CN103961715A - Self-assembled adriamycin nano medicine precursor and preparation method and application thereof - Google Patents
Self-assembled adriamycin nano medicine precursor and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a self-assembled adriamycin nano medicine precursor. According to the invention, a hydrophilic poly ethylene glycol segment is used as a carrier, and the self-assembled Adriamycin nano medicine precursor has the structure shown in formula I; the segment (shown in the description) has the weight-average molecular weight of 2,000-20,000. The invention further discloses a preparation method and application of the self-assembled adriamycin nano medicine precursor. The prepared nano precursor is high in drug-loading capacity, high in yield, and suitable for industrial production; molecular weight of the hydrophilic poly ethylene glycol segment bonded with hydrophobic DOX molecule is controlled within the range of 2,000-20,000, so that the blood cycling time of the nano medicine is remarkably prolonged, and the tumor targeting capacity of the medicine is improved; high antineoplastic activity is achieved with respect to various sensitive and drug-resisting tumor cell series such as BCap37, KB, MCF-7, MCF-7/DAR and KBv200; particularly, remarkable antineoplastic activity is achieved in drug-resisting tumor cell series which are difficult to handle in clinical trials.
Description
Technical field
The present invention relates to biological medicine technology field, be specifically related to doxorubicin nanometer medicament precursor of a kind of self assembly and its preparation method and application.
Background technology
Amycin (being called for short DOX), another name doxorubicin, doxorubicin or Adriamycin, English Doxorubicin by name or Adriamycin, the commercially available doxorubicin hydrochloride that mostly is, molecular formula is C
27h
29nO
11hCl, molecular weight is that 580.02, CAS is numbered 25316-40-9, structural formula is as follows:
Amycin is a kind of anthracycline broad-spectrum anti-cancer drug, is non-specific antibiotic of cycle, and it is macromolecular synthetic that the main activity by the intercalation of DNA, inhibition typeⅡtopoisomerase suppresses DNA, RNA, protein etc., thus the apoptosis of induced tumor cell.But, due to its water solublity extreme difference, can bring out serious toxic and side effects, and can produce drug resistance by induced tumor, its clinical practice is greatly limited.Drug resistance of tumor is the one of the main reasons that causes chemotherapy failure, tumor recurrence and transfer.
Nano medication has broad application prospects, can utilize high-permeability and retention effect (being EPR effect) the passive target tumor tissues of tumor, make optionally enrichment in tumor tissues of cancer therapy drug, reach the object (H.Maeda that effective killing off tumor cells reduces toxic and side effects simultaneously, H.Nakamura and J.Fang, the EPR effect for macromolecular drug delivery tosolid tumors:Improvement of tumor uptake, lowering of systemic toxicity anddistinct tumor imaging in vivo, Adv Drug Deliv Rev, 2013, 65, 71).
Now widely used Nano medication preparation method is for utilizing liposome or nano-micelle to carry out physically trapping to medicine.The people such as D.Kim (D.Kim, E.S.Lee, K.T.Oh, Gao Z.G.and Y.H.Bae, Doxorubicin-loaded polymeric micelle overcomes multidrug resistance ofcancer by double-targeting folate receptor and early endosomal pH, Small, 2008,4,2043) with polymer bag, carry DOX and form nano-micelle, utilize receptor target tumor tissues simultaneously, and further studied the overcome effect of this adriamycin nano micelle to multi-drug resistance of the tumor.The people such as R.I.Pakunlu (R.I.Pakunlu, Wang Y., M.Saad, J.J.Khandare, V.Starovoytov and T.Minko, In vitro and in vivo intracellular liposomal deliveryof antisense oligonucleotides and anticancer drug, J Control Release, 2006,114,153) with liposome entrapment antisense oligonucleotide and cancer therapy drug, and in two aspects, studied in vitro transportation in the born of the same parents of gained liposome in body.The liposome medicament of amycin has entered clinical use.
(Y.Barenholz,
--the first FDA-approved nano-drug:Lessonslearned, J Control Release, 2012,160,117) be first Nano medication of U.S. FDA approval, for the polyoxyl 40 stearate plastid of DOX, develop the earliest to treat Kaposi sarcoma, now also by FDA approval, be used for the treatment of ovarian cancer and multiple myeloma.Yet " the red and swollen disease of acra " (claiming again " brothers' syndrome ") that Doxil causes limited the effectiveness of its treatment tumor.
be non-polyoxyl 40 stearate plastid amycin, in Europe and Canada, go through and cyclophosphamide combined treatment metastatic breast cancer.But these nanometer formulations of preparing by physically trapping all need high amount of drug carrier, there is the defects such as drug loading is low, burst drug release.
It is the another kind of effective means (R.Duncan, Polymer conjugates as anticancer nanomedicines, 2006, Nat RevCancer6,688) that builds Nano medication that cancer therapy drug is bonded on hydrophilic support.People (the Zhu S. such as Zhu S., Qian L., Hong M., Zhang L., Pei Y., andJiang Y., RGD-modified PEG-PAMAM-DOX conjugate:In vitro and in vivotargeting to both tumor neovascular endothelial cells and tumor cells, 2011, Adv Mater23, H84) by DOX and PEG, PAMAM keyed jointing forms key compound PEG-PAMAM-DOX, and modified with functional protein RGD, the Nano medication of gained is target tumor cell and endothelial cells in tumor neogenetic blood vessels simultaneously, its in vivo and in vitro has all obtained good result.The people such as M.Sirova (M.Sirova, T.Mrkvan, T.Etrych, P.Chytil, P.Rossmann, M.Ibrahimova, L.Kovar, K.Ulbrich and B.Rihova, Preclinicalevaluation of linear HPMA-doxorubicin conjugates with pH-sensitive drugrelease:Efficacy, safety, and immunomodulating activity in murine model, 2010, Pharmaceutical Research27, 200) by DOX and hydrophile linearity polymer HPMA keyed jointing, the HPMA-DOX key compound of gained has the responsive release characteristics of pH, and it is active to the anti-tumor in vivo of this key compound to set up animal model, biological safety and immunoregulatory activity have carried out clinical front evaluation.The Nano medication obtaining by chemical bonding mode, structure is more stable conventionally, forms clearly simultaneously, and technique is also more stable, and can avoid burst drug release phenomenon.But, similar with embedded nano medicine, the also not high shortcoming of ubiquity drug loading.
Publication number is that the Chinese patent literature of CN102188717B discloses a kind of self-emulsifying doxorubicin nanometer medicament and preparation method thereof; by hydrophilic small molecules group or hydrophilic polymer short chain (PEG200-1900) are incorporated on hydrophobic DOX molecule and become hydrophilic-hydrophobic type molecule, granule or vesicle that can self-assembled nanometer size in water.Although the drug loading of self-emulsifying doxorubicin nanometer medicament prepared by the method is very high, but because the molecular weight of the hydrophilic polymer short chain of introducing is low, make the time of its blood circulation shorter, the cancer target effect of EPR effect mediation is limited, and not obvious to the therapeutic effect of drug-resistant tumor cell.And antitumor drug induction system efficient, low toxicity not only wants to overcome some inherent shortcomings of original small-molecule drug, also need to there is desirable drug loading simultaneously, particle diameter, controllable release, blood circulation time, cancer target ability, and can kill and wound the physics and chemistry such as drug-resistant tumor and biological characteristics.
Summary of the invention
The invention provides a kind of doxorubicin nanometer medicament precursor of self assembly, by by the molecular weight control of the hydrophilic polyglycol segment with hydrophobic DOX molecular linkage 2000～20000, significant prolongation the blood circulation time of Nano medication, improved EPR effect, and confirm that this Nano medication precursor all shows good anti-tumor activity in the multiple sensitivities such as BCap37, MCF-7, MCF-7/DAR, KB and KBv200 and drug-resistant tumor cell line, especially significantly improves compared with the former medicine of amycin the anti-tumor activity of drug-resistant tumor cell line.
The invention discloses a kind of doxorubicin nanometer medicament precursor of self assembly, take hydrophilic polyglycol segment as carrier, the doxorubicin nanometer medicament precursor of described self assembly has the structure as shown in formula I:
Ⅰ；
In formula, described segment
weight average molecular weight be 2000～20000.
The doxorubicin nanometer medicament precursor of self assembly prepared by the present invention, take amycin as active medicine, take hydrophilic polyglycol segment as carrier, by the carbonyl of 8 glycolyls on amycin, by degradable chemical bond and hydrophilic group, is formed by connecting.Degradable chemical bond is convenient to doxorubicin nanometer medicament precursor degradation in vivo slow release DOX.Degradable chemical bond described herein is selected from ester bond, carbonic acid ester bond, phosphoric acid ester bond, hydrazone key, imine linkage, amido link etc.
As preferably, described segment
weight average molecular weight be 2000～5000; Further preferably, described segment
weight average molecular weight be 2000.Result of study confirms the weight average molecular weight of described segment preferably in above-mentioned scope, not only makes nano-prodrug have the advantages such as high drug load, blood circulation time be long, also makes this nano-prodrug have characteristic and the advantage of Efficient killing effect several drug resistance tumor cell.
The preparation method that the invention also discloses a kind of doxorubicin nanometer medicament precursor of described self assembly, step is as follows:
(1) the first organic solvent by terminal hydroxy group Polyethylene Glycol, metal hydride and after dewatering mixes, and room temperature reaction is to without Bubble formation, then adds anhydrous halogenated acetic acids ethyl ester, through post processing, obtains intermediate product A, and structural formula is suc as formula shown in a;
(2) intermediate product A step (1) being obtained and hydrazine hydrate back flow reaction 5～10h in the second organic solvent, standing cooling after, after rotary evaporation, precipitation and sucking filtration, obtain intermediate product B, structural formula is suc as formula shown in b;
(3), under weak acid catalyst, in the intermediate product B that step (2) is obtained, amycin the 3rd organic solvent after dewatering, react, then through dialysis, lyophilization, obtain the doxorubicin nanometer medicament precursor of described self assembly;
formula a;
formula b;
Wherein, described segment
weight average molecular weight be 2000～20000.
As preferably, described segment
weight average molecular weight be 2000～5000; Further preferably, described segment
weight average molecular weight be 2000.
As preferably, the metal hydride that step (1) is described and the mol ratio of terminal hydroxy group Polyethylene Glycol are 1～1.5:1; Described halogenated acetic acids ethyl ester is bromoacetate, with the mol ratio of terminal hydroxy group Polyethylene Glycol be 1～2:1.
The last handling process that step (1) is described, is specially:
After rotary evaporation and extraction, get organic facies and remove extractant precipitation, then process through rotary evaporation.
Intermediate product A prepared by step (1) is for take the Polyethylene Glycol that ethyl acetate is end group, and molecular formula can be expressed as PEG-OCH
2cOOEt, the weight average molecular weight of PEG is 2000～20000, concrete structure can be characterized and be confirmed by nuclear-magnetism.
As preferably, the described hydrazine hydrate of step (2) is 80～120:1 with the mol ratio of intermediate product A, and the intermediate product B of preparation be take the Polyethylene Glycol that hydrazide group is end group, and molecular formula can be expressed as PEG-OCH
2cONHNH
2, concrete structure also can be characterized and be confirmed by nuclear-magnetism.
As preferably, the amycin that step (3) is described and the mol ratio of intermediate product B are 0.5～2:1; Described weak acid catalyst is selected from phosphoric acid, acetic acid or trifluoroacetic acid, is preferably trifluoroacetic acid.
The doxorubicin nanometer medicament precursor that step (3) prepares, molecular formula can be expressed as PEG-DOX, and concrete structure can be characterized and be confirmed by nuclear-magnetism and high performance liquid chromatography (HPLC).
First, second, and third described organic solvent is independently selected from methanol, ethanol, oxolane, dimethyl sulfoxide or N, N '-dimethyl formamide, as preferably, described the first organic solvent and the second organic solvent are oxolane, and the 3rd organic solvent is methanol.
Described preparation method, to temperature and not too much restriction of pressure, all can be carried out the chemical reaction that degradable in vivo chemical bond is connected with hydrophilic group under normal temperature and pressure, aerobic or oxygen free condition.
As preferably, described degradable in vivo chemical bond is hydrazone key, can obtain better pH response release function, guarantees that doxorubicin nanometer medicament precursor effectively discharges medicine at tumor tissues place.
Except the preparation method providing in the present invention, described hydrazone key also can be by the amino (PEG-NH of Polyethylene Glycol
2) or Polyethylene Glycol hydrazine (PEG-NHNH
2) react and make with amycin.
The application of the doxorubicin nanometer medicament precursor that the invention also discloses described self assembly in preparing overriding resistance tumour medicine.
The PEG-DOX that the present invention is prepared is added to the water, and through self assembly, can form micelle or vesicle (or claiming nano-particle).
Because described amycin self assembly prodrug is amphiphilic macromolecular, thereby both can in the hydrophilic solvents such as water, buffer salt solution, normal saline, carry out self assembly formation micelle or vesicle, also can in hydrophobic solvent, be self-assembled into micelle or vesicle.From medical usage, consider, preferred solvent harmless, that be applicable to Human Physiology environment, as water, physiological buffer saline etc.
Compared with prior art, the present invention has following beneficial effect:
(1) the present invention, by introduce hydrophilic PEG on DOX surface, has greatly increased the dissolubility of DOX in water, overcomes its tissue distribution poor selectivity, defect that toxic and side effects is large;
(2) the present invention prepares has amphipathic Nano medication precursor PEG-DOX, can form nano-particle in self assembly under physiological condition, and have good stability;
(3) the PEG-DOX Nano medication precursor that the present invention prepares has not only kept high drug loading, can also in weakly acidic tumor tissues, by pH, respond and discharge DOX, the body inner blood circulation time of medicine effectively extends, and this is beneficial to the accumulation of medicine in tumor tissues;
(4) the present invention limits the weight average molecular weight of the PEG as carrier, the PEG-DOX Nano medication precursor preparing has shown extremely strong anti-tumor activity, especially for drug-resistant tumor cell, its active anticancer is far above DOX, the drug resistance that can efficiently overcome tumor cell and tissue, has wide development and application prospect.
Accompanying drawing explanation
Fig. 1 is the intermediate product A of embodiment 1 preparation
1hNMR spectrogram;
Fig. 2 is the intermediate product B of embodiment 1 preparation
1hNMR spectrogram;
Fig. 3 is the doxorubicin nanometer medicament precursor of embodiment 1 preparation
1hNMR spectrogram;
Fig. 4 is the HPLC spectrogram of the doxorubicin nanometer medicament precursor of embodiment 1 preparation, and the HPLC spectrogram that provides DOX as a comparison;
Fig. 5 is the dynamic light scattering figure of the nano-particle that forms in water of the doxorubicin nanometer medicament precursor PEG-DOX of embodiment 1 preparation;
Fig. 6 is the transmission electron microscope picture of the nano-particle that forms in water of the doxorubicin nanometer medicament precursor PEG-DOX of embodiment 1 preparation;
Fig. 7 is doxorubicin nanometer medicament precursor PEG-DOX and the toxic effect figure of DOX to MCF-7 human breast cancer cell of embodiment 1 preparation;
Fig. 8 is doxorubicin nanometer medicament precursor PEG-DOX and the toxic effect figure of DOX to MCF-7/ADR human breast carcinoma mdr cell of embodiment 1 preparation;
Fig. 9 is doxorubicin nanometer medicament precursor PEG-DOX and the toxic effect figure of DOX to KBv200 human oral cavity epithelial cancer mdr cell of embodiment 1 preparation;
Figure 10 is the situation that doxorubicin nanometer medicament precursor PEG-DOX and the DOX of embodiment 1 preparation absorbed by KBv200 human oral cavity epithelial cancer mdr cell and MCF-7/ADR human breast carcinoma mdr cell;
Figure 11 is doxorubicin nanometer medicament precursor PEG-DOX and the form impact of DOX on MCF-7/ADR human breast carcinoma mdr cell of embodiment 1 preparation;
Figure 12 is that the embodiment 1 doxorubicin nanometer medicament precursor PEG-DOX of preparation and the blood of DOX are removed correlation curve figure.
The specific embodiment
The invention provides specific embodiment, but the present invention is not limited by the following examples.
Embodiment 1
(1) PEG-O-CH
2cOOEt's is synthetic
Anhydrous short chain polyalkylene glycol monomethyl ether (PEG-OH, Mw=2000,5mmol) is added in dry there-necked flask, add dry oxolane (THF) 30mL, add fast sodium hydride (NaH, Mw=24,6mmol).Room temperature reaction is extremely without Bubble formation.Centrifugal, to get supernatant and add anhydrous bromoacetate (Mw=167,8mmol), under room temperature, reaction is extremely without bubble.Rotary evaporation, extraction, precipitation, dry after, obtain the product A-PEG-O-CH of structure shown in formula a
2cOOEt (4.2mmol, productive rate 84%).
Warp
1hNMR (400MHz, CDCl
3) characterize: δ (ppm) 4.26 (m), 4.13 (s), 3.63 (m), 3.36 (s), 1.26 (t), as shown in Figure 1.
(2) PEG-O-CH
2cONHNH
2synthetic
By product A (PEG-OCH
2cOOEt, Mw=2086,2mmol) drop into single port bottle, add hydrazine hydrate (NH
2nH
2h
2o, Mw=50,200mmol) and 100mL THF, back flow reaction 7h.Stratification, gets THF through rotary evaporation, with absolute ether precipitation repeatedly, obtains the product B-PEG-OCH of structure shown in formula b
2cONHNH
2, Mw=2072,1.6mmol, productive rate 80%).
Warp
1hNMR (400MHz, CDCl
3) characterize: δ (ppm) 8.4 (br), 4.08 (s), 3.73 (m), 3.46 (s), 2.63 (br), as shown in Figure 2.
(3) PEG-DOX's is synthetic
Accurately weigh above-mentioned product B (PEG-OCH
2cONHNH
2, Mw=2072,0.1mmol) and doxorubicin hydrochloride (DOXHCl, Mn=580,0.1mmol) add 10mL absolute methanol, then add a small amount of trifluoroacetic acid catalysis, room temperature lucifuge reaction 48h.Then add appropriate triethylamine desalination hydrochlorate, after dialysis lyophilizing, obtain PEG-DOX (0.096mmol, productive rate 96%).
Product P EG-DOX's prepared by the present embodiment
1as shown in Figure 3, chemical shift is that δ (ppm) 8.0,7.7,5.3,1.2 left and right are amycin characteristic peak to the spectrogram of HNMR (400MHz, deuterated DMSO), and δ (ppm) 3.7-3.4,3.3 left and right are Polyethylene Glycol characteristic peak.
The HPLC of PEG-DOX characterizes as shown in Figure 4, and the HPLC spectrogram that provides DOX as a comparison, as a figure.
Result proof has prepared the object key compound PEG-DOX of structure shown in formula I, i.e. doxorubicin nanometer medicament precursor.
Said synthesis route is as follows:
Application examples
PEG-DOX prepared by the present embodiment is added to the water (concentration is 0.5mg/mL), in water, self assembly forms Nano medication, and as shown in Figure 5, mean diameter is 91.5nm ± 2.5nm to its dynamic light scattering figure, particle size distribution PDI=0.14, illustrates that the particle diameter of PEG-DOX has good stability; As shown in Figure 6, the scale providing in figure is 200nm to its transmission electron microscope picture.
One, mtt assay is evaluated the anti tumor activity in vitro of PEG-DOX
With MTT, test to evaluate the anti tumor activity in vitro of PEG-DOX Nano medication, and using PEG and amycin solution as reference.Specific experiment method is as follows: by well-grown human breast cancer cell strain (MCF-7) in culture bottle, human breast carcinoma drug-resistant cell strain (MCF-7/ADR), human oral cavity epithelial cancer drug-resistant cell strain (KBv200) respectively after trypsinization counting, by the density of 4500 cells/well, be inoculated in 96 orifice plates, be placed in incubator and cultivate 18～24h (to cell attachment).By the amycin equivalent final concentration of setting, make up a prescription, by the culture fluid sucking-off dosing in 96 orifice plates, every concentration is established three parallel hole samples.After drug effect 48h, by the centrifugal 6min of 96 orifice plate 1100rpm, supernatant discarded, adds MTT solution (0.75mg/mL, 100 μ L/ holes) under lucifuge condition, is placed in incubator and continues to cultivate 3h.By the centrifugal 8min of cell plates 2800rpm, supernatant discarded, adds DMSO (100 μ L/ hole), slowly rocks to MTT crystallization and dissolves completely.With microplate reader, read the O.D. value at every hole 562nm and 620nm place, two O.D. values are subtracted each other (the O.D. value at 620nm place is the O.D. value that cell plates record, and deducts to get rid of the background absorption of cell plates), are calculated as follows cell survival rate:
Comparative survival rate of cells (%)=(the average O.D. value of the average O.D. value of test group/blank group) * 100% inhibitory rate of cell growth (%)=100%-comparative survival rate of cells
Fig. 7 is PEG-DOX and the toxic effect figure of DOX to MCF-7 cell of preparation, and as can be seen from Figure 7, the cytotoxicity of PEG-DOX is stronger than DOX, and PEG-DOX is 0.43 mcg/ml to the half lethal dose of MCF-7 cell, can be used as cancer therapy drug.Fig. 8 is PEG-DOX and the toxic effect figure of DOX to MCF-7/ADR cell of preparation, and as can be seen from Figure 8, PEG-DOX is to the antitumor activity of MCF-7/ADR cell far above DOX, and its half lethal dose is only 1/50 of DOX.Fig. 9 is PEG-DOX and the toxic effect figure of DOX to KBv200 cell of preparation, and as can be seen from Figure 9, PEG-DOX is also significantly higher than DOX to the antitumor activity of drug-resistant tumor cell KBv200, and its half lethal dose is about 1/60 of DOX.
Above data declaration, the doxorubicin nanometer medicament precursor PEG-DOX of self assembly prepared by the present invention can be used as cancer therapy drug, especially for drug-resistant tumor cell, there is significant cytotoxicity, and semilethal dosage is low, the function also with the drug resistance that can effectively reverse human breast carcinoma mdr cell and human oral cavity epithelial cancer mdr cell, has wide development and application prospect.
Two, flow cytometer detects the cellular uptake of PEG-DOX Nano medication precursor
Select well-grown MCF-7/ADR cell and KBv200 cell, by 2 * 10
5the density inoculating cell of cells/well, in six orifice plates, is placed in cell culture incubator and cultivates 24h.After cell is completely adherent, by 5 μ g/mL amycin equivalent concentration, add medicinal liquid.After drug effect 5h, 24h, with trypsinization collecting cell, the centrifugal 5min of 1000rpm, supernatant discarded, after the PBS washing with 4 ℃ of pre-coolings, resuspended with 0.8ml PBS.With BD FACSCalibur flow cytometer, sample is detected, adopt 480nm argon laser, under FL1 passage, collect signal, 10000 of cell countings, with CellQuest Pro software analysis data.PEG-DOX and DOX are by the situation of human oral cavity epithelial cancer mdr cell and the picked-up of human breast carcinoma mdr cell as shown in figure 10.
In Figure 10, after A figure is administration 5h, the picked-up situation of KBv200 cell to PEG-DOX and DOX; After B figure is administration 24h, the picked-up situation of KBv200 cell to PEG-DOX and DOX; After C figure is administration 5h, the picked-up situation of MCF-7/ADR cell to PEG-DOX and DOX; After D figure is administration 24h, the picked-up situation of MCF-7/ADR cell to PEG-DOX and DOX.
Observation Figure 10 is known, and two kinds of different drug-resistant tumor cells are all remarkable in the picked-up to DOX to the picked-up of PEG-DOX.
Three, PEG-DOX and the DOX form impact on human breast carcinoma mdr cell
Light microscopic photo after MCF-7/ADR two kinds of Experimental agents of acceptance (DOX3 μ g/ml equivalent) processing 5,12 and 24h as shown in figure 11, as shown in Figure 11, the lethal effect of PEG-DOX significantly strengthens compared with DOX: PEG-DOX processes and to cause that significant morphocytology changes (in endochylema the change of cavity sample etc.) after drug-resistant tumor cell 5h, and after administration 12h, cell is all dead and lose normal morphology.
Four, the plasma clearance of PEG-DOX Nano medication precursor test
DOX solution and PEG-DOX solution are organized to mice administration by tail vein injection mode respectively to each, injection volume is about 200 μ L, dosage is 6mg amycin/kg body weight, and after administration, 0.05h, 0.5h, 1h, 2h, 4h, 7h, 24h take out blood sample by eye socket blood taking method respectively.With the centrifuge tube of processing through heparin, accept the blood sample of taking-up, get 75 μ L blood samples, add 450 μ L acid isopropyl alcohol extractions, whirlpool concussion is placed on overnight incubation in 4 ℃ of refrigerators.After hatching, each sample, at 4 ℃, with the centrifugal 10min of rotating speed of 5000rpm, is respectively got to 100 μ L supernatants and added fluorescent screen.Microplate reader arranges excitation wavelength 480nm, and emission wavelength 600nm surveys fluorescent value RFU.According to the doxorubicin concentration in the quantitative blood sample of amycin fluorescence standard curve obtaining.
Figure 12 is that the blood of PEG-DOX and DOX is removed correlation curve figure, and as shown in Figure 12, PEG-DOX has than the blood of DOX significant prolongation half checkout time t
1/2its area under curve (AUC) enlarges markedly compared with DOX, and clearance rate is lower than 1/10 of DOX, confirms the effectively blood circulation time of prolong drug of this doxorubicin nanometer medicament precursor, this is conducive to increase the cancer target ability of medicine, improves antitumor curative effect and reduces toxic and side effects.
Claims (9)
1. a doxorubicin nanometer medicament precursor for self assembly, is characterized in that, take hydrophilic polyglycol segment as carrier, and the doxorubicin nanometer medicament precursor of described self assembly has the structure as shown in formula I:
Ⅰ；
In formula, described segment
weight average molecular weight be 2000～20000.
2. doxorubicin nanometer medicament precursor as claimed in claim 1, is characterized in that, described segment
weight average molecular weight be 2000～5000.
3. doxorubicin nanometer medicament precursor as claimed in claim 2, is characterized in that, described segment
weight average molecular weight be 2000.
4. a preparation method for doxorubicin nanometer medicament precursor as claimed in claim 1, is characterized in that, step is as follows:
(1) the first organic solvent by terminal hydroxy group Polyethylene Glycol, metal hydride and after dewatering mixes, and room temperature reaction is to without Bubble formation, then adds anhydrous halogenated acetic acids ethyl ester, through post processing, obtains intermediate product A, and structural formula is suc as formula shown in a;
(2) intermediate product A step (1) being obtained and hydrazine hydrate back flow reaction 5～10h in the second organic solvent, standing cooling after, after rotary evaporation, precipitation and sucking filtration, obtain intermediate product B, structural formula is suc as formula shown in b;
(3), under weak acid catalyst, in the intermediate product B that step (2) is obtained, amycin the 3rd organic solvent after dewatering, react, then through dialysis, lyophilization, obtain the doxorubicin nanometer medicament precursor of described self assembly;
formula a;
formula b;
Wherein, segment
weight average molecular weight be 2000～20000.
5. preparation method as claimed in claim 4, is characterized in that, the metal hydride that step (1) is described and the mol ratio of terminal hydroxy group Polyethylene Glycol are 1～1.5:1;
Described halogenated acetic acids ethyl ester is bromoacetate, with the mol ratio of terminal hydroxy group Polyethylene Glycol be 1～2:1.
6. preparation method as claimed in claim 4, is characterized in that, the hydrazine hydrate that step (2) is described and the mol ratio of intermediate product A are 80～120:1.
7. preparation method as claimed in claim 4, is characterized in that, the amycin that step (3) is described and the mol ratio of intermediate product B are 0.5～2:1; Described weak acid catalyst is selected from phosphoric acid, acetic acid or trifluoroacetic acid.
8. preparation method as claimed in claim 4, is characterized in that, first, second, and third described organic solvent is independently selected from methanol, ethanol, oxolane, dimethyl sulfoxide or N, N '-dimethyl formamide.
9. the doxorubicin nanometer medicament precursor as described in claim 1～3 application in preparing overriding resistance tumour medicine.
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| CN108434457A (en) * | 2018-04-20 | 2018-08-24 | 湖南华腾制药有限公司 | A kind of adriamycin polyethylene glycol epothilone B conjugate and preparation method thereof |
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| CN102188717A (en) * | 2011-05-04 | 2011-09-21 | 浙江大学 | Self-emulsifying doxorubicin nanometer medicament and preparation method thereof |
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2014
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102188717A (en) * | 2011-05-04 | 2011-09-21 | 浙江大学 | Self-emulsifying doxorubicin nanometer medicament and preparation method thereof |
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| CN106139159A (en) * | 2016-08-30 | 2016-11-23 | 四川大学 | The polyethyleneglycol derivative small-molecule drug conjugate polymer micelle sensitive for pH purposes in inflammation targeted delivery system |
| CN107375213A (en) * | 2017-07-05 | 2017-11-24 | 西安交通大学 | PH response type carrier-free nano-drug preparations and preparation method, application |
| CN108434457A (en) * | 2018-04-20 | 2018-08-24 | 湖南华腾制药有限公司 | A kind of adriamycin polyethylene glycol epothilone B conjugate and preparation method thereof |
| CN113747906A (en) * | 2019-08-26 | 2021-12-03 | 真共生株式会社 | Biointroduction aid and method for using same |
| CN112618728A (en) * | 2020-12-18 | 2021-04-09 | 绍兴文理学院 | Dual-response prodrug containing polysialic acid group, synthetic method thereof and application thereof in pharmaceutical preparation |
| CN113995850A (en) * | 2021-09-30 | 2022-02-01 | 浙江大学杭州国际科创中心 | Tyrosinase response cascade amplification nano-drug and preparation and application thereof |
| CN113995850B (en) * | 2021-09-30 | 2023-08-01 | 浙江大学杭州国际科创中心 | Cascade amplification nano-drug with tyrosinase response and preparation and application thereof |
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Application publication date: 20140806 |