CN109762170A - A kind of polyphosphate polymer and preparation method thereof, modified porous silicon nanoparticle and its preparation method and application - Google Patents
A kind of polyphosphate polymer and preparation method thereof, modified porous silicon nanoparticle and its preparation method and application Download PDFInfo
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- CN109762170A CN109762170A CN201910062619.8A CN201910062619A CN109762170A CN 109762170 A CN109762170 A CN 109762170A CN 201910062619 A CN201910062619 A CN 201910062619A CN 109762170 A CN109762170 A CN 109762170A
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Abstract
The present invention relates to technical field of medicine, and in particular to a kind of polyphosphate polymer and preparation method thereof, modified porous silicon nanoparticle and its preparation method and application.The main chain of polyphosphate polymer provided by the invention is polyphosphate segment, side chain is amphoteric ion segment, with good biocompatibility and degradability, nanoparticle colloidal stability can be dramatically increased by being introduced into porous silicon nanoparticle surface, mucus is passed through faster and reduces nanoparticle in the hold-up of rete malpighii, there is better compatibility to epithelial cell, greatly improve the endocytosis amount of cell, and effectively across cell intraor extracellular drug release can be carried out, be conducive to the bioavilability for improving drug.Simultaneously, polyphosphate segment active site is easy to be coupled with targeting factor more in polyphosphate polymer provided by the invention, and hydrophily polyphosphate segment and cell membrane affinity are high, compared with the nanoparticle of existing promotion mucus diffusion, can effectively improve the safety of nanoparticle.
Description
Technical field
The present invention relates to technical field of medicine, and in particular to a kind of polyphosphate polymer and preparation method thereof changes
Property porous silicon nanoparticle and its preparation method and application.
Background technique
Porous silicon nanoparticle has unique physicochemical properties, in drug delivery system, catalysis and absorption, Protein Separation
Etc. have a wide range of applications.It is that a kind of specific surface area is high, aperture and homogeneous grain diameter are controllable, drugloading rate is high, surface is easily repaired
It is decorated with and the good nano material of biocompatibility, in drug delivery system, controllable duct, partial size and surface modification,
Be conducive to load various drugs, and realize the controllable load medicine of drug and release, final targeting reaches tissue.
The behavior of nanoparticle in vivo and its Surface Physical Chemistry property such as partial size, form, hydrophobe, charge type etc.
It is closely bound up.Intestinal mucus layer is filled with water, protein, lipid, electrolyte, bacterium and cell fragment etc., and hole is 50~
The purge mechanism of 1800nm, continuous renewal can quickly remove pathogen and foreign substance.Many studies have shown that hydrophilic and electroneutral
Nanoparticle can pass through rete malpighii and avoid being adsorbed removing.Such as carrying out surface modification to nanoparticle using polyethylene glycol can effectively keep away
The Electrostatic Absorption of non-sticking albumen, but the hydrophily of polyethylene glycol limits contacting with each other between nanoparticle and epithelial cell and then subtracts
Effective intake of cell is lacked.Meanwhile polyethylene glycol is difficult to degrade in vivo, into cell after drug release it is slow.
Summary of the invention
The purpose of the present invention is to provide a kind of polyphosphate polymer and preparation method thereof, modified porous silicon nanoparticle and
Preparation method and application, polyphosphate polymer provided by the invention have good biocompatibility and degradability, will
It, which introduces porous silicon nanoparticle surface, can dramatically increase nanoparticle colloidal stability, pass through mucus faster and reduction nanoparticle exists
The hold-up of rete malpighii has better compatibility to epithelial cell, greatly improves the endocytosis amount of cell, and can effectively across
Cell carries out intraor extracellular drug release, is conducive to the bioavilability for improving drug.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of polyphosphate polymer, have structure shown in Formulas I:
In Formulas I, 0 < x≤100,10≤y≤100, and 0.08 < x/y≤2；R is amphoteric ion group.
Preferably, the amphoteric ion monomer for forming the amphoteric ion group includes cysteine, cysteamine paddy ammonia
Acid, carboxybetaine, sulfobetaines or phosphorus base glycine betaine.
The present invention provides the preparation methods of polyphosphate polymer described in above-mentioned technical proposal, comprising the following steps:
N- (tertbutyloxycarbonyl) ethanol amine, two thiopyridines phosphate of ethoxy and the first solvent are mixed, in organic gold
Metal catalyst effect is lower to carry out ring-opening polymerization, obtains ring opening polymerization product；
The ring opening polymerization product is mixed with go back original reagent and the second solvent, de- pyridine reaction is carried out, obtains de- pyridine
Product；
The de- pyridine product is mixed with amphoteric ion monomer and third solvent, is catalyzed and makees in 4-dimethylaminopyridine
Click chemistry reaction is carried out under, obtains the polyphosphate polymer with structure shown in Formulas I.
The present invention provides a kind of modified porous silicon nanoparticle, the polyphosphate polymer as described in above-mentioned technical proposal or on
It states the polyphosphate polymer that preparation method described in technical solution is prepared porous silicon nanoparticle is modified and handle.
Preferably, the partial size of the modified porous silicon nanoparticle is 50~500nm, and Zeta potential is -30~+30mV, than
Surface area is 50~2000m2/ g, aperture size are 5~20nm.
The present invention provides the preparation methods of modified porous silicon nanoparticle described in above-mentioned technical proposal, comprising the following steps:
At N, N'- carbonyl dimidazoles effect, processing is modified to porous silicon nanoparticle using polyphosphate polymer,
Obtain modified porous silicon nanoparticle.
Preferably, the amount ratio of the porous silicon nanoparticle, polyphosphate polymer and N, N'- carbonyl dimidazoles is 1g:
(0.005~0.05) mmol:(0.5~5) mmol.
Preferably, the porous silicon nanoparticle is carrying out amination processing using amino silicane coupling agent using preceding.
The present invention provides preparation sides described in modified porous silicon nanoparticle described in above-mentioned technical proposal or above-mentioned technical proposal
Application of the modified porous silicon nanoparticle that method is prepared as the carrier of oral administration.
Preferably, the mass ratio of the active medicine in the oral administration and carrier is (0.01~9): 10.
The present invention provides a kind of polyphosphate polymer with structure shown in Formulas I, polyphosphates provided by the invention
The main chain of polymer is polyphosphate segment, and side chain is amphoteric ion segment, has good biocompatibility and degradable
Property, nanoparticle colloidal stability can be dramatically increased by being introduced into porous silicon nanoparticle surface, passed through mucus faster and reduced and receive
The grain of rice has better compatibility to epithelial cell in the hold-up of rete malpighii, greatly improves the endocytosis amount of cell, and can have
The across cell of effect carries out intraor extracellular drug release, is conducive to the bioavilability for improving drug.Wherein, in neutral mucus layer condition
Under, the amphoteric ion side chain of modified porous silicon nanoparticle outer layer shows electroneutral and hydrophilic, can quickly pass through rete malpighii；And on reaching
The part polyphosphate main chain of endothelial cell surface, modified porous silicon nanoparticle outer layer is degraded by alkaline phosphatase or phosphatidase, outside
Layer electropositive is reinforced, and the compatibility with cell membrane is increased, and achievees the purpose that promote cellular uptake；Strong acid and phosphoric acid in the cell
Under enzyme, outer layer is further degraded, and the rock-steady structure of carrier surface polymer is destroyed, to trigger drug release.Meanwhile the present invention
Polyphosphate segment active site is easy to be coupled with targeting factor more in the polyphosphate polymer of offer, and hydrophily polyphosphoric acid
Ester segment and cell membrane affinity are high, compared with the nanoparticle of existing promotion mucus diffusion, can effectively improve the safety of nanoparticle
Property.
Detailed description of the invention
Fig. 1 is the ring opening polymerization product that the embodiment of the present invention 4 is prepared and polyphosphate polymer in DMSO1H-
NMR nuclear magnetic spectrum；
Fig. 2 is drug-carrying nanometer particle scanning electron microscope (SEM) photograph prepared by the embodiment of the present invention 4；
Fig. 3 is nanoparticle infrared spectrum prepared by the embodiment of the present invention 1,2 and 5；
Fig. 4 is the vitro drug release figure of drug-carrying nanometer particle prepared by the embodiment of the present invention 2,4,5,6,8 and 9；
Fig. 5 is the absorption figure that mucoprotein prepares nanoparticle to the embodiment of the present invention 2,3,4,5 and 6；
Fig. 6 be the embodiment of the present invention 2,3,4,5,6,8 and 9 prepare nanoparticle wear mucus rate diagram；
Fig. 7 is the drug transport rate diagram that the embodiment of the present invention 2,4,5,6,8 and 9 prepares drug-carrying nanometer particle.
Specific embodiment
The present invention provides a kind of polyphosphate polymer, have structure shown in Formulas I:
In Formulas I, 0 < x≤100,10≤y≤100, and 0 < x/y≤2；R is amphoteric ion group.
In the present invention, it is preferred to, in Formulas I, 0 < x≤40,10≤y≤50, and 0.08 < x/y≤2.
In the present invention, the amphoteric ion monomer for forming the amphoteric ion group preferably includes cysteine, half
Cystamine glutamic acid, carboxybetaine, sulfobetaines or phosphorus base glycine betaine.
The present invention provides the preparation methods of polyphosphate polymer described in above-mentioned technical proposal, comprising the following steps:
N- (tertbutyloxycarbonyl) ethanol amine, two thiopyridines phosphate of ethoxy and the first solvent are mixed, in organic gold
Metal catalyst effect is lower to carry out ring-opening polymerization, obtains ring opening polymerization product；
The ring opening polymerization product is mixed with go back original reagent and the second solvent, de- pyridine reaction is carried out, obtains de- pyridine
Product；
The de- pyridine product is mixed with amphoteric ion monomer and third solvent, is catalyzed and makees in 4-dimethylaminopyridine
Click chemistry reaction is carried out under, obtains the polyphosphate polymer with structure shown in Formulas I.
The present invention mixes N- (tertbutyloxycarbonyl) ethanol amine, two thiopyridines phosphate of ethoxy and the first solvent,
Organo-metallic catalyst effect is lower to carry out ring-opening polymerization, obtains ring opening polymerization product.The present invention is for the ethoxy two
The source of thiopyridines phosphate (Py-EAOP) does not have special restriction, is prepared using method well known to those skilled in the art
It obtains；In the present invention, the preparation method of the two thiopyridines phosphate of ethoxy preferably includes following steps:
Two sulphur, two pyridine, mercaptoethanol and methanol are mixed, it is anti-that sulfydryl-dimercapto exchange is carried out under acetic acid catalysis effect
It answers, obtains two thiopyridines of ethoxy；
By two thiopyridines of ethoxy and the chloro- 2- oxygen -1,3,2- dioxaphospholane of 2-, organic basic substance and non-
Proton-organic solvent mixing, carries out substitution reaction, obtains two thiopyridines phosphate of ethoxy.
The present invention preferably by two sulphur, two pyridine, mercaptoethanol and methanol mix, acetic acid catalysis effect under carry out sulfydryl and
Dimercapto exchange reaction obtains two thiopyridines of ethoxy.In the present invention, two sulphur, two pyridine, mercaptoethanol, methanol and second
The amount ratio of acid is preferably (15~25) g:(2~3) g:(40~60) mL:(4~6) mL, more preferably (18~22) g:(2.4
~2.8) g:(45~55) mL:(4.5~5.5) mL.In the present invention, the sulfydryl and dimercapto exchange reaction are preferably in room temperature
Under the conditions of carry out；The time of the sulfydryl and dimercapto exchange reaction is preferably 2~8h, more preferably 2~5h.
After completing the sulfydryl and dimercapto exchange reaction, gained system is preferably carried out rotary evaporation to remove by the present invention
Then gained residue is carried out column chromatography separating purification by organic reagent, gained eluent is finally carried out vacuum freeze drying,
Obtain two thiopyridines of ethoxy.In the present invention, eluant, eluent used by the column chromatography separating purification is preferably ethyl acetate
With the mix reagent of petroleum ether；The volume ratio of the ethyl acetate and petroleum ether is preferably (10~25): 100.The present invention for
The vacuum freeze drying does not have special restriction, and it is sufficiently dry to can be realized material.
After obtaining two thiopyridines of ethoxy, the present invention preferably by two thiopyridines of ethoxy and chloro- oxygen -1 2- 2-,
3,2- dioxaphospholane, organic basic substance and aprotic organic solvent mixing, carry out substitution reaction, obtain ethoxy two
Thiopyridines phosphate.In the present invention, two thiopyridines of ethoxy and the chloro- 2- oxygen -1,3 of 2-, 2- dioxaphospholane,
Organic basic substance and aprotic organic solvent mixing are preferably to the chloro- 2- oxygen -1,3,2- dioxaphospholane of 2-, organic base
Property substance and aprotic organic solvent mixture in two thiopyridines of ethoxy are added dropwise, to guarantee that the substitution reaction is steady, peace
It carries out entirely and reacts two thiopyridines of ethoxy sufficiently；The present invention does not have the drop rate of two thiopyridines of ethoxy
There is special restriction, using drop rate well known to those skilled in the art.
In the present invention, the organic basic substance preferably includes one or both of triethylamine, ethylenediamine and pyridine；
The present invention add organic alkaline matter be in order to remove the by-product hydrochloric acid generated during substitution reaction, guarantee reaction smoothly into
Row.In the present invention, the aprotic organic solvent preferably includes chloroform, methylene chloride, tetrahydrofuran or ethyl acetate.
In the present invention, two thiopyridines of ethoxy, the chloro- 2- oxygen -1,3 of 2-, 2- dioxaphospholane (COP) have
The amount ratio of machine alkaline matter and aprotic organic solvent is preferably (110~130) mg:(90~110) mg:(40~60) mg:
(10~30) mL；More preferably (115~125) mg:(95~105) mg:(45~55) mg:(10~20) mL.
In the present invention, the temperature of the substitution reaction is preferably -15~0 DEG C, more preferably -5~0 DEG C；Time is preferred
For 6~18h, more preferably 6~10h；In the present invention, the substitution reaction preferably carries out under the protection of argon gas.
After completing the substitution reaction, the present invention preferably filters gained system to remove desalination by-product, then by gained
Filtrate carries out rotary evaporation to remove solvent, and residue is redissolved in methanol, and precipitation reagent is added and carries out precipitation process, collects
Sediment carries out vacuum freeze drying, obtains two thiopyridines phosphate of ethoxy.In the present invention, the precipitation reagent is preferred
Including ether, petroleum ether, chloroform or n-hexane；The present invention does not have the usage amount of the precipitation reagent special restriction, energy
Enough guarantee that the precipitation process sufficiently carries out.The present invention preferably repeats precipitation process-filtering-methanol and redissolves operation,
To guarantee impurity in abundant removal system；The present invention does not have the number of repetitive operation special restriction, preferably 2~5 times.
The present invention does not have the vacuum freeze drying special restriction, and it is sufficiently dry to can be realized material；The present invention is preferred
Vacuum freeze drying is for 24 hours under the conditions of -20 DEG C.
After obtaining two thiopyridines phosphate of ethoxy, the present invention preferably by the two thiopyridines phosphate of ethoxy with
N- (tertbutyloxycarbonyl) ethanol amine, organo-metallic catalyst and the mixing of the first solvent, carry out ring-opening polymerization, it is poly- to obtain open loop
Close product.In the present invention, the organo-metallic catalyst is preferably organotin catalysts, more preferably stannous octoate；It is described
First solvent is preferably organic solvent, more preferably tetrahydrofuran, ethyl acetate or methylene chloride.In the present invention, the N-
(tertbutyloxycarbonyl) ethanol amine, two thiopyridines phosphate of ethoxy, the mass ratio of the first solvent and organo-metallic catalyst are excellent
It is selected as 1mg:(18~220) mg:(1~15) mL:(1~5) mg, more preferably 1mg:(18~130) mg:(5~11) mL:(2~
3)mg.In the present invention, the temperature of the ring-opening polymerization is preferably 30~50 DEG C, and more preferably 35~45 DEG C；Time is excellent
It is selected as 3~5h, more preferably 3~4h.
After completing the ring-opening polymerization, the present invention, will preferably by gained system progress rotary evaporation to remove solvent
Residue redissolves in methanol, and precipitation reagent is added and carries out precipitation process, collects sediment and carries out vacuum freeze drying, is opened
Cyclopolymerization product (N-Boc-P (PyEP)).In the present invention, the precipitation reagent preferably includes ether, petroleum ether, chloroform or just
Hexane；The present invention does not have the usage amount of the precipitation reagent special restriction, can guarantee the precipitation process sufficiently into
Row.The present invention preferably repeats precipitation process-filtering-methanol and redissolves operation, to guarantee impurity in abundant removal system；
The present invention does not have the number of repetitive operation special restriction, preferably 2~5 times.The present invention is dry for the vacuum refrigeration
It is sufficiently dry to can be realized material for dry not special restriction；The present invention preferably vacuum freeze drying under the conditions of -20 DEG C
24h。
After obtaining ring opening polymerization product, the present invention mixes the ring opening polymerization product with go back original reagent and the second solvent,
De- pyridine reaction is carried out, de- pyridine product is obtained.In the present invention, the go back original reagent preferably includes dithiothreitol (DTT), β-mercapto
Base ethyl alcohol or three (2- carboxyethyl) phosphines；Second solvent is preferably organic solvent, more preferably tetrahydrofuran, ethyl acetate or
Methylene chloride.In the present invention, the amount ratio of the ring opening polymerization product, go back original reagent and the second solvent is preferably 100mg:
(20~100) mg:(1~10) mL, more preferably 100mg:(30~80) mg:(2~6) mL.In the present invention, the de- pyridine
Reaction preferably carries out at room temperature；The time of the de- pyridine reaction is preferably 8~for 24 hours, more preferably 12~20h；?
In the present invention, the de- pyridine reaction is preferably carried out under the conditions of argon gas is protected, is protected from light.
After completing the de- pyridine reaction, gained system is preferably carried out rotary evaporation to remove solvent by the present invention, will be remained
Excess redissolves in methanol, and precipitation reagent is added and carries out precipitation process, collects sediment and carries out vacuum freeze drying, obtains de- pyrrole
Pyridine product.In the present invention, the precipitation reagent preferably includes ether, petroleum ether, chloroform or n-hexane；The present invention is for described
The usage amount of precipitation reagent does not have special restriction, can guarantee that the precipitation process sufficiently carries out.The present invention is preferably heavy
Precipitation process-filtering-methanol is carried out again and redissolves operation, to guarantee impurity in abundant removal system；The present invention is for repetitive operation
Number there is no special restriction, preferably 2~5 times.The present invention does not have the vacuum freeze drying special restriction, energy
Enough realize that material is sufficiently dry；Preferably vacuum freeze drying is for 24 hours under the conditions of -20 DEG C by the present invention.
After obtaining de- pyridine product, the present invention mixes the de- pyridine product with amphoteric ion monomer and third solvent
It closes, click chemistry reaction is carried out under 4-dimethylaminopyridine catalytic action, obtain having the polyphosphate of structure shown in Formulas I poly-
Close object.The present invention preferably mixes the amphoteric ion monomer and third solvent, stir 5 under the protection of argon gas~
4-dimethylaminopyridine (DMAP) and the de- pyridine product, vacuum freeze thawing three is added in liquid nitrogen flash freezer after 15min simultaneously immediately
Secondary, after 0.5~3h of ultraviolet irradiation, sustained response 1~for 24 hours, blowing air terminate reaction, are had under the protection of argon gas
The polyphosphate polymer of structure shown in Formulas I.
In the present invention, the amphoteric ion monomer preferably includes cysteine (Cys), cysteamine glutamic acid (Glu-
Cya), carboxybetaine (CB), sulfobetaines (SB) or phosphorus base glycine betaine (PB).In the present invention, the third solvent is excellent
It is selected as the mixed solvent of organic solvent and water；The organic solvent preferably includes methanol, ethyl alcohol or dimethyl sulfoxide；It is described organic
The volume ratio of solvent and water is preferably (0.5~2): 1.In the present invention, the de- pyridine product, amphoteric ion monomer and 4-
Dimethylamino naphthyridine mole preferably than 1:(0,50]: (0,50], more preferably 1:(2~20): (1~20), further preferably
For 1:(2~10): (1~10).The present invention does not have special restriction for the usage amount of the third solvent, can guarantee described
Click chemistry reaction is gone on smoothly.In the present invention, the time of ultraviolet irradiation is preferably 0.5 in the click chemistry reaction
~1h, the time of sustained response is preferably 8~12h in argon gas.
After completing the click chemistry reaction, gained system is successively preferably carried out dialysis by the present invention and vacuum refrigeration is done
It is dry, obtain polyphosphate polymer.It is special that the present invention does not have the molecular cut off of bag filter used by the dialysis
It limits, can guarantee flux matched with the molecule of subject polymer；The molecular cut off of heretofore described bag filter is preferred
For 1.5~20KDa, more preferably 5~15KDa.The present invention does not have the vacuum freeze drying special restriction, Neng Goushi
Existing material is sufficiently dry；Preferably vacuum freeze drying is for 24 hours under the conditions of -20 DEG C by the present invention.
The present invention provides a kind of modified porous silicon nanoparticles, and polyphosphate polymer is to more as described in above-mentioned technical proposal
Hole silicon nanoparticle, which is modified, to be handled.In the present invention, the partial size of the modified porous silicon nanoparticle be preferably 50~
500nm, Zeta potential are preferably -30~+30mV, and specific surface area is preferably 50~2000m2/ g, aperture size is preferably 5~
20nm。
The present invention provides the preparation methods of modified porous silicon nanoparticle described in above-mentioned technical proposal, comprising the following steps:
At N, N'- carbonyl dimidazoles effect, processing is modified to porous silicon nanoparticle using polyphosphate polymer,
Obtain modified porous silicon nanoparticle.
In the present invention, the porous silicon nanoparticle is preferably carried out at amination using amino silicane coupling agent using preceding
Reason.The present invention does not have special restriction for the source of the porous silicon nanoparticle, using city well known to those skilled in the art
It sells commodity or preparation method (such as ball-milling method, reduction method or chemical etching method) is prepared；In an embodiment of the present invention,
The porous silicon nanoparticle is specifically prepared using chemical etching method, preferably includes following steps:
Silicon wafer is subjected to surface cleaning processing, obtains cleaning silicon chip；
The cleaning silicon chip is successively subjected to the first electrochemical etching and the second electrochemical etching, obtains porous silicon film；
The porous silicon film is ultrasonically treated in ethanol, obtains porous silicon nanoparticle.
Silicon wafer is preferably carried out surface cleaning processing by the present invention, obtains cleaning silicon chip.In the present invention, the silicon wafer is preferred
For the monocrystalline silicon of p-type doping, the monocrystalline silicon of n-type doping, polysilicon or silicon point, the more preferably monocrystalline silicon of n-type doping；The n
The resistivity of the monocrystalline silicon of type doping is preferably 0.01~15 Ω cm, more preferably 0.1~1 Ω cm.The present invention is for institute
Stating surface cleaning processing does not have special restriction, using technical solution well known to those skilled in the art；Of the invention
In embodiment, preferably by silicon wafer be placed in hydrogen peroxide and the concentrated sulfuric acid mix reagent (mass concentration of the hydrogen peroxide preferably >=
40.0%, more preferably 40.0%；The mass concentration of the concentrated sulfuric acid is preferably 95.0~98.0%；The hydrogen peroxide and dense sulphur
The volume ratio of acid is preferably 3:1) in be cleaned by ultrasonic 5min, then successively use acetone, dehydrated alcohol and deionized water to clean with
Remove surface impurity.
After obtaining cleaning silicon chip, the cleaning silicon chip is preferably successively carried out the first electrochemical etching and the second electricity by the present invention
Chemical etching obtains porous silicon film.In the present invention, etching solution used by first electrochemical etching is preferably hydrogen
The mix reagent of fluoric acid and ethyl alcohol, the mass concentration of the hydrofluoric acid preferably >=40.0%, more preferably 40.0%, the hydrogen fluorine
The volume ratio of acid and ethyl alcohol is (1~3): 1；The current strength of first electrochemical etching is preferably 80~120mA/cm2, when
Between preferably 10~30min.In the present invention, etching solution used by second electrochemical etching be preferably hydrofluoric acid with
The mix reagent of ethyl alcohol, the mass concentration of the hydrofluoric acid preferably >=40.0%, more preferably 40.0%, the hydrofluoric acid and second
The volume ratio of alcohol is (5~15): 100；The current strength of second electrochemical etching is preferably 5~20mA/cm2, the time is excellent
It is selected as 1~3min.
After completing second electrochemical etching, the present invention preferably sucks the mix reagent in system, using dehydrated alcohol
Cleaning porous silicon film 2 times, the porous silicon film after collecting cleaning, then carries out ultrasound for the porous silicon film in ethanol
Processing, obtains porous silicon nanoparticle.In the present invention, the time of the ultrasonic treatment is preferably 8~48h, more preferably 12~
36h。
After completing the ultrasonic treatment, gained system is preferably carried out the first centrifugation by the present invention, collects supernatant i.e. much
The alcohol dispersion liquid of hole silicon nanoparticle saves under the conditions of 4 DEG C stand-by.In the present invention, the revolving speed of first centrifugation is preferably
3000~15000rpm, more preferably 6000~12000rpm；The time of first centrifugation is preferably 5~40min, more preferably
For 15~40min.
After obtaining the alcohol dispersion liquid of the porous silicon nanoparticle, the present invention preferably carried out when in use second from
The heart carries out amination processing after directly mixing the porous silicon nanoparticle of collection with organic reagent and amino silicane coupling agent.?
In the present invention, the revolving speed of second centrifugation is preferably 10000~22000rpm, more preferably 12000~18000rpm；It is described
The time of second centrifugation is preferably 2~20min, more preferably 5~15min.The present invention by control first centrifugation and second from
The condition of the heart is conducive to the porous silicon nanoparticle for obtaining uniform particle sizes.
In the present invention, organic reagent preferably includes ethyl alcohol or methanol；The amino silicane coupling agent preferably includes 3- ammonia
Propyl trimethoxy silicane, 3- aminopropyl triethoxysilane, 3- aminopropyltriethoxy diethoxy silane, 3- aminopropyltriethoxy two
Methoxy silane, N- (2- aminoethyl) -3- aminopropyl trimethoxysilane, N- (2- aminoethyl) -3- aminopropyl-triethoxy silicon
One or more of alkane and N- normal-butyl -3- aminopropyl trimethoxysilane, more preferably 3- aminopropyl trimethoxysilane
Or 3- aminopropyl triethoxysilane.The present invention does not have the usage amount of the amino silicane coupling agent special restriction, energy
Enough guarantee that the porous silicon nanoparticle sufficiently carries out amination processing.In the present invention, the amino silicane coupling agent with
The mass ratio of organic reagent is preferably (0.1~10): 100, more preferably (2~6): 100.In the present invention, the amination
The temperature of processing is preferably 25~130 DEG C, and more preferably 25~60 DEG C；In an embodiment of the present invention, specifically in system
The amination processing is carried out under reflux temperature；The time of amination processing is preferably 0.5~for 24 hours, more preferably 3~
12h。
After completing the amination processing, the present invention is preferably by reaction system cooled to room temperature, through anhydrous dimethyl base
Then gained amination porous silicon nanoparticle is stored in anhydrous dimethyl sulphoxide, 4 DEG C by sulfoxide (DMSO) multiple centrifuge washing
Under the conditions of save it is stand-by.
After obtaining amination porous silicon nanoparticle, the present invention is poly- using polyphosphate at N, N'- carbonyl dimidazoles effect
It closes object and processing is modified to the amination porous silicon nanoparticle, obtain modified porous silicon nanoparticle.In the present invention, described
N, N'- carbonyl dimidazoles (CDI) are to pass through the amino and polyphosphate in connection amination porous silicon nanoparticle as activator
Sulfydryl in polymer, to realize modification of the polyphosphate polymer to amination porous silicon nanoparticle；The present invention is preferably
Under the protection of argon gas by amination porous silicon nanoparticle, N, N'- carbonyl dimidazoles and anhydrous dimethyl sulphoxide mixing are laggard
Row first reacts, and polyphosphate polymer is then added into system and carries out the second reaction, obtains modified porous silicon nanoparticle.?
In the present invention, the amount ratio of the amination porous silicon nanoparticle, polyphosphate polymer and N, N'- carbonyl dimidazoles is preferably
1g:(0.01~1) mmol:(0.5~5) mmol, more preferably 1g:(0.1~1) mmol:(1~4) mmol.In the present invention,
First reaction preferably carries out at room temperature；The time of first reaction is preferably 3~7h, more preferably 4~5h.
In the present invention, second reaction preferably carries out at room temperature；The time of second reaction is preferably 8~15h, more
Preferably 10~12h.
After completing the modification, present invention preferably employs anhydrous dimethyl sulphoxides to be repeatedly centrifuged gained system
The modified porous silicon nanoparticle of gained, is then stored in anhydrous dimethyl sulphoxide by washing, saves under the conditions of 4 DEG C stand-by.
The present invention provides preparation sides described in modified porous silicon nanoparticle described in above-mentioned technical proposal or above-mentioned technical proposal
Application of the modified porous silicon nanoparticle that method is prepared as the carrier of oral administration.In the present invention, the oral administration
In active medicine and carrier mass ratio be preferably (0.01~9): 10, more preferably (0.05~6): 10, further preferably
For (0.1~4): 10.In the present invention, the active medicine preferably includes chemical classes drug or protein and peptide drugs, described
Chemical classes drug preferably includes taxol, docetaxel, hydroxycamptothecin or adriamycin, and the protein and peptide drugs preferably wrap
Include insulin, Octreotide, Lovastatin, calcitonin, thymopeptide-5, luteinizing hormone-releasing hormone, Buserelin, Ai Saina
Peptide or glucagon-like-peptide-1.
In the present invention, the preparation method of the oral administration, preferably includes following steps:
Modified porous silicon nanoparticle and active medicine are subjected to total incubation, obtained load medicine porous silicon nanoparticle in hydrochloric acid
The as described oral administration.
In the present invention, the concentration of the hydrochloric acid is preferably 0.01~0.05mol/L, more preferably 0.02~0.03mol/
L.In the present invention, the temperature being incubated for altogether is preferably 15~35 DEG C, and more preferably 20~25 DEG C；The time being incubated for altogether
Preferably 0.5~for 24 hours, more preferably 2~10h；The total incubation preferably carries out under agitation, and the present invention is stirred for described
Mixing rate does not have special restriction, using stirring rate well known to those skilled in the art.
After completing the total incubation, gained system is preferably centrifuged by the present invention, and gained carries the storage of medicine porous silicon nanoparticle
It is stand-by there are being saved in ultrapure water, under the conditions of 4 DEG C.In the present invention, the revolving speed of the centrifugation is preferably 8000~12000rpm,
The time of centrifugation is preferably 2~20min.
Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Embodiment 1
Silicon wafer (monocrystalline silicon of n-type doping, resistivity are 0.01~1 Ω cm) is placed in the mixing of hydrogen peroxide and the concentrated sulfuric acid
Reagent (mass concentration of the hydrogen peroxide is 40.0%, and the mass concentration of the concentrated sulfuric acid is 98.0%, the hydrogen peroxide with it is dense
The volume ratio of sulfuric acid then successively uses acetone, dehydrated alcohol and deionized water cleaning to remove to be cleaned by ultrasonic 5min in 3:1)
Remove surface impurity；Silicon wafer after cleaning is put into electro chemical etching apparatus, the mix reagent that hydrofluoric acid and ethyl alcohol is added is (described
The mass concentration of hydrofluoric acid is 40.0%, and the volume ratio of the hydrofluoric acid and ethyl alcohol is 2:1), in 100mA/cm2Current strength
20min is persistently etched down, adds washes of absolute alcohol, continuously adds the mix reagent (matter of the hydrofluoric acid of hydrofluoric acid and ethyl alcohol
Measuring concentration is 40.0%, and the volume ratio of the hydrofluoric acid and ethyl alcohol is 8:100), in 10mA/cm2Current strength under persistently carve
2min is lost, the porous silicon film in silicon wafer is removed from silicon matrix；Mix reagent in system is sucked, it is more using washes of absolute alcohol
Mesoporous silica film 2 times, the porous silicon film after collecting cleaning, and transfer them to ultrasound 12h in dehydrated alcohol, 6000rpm centrifugation
30min collects supernatant up to the alcohol dispersion liquid of porous silicon nanoparticle, saves under the conditions of 4 DEG C stand-by.
The partial size of porous silicon nanoparticle when using 4 DEG C of laser particle analyzer measurement, measuring average grain diameter is 95 ± 30nm,
PDI is 0.163, and Zeta potential is -27.3 ± 0.3mV, it is seen that the strong negative electricity of unmodified porous silicon nanoparticle surface band of brand-new
Lotus, this is mainly due to surface, there are a large amount of silanol hydroxyls.
Embodiment 2
(1) alcohol dispersion liquid of porous silicon nanoparticle is prepared according to the method for embodiment 1, after 12000rpm is centrifuged 15min
Porous silicon nanoparticle is collected, 100mg porous silicon nanoparticle is dispersed in 20mL anhydrous methanol, 1.5mL3- aminopropyl three is added
Methoxy silane is warming up to system and reflux (40 DEG C) occurs, and carries out amination and handles 10h, cooled to room temperature, through anhydrous
More centrifuge washings of DMSO, gained amination porous silicon nanoparticle are stored in anhydrous DMSO, and 4 DEG C save for use.
(2) that amination is collected after centrifugation in the anhydrous DMSO dispersion liquid of amination porous silicon nanoparticle in step (1) is porous
Silicon nanoparticle mixes 100mg amination porous silicon nanoparticle, 60mg insulin and hydrochloric acid (6mL, concentration 0.02mol/L),
Total incubation 1.2h is carried out under the conditions of stirring, 25 DEG C；Then 9000rpm is centrifuged 5min, is repeated 3 times centrifuge washing through ultrapure water
Except extra medicinal, gained carries medicine porous silicon nanoparticle and is stored in ultrapure water, and 4 DEG C save for use.
The partial size of amination porous silicon nanoparticle when using 4 DEG C of laser particle analyzer measurement, measuring average grain diameter is 148
± 23nm, PDI 0.210, Zeta potential are 15.3 ± 2.2mV, it is seen then that the surface of amination porous silicon nanoparticle is positively charged
Lotus.
Accurately weigh 100.000mg amination porous silicon nanoparticle, using CHNS elemental analyser analysis organic element N,
C, the mass percent of H and S, the results are shown in Table 1, and can calculate its surface amino groups amount is 1~5mmol/g.
Specific surface area and the aperture of the amination porous silicon nanoparticle are analyzed using specific surface area adsorption desorption curve (BET)
Volume, the results show that the specific surface area of the amination porous silicon nanoparticle is 219 ± 21m2/ g, pore volume be 0.63 ±
0.09cm3/ g, it is 9.53 ± 0.08nm that its aperture can be calculated with through hole diametal curve (BJH).
The CHN elemental analysis of the every 100mg amination porous silicon nanoparticle of table 1
Embodiment 3
(1) amination porous silicon nanoparticle is prepared according to the method for embodiment 2.
(2) 2.65g mercaptoethanol, bis- sulphur of 20g, two pyridine, 5mL acetic acid and 50mL methanol are mixed, is stirred at room temperature
2h is mixed, rotary evaporation is to remove organic reagent, and then (used eluant, eluent is ethyl acetate and stone through column chromatography separating purification
The volume ratio of the mix reagent of oily ether, the ethyl acetate and petroleum ether is 18:100), vacuum freeze drying obtains two sulphur of ethoxy
Pyridine；
The chloro- 2- oxygen -1,3,2- dioxaphospholane (COP) of 100mg2- and 50mg anhydrous triethylamine are dissolved in 10mL
In anhydrous tetrahydro furan, two thiopyridines of 120mg ethoxy are slowly added dropwise under protection of argon gas, carry out substitution reaction under the conditions of 0 DEG C
15h；Gained system is filtered to remove desalination by-product, it, will be remaining then by gained filtrate progress rotary evaporation to remove solvent
Object redissolves in methanol, and petroleum ether is added and carries out precipitation process, collects sediment and carries out vacuum freeze drying under the conditions of -20 DEG C
For 24 hours, two thiopyridines phosphate (Py-EAOP) of ethoxy is obtained；
By amount ratio 1mg:36mg:8mL:2.5mg, by N- (tertbutyloxycarbonyl) ethanol amine, two thiopyridines phosphorus of ethoxy
Acid esters, tetrahydrofuran and stannous octoate mixing, carry out ring-opening polymerization 4h under the conditions of 40 DEG C；Gained system is rotated
Evaporation is redissolved residue in methanol with removing solvent, and ether is added and carries out precipitation process, collects sediment in -20 DEG C of items
Vacuum freeze drying is carried out for 24 hours under part, obtains N-Boc-P (PyEP)20Polymer；
It is 100mg:50mg:6mL by amount ratio, by the N-Boc-P (PyEP)20Polymer, dithiothreitol (DTT) and acetic acid
Ethyl ester mixing protect in argon gas, is protected from light, carrying out de- pyridine under room temperature and react 12h；By gained system carry out rotary evaporation with
Remove solvent, by residue redissolve in methanol, be added ether carry out precipitation process, collect sediment under the conditions of -20 DEG C into
Row vacuum freeze drying for 24 hours, obtains P (PPyEP-g-SB0)20Polymer.
(3) under protection of argon gas, by 0.1g amination porous silicon nanoparticle, 0.18mmolN, N'- carbonyl dimidazoles and nothing
Water DMSO mixing, reacts 5h at room temperature, and 1 μm of olP (PPyEP-g-SB is then added0)20Polymer reaction 12h, through nothing
More centrifuge washings of water DMSO, the modified porous silicon nanoparticle (i.e. P (PPyEP-g-SB of gained0)20Polymer-modified porous silicon is received
The grain of rice, P (PPyEP-g-SB0)20- PSiNPs) it is stored in anhydrous DMSO, it is saved under the conditions of 4 DEG C stand-by.
(4) modified porous silicon is collected after centrifugation in the anhydrous DMSO dispersion liquid of silicon nanoparticle modified porous in step (3) to receive
The modified porous silicon nanoparticle of 100mg, 40mg insulin and hydrochloric acid (3mL, concentration 0.01mol/L) are mixed, are being stirred by the grain of rice
It mixes, carry out total incubation 2h under the conditions of 25 DEG C；Then 10000rpm is centrifuged 5min, and it is more to be repeated 3 times centrifuge washing removal through ultrapure water
Remaining drug, gained carry medicine porous silicon nanoparticle and are stored in ultrapure water, and 4 DEG C save for use.
Embodiment 4
(1) amination porous silicon nanoparticle is prepared according to the method for embodiment 2；Open loop is prepared according to the method for embodiment 3
Polymerizate (i.e. N-Boc-P (PyEP)20Polymer), wherein the N- (tertbutyloxycarbonyl) ethanol amine, two thiopyridines of ethoxy
The amount ratio for changing phosphate, tetrahydrofuran and stannous octoate is 1mg:41mg:9mL:2.5mg.
It (2) is 100mg:60mg:5mL by amount ratio, by N-Boc-P (PyEP)20Polymer, dithiothreitol (DTT) and acetic acid second
Ester mixing protect in argon gas, is protected from light, carrying out de- pyridine under room temperature and react 12h；Gained system is subjected to rotary evaporation to go
Except solvent, residue is redissolved in methanol, ether is added and carries out precipitation process, sediment is collected and is carried out under the conditions of -20 DEG C
Vacuum freeze drying for 24 hours, obtains de- pyridine product (i.e. P (PPyEP-g-SB0)20Polymer)；
Then by 0.1mmol sulfobetaines (SB) and 10mL methanol-water mix reagent (volume ratio of methanol and water is 3:
1) it mixes, stirs liquid nitrogen flash freezer after 10min under the protection of argon gas, 0.1mmol4- dimethylamino naphthyridine is added simultaneously immediately
With de- pyridine product described in 0.1mmol, vacuum freeze thawing three times, after ultraviolet irradiation 0.5h, continues anti-under the protection of argon gas
10h is answered, blowing air terminates reaction；By gained system after bag filter (molecular cut off 10KDa) dialysis under the conditions of -20 DEG C
Vacuum freeze drying is for 24 hours to get the polyphosphate polymer (P (PPyEP-g-SB with sulfobetaines side chain1)20Polymerization
Object).
(3) under protection of argon gas, by 0.1g amination porous silicon nanoparticle, 0.1mmolN, N'- carbonyl dimidazoles and anhydrous
DMSO mixing, reacts 5h at room temperature, and 0.4mmolP (PPyEP-g-SB is then added1)20Polymer reaction 12h, through nothing
More centrifuge washings of water DMSO, the modified porous silicon nanoparticle (i.e. P (PPyEP-g-SB of gained1)20Polymer-modified porous silicon is received
The grain of rice, P (PPyEP-g-SB1)20- PSiNPs) it is stored in anhydrous DMSO, it is saved under the conditions of 4 DEG C stand-by.
(4) modified porous silicon is collected after centrifugation in the anhydrous DMSO dispersion liquid of silicon nanoparticle modified porous in step (3) to receive
The modified porous silicon nanoparticle of 100mg, 50mg insulin and hydrochloric acid (5mL, concentration 0.03mol/L) are mixed, are being stirred by the grain of rice
It mixes, carry out total incubation 1h under the conditions of 25 DEG C；Then 12000rpm is centrifuged 3min, and it is more to be repeated 3 times centrifuge washing removal through ultrapure water
Remaining drug, gained carry medicine porous silicon nanoparticle and are stored in ultrapure water, and 4 DEG C save for use.
Fig. 1 is the ring opening polymerization product that embodiment 4 is prepared and polyphosphate polymer in DMSO1H-NMR core
Magnetic chart spectrum, the methyl (- OC (CH that the Boc group end of N- (tertbutyloxycarbonyl) ethanol amine connects as can be seen from Figure 13)3, 1.3~
1.4ppm), the pyridine ring (7.26ppm, 7.82ppm and 8.46ppm) of two thiopyridines phosphate of ethoxy, two sulphur pyrrole of ethoxy
The methylene (- POCH of pyridine phosphate backbone2CH2O-, 4.2~4.3ppm) and side chain methylene (- POCH2CH2SS-, 4.2
~4.0ppm)；The methyl (- CH of sulfobetaines3, 1.1ppm) and methylene (- OCH2CH2NH3 +, 4.5ppm).By nuclear-magnetism point
Analysis is it is found that the polyphosphate of two thiopyridines side chain of ethoxy and sulfobetaines side chain synthesizes success, final goal product
For P (PPyEP-g-SB1)20。
Fig. 2 is that the SEM figure of load medicine porous silicon nanoparticle prepared by embodiment 4 carries medicine porous silicon nanoparticle as shown in Figure 2
More uniform spherical morphology is presented (granularity is 190~230nm).
Fig. 3 be embodiment 1,2,5 prepare nanoparticle infrared spectrum, from the figure 3, it may be seen that amino porous silicon nanoparticle in addition to
Retain 977cm possessed by porous silicon-1(Si-OH) and 1058cm-1(Si-O-Si) outside characteristic peak, there is-NH2Flexible vibration
Dynamic peak 1620cm-1And 1502cm-1, it is seen that amino is successfully modified on porous silicon nanoparticle surface；Polyphosphate polymer is repaired
The porous silicon nanoparticle of decorations, the characteristic peak for remaining porous silicon simultaneously, in 1709cm-1There is new peak in place, this is attributed to carbonyl
The stretching vibration peak of base；2926cm-1、2854cm-1、1631cm-1And 1435cm-1There is absorption peak, these absorption peaks and-CH3
With-CH2The stretching vibration of middle C-H is related with bending vibration, shows that the carbochain of polyphosphate polymer is already connected to porous silicon and receives
Grain of rice surface.
Embodiment 5
It is prepared according to embodiment 4 and carries medicine P (PPyEP-g-SB2)20- PSiNPs, wherein the N- (tertbutyloxycarbonyl) ethyl alcohol
Amine, two thiopyridines phosphate of ethoxy, tetrahydrofuran and stannous octoate amount ratio be 1mg:30mg:7.5mL:2.5mg, take off
The molar ratio of pyridine product, sulfobetaines and 4-dimethylaminopyridine is 1:2.8:2.8.
Embodiment 6
It is prepared according to embodiment 4 and carries medicine P (PPyEP-g-SB3)20- PSiNPs, wherein the N- (tertbutyloxycarbonyl) ethyl alcohol
Amine, two thiopyridines phosphate of ethoxy, tetrahydrofuran and stannous octoate amount ratio be 1mg:38mg:11mL:2.5mg, take off
The molar ratio of pyridine product, sulfobetaines and 4-dimethylaminopyridine is 1:3.1:3.1.
Embodiment 7
It is prepared according to embodiment 4 and carries medicine P (PPyEP-g-SB4)20- PSiNPs, wherein the N- (tertbutyloxycarbonyl) ethyl alcohol
Amine, two thiopyridines phosphate of ethoxy, tetrahydrofuran and stannous octoate amount ratio be 1mg:48mg:9mL:2.5mg, take off pyrrole
The molar ratio of pyridine product, sulfobetaines and 4-dimethylaminopyridine is 1:4.2:4.2.
Embodiment 8
It is prepared according to embodiment 4 and carries medicine P (PPyEP-g-SB3)10- PSiNPs, wherein the N- (tertbutyloxycarbonyl) ethyl alcohol
Amine, two thiopyridines phosphate of ethoxy, tetrahydrofuran and stannous octoate amount ratio be 1mg:22mg:7mL:2.5mg, take off pyrrole
The molar ratio of pyridine product, sulfobetaines and 4-dimethylaminopyridine is 1:3.5:3.5.
Embodiment 9
It is prepared according to embodiment 4 and carries medicine P (PPyEP-g-SB3)30- PSiNPs, wherein the N- (tertbutyloxycarbonyl) ethyl alcohol
Amine, two thiopyridines phosphate of ethoxy, tetrahydrofuran and stannous octoate amount ratio be 1mg:70mg:10mL:2.5mg, take off
The molar ratio of pyridine product, sulfobetaines and 4-dimethylaminopyridine is 1:3.2:3.2.
Embodiment 10
It is prepared according to embodiment 4 and carries medicine P (PPyEP-g-SB3)40- PSiNPs, wherein the N- (tertbutyloxycarbonyl) ethyl alcohol
Amine, two thiopyridines phosphate of ethoxy, tetrahydrofuran and stannous octoate amount ratio be 1mg:100mg:8mL:2.5mg, take off
The molar ratio of pyridine product, sulfobetaines and 4-dimethylaminopyridine is 1:3.5:3.5.
Embodiment 11
The load medicine porous silicon of porous silicon nanoparticle after surface modification and its preparation that the embodiment of the present invention 2~10 is provided
The property of nanoparticle is analyzed, and the results are shown in Table 2.As shown in Table 2, the Zeta potential of amination porous silicon nanoparticle is
15.35mV, Zeta potential drops to 3~8mV after polyphosphate is polymer-modified, without the porous of glycine betaine side chain modification
The Zeta potential of silicon nanoparticle is -4.84mV.Meanwhile polymer-modified nanoparticle its partial size of polyphosphate dramatically increases 50nm
More than, this may be attributed to the protonation of surface polymer, lead to the stretching, extension of glycine betaine side chain section.From partial size and Zeta potential
Variation demonstrates the polymer-modified porous silicon nanoparticle of polyphosphate and successfully prepares.
Partial size, Zeta potential and the drugloading rate of the porous silicon nanoparticle of table 2 after surface modification
The present invention further investigates tablets in vitro situation, as shown in Figure 4.It is porous to medicine is carried in order to investigate different gastroenteric environments
Load medicine porous silicon nanoparticle prepared by embodiment is placed in different simulation gastro-intestinal Fluids (containing enzyme) by the influence of silicon nanoparticle Release Properties
Middle incubation, simulation gastro-intestinal Fluid is according to USP-34 standard preparation.It will first be carried in 100mg embodiment 2,4,5,6,8 and 9 before experiment
Medicine porous silicon nanoparticle distinguishes ultrasonic disperse in 10mL simulate the gastric juice, shakes 2h in 37 DEG C of constant-temperature tables.After 2h, 1500rpm
Ultrafiltration centrifugation removal enzyme solutions, then be scattered in 10mL simulated intestinal fluid again, 4h is shaken in 37 DEG C of constant-temperature tables.It is different being incubated for
500 μ L solution are successively taken out when time interval, the blank simulated solution for accordingly adding same volume keeps constant the volume of system.
The sample 22000rpm of taking-up is centrifuged, and supernatant analyzes drug concentration using HPLC, and using drug pH value be 2 hydrochloric acid is molten
Liquid and pH value are that 6.8 standard curves obtained in buffer solution calculate its concentration.The result shows that only seldom in simulate the gastric juice
The insulin (less than 1%) of amount is released, but when these embodiment samples enter simulated intestinal fluid environment, and preceding 1h occurs 30
~40% burst release.This may be related with the electrostatic adsorption of drug and porous silicon surface, cholate etc. in simulated intestinal fluid
Salt ion promotes drug release as the natural wetting agent on porous silicon nanoparticle surface.And Fig. 4 is shown, in simulated intestinal fluid environment
The drug release patterns of the polymer-modified porous silicon nanoparticle of lower polyphosphate are substantially less than amination porous silicon nanoparticle
Release profiles (p < 0.05).Wherein the final burst size of drug is more higher than medicine is carried in embodiment 9 in amination porous silicon nanoparticle
The 20% of hole silicon nanoparticle burst size.The incomplete release of drug be due to some drugs with porous silicon nanoparticle silicon face
Irreversible Adsorption is formed, polyphosphate polymer also inhibits the salt such as cholate in the dizzy gate valve of water that porous silicon nanoparticle surface is formed
The influence of ion pair drug and silicon suction-operated, and then hinder the release of drug.Therefore, drug release patterns in vitro shows this
The polymer-modified porous silicon nanoparticle of polyphosphate effectively controls the release of drug as carrier in simulated intestinal fluid environment.
The polyphosphate polymer protective layer of the present invention performance testing that falls off is as shown in table 3, weigh respectively 5.0mg embodiment 2,
The load medicine porous silicon nanoparticle of 3 and 5 preparations is added to phosphate (5 μm of ol/L) buffer solution of different pH value (6.5 and 7.4)
In, 3h is shaken in 37 DEG C of constant-temperature tables.It is final carry the centrifugation of medicine porous silicon nanoparticle and with milli-Q water three times, take and a small amount of carry medicine
Porous silicon nanoparticle is dispersed in ultrapure water, tests Zeta potential.The result shows that carrying medicine porous silicon nanoparticle (no in embodiment 3
Side chain containing glycine betaine) Zeta potential improved by -4.84mV to+7.14mV (P < 0.05)；Medicine porous silicon nanometer is carried in embodiment 5
The Zeta potential of grain (side chain containing glycine betaine) is improved by 5.37mV to 10.35mV (P < 0.05)；Medicine porous silicon is carried in embodiment 2 to receive
The Zeta potential of the grain of rice (amination) is without significant change.As it can be seen that the behavior energy of phosphate partial hydrolysis polyphosphate polymer
Enough change the charge of nanoparticle neutral-surface.Physiological environment midgut epithelial cell surface is also enriched in phosphate, can promote and receives
The grain of rice reaches epithelial cell after reaching electropositive, and then increases the compatibility of nanoparticle and cell membrane.In addition, both sexes glycine betaine side
The introducing of chain has resistant function to the charge of nanoparticle surface, and further demonstrating charge reversal behavior is by polyphosphate master
Caused by chain and side chain.
Zeta potential of the 3 porous silicon nanoparticle of table before and after the incubation of different pH phosphate solution
The present invention has investigated mucoprotein to the absorption property of the polymer-modified front and back porous silicon nanoparticle of polyphosphate, specifically
Be mucoprotein (specifically use pig mucoprotein) is dissolved in pH value in 7.4 phosphate buffer, be made into 0.1mg/mL,
The solution of 0.5mg/mL, 2mg/mL, 5mg/mL and 8mg/mL various concentration.Embodiment 2,3,4,5 and 6 is added in 1:1 by volume
(wherein, embodiment 2 is amination porous silicon nanoparticle to the nanoparticle of middle preparation；Embodiment 3~6 is modified porous silicon nanoparticle,
I.e. polyphosphate it is polymer-modified after porous silicon nanoparticle), shake 2h in 37 DEG C of constant-temperature tables, 2000rpm is centrifuged 2min, sinks
Starch with milli-Q water three times.The sediment of cleaning utilizes dimethyl sulfoxide-phosphate buffer (dimethyl sulfoxide and phosphoric acid
The volume ratio of salt buffer is 3:1) solution dispersion 20min, disintegrates aggregation, 10000rpm is centrifuged 10min, measures in supernatant
Fluorescence intensity, as a result as shown in Figure 5.As seen from Figure 5, as the concentration of mucoprotein increases, what all embodiment groups were adsorbed
Nanoparticle amount both increases, but the concentration of mucoprotein reaches adsorbance after 2mg/mL and reaches saturation and do not change.In particular, will
In embodiment 2 adsorbance of amination porous silicon nanoparticle to mucoprotein concentration (0.1mg/mL, 0.5mg/mL, 2mg/mL and
5mg/mL) make linear regression analysis, gained linear equation is Y=19.079X+13.311, wherein Y is that amination porous silicon is received
The adsorbance of the grain of rice, X are mucoprotein concentration, R2It is 0.715, this illustrates amination porous silicon nanoparticle and glutinous egg in embodiment 2
Its most fast adsorbance of white absorption is also maximum；Meanwhile as can be seen from FIG. 5, no matter the concentration level of mucoprotein, polyphosphate polymerization
Absorption of the mucoprotein to nanoparticle can be significantly reduced after object modification, and glycine betaine side chain content is higher, to nanoparticle
Adsorbance is lower.
Mucus energy is worn using the polymer-modified front and back nanoparticle of Transwell experimental evaluation polyphosphate in the present invention
100 μ L pig mucoprotein solution (2mg/ are specifically added in power on the polycarbonate membrane of 4.0 μm of the cell 12-Transwell aperture
ML), it is then respectively adding nanoparticle prepared by the embodiment 2,3,4,5,6,8 and 9 of the identical fluorescence intensity of 2mL, phase is added in mistress
The blank PBS of same volume is strong from mistress's absorption 200 μ L samples progress fluorescence in different time (30,60,90,120 and 180min)
Degree analysis, and supplement the blank PBS of same volume.Mucus rate is worn using what apparent permeability coefficients (Papp) compared nanoparticle,
Papp=(dQ/dt) × [1/ (A × C0)], dQ/dt indicates the diffusion rate of nanoparticle, and A is polycarbonate membrane area, C0To receive
Grain of rice initial concentration.As a result as shown in fig. 6, as seen from Figure 6, nanoparticle can be significantly greatly increased after polyphosphate is polymer-modified
Wear mucus ability, especially embodiment 4,5,6,8 and 9 (side chain containing glycine betaine) apparent permeability coefficients (Papp) are 2 groups of embodiment
3~5 times of (amination) are 1.2~2 times of embodiment 3 (without glycine betaine side chain).The higher implementation of glycine betaine side chain content
Example 5,6 and 8, apparent permeability coefficients are bigger, nanoparticle to wear mucus ability higher.
The present invention uses the drug transport of the polymer-modified front and back drug-carrying nanometer particle of drug transport experimental evaluation polyphosphate
Behavior.In drug transport experiment, CaCO-2 and HT29-MTX cell (9:1) is with 5 × 104Cell number/hole plantation is in 12-
Transwell tissue culture plate is cultivated 21 days or so under the conditions of 37 DEG C.Interior room cell culture medium is removed before transport experiment,
Phosphate buffer cleans 3 times, and fresh phosphoric salt buffer is added in 37 DEG C of incubation 30min.In the upper end Transwell, interior room adds
Enter drug-carrying nanometer particle in 2mL embodiment 2,4,5,6,8 and 9 (200 μ g/mL), 3h is incubated in 37 DEG C of shaking tables (100rpm).Not
Same time point (30,60,90,120 and 180min) draws 200 μ L samples from mistress, while adding same volume blank PBS.It inhales
Sample 22000rpm centrifuging and taking supernatant is taken, drug concentration is detected using actrapid monotard's Elisa kit, and using apparent infiltration system
Number (Papp) explains the transport speed of drug.As a result as shown in fig. 7, as seen from Figure 7, can be significantly greatly increased after polymer-modified
Across the born of the same parents turn-over capacities of the drug of drug-carrying nanometer particle, embodiment 4,5,6,8 and 9 nanoparticles (side chain containing glycine betaine) apparent permeability coefficients
(Papp) it is 2.8~4.1 times of embodiment 2 (amination).Drug transport tests overall merit nanoparticle and meets with rete malpighii
With the final capacity of cellular layer physiological environment.As can be seen from the results, medicament-carried nano is increased after polyphosphate is polymer-modified
Across the born of the same parents turn-over capacities of drug of grain, what the high glycine betaine side chain modification of content was conducive to nanoparticle in wearing mucus experiment wears mucus
Ability, but its subsequent power is insufficient in drug transport experiment, and polyphosphate main chain is long and Optimum Contents glycine betaine side chain is modified
Nanoparticle be conducive to drug transport.Because charge reversal behavior will affect nanoparticle surface charge, excessive both sexes glycine betaine side
The introducing of chain can inhibit to reduce the affinity with cell membrane, and then reduce nanometer to the electropositive increasing action in nanoparticle surface
The cell endocytic amount of grain, across born of the same parents turn-over capacities of drug will reduce.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of polyphosphate polymer has structure shown in Formulas I:
In Formulas I, 0 < x≤100,10≤y≤100, and 0.08 < x/y≤2；R is amphoteric ion group.
2. polyphosphate polymer according to claim 1, which is characterized in that form the two of the amphoteric ion group
The ionic monomer of property includes cysteine, cysteamine glutamic acid, carboxybetaine, sulfobetaines or phosphorus base glycine betaine.
3. the preparation method of polyphosphate polymer as claimed in claim 1 or 2, comprising the following steps:
N- (tertbutyloxycarbonyl) ethanol amine, two thiopyridines phosphate of ethoxy and the first solvent are mixed, urged in organic metal
Agent effect is lower to carry out ring-opening polymerization, obtains ring opening polymerization product；
The ring opening polymerization product is mixed with go back original reagent and the second solvent, de- pyridine reaction is carried out, obtains de- pyridine product；
The de- pyridine product is mixed with amphoteric ion monomer and third solvent, under 4-dimethylaminopyridine catalytic action
Click chemistry reaction is carried out, the polyphosphate polymer with structure shown in Formulas I is obtained.
4. a kind of modified porous silicon nanoparticle is made as described in polyphosphate polymer as claimed in claim 1 or 2 or claim 3
The polyphosphate polymer that Preparation Method is prepared, which is modified porous silicon nanoparticle, to be handled.
5. modified porous silicon nanoparticle according to claim 4, which is characterized in that the grain of the modified porous silicon nanoparticle
Diameter is 50~500nm, and Zeta potential is -30~+30mV, and specific surface area is 50~2000m2/ g, aperture size are 5~20nm.
6. the preparation method of the modified porous silicon nanoparticle of claim 4 or 5, comprising the following steps:
At N, N'- carbonyl dimidazoles effect, processing is modified to porous silicon nanoparticle using polyphosphate polymer, is obtained
Modified porous silicon nanoparticle.
7. preparation method according to claim 6, which is characterized in that the porous silicon nanoparticle, polyphosphate polymer
And the amount ratio of N, N'- carbonyl dimidazoles is 1g:(0.005~0.05) mmol:(0.5~5) mmol.
8. preparation method according to claim 6 or 7, which is characterized in that the porous silicon nanoparticle is using preceding use
Amino silicane coupling agent carries out amination processing.
9. any one of the modified porous silicon nanoparticle of claim 4 or 5 or claim 6~8 preparation method are prepared into
Application of the modified porous silicon nanoparticle arrived as the carrier of oral administration.
10. application according to claim 9, which is characterized in that the matter of active medicine and carrier in the oral administration
Amount is than being (0.01~9): 10.
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| CN105820334A (en) * | 2016-05-19 | 2016-08-03 | 江南大学 | Preparing method for poly-zwitter-ion nanoparticles based on amino acid |
| US20180258257A1 (en) * | 2017-03-08 | 2018-09-13 | Guangdong Provincial Hospital of TCM | Preparation method of new-type nanoparticles for loading emodin |
| CN108210932A (en) * | 2017-12-18 | 2018-06-29 | 温州医科大学 | A kind of preparation method of charge driving self-assembly chitosan base medicine-carried nano particles |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113105614A (en) * | 2021-04-08 | 2021-07-13 | 湘潭大学 | Easily degradable responsive core-crosslinkable amphiphilic block polymer, preparation method thereof and application of polymer as drug carrier |
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