CN104667292B - A kind of preparation and its application for reducing response type drug conjugates nanoparticle - Google Patents
A kind of preparation and its application for reducing response type drug conjugates nanoparticle Download PDFInfo
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Abstract
The present invention relates to a kind of preparation and its application for reducing response type drug conjugates nanoparticle, the drug conjugates include medication medication conjugate and pharmaceutical polypeptide conjugate, antineoplastic and drug molecule or polypeptide are coupled using reduction response type disulfide bond and nanoparticle is made as carrier material, nanoparticle particle diameter prepared by the present invention is in 100nm or so.It is an object of the invention to a part for small molecule free drug as carrier or carrier without site selective originally is prepared into the nanoparticle with certain particle diameter.The nanoparticle drugloading rate is high, and using passive target principle make drug accumulation tumor locus, using reduction sensitive disulfide bond selective degradation in the cell the characteristics of, it is ensured that medicine discharges completely, plays drug effect.
Description
Technical field
The present invention relates to a kind of preparation method of drug conjugates as the nanoparticle of carrier, and its in anti-tumor aspect
Using belonging to field of pharmaceutical preparations.
Background technology：
Malignant tumour is the significant threat of human life and health, is fatal rate highest disease.In the clinical treatment of tumour
In, chemotherapy is still one of Main Means.But, many chemotherapeutics exist selectivity it is low, easily by reticuloendothelial system
Remove, the problems such as fast, toxic side effect is big be metabolized in vivo, so as to limit clinical practice.
Anthracycline antibiotic is that the more faster series antineoplastic medicament of development is studied over nearly 30 years, and adriamycin is wherein most
One of conventional, most important antibiotic.Adriamycin is the zymotic fluid from streptomyces peucerius var.caesius
A kind of glycoside antibiotic of middle extraction, its activity is strong, and has a broad antifungal spectrum occupies critical role in oncotherapy.Adriamycin
Anticancer Effect and Mechanism mainly includes passing through in intercalation of DNA double-strand, forms stable compound, influences DNA function, prevents
DNA replication dna and rna transcription；Topo II function is influenceed, by being combined with-DNA the compounds of topo II, suppresses connecting again for DNA
Connect, so that inducing cell apoptosis.
Although adriamycin has stronger antitumor activity, adverse reaction is as serious caused by its nonspecific action
Cardiac toxic limits it in clinical extensive use, and liposome due to can solve to a certain extent with passive target effect
The problem of certainly selectivity is low, so as to reduce adverse reaction, improves curative effect.But this strategy still suffers from certain defect：Lipid
Body can be only achieved drug effect to the drugloading rate relatively low (being less than 10%) of adriamycin, it is necessary to increase consumption, but while increase consumption
Toxic side effect can be increased again, therefore for existing targeting, the demand of drugloading rate high delivery system again is particularly urgent.
Medicine-cell-penetrating peptide conjugate has turned into study hotspot in recent years as prodrug.Cell-penetrating peptide is that length is less than 20
The peptide sequence of amino acid, the ability with penetration cell film, cell-penetrating peptide transmembrane mechanism may have electrostatic interaction, hydrogen bond action or
Hydrophobic effect.The antitumor action of medicine is combined by medicine-cell-penetrating peptide conjugate with the membrane penetration effect of cell-penetrating peptide, improves tumour
Intake of the cell to medicine, increases drug effect, but is due to that cell-penetrating peptide does not have specificity, has to tumour cell and normal cell
Membrane penetration effect, therefore conjugate also has lethal effect while killing tumor cell to normal cell.If but being prepared into
Nanoparticle, then can because it is enhanced infiltration and be detained (EPR) effect tumor tissues are played passive target effect, specific enrichment
To tumor locus, its membrane penetration effect and CDCC are played.In addition, this delivery system also has a maximum advantage to be,
Due to a part of the medicine as carrier, drugloading rate can be increased, toxic side effect is reduced, strengthen drug effect.There is research will recently
Drug conjugates prepare nanoparticle as carrier, and this more considerably increases drugloading rate, carried while passive target effect is kept
The drug concentration of high tumor locus, so as to increase intake, improves drug effect, in addition, drug conjugates also avoid because synthesis is carried
Body material degradation in vivo produces the possibility of toxicity.
Because carrier is drug conjugates or pharmaceutical polypeptide conjugate, thus after nanoparticle reaches tumour cell, it is necessary to
By the insoluble drug release competence exertion effect in carrier, this just proposes requirement to the connecting key between medicine and medicine or polypeptide：
It can keep stable in blood circulation, but can be broken in inside tumor cells, discharge medicine.Research discovery, zooblast
It is inside and outside to there is oxidation-reduction potential：Intracellular glutathione (GSH) concentration (2~10mmolL-1) be EC (2~
10μmol·L-1) more than 200 times.There is also reducing environment in endosome, lysosome sulfydryl is mainly induced also by IFN-γ
Protoenzyme (GILT) and reducing agent (such as cysteine) are adjusted jointly；Contain low price Fe in lysosome2+With the mercaptan (such as half of high concentration
Cystine), make that also there is reducing environment in lysosome.In addition, tumor tissue cell is than normal tissue cell anoxic, with more also
Originality environment.Using this point, we select the disulfide bond with reduction-sensitive that medicine is connected into obtained with medicine or polypeptide
Medicine-disulfide bond-medicine or polypeptide-conjugate, and it is prepared into nanoparticle.Because the environment in extracellular and blood circulation is not enough
So that disulfide bonds, therefore carrier can keep stable in extracellular and blood circulation, and intracellular reducing environment can be fast
Speed is degraded to sulfydryl, causes nanoparticle to reach after target cell, disulfide bond is broken, and discharges free drug, plays medicine
Effect.
Based on background above, antineoplastic is worn with antineoplastic or cell with the disulfide bond sensitive to reducing environment
Film peptide is coupled as carrier material, is prepared into nanoparticle, has both been remained the passive target advantage of nano-delivery system, added again
Drugloading rate, while also having played disulfide bond the characteristics of tumor locus selective degradation, can reduce toxic side effect, improve and treat
Effect.
The content of the invention
An object of the present invention is to provide a kind of antineoplastic and is coupled the reduction response type to be formed with medicine or polypeptide
Conjugate nanoparticle, realizes and improves drugloading rate, cancer target, reduces the effect of toxicity.
Drug conjugates of the present invention, pass through the company containing disulfide bond between medicine and medicine or medicine and polypeptide
Connect key coupling to obtain, the formula of the drug conjugates is as follows：
Medicine-connecting key-medicine or polypeptide,
Wherein, the medicine is the antineoplastic of hydroxyl or amino structure group.
Wherein, the connecting key is the small molecule linking arm containing disulfide bond.
Wherein, the polypeptide, is to contain cysteine in cell-penetrating peptide, and sequence, mean molecule quantity is 800~5000 roads
Er Dun.
The drug conjugates of the present invention, wherein containing hydroxyl or amino in described anti-tumor medicament structure.
The drug conjugates of the present invention, wherein described antineoplastic is selected from：Taxol, Docetaxel, adriamycin.
The drug conjugates of the present invention, wherein containing disulfide bond in the small molecule linking arm.
The present invention drug conjugates, wherein described polypeptide be cell-penetrating peptide, and sequence in contain cysteine.
Be prepared into the drug conjugates of the present invention, preferably nanoparticulate form, preparation process particle diameter 80~
200nm nanoparticle.
It is preferred that, described drug conjugates of the invention are selected from：Adriamycin-connecting key-adriamycin, adriamycin-connection
Key-hydrophobic peptide, taxol-connecting key-taxol, taxol-connecting key-hydrophobic peptide, Docetaxel-connecting key-polyenoid are purple
China fir alcohol, Docetaxel-connecting key-hydrophobic peptide.
Most preferably, conjugate of the present invention is selected from：DOX-SS-DOX、PFV-SS-DOX.
The second object of the present invention is to provide the preparation method of said medicine conjugate.
The preparation method of drug conjugates of the present invention, including
(1) medicine-connecting key-medicines structure is the conjugate of representative, hereinafter referred to as drug-drug conjugate；
(2) medicine-connecting key-polypeptide structure is the conjugate of representative, hereinafter referred to as medicine-polypeptide-conjugate.
The preparation method of drug-drug conjugate, comprises the following steps：
Medicine and crosslinking agent 3,3'- dithiodipropionic acids-two (N- hydroxysuccinimides ester) reaction, through ether place of settling
Reason obtains drug-drug conjugate.
Optimization, the preparation method of drug-drug conjugate comprises the following steps：
In the presence of organic amine catalyst, in dimethylformamide, under nitrogen protection, by medicine and containing disulfide bond
Crosslinking agent 3,3'- dithiodipropionic acids-two (N- hydroxysuccinimides ester) press 1-4:1 mol ratio is esterified or acylated anti-
Should, drug-drug conjugate is obtained after ether precipitation process.
Wherein, medicine and the crosslinking agent 3 containing disulfide bond, 3'- dithiodipropionic acids-two (N- hydroxysuccinimides ester) are pressed
2.1:1 mol ratio is esterified or acylation reaction.
Wherein, the medicine is selected from：The antineoplastics such as taxol, Docetaxel, adriamycin.
It is further preferred that the synthesis of adriamycin-connecting key-adriamycin, comprises the following steps：
DOXHCl (60.9mg, 0.105mmol) is dissolved in 3mL DMF, the DSP in 0.5mL DMF will be dissolved in
(20.2mg, 0.05mmol) is added dropwise in DOX, is subsequently added catalyst TEA (14.6 μ L), room temperature lucifuge reaction 30min
Afterwards, ice bath is stayed overnight, and transfers them within second day precipitation to be precipitated in ice ether, and suction filtration obtains product for Orange red solid powder.
The preparation method of medicine-polypeptide-conjugate, comprises the following steps：
The pyridine of (1) two sulphur two and mercaptopropionic acid are reacted in ethanol/acetum, and Py-SS- is obtained after crossing post separation processing
COOH。
(2) Py-SS-COOH is activated using activator HBTU, carboxyl and drug response after activation are obtained by post separation
To Py-SS- medicines.
(3) Py-SS- medicines and polypeptide containing sulfydryl pass through disulfide bond exchange reaction, obtained after dialysis frozen dried medicine-
Polypeptide-conjugate.
Optimization, the preparation method of medicine-polypeptide-conjugate comprises the following steps：
(1) 1 is pressed:1.5~1:3 molar ratio, be stirred vigorously it is lower by thiolic acid be added dropwise the ethanol of the pyridine of two sulphur two/
In acetum, 2~3h is reacted, the pyridine acid containing disulfide bond is obtained after being handled through neutral alundum (Al2O3) post separation.
(2) HBTU is activated after the carboxyl in the pyridine acid containing disulfide bond, by 1:1~1:1.2 molar ratio, nitrogen protection
It is lower that the antineoplastic containing amino is added dropwise in above-mentioned reaction solution, 5~7h is reacted, contains two through silicagel column is isolated
The medicaments derivative of sulfide linkage.
(3) under nitrogen protection, medicaments derivative and the polypeptide containing sulfydryl are pressed 2:1 mol ratio carries out disulfide bond exchange
Reaction, through dialysis, medicine-polypeptide-conjugate is obtained after frozen dried.
It is further preferred that the synthesis of hydrophobic peptide-connecting key-adriamycin, comprises the following steps：
(1) Py-SS-COOH preparation：
The pyridine of two sulphur two (Py-SS-Py, 2g, 9.06mmol) is dissolved in 16mL ethanol, glacial acetic acid (AcOH) is added
213 μ L, with vigorous stirring, the ethanol solution of 10.6mL 3- mercaptopropionic acids (0.48g, 4.53mmol) is added dropwise.Reaction
After 2h, ethanol is removed under reduced pressure, through neutral alumina post separation, concentration adds cold water and separates out solid, suction filtration is dried in vacuum overnight,
It is white solid to obtain product,
(2) Py-SS-DOX preparation：
Py-SS-COOH (29.1mg, 0.135mmol) is dissolved in 1mL dry DMFs, addition HBTU (102.6mg,
0.27mmol) activate 30min.DOXHCl (93.96mg, 0.162mmol) is added, DIEA (71.lmg, 0.43mmol) uses pH
Test paper determines its pH for 9-10, reacts at room temperature 3h.Product is concentrated to give product for Orange red solid powder through silica gel post separation.
(3) PFV-SS-DOX preparation：
Py-SS-DOX (38.5mg, 0.052mmol) is dissolved in 0.5mL DMF, is stirred vigorously down and PFV is added dropwise
The DMF solution of (25mg, 0.026mmol).Room temperature lucifuge reacts 48h.Reaction is dialysed after terminating, and is freezed, and obtains product solid for red
Body.
Another object of the present invention is to provide a kind of nanoparticle of drug conjugates formation, even with medicine of the present invention
Nanoparticle is made as carrier in connection.
The preparation method of nanoparticle of the present invention, step is as follows：Drug conjugates are dissolved in DMSO, are made into organic
Phase solution, takes and is scattered in right amount in aqueous phase solution, and Probe Ultrasonic Searching obtains the nanoparticle solution of uniform particle diameter.Its particle diameter 80~
200nm.Granularity and the distribution that nanoparticle is made are determined using dynamic light scattering.
Above involved english abbreviation is explained：
DOX：Adriamycin.
Py-SS-COOH：3- (2- pyridyidithios) propionic acid
Py-SS-DOX：Pyridyldithiol adriamycin
HBTU：BTA-N, N, N' ', N '-tetramethylurea hexafluorophosphoric acid ester
DMSO：Dimethyl sulfoxide (DMSO)
DIEA：Diisopropylethylamine
PFV：Small molecule hydrophobic peptide PFVYLIGC
TEA：Triethylamine
DSP：3,3'- dithiodipropionic acids-two (N- hydroxysuccinimides ester)
DOX·HCl：Doxorubicin hydrochloride
DMF：Dimethylformamide
The three of the object of the invention are to provide a kind of pharmaceutical composition containing drug conjugates.
Pharmaceutical composition of the present invention is if necessary containing conventional pharmaceutic adjuvant, and appropriate formulation is made
The pharmaceutical composition of the present invention can be prepared into any pharmaceutically useful formulation, and these formulations include：Tablet, sugar coated tablet
Agent, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, mouth containing agent, granule, electuary,
Pill, powder, paste, sublimed preparation, supensoid agent, pulvis, solution, injection, suppository, ointment, emplastrum, creme, spray,
Drops, patch.The preparation of the present invention, it is preferred that such as liquid drugs injection, freeze dried powder, tablet and capsule.The administration of the preparation
Mode includes intravenous injection, intra-tumoral injection and oral administration.It can be applied to oncotherapy.
The pharmaceutical composition of the present invention, its preparation being administered orally can contain conventional excipient, such as adhesive, filling
Agent, diluent, tablet agent, lubricant, disintegrant, colouring agent, flavor enhancement and wetting agent, can be coated to tablet if necessary.
Applicable filler includes cellulose, mannitol, lactose and other similar fillers.Suitable disintegrant bag
Include starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycollate.Suitable lubricant includes, for example firmly
Fatty acid magnesium.Suitable pharmaceutically acceptable wetting agent includes lauryl sodium sulfate.
It can be filled by mixing, the conventional method such as tabletting prepares solid oral composition.Work can be made by carrying out mixing repeatedly
Property material be distributed in entirely using a large amount of fillers those compositions in.
The form of oral liquid for example can be aqueous or oily suspensions, solution, emulsion, syrup or elixir,
Or can be a kind of dry products that can be compounded before use with water or other suitable carriers.This liquid preparation can contain
Conventional additive, such as suspending agent, such as sorbierite, syrup, methylcellulose, gelatin, hydroxyethyl cellulose, carboxymethyl are fine
Dimension element, aluminium stearate gel or hydrogenated edible fats, emulsifying agent, such as lecithin, anhydro sorbitol monooleate or Arab
Glue；Non-aqueous carrier (they can include edible oil), such as apricot kernel oil, fractionated coconut oil, the oily ester of the ester of such as glycerine,
Propane diols or ethanol；Preservative, such as para hydroxybenzene methyl esters or propylparaben or sorbic acid, and if desired,
Conventional flavouring agent or colouring agent can be contained.
For injection, the fluid unit dosage form of preparation contains the active material and sterile carrier of the present invention.According to carrier
And concentration, this compound can be suspended or be dissolved.The preparation of solution is dissolved in a kind of load typically by by active material
In body, sterilization is filtered before a kind of suitable bottle or ampoule is loaded into, is then sealed.For example a kind of local anaesthesia of auxiliary material
Agent, preservative and buffer can also be dissolved in this carrier., can be after bottle be loaded by this in order to improve its stability
Composition frost is planted, and under vacuo removes water.
The pharmaceutical composition of the present invention, suitable pharmaceutically acceptable load is optionally added when being prepared into medicament
Body, the pharmaceutically acceptable carrier is selected from：Mannitol, sorbierite, sodium pyrosulfite, sodium hydrogensulfite, sodium thiosulfate, salt
Sour cysteine, TGA, methionine, injection Vitamin B_6 DTA disodiums, Ethylenediaminetetraacetic Acid Calcium Salt, carbonate, the acetic acid of monovalence alkali metal
Salt, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, wheat
Bud sugar, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and its
Derivative, alginates, gelatin, polyvinylpyrrolidone, glycerine, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surface-active
Agent, polyethylene glycol, cyclodextrin, beta-schardinger dextrin, phospholipid material, kaolin, talcum powder, calcium stearate, magnesium stearate etc..
The drug conjugates or nanoparticle that the fourth object of the present invention is to provide the present invention are preparing anti-tumor drug
In application.
Drug conjugates or medicament nano granule of the present invention are as pharmaceutical carrier, in antineoplastic is prepared
Using.
The conjugate of the present invention is by passive target, fixed-point drug releasing and reduces the advantage of toxicity, realizes treatment tumour
Purpose.
Application of the present invention, is the disulfide bond by drug conjugates under human body cell glutathion inside reduction
It is broken, medicine therefrom discharges, assigns antineoplastic reduction response, controllable, the toxicity reduction that releases the drug and realize antitumous effect
's.
The sign of response type drug conjugates nanoparticle is reduced in the present invention：By dynamic light scattering to its particle diameter and distribution
Investigated；The form of conjugate nanoparticle is intuitively observed by transmission electron microscope；On cellular level, by investigating cell
The inhibiting rate of intake, conjugate nanoparticle cell growth to conjugate nanoparticle evaluates drug effect；In animal level, pass through idol
Its drug effect is evaluated in terms of inhibiting rate of the connection thing nanoparticle on tumour growth, the influence of the changes of weight on animal.
The advantage of the invention is that：
1st, medicine is connected and obtains prodrugs by the present invention with drug molecule or polypeptide by disulfide bond, and can be in water
Nanoparticle is formed, passive target advantage is played using EPR effects, drug effect is improved, toxicity is reduced.
2nd, the present invention increases drugloading rate using antineoplastic as a part for nanoparticulate carriers or carrier, increases medicine
While effect, dosage is reduced, so as to reduce toxic side effect.
3rd, present invention employs the thinking of tumor microenvironment target administration, introduced in drug conjugates containing the small of disulfide bond
Molecule linking arm.Disulfide bond is sensitive to reducing environment, can be degraded in the distinctive reproducibility environment of tumor tissues, quick release goes out
Medicine, so as to play specific antitumous effect.
4th, drug conjugates of the invention, are shown by cell experiment and zoopery, can improve the treatment of antineoplastic
Effect, reduces toxicity, with preferable clinical value.
Brief description of the drawings：
The DOX-SS-DOX synthesized in Fig. 1, embodiment 1 TOF MS ESI mass spectrograms
The DOX-SS-DOX synthesized in Fig. 2, embodiment 1 HPLC collection of illustrative plates
The Py-SS-COOH's synthesized in Fig. 3, embodiment 21H NMR scheme
The Py-SS-COOH synthesized in Fig. 4, embodiment 2 TOF MS ESI mass spectrograms
The Py-SS-DOX's synthesized in Fig. 5, embodiment 21H NMR mass spectrograms
The Py-SS-DOX synthesized in Fig. 6, embodiment 2 HPLC collection of illustrative plates
The Py-SS-DOX synthesized in Fig. 7, embodiment 2 TOF MS ESI mass spectrograms
The Py-SS-DOX synthesized in Fig. 8, embodiment 2 FTIR collection of illustrative plates
The PFV-SS-DOX synthesized in Fig. 9, embodiment 2 HPLC collection of illustrative plates
The grain size distribution of the DOX-SS-DOX nanoparticles prepared in Figure 10, embodiment 3
The TEM figures of the DOX-SS-DOX nanoparticles prepared in Figure 11, embodiment 3
The grain size distribution of the PFV-SS-DOX nanoparticles prepared in Figure 12, embodiment 3
Figure 13, free DOX, DOX liposome and DOX-SS-DOX nanoparticles streaming intake are tested
Figure 14, free DOX, DOX liposome and DOX-SS-DOX nanoparticles vitro cytotoxicity
Figure 15, DOX liposome and DOX-SS-DOX nanoparticles are through the pretreated vitro cytotoxicities of GSH-OEt
Figure 16, the DOX-SS-DOX grains and free DOX, DOX liposome of the present invention antitumor comparative study of pharmacodynamics.A：
Tumor growth curve；B：Tumor weight；C：Nude mice body weight change curve after administration；D：Tumor size figure
The freeze-dried granularity of Figure 17, DOX-SS-DOX nanoparticle and distribution
Embodiment
The present invention is further illustrated and explains by the following examples, but not as the limitation that the present invention is carried out.
The synthesis of embodiment 1, adriamycin-disulfide bond-adriamycin
DOXHCl (60.9mg, 0.105mmol) is dissolved in 3mL DMF, the DSP in 0.5mL DMF will be dissolved in
(20.2,0.05mmol) are added dropwise in DOX, are subsequently added the TEA (14.6 μ L) of catalytic amount, the reaction of room temperature lucifuge
After 30min, ice bath is stayed overnight, and transfers them within second day precipitation to be precipitated in ice ether, and suction filtration obtains product for Orange red solid powder
End.Yield is 76.7%.Mass spectrum, efficient liquid phase detection, as a result such as Fig. 1 and Fig. 2.
The synthesis of embodiment 2, hydrophobic peptide (PFV)-disulfide bond-adriamycin
Py-SS-COOH preparation：
The pyridine of two sulphur two (Py-SS-Py, 2g, 9.06mmol) is dissolved in 16mL ethanol, glacial acetic acid (AcOH) is added
213 μ L, with vigorous stirring, the ethanol solution of 10.6mL 3- mercaptopropionic acids (0.48g, 4.53mmol) is added dropwise.Reaction
After 2h, ethanol is removed under reduced pressure, through neutral alumina post separation, concentration adds cold water and separates out solid, suction filtration is dried in vacuum overnight,
It is white solid to obtain product, and yield is 44.68%.Nuclear-magnetism, Mass Spectrometer Method, as a result such as Fig. 3 and Fig. 4.
Py-SS-DOX preparation：
Py-SS-COOH (29.1mg, 0.135mmol) is dissolved in 1mL dry DMFs, addition HBTU (102.6mg,
0.27mmol) activate 30min.DOXHCl (93.96mg, 0.162mmol) is added, DIEA (71.l, 0.43mmol) is tried with pH
Paper determines its pH for 9-10, reacts at room temperature 3h.Product is concentrated to give product for Orange red solid powder through silica gel post separation.Yield
For 29.9%.Nuclear-magnetism, efficient liquid phase, mass spectrum, infrared detection, as a result such as Fig. 5, Fig. 6, Fig. 7 and Fig. 8.
PFV-SS-DOX preparation：
Py-SS-DOX (38.5mg, 0.052mmol) is dissolved in 0.5mL DMF, is stirred vigorously down and PFV is added dropwise
The DMF solution of (25mg, 0.026mmol).Room temperature lucifuge reacts 48h.Reaction is dialysed (DMSO → water) after terminating, and is freezed, must be produced
Thing is red solid, and yield is 18.6%.Efficient liquid phase is detected, as a result such as Fig. 9.
Embodiment 3, the preparation of reduction response type nano grain and sign
The preparation of DOX-SS-DOX nanoparticles and sign：Appropriate amount of drug conjugate is weighed, the DMSO for being configured to 5mg/mL is molten
Liquid takes 50 μ L mother liquors to be added in the 1950 μ L aqueous solution as mother liquor, Probe Ultrasonic Searching 10min (work 2s, interval 2s, power
200w)。
Granularity and the distribution that nanoparticle is made are determined using dynamic light scattering.Observed using transmission electron microscope (TEM)
The form of nanoparticle.Its nanoparticle size distribution is shown in Figure 10.Its TEM result is shown in Figure 11.
The preparation of PFV-SS-DOX nanoparticles and sign：Appropriate amount of drug conjugate is weighed, the DMSO for being configured to 20mg/mL is molten
Liquid takes 50 μ L mother liquors to be added in the 1950 μ L aqueous solution as mother liquor, visits super 10min (work 2s, interval 2s, power 200w).
Granularity and the distribution that nanoparticle is made are determined using dynamic light scattering (DLS).As a result such as Figure 12.
The streaming intake experiment of embodiment 4, reduction response type nano grain
Intake of the MCF-7 cells to conjugate nanoparticle is quantitative determined using Fluorescein activated cell sorter.MCF-7 with
2.5×105The density in individual/hole is inoculated in 12 orifice plates, and cell attachment grows into individual layer after 24h.Original fluid is abandoned in suction, with without blood
Clear nutrient solution rinsing.DOX solution, DOX liposomes or the DOX-SS-DOX nanoparticles that addition serum-free medium dilutes (contain 20
μ g/mL DOX) each 1mL, puts 37 DEG C of incubators and is incubated 3h.After incubation terminates, washed 2-3 times with the PBS of precooling, per hole 300mL
0.25% trypsin digestion and cell, is terminated with 1mL serum-containing mediums and digested, and is neutralized with precooling PBS and is blown and beaten into cell and hanged
Liquid, centrifuges 5min in 1000rpm, is rinsed with the PBS of 350 μ L precoolings and cell is resuspended, and blows and beats into single cell suspension, crosses cell
Sieve.With the DOX fluorescence intensities of flow cytometer measure cellular uptake, (excitation wavelength of measure is 480nm, determines wavelength and is
560nm).Analysis cell number used is no less than 10 every time5Individual, the cell number of collection is 10,000.Data use FCS
Express V3 softwares are analyzed.
As a result as shown in figure 13, the intake of DOX-SS-DOX nanoparticles is significantly larger than the intake of DOX liposomes, this be by
It is big in the drugloading rate of DOX-SS-DOX nanoparticles, therefore add the intake of cell.
The vitro cytotoxicity experiment of embodiment 5, reduction response type nano grain
The cytotoxic activity of nanoparticle is investigated using SRB (Sulforhodamine B) method.By the MCF-7 cells of exponential phase
It is inoculated in 96 porocyte culture plates, per about 5000, hole cell, if 6 multiple holes, are placed in CO2gas incubator, 37
DEG C, be incubated 24h under conditions of 5%CO2, treat that cell attachment grows.Series concentration is prepared with the nutrient solution dilution containing serum
DOX, DOX liposome and DOX-SS-DOX nanoparticles solution (0.0625,0.125,0.25,0.5,1,2.5,5,10,20 μ g/mL,
Administration concentration is in terms of DOX).The drug solution diluted is added separately in above-mentioned cell culture well, six blank ginsengs are separately set
Control is used as than hole.The CO2gas incubator that Tissue Culture Plate is placed in into 37 DEG C is incubated 48h.After incubation terminates, pastille is discarded
Nutrient solution, 200 μ L 10% trichloroacetic acid is added per hole, stands 5min, is moved into 4 DEG C of refrigerators and is stood more than 1 hour, takes out and use
Deionization is washed 5 times, is dried 5 hours in air-dried overnight or 30 DEG C of baking ovens, after being completely dried, and 1% vinegar is added per hole
The μ L of 0.4%SRB 100 that acid is prepared, room temperature dyeing 30min, outwell dyeing liquor, uncombined dyestuff are washed away with 1% acetic acid, empty
Dried in gas, dissolved with the pH10.5 10mM μ L of Tris alkali lye 200, vibrate 30min, determined on ELIASA at 540nm
Absorbance.
As a result as shown in figure 14, cytotoxicity power is free DOX ＞ DOX liposome ＞ DOX-SS-DOX nanoparticles.This
It is opposite with intake result, it may be possible to because the fracture of disulfide bond is relatively slow caused.DOX-SS-DOX nanoparticles are needed in reduction ring
Disulfide bond is disconnected under border and discharges free adriamycin competence exertion effect, and the fracture of disulfide bond is reduction dependence process
It is time dependence process, i.e. reduction degree increase breaking degree is bigger, and cytotoxicity is more obvious, and the time is longer, and breaking degree is got over
Greatly, cytotoxicity is more obvious.In incubation time, not fully, therefore its cytotoxic effect is not pre- for the fracture of disulfide bond
Phase it is so obvious.
The reduction response of embodiment 6, the vitro cytotoxicity of reduction response type nano grain
The cytotoxic activity of nanoparticle is investigated using SRB (Sulforhodamine B) method.By the MCF-7 cells of exponential phase
It is inoculated in 96 porocyte culture plates, per about 5000, hole cell, if 6 multiple holes, are placed in CO2gas incubator, 37
DEG C, 5%CO2Under conditions of be incubated 24h, treat cell attachment grow.Original fluid is abandoned in suction, is added 10mM GSH-OEt and is incubated 2h.
GSH-OEt is sucked, PBS is washed.DOX, DOX liposome and DOX-SS-DOX of series concentration are prepared with the nutrient solution dilution containing serum
Nanoparticle solution (0.25,1,2.5,5 μ g/mL, administration concentration is in terms of DOX).The drug solution diluted is added separately to
State in cell culture well, separately set six blank reference holes as control.Tissue Culture Plate is placed in 37 DEG C of carbon dioxide culture
Case is incubated 48h.After incubation terminates, pastille nutrient solution is discarded, 200 μ L 10% trichloroacetic acid is added per hole, 5min is stood, moved
Enter 4 DEG C of refrigerators and stand more than 1 hour, taking-up is washed with deionized water 5 times, 5 are dried in air-dried overnight or 30 DEG C of baking ovens small
When, after being completely dried, the μ L of 0.4%SRB 100 that 1% acetic acid is prepared, room temperature dyeing 30min are added per hole, dyeing liquor is outwelled,
Uncombined dyestuff is washed away with 1% acetic acid, air drying is dissolved, vibration with the pH10.5 10mM μ L of Tris alkali lye 200
30min, determines the absorbance at 540nm on ELIASA.
As a result Figure 15 is seen, GSH-OEt is incubated in advance to be increased after intracellular reduction degree, the cell of DOX-SS-DOX nanoparticles
Toxicity is dramatically increased, and the cytotoxicity to DOX liposomes does not have remarkable effect.This result explanation, DOX-SS-DOX nanometers
Small toxicity of the toxicity than DOX liposome of grain, is strictly that, because disulfide bonds are not exclusively caused, increase reduction degree accelerates two
The fracture of sulfide linkage, can increase the cytotoxicity of nanoparticle.
Embodiment 7, antitumor activity experiment
From MCF-7 cells, it is inoculated with the right side oxter of female nu/nu nude mices, every mouse inoculation 106Individual cell,
Inoculation is administered after 7 days.Administration group is 4 groups：PBS, DOX, DOX liposome, DOX-SS-DOX nanoparticles or PFV-SS-DOX nanometers
Grain, every group 5.DOX contents are 0.2mg/mL in drug solution, altogether administration 4 times, every time 200 μ L, and delivery time is 1
My god, every other day survey nude mice body weight and knurl volume.Put to death in the 12nd day after administration, take tumor tissue, weigh, take pictures.
As shown in figure 16, the antitumous effect that DOX-SS-DOX nanoparticles are produced is significantly larger than with the free DOX under dosage
With DOX liposomes, and its toxicity is relatively low compared with free DOX and DOX liposomes.Internal pharmacodynamic results and cell in vitro poison
As a result on the contrary, this is probably because internal intracellular reducing degree is higher, quickly the nanometer for arriving cell interior will can be absorbed
Disulfide bond in grain disconnects, and discharges medicine, plays drug effect.
In addition, the present invention is also by DOX-SS-DOX nanoparticles and PFV-SS-DOX nanoparticles, with PEG-SS-DOX nanoparticles
The comparison of antitumor activity is carried out, is as a result shown, the medicine of the invention effect in terms of antitumor activity is more preferable.
Embodiment 8, freeze-dried preparation (by taking DOX-SS-DOX nanoparticles as an example)
Drug conjugates are dissolved in DMSO, certain density organic phase solution is made into, take that appropriate to be scattered in aqueous phase molten
In liquid, Probe Ultrasonic Searching obtains the nanoparticle solution of uniform particle diameter.10% trehalose is added into nanoparticle solution, freeze-drying
It is freeze-dried.Freeze-dried distilled water redissolves, and determines with dynamic light scattering its granularity and distribution.As a result such as Figure 17.
Claims (4)
1. a kind of nanoparticle of drug conjugates formation, it is characterised in that nanoparticle is made using conjugate as carrier,
Wherein, the drug conjugates：Polypeptide-connecting key-adriamycin, adriamycin-connecting key-adriamycin,
Wherein, the connecting key is the small molecule linking arm containing disulfide bond,
Wherein, the polypeptide is contains cysteine in cell-penetrating peptide, and sequence, and mean molecule quantity is 800~5000 dalton.
2. nanoparticle as claimed in claim 1, it is characterised in that the preparation method of nanoparticle is as follows：
Drug conjugates are dissolved in DMSO, organic phase solution is made into, takes and is scattered in right amount in aqueous phase solution, Probe Ultrasonic Searching is obtained
To the nanoparticle solution of uniform particle diameter, its particle diameter is in 80~200nm.
3. the application using the nanoparticle described in claim 1 as pharmaceutical carrier in anti-tumor drug is prepared.
4. nanoparticle as claimed in claim 1, it is characterised in that the polypeptide is swum to contain in cell-penetrating peptide, and sequence
From sulfydryl.
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| CN105251013B (en) * | 2015-09-28 | 2018-08-14 | 湘潭大学 | One kind having degradable water-soluble antitumor polymeric prodrugs of redox response and preparation method thereof |
| CN105560178B (en) * | 2015-12-30 | 2019-02-01 | 浙江工业大学 | A kind of reduction responsive type polypeptide prodrug nano-micelle and its preparation and application |
| CN106420664A (en) * | 2016-11-03 | 2017-02-22 | 福州大学 | Application of adopting aspirin conjugate with antitumor activity as drug carrier or molecular probe carrier |
| CN107735108A (en) * | 2016-12-06 | 2018-02-23 | 江苏大学 | A kind of PEGylation vitamin E periplocymarin conjugate nanoparticle and its preparation method and purposes |
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