegradable polymer according to claim 3 based on hydrophobic function small molecule-hydrophilic polyaminoacid Preparation method, it is characterised in that：It is with hydrophobicity hydroxy compound to prepare hydrophobic amine based compound by hydrophobicity hydroxy compounds Thing, p-nitrophenyl chloro-formate are raw material, and in anhydrous methylene chloride and pyridine, reaction obtains p-nitrophenyl chloro-formate The feature Hydrophobic small molecules of activation；Then with the feature Hydrophobic small molecules of p-nitrophenyl chloroformate activation and two aminations Compound is reactant, and in anhydrous methylene chloride and pyridine, reaction prepares hydrophobic amine based compound.
- 5. biodegradable polymer according to claim 4 based on hydrophobic function small molecule-hydrophilic polyaminoacid Preparation method, it is characterised in that：Hydrophobicity hydroxy compounds include vitamin e, cholesterol, cumarin, cholic acid, camptothecine, she It is vertical to replace health；The one kind of diamine compound in following compound：Ethylenediamine, butanediamine, octamethylenediamine, lysine methyl ester, bad ammonia Acetoacetic ester, omithine methyl ester, ornithine ethyl ester, cystine methyl esters, cystine ethyl ester, cystamine.
- 6. biodegradable polymer according to claim 4 based on hydrophobic function small molecule-hydrophilic polyaminoacid Preparation method, it is characterised in that：The mol ratio of hydrophobicity hydroxy compounds, p-nitrophenyl chloro-formate and pyridine is 1: 2: 5； The mol ratio of the feature Hydrophobic small molecules of p-nitrophenyl chloroformate activation, ethylenediamine and pyridine is 1: 20: 20.
- 7. the biodegradable polymer based on hydrophobic function small molecule-hydrophilic polyaminoacid described in claim 1 is hated in solubilising Application in aqueous pharmaceutical.
- 8. the biodegradable polymer based on hydrophobic function small molecule-hydrophilic polyaminoacid described in claim 1 is as high score The application of sub- surfactant.
- 9. the biodegradable polymer based on hydrophobic function small molecule-hydrophilic polyaminoacid described in claim 1 is more in preparation Application in functional nano pharmaceutical carrier.
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| WO2020014911A1 (en) * | 2018-07-18 | 2020-01-23 | 苏州大学张家港工业技术研究院 | Polyethylene glycol-b-polytyrosine-lipoic acid copolymer, polypeptide micelle, method for preparation thereof, and application thereof |
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| CN111454443B (en) * | 2020-03-05 | 2021-04-06 | 中国药科大学 | Functional biodegradable polymer with high NO content and application thereof |
| CN115433291B (en) * | 2022-07-27 | 2024-04-05 | 海南师范大学 | Synthesis of alginic acid-g-coumarin derivative and method for preparing Pickering emulsion loaded with doxorubicin |
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| CN101215377A (en) * | 2007-12-27 | 2008-07-09 | 中国科学院长春应用化学研究所 | Aliphatic polyester-polyamino acid Y-type three-arm block copolymers and synthetic method thereof |
| CN102300556A (en) * | 2008-12-26 | 2011-12-28 | 株式会社三养社 | Pharmaceutical composition containing an anionic drug, and a production method therefor |
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| CN101215377A (en) * | 2007-12-27 | 2008-07-09 | 中国科学院长春应用化学研究所 | Aliphatic polyester-polyamino acid Y-type three-arm block copolymers and synthetic method thereof |
| CN102300556A (en) * | 2008-12-26 | 2011-12-28 | 株式会社三养社 | Pharmaceutical composition containing an anionic drug, and a production method therefor |
| CN103588933A (en) * | 2013-10-10 | 2014-02-19 | 西北大学 | Multi-bionic anti-biological pollution copolymer, and preparation method and application thereof |
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