CN102743762A - Receptor-mediated quantum dot tracing targeted drug delivery system, preparation method thereof and application - Google Patents
Receptor-mediated quantum dot tracing targeted drug delivery system, preparation method thereof and application Download PDFInfo
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Abstract
Disclosed are a receptor-mediated quantum dot tracing targeted drug delivery system, a preparation method thereof and application. The targeted drug delivery system comprises polyethylene glycol, hyaluronic acid, quantum dots and melphalan complex which are abbreviated as PEG-HA-QDs-MEL. The PEG is connected with HA amide in a dewatered and condensed manner, the water-soluble CdTe/CdS quantum dots are connected into the HA, and finally, the PEG, the HA and the QDs are prepared with the MEL complex to obtain the targeted drug delivery system. The component content of the PEG-HA-QDs-MEL includes, by weight, 30-100 parts of the MEL, 600-1500 parts of the PEG, 200-500 parts of the HA, 5-50 parts of the CdTe/CdS quantum dots, 20-50 parts of DCC (dicyclohexylcarbodiimide), 1-10 parts of DMAP (dimethylamino-pyridine), 100-500 parts of succinic anhydride, 20-50 parts of NHS, 20-50 parts of EDC (carbodiimide) and 10-100 parts of sodium bisulfite. The targeted drug delivery system is applied to preparing antitumor drugs.
Description
Technical field
The present invention relates to medicine field, be specifically related to a kind of receptor-mediated quantum dot spike targeting drug delivery system.
Background technology
Malignant tumor has become one of threat human health and the topmost disease of life.The Comprehensive Treatment that at present adopts operation to combine with chemotherapy to malignant tumor is main more, wherein chemotherapy be must use treatment means, powerful because of its efficacy of a drug, can kill and wound fast or the kill tumor cell, in clinical treatment, bringing into play important effect.But chemotherapeutics ubiquity cell selective is poor, toxic and side effects is big, untoward reaction is serious, (Multi-drug resistance MDR), causes chemotherapy effect undesirable be prone to produce serious adverse reaction and multidrug resistance.Therefore, how to improve the tissue or the cell selective of medicine, medicine accurately is transported to tumor tissues or cell and at the directed performance therapeutical effect that discharges of target site, become one of key issue that oncotherapy needs to be resolved hurrily.
Along with medical science and the new achievement in research of material science continue to bring out, the pharmaceutical preparation theory and practice has obtained to develop rapidly, has brought into play important function for improving tumor treatment effect, reduction toxic and side effects, reversion MDR etc.Especially the achievement of " target administration " theory and practice research; With common problems such as chemotherapeutics selectivity guiding specific tumors cell, the ubiquitous poor selectivity of solution chemotherapeutic preparation new thinking is provided, the target administration of chemotherapeutics is expected to become the new way of treatment malignant tumor.(Targeting Drug Delivery System TDDS) can accurately deliver to lesions position with medicine to targeting drug delivery system, improves the drug level of lesions position, thereby improves medication effect, avoids or alleviates poisonous side effect of medicine.Targeting property is divided into initiatively targeting property and passive targeting.Initiatively targeting property is meant that this system has the ability of initiatively discerning target cell and getting into target cell, like the receptor target drug-supplying system etc.The passive target ability is relevant with the size of system, and the granule below 100 nanometers is prone to see through the blood vessel of tumor tissues, makes effect molecules such as medicine or gene concentrate around tumor cell and the generation effect.
CD44 is a kind of transmembrane receptor glycoprotein, can with hyaluronic acid (Hyaluronan acid, HA) etc. multiple part combines; Nearest research shows; Interaction between HA and its specific receptor CD44 can be used as the research basis of HA targeted therapy malignant tumor, and CD44 had expression at multiple malignant tumor cell surface, so utilize HA as carrier; Small-molecule drug is carried on it; Can when strengthening drug selectivity, reduce medicine, and the dissolubility that is connected the back medicine with HA can improve greatly with stability for Normocellular lethal effect.Therefore utilize HA to be connected to medicine or its carrier surface, can reach the purpose of targeted therapy through receptor-mediated effect with killing and wounding in the medicine transfered cell or suppressing tumor cell as the targeting group of medicine.But HA is soluble in water, be prone to be absorbed, in tissue the time of staying short, degradation speed is fast, this has just limited his application at medical domain.
Polyethylene Glycol (PEG) is the hypotoxicity linear molecule, and no antigen has water solublity, immunology inertia, and its biocompatibility has obtained the FDA of food and drug administration authentication.The terminal hydroxyl of PEG is the functional group of chemical reaction; But chemical property torpescence; Must under fierce condition, could react with other group; For make HA can than under the temperate condition with higher rate and PEG bonding, have only through specific activator and PEG reaction to make it activation, with the activatory PEG carboxyl and the amino of the HA acetylation processing binary conjugate that formation ester chain or ether chain be connected that reacts.As the medicine transmission purposes time, the ester chain connects in vivo under the suitable condition, can be opened by hydrolysis gradually.
Hyaluronic acid (Hyaluronan acid; HA) be by repeating the linear mucopolysaccharide that disaccharide construction unit β-D-glucuronic acid and N-acetylglucosamine alternately connect and compose each other; Having reduced immunogenicity, biodegradability, is a kind of ideal biological medical polymer material.Bibliographical information is arranged, have good active and passive targeting as the prodrug of pharmaceutical carrier, antitumor drug and HA coupling can be improved dissolubility, stability, the controlled capability of medicine with HA; And the toxicity of medicine is reduced, reach the purpose of targeted therapy tumor.CD44 is the most important HA receptor of cell surface; Its N end combines with HA is non-covalent; Binding site can extend to several repetition disaccharide unit of HA, and it is in the expression of malignant cell apparent height, thereby HA is suitable as the active targeting factor of malignant tumor targeting drug delivery system.
Quantum dot (quantum dots; QDs; Be called semiconductor nano again) as a kind of novel fluorescence probe, aspect the fluorescence imaging of cell, live body and bioactive substance qualitative, quantitative, application promise in clinical practice is arranged all with unique optics and chemical property, compare with traditional organic fluorescence raw material; Has incomparable advantage: exciting light spectrum width, the quantum dot of available single excitation wavelength simultaneous excitation different colours; Emission spectra is narrow and symmetrical, can reduce the spectra overlapping in the detection, improves the sensitivity that detects; The fluorescent emission wavelength can be by quantum dot size and component regulation and control; Luminous strength ratio conventional fluorescent material is strong more than 10 times, and fluorescence lifetime is strong.Though it is virulent forming the element pair cell such as chemical constituent Cd and Se and the tissue of quantum dot, then show excellent biological compatibility (biocompatibility) through the quantum dot of hydrophilic and functionalization.To labelling the cultured cell of quantum dot observe with the long-term follow of the living animal of having injected quantum dot and show that quantum dot does not cause physiological damage also can keep the quantum dot physicochemical property to experimental cell and animal body.Up to now, existing Chinese scholars replaces traditional organic fluorescent dye molecule with the QDs and the biomolecule coupling of different materials, is widely used in bioactive molecule and detects aspects such as tumor cell detection and tumor imaging and biological living imaging.
Summary of the invention
Technical problem to be solved by this invention provides a kind of receptor-mediated quantum dot spike targeting drug delivery system.This drug-supplying system is used for lymphoma, breast carcinoma, ovarian cancer, spermocytoma, multiple myeloma and thyroid carcinoma chemotherapy and has remarkable advantages; Special in the breast carcinoma of killing and wounding high expressed CD44 receptor; Ovarian cancer, the cervical cancer tumor cell is lower to the toxicity of normal structure; And this targeting drug delivery system is read the information of many bio-molecular interactions of medicine and cell and cell in vivo in real time, can study transport process and many target spots of dynamic monitoring medicine process of medicine.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of receptor-mediated quantum dot spike melphalan targeting drug delivery system; It is Polyethylene Glycol-hyaluronic acid-quantum dot-melphalan complex; Be abbreviated as PEG-HA-QDs-MEL, be connected with the condensation of HA dehydration of amide, and insert water-soluble CdTe/CdS quantum dot at HA by PEG; Last and medicine MEL dehydration of amide condensation prepared obtains; Wherein, the constituent content of PEG-HA-QDs-MEL is counted as follows by weight, and each weight portion is 1 milligram:
Melphalan (MEL) 30-100 part, Polyethylene Glycol (PEG) 600-1500 part, hyaluronic acid (HA) 200-500 part; CdTe/CdS quantum dot 5-50 part, N, N' dicyclohexylcarbodiimide (DCC) 20-50 part; 4-dimethylamino naphthyridine (DMAP) 1-10 part, succinic anhydride 100-500 part, N-hydroxy thiosuccinimide (NHS) 20-50 part; 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) 20-50 part, sodium sulfite 10-100 part, benzyl alcohol (BA) 50-200 part.
The method for preparing of receptor-mediated quantum dot spike melphalan targeting drug delivery system of the present invention may further comprise the steps, and following each weight portion is 1 milligram:
1) the synthetic and purification of end carboxy polyethylene glycol
600-1500 weight portion Polyethylene Glycol (PEG) is placed the there-necked flask that has condensing tube, agitator and thermometer, add dissolved in chloroform, treat to add 100-500 weight portion succinic anhydride, heating for dissolving after dissolving finishes; Add exsiccant pyridine simultaneously, stir refluxed reaction 2-10 hour, the stopped reaction postcooling is to room temperature; Reaction gained mixture reduction vaporization adds the absolute ether deposition again and obtains white product, sucking filtration to being thick; Reuse dichloromethane lysate, the elimination insoluble matter adds the absolute ether deposition and obtains white product; Repeat more than 2 times, the sucking filtration final vacuum is dried to constant weight, obtains the end carboxy polyethylene glycol of purification;
2) preparation of PEG-HA
The end carboxy polyethylene glycol that step 1) is obtained is dissolved in the hyaluronic hydrolyzed solution of 200-500 weight portion; Pour the there-necked flask that dephlegmator and agitator are housed into; Add catalyst 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide 10-50 weight portion and 4-dimethylamino naphthyridine 0.5-5 weight portion; Collect 100 ℃ of fractions in reaction under 60-140 ℃ after 2-10 hour, reaction finishes postcooling; Add dehydrated alcohol and do not generate, filter collecting precipitation to there being deposition, washing precipitation, dry sediment promptly gets PEG-HA;
3) preparation of water-soluble CdTe/CdS quantum dot
5-20 weight portion tellurium powder and 2-10 weight portion sodium borohydride are added in the entry, and under the nitrogen protection condition, the room temperature lower magnetic force stirs 2-5h, obtains the NaHTe precursor liquid that kermesinus contains Te; Take by weighing 2-10 weight portion CdCl
2Be dissolved in the distilled water, add 2-10 weight portion mercaptopropionic acid, with the pH value of the NaOH solution regulator solution of concentration 1mol/L-2mol/L to 8-10; Add solution in the three-necked bottle, join the CdCl under the nitrogen protection to freshly prepd NaHTe
2In the solution, the color of solution becomes the transparent orange yellow, and stirring at room is transferred in 90 ℃ the water bath with thermostatic control behind the reaction 0.5-2h then, splashes into concentration 1 μ mol/L-20 μ mol/L Na with 0.1ml/min speed
2The solution 1ml-15ml of S obtains the CdTe/CdS core-shell quanta dots; Total overall reaction is accomplished under nitrogen protection, behind the reaction terminating, adds ethanol; Centrifugalize 10-15min discards the supernatant under 5000r/min, adds water deposition is suspended again; Repeat this process 2 times; The solid that obtains at room temperature dries naturally, grinds and obtains CdTe/CdS quantum dot pressed powder, is abbreviated as QDs;
4) preparation of PEG-HA-QDs
It is soluble in water to take by weighing 5-50 weight portion QDs, and room temperature lucifuge magnetic agitation is all dissolved to QDs, adding 5-50 weight portion N-hydroxy thiosuccinimide (NHS) and 5-50 weight portion 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC).Behind room temperature lucifuge magnetic agitation 1h with step 2) the aqueous solution 20ml of PEG-HA of the 0.1g/ml-1g/ml of preparation; Add in the above-mentioned mixed liquor; Room temperature lucifuge magnetic agitation 12h; The products therefrom mixed liquor is that 7.4 phosphate buffer is dialysed with bag filter at pH, and lyophilization gets the PEG-HA-QDs complex;
5) preparation of American and French human relations benzyl ester (BA-MEL)
In the three-neck flask that thermometer, water knockout drum are housed, add melphalan 30-100 weight portion, sodium sulfite 10-100 weight portion and excessive benzene methanol react under magnetic agitation, and the mixed liquor of reaction back gained is used distilled water, saturated sodium bicarbonate and distilled water wash successively; Obtain grease; Grease after the washing is evaporated with Rotary Evaporators, remove unreacted benzene methanol, the temperature of setting rotary evaporation is 30-60 ℃; Evaporation is till no distillation; The material that evaporation obtains carries out recrystallization with alcohol-ether, and its ethanol and ether volume ratio are 1:2, obtain milky solid BA-MEL;
6) preparation of PEG-HA-QDs-MEL
100-500 weight portion PEG-HA-QDs complex, 10-50 weight portion N that step 4) is obtained; N' dicyclohexylcarbodiimide and 0.5-5 weight portion 4-dimethylamino naphthyridine are dissolved in the anhydrous dimethyl sulphoxide, and room temperature lucifuge magnetic agitation is all dissolved to PEG-HA-QDs, the 10-50 weight portion BA-MEL aqueous solution of the pH4.7 of adding step 5) gained; Room temperature lucifuge magnetic agitation 12h; It is carried palladium 0.5wt%-15wt% catalyst hydrogenation through carbon remove benzyl alcohol in the tert-butyl alcohol, using pH then is 7.4 phosphate buffer dialysis 24-72h, reuse distilled water dialysis 24-72h; Lyophilization gets reactant powders, i.e. the PEG-HA-QDs-MEL product.
Preparation technology of the present invention is following:
This targeting drug delivery system is applied to prepare antitumor drug.
Beneficial effect of the present invention is:
1) native system has good active targeting property and reversing drug resistance effect preferably
CD44 is a kind of efficient endocytosis HA receptor, but HA specific recognition receptor in this system and combination with it, and the messenger drug object height is imitated initiatively target tumor cell; Through the particle diameter of control particle, the passive targeting of feasible system effectively improves the drug level in the tumor cell, has reversing drug resistance effect preferably simultaneously.
2) native system has good blood stability.
HA is in vivo mainly by the hyaluronic acid enzymatic degradation; Hyaluronidase is a kind of β-1-4 inscribe glucose amino acid enzyme; It is bigger that its activity is influenced by the environment PH, and the hyaluronidase in human serum, urine, liver and the tumor tissues is acid type hyaluronidase, and its optimum pH is 3.5-4.0.In blood, higher pH (7.35-7.45) makes acid type hyaluronic acid enzyme deactivation, and HA is not degraded, and the chemical bond that MEL, PEG are connected with HA is all stable, can ensure that drug-supplying system is not released at blood circulation process Chinese medicine.
3) good hidden property, release property and controlled release properties are arranged.
This system's coupling PEG can avoid engulfing of reticuloendothelial system and have hidden property preferably.Acid type hyaluronidase only has activity in tumor tissues or around it.After this system got into tumor cell, under the hyaluronidase effect, HA was degraded, and simultaneously, lower pH value also makes the PEG-HA ester bond be hydrolyzed disconnection, caused drug release.
4) tracking function in the good body is arranged.Quantum dot has the tumor imaging function, can dynamically observe pharmaceutical carrier and whether reach tumor cell and performance curative effect.This drug-supplying system is expected to solve that aminoaryl chlormethine series pharmaceuticals preparation is poor at the cell selective that exists aspect the treatment tumor, problems such as the low and drug resistance of concentration seriously, are effectively treated in untoward reaction.
5) broad applicability of native system
Targeting drug delivery system of the present invention connects through the HA active group and contains-NH
2Or-the different activities material of COOH reactive group, like antitumor monoclonal antibody, anti-tumor chemotherapeutic medicine and absorption gene etc., thereby further enlarge its utilization scope, have broad applicability.
The specific embodiment
In order to understand the present invention better, further illustrate content of the present invention below in conjunction with embodiment, but content of the present invention not only is confined to following embodiment.Each weight portion is 1 milligram among the following embodiment.
Embodiment 1
1.PEG-HA-QDs-MEL the preparation of component
(1) the synthetic and purification of end carboxy polyethylene glycol
Place 250mL to have the there-necked flask of condensing tube, agitator and thermometer 10g (5mmol) PEG-1500, add 100mL chloroform (CaH
2Steam under existing) dissolving, to treat to add 2.5g (25mmol) succinic anhydride after dissolving finishes, heating for dissolving adds the 2mL dry pyridine simultaneously; Stir refluxed reaction 6 hours, the stopped reaction postcooling is to room temperature, and reaction gained mixture reduction vaporization is to being thick, and residue adds a large amount of absolute ethers; Separate out the white precipitate product, sucking filtration gets filter cake, and filter cake is with 30mL dichloromethane lysate, elimination insoluble matter; Add the absolute ether deposition again, separate out the white precipitate product, sucking filtration gets filter cake, reuse dichloromethane lysate; Repeat aforesaid operations 3 times, the sucking filtration final vacuum is dried to constant weight, must hold carboxy polyethylene glycol (PEG-SA-NHS), weighs;
(2) preparation of PEG-HA
The NaOH aqueous solution of getting 3.0g hyaluronic acid and 15mL concentration 20wt% mechanical agitation reaction at normal temperatures 12 hours, deacetylate; Solution was dialysed in distilled water 2 days, and lyophilization obtains deacetylated hyaluronic acid freeze-dried powder; The 0.500g end carboxy polyethylene glycol that step (1) is obtained is dissolved in the hyaluronic acid hydrolyzed solution, pours in the 100mL there-necked flask that dephlegmator and agitator are housed, and adds EDC catalyst 0.250g, DMAP0.020g; 100 ℃ of water-baths were reacted 6 hours down; Collect 100 ℃ of fractions, reaction finishes, and it is brown that solution is; Cooling adds dehydrated alcohol and does not generate to there being deposition; Filter collecting precipitation, washing is drying to obtain PEG-HA;
(3) preparation of water-soluble CdTe/CdS quantum dot
Synthesizing of water-soluble CdTe/CdS core-shell type nano quantum dot mainly is divided into two steps: at first synthetic CdTe nuclear Nano sol; And then outside CdTe, coat the CdS shell.0.1200g tellurium powder and 0.0700g sodium borohydride are added in the aqueous solution of 5mL, and under the condition of nitrogen protection, the room temperature lower magnetic force stirs 3h, obtains the precursor liquid of the bolarious Te of containing.Take by weighing CdCl
20.040g, be dissolved in the 15mL distilled water, add 50 μ L mercaptopropionic acids, with the pH value of the NaOH solution regulator solution of concentration 1mol/L to 8-10; Solution is added in the 100mL three-necked bottle, behind the logical nitrogen 20min, join the CdCl under the nitrogen protection to freshly prepd NaHTe
2In the solution, the color of solution becomes the transparent orange yellow, and stirring at room 20min is transferred in 90 ℃ the water bath with thermostatic control then, behind the reaction 0.5h, splashes into the Na of 5 μ mol/L with the speed of 0.1mL/min
2S solution 1ml obtains the CdTe/CdS core-shell quanta dots; Total overall reaction is accomplished under nitrogen protection, behind the reaction terminating, adds the ethanol of 10mL; Centrifugalize 10min under 5000r/min; Discard the supernatant, add 10mL water deposition is suspended again, repeat this process 2 times; The solid that obtains at room temperature dries naturally, and obtains CdTe/CdS quantum dot (QDs) pressed powder with the crucible grinding;
(4) preparation of PEG-HA-QDs
It is soluble in water to take by weighing 0.100g QDs; Room temperature lucifuge magnetic agitation is all dissolved to QDs; Add 0.25g NHS and 0.15gEDC, behind room temperature lucifuge magnetic agitation 1h with step 2) the aqueous solution 20ml of the PEG-HA of prepared 0.1g/ml, room temperature lucifuge magnetic agitation 12h; The products therefrom mixed liquor is that 7.4 phosphate buffer is dialysed with bag filter at pH, and lyophilization gets the PEG-HA-QDs complex;
(5) preparation of BA-MEL
In the three-neck flask that thermometer, water knockout drum are housed, add melphalan 0.600g, benzyl alcohol 10ml, sodium sulfite 0.394g reacts under magnetic agitation; Reacted mixed liquor is used distilled water, saturated sodium bicarbonate, distilled water wash successively, obtain grease; Grease after the washing is evaporated with Rotary Evaporators; Remove unreacted benzyl alcohol in the mixture; The temperature of setting rotary evaporation is 60 ℃, and evaporation is till no distillation, and the material that obtains carries out recrystallization with alcohol-ether; Its ethanol and ether volume ratio are 1:2, obtain milky solid BA-MEL;
(6) preparation of PEG-HA-QDs-MEL
The PEG-HA-QDs300mg that step (2) is obtained; Get 260mg DCC; 0.05mmol4-dimethylamino naphthyridine (DMAP) is dissolved in the 15mL anhydrous dimethyl sulphoxide, room temperature lucifuge magnetic agitation is all dissolved (about 1 hour) to PEG-HA-QDs, BA-MEL aqueous solution (pH4.7) 30mL of adding 5mg/mL; Room temperature lucifuge magnetic agitation 12h; It is carried palladium 5wt% hydrogen catalyst through carbon remove benzyl alcohol in the tert-butyl alcohol, pH is 7.4 phosphate buffer dialysis 3 days, reuse distilled water dialysis 3 days.Lyophilization gets reactant powders, promptly gets product P EG-HA-QDs-MEL.
2. MTT experiment (the ovarian cancer SKOV of receptor-mediated quantum dot spike targeting drug delivery system
3Cell)
(1) uses behind the trypsinization preparation to become concentration in the cell of exponential phase and be 2-10 * 10
4The cell suspending liquid of individual/ml is inoculated in 96 orifice plates through after the accurate cell technology by 500 cells/well, and every hole adds 100 μ l.
(2) flat board is put 37 ℃, contained volumetric concentration 5%CO
2And the saturated humidity condition is after following 24 hours; Add each cell sample corresponding drug serum, normal saline serum and diluted good medicine; Each sample concentration is established three parallel holes, and matched group adds the culture fluid 100 μ l that do not contain sample, puts into incubator again and hatches 72 hours.
(3) every hole adds the MTT solution 50 μ l that press the new preparation of 5mg/ml, and incubation 4 hours makes MTT be reduced to the first a ceremonial jade-ladle, used in libation; When seeing that under inverted microscope thread purple crystal body appears in cell peripheral in the orifice plate, outwell supernatant; Every hole adds dimethyl sulfoxide 220 μ l, after shaking up with dull and stereotyped shaking table, uses ELIASA to measure OD value (OD) (detecting wavelength 570nm); Handling cell with solvent control is matched group, by formula the suppression ratio of computerized compound pair cell.
The MTT experimental result
3. this drug-supplying system range of application
Melphalan also is Melphalan, L-Sarcolysinum, L-sarcolysin; English name: Melphalan or Alkeran Melphalanum; Malignant lymphoma, breast carcinoma, ovarian cancer, spermocytoma, chronic leukemia, polycythemia vera, child's neuroblastoma in late period, thyroid carcinoma there is better curative effect; Arterial perfusion can also be treated limb malignant tumor, like malignant melanoma, osteosarcoma and soft tissue sarcoma etc.
Embodiment 2
1.PEG-HA-QDs-MEL the preparation of component
(1) the synthetic and purification of end carboxy polyethylene glycol
Place 500mL to have the there-necked flask of condensing tube, agitator and thermometer 40g (5mmol) PEG-1500, add 200mL chloroform (CaH
2Steam under existing) dissolving, to treat to add 5.0g (25mmol) succinic anhydride after dissolving finishes, heating for dissolving adds the 8mL dry pyridine simultaneously; Stir refluxed reaction 6 hours, the stopped reaction postcooling is to room temperature, and reaction gained mixture reduction vaporization is to being thick, and residue adds a large amount of absolute ethers; Separate out the white precipitate product, sucking filtration is with 120mL dichloromethane lysate, elimination insoluble matter; Add the absolute ether deposition again, separate out the white precipitate product, sucking filtration repeats aforesaid operations 3 times; The sucking filtration final vacuum is dried to constant weight, must hold carboxy polyethylene glycol (PEG-SA-NHS), weighs;
(2) preparation of PEG-HA
The NaOH aqueous solution of getting 12.0g hyaluronic acid and 30mL concentration 20wt% mechanical agitation reaction at normal temperatures 12 hours, deacetylate; Solution was dialysed in distilled water 2 days, and lyophilization obtains deacetylated hyaluronic acid freeze-dried powder; The 2.000g end carboxy polyethylene glycol that step (1) is obtained is dissolved in the hyaluronic acid hydrolyzed solution, pours in the 100mL there-necked flask that dephlegmator and agitator are housed, and adds 1.000g catalyst EDC, 0.080g DMAP; 100 ℃ of water-baths were reacted 6 hours down; Collect 100 ℃ of fractions, reaction finishes, and it is brown that solution is; Cooling adds dehydrated alcohol and does not generate to there being deposition; Filter collecting precipitation, washing is drying to obtain PEG-HA;
(3) preparation of water-soluble CdTe/CdS quantum dot
Synthesizing of water-soluble CdTe/CdS core-shell type nano quantum dot mainly is divided into two steps: at first synthetic CdTe nuclear Nano sol; And then outside CdTe, coat the CdS shell; 0.4800g tellurium powder and 0.2800g sodium borohydride are added in the aqueous solution of 20mL, and under the condition of nitrogen protection, the room temperature lower magnetic force stirs 3h, obtains the precursor liquid of the bolarious Te of containing; Take by weighing CdCl
20.160g, be dissolved in the 40mL distilled water, add 200 μ L mercaptopropionic acids, with the pH value of the NaOH solution regulator solution of concentration 1mol/L to 8-10; Solution is added in the 100mL three-necked bottle, behind the logical nitrogen 20min, join the CdCl under the nitrogen protection to freshly prepd NaHTe
2In the solution, the color of solution becomes the transparent orange yellow, and stirring at room 20min is transferred in 90 ℃ the water bath with thermostatic control then, behind the reaction 0.5h, splashes into the Na of 5 μ mol/L with the speed of 0.1mL/min
2S solution 2ml obtains the CdTe/CdS core-shell quanta dots; Total overall reaction is accomplished under nitrogen protection; Behind the reaction terminating, add the ethanol of 15mL, centrifugalize 10min under 5000r/min; Discard the supernatant, add 10mL water deposition is suspended again, repeat this process 2 times; The solid that obtains at room temperature dries naturally, and obtains CdTe/CdS quantum dot (QDs) pressed powder with the crucible grinding;
(4) preparation of PEG-HA-QDs
It is soluble in water to take by weighing 0.400gQDs; Room temperature lucifuge magnetic agitation is all dissolved to QDs; Add 1.0g NHS and 0.60gEDC, add the aqueous solution 20ml of PEG-HA of the 0.2g/ml of step (2) preparation behind the 1h, room temperature lucifuge magnetic agitation 12h; The products therefrom mixed liquor is that 7.4 phosphate buffer is dialysed with bag filter at pH, and lyophilization gets the PEG-HA-QDs complex;
(5) preparation of BA-MEL
In the three-neck flask that thermometer, water knockout drum are housed, add melphalan 2.400g, benzyl alcohol 10ml, sodium sulfite 1.600g reacts under magnetic agitation; Reacted mixed liquor is used distilled water, saturated sodium bicarbonate, distilled water wash successively, obtain grease; Grease after the washing is evaporated with Rotary Evaporators; Remove unreacted benzyl alcohol in the mixture, the temperature of setting rotary evaporation is 60 ℃, and evaporation is till no distillation; The material that obtains carries out recrystallization with alcohol-ether; Its ethanol and ether volume ratio are 1:2, and its ethanol and ether volume ratio are 1:2, obtain milky solid BA-MEL;
(6) preparation of PEG-HA-QDs-MEL
PEG-HA-QDs1200mg, 1040mg DCC and 0.20mmol DMAP that step (2) is obtained are dissolved among the anhydrous dimethyl sulphoxide 40mL, and room temperature lucifuge magnetic agitation is all dissolved (about 1 hour) to PEG-HA-QDs; Add 20mg/mL BA-MEL aqueous solution (pH4.7) 30mL, room temperature lucifuge magnetic agitation is spent the night, and adds the 40ml tert-butyl alcohol, and through the hydrocarbon catalytic eliminating benzyl alcohol of 5wt% palladium, using pH is 7.4 phosphate buffer dialysis 3 days, reuse distilled water dialysis 3 days; Lyophilization gets reactant powders, promptly gets product P EG-HA-QDs-MEL.
2. MTT experiment (the ovarian cancer SKOV of receptor-mediated quantum dot spike targeting drug delivery system
3Cell)
(1) uses behind the trypsinization preparation to become concentration in the cell of exponential phase and be 2-10 * 10
4The cell suspending liquid of individual/ml is inoculated in 96 orifice plates through after the accurate cell technology by 500 cells/well, and every hole adds 100 μ l.
(2) flat board is put 37 ℃, contained volumetric concentration 5%CO
2And the saturated humidity condition is after following 24 hours; Add each cell sample corresponding drug serum, normal saline serum and diluted good medicine; Each concentration of specimens is established three parallel holes, and matched group adds the culture fluid 100 μ l that do not contain sample, puts into incubator again and hatches 72 hours.
(3) every hole adds with pressing the freshly prepared MTT solution 50 μ l of 5mg/ml, and incubation 4 hours makes MTT be reduced to the first a ceremonial jade-ladle, used in libation; When seeing that under inverted microscope thread purple crystal body appears in cell peripheral in the orifice plate, outwell supernatant; Every hole adds dimethyl sulfoxide (DMSO) 220 μ l, after shaking up with dull and stereotyped shaking table, uses ELIASA to measure OD value (OD) (detecting wavelength 570nm); Handling cell with solvent control is matched group, by formula the suppression ratio of computerized compound pair cell.
The MTT experimental result
3. this drug-supplying system range of application
Melphalan also is Melphalan, L-Sarcolysinum, L-sarcolysin; English name: Melphalan or Alkeran Melphalanum; Malignant lymphoma, breast carcinoma, ovarian cancer, spermocytoma, chronic leukemia, polycythemia vera, child's neuroblastoma in late period, thyroid carcinoma there is better curative effect; Arterial perfusion can also be treated limb malignant tumor, like malignant melanoma, osteosarcoma and soft tissue sarcoma etc.
Embodiment 3
1.PEG-HA-QDs-MEL the preparation of component
(1) the synthetic and purification of end carboxy polyethylene glycol
Place 250mL to have the there-necked flask of condensing tube, agitator and thermometer 20g (5mmol) PEG-1500, add 100mL chloroform (CaH
2Steam under existing) dissolving, to treat to add 5.0g (25mmol) succinic anhydride after dissolving finishes, heating for dissolving adds the exsiccant pyridine of 4mL simultaneously; Stir refluxed reaction 6 hours, the stopped reaction postcooling is to room temperature, and reaction gained mixture reduction vaporization is to being thick, and residue adds a large amount of absolute ethers; Separate out the white precipitate product, sucking filtration is with 60mL dichloromethane lysate, elimination insoluble matter; Add the absolute ether deposition again, separate out the white precipitate product, sucking filtration repeats aforesaid operations 3 times; The sucking filtration final vacuum is dried to constant weight, must hold carboxy polyethylene glycol (PEG-SA-NHS), weighs;
(2) preparation of PEG-HA
The NaOH aqueous solution of getting 6.0g hyaluronic acid and 30mL concentration 20wt% mechanical agitation reaction at normal temperatures 12 hours, deacetylate; Solution was dialysed in distilled water 2 days, and lyophilization obtains deacetylated hyaluronic acid freeze-dried powder; The 1.000g end carboxy polyethylene glycol that step (1) is obtained is dissolved in the hyaluronic acid hydrolyzed solution, pours in the 100mL there-necked flask that dephlegmator and agitator are housed, and adds 0.500g catalyst EDC, 0.040g DMAP; 100 ℃ of water-baths were reacted 6 hours down; Collect 100 ℃ of fractions, reaction finishes, and it is brown that solution is; Cooling adds dehydrated alcohol and does not generate to there being deposition; Filter collecting precipitation, washing is drying to obtain PEG-HA;
(3) preparation of water-soluble CdTe/CdS quantum dot
Synthesizing of water-soluble CdTe/CdS core-shell type nano quantum dot mainly is divided into two steps: at first synthetic CdTe nuclear Nano sol; And then outside CdTe, coat the CdS shell; 0.2400g tellurium powder and 0.1400g sodium borohydride are added in the aqueous solution of 10mL, and under the condition of nitrogen protection, the room temperature lower magnetic force stirs 3h, obtains the precursor liquid of the bolarious Te of containing; Take by weighing 0.080g CdCl
2Be dissolved in the 30mL distilled water, add 100 μ L mercaptopropionic acids, with the pH value of the NaOH solution regulator solution of concentration 1mol/L to 8-10; Solution is added in the 100mL three-necked bottle, behind the logical nitrogen 20min, join the CdCl under the nitrogen protection to freshly prepd NaHTe
2In the solution, the color of solution becomes the transparent orange yellow, and stirring at room 20min is transferred in 90 ℃ the water bath with thermostatic control then, behind the reaction 0.5h, splashes into the Na of 5 μ mol/L with the speed of 0.1mL/min
2S solution 3ml obtains the CdTe/CdS core-shell quanta dots; Total overall reaction is accomplished under nitrogen protection; Behind the reaction terminating, add the ethanol of 15mL, centrifugalize 10min under 5000r/min; Discard the supernatant, add 10mL water deposition is suspended again, repeat this process 2 times; The solid that obtains at room temperature dries naturally, and obtains CdTe/CdS quantum dot (QDs) pressed powder with the crucible grinding;
(4) preparation of PEG-HA-QDs
It is soluble in water to take by weighing 0.200g QDs, and room temperature lucifuge magnetic agitation is all dissolved to QDs, adds 0.50g NHS and 0.30gEDC, adds the aqueous solution 20ml of PEG-HA of the 0.3g/ml of step (2) preparation behind the 1h, room temperature lucifuge magnetic agitation 12h; The products therefrom mixed liquor is that 7.4 phosphate buffer is dialysed with bag filter at pH, and lyophilization gets the PEG-HA-QDs complex;
(5) preparation of BA-MEL
In the three-neck flask that thermometer, water knockout drum are housed, add melphalan 1.200g, benzyl alcohol 10ml, sodium sulfite 0.800g reacts under magnetic agitation; Reacted mixed liquor is used distilled water, saturated sodium bicarbonate, distilled water wash successively, obtain grease; Grease after the washing is evaporated with Rotary Evaporators; Remove unreacted benzyl alcohol 1 in the mixture; The temperature of setting rotary evaporation is 60 ℃, and evaporation is till no distillation, and the material that obtains carries out recrystallization with alcohol-ether; Its ethanol and ether volume ratio are 1:2, obtain milky solid BA-MEL;
(6) preparation of PEG-HA-QDs-MEL
PEG-HA-QDs600mg, DCC520mg and 0.30mmol4-dimethylamino naphthyridine (DMAP) that step (2) is obtained are dissolved in the 30mL anhydrous dimethyl sulphoxide, and room temperature lucifuge magnetic agitation is all dissolved (about 1 hour) to PEG-HA-QDs; Add 10mg/mL BA-MEL aqueous solution (pH4.7) 30mL, room temperature lucifuge magnetic agitation is spent the night, and adds the 40ml tert-butyl alcohol, through the hydrocarbon catalytic eliminating benzyl alcohol of 5wt% palladium; Using pH is 7.4 phosphate buffer dialysis 3 days, reuse distilled water dialysis 3 days; Lyophilization gets reactant powders, promptly gets product P EG-HA-QDs-MEL.
2. MTT experiment (the ovarian cancer SKOV of receptor-mediated quantum dot spike targeting drug delivery system
3Cell)
(1) uses behind the trypsinization preparation to become concentration in the cell of exponential phase and be 2-10 * 10
4The cell suspending liquid of individual/ml is inoculated in 96 orifice plates through after the accurate cell technology by 500 cells/well, and every hole adds 100 μ l.
(2) flat board is put 37 ℃, contained volumetric concentration 5%CO
2And the saturated humidity condition is after following 24 hours; Add each cell sample corresponding drug serum, normal saline serum and diluted good medicine; Each concentration of specimens is established three parallel holes, and matched group adds the culture fluid 100 μ l that do not contain sample, puts into incubator again and hatches 72 hours.
(3) every hole adds with pressing the freshly prepared MTT solution 50 μ l of 5mg/ml, and incubation 4 hours makes MTT be reduced to the first a ceremonial jade-ladle, used in libation; When seeing that under inverted microscope thread purple crystal body appears in cell peripheral in the orifice plate, outwell supernatant; Every hole adds dimethyl sulfoxide (DMSO) 220 μ l, after shaking up with dull and stereotyped shaking table, uses ELIASA to measure OD value (OD) (detecting wavelength 570nm); Handling cell with solvent control is matched group, by formula the suppression ratio of computerized compound pair cell.
The MTT experimental result
3. this drug-supplying system range of application
Melphalan also is Melphalan, L-Sarcolysinum, L-sarcolysin; English name: Melphalan or Alkeran Melphalanum; Malignant lymphoma, breast carcinoma, ovarian cancer, spermocytoma, chronic leukemia, polycythemia vera, child's neuroblastoma in late period, thyroid carcinoma there is better curative effect; Arterial perfusion can also be treated limb malignant tumor, like malignant melanoma, osteosarcoma and soft tissue sarcoma etc.
Embodiment 4
1.PEG-HA-QDs-MEL the preparation of component
(1) the synthetic and purification of end carboxy polyethylene glycol
Place 500mL to have the there-necked flask of condensing tube, agitator and thermometer 60g (5mmol) PEG-1500, add 300mL chloroform (CaH
2Steam under existing) dissolving, to treat to add 7.5g (25mmol) succinic anhydride after dissolving finishes, heating for dissolving adds the 12mL dry pyridine simultaneously; Stir refluxed reaction 6 hours, the stopped reaction postcooling is to room temperature, and reaction gained mixture reduction vaporization is to being thick, and residue adds a large amount of absolute ethers; Separate out the white precipitate product, sucking filtration is with 180mL dichloromethane lysate, elimination insoluble matter; Add the absolute ether deposition again, separate out the white precipitate product, sucking filtration repeats aforesaid operations 3 times; The sucking filtration final vacuum is dried to constant weight, must hold carboxy polyethylene glycol (PEG-SA-NHS), weighs;
(2) preparation of PEG-HA
The NaOH aqueous solution of getting 18.0g hyaluronic acid and 40mL concentration 20wt% mechanical agitation reaction at normal temperatures 12 hours, deacetylate; Solution was dialysed in distilled water 2 days, and lyophilization obtains deacetylated hyaluronic acid freeze-dried powder; The 3.000g end carboxy polyethylene glycol that step (1) is obtained is dissolved in the hyaluronic acid hydrolyzed solution, pours in the 100mL there-necked flask that dephlegmator and agitator are housed, and adds catalyst EDC1.500g, DMAP0.120g; 100 ℃ of water-baths were reacted 6 hours down; Collect 100 ℃ of fractions, reaction finishes, and it is brown that solution is; Cooling adds dehydrated alcohol and does not generate to there being deposition; Filter collecting precipitation, washing is drying to obtain PEG-HA;
(3) preparation of water-soluble CdTe/CdS quantum dot
Synthesizing of water-soluble CdTe/CdS core-shell type nano quantum dot mainly is divided into two steps: at first synthetic CdTe nuclear Nano sol; And then outside CdTe, coat the CdS shell; 0.7200g tellurium powder and 0.4200g sodium borohydride are added in the aqueous solution of 20mL, and under the condition of nitrogen protection, the room temperature lower magnetic force stirs 3h, obtains the precursor liquid of the bolarious Te of containing; Take by weighing 0.240g CdCl
2, be dissolved in the 40mL distilled water, add 250 μ L mercaptopropionic acids, with the pH value of the NaOH solution regulator solution of concentration 1mol/L to 8-10; Solution is added in the 100mL three-necked bottle, behind the logical nitrogen 20min, join the CdCl under the nitrogen protection to freshly prepd NaHTe
2In the solution, the color of solution becomes the transparent orange yellow, and stirring at room 20min is transferred in 90 ℃ the water bath with thermostatic control then, behind the reaction 0.5h, splashes into the Na of 5 μ mol/L with the speed of 0.1mL/min
2S solution 4ml obtains the CdTe/CdS core-shell quanta dots; Total overall reaction is accomplished under nitrogen protection; Behind the reaction terminating, add the ethanol of 15mL, centrifugalize 10min under 5000r/min; Discard the supernatant, add 10mL water deposition is suspended again, repeat this process 2 times; The solid that obtains at room temperature dries naturally, and obtains CdTe/CdS quantum dot pressed powder with the crucible grinding;
(4) preparation of PEG-HA-QDs
It is soluble in water to take by weighing 0.500g QDs; Room temperature lucifuge magnetic agitation is all dissolved to QDs; Add 1.25g NHS and 0.80gEDC, add the aqueous solution 20ml of PEG-HA of the 0.4g/mll of step (2) preparation behind the 1h, room temperature lucifuge magnetic agitation 12h; The products therefrom mixed liquor is that 7.4 phosphate buffer is dialysed with bag filter at pH, and lyophilization gets the PEG-HA-QDs complex;
(5) preparation of BA-MEL
In the three-neck flask that thermometer, water knockout drum are housed, add melphalan 3.000g, benzyl alcohol 10ml, sodium sulfite 1.600g reacts under magnetic agitation; Reacted mixed liquor is used distilled water, saturated sodium bicarbonate, distilled water wash successively, obtain grease; Grease after the washing is evaporated with Rotary Evaporators; Remove unreacted benzyl alcohol 1 in the mixture; The temperature of setting rotary evaporation is 60 ℃, and evaporation is till no distillation, and the material that obtains carries out recrystallization with alcohol-ether; Its ethanol and ether volume ratio are 1:2, obtain milky solid BA-MEL;
(6) preparation of PEG-HA-QDs-MEL
PEG-HA-QDs1800mg, DCC1560mg that step (2) is obtained; 0.35mmol4-dimethylamino naphthyridine is dissolved among the anhydrous dimethyl sulphoxide 50mL; Room temperature lucifuge magnetic agitation is all dissolved (about 1 hour) to PEG-HA-QDs, adding 20mg/mL BA-MEL aqueous solution (pH4.7) 30mL, and room temperature lucifuge magnetic agitation is spent the night; Add the 40ml tert-butyl alcohol, through the hydrocarbon catalytic eliminating benzyl alcohol of 5wt% palladium; Using pH is 7.4 phosphate buffer dialysis 3 days, reuse distilled water dialysis 3 days; Lyophilization gets reactant powders, promptly gets product P EG-HA-QDs-MEL;
2. MTT experiment (the ovarian cancer SKOV of receptor-mediated quantum dot spike targeting drug delivery system
3Cell)
(1) uses behind the trypsinization preparation to become concentration in the cell of exponential phase and be 2-10 * 10
4The cell suspending liquid of individual/ml is inoculated in 96 orifice plates through after the accurate cell technology by 500 cells/well, and every hole adds 100 μ l.
(2) flat board is put 37 ℃, contained volumetric concentration 5%CO
2And the saturated humidity condition is after following 24 hours; Add each cell sample corresponding drug serum, normal saline serum and diluted good medicine; Each concentration of specimens is established three parallel holes, and matched group adds the culture fluid 100 μ l that do not contain sample, puts into incubator again and hatches 72 hours.
(3) every hole adds with pressing the freshly prepared MTT solution 50 μ l of 5mg/ml, and incubation 4 hours makes MTT be reduced to the first a ceremonial jade-ladle, used in libation; When seeing that under inverted microscope thread purple crystal body appears in cell peripheral in the orifice plate, outwell supernatant; Every hole adds dimethyl sulfoxide (DMSO) 220 μ l, after shaking up with dull and stereotyped shaking table, uses ELIASA to measure OD value (OD) (detecting wavelength 570nm); Handling cell with solvent control is matched group, by formula the suppression ratio of computerized compound pair cell.
The MTT experimental result
3. this drug-supplying system range of application
Melphalan also is Melphalan, L-Sarcolysinum, L-sarcolysin; English name: Melphalan or Alkeran Melphalanum; Malignant lymphoma, breast carcinoma, ovarian cancer, spermocytoma, chronic leukemia, polycythemia vera, child's neuroblastoma in late period, thyroid carcinoma there is better curative effect; Arterial perfusion can also be treated limb malignant tumor, like malignant melanoma, osteosarcoma and soft tissue sarcoma etc.
Embodiment 5
1.PEG-HA-QDs-MEL the preparation of component
(1) the synthetic and purification of end carboxy polyethylene glycol
Place 500mL to have the there-necked flask of condensing tube, agitator and thermometer 120g (5mmol) PEG-1500, add 350mL chloroform (CaH
2Steam under existing) dissolving, to treat to add 7.5g (25mmol) succinic anhydride after dissolving finishes, heating for dissolving adds the exsiccant pyridine of 24mL simultaneously; Stir refluxed reaction 6 hours, the stopped reaction postcooling is to room temperature, and reaction gained mixture reduction vaporization is to being thick, and residue adds a large amount of absolute ethers; Separate out the white precipitate product, sucking filtration, usefulness, 270mL dichloromethane lysate; The elimination insoluble matter adds the absolute ether deposition again, separates out the white precipitate product, sucking filtration; Repeat aforesaid operations 3 times, the sucking filtration final vacuum is dried to constant weight, must hold carboxy polyethylene glycol (PEG-SA-NHS), weighs;
(2) preparation of PEG-HA
The NaOH aqueous solution of getting 36.0g hyaluronic acid and 80mL concentration 20wt% mechanical agitation reaction at normal temperatures 12 hours, deacetylate; Solution was dialysed in distilled water 2 days, and lyophilization obtains deacetylated hyaluronic acid freeze-dried powder.The 6.000g end carboxy polyethylene glycol that step (1) is obtained is dissolved in the hyaluronic acid hydrolyzed solution, pours in the 100mL there-necked flask that thorn shape dephlegmator and agitator are housed, and adds catalyst EDC3.000g, DMAP0.240g; 100 ℃ of water-baths were reacted 6 hours down; Collect 100 ℃ of fractions, reaction finishes, and it is brown that solution is; Cooling adds dehydrated alcohol and does not generate to there being deposition; Filter collecting precipitation, washing is drying to obtain PEG-HA;
(3) preparation of water-soluble CdTe/CdS quantum dot
Synthesizing of water-soluble CdTe/CdS core-shell type nano quantum dot mainly is divided into two steps: at first synthetic CdTe nuclear Nano sol; And then outside CdTe, coat the CdS shell.1.400g tellurium powder and 0.8400g sodium borohydride are added in the aqueous solution of 30mL, and under the condition of nitrogen protection, the room temperature lower magnetic force stirs 3h, obtains the precursor liquid of the bolarious Te of containing; Take by weighing CdCl
20.480g, be dissolved in the 80mL distilled water, add 250 μ L mercaptopropionic acids, with the pH value of the NaOH solution regulator solution of concentration 1mol/L to 8-10; Solution is added in the 250mL three-necked bottle, behind the logical nitrogen 20min, join the CdCl under the nitrogen protection to freshly prepd NaHTe
2In the solution, the color of solution becomes the transparent orange yellow, and stirring at room 20min is transferred in 90 ℃ the water bath with thermostatic control then, behind the reaction 0.5h, splashes into the Na of 5 μ mol/L with the speed of 0.1mL/min
2S solution 5ml obtains the CdTe/CdS core-shell quanta dots; Total overall reaction is accomplished under nitrogen protection; Behind the reaction terminating, add the ethanol of 20mL, centrifugalize 10min under 5000r/min; Discard the supernatant, add 20mL water deposition is suspended again, repeat this process 2 times; The solid that obtains at room temperature dries naturally, and obtains CdTe/CdS quantum dot pressed powder with the crucible grinding;
(4) preparation of PEG-HA-QDs
It is soluble in water to take by weighing 1.000g QDs; Room temperature lucifuge magnetic agitation is all dissolved to QDs; Add 2.5.g NHS and 1.60gEDC, add the aqueous solution 20ml of PEG-HA of the 0.5g/ml of step (2) preparation behind the 1h, room temperature lucifuge magnetic agitation 12h; The products therefrom mixed liquor is that 7.4 phosphate buffer is dialysed with bag filter at pH, and lyophilization gets the PEG-HA-QDs complex;
(5) preparation of BA-MEL
In the three-neck flask that thermometer, water knockout drum are housed, add melphalan 6.000g, benzyl alcohol 20ml, sodium sulfite 3.200g reacts under magnetic agitation; Reacted mixed liquor is used distilled water, saturated sodium bicarbonate, distilled water wash successively, obtain grease; Grease after the washing is evaporated with Rotary Evaporators; Remove unreacted benzyl alcohol 1 in the mixture; The temperature of setting rotary evaporation is 60 ℃, and evaporation is till no distillation, and the material that obtains carries out recrystallization with alcohol-ether; Its ethanol and ether volume ratio are 1:2, obtain milky solid BA-MEL;
(6) preparation of PEG-HA-QDs-MEL
PEG-HA-QDs3.60g, 3.02g DCC and 0.70mmol 4-dimethylamino naphthyridine (DMAP) that step (2) is obtained are dissolved in the 50mL anhydrous dimethyl sulphoxide; Room temperature lucifuge magnetic agitation is all dissolved (about 1 hour) to PEG-HA-QDs; Add 20mg/mL BA-MEL aqueous solution (pH4.7) 30mL; Room temperature lucifuge magnetic agitation is spent the night, and adds the 40ml tert-butyl alcohol, through the hydrocarbon catalytic eliminating benzyl alcohol of 5wt% palladium; Using pH is 7.4 phosphate buffer dialysis 3 days, reuse distilled water dialysis 3 days; Lyophilization gets reactant powders, promptly gets product P EG-HA-QDs-MEL.
2. MTT experiment (the ovarian cancer SKOV of receptor-mediated quantum dot spike targeting drug delivery system
3Cell)
(1) uses behind the trypsinization preparation to become concentration in the cell of exponential phase and be 2-10 * 10
4The cell suspending liquid of individual/ml is inoculated in 96 orifice plates through after the accurate cell technology by 500 cells/well, and every hole adds 100 μ l.
(2) flat board is put 37 ℃, contained volumetric concentration 5%CO
2And the saturated humidity condition is after following 24 hours; Add each cell sample corresponding drug serum, normal saline serum and diluted good medicine; Each concentration of specimens is established three parallel holes, and matched group adds the culture fluid 100 μ l that do not contain sample, puts into incubator again and hatches 72 hours.
(3) every hole adds with pressing the freshly prepared MTT solution 50 μ l of 5mg/ml, and incubation 4 hours makes MTT be reduced to the first a ceremonial jade-ladle, used in libation; When seeing that under inverted microscope thread purple crystal body appears in cell peripheral in the orifice plate, outwell supernatant; Every hole adds DMSO (dimethyl sulfoxide) 220 μ l, after shaking up with dull and stereotyped shaking table, uses ELIASA to measure OD value (OD) (detecting wavelength 570nm); Handling cell with solvent control is matched group, by formula the suppression ratio of computerized compound pair cell.
The MTT experimental result
3. this drug-supplying system range of application
Melphalan also is Melphalan, L-Sarcolysinum, L-sarcolysin; English name: Melphalan or Alkeran Melphalanum; Malignant lymphoma, breast carcinoma, ovarian cancer, spermocytoma, chronic leukemia, polycythemia vera, child's neuroblastoma in late period, thyroid carcinoma there is better curative effect; Arterial perfusion can also be treated limb malignant tumor, like malignant melanoma, osteosarcoma and soft tissue sarcoma etc.
Claims (3)
1. receptor-mediated quantum dot spike melphalan targeting drug delivery system is characterized in that it is Polyethylene Glycol-hyaluronic acid-quantum dot-melphalan complex; Be abbreviated as PEG-HA-QDs-MEL, be connected with the condensation of HA dehydration of amide, and insert water-soluble CdTe/CdS quantum dot at HA by PEG; Last and medicine MEL dehydration of amide condensation prepared obtains; Wherein, the constituent content of PEG-HA-QDs-MEL is counted as follows by weight, and each weight portion is 1 milligram:
Melphalan 30-100 part, PEG 600-1500 part, HA 200-500 part; CdTe/CdS quantum dot 5-50 part, N, N' dicyclohexylcarbodiimide 20-50 part; 4-dimethylamino naphthyridine 1-10 part, succinic anhydride 100-500 part, N-hydroxy thiosuccinimide 20-50 part; 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide 20-50 part, sodium sulfite 10-100 part.
2. the method for preparing of receptor-mediated quantum dot spike melphalan targeting drug delivery system as claimed in claim 1 is characterized in that, may further comprise the steps, and following each weight portion is 1 milligram:
1) the synthetic and purification of end carboxy polyethylene glycol
600-1500 weight portion Polyethylene Glycol is placed the there-necked flask that has condensing tube, agitator and thermometer, add dissolved in chloroform, treat to add 100-500 weight portion succinic anhydride, heating for dissolving after dissolving finishes; Add exsiccant pyridine simultaneously, stir refluxed reaction 2-10 hour, the stopped reaction postcooling is to room temperature; Reaction gained mixture reduction vaporization adds the absolute ether deposition again and obtains white product to being thick, and sucking filtration gets filter cake; Reuse dichloromethane dissolving filter cake, the elimination insoluble matter adds the absolute ether deposition and obtains white product; Repeat aforesaid operations more than 2 times, the filter cake vacuum drying obtains the end carboxy polyethylene glycol of purification to constant weight behind the sucking filtration;
2) preparation of PEG-HA
The end carboxy polyethylene glycol that step 1) is obtained is dissolved in the hyaluronic hydrolyzed solution of 200-500 weight portion; Pour the there-necked flask that dephlegmator and agitator are housed into; Add catalyst 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide 10-50 weight portion and 4-dimethylamino naphthyridine 0.5-5 weight portion; Collect 100 ℃ of fractions in reaction under 60-140 ℃ after 2-10 hour, reaction finishes postcooling; Add dehydrated alcohol and do not generate, filter collecting precipitation to there being deposition, washing precipitation, dry sediment promptly gets PEG-HA;
3) preparation of water-soluble CdTe/CdS quantum dot
5-20 weight portion tellurium powder and 2-10 weight portion sodium borohydride are added in the entry, and under the nitrogen protection condition, the room temperature lower magnetic force stirs 2-5h, obtains the NaHTe precursor liquid that kermesinus contains Te; Take by weighing 2-10 weight portion CdCl
2Be dissolved in the distilled water, add 2-10 weight portion mercaptopropionic acid, with the pH value of the NaOH solution regulator solution of concentration 1mol/L-2 mol/L to 8-10; Add solution in the three-necked bottle, join the CdCl under the nitrogen protection to freshly prepd NaHTe
2In the solution, the color of solution becomes the transparent orange yellow, and stirring at room is transferred in 90 ℃ the water bath with thermostatic control behind the reaction 0.5-2 h then, splashes into concentration 1 μ mol/L-20 μ mol/L Na with 0.1ml/min speed
2The solution 1 ml-15 ml of S obtains the CdTe/CdS core-shell quanta dots; Total overall reaction is accomplished under nitrogen protection, behind the reaction terminating, adds ethanol; Centrifugalize 10-15 min discards the supernatant under 5 000 r/min, adds water deposition is suspended again; Repeat this process 2 times; The solid that obtains at room temperature dries naturally, grinds and obtains CdTe/CdS quantum dot pressed powder, is abbreviated as QDs;
4) preparation of PEG-HA-QDs
It is soluble in water to take by weighing 5-50 weight portion QDs; Room temperature lucifuge magnetic agitation is all dissolved to QDs; Add 5-50 weight portion N-hydroxy thiosuccinimide and 5-50 weight portion 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide, add step 2 behind the 1h) the aqueous solution 20ml of the PEG-HA of the 0.1g/ml-1g/ml for preparing, room temperature lucifuge magnetic agitation 12 h; The products therefrom mixed liquor is that 7.4 phosphate buffer is dialysed with bag filter at pH, and lyophilization gets the PEG-HA-QDs complex;
5) preparation of American and French human relations benzyl ester
In the three-neck flask that thermometer, water knockout drum are housed, add melphalan 30-100 weight portion, sodium sulfite 10-100 weight portion and excessive benzene methanol react under magnetic agitation, and the mixed liquor of reaction back gained is used distilled water, saturated sodium bicarbonate and distilled water wash successively; Obtain grease, the grease after the washing is evaporated with Rotary Evaporators, remove unreacted benzene methanol; The temperature of setting rotary evaporation is 30-60 ℃; Evaporation is till no distillation, and the material that evaporation obtains carries out recrystallization with alcohol-ether, and its ethanol and ether volume ratio are 1:2; Obtain the American and French human relations benzyl of milky solid ester, be abbreviated as BA-MEL;
6) preparation of PEG-HA-QDs-MEL
100-500 weight portion PEG-HA-QDs complex, 10-50 weight portion N that step 4) is obtained; N' dicyclohexylcarbodiimide and 0.5-5 weight portion 4-dimethylamino naphthyridine are dissolved in the anhydrous dimethyl sulphoxide, and room temperature lucifuge magnetic agitation is all dissolved to PEG-HA-QDs, the 10-50 weight portion BA-MEL aqueous solution of the pH4.7 of adding step 5) gained; Room temperature lucifuge magnetic agitation 12 h; It is carried the hydrogenation of palladium 0.5wt%-15wt% catalyst through carbon remove benzyl alcohol in the tert-butyl alcohol, using pH then is 7.4 phosphate buffer dialysis 24-72h, reuse distilled water dialysis 24-72h; Lyophilization gets reactant powders, i.e. the PEG-HA-QDs-MEL product.
3. the application of receptor-mediated quantum dot spike melphalan targeting drug delivery system as claimed in claim 1 is characterized in that, is applied to prepare antitumor drug.
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