CN108478531A - Folate-targeted restores sensitive medicament-carried polymer nano micelle and its preparation method and application - Google Patents
Folate-targeted restores sensitive medicament-carried polymer nano micelle and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of folate-targeteds to restore sensitive medicament-carried polymer nano micelle and its preparation method and application.The delivery vector is using amphipathic three block copolymer PCL ss PEG ss PCL as material preparation, and contain chemotherapeutic drugs Doxorubicin and photosensitizer indocyanine green in the hydrophobic inner core of the micella, while the phosphatide DSPE PEG NH of active group are introduced in preparation process2The ends the DSPE hydrophobicity of the phosphatide can be inserted into more by force in the hydrophobicity PCL kernels of polymer micelle, submissive hydrophily PEG long-chains are in the outer surface of micella, FA with targeting is connected to the PEG activity distal end on polymer micelle surface, integrates active targeting tumour, reduction response drug release function.The present invention has the advantages that the high and preferable photothermal conversion effect of small grain size, good dispersion, drugloading rate, encapsulation rate, reduction triggering drug release, the administration of fluorescence imaging tumor locus, cancer target, chemotherapy photo-thermal combination therapy raising tumor killing effect can be achieved, have broad application prospects in terms of the targeting combination therapy of tumour.
Description
Technical field
The present invention relates to a kind of folate-targeteds to restore sensitive medicament-carried polymer nano micelle and its preparation method and application, tool
Body is target polymer micella that is folate-mediated, can having reduction response to tumor microenvironment, can be efficiently in polymer micelle
Kernel contains chemotherapeutics and photosensitizer, and has long cycle characteristics, can efficiently enter tumour cell, realizes reduction sensitive trigger
Discharge chemotherapy-photo-thermal combination therapy of drug and tumour.
Background technology
It is by amphipathic nature polyalcohol in selective solvent, due to intermolecular hydrogen before polymer micelle is developed in 30 years
A kind of thermodynamically stable colloidal solution that key, electrostatic interaction and Van der Waals force and spontaneous assembling are formed has nucleocapsid,
Hydrophilic block forms shell, and hydrophobic block forms kernel.As a kind of efficient pharmaceutical carrier, polymer micelle can incite somebody to action
Hydrophobic drug packet reduces its toxic side effect to human body in hydrophobic cores, and the stabilization of drug can be improved in hydrophily shell
Property, and influence the inside and outside behavior of micella.Common hydrophobic chain segment for building polymer micelle hydrophobic cores has poly- breast
Acid（PLA）, polycaprolactone（PCL）, poly-aspartate, polyglutamic acid, polyoxypropylene etc..Hydrophilic chain selected by hydrophily shell
Section directly affects the properties such as biocompatibility and the biodynamics of polymer micelle, and the most commonly used is with excellent biocompatibility
With the polyethylene glycol of safety（PEG）, other hydrophilic segments also have polylysine, polyoxyethylene, polyvinylpyrrolidone, poly- third
Olefin(e) acid class etc..Polymer micelle grain size is generally less than 200 nm, have stability it is good, carry medicine range it is wide, drugloading rate is high, in vivo it is stagnant
Stay the features such as time is long, medicine stability is good, toxic side effect is low and bioavilability is high.
Folic acid（Folic acid）Belong to small molecule vitamin, hardly possible penetrates cell membrane under physiological condition, needs by internalization
It could be absorbed.The existing mechanism in two kinds of folic acid internalization：First, by the transmembrane protein of low-affinity, transhipment folic acid is gone back
Ortho states enters intracellular；Second is that by the folacin receptor of high-affinity, it can absorb folic acid and enters into the cell.Folacin receptor（FR）For
A kind of glycoprotein membrane receptor, the low expression in normal structure, and expressed in certain solid tumor cell film surface height, to folic acid
Or the conjugate of folic acid has higher affinity, therefore the carrier containing folic acid or folate conjugate can be mediated to enter tumour cell
It is interior.The characteristics of folic acid is connected to polymer micelle surface, is specifically bound with folacin receptor using it, makes polymer micelle have
The characteristic of standby targets neoplastic cells, to reduce toxic side effect and improve the bioavilability of tumour medicine in vivo.
In order to improve the cancer target efficiency of drug delivery system, the targeting ligand for finding specificity is not only needed, is also needed
Enhance the body-internal-circulation stability of nanoparticle, reduces leakage, accumulation of the chemotherapeutics in non-target area, it is secondary to reduce its poison
Reaction.Tumour cell microenvironment is there are the different variation characteristics of pH, redox characteristic, temperature or enzyme, therefore Many researchers
The drug delivery vehicle of design environment sensitivity.In intracellular, the glutathione of animal and human body（Glutathione, GSH）It is most
Abundant low molecule bioactive sulfhydryl substance and most important reducing substances.A concentration of 2- of intracellular reproducibility GSH
10 mM, than extracellular（2-20 μM）It is hundreds times high.The content of tumor tissues GSH is then at least more than 4 times of normal structure.
Especially in some drug-resistant tumors, the GSH of overexpression even will be higher by 10 times.Using this characteristic, can design has
The drug delivery system of GSH triggerings, so that it is recycled in vivo has stability, and reaches tumor by local or enter tumour cell
Afterwards, high concentration GSH triggers drug release and is remarkably improved drug to the lethal effect of tumour and reduces the poison pair of normal tissue
Effect.
Breast cancer is to endanger one of most common tumour of women's health, and essential therapeutic arsenals include operation, chemotherapy, put
Treatment and immunotherapy.However, each way has the limitation of its clinical application.It may be led since chemotherapy lacks tumour-specific
Cause serious toxic side effect.Long-term chemotherapy can lead to the problem of multidrug resistance and therapeutic efficiency is caused to reduce.Recently, it is immunized
It treats since its strong antitumor efficiency causes the concern extensively sent out, but this therapeutic modality is not suitable for all patients.Cause
This, a kind of safely and effectively therapeutic modality for the treatment of searching for breast cancer is the hot spot of clinical research.Photo-thermal therapy is a kind of non-
The oncotherapy means of intrusion can convert near infrared light to thermal energy using photosensitizer, tumor by local is made to be heated to effectively treating
Temperature simultaneously kills tumour cell.Thermotherapy can cause DNA damage and DNA is inhibited to synthesize；Tumoricidal endoplasmic reticulum, line grain
Body film and lysosome membrane cause after birth rupture and endochylema excessive；The expression of related gene is influenced, cancer cell-apoptosis is promoted.It compares
Compared with traditional means, the advantages of laserthermia has minimally invasive or even noninvasive, recovery time short and few intercurrent disease.But inside tumor
Heat distribution is uneven, and simple thermotherapy is difficult to kill all cancer cells, and the tumour cell of survival may cause to recur or shift.Cause
This, needs joint other treatment means to improve the effect of thermotherapy, the side that clinic is generally combined using chemotherapy with thermotherapy at present
Method, studies have shown that chemotherapy-thermotherapy combination therapy can significantly increase antitumor curative effect.
Invention content
The present invention is intended to provide a kind of folate-targeted restores sensitive medicament-carried polymer nano micelle and preparation method thereof and answers
With can be with overcome the deficiencies in the prior art.The present invention be it is a kind of have the function of active targeting and meanwhile it is efficient contain chemotherapeutics and
Photosensitizer simultaneously there is the newtype drug of tumor microenvironment reduction intelligent response to pass release system.This passs release system can be by hydrophobic work
Firmly chemotherapeutics and photosensitizer are effectively contained in the hydrophobic cores of polymer micelle, the PEG layers of shell assign polymerization
Object micella long circulating and spatial stability, targeting group pass through DSPE-PEG-NH2It is connected to the shell of micella, grain size is smaller（<
200 nm）, can realize tumor locus efficiently concentrating by EPR passive targets effect and the effect of folacin receptor active targeting, and lead to
It crosses tumour micro-loop reduction response quickly release drug and plays drug effect.
The present invention by by broad-spectrum anti-cancer drug adriamycin and can be used for clinical photosensitizer ICG carries altogether, cancer target delivering
With tumor locus medicine controlled releasing, realize that the combination therapy effect of chemotherapy and photo-thermal therapy maximizes, after one side photosensitizer contains
With good photothermal conversion efficiency, the photo-thermal generated under laser irradiation can enhance intake, acceleration of the tumour cell to carrier
Drug release, Chemotherapy drug, another aspect hyperthermia can with direct killing tumour cell, to realize the chemotherapy of tumour with
The combination therapy of photo-thermal therapy.Containing altogether, target conveying, environmental response triggering for chemotherapeutics and photosensitizer can be achieved in the present invention
It releases the drug and photo-thermal-chemotherapy combined of tumour is treated.
It is to have reduction sensitivity characteristic that the present invention, which provides a kind of sensitive medicament-carried polymer nano micelle of folate-targeted reduction,
PCL-ss-PEG-ss-PCL polymer and active group function phosphatide DSPE-PEG-NH2It is super by film for raw material
It is carrier, chemotherapeutics and photosensitizer that the polymer nano micelle with reduction intelligent response is prepared in sound dispersion method self assembly
The hydrophobic cores in micella are contained, shell is hydrophily PEG, has long circulating and spatial stability characteristic, folate-targeted group
It is modified in micellar surface by covalent bond；The PCL-ss-PEG-ss-PCL polymer that there is reduction sensitivity characteristic（Altogether
It is poly-）Structure it is as follows：
The molecular weight of polymer is 10000-24000, wherein PEG hydrophilic segments mass percent>45%, point of preferred polymers
Son amount is 15000, functionalization phosphatide DSPE-PEG-NH2PEG molecular weight be 2000, wherein m 114-273, n 22-53.
PCL-ss-PEG-ss-PCL is between polycaprolactone and polyethyleneglycol block copolymer with reduction-sensitive
Disulfide bond connection, therefore there is reduction-sensitive.
The load medicine quality is 10%, and the mass ratio of chemotherapeutics and photosensitizer is 1:1；Grain size is in 200 nm or less.
PCL-ss-PEG-ss-PCL polymer molecular weights of the present invention are in 10000-24000, wherein PEG hydrophilic chains
Section mass percent>45% is that can prepare reduction sensitive polymer micella, with PCL3750-ss-PEG7500-ss-PCL3750
（Its n=33, m=170）For, preparation method is as follows：
1）Weigh 1g Py-ss-PEG7500- ss-Py is dissolved in 12 mL dimethyl sulfoxide (DMSO)s（DMSO）In, it is to be dissolved fully after, add
Enter 25 μ L mercaptoethanols, 20 μ L acetic acid；Magnetic agitation reacts 24 h at ambient temperature；After reaction by reaction mixture
It is transferred in the bag filter of MWCO8000-10000, and is positioned in distilled water 24 h that dialyse；It is freeze-dried, obtains pure white later
Color powdered product HO-ss-PEG7500-ss-OH。
2）It is accurate that 6-caprolactone monomer, the 1 g HO-ss-PEG of 1 g after purification is added7500- ss-OH adds in polymerization pipe
Enter a drop stannous octoate as catalyst；Reaction system is vacuumized into 5 min, 5 min of inflated with nitrogen, so in triplicate.Finally
Polymerization pipe is closed under the protection of nitrogen, 12-24 h are reacted in 100-130 DEG C of oil bath；After reaction, with a small amount of two
Chloromethanes dissolves, and is poured into later in 200-400 mL anhydrous ethers；Suction filtration takes precipitation to obtain pure white product.
3）It repeats purifying three times, is dried in vacuo under conditions of 35 DEG C and obtains pure white granular disintegration PCL3750-ss-
PEG7500-ss-PCL3750；Its reactional equation is as follows：
The PCL-ss-PEG-ss-PCL of other molecular weight is synthesized：Can by synthesize different molecular weight HO-SS-PEG-SS-OH,
Using stannous octoate as catalyst, cause 6-caprolactone monomer ring-opening polymerisation, and pass through 6-caprolactone monomer and HO-ss-PEG-ss-
Polymer molecular weight synthesized by the rate of charge control of OH.
The chemotherapeutics is adriamycin, daunorubicin, idarubicin, Aclarubicin, valrubicin, taxol or rice
Hold in the palm anthraquinone or combination thereof；
The photosensitizer is indocyanine green（ICG）Or chlorin（Chlorin e6）.
The function phosphatide is DSPE-PEG-NH2, can also be the function phosphatide DSPE-PEG- of maleimation
Mal。
The targeting group is folic acid group, and when function phosphatide is DSPE-PEG-Mal, the targeting group also may be used
To be the polypeptide LHRH, monoclonal antibody anti-Her2-Fab, cell-penetrating peptide TAT etc. of sulfhydrylation.
The system of the tumour target polymer micella of reduction of the present invention sensitive total load chemotherapeutics and photosensitizer
Preparation Method includes the following steps：
1）It will be by metering by PCL-ss-PEG-ss-PCL, chemotherapeutics, photosensitizer, DSPE-PEG-NH2Be dissolved in dichloromethane or
Methanol and dichloromethane（Mass ratio 1:1）Uniform mixed solvent after, revolving removes organic solvent plastic film mulch, and vacuum drying is 16 small
When；
2）With the phosphate buffer solution of pH=7.4（PBS）Dispersion, 60-65 DEG C of aquation, are cooled to room temperature, ultrasonic under ice bath, obtain
It is positioned in the bag filter of MWCO8000-14000 Da and dialyses 4 hours to micella dispersion liquid, remove free drug, dialysed
The tumour target polymer micella dispersion liquid of the sensitive total load chemotherapeutics and photosensitizer of reduction afterwards；
3）Pass through EDC/NHS reaction forming folate-targeted molecules again；Specifically reaction step is：It is about DSPE- to weigh molar ratio
PEG-NH25 times of folic acid, be dissolved in deionized water；Addition molar ratio is about the EDC of 5 times of folic acid and molar ratio is 2 times of EDC
NHS activate 15 min；Above-mentioned amidized nanoparticle suspension is added, is reacted 3 hours under room temperature magnetic agitation.
The present invention provides a kind of folate-targeteds to restore sensitive medicament-carried polymer nano micelle and its preparation method and application,
The tumour target polymer micella for restoring sensitive total load chemotherapeutics and photosensitizer is being prepared for chemotherapy of tumors and light
Application in heat integration treatment.With it is folate-mediated, can to tumor microenvironment have reduction response target polymer micella, as
The total load chemotherapeutic of target tumor and the intelligent response type nano-delivery system of photosensitizer, can be efficiently in polymer micelle
Core contains chemotherapeutics and photosensitizer, and has long cycle characteristics, can efficiently enter tumour cell, realizes that reduction sensitive trigger is released
Put chemotherapy-photo-thermal combination therapy of drug and tumour.Specifically, prominent substantive features of the present invention are：
1）Amphipathic carrier material used in the present invention has good biodegradability, biocompatibility and without immunogene
Property.The nano-micelle grain size of preparation is small, good dispersion degree, is suitable for intravenous systemic administration.
2）Dewatering medicament is effectively packed in micellar hydrophobic by the polymer micelle designed by the present invention by hydrophobic effect
Core has compared with high drug load and encapsulation rate.It overcomes the internal unstability of free adriamycin and photosensitizer and is easily eliminated
Disadvantage.
3）The hydrophilic layer PEG of micellar surface has flexibility so that polymer micelle has long cycle characteristics and space is steady
It is qualitative, extend its circulation time in blood.Suitably sized size is effectively rich so as to using EPR effects
Collect tumor locus, reaches killing function of tumor.
4）Functionalization phosphatide DSPE-PEG-NH employed in preparation2, it is thin that DSPE hydrophobicitys can be inserted into more by force micella
Aqueous kernel, folic acid are combined with micellar surface PEG distal aminos, can improve the active targeting of medicament-carried nano micelle.
5）Long circulating is stablized in polymer micelle body, can be sensitive by being restored to tumor microenvironment after reaching tumor by local
Intelligent response and rapid delivery of pharmaceuticals, to improve therapeutic effect and reduce toxic side effect.
6）The micella preparation method is simple and practicable, and preparation process is at low cost without using surfactants such as polyvinyl alcohol,
Period is short.
7）After free ICG is wrapped in nanoparticle micella, its internal stability can not only be improved, and pass through laser irradiation
After can generate higher thermal energy, heat dissipation less, thermal transition it is efficient, effectively improve photo-thermal therapy effect.
Description of the drawings
Fig. 1 is the particle diameter distribution variation diagram of carrier micelle of the present invention different time in pH7.4,10 mM GSH.
Fig. 2 is to restore sensitive medicament-carried polymer micelle（FA-DINPs）Characterization, A）The grain size of DINPs and FA-DINPs is steady
It is qualitative.B）The time-temperature curve of laser irradiation FA-DINPs, INPs, free ICG, free DOX and PBS.C）Laser irradiation
Near infrared imaging figure when FA-DINPs, INPs, free ICG, free DOX and PBS 5 minutes.D）FA-DINPs is in laser irradiation
Front and back transmission electron microscope picture.
Fig. 3 is carrier micelle of the present invention in pH7.4, pH7.4+10 mM GSH, pH7.4+ laser and pH7.4+10 mM
Adriamycin release under GSH+ laser different conditions.
Fig. 4, which is various concentration reduction sensitive polymer micella, laser A）, without laser B）To EMT-6 breast cancer when irradiation
The cytotoxicity of cell.
Fig. 5, which is different load medicines reduction sensitive polymer micella, laser（a）, without laser（b）Cell phagocytosis under irradiation
With the distribution map of drug in the cell.
Fig. 6 is the in-vivo imaging and bio distribution figure of the polymer micelle shot using bioluminescence imaging technology, wherein A)
The ICG fluorescence imaging figures of sensitive polymer micella and free drug in lotus knurl BALB/C mice body are restored for different load medicines；B)
For tumor by local ICG fluorescence intensity semi-quantitative results；C it is) that different load medicines restore sensitive polymer micella and free drug in lotus
The DOX fluorescence imaging figures of tumor BALB/C mice isolated viscus；D it is) that different load medicines restore sensitive polymer micella and free drug
In the DOX fluorescence intensity semi-quantitative results of lotus knurl BALB/C mice isolated viscus.
Specific implementation mode
With reference to specific embodiment, the present invention is further illustrated.The experiment of actual conditions is not specified in embodiment
Method, the condition usually according to normal condition and described in handbook, or according to the normal condition proposed by manufacturer；Used is logical
With equipment, material, reagent etc., it is commercially available unless otherwise specified.
Embodiment 1
The synthesis of PCL-ss-PEG-ss-PCL.
A kind of amphipathic three block copolymer that reduction is sensitive（PCL-ss-PEG-ss-PCL）Synthetic method, with
PCL3750-ss-PEG7500-ss-PCL3750Synthetic method for, include the following steps：
（1）Accurately weigh 1g Py-ss-PEG7500-ss-Py（Purchased from Beijing Jian Kai Science and Technology Co., Ltd.）It is dissolved in 12 mL
Dimethyl sulfoxide (DMSO)（DMSO）In.It is to be dissolved fully after, be added 25 μ L mercaptoethanols, 20 μ L acetic acid.Magnetic force under the conditions of 25 DEG C
It is stirred to react 24 h.Reaction mixture is transferred in the bag filter of MWCO8000-14000Da after reaction, and is positioned over
Dialyse 24 h in distilled water.It is freeze-dried later, obtains fine white powder product HO-SS-PEG7500-SS-OH。
（2）It is accurate that 6-caprolactone monomer, the 1 g HO-ss-PEG of 1 g after purification is added7500- ss-OH in polymerization pipe,
A drop stannous octoate is added as catalyst.Reaction system is vacuumized into 5 min, 5 min of inflated with nitrogen, so in triplicate.Most
Polymerization pipe is closed under the protection of nitrogen afterwards, 24 h are reacted in 100 DEG C of oil bath.After reaction, with a small amount of dichloromethane
Alkane dissolves, and is poured into later in 500 mL anhydrous ethers.Suction filtration takes precipitation to obtain pure white product.Repeat purifying three times.35
It is dried in vacuo under conditions of DEG C.Obtain pure white granular disintegration PCL3750-ss-PEG7500-ss-PCL3750。
The synthesis of this embodiment is PCL3750-ss-PEG7500-ss-PCL3750, but it is total in PCL-ss-PEG-ss-PCL
Polymers molecular weight is 10000-24000, wherein PEG hydrophilic segments mass percent>It is sensitive that reduction can be prepared when 45%
Polymer micelle.
A kind of reduction sensitivity blank polymer micella（Blank NPs）Preparation method, include the following steps：
（1）PCL-ss-PEG-ss-PCL amphipathic three block copolymers are substantially soluble in dichloromethane, are removed with Rotary Evaporators
Organic solvent is removed, is allowed to form one layer of uniform film in eggplant-shape bottle inner wall, residual dichloromethane is dried up with nitrogen, is put into vacuum
In drying box, it is dried in vacuo 16 hours；
（2）The phosphate buffer solution of pH 7.4 is added into eggplant-shape bottle（PBS）, make step（1）Product mass percent
For 10 %, rocking makes film dispersion in the solution, and after 65 DEG C of 5 h of aquation, mixing is placed to room temperature, ultrasound under ice bath（5 mm
Probe, 30 % of Ampl）20 minutes, stablized, uniform blank polymer micella dispersion liquid, 4 DEG C of preservations.Its grain size, electricity
Position, polydispersity are shown in Table 1.
（3）Investigation sensitive to reducing environment Blank NPs
Blank NPs obtained are scattered in the solution of different reducing conditions, including PBS (pH7.4) and 10mM GSH PBS
(pH7.4).24 h are placed in 37 DEG C of shaking tables detects its interior change of size during this period.As shown in Figure 1, Blank NPs are 10
Nanoparticle in mM GSH (pH7.4) solution shows two wave crests, wherein smaller peak（~150 nm）It may be due to reduction
The disulfide bond reduction of amphipathic three block is caused to be broken by solution so that the PEG of micellar surface falls off.Larger peak（400~900
nm）Appearance probably due to polymer micelle structure destroy caused by particle buildup.On the contrary, polymer micelle is in PBS
(pH7.4) be always maintained in one it is unimodal, show its stability in physiological conditions.
Embodiment 2
A kind of reduction sensitivity contains the polymer micelle of DOX and ICG altogether（DINPs）Preparation method, include the following steps：
（1）Doxorubicin hydrochloride and ICG are first turned hydrophobic respectively：By 1 %（With quality of materials percentage）Doxorubicin hydrochloride addition contains
There is triethylamine（The mg adriamycins of 20 μ L triethylamines/1）Methanol solution in, reacted at room temperature 20 hours under magnetic agitation；By ICG with
Tetrabutylammonium iodide is mixed in（1:5.72 mass ratio）In dichloromethane, reacted at room temperature 20 hours under magnetic agitation.
（2）PCL-ss-PEG-ss-PCL is substantially soluble in organic solvent dichloromethane, desalination adriamycin is added and is turned
Hydrophobic ICG solution mixing removes organic solvent with Rotary Evaporators, is allowed to form one layer of uniform film in eggplant-shape bottle inner wall,
Residual dichloromethane is dried up with nitrogen, is put into vacuum drying chamber（25℃）It is interior, it is dried in vacuo 12 hours；
It should be noted that as long as hydrophobic drug, which can be transferred through hydrophobic effect, contains dredging in the mixed polymer micella
In aqueous kernel, because this patent is mainly used for the reduction sensitivity micella of photo-thermal and chemotherapy combined treatment at present, it is negative altogether
Chemotherapeutics and photosensitizer are carried, photosensitizer ICG itself is hydrophilic, in patent of the present invention is converted to tetrabutylammonium iodide
For hydrophobic ICG- tetrabutylammonium iodides compound.If itself it is exactly hydrophobicity using chlorin Ce6 as photosensitizer
, without carrying out hydrophobicity conversion.
（3）The phosphate buffer solution of pH7.4 is added into eggplant-shape bottle（PBS）, make step（2）Product quality percentage
Make film dispersion in the solution than for 10 %, rocking, after 65 DEG C of 5 h of aquation, mixing is placed to room temperature, ultrasound 10 under ice bath
Minute, stablized, uniform blank polymer micella dispersion liquid, 4 DEG C of preservations.Sample is taken to survey grain size, particle diameter distribution, current potential,
It the results are shown in Table 1.
Table 1. contains the characterization of the reduction sensitive polymer micella of DOX and ICG altogether
4）A kind of targeting reduction is sensitive to contain DOX and ICG polymer micelles altogether（FA-DINPs）Preparation method.Including walking as follows
Suddenly：
It is in embodiment 2 when preparing the polymer micelle that there is folate-targeted to carry medicine（2）Add when rotary evaporation plastic film mulch in step
Enter 0.5 %-5 %（Mass percent）DSPE-PEG（2000）-NH2.According to embodiment 2（1）、（2）、（3）With（4）It is identical
The step of prepare surface carry-NH2After the drug-carrying polymer micelle of active group, pass through EDC/NHS reaction forming folic acid targets
To molecule.Specifically reaction step is：（1）It is about DSPE-PEG-NH to weigh molar ratio25 times of folic acid, be dissolved in 500 μ L go from
In sub- water.（2）The EDC that molar ratio is about 5 times of folic acid is added and the NHS that molar ratio is 2 times of EDC activates 15 min.（3）It is added
Above-mentioned amidized nanoparticle reacts 3 hours under room temperature magnetic agitation.
（5）The drugloading rate and encapsulation rate of DINPs and FA-DINPs
By embodiment 2（3）Prepared sample centrifugation is three times（30 min/ times, 23000 rpm, deionized water is resuspended）, finally from
The heart is complete to be resuspended with 5 mL deionized waters and sample is lyophilized.A certain amount of sample is weighed to be dissolved in acetonitrile and methanol mixed solvent, it is ultraviolet
Spectrophotometer quantifies the medicament contg of adriamycin and ICG, calculates drugloading rate and encapsulation rate later, the results are shown in Table 1.Drugloading rate and
Encapsulation rate is calculated according to following formula：
Encapsulation rate (%)=(actually containing medication amount)/(practical dosage) × 100%;
Drugloading rate (%)=(actually containing medication amount)/(total polymer micell weight) × 100%
It is demonstrated experimentally that for DINPs, the drugloading rate of ICG and DOX are 13.48 % and 15.28 % respectively, and encapsulation rate is respectively
94.71 % and 97.05 %.Drugloading rate for targeting micella FA-DINPs, ICG and DOX is 15.05 % and 17.32 % respectively,
Encapsulation rate is respectively 96.54 % and 98.67 %, shows to restore the higher drug encapsulation ability of sensitive polymer micella, as a result see
Table 1.Clinically common other hydrophobic drug polymer micelles can also be prepared with the method for the present invention.Ce 6 is carried as double
With Aclarubicin, double load Ce 6 and taxol, double load ICG and taxol etc..
（6）The grain size stability of DINPs and FA-DINPs is investigated
Prepared sample dispersion liquid is stored in 4 DEG C, the daily same time measures its grain size, METHOD FOR CONTINUOUS DETERMINATION 7 days.
The result shows that the grain size of FA-DINPs, DINPs after a week without significant change, do not occur aggregation and
Precipitation, it was demonstrated that the reduction sensitive polymer micella for containing chemotherapeutics and photosensitizer altogether has high stability.As a result see figure
2,A)。
（7）The photo-thermal efficiency rating of FA-DINPs under laser irradiation
The results show that in 1 W/cm2Laser irradiation after 8 minutes, the maximum heating difference of FA-DINPs, INPs and free ICG
It is 34.3,30.4 and 12.4 DEG C, and PBS and free DOX has only heated up 3.4 and 4.3 DEG C under the same terms（Fig. 2, B）.FA-
The final temperature of DINPs and INPs generates irreversible damage more than 43 DEG C to tumour cell.Infrared thermal imaging figure is aobvious
Show, after 5 minutes laser irradiations, FA-DINPs, INPs, free ICG, free DOX and PBS temperature respectively reach 65.2,
59.5,41.5,33.4 and 32.5 DEG C, as a result see Fig. 2, C）.Transmission electron microscope is used for observing FA-DINPs in 1 W/cm2Power
The metamorphosis of 808 nm laser irradiations after five minutes.The results show that FA-DINPs is cracked into small under the destruction of thermal energy
Grain.
Embodiment 3
The vitro drug release of FA-DINPs is investigated
By embodiment 2（3）Prepared sample is divided into 12 groups and is discharged in MWCO 8000-14000Da bag filters.Release liquid point
Not Wei PBS (pH 7.4) and 10mM GSH PBS (pH7.4) release liquid be 20 mL, be positioned on shaking table, 37 DEG C 120
Rpm shakes, and all takes out release liquid survey medicament contg after a certain period of time, mend the fresh release liquid of same volume.Laser irradiation
Group needs before being put into bag filter with 1 W/cm2808 nm laser in advance shine 5 min.
Releasing result is shown in Fig. 3, shows the adriamycin release in FA-DINPs by release liquid reproducibility and laser irradiation
It influences.By incubation in 2 days, FA-DINPs was in PBS（pH 7.4）It is released with adriamycin in 10 mM GSH PBS (pH 7.4)
It is respectively high-volume 21.59 % and 48.77 %；Under laser irradiation, DOX at 2 days in FA-DINPs is in PBS（pH 7.4）With 10
Burst size in mM GSH PBS (pH 7.4) is respectively 24.11 % and 52.05 %.FA-DINPs is in PBS after 24 days（pH
7.4）Burst size with adriamycin in 10 mM GSH PBS (pH 7.4) is respectively 37.14 % and 76.5 %；And laser compares
Under, FA-DINPs is in PBS after 24 days（pH 7.4）Burst size with adriamycin in 10 mM GSH PBS (pH 7.4) is respectively
43.14 % and 82.82 %.This illustrates that the release of drug in FA-DINPs can be regulated and controled by reducing substances and laser irradiation.
Embodiment 4
FA is to the modification of DINPs and laser irradiation to the cytotoxicity of EMT-6 breast cancer cells.
With the EMT-6 breast cancer cells in 0.25 % trypsin digestions exponentially growth period, then with 10 % fetal calf serums
No folic acid RPMI 1640 culture mediums are made into individual cells suspension, with 3500, every hole cell inoculation in 96 well culture plates, per hole
100 μ L of volume.The diluted various concentration Blank NPs of addition culture medium, Free DOX ＆ ICG, DINPs and FA-
DINPs（20,10,5,2.5,1.25,0.5,0.05 μ g/mL of adding consistency（Doxorubicin concentration））, using MTS quantitative assessments
The chemotherapy effect of DINPs and FA-DINPs.Laser irradiation group is set simultaneously and investigates chemotherapy-killing of the photo-thermal synergy to cell
Ability, after DINPs and FA-DINPs is added and is incubated two hours, drug concentration 2.5,5 and 10 μ g/mL（Adriamycin is dense
Degree）1.6 W/cm of group2808 nm laser irradiations, 5 min after continue be incubated to 24 h, then according to the method for MTS reagent box
Detect cell activity.Cell survival rate is calculated according to following formula：
Cell survival rate=（Experimental group OD values-blank group OD values）/（Feminine gender group OD values-blank group OD values）
Cytotoxicity result is shown in Fig. 4, shows after being incubated 24 h, DINPs and FA-DINPs（Doxorubicin concentration is 20 μ g/mL）It leads
It is respectively 59.61 % and 75.86 % to cause the death rate of EMT-6 cells.With the death rate of cell caused by free DOX（93.75
%）It is slightly lower compared to toxicity.For blank polymer micella at the concentration tested without apparent cytotoxicity, this shows unentrapped drug
Reduction sensitive polymer micella have good biocompatibility.1.6 W/cm of further quantitative analysis2Laser irradiation under
The cell survival rate of each group.The result shows that compared with simple chemotherapy or photo-thermal therapy, chemotherapy-photo-thermal combination therapy can be significantly
Reduce the survival rate of cell.And individual laser irradiation cell survival rate is 100 %, shows 1.6 W/cm2808 nm swash
Light, which irradiates 5 min, does not influence the survival rate of cell.It is individually added into INPs and is maintained at 100 %, table without laser irradiation cell survival rate
Bright single polymer micelle for carrying photosensitizer has good biocompatibility.DINPs and FA-DINPs（10 µg/mL DOX）Change
After treatment, cell activity is substantially reduced, and activity is about 60.38 % and 47.76 % compared with laser control group, cell and only contains light
It is about 76.06 % that quick dose of nanoparticle FA-INPs is incubated and carries out the cell activity after laser irradiation altogether, illustrates independent thermotherapy pair
Cell has certain lethality.It is worth noting that, after DINPs and FA-DINPs chemotherapy while laser irradiation, cell activity drop
Down to 34.14 % and 12.15 %.The modification and laser irradiation synergy that these results suggest that folate ligand can significantly improve
Cytotoxicity of the polymer micelle to EMT-6.
Embodiment 5
Distribution of the EMT-6 breast cancer cells to the phagocytosis of FA-DINPs and DINPs and drug in the cell.
With 0.25 % trypsin digestion EMT-6 breast cancer cells, then with 10 % fetal calf serums without folic acid RPMI
1640 culture medium is made into individual cells suspension, with 5000, every hole cell inoculation in laser co-focusing ware（Mattek copolymerization is burnt
Culture vessel with glass bottom, diameter 35 mm, cover-glass thickness 0.085-0.13 mm）, per 1 mL of pore volume.It, will after culture 24 hours
Culture medium suction in laser co-focusing culture dish is abandoned, each culture dish be added the diluted polymer micelle of 2 mL culture mediums and
Free drug, drug concentration are 15 μ g/mL（Doxorubicin concentration）.
Cultivate 1.6 W/cm of FA-DINPs administration groups after 2 h2808 nm laser irradiations, 5 min after continue be incubated 2 h,
Remaining group is not added with laser and is incubated 2 h again.The culture solution containing nanoparticle or free drug is abandoned in suction, and cell 2 is washed with 1 mL PBS
It is secondary；With 1 mL of immunostaining fixer in the green skies（P0098）Room temperature fixes cell 20 minutes；It is washed with the immunostaining in the green skies
1 mL of liquid (P0106) is washed to wash 3 times, every time about 5 minutes；1 mL of DAPI in the green skies, 20 min of incubated at room temperature is added, uses
PBS is washed 3 times（Each 3-5 min）.It is observed with laser co-focusing after being eventually adding 600 μ L PBS.
The result shows that by incubation in 4 hours, the DOX fluorescence of free DOX, DINPs and FA-DINPs are present not only in carefully
Karyon, cytoplasm also appear on cell membrane.For DINPs and FA-DINPs, nanoparticle by phagocytosis enter cell after
DOX is released under the sensitive environment of cell reduction and enters nucleus, and the DOX not discharged also inside polymer micelle due to stopping
In cytoplasm, therefore DOX fluorescence appears in cell membrane, cytoplasm and nucleus.The machine that free adriamycin passes through diffusion
System rapidly enters nucleus and is inserted into the synthesis that DNA inhibits nucleic acid.And the DOX fluorescence occurred on cell membrane speculates that its reason may be
Since DOX is combined with electronegative cell membrane.Therefore when 4 h, the fluorescence intensity of DOX into core is higher than in free DOX groups
DINPs and FA-DINPs groups.But FA-DINPs groups show DOX fluorescence more stronger than DINPs groups, show the polymer of FA modifications
Micella can be combined with the EMT-6 breast cancer cells that folacin receptor height is expressed and enhance its endocytosis efficiency.ICG is mainly and cell
Interior albumen（Glutathione S-transferase）In conjunction with to be uniformly distributed in the cytoplasm of EMT-6 cells.With nanoparticle group phase
Than the fluorescence of ICG almost not being seen in ICG groups of dissociating, this is because the negative electricity of free ICG hinders it to enter cell；But ICG
It is contained into after nanoparticle, since cell enhances intracellular ICG amounts to the intake of DINPs and FA-DINPs, to make
ICG is uniformly distributed in cytoplasm.In addition under laser irradiation, intake of the cell to nanoparticle, while DINPs can be effectively improved
It can be broken to little particle and promote the release of drug so that DOX is easier to enter nucleus.These comprehensive factors, thermotherapy energy
Significantly improve intake of the EMT-6 cells to DOX and ICG.As a result see Fig. 5.
Embodiment 6
In-vivo imagings and bio distribution of the FA-DINPs and DINPs in lotus knurl BALB/C mice
The near-infrared fluorescent having using ICG and DOX itself, with small animal living body imager（Maestro™ 2 Maestro™
EX-RRO, U.S. CRi Maestro）The cancer target effect of observation reduction sensitive polymer micella collaboration drug delivery in vivo
It answers.Since the excitation wavelength (480 nm) of DOX is not suitable for living body fluorescent imaging, carried out in vitro with isolated viscus after dissection
DOX fluorescence signals detect its distribution in each internal organs.As shown in A in Fig. 6, fluorescence is predominantly located at liver and intestines after injecting 3 h
In, fluorescence starts to accumulate toward at tumour after injecting 8h, and free DOX＆ICG groups ratio DINPs and FA-DINPs groups are high, still
With the extension of administration time, free DOX＆ICG group fluorescence intensities are remarkably decreased, until do not observe drug fluorescence after the 6th day,
Speculate the reason is that free drug be easy in physiological environment assemble and be metabolized, make its fluorescence decrease fast in vivo.In injection 1
After it, the tumor locus fluorescence intensity of DINPs and FA-DINPs groups reaches maximum, hence it is evident that is better than free DOX＆ICG groups.This may
Being attributed to polymer micelle, circulation time extends in vivo, reduces the phagocytosis of RES, passes through EPR effects passive cogregation to tumour
Part.It was noted that FA-DINPs ratio DINPs groups have stronger fluorescence intensity, this is the active targeting function of folate ligand
Promote accumulation of the polymer micelle in tumor locus.Administration 6 days after DINPs and FA-DINPs groups tumor locus still keep compared with
Strong fluorescence intensity, and FA-DINPs is higher than DINPs.Fig. 6 C are BALB/C mice major organs and tumour after being administered 6 days
In vitro DOX fluorograms.For the mouse of free drug treatment group, tumor locus is almost without DOX fluorescence signals.In contrast,
The tumour portion DOX fluorescence of FA-DINPs and DINPs treatment groups is all very strong, and targeting group is obviously strong in the fluorescence intensity of tumor locus
In non-targeted group.Fig. 6 D are the in vitro DOX fluorograms semi-quantitative analyses of the BALB/C mice major organs and tumour after being administered 6 days
Figure.FA-DINPs significantly increases its accumulation within the tumor, mainly liver, kidney and tumour；Its fluorescence intensity ratio in tumour
DINPs is higher by 1.37 times.These results indicate that FA-DINPs has good internal stability, grain size smaller, it can be by EPR
Drug accumulation in effect and folacin receptor mediated endocytosis enhancing tumour.
Claims (9)
1. a kind of folate-targeted restores sensitive medicament-carried polymer nano micelle, it is with the PCL-ss- with reduction sensitivity characteristic
The function phosphatide DSPE-PEG-NH of PEG-ss-PCL polymer and active group2Pass through membrane-sonic method for raw material
It is carrier that the polymer nano micelle with reduction intelligent response is prepared in self assembly, and chemotherapeutics is contained with photosensitizer in glue
The hydrophobic cores of beam, shell are hydrophily PEG, have long circulating and spatial stability characteristic, folate-targeted group passes through covalent
Key is modified in micellar surface；It is characterized in that：
The PCL-ss-PEG-ss-PCL polymer that there is reduction sensitivity characteristic（Copolymerization）Structure it is as follows：
The molecular weight of polymer is 10000-24000, wherein PEG hydrophilic segments mass percent>45%, wherein m 114-
The molecular weight of 273, n 22-53, preferred polymers are 15000, functionalization phosphatide DSPE-PEG-NH2PEG molecular weight be
2000。
2. polymer micelle described in accordance with the claim 1, it is characterised in that the load medicine quality is 10%, chemotherapeutics with
The mass ratio of photosensitizer is 1:1.
3. polymer micelle described in accordance with the claim 1, it is characterised in that grain size is between 70-200 nm.
4. polymer micelle described in accordance with the claim 1, it is characterised in that the chemotherapeutics is adriamycin, soft red mould
Element, idarubicin, Aclarubicin, valrubicin, taxol or mitoxantrone or combination thereof；The photosensitizer is Yin
Diindyl cyanines are green（ICG）Or chlorin（Chlorin e6）.
5. polymer micelle described in accordance with the claim 1, it is characterised in that the function phosphatide DSPE-PEG-NH2Use Malaysia
The function phosphatide DSPE-PEG-Mal of imidizate is replaced, and then connects the monoclonal antibody anti-Her2-Fab, more of sulfhydrylation
Peptide LHRH, cell-penetrating peptide TAT are as targeting group.
6. polymer micelle described in accordance with the claim 1, it is characterised in that the PCL-ss-PEG-ss-PCL polymer is
PCL3750-ss-PEG7500-ss-PCL3750。
7. polymer micelle according to claim 6, it is characterised in that PCL3750-ss-PEG7500-ss-PCL3750Preparation
Method is as follows：
1）It weighs 1 g Py-ss-PEG7500-ss-Py and is dissolved in 12 mL dimethyl sulfoxide (DMSO)s（DMSO）In, it is to be dissolved fully after,
25 μ L mercaptoethanols, 20 μ L acetic acid are added；Magnetic agitation reacts 24 h under the conditions of 25 DEG C；Reaction is mixed after reaction
Object is transferred in the bag filter of MWCO8000-14000 Da, and is positioned in distilled water and is dialysed for 24 hours；It is freeze-dried, obtains later
Fine white powder product HO-ss-PEG7500-ss-OH；
2）Accurate 1 g 6-caprolactone monomers after purification, the 1 g HO-ss-PEG7500-ss-OH of being added is added in polymerization pipe
One drop stannous octoate is as catalyst；Reaction system is vacuumized into 5 min, 5 min of inflated with nitrogen, so in triplicate；Finally exist
Closing polymerization pipe under the protection of nitrogen, reacts 24 h in 100 DEG C of oil bath；After reaction, molten with a small amount of dichloromethane
Solution, is poured into later in 500 mL anhydrous ethers；Suction filtration takes precipitation to obtain pure white product；
3）It repeats purifying three times, is dried in vacuo under conditions of 35 DEG C and obtains pure white granular disintegration PCL3750-ss-PEG7500-
ss-PCL3750；Its reactional equation is as follows：
。
8. the preparation method of polymer micelle described in claim 1：It is characterized by comprising following steps：
1）It will be by metering by PCL-ss-PEG-ss-PCL, chemotherapeutics, photosensitizer, DSPE-PEG-NH2It is dissolved in dichloromethane or first
Alcohol and dichloromethane（Mass ratio 1:1）Uniform mixed solvent after, revolving remove organic solvent plastic film mulch, be dried in vacuo 16 hours；
2）With the phosphate buffer solution of pH7.4（PBS）Dispersion, 60-65 DEG C of aquation, are cooled to room temperature, ultrasonic under ice bath, obtain
It is positioned in the bag filter of MWCO8000-14000 Da and dialyses 4 hours to micella dispersion liquid, remove free drug, dialysed
The tumour target polymer micella dispersion liquid of the sensitive total load chemotherapeutics and photosensitizer of reduction afterwards；
3）Pass through EDC/NHS reaction forming folate-targeted molecules again；Specifically reaction step is：It is about DSPE- to weigh molar ratio
PEG-NH25 times of folic acid, be dissolved in deionized water；Addition molar ratio is about the EDC of 5 times of folic acid and molar ratio is 2 times of EDC
NHS activate 15 min；Above-mentioned amidized nanoparticle suspension is added, is reacted 3 hours under room temperature magnetic agitation.
9. the polymer micelle described in claim 1 to 6 any one is being prepared for chemotherapy of tumors and photo-thermal combination therapy medicine
Application in object.
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