CN104667292A - Preparation of reducing response type drug conjugate nanoparticle and application of reducing response type drug conjugate nanoparticle - Google Patents
Preparation of reducing response type drug conjugate nanoparticle and application of reducing response type drug conjugate nanoparticle Download PDFInfo
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Abstract
The invention relates to preparation of a reducing response type drug conjugate nanoparticle and an application of the reducing response type drug conjugate nanoparticle. The drug conjugate comprises a drug-drug conjugate and a drug-polypeptide conjugate; antitumor drugs and a medicine molecule or polypeptide are coupled as a carrier material by virtue of a reducing response type disulfide bond to prepare the nanoparticle; and the particle size of the prepared nanoparticle is about 100nm. The invention aims at preparing the nanoparticle with a certain particle size from an original micromolecular free drug without part selectivity as a carrier or one part of the carrier. The nanoparticle is high in drug loading capacity; full release of the drug is ensured on the basis of the characteristics that the drug is accumulated in the tumor part and is specifically degraded in the cell by virtue of the reducing sensitive disulfide bond by the passive targeting principle; and the drug effect is put into a full play.
Description
Technical field
The present invention relates to the preparation method of a kind of drug conjugates as the nanoparticle of carrier, and in the application of anti-tumor aspect, belong to field of pharmaceutical preparations.
Background technology:
Malignant tumor is the significant threat of human life's health, is the highest disease of fatality rate.In the clinical treatment of tumor, chemotherapy is still one of Main Means.But, many chemotherapeutics also exist selectivity low, easily removed by reticuloendothelial system, the problem such as internal metabolism is fast, toxic and side effects is large, thus limit clinical practice.
Anthracycline antibiotics studies a more development series antineoplastic medicament faster over nearly 30 years, and amycin is wherein one of the most frequently used, most important antibiotic.Amycin is the one sugar tobramycin antibiotic extracted from the fermentation liquid of streptomyces peucerius var.caesius, and it is active strong, and has a broad antifungal spectrum, occupies critical role in oncotherapy.The Anticancer Effect and Mechanism of amycin mainly comprises by intercalation of DNA double-strand, forms stable complex, affects the function of DNA, stops DNA replication dna and rna transcription; Affecting the function of topo II, by being combined with topo II-DNA complex, suppressing reconnecting of DNA, thus cell death inducing.
Although amycin has stronger anti-tumor activity, but the cardiac toxicity of the untoward reaction that its nonspecific action causes as serious limits it in clinical extensive use, liposome can solve the low problem of selectivity to a certain extent owing to having passive target effect, thus reduction untoward reaction, improve curative effect.But still there is certain defect in this strategy: the drug loading of liposome to amycin lower (being less than 10%), need to increase consumption and just can reach drug effect, but can toxic and side effects be increased again while increasing consumption, therefore for existing targeting, the demand of the delivery system that drug loading is high is again particularly urgent.
Medicine-wear film peptide conjugate has become study hotspot in recent years as prodrug.Wearing film peptide is that length is less than 20 amino acid whose peptide sequences, and have the ability of permeates cell membranes, wearing film peptide transmembrane mechanism may have electrostatic interaction, hydrogen bond action or hydrophobic interaction.Medicine-wear film peptide conjugate the antitumor action of medicine is combined with the membrane penetration effect wearing film peptide, improve tumor cell to the picked-up of medicine, increase drug effect, but not there is specificity owing to wearing film peptide, all have membrane penetration effect to tumor cell and normal cell, therefore conjugate also has lethal effect to normal cell while killing tumor cell.If but be prepared into nanoparticle, then can play passive target effect because of the infiltration that strengthens and delay (EPR) effect to tumor tissues, specificly be enriched to tumor locus, play its membrane penetration effect and cytotoxicity.In addition, this drug-supplying system also has a maximum advantage to be, because medicine is as a part for carrier, can increase drug loading, reduces toxic and side effects, strengthens drug effect.Also have research that drug conjugates is prepared nanoparticle as carrier recently, this is while the effect of maintenance passive target, more considerably increase drug loading, improve the drug level of tumor locus, thus increase picked-up, improve drug effect, in addition, drug conjugates also avoid due to the toxigenous possibility of synthesis of carrier material degradation in vivo.
Because carrier is drug conjugates or pharmaceutical polypeptide conjugate, therefore after nanoparticle arrives tumor cell, need the drug release competence exertion effect in carrier, this just proposes requirement to the connecting key between medicine and medicine or polypeptide: can keep stable in blood circulation, but can rupture at inside tumor cells, release medicine.Research finds, there is oxidation-reduction potential: intracellular glutathion (GSH) concentration (2 ~ 10mmolL inside and outside zooblast
-1) be EC (2 ~ 10 μm of olL
-1) more than 200 times.Also there is reducing environment in endosome, mainly induce lysosome sufhydryl reductase (GILT) and reducing agent (as cysteine) jointly to regulate by IFN-γ; Containing low price Fe in lysosome
2+mercaptan (as cysteine) with high concentration, makes also have reducing environment in lysosome.In addition, tumor tissue cell, than normal tissue cell anoxia, has more reproducibility environment.Utilize this point, we select the disulfide bond with reduction-sensitive medicine to be connected obtained medicine-disulfide bond-medicine or polypeptide-conjugate with medicine or polypeptide, and are prepared into nanoparticle.Because the environment in extracellular and blood circulation is not enough to make disulfide bonds, therefore carrier can keep stable in extracellular and blood circulation, and intracellular reducing environment can be degraded to rapidly sulfydryl, after causing nanoparticle to arrive target cell, disulfide bond ruptures, discharge free drug, play drug effect.
Based on above background, with to the disulfide bond of reducing environment sensitivity using antitumor drug and antitumor drug or cell-penetrating peptide coupling as carrier material, be prepared into nanoparticle, both the passive target advantage of nano-delivery system had been remained, turn increase drug loading, also play the feature of disulfide bond at tumor locus selective degradation simultaneously, can toxic and side effects have been reduced, improve curative effect.
Summary of the invention
An object of the present invention is to provide the reduction response type conjugate nanoparticle that a kind of antitumor drug and medicine or polypeptide coupling are formed, and realizes improving drug loading, cancer target, falling hypotoxic effect.
Drug conjugates of the present invention, by medicine and medicine or obtained by the connecting key coupling containing disulfide bond between medicine and polypeptide, the general formula of this drug conjugates is as follows:
Medicine-connecting key-medicine or polypeptide,
Wherein, described medicine is the antitumor drug of hydroxyl or amino structure group.
Wherein, described connecting key is the micromolecule linking arm containing disulfide bond.
Wherein, described polypeptide is cell-penetrating peptide, and containing cysteine in sequence, mean molecule quantity is 800 ~ 5000 dalton.
Drug conjugates of the present invention, containing hydroxyl or amino in wherein said anti-tumor medicament structure.
Drug conjugates of the present invention, wherein said antitumor drug is selected from: paclitaxel, Docetaxel, amycin.
Drug conjugates of the present invention, containing disulfide bond in wherein said micromolecule linking arm.
Drug conjugates of the present invention, wherein said polypeptide is cell-penetrating peptide, and containing cysteine in sequence.
Drug conjugates of the present invention, preferably nanoparticulate form, be prepared into the nanoparticle of particle diameter at 80 ~ 200nm in preparation process.
Preferably, described drug conjugates of the present invention is selected from: amycin-connecting key-amycin, amycin-connecting key-hydrophobic peptide, paclitaxel-connecting key-paclitaxel, paclitaxel-connecting key-hydrophobic peptide, Docetaxel-connecting key-Docetaxel, Docetaxel-connecting key-hydrophobic peptide.
Most preferred, conjugate of the present invention is selected from: DOX-SS-DOX, PFV-SS-DOX.
Two of 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, comprises
(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 cross-linking agent 3,3'-dithiodipropionic acid-two (N-hydroxysuccinimide ester) react, and obtain drug-drug conjugate through ether sedimentation process.
Optimize, the preparation method of drug-drug conjugate, comprises the following steps:
Under the existence of organic amine catalyst; in dimethyl formamide; under nitrogen protection; by medicine and the cross-linking agent 3 containing disulfide bond; 3'-dithiodipropionic acid-two (N-hydroxysuccinimide ester) carries out esterification or acylation reaction by the mol ratio of 1-4:1, obtains drug-drug conjugate after ether sedimentation process.
Wherein, medicine carries out esterification or acylation reaction with cross-linking agent 3, the 3'-dithiodipropionic acid-two (N-hydroxysuccinimide ester) containing disulfide bond by the mol ratio of 2.1:1.
Wherein, described medicine is selected from: the antitumor drug such as paclitaxel, Docetaxel, amycin.
Preferred further, the synthesis of amycin-connecting key-amycin, comprises the following steps:
By DOXHCl (60.9mg, 0.105mmol) be dissolved in 3mL DMF, DSP (20.2mg in 0.5mL DMF will be dissolved in, 0.05mmol) dropwise join in DOX, add catalyst TEA (14.6 μ L) subsequently, after room temperature lucifuge reaction 30min, ice bath spends the night, within second day, transfer them to precipitation to be precipitated in ice ether, it is Orange red solid powder that sucking filtration obtains product.
The preparation method of medicine-polypeptide-conjugate, comprises the following steps:
(1) two sulfur two pyridine and mercaptopropionic acid react in ethanol/acetum, obtain Py-SS-COOH after crossing post separating treatment.
(2) adopt activator HBTU to be activated by Py-SS-COOH, the carboxyl after activation and drug reaction, be separated through post and obtain Py-SS-medicine.
(3) Py-SS-medicine reacts through disulfide exchange with containing sulfydryl polypeptide, after dialysis frozen dried, obtain medicine-polypeptide-conjugate.
Optimize, the preparation method of medicine-polypeptide-conjugate, comprises the following steps:
(1) by the molar ratio of 1:1.5 ~ 1:3, dropwise added by thiolic acid in the ethanol/acetum of two sulfur two pyridines under vigorous stirring, reaction 2 ~ 3h, obtains the pyridine acid containing disulfide bond after neutral aluminium sesquioxide post separating treatment.
(2) HBTU activation is containing after the carboxyl in the pyridine acid of disulfide bond; by the molar ratio of 1:1 ~ 1:1.2; dropwise will add in above-mentioned reaction solution containing amino antitumor drug under nitrogen protection, reaction 5 ~ 7h, is separated the medicaments derivative obtained containing disulfide bond through silicagel column.
(3) under nitrogen protection, medicaments derivative and the polypeptide containing sulfydryl are carried out disulfide exchange reaction by the mol ratio of 2:1, through dialysis, after frozen dried, obtain medicine-polypeptide-conjugate.
Preferred further, the synthesis of hydrophobic peptide-connecting key-amycin, comprises the following steps:
(1) preparation of Py-SS-COOH:
Two sulfur two pyridines (Py-SS-Py, 2g, 9.06mmol) are dissolved in 16mL ethanol, add glacial acetic acid (AcOH) 213 μ L, with vigorous stirring, the alcoholic solution of 10.6mL 3-mercaptopropionic acid (0.48g, 4.53mmol) is dropwise added.After reaction 2h, decompression removing ethanol, is separated through neutral alumina column, concentrated, and add cold water and separate out solid, sucking filtration, dried in vacuo overnight, obtaining product is white solid,
(2) preparation of Py-SS-DOX:
Py-SS-COOH (29.1mg, 0.135mmol) is dissolved in 1mL dry DMF, adds HBTU (102.6mg, 0.27mmol) and activate 30min.Add DOXHCl (93.96mg, 0.162mmol), it is 9-10 that DIEA (71.lmg, 0.43mmol) measures its pH with pH reagent paper, room temperature reaction 3h.Product is separated through silicagel column, concentrated that product is Orange red solid powder.
(3) preparation of PFV-SS-DOX:
Py-SS-DOX (38.5mg, 0.052mmol) is dissolved in 0.5mL DMF, under vigorous stirring, dropwise adds the DMF solution of PFV (25mg, 0.026mmol).Room temperature lucifuge reaction 48h.Reaction terminates rear dialysis, lyophilizing, and obtaining product is red solid.
Another object of the present invention is the nanoparticle providing a kind of drug conjugates to be formed, and makes nanoparticle using drug coupling of the present invention as carrier.
The preparation method of nanoparticle of the present invention, step is as follows: be dissolved in by drug conjugates in DMSO, be made into organic phase solution, gets and is scattered in aqueous phase solution in right amount, and Probe Ultrasonic Searching obtains the nanoparticle solution of uniform particle diameter.Its particle diameter is at 80 ~ 200nm.Dynamic Light Scattering Determination is utilized to obtain the Particle size and distribution of nanoparticle.
Above involved english abbreviation is made an explanation:
DOX: amycin.
Py-SS-COOH:3-(2-pyridyidithio) propanoic acid
Py-SS-DOX: pyridyldithiol amycin
HBTU: BTA-N, N, N' ', N '-tetramethylurea hexafluorophosphoric acid ester
DMSO: dimethyl sulfoxide
DIEA: diisopropylethylamine
PFV: micromolecule hydrophobic peptide PFVYLIGC
TEA: triethylamine
DSP:3,3'-dithiodipropionic acid-two (N-hydroxysuccinimide ester)
DOXHCl: doxorubicin hydrochloride
DMF: dimethyl formamide
Three of the object of the invention is to provide a kind of pharmaceutical composition containing drug conjugates.
Pharmaceutical composition of the present invention if desired containing conventional pharmaceutic adjuvant, and makes suitable dosage form
Pharmaceutical composition of the present invention can be prepared into any pharmaceutically useful dosage form, and these dosage forms comprise: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch.Preparation of the present invention, preferably, as aqueous injection, freeze dried powder, Tablet and Capsula agent.The administering mode of described preparation comprises intravenous injection, intra-tumoral injection and oral administration.Can be applicable to oncotherapy.
Pharmaceutical composition of the present invention, the preparation of its oral administration can containing conventional excipient, and such as binding agent, filler, diluent, tablet agent, lubricant, disintegrating agent, coloring agent, flavoring agent and wetting agent, can carry out coating to tablet if desired.
The filler be suitable for comprises cellulose, mannitol, lactose and other similar filler.Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycollate.Suitable lubricant comprises, such as magnesium stearate.The suitable acceptable wetting agent of medicine comprises sodium lauryl sulphate.
By mixing, fill, the method that tabletting etc. are conventional prepares solid oral composition.Repeatedly mix and active substance can be made to be distributed in those compositionss of a large amount of filler of whole use.
The form of oral liquid can be such as aqueous or oily suspensions, solution, Emulsion, syrup or elixir, or can be the composite dry products of a kind of available water before use or other suitable carrier.This liquid preparation can containing conventional additive, such as suspending agent, such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethyl-cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agent, such as lecithin, anhydro sorbitol monooleate or arabic gum; Non-aqueous carrier (they can comprise edible oil), the oily ester of the such as ester of almond oil, fractionated coconut oil, such as glycerol, propylene glycol or ethanol; Antiseptic, such as para hydroxybenzene methyl ester or propyl p-hydroxybenzoate or sorbic acid, and if need, can containing conventional flavouring agent or coloring agent.
For injection, the fluid unit dosage form of preparation contains active substance of the present invention and sterile carrier.According to carrier and concentration, this compound can be suspended or dissolve.The preparation of solution is normally by being dissolved in a kind of carrier by active substance, filter-sterilized before being loaded a kind of suitable bottle or ampoule, then seals.Adjuvant such as a kind of local anesthetic, antiseptic and buffer agent also can be dissolved in this carrier.In order to improve its stability, by freezing for this compositions after loading bottle, and under vacuo water can be removed.
Pharmaceutical composition of the present invention, applicable medicine acceptable carrier is optionally added when being prepared into medicament, described medicine acceptable carrier is selected from: mannitol, sorbitol, sodium pyrosulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, vitamin C, EDETATE SODIUM, Ethylenediaminetetraacetic Acid Calcium Salt, the alkali-metal carbonate of monovalence, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid, aminoacid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and its derivates, alginate, gelatin, polyvinylpyrrolidone, glycerol, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surfactant, Polyethylene Glycol, cyclodextrin, beta-schardinger dextrin-, phospholipid material, Kaolin, Pulvis Talci, calcium stearate, magnesium stearate etc.
Four of object of the present invention is to provide drug conjugates of the present invention or nanoparticle is preparing the application in anti-tumor drug.
Drug conjugates of the present invention or medicament nano granule are as pharmaceutical carrier, are preparing the application in antitumor drug.
Conjugate of the present invention is by passive target, fixed-point drug releasing and fall hypotoxic advantage, realizes the object for the treatment of tumor.
Application of the present invention is that disulfide bond ruptures, and medicine therefrom discharges by drug conjugates under human body cell glutathion inside reduction, gives antitumor drug reduction response, controlled, the toxicity of release reduces and realize antitumous effect.
The sign of response type drug conjugates nanoparticle is reduced: by dynamic light scattering, its size and distribution is investigated in the present invention; By the form of transmission electron microscope direct vision conjugate nanoparticle; On cellular level, by investigating the picked-up of cell to conjugate nanoparticle, the suppression ratio evaluate efficacy of conjugate nanoparticle cell growth; In animal level, evaluate its drug effect by aspects such as the suppression ratio of conjugate nanoparticle on tumor growth, body weight change impacts on animal.
The invention has the advantages that:
1, medicine is connected by disulfide bond with drug molecule or polypeptide and obtains prodrugs by the present invention, and can form nanoparticle in water, utilizes EPR effect to play passive target advantage, improves drug effect, reduce toxicity.
2, the present invention is using the part of antitumor drug as nanoparticulate carriers or carrier, increases drug loading, while increasing drug effect, reduces dosage, thus reduces toxic and side effects.
3, present invention employs the thinking of tumor microenvironment target administration, in drug conjugates, introduce the micromolecule linking arm containing disulfide bond.Disulfide bond is responsive to reducing environment, can degrade, discharge medicine fast, thus play specific antitumous effect in the distinctive reproducibility environment of tumor tissues.
4, drug conjugates of the present invention, is shown by cell experiment and zoopery, can improve the curative effect of antitumor drug, reduces toxicity, has good clinical value.
Accompanying drawing illustrates:
The TOF MS ESI mass spectrum of the DOX-SS-DOX of synthesis in Fig. 1, embodiment 1
The HPLC collection of illustrative plates of the DOX-SS-DOX of synthesis in Fig. 2, embodiment 1
The Py-SS-COOH's synthesized in Fig. 3, embodiment 2
1h NMR schemes
The TOF MS ESI mass spectrum of the Py-SS-COOH of synthesis in Fig. 4, embodiment 2
The Py-SS-DOX's synthesized in Fig. 5, embodiment 2
1h NMR mass spectrum
The HPLC collection of illustrative plates of the Py-SS-DOX of synthesis in Fig. 6, embodiment 2
The TOF MS ESI mass spectrum of the Py-SS-DOX of synthesis in Fig. 7, embodiment 2
The FTIR collection of illustrative plates of the Py-SS-DOX of synthesis in Fig. 8, embodiment 2
The HPLC collection of illustrative plates of the PFV-SS-DOX of synthesis in Fig. 9, embodiment 2
The grain size distribution of the DOX-SS-DOX nanoparticle of preparation in Figure 10, embodiment 3
The TEM figure of the DOX-SS-DOX nanoparticle of preparation in Figure 11, embodiment 3
The grain size distribution of the PFV-SS-DOX nanoparticle of preparation in Figure 12, embodiment 3
The streaming of Figure 13, free DOX, DOX liposome and DOX-SS-DOX nanoparticle is absorbed and is tested
The vitro cytotoxicity of Figure 14, free DOX, DOX liposome and DOX-SS-DOX nanoparticle
Figure 15, DOX liposome and DOX-SS-DOX nanoparticle are through the pretreated vitro cytotoxicity of GSH-OEt
The antitumor comparative study of pharmacodynamics of Figure 16, DOX-SS-DOX grain of the present invention and free DOX, DOX liposome.A: tumor growth curve; B: tumor weight; C: nude mice body weight change curve after administration; D: tumor size figure
The Particle size and distribution of Figure 17, DOX-SS-DOX nanoparticle lyophilized preparation
Detailed description of the invention
Further illustrate by the following examples and explain the present invention, but not as the restriction that the present invention carries out.
The synthesis of embodiment 1, amycin-disulfide bond-amycin
By DOXHCl (60.9mg, 0.105mmol) be dissolved in 3mL DMF, by the DSP (20.2 be dissolved in 0.5mL DMF, 0.05mmol) dropwise join in DOX, add the TEA (14.6 μ L) of catalytic amount subsequently, after room temperature lucifuge reaction 30min, ice bath spends the night, within second day, transfer them to precipitation to be precipitated in ice ether, it is Orange red solid powder that sucking filtration obtains product.Yield is 76.7%.Mass spectrum, efficient Liquid Detection, result is as Fig. 1 and Fig. 2.
The synthesis of embodiment 2, hydrophobic peptide (PFV)-disulfide bond-amycin
The preparation of Py-SS-COOH:
Two sulfur two pyridines (Py-SS-Py, 2g, 9.06mmol) are dissolved in 16mL ethanol, add glacial acetic acid (AcOH) 213 μ L, with vigorous stirring, the alcoholic solution of 10.6mL 3-mercaptopropionic acid (0.48g, 4.53mmol) is dropwise added.After reaction 2h, decompression removing ethanol, is separated through neutral alumina column, concentrated, and add cold water and separate out solid, sucking filtration, dried in vacuo overnight, obtaining product is white solid, and yield is 44.68%.Nuclear-magnetism, Mass Spectrometer Method, result is as Fig. 3 and Fig. 4.
The preparation of Py-SS-DOX:
Py-SS-COOH (29.1mg, 0.135mmol) is dissolved in 1mL dry DMF, adds HBTU (102.6mg, 0.27mmol) and activate 30min.Add DOXHCl (93.96mg, 0.162mmol), DIEA (71.l, 0.43mmol) with pH reagent paper measure its pH be 9 ?10, room temperature reaction 3h.Product is separated through silicagel column, concentrated that product is Orange red solid powder.Yield is 29.9%.Nuclear-magnetism, efficient liquid phase, mass spectrum, infrared detection, result is as Fig. 5, Fig. 6, Fig. 7 and Fig. 8.
The preparation of PFV-SS-DOX:
Py-SS-DOX (38.5mg, 0.052mmol) is dissolved in 0.5mL DMF, under vigorous stirring, dropwise adds the DMF solution of PFV (25mg, 0.026mmol).Room temperature lucifuge reaction 48h.Reaction terminates rear dialysis (DMSO → water), lyophilizing, and obtaining product is red solid, and yield is 18.6%.Efficient Liquid Detection, result is as Fig. 9.
The preparation and characterization of embodiment 3, reduction response type nano grain
The preparation and characterization of DOX-SS-DOX nanoparticle: take appropriate amount of drug conjugate, the DMSO solution being mixed with 5mg/mL, as mother solution, is got 50 μ L mother solutions and is joined in 1950 μ L aqueous solutions, Probe Ultrasonic Searching 10min (work 2s, interval 2s, power 200w).
Dynamic Light Scattering Determination is utilized to obtain the Particle size and distribution of nanoparticle.Transmission electron microscope (TEM) is utilized to observe the form of nanoparticle.Its nanoparticle particle size distribution is shown in Figure 10.Its TEM the results are shown in Figure 11.
The preparation and characterization of PFV-SS-DOX nanoparticle: take appropriate amount of drug conjugate, the DMSO solution being mixed with 20mg/mL, as mother solution, is got 50 μ L mother solutions and is joined in 1950 μ L aqueous solutions, visits super 10min (work 2s, interval 2s, power 200w).
Dynamic light scattering (DLS) is utilized to measure the Particle size and distribution of obtained nanoparticle.Result is as Figure 12.
The streaming picked-up experiment of embodiment 4, reduction response type nano grain
Adopt Fluorescein activated cell sorter quantitative assay MCF-7 cell to the intake of conjugate nanoparticle.MCF-7 is with 2.5 × 10
5the density in individual/hole is inoculated in 12 orifice plates, and after 24h, cell attachment grows into monolayer.Original fluid is abandoned in suction, uses serum-free medium rinsing.Add the DOX solution with serum-free medium dilution, DOX liposome or DOX-SS-DOX nanoparticle (containing 20 μ g/mL DOX) each 1mL, put 37 DEG C of incubators and hatch 3h.After hatching end, wash 2-3 time with the PBS of pre-cooling, every hole 300mL 0.25% trypsin digestion and cell, digestion is stopped containing serum free culture system liquid with 1mL, neutralize with pre-cooling PBS and blow and beat into cell suspension, in the centrifugal 5min of 1000rpm, with the PBS rinsing of 350 μ L pre-coolings and re-suspended cell, blow and beat into single cell suspension, cross cell sieve.By the DOX fluorescence intensity (excitation wavelength of mensuration is 480nm, and mensuration wavelength is 560nm) of cells were tested by flow cytometry cellular uptake.Each analysis cell number used is no less than 10
5individual, the cell number of collection is 10,000.Data use FCS Express V3 software to analyze.
As shown in figure 13, the intake of DOX-SS-DOX nanoparticle is far away higher than the picked-up of DOX liposome, and this is because the drug loading of DOX-SS-DOX nanoparticle is large, because this increasing the picked-up of cell for result.
The vitro cytotoxicity experiment of embodiment 5, reduction response type nano grain
SRB (Sulforhodamine B) method is adopted to investigate the cytotoxic activity of nanoparticle.The MCF-7 cell of exponential phase is inoculated in 96 porocyte culture plates, about 5000, every hole cell, if 6 multiple holes, is placed in CO2 gas incubator, 37 DEG C, hatch 24h under the condition of 5%CO2, treat that cell attachment grows.With DOX, DOX liposome and the DOX-SS-DOX nanoparticle solution (0.0625,0.125,0.25,0.5,1,2.5,5,10,20 μ g/mL, administration concentration is in DOX) of the culture fluid dilution preparation series concentration containing serum.The drug solution diluted is joined respectively in above-mentioned cell culture well, separately establish six blank reference holes in contrast.CO2 gas incubator Tissue Culture Plate being placed in 37 DEG C hatches 48h.After hatching end, discard pastille culture fluid, every hole adds the trichloroacetic acid of 10% of 200 μ L, leave standstill 5min, move into 4 DEG C of refrigerators and leave standstill more than 1 hour, taking-up deionized water washes 5 times, in air-dried overnight or 30 DEG C of baking ovens dry 5 hours, after bone dry, every hole adds the 0.4%SRB 100 μ L of 1% acetic acid preparation, room temperature dyeing 30min, outwell dyeing liquor, unconjugated dyestuff is washed away with 1% acetic acid, air drying, dissolve with the Tris alkali liquor 200 μ L of the 10mM of pH10.5, vibration 30min, microplate reader is determined at the absorbance at 540nm place.
As shown in figure 14, cytotoxicity power is free DOX > DOX liposome > DOX-SS-DOX nanoparticle to result.This is contrary with picked-up result, may be because the fracture of disulfide bond causes more slowly.DOX-SS-DOX nanoparticle needs to disconnect disulfide bond in a reducing environment and discharges the effect of free amycin competence exertion, and the fracture of disulfide bond be reduction dependency process be also time dependence process, namely reduction degree increase breaking degree is larger, cytotoxicity is more obvious, time is longer, breaking degree is larger, and cytotoxicity is more obvious.In incubation time, the fracture of disulfide bond not exclusively, therefore its cytotoxic effect do not expect so obvious.
The reduction response of the vitro cytotoxicity of embodiment 6, reduction response type nano grain
SRB (Sulforhodamine B) method is adopted to investigate the cytotoxic activity of nanoparticle.The MCF-7 cell of exponential phase is inoculated in 96 porocyte culture plates, about 5000, every hole cell, if 6 multiple holes, is placed in CO2 gas incubator, at 37 DEG C, 5%CO
2condition under hatch 24h, treat that cell attachment grows.Original fluid is abandoned in suction, adds 10mM GSH-OEt and hatches 2h.Suck GSH-OEt, PBS washes.With DOX, DOX liposome and the DOX-SS-DOX nanoparticle solution (0.25,1,2.5,5 μ g/mL, administration concentration is in DOX) of the culture fluid dilution preparation series concentration containing serum.The drug solution diluted is joined respectively in above-mentioned cell culture well, separately establish six blank reference holes in contrast.CO2 gas incubator Tissue Culture Plate being placed in 37 DEG C hatches 48h.After hatching end, discard pastille culture fluid, every hole adds the trichloroacetic acid of 10% of 200 μ L, leave standstill 5min, move into 4 DEG C of refrigerators and leave standstill more than 1 hour, taking-up deionized water washes 5 times, in air-dried overnight or 30 DEG C of baking ovens dry 5 hours, after bone dry, every hole adds the 0.4%SRB 100 μ L of 1% acetic acid preparation, room temperature dyeing 30min, outwell dyeing liquor, unconjugated dyestuff is washed away with 1% acetic acid, air drying, dissolve with the Tris alkali liquor 200 μ L of the 10mM of pH10.5, vibration 30min, microplate reader is determined at the absorbance at 540nm place.
The results are shown in Figure 15, hatch after GSH-OEt increases intracellular reduction degree in advance, the cytotoxicity of DOX-SS-DOX nanoparticle significantly increases, and does not have remarkable effect to the cytotoxicity of DOX liposome.This result illustrates, the toxicity of the toxicity ratio DOX liposome of DOX-SS-DOX nanoparticle is little, really because disulfide bonds not exclusively causes, increases the fracture that reduction degree accelerates disulfide bond, can increase the cytotoxicity of nanoparticle.
Embodiment 7, anti-tumor activity are tested
Select MCF-7 cell, inoculate in the oxter, right side of female nu/nu nude mice, every Mus inoculation 10
6individual cell, inoculates administration after 7 days.Administration group is 4 groups: PBS, DOX, DOX liposome, DOX-SS-DOX nanoparticle or PFV-SS-DOX nanoparticle, often organizes 5.In drug solution, DOX content is 0.2mg/mL, altogether administration 4 times, each 200 μ L, and delivery time is 1 day, every other day surveys nude mice body weight and tumor volume.Within the 12nd day after administration, put to death, get tumor tissue, weigh, take pictures.
As shown in figure 16, the antitumous effect that DOX-SS-DOX nanoparticle produces is far away higher than free DOX and the DOX liposome under same dosage, and its toxicity is compared also lower with free DOX with DOX liposome.In body, pharmacodynamic results is contrary with cell in vitro poison result, and this may be because in the cell in body, reducing degree is higher, picked-up can be disconnected to the disulfide bond in the nanoparticle of cell interior fast, discharge medicine, play drug effect.
In addition, the present invention also by DOX-SS-DOX nanoparticle and PFV-SS-DOX nanoparticle, carries out comparing of anti-tumor activity with PEG-SS-DOX nanoparticle, and result shows, medicine of the present invention better effects if in anti-tumor activity.
The preparation (for DOX-SS-DOX nanoparticle) of embodiment 8, lyophilized preparation
Be dissolved in by drug conjugates in DMSO, be made into certain density organic phase solution, get and be scattered in aqueous phase solution in right amount, Probe Ultrasonic Searching obtains the nanoparticle solution of uniform particle diameter.10% trehalose is added, cryodesiccated lyophilized preparation in nanoparticle solution.Lyophilized preparation distilled water redissolves, and with its Particle size and distribution of Dynamic Light Scattering Determination.Result is as Figure 17.
Claims (10)
1. a drug conjugates, is characterized in that, by medicine and medicine or obtained by the connecting key coupling containing disulfide bond between medicine and polypeptide, the general formula of this drug conjugates is as follows:
Medicine-connecting key-medicine or polypeptide.
2. drug conjugates as claimed in claim 1, it is characterized in that, described medicine is the antitumor drug of hydroxyl or amino structure group;
Described connecting key is the micromolecule linking arm containing disulfide bond,
Described polypeptide is cell-penetrating peptide, and containing cysteine in sequence, mean molecule quantity is 800 ~ 5000 dalton.
3. drug conjugates as claimed in claim 1, it is characterized in that, described medicine is selected from: paclitaxel, Docetaxel, amycin.
4. drug conjugates as claimed in claim 1, it is characterized in that, described polypeptide is cell-penetrating peptide, and containing free sulfhydryl groups in sequence.
5. drug conjugates as claimed in claim 1, it is characterized in that, described drug conjugates is selected from: amycin-connecting key-amycin or polypeptide-connecting key-amycin.
6. the preparation method of drug conjugates as claimed in claim 1, it is characterized in that, the preparation of described drug-drug conjugate, comprises the following steps:
Under the existence of organic amine catalyst; in dimethyl formamide; under nitrogen protection; by medicine and the cross-linking agent 3 containing disulfide bond; 3'-dithiodipropionic acid-two (N-hydroxysuccinimide ester) carries out esterification or acylation reaction, obtains drug-drug conjugate after ether sedimentation process.
7. the preparation method of drug conjugates as claimed in claim 1, it is characterized in that, the preparation of described medicine-polypeptide-conjugate, comprises the following steps:
(1) by the molar ratio of 1:1.5 ~ 1:3, dropwise added by thiolic acid in the ethanol/acetum of two sulfur two pyridines under vigorous stirring, reaction 2 ~ 3h, obtains the pyridine acid containing disulfide bond after neutral aluminium sesquioxide post separating treatment;
(2) HBTU activation is containing after the carboxyl in the pyridine acid of disulfide bond, by the molar ratio of 1:1 ~ 1:1.2, dropwise will add in above-mentioned reaction solution containing amino antitumor drug amycin under nitrogen protection, reaction 5 ~ 7h, is separated the medicaments derivative obtained containing disulfide bond through silicagel column;
(3) under nitrogen protection, medicaments derivative and the polypeptide containing sulfydryl are carried out disulfide exchange reaction by the mol ratio of 2:1, through dialysis, after frozen dried, obtain medicine-polypeptide-conjugate.
8. a nanoparticle for drug conjugates formation, is characterized in that, make nanoparticle using drug conjugates according to claim 1 as carrier.
9. nanoparticle as claimed in claim 8, it is characterized in that, the preparation method of nanoparticle is as follows:
Be dissolved in by drug conjugates in DMSO, be made into organic phase solution, get and be scattered in aqueous phase solution in right amount, Probe Ultrasonic Searching obtains the nanoparticle solution of uniform particle diameter, and its particle diameter is at 80 ~ 200nm.
10. preparing the application in antitumor drug using drug conjugates described in claim 1 or nanoparticle according to claim 8 as pharmaceutical carrier.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105251013A (en) * | 2015-09-28 | 2016-01-20 | 湘潭大学 | Degradable water-soluble antitumor polymer prodrug with redox responsiveness and preparation method thereof |
| CN105560178A (en) * | 2015-12-30 | 2016-05-11 | 浙江工业大学 | Reducing sensitive polypeptide prodrug nanomicelle, and preparation method and application thereof |
| WO2017025057A1 (en) * | 2015-08-11 | 2017-02-16 | Coherent Biopharma | Multi-ligand drug conjugates and uses thereof |
| 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 |
| CN106466484A (en) * | 2015-08-11 | 2017-03-01 | 同宜医药开曼有限公司 | A kind of many targeting ligand-drug coupling body with cell endocytic mediation function |
| CN106466485A (en) * | 2015-08-11 | 2017-03-01 | 同宜医药开曼有限公司 | A kind of targeting ligand-drug coupling body with cell endocytic mediation function |
| 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 |
| CN109999001A (en) * | 2019-05-21 | 2019-07-12 | 中国药科大学 | A kind of Nano medication and preparation method thereof |
| CN111729088A (en) * | 2020-08-21 | 2020-10-02 | 江苏申基生物科技有限公司 | Micro ribonucleic acid miR-34a coupling probe and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103705943A (en) * | 2014-01-03 | 2014-04-09 | 北京大学 | Preparation method and application of reduction-response-type pegylation (PEG) nanomedicine composition |
| CN103804472A (en) * | 2014-01-23 | 2014-05-21 | 浙江大学 | Taxane medicinal precursor |
-
2015
- 2015-02-02 CN CN201510053565.0A patent/CN104667292B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103705943A (en) * | 2014-01-03 | 2014-04-09 | 北京大学 | Preparation method and application of reduction-response-type pegylation (PEG) nanomedicine composition |
| CN103804472A (en) * | 2014-01-23 | 2014-05-21 | 浙江大学 | Taxane medicinal precursor |
Non-Patent Citations (2)
| Title |
|---|
| JAKOB REGBERG ET AL.: "Applications of Cell-Penetrating Peptides for Tumor Targeting and Future Cancer Therapies", 《PHARMACEUTICALS》 * |
| SUJIN YOON ET AL.: "Dual-Responsive Breakdown of Nanostructures with High Doxorubicin Payload for Apoptotic Anticancer Therapy", 《SMALL》 * |
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| CN106466484B (en) * | 2015-08-11 | 2021-05-04 | 同宜医药（苏州）有限公司 | Multi-target ligand-drug conjugate with function of cell endocytosis mediation |
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