CN103251573B - Protein micro/nano sphere carrying antitumor chemotherapeutic medicine and preparation method of protein micro/nano sphere - Google Patents
Protein micro/nano sphere carrying antitumor chemotherapeutic medicine and preparation method of protein micro/nano sphere Download PDFInfo
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Abstract
The invention relates to a protein micro/nano sphere carrying an antitumor chemotherapeutic medicine. The composition preferably comprises a housing and a core on the premise that the protein is TNFSF (Tumor Necrosis Factor Superfamily) protein, and the antitumor chemotherapeutic medicine is an indissolvable medicine; the housing is composed of the TNFSF protein and is basically completely closed; the core is placed into the housing and contains the antitumor chemotherapeutic medicine; and the maximum diameter of the housing is less than 500nm. The protein micro/nano sphere carrying the antitumor chemotherapeutic medicine has the beneficial effects that the protein micro/nano sphere which is prepared based on the membrane emulsification technology and can carry an indissolvable antitumor chemotherapeutic medicine is provided; and the invention particularly provides a preparation method of the protein micro/nano sphered of the TNFSF protein and HAS (Human Serum Albumin) protein, represented by TRAIL (Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand).
Description
Technical field
The invention belongs to field of antineoplastic medicaments, particularly a kind of albumen micro-nano ball and method for making thereof of carrying antineoplastic chemotherapy medicine.
Background technology
Micro-nano ball, refer to that medicine disperses or is attracted in macromolecule, polymeric matrix and forms, size, at the nanoscale dispersion of 10-1000nm, can be divided into polybutylcyanoacrylate nanoparticle, albumin nanoparticles, polylactic acid-based nanoparticle, lipid nanoparticle etc. according to the difference of carrier dissimilar.
Protein or polypeptide are made to micro-nano ball and be administered systemically, can not only effectively prevent medicine degraded very soon in vivo, also may send to effective site in body by targeting, reach slow release long-acting object.But the poor stability of protein, envelop rate is low, drug loading is little, easily assemble and biological activity is reduced, and may cause that immunoreation, inside and outside have the development that the problems such as obvious burst effect are having a strong impact on this class preparation while discharging.The common method of preparing polypeptide and protein medicaments micro-nano ball comprises emulsion solvent evaporation/extraction, atomizing freeze drying method, phase separation method, spray drying method etc., but the whole bag of tricks defectiveness all.
While preparing micro-nano ball such as emulsion solvent evaporation/extraction, protein is easily assembled and degeneration at oil-water interfaces.Such as biodegradable polymers PELA, lipotropy is strong again, not high to the affinity of water soluble polypeptide, protein and vaccine.In prior art, the also report of preparing protein micro-nano ball of useful film emulsifying technology.
Film emulsion process is considered to obtain the effective ways of high-quality list stably dispersing emulsion, can be used for functional single preparation that disperses micro-nano ball and microcapsule.But very many owing to affecting porosity, film surface type, emulsifier type and content, decentralized photo flow, continuous phase speed and the operation pressure reduction etc. of footpath that the parameter of film emulsion process mainly comprises that film is micro-and distribution, film, the protein micro-nano ball that very multifunctional membrane emulsion process makes cannot practical application, particularly for the wider HSA-DOC(human albumin-paclitaxel of application) micro-nano ball, in prior art, also with film emulsifying technology, successfully do not obtain the report of HSA-DOC.
Tumor necrosis factor superfamily (tumor necrosis factor superfamily; TNFSF) belong to II type transmembrane protein, its extracellular region C end is containing being about 150 amino acid whose sequences (THD), the conservative skeleton that comprises aromatic series and hydrophobic residue.
The TNFSF albumen that the TRAIL of take is representative can be induced kinds of tumor cells apoptosis specifically, also can with other tumor chemotherapeutic drug use in conjunction." TRAIL and associating amycin are treated osteosarcomatous animal experiment study " of delivering on Chinese science and technology paper online (www.paper.edu.cn) such as Wu Gang etc.; The magnificent grade of the happy mountain valley with clumps of trees and bamboo is published in Anhui medicine (2009,12; " progress of TRAIL coupling chemotherapeutics gynecological tumor " 13(12)).Liu Zheng etc. are published in Central China University of Science and Technology's journal (medicine) " inhibitory action of paclitaxel plus tumor necrosin relative death inducing ligand to prostate gland cancer cell " of (the 36th the 352nd page of the 3rd phase of volume).Chou Bo etc. are published in " depression effect of TRAIL associating Paclitaxel on Human cerebral glioma U87 cell and the possible mechanism thereof " on Chinese tumor biotherapy magazine (Feb.2013, Vol.20, No.1).Liu Yanhou etc. are published in Shandong medicine (the 51st the 17th phase of volume in 2011) " paclitaxel is on the apoptotic impact of TRAIL induction gastric cancer SGC7901 ", also have english literature to be published in JOURNAL OFCLINICALONCOLOGY(VOLUME 26 NUMBER 21 JULY 20 2008 as Avi Ashkenazi etc.) " Ligand-Based Targeting of Apoptosis in Cancer:The Potential of Recombinant Human Apoptosis Ligand 2/Tumor Necrosis Factor – Related Apoptosis-Inducing Ligand (rhApo2L/TRAIL) " and Frank A.E.Kruyt " the TRAIL and cancer therapy " that be published in Cancer Letters 263 (2008) 14 – 25.The method of above use in conjunction, TRAIL and chemotherapeutics have been related in the use in conjunction of osteosarcoma, gynecological tumor, carcinoma of prostate, cerebral glioma, gastric cancer etc., common administering mode is all to belong to drug combination, namely TRAIL belongs to different pharmaceutics unit from chemotherapeutics, and during clinical practice, occasional combination together.Although those of ordinary skill, according to above-mentioned document, may be made Yi Ge preparation unit by TRAIL and slightly solubility chemotherapeutics, because TRAIL is water miscible, itself and insoluble drug are mixed and make compound medicine and almost cannot realize.
Although for the antineoplastic chemotherapy medicine of slightly solubility, those of ordinary skill can consider to make nanosphere, can for preparing suspension for injection or for oral use.Such as Chinese patent CN101352420B is disclosed with polylactic acid enclose hydroxy camptothecin.But in prior art, still the antitumor drug of TNFSF albumen and slightly solubility is not made to the report of suitable micro-nano ball.
In addition, also there is too short defect of half-life in the TNFSF albumen that TRAIL is representative, thereby cause affecting the interior Pharmacokinetic Characteristics of body of medicine.Paclitaxel is the antineoplastic chemotherapy medicine of representative, need to enter competence exertion optimum curative effect in cell, and how research strengthens local taxanes drug level and then make paclitaxel is that the antineoplastic chemotherapy medicine targeted cells that enter of representative significant more.
Summary of the invention
The present invention is directed to many technical problems of the prior art, a kind of albumen micro-nano ball that can carry antineoplastic chemotherapy medicine that utilizes film emulsifying technology to make is provided.Specifically, first object of the present invention has been to provide a kind of TNFSF albumen micro-nano ball that can carry antineoplastic chemotherapy medicine, this micro-nano ball can bring into play TNFSF albumen in vivo with corresponding receptors bind, in the time of performance antitumaous effect, the antineoplastic chemotherapy medicine that also can carry slightly solubility arrives in body, has brought into play synergism.Second object of the present invention has been to provide a kind of HSA albumen micro-nano ball that utilizes film emulsifying can carry paclitaxel.The 3rd object of the present invention has been to provide the above-mentioned preparation method that can carry the albumen micro-nano ball of antineoplastic chemotherapy medicine.
A kind of albumen micro-nano ball that carries antineoplastic chemotherapy medicine of the present invention, preferred the first albumen is TNFSF albumen, and antineoplastic chemotherapy medicine is insoluble drug, compositions comprises the complete totally enclosed housing substantially being formed by described TNFSF albumen, and the kernel that contains antineoplastic chemotherapy medicine that is positioned at enclosure interior, the maximum gauge of housing is less than 500nm.
The preferred particle diameter scope of the present invention is at 340nm-400nm, and most preferred particle diameter scope is below 200nm.
The albumen micro-nano ball that carries antineoplastic chemotherapy medicine of the present invention, the antineoplastic chemotherapy medicine of preferred slightly solubility, includes but not limited to platinum class, vinca, taxanes, camptothecin, AC, D actinomycin D, bleomycin, daunorubicin, epirubicin, mitomycin, methotrexate, fluorouracil, carmustine, etoposide, interferon, phenesterin, hormones, tamoxifen; Vinca comprises that vincaleucoblastine, vincristine and other are similar to the medicine of vinblastine; Taxanes comprises that paclitaxel and derivant thereof, paclitaxel prodrug, taxane and other are similar to the medicine of paclitaxel; Camptothecin comprises camptothecine and analog, hydroxy camptothecin and analog thereof; Platinum class comprises carboplatin, cisplatin; Hormones comprises estrogen, progestogen.
Special needs to be pointed out is, albumen micro-nano ball of the present invention is applicable to carrying paclitaxel very much.
The albumen micro-nano ball that carries antineoplastic chemotherapy medicine of the present invention, TNFSF albumen comprises one or more wild type and the allosteric type among EDA-A1, TNFSF1, TNFSF2, TNFSF3, TNFSF3L, TNFSF4, TNFSF5 4, TNFSF6, TNFSF7, TNFSF8, TNFSF9, TRAIL, TNFSF11, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TNFSF18.
Particularly preferably be, TNFSF albumen is TRAIL.TRAIL (TNF – related apop tosis inducing ligand) is an important apoptosis regulatory protein, TRAIL molecule is present in most of organs and the cell of human body with transcript form, the more important thing is that the T, bone-marrow-derived lymphocyte and the NK cell that are activated also express trail protein.Ripe trail protein mainly exists with the form of II type transmembrane protein or solubility.Its extracellular region is comprised of 747 aminoacid.TRAIL extracellular region is combined with its specific receptor, can promptly induce the apoptosis of kinds of tumor cells system, and normal tissue cell is without apoptosis-induced effect.
The present invention has found a kind of method for making of carrying the albumen micro-nano ball of antineoplastic chemotherapy medicine especially unexpectedly, comprises the steps:
A. antineoplastic chemotherapy medicine is dissolved in to organic solvent as oil phase;
B. albumen is water-soluble as water;
C. adopt film emulsification method that water and oil phase are made to film emulsion;
D. by the curing micro-nano ball that makes of film emulsion;
E. by micro-nano ball lyophilization.
The first is improved to the method for making of albumen micro-nano ball, when albumen is TNFSF albumen, and preparation as follows:
A. antineoplastic chemotherapy medicine is dissolved in to organic solvent as oil phase;
B. using TNFSF protein solution as water;
C. oil phase and water are made to oil/water colostric fluid;
D. oil/water colostric fluid is carried out to film emulsifying, obtain film emulsion;
E. by the curing micro-nano ball that makes of film emulsion;
F. by micro-nano ball lyophilization.
In this experiment, without continuous phase, wash away, once get product.Principle is that colostric fluid is the oil-in-water system that granule is larger, by being squeezed into nano level oil-in-water structure after film, and then solidify to form granule
Furtherly, when antitumor drug is that paclitaxel and TNFSF albumen are TRAIL, by following steps, made:
A. the paclitaxel of 20-40 weight portion is dissolved in to ethyl acetate as oil phase;
B. using 50 weight portion TRAIL aqueous solutions as water;
C. under ice-water bath condition, adopt ultrasonication that oil phase is scattered in and in water, prepares oil/water colostric fluid;
D. colostric fluid is poured in fast film emulsifier unit, under room temperature, under 0.5-1.0MPa nitrogen pressure, repeatedly pressed fenestra to carry out film emulsifying colostric fluid, obtain film emulsion;
E. the magnetic agitation volatilization at normal temperatures of film emulsion is removed to organic solvent and make its curing micro-nano ball that makes;
F. by deionized water centrifuge washing 5 times for the micro-nano ball after solidifying, and described compositions is made in lyophilization.
Surprisingly, in the weight portion proportioning of ethyl acetate and water, be 20: 50 o'clock, effect is ideal.
The second is improved to the method for making of albumen micro-nano ball, when albumen is TNFSF albumen, also can prepare as follows:
A. antineoplastic chemotherapy medicine is dissolved in to organic solvent as oil phase;
B. using TNFSF protein solution as water;
C. using oil phase as decentralized photo, using water as continuous phase, carry out film emulsifying, obtain film emulsion;
D. by the curing micro-nano ball that makes of film emulsion;
E. by micro-nano ball lyophilization.
The method for making of the third albumen micro-nano ball, be when albumen be that HSA albumen and antineoplastic chemotherapy medicine are paclitaxel, comprise the steps:
A. paclitaxel is dissolved in to organic solvent as oil phase;
B. HAS albumen is water-soluble as water;
C. oil phase and described water are made to oil/water colostric fluid;
D. oil/water colostric fluid is carried out to film emulsifying, obtain film emulsion;
E. by the curing micro-nano ball that makes of film emulsion;
F. by micro-nano ball lyophilization.
Human serum albumin of the present invention (Human Serum Albumin is called for short HSA) is the protein in human plasma, and its nonglycosylated single chain polypeptide comprises 585 aminoacid, and molecular weight is 66kD.In blood plasma, its concentration is 42g/L, accounts for 60% of blood plasma total protein.In body fluid, human serum albumin can transport fatty acid, bile pigments, aminoacid, steroid hormone, metal ion and many treatment molecules etc.: maintain the normal osmotic pressure of blood simultaneously.
Beneficial effect of the present invention is, provide a kind of preparation based on film emulsifying technology can carry the albumen micro-nano ball of insoluble anti-tumor medicament, particularly provide and take the preparation method of albumen micro-nano ball of TNFSF albumen that TRAIL is representative, HSA albumen, high, the rational burst effect of having controlled of envelop rate of the method.The TRAIL of take below illustrates beneficial effect of the present invention as example helps to understand, but should not be subject to this to limit for example beneficial effect of the present invention, and TRA-DOC micro-nano ball can, in the associating synergistic while of anticancer generation, possess again following features:
1, TRA-DOC is micro-nano ball, can make unitary agent; And " application of TRAIL-paclitaxel plus " just face with now joining, it not unitary agent;
2, TRA-DOC is target administration, and medicine can be brought in the cell that has TRA receptor (death receptor Death receptor), and " application of TRAIL-paclitaxel plus " is without this effect;
3, TRA-DOC micro-nano ball solves the not diffluent problem of slightly solubility chemotherapeutics, the dual curative effect of performance TRI and DOC, and in " application of TRAIL-paclitaxel plus ", paclitaxel needs other macromolecular material enclose.
4, the synergism of TRA-DOC micro-nano ball, be higher than the synergism of " application of TRAIL-paclitaxel plus ".
5, the TNFSF that more independent TRAIL of the half-life of TRA-DOC micro-nano ball is representative extends about 20%-200%.
Below by test example, further illustrate the present invention.
The test of test example 1:H460 cell cytotoxicity
Test method: H460 cell is inoculated in 96 orifice plates, is placed in after incubator 24h, culture fluid in each hole of careful sucking-off after the attached wall of cell.Arranging blank group is that H460 cell does not add any medicine, and matched group is respectively TRA-IBU (TRA is TRAIL, and IBU is ibuprofen), HSA-DOC(HSA human serum albumin, DOC is paclitaxel, non-film emulsifying technology HAS-DOC of the present invention), experimental group is TRA-DOC.Test sample (calculate with DOC, initial concentration is 1mg/mL) and matched group are added in 96 hole versions, and it is 100 times, 1000 times, 4000,16000,64000 times that every group of medicine dilutes respectively.
Result of the test: this nanoparticle all presents significant inhibitory action to H460 cell in each concentration, and be all better than matched group effect.Wherein fragmentation effect is optimum when diluting 4000 times, and as Fig. 7, TRA-DOC is 87.4 ± 1.6% to the suppression ratio of H460, significantly better than HSA-DOC group 65.4 ± 1.3%, TRA-IBU group 31.1 ± 0.6%.
The test of test example 2:L929 cell cytotoxicity
Test method is with test example 1.
Result of the test: this nanoparticle all presents significant inhibitory action to L929 cell in each concentration, and be all better than matched group effect.Wherein fragmentation effect is optimum when diluting 4000 times, and as Fig. 8, TRA-DOC is 49.1 ± 2.2% to the suppression ratio of L929, significantly better than HSA-DOC group 5.3 ± 1.8%, TRA-IBU group 7.0 ± 1.3%.
3 pairs of S180 ascites tumor mice inhibitory action of test example
Test method: get S180 ascites tumor mouse tumor liquid under aseptic condition, preparation cell suspension, with 1 * 10
6/ concentration is only inoculated in after the axil of experiment mice right side subcutaneous.Next day, by animal random packet, 10 every group.
If negative control group, the non-film emulsifying technology HAS-DOC of the present invention of matched group HSA-DOC() and test group TRA-DOC, with 10mg/kg/ daily dose lumbar injection; With same dose (10mg/kg) same volume (0.2ml/20g) and the administration simultaneously of laboratory observation sample.Successive administration 7 times.Treat that negative control group animal tumor grows to certain volume and finishes to observe.
When experiment finishes, scale body weight, peels off tumor, and scale tumor weight is used the relatively statistics difference of each treated animal tumor weight of t method of inspection.
The computing formula of inhibition rate of tumor growth is:
Experimental result: as shown in the table, the tumour inhibiting rate (%) of TRA-DOC group, HSA-DOC group is respectively 54.58% and 33.47%, and test group tumor killing effect is significantly better than matched group.
Test example 4 targeting tests
Experimental apparatus flow cytometer
Test method is got two culture dishs, when cell reaches exponential phase, adds respectively HSA-DOC and TRA-DOC, and HeLa cell co-culture 24 hours (5 hours).Then with PBS buffer solution, rinse three times to remove not by the nanoparticle of endocytosis and part dead cell.Then use pancreatin solution to digest and be prepared into cell suspension, using flow cytometer to detect the endocytosis efficiency of cell to two kinds of particles.With the cell that does not pass through any processing as reference frame.HSA-DOC and TRA-DOC nanoparticle are passed through to fluorochrome label.
Result of the test is in conjunction with shown in Fig. 9, and the abscissa of Fig. 9 is fluorescence intensity signals, and vertical coordinate is relative number of cells; Curve moves right and represents that intracellular fluorescence intensity increases, and nanoparticle is with fluorescence, and fluorescence enhancing can only be that the particle of cell endocytic causes more.Curve moves to right and represents that the interior TRA-DOC particle of cell is more than HSA-DOC particle, thereby the targeting of proof TRA-DOC particle is stronger.
Accompanying drawing explanation
Fig. 1. particle structure schematic diagram of the present invention; In figure, 1 represents nanoparticle; 2 represent insoluble anti-tumor medicament kernel; 3 represent to form shell by TNFSF albumen, and the thickness of shell is 5-50nm, and the diameter of shell is 1-500nm.
Fig. 2. scanning electron microscope diagram of the present invention; In figure, can find out that the surperficial enclose of micro-nano ball of the present invention is complete, even size distribution.
Fig. 3 .Zeta potentiometer; In figure, vertical coordinate Intensity represents intensity, and abscissa size represents particle diameter, and statistics graph represents statistical graph; Measurements represents to measure.In figure, show, the distribution of particles of the particle size distribution 90% of micro-nano ball of the present invention is between 160-190nm.
Fig. 4 .X X-ray photoelectron spectroscopy X; In figure, counts/s represents counts/second, binding energy(ev) represent in conjunction with energy, survey represents survey map, in figure, show, micro-nano ball of the present invention detects the characteristic energy peak of N element at 399eV place, show to have nitrogen element at this particle surface, and then contain TNFSF albumen in proof nanoparticle.
Fig. 5. circular dichroism spectra; In figure, intensitiy represents intensity, and wavelength represents wavelength; In figure, show, in micro-nano ball of the present invention, have protein, and the secondary structure of this albumen does not significantly change.
Fig. 6. laser confocal microscope; 1 represents micro-nano ball; 2 represent HeLa tumor cell, in figure, show, micro-nano ball 1, by the process of the cell membrane endocytosis of HeLa tumor cell 2, proves that micro-nano ball of the present invention easily enters into cell interior, thus the effect of performance paclitaxel;
The cell toxicity test cell killing situation cartogram of Fig. 7 .H460 cell;
In figure, vertical coordinate Inhibition Ratio represents suppression ratio; Control represents blank group;
The cell toxicity test cell killing situation cartogram of Fig. 8 .L929 cell;
In figure, vertical coordinate Inhibition Ratio represents suppression ratio; Control represents blank group;
Fig. 9. targeting test;
Figure 10. scanning electron microscope diagram sheet: HSA-DOC;
Figure 11 .Zeta potentiometer: the distribution of particles of particle size distribution 80% is between 300-400nm.
The specific embodiment
Embodiment 1 TRA-DOC micro-nano ball
Experiment material:
Recombinant soluble human TRAIL (TRAIL), by escherichia coli expression, extraction.Paclitaxel (purity >=98.00%); Ethyl acetate; PBS buffer solution; Deionized water.
Experimental apparatus:
Ultrasonication machine, high speed centrifuge, with the zeta potential instrument of submicron particle diameter analytic function, cold field emission scanning electron microscope, flow cytometer, laser confocal microscope, x-ray photoelectron power spectrum diffractometer, high performance liquid chromatography, circular dichroism spectrometer, SPG membrane emulsifier.
Experimentation:
Using 20mg paclitaxel (DOC) be dissolved in 20ml ethyl acetate, be made into 1mg/mL solution as oil phase, using the TRAIL of 50mg be dissolved in 50ml water, be made into 1mg/mL solution as water, under ice-water bath condition, adopt ultrasonication that oil phase is scattered in and in water, prepares oil/water colostric fluid.This colostric fluid is poured in fast film emulsifier unit, nitrogen pressure with 0.5-1.0MPa under room temperature was pressed the fenestra (aperture 800-1000nm) of SPG film repeatedly by it, the magnetic agitation volatilization at normal temperatures of gained emulsion is removed to organic solvent makes it be solidified into micro-nano ball, by micro-nano ball deionized water centrifuge washing 5 times after solidifying, and finished product is made in lyophilization.
Result of the test: as shown in Figure 1, TRA-DOC micro-nano ball 1 comprises the complete totally enclosed housing 3 substantially that trail protein forms, and the kernel that contains paclitaxel that is positioned at enclosure interior.Scanning electron microscope diagram as shown in Figure 2 shows, the surperficial enclose of this micro-nano ball is complete, even size distribution.In conjunction with the zeta potential instrument test result of Fig. 3, the distribution of particles of the particle size distribution 90% of this micro-nano ball is between 160-190nm.
As the x-ray photoelectron power spectrum of Fig. 4 shows, micro-nano ball of the present invention detects the characteristic energy peak of N element at 399eV place, shows to have nitrogen element at this particle surface, and then contains trail protein in proof nanoparticle.As the circular dichroism spectra of Fig. 5 shows, in micro-nano ball of the present invention, have protein, and the secondary structure of this albumen does not significantly change.
Laser confocal microscope has as shown in Figure 6 proved that micro-nano ball 1 is by the process of the cell membrane endocytosis of HeLa tumor cell 2, thereby micro-nano ball of the present invention easily enters into cell interior, thus the effect of performance paclitaxel.
Embodiment 2 HSA-DOC micro-nano balls
Experiment material:
Human serum albumin (HSA), by escherichia coli expression, extraction.Paclitaxel (purity >=98.00%); Ethyl acetate; PBS buffer solution; Deionized water.
Experimental apparatus:
Ultrasonication machine, high speed centrifuge, with the zeta potential instrument of submicron particle diameter analytic function, cold field emission scanning electron microscope, SPG membrane emulsifier.
Experimentation:
Using 20mg paclitaxel (DOC) be dissolved in 20ml ethyl acetate, be made into 1mg/mL solution as oil phase, using the HSA of 50mg be dissolved in 50ml water, be made into 1mg/mL solution as water, under ice-water bath condition, adopt ultrasonication that oil phase is scattered in and in water, prepares oil/water colostric fluid.This colostric fluid is poured in fast film emulsifier unit, nitrogen pressure with 0.5-1.0MPa under room temperature was pressed the fenestra (aperture 800-1000nm) of SPG film repeatedly by it, the magnetic agitation volatilization at normal temperatures of gained emulsion is removed to organic solvent makes it be solidified into micro-nano ball, by micro-nano ball deionized water centrifuge washing 5 times after solidifying, and finished product is made in lyophilization.
Result of the test: scanning electron microscope diagram sheet as shown in figure 10 shows that the surperficial enclose of HSA-DOC micro-nano ball is complete, even size distribution; Zeta potential instrument result as shown in figure 11 shows, the distribution of particles of particle size distribution 80% is between 300-400nm.
Embodiment 3 TNF-α and DOC micro-nano ball
Experiment material:
TNF-α, by escherichia coli expression, extraction.Paclitaxel (purity >=98.00%); Ethyl acetate; PBS buffer solution; Deionized water.
Experimental apparatus:
Ultrasonication machine, high speed centrifuge, with the zeta potential instrument of submicron particle diameter analytic function, cold field emission scanning electron microscope, SPG membrane emulsifier.
Experimentation:
Using 20mg paclitaxel (DOC) be dissolved in 20ml ethyl acetate, be made into 1mg/mL solution as oil phase, using the TNF-α of 50mg be dissolved in 50ml water, be made into 1mg/mL solution as water, under ice-water bath condition, adopt ultrasonication that oil phase is scattered in and in water, prepares oil/water colostric fluid.This colostric fluid is poured in fast film emulsifier unit, the nitrogen pressure of 0.8MPa of take under room temperature pressed by it SPG film that aperture is 800nm repeatedly, the magnetic agitation volatilization at normal temperatures of gained emulsion is removed to organic solvent makes it be solidified into micro-nano ball, by micro-nano ball deionized water centrifuge washing 5 times after solidifying, and finished product is made in lyophilization.
Result of the test: the surperficial enclose of TNF-α-DOC micro-nano ball is complete, even size distribution; The distribution of particles of particle size distribution 80% is between 350-400nm.
The above embodiment is only that the preferred embodiment of the present invention is described; not scope of the present invention is limited; design under the prerequisite of spirit not departing from the present invention; various distortion and improvement that those of ordinary skills make technical scheme of the present invention, all should fall in the definite protection domain of claims of the present invention.
Claims (5)
1. an albumen micro-nano ball that carries antineoplastic chemotherapy medicine, it is characterized in that, described albumen is TNFSF albumen, described antineoplastic chemotherapy medicine is insoluble drug, described albumen micro-nano ball comprises the complete totally enclosed housing substantially being formed by described TNFSF albumen, and the kernel that contains described antineoplastic chemotherapy medicine that is positioned at described enclosure interior, the maximum gauge of described housing is less than 500nm; The antineoplastic chemotherapy medicine of described slightly solubility is paclitaxel; Described TNFSF albumen is TRAIL.
2. prepare a method of carrying the albumen micro-nano ball of antineoplastic chemotherapy medicine claimed in claim 1, it is characterized in that, comprise the steps:
A. described antineoplastic chemotherapy medicine is dissolved in to organic solvent as oil phase;
B. described albumen is water-soluble as water;
C. adopt film emulsification method that described water and described oil phase are made to film emulsion;
D. by the curing micro-nano ball that makes of film emulsion;
E. by micro-nano ball lyophilization.
3. the method for making of albumen micro-nano ball as claimed in claim 1, is characterized in that, comprises the steps:
A. described antineoplastic chemotherapy medicine is dissolved in to organic solvent as oil phase;
B. using described TNFSF protein solution as water;
C. described oil phase and described water are made to oil/water colostric fluid;
D. oil/water colostric fluid is carried out to film emulsifying, obtain film emulsion;
E. by the curing micro-nano ball that makes of film emulsion;
F. by micro-nano ball lyophilization.
4. the method for making of albumen micro-nano ball as claimed in claim 1, is characterized in that, comprises the steps:
A. described antineoplastic chemotherapy medicine is dissolved in to organic solvent as oil phase;
B. using TNFSF protein solution as water;
C. using described oil phase as decentralized photo, using described water as continuous phase, carry out film emulsifying, obtain film emulsion;
D. by the curing micro-nano ball that makes of film emulsion;
E. by micro-nano ball lyophilization.
5. the method for making of albumen micro-nano ball as claimed in claim 1, is characterized in that, comprises the steps:
A. the paclitaxel of 20-40 weight portion is dissolved in to ethyl acetate as oil phase;
B. using 50 weight portion TRAIL aqueous solutions as water;
C. under ice-water bath condition, adopt ultrasonication that oil phase is scattered in and in water, prepares oil/water colostric fluid;
D. described colostric fluid is poured in fast film emulsifier unit, under room temperature, under 0.5-1.0MPa nitrogen pressure, repeatedly pressed fenestra to carry out film emulsifying described colostric fluid, obtain film emulsion;
E. the magnetic agitation volatilization at normal temperatures of described film emulsion is removed to organic solvent and make its curing micro-nano ball that makes;
F. by deionized water centrifuge washing 5 times for the described micro-nano ball after solidifying, and described compositions is made in lyophilization.
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| CN112089704B (en) * | 2020-09-27 | 2022-04-26 | 中国药科大学 | Bionic nano-carrier and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2209523A (en) * | 1987-09-07 | 1989-05-17 | Glaverbel | Glass microbeads having bacteriostatic properties and process for manufacturing such microbeads |
| CN101352420A (en) * | 2008-09-08 | 2009-01-28 | 厦门大学 | Hydroxycamptothecin sustained-release microsphere and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2209523A (en) * | 1987-09-07 | 1989-05-17 | Glaverbel | Glass microbeads having bacteriostatic properties and process for manufacturing such microbeads |
| CN101352420A (en) * | 2008-09-08 | 2009-01-28 | 厦门大学 | Hydroxycamptothecin sustained-release microsphere and preparation method thereof |
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