CN103251573A - 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, the polymeric matrix and forms, size is at the nanoscale dispersion of 10-1000nm, and it is dissimilar to be divided into polybutylcyanoacrylate nanoparticle, albumin nanoparticle, polylactic acid-based nanoparticle, lipid nanoparticle etc. according to the difference of carrier.
Protein or polypeptide are made micro-nano ball be administered systemically, can not only effectively prevent medicine very fast degraded in vivo, also may send to effective site in the body by targeting, reach the slow release long-acting purpose.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 problem such as obvious burst effect is having a strong impact on this class preparation when discharging.The common method of preparation 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.
When 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, and is not high to the affinity of water soluble polypeptide, protein and vaccine.In the prior art, the also report of the preparation protein micro-nano ball of useful film emulsifying technology.
The 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 because that the parameter that influence the film emulsion process mainly comprises porosity, film surface type, emulsifier type and content, decentralized photo flow, the continuous phase speed of footpath that film is little and distribution, film and operates pressure reduction etc. is very many, make very much protein micro-nano ball of multifunctional membrane emulsion process can't practical application, particularly for using wider HSA-DOC(human albumin-paclitaxel) micro-nano ball, in the prior art also film emulsifying technology of no use successfully 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 holds to contain and is about 150 amino acid whose sequences (THD), comprises the conservative skeleton of aromatic series and hydrophobic residue.
The TNFSF albumen that with TRAIL is representative can be induced the 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 at Chinese science and technology paper online (www.paper.edu.cn) such as Wu Gang etc.; Happy mountain valley with clumps of trees and bamboo China etc. 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) " paclitaxel combination tumor necrosin relative death inducing ligand is to the inhibitory action of prostate gland cancer cell " of (the 352nd page of the 36th the 3rd phase of volume).Chou Bo etc. are published in Chinese tumor biotherapy magazine (Feb.2013, Vol.20, No.1) on " TRAIL associating paclitaxel is to human glioma U87 cell inhibiting effect and possible mechanism thereof ".Liu Yanhou, etc. published in Shandong Medical (2011 51 No. 17) of the "paclitaxel on TRAIL-induced apoptosis of gastric cancer SGC7901 cells", as well as Avi Ashkenazi, etc. English literature published in the JOURNAL OFCLINICALONCOLOGY (VOLUME 26 NUMBER 21 JULY 20 2008) of "Ligand-Based Targeting of Apoptosis in Cancer: The Potential of Recombinant Human Apoptosis Ligand 2/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (rhApo2L/TRAIL)" as well as Frank AEKruyt published in Cancer Letters 263 (2008) 14 -25 "TRAIL and cancer therapy".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 all is to belong to drug combination, just TRAIL belongs to different pharmaceutics unit with chemotherapeutics, and occasional combination together during clinical practice.Though those of ordinary skill according to above-mentioned document, may be made a preparation unit with TRAIL and slightly solubility chemotherapeutics, because TRAIL is water miscible, itself and insoluble drug mixed make compound medicine and almost can't realize.
Though for the antineoplastic chemotherapy medicine of slightly solubility, those of ordinary skill can consider to make nanosphere, can supply injection or for oral use for the preparation suspension.Disclosed with polylactic acid enclose hydroxy camptothecin such as Chinese patent CN101352420B.But still the antitumor drug of TNFSF albumen and slightly solubility is not made the report of suitable micro-nano ball in the prior art.
In addition, TRAIL is that the TNFSF albumen of representative also exists too short defective of half-life, thereby causes influencing the body giving drugs into nose of medicine for dynamic characteristic.Paclitaxel is the antineoplastic chemotherapy medicine of representative, need enter competence exertion optimum curative effect in the 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 the film emulsifying technology to make is provided.Specifically, first purpose of the present invention has provided 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 the performance antitumaous effect, the antineoplastic chemotherapy medicine that also can carry slightly solubility arrives in the body, has brought into play synergism.Second purpose of the present invention provided a kind of HSA albumen micro-nano ball that utilizes film emulsifying can carry paclitaxel.The 3rd purpose of the present invention provided 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 first kind of albumen is TNFSF albumen, and antineoplastic chemotherapy medicine is insoluble drug, compositions comprises the housing of the complete closed basically that is 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; The platinum class comprises carboplatin, cisplatin; Hormones comprises estrogen, progestogen.
Special needs to be pointed out is that albumen micro-nano ball of the present invention is fit to carry paclitaxel very much.
The albumen micro-nano ball that carries antineoplastic chemotherapy medicine of the present invention, TNFSF albumen comprise 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, the TNFSF18.
Particularly preferably be, TNFSF albumen is TRAIL.TRAIL (TNF – related apop tosis inducing ligand) is an important apoptosis regulatory protein, the TRAIL molecule is present in most of organs and the cell of human body with the 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 made up of 747 aminoacid.The 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 not had 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 organic solvent as oil phase;
B. albumen is water-soluble as water;
C. adopt the film emulsification method that water and oil phase are made the film emulsion;
D. the curing with the film emulsion makes micro-nano ball;
E. with the micro-nano ball lyophilization.
First kind to being improved to of 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 organic solvent as oil phase;
B. with the TNFSF protein solution as water;
C. oil phase and water are made oil/water colostric fluid;
D. oil/water colostric fluid is carried out film emulsifying, get the film emulsion;
E. the curing with the film emulsion makes micro-nano ball;
F. with the micro-nano ball lyophilization.
In this experiment, wash away without continuous phase, once get product.Principle is that colostric fluid is the bigger oil-in-water system of granule, by being squeezed into nano level oil-in-water structure behind the film, and then solidify to form granule
Furtherly, when antitumor drug is that paclitaxel and TNFSF albumen are TRAIL, made by following steps:
A. the paclitaxel with the 20-40 weight portion is dissolved in ethyl acetate as oil phase;
B. with 50 weight portion TRAIL aqueous solutions as water;
C. under the ice-water bath condition, adopt ultrasonication that oil phase is scattered in aqueous phase and prepare oil/water colostric fluid;
D. colostric fluid is poured in the quick film emulsifier unit, under room temperature, under the 0.5-1.0MPa nitrogen pressure, pressed fenestra to carry out film emulsifying repeatedly colostric fluid, get the film emulsion;
E. organic solvent being removed in the magnetic agitation volatilization at normal temperatures of film emulsion makes its curing make micro-nano ball;
F. deionized water centrifuge washing 5 times of the micro-nano ball after will solidifying, and described compositions is made in lyophilization.
Surprisingly, be 20: 50 o'clock in the weight portion proportioning of ethyl acetate and water, effect is ideal.
Second kind to being improved to of the method for making of albumen micro-nano ball, and when albumen is TNFSF albumen, also can prepare as follows:
A. antineoplastic chemotherapy medicine is dissolved in organic solvent as oil phase;
B. with the TNFSF protein solution as water;
C. with oil phase as decentralized photo, water is carried out film emulsifying as continuous phase, get the film emulsion;
D. the curing with the film emulsion makes micro-nano ball;
E. with the 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 organic solvent as oil phase;
B. HAS albumen is water-soluble as water;
C. oil phase and described water are made oil/water colostric fluid;
D. oil/water colostric fluid is carried out film emulsifying, get the film emulsion;
E. the curing with the film emulsion makes micro-nano ball;
F. with the micro-nano ball lyophilization.
Human serum albumin of the present invention (Human Serum Albumin is called for short HSA) is the protein in the human plasma, and its nonglycosylated single chain polypeptide comprises 585 aminoacid, and molecular weight is 66kD.Its concentration is 42g/L in blood plasma, accounts for 60% of blood plasma total protein.The human serum albumin can transport fatty acid, bile pigments, aminoacid, steroid hormone, metal ion and many treatment molecules etc. in body fluid: keep the normal osmotic pressure of blood simultaneously.
Beneficial effect of the present invention is, provide a kind of preparation based on the film emulsifying technology can carry the albumen micro-nano ball of insoluble anti-tumor medicament, the preparation method of albumen micro-nano ball of the TNFSF albumen, the HSA albumen that with TRAIL are representative particularly is provided, the envelop rate height of this method, has reasonably controlled burst effect.Be that example helps to understand explanation beneficial effect of the present invention below with TRAIL, but should be subjected to this to limit beneficial effect of the present invention for example, the TRA-DOC micro-nano ball can possess following characteristics again in the associating synergistic while of anticancer generation:
1, TRA-DOC is micro-nano ball, can make unitary agent; And " TRAIL-paclitaxel use in conjunction " just face with now joining, and is 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 " TRAIL-paclitaxel use in conjunction " do not have this effect;
3, the TRA-DOC micro-nano ball solves the not diffluent problem of slightly solubility chemotherapeutics, the dual curative effect of performance TRI and DOC, and paclitaxel needs other macromolecular material enclose in " TRAIL-paclitaxel use in conjunction ".
4, the synergism of TRA-DOC micro-nano ball will be higher than the synergism of " TRAIL-paclitaxel use in conjunction ".
5, more independent TRAIL of the half-life of TRA-DOC micro-nano ball is the about 20%-200% of TNFSF prolongation of representative.
Further specify the present invention below by the test example.
Test routine 1:H460 cell cytotoxicity test
Test method: the H460 cell is inoculated in 96 orifice plates, place incubator 24h after, treat behind the attached wall of cell culture fluid in careful each hole of sucking-off.It is that the H460 cell does not add any medicine that blank group is set, and matched group is respectively TRA-IBU (TRA is TRAIL, and IBU is ibuprofen), the 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 the 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 effect to the H460 cell on each concentration, and all is better than the matched group effect.Fragmentation effect optimum when diluting 4000 times wherein, as Fig. 7, the suppression ratio of the H460 of TRA-DOC is 87.4 ± 1.6%, significantly better than HSA-DOC group 65.4 ± 1.3%, TRA-IBU group 31.1 ± 0.6%.
Test routine 2:L929 cell cytotoxicity test
Test method is with test example 1.
Result of the test: this nanoparticle all presents significant inhibitory effect to the L929 cell on each concentration, and all is better than the matched group effect.Fragmentation effect optimum when diluting 4000 times wherein, as Fig. 8, the suppression ratio of the L929 of TRA-DOC is 49.1 ± 2.2%, significantly better than HSA-DOC group 5.3 ± 1.8%, TRA-IBU group 7.0 ± 1.3%.
3 pairs of S180 ascites tumors of test example mice inhibitory action
Test method: get S180 ascites tumor mouse tumor liquid under the aseptic condition, preparation cell suspension is with 1 * 10
6/ only concentration is inoculated in behind the axil of experiment mice right side subcutaneous.Next day, with the 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) equal volume (0.2ml/20g) and the administration simultaneously of laboratory observation sample.Successive administration 7 times.Treat that the negative control group animal tumor grows to certain volume and finishes to observe.
When experiment finished, the scale body weight was peeled off tumor, and the scale tumor is heavy, compared the statistics difference of each treated animal tumor weight with the 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 the test group tumor killing effect is significantly better than matched group.
The test of test example 4 targetings
The experimental apparatus flow cytometer
Test method is got two culture dishs, when cell reaches exponential phase, adds HSA-DOC and TRA-DOC and HeLa cell co-cultivation 24 hours (5 hours) respectively.Wash three times to remove not by the nanoparticle of endocytosis and part dead cell with PBS buffer solution then.Then use the digestion of pancreatin solution and be prepared into cell suspension, use flow cytometer to detect cell to the endocytosis efficient of 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 fluorochrome label.
Result of the test is in conjunction with shown in Figure 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 band fluorescence, and the fluorescence enhancing can only be that the particle of cell endocytic causes more.Curve moves to right and represents the interior TRA-DOC particle of cell more than the HSA-DOC particle, thereby the targeting of proof TRA-DOC particle is stronger.
Description of drawings
Fig. 1. particle structure sketch map of the present invention; Among the figure, 1 expression nanoparticle; 2 expression insoluble anti-tumor medicament kernels; 3 expressions are formed 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; Among the figure, the surperficial enclose of micro-nano ball of the present invention is complete as can be seen, even size distribution.
Fig. 3 .Zeta potentiometer; Among the figure, vertical coordinate Intensity represents intensity, and abscissa size represents particle diameter, and statistics graph represents statistical graph; Measurements represents to measure.Show among the figure that 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; Among the figure, counts/s represents counts/second, binding energy(ev) expression binding energy, survey represents survey map, show among the figure, micro-nano ball of the present invention detects the characteristic energy peak of N element at the 399eV place, show at this particle surface to have the nitrogen element, and then contain TNFSF albumen in the proof nanoparticle.
Fig. 5. circular dichroism spectra; Among the figure, intensitiy represents intensity, and wavelength represents wavelength; Show among the figure, 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 expression micro-nano ball; 2 expression HeLa tumor cells show among the figure that micro-nano ball 1 is proved that by the process of the cell membrane endocytosis of HeLa tumor cell 2 micro-nano ball of the present invention enters into cell interior easily, thus the effect of performance paclitaxel;
The cell toxicity test cell killing situation cartogram of Fig. 7 .H460 cell;
Among the 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;
Among the figure, vertical coordinate Inhibition Ratio represents suppression ratio; Control represents blank group;
Fig. 9. the 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
Experiment material:
Recombinant soluble human TRAIL (TRAIL) is by escherichia coli expression, extraction.Paclitaxel (purity 〉=98.00%); Ethyl acetate; PBS buffer solution; Deionized water.
Experimental apparatus:
The ultrasonication machine, high speed centrifuge has the zeta potential instrument of submicron particle diameter analytic function, awkward silence at a meeting emission scan ultramicroscope, flow cytometer, laser confocal microscope, x-ray photoelectron power spectrum diffractometer, high performance liquid chromatography, circular dichroism spectrometer, SPG membrane emulsifier.
Experimentation:
With 20mg paclitaxel (DOC) be dissolved in be made into 1mg/mL in the 20ml ethyl acetate solution as oil phase, with the TRAIL of 50mg be dissolved in be made into 1mg/mL in the 50ml water solution as water, under the ice-water bath condition, adopt ultrasonication that oil phase is scattered in aqueous phase and prepare oil/water colostric fluid.This colostric fluid is poured in the quick film emulsifier unit, nitrogen pressure with 0.5-1.0MPa under the room temperature was pressed the fenestra (aperture 800-1000nm) of SPG film repeatedly with it, organic solvent is removed in the magnetic agitation volatilization at normal temperatures of gained emulsion makes it be solidified into micro-nano ball, with 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 housing 3 of the complete closed basically 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 that 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.
X-ray photoelectron power spectrum as Fig. 4 shows that micro-nano ball of the present invention detects the characteristic energy peak of N element at the 399eV place, shows at this particle surface to have the nitrogen element, and then contains trail protein in the proof nanoparticle.Circular dichroism spectra as Fig. 5 shows, has protein in micro-nano ball of the present invention, and the secondary structure of this albumen does not significantly change.
Laser confocal microscope has as shown in Figure 6 proved micro-nano ball 1 by the process of the cell membrane endocytosis of HeLa tumor cell 2, thereby micro-nano ball of the present invention enters into cell interior easily, thus the effect of performance paclitaxel.
Experiment material:
Human serum albumin (HSA) is by escherichia coli expression, extraction.Paclitaxel (purity 〉=98.00%); Ethyl acetate; PBS buffer solution; Deionized water.
Experimental apparatus:
The ultrasonication machine, high speed centrifuge has the zeta potential instrument of submicron particle diameter analytic function, awkward silence at a meeting emission scan ultramicroscope, SPG membrane emulsifier.
Experimentation:
With 20mg paclitaxel (DOC) be dissolved in be made into 1mg/mL in the 20ml ethyl acetate solution as oil phase, with the HSA of 50mg be dissolved in be made into 1mg/mL in the 50ml water solution as water, under the ice-water bath condition, adopt ultrasonication that oil phase is scattered in aqueous phase and prepare oil/water colostric fluid.This colostric fluid is poured in the quick film emulsifier unit, nitrogen pressure with 0.5-1.0MPa under the room temperature was pressed the fenestra (aperture 800-1000nm) of SPG film repeatedly with it, organic solvent is removed in the magnetic agitation volatilization at normal temperatures of gained emulsion makes it be solidified into micro-nano ball, with 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 that the distribution of particles of particle size distribution 80% is between 300-400nm.
Experiment material:
TNF-α is by escherichia coli expression, extraction.Paclitaxel (purity 〉=98.00%); Ethyl acetate; PBS buffer solution; Deionized water.
Experimental apparatus:
The ultrasonication machine, high speed centrifuge has the zeta potential instrument of submicron particle diameter analytic function, awkward silence at a meeting emission scan ultramicroscope, SPG membrane emulsifier.
Experimentation:
With 20mg paclitaxel (DOC) be dissolved in be made into 1mg/mL in the 20ml ethyl acetate solution as oil phase, with the TNF-α of 50mg be dissolved in be made into 1mg/mL in the 50ml water solution as water, under the ice-water bath condition, adopt ultrasonication that oil phase is scattered in aqueous phase and prepare oil/water colostric fluid.This colostric fluid is poured in the quick film emulsifier unit, with the nitrogen pressure of 0.8MPa it being pressed the aperture repeatedly under the room temperature is the SPG film of 800nm, organic solvent is removed in the magnetic agitation volatilization at normal temperatures of gained emulsion makes it be solidified into micro-nano ball, with 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 only is that preferred implementation of the present invention is described; be not that scope of the present invention is limited; design under the prerequisite of spirit not breaking away 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 (11)
1. 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 compositions comprises the housing of the complete closed basically that is 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.
2. the albumen micro-nano ball that carries antineoplastic chemotherapy medicine as claimed in claim 1, it is characterized in that, the antineoplastic chemotherapy medicine of described slightly solubility comprises platinum class, vinca, taxanes, camptothecin, AC, D actinomycin D, bleomycin, daunorubicin, epirubicin, mitomycin, methotrexate, fluorouracil, carmustine, etoposide, interferon, phenesterin, hormones, tamoxifen; Described vinca comprises that vincaleucoblastine, vincristine and other are similar to the medicine of vinblastine; Described taxanes comprises that paclitaxel and derivant thereof, paclitaxel prodrug, taxane and other are similar to the medicine of paclitaxel; Described camptothecin comprises camptothecine and analog, hydroxy camptothecin and analog thereof; Described platinum class comprises carboplatin, cisplatin; Described hormones comprises estrogen, progestogen.
3. the albumen micro-nano ball that carries antineoplastic chemotherapy medicine as claimed in claim 2 is characterized in that, the antineoplastic chemotherapy medicine of described slightly solubility is paclitaxel.
4. the albumen micro-nano ball that carries antineoplastic chemotherapy medicine as claimed in claim 1, it is characterized in that described TNFSF albumen comprises one or more wild type and the allosteric type among EDA-A1, TNFSF1, TNFSF2, TNFSF3, TNFSF3L, TNFSF4, TNFSF54, TNFSF6, TNFSF7, TNFSF8, TNFSF9, TRAIL, TNFSF11, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, the TNFSF18.
5. the albumen micro-nano ball that carries antineoplastic chemotherapy medicine as claimed in claim 4 is characterized in that, described TNFSF albumen is TRAIL.
6. a method for making of carrying the albumen micro-nano ball of antineoplastic chemotherapy medicine is characterized in that, comprises the steps:
A. described antineoplastic chemotherapy medicine is dissolved in organic solvent as oil phase;
B. described albumen is water-soluble as water;
C. adopt the film emulsification method that described water and described oil phase are made the film emulsion;
D. the curing with the film emulsion makes micro-nano ball;
E. with the micro-nano ball lyophilization.
7. the method for making of albumen micro-nano ball as claimed in claim 6 is characterized in that, described albumen is TNFSF albumen, comprises the steps:
A. described antineoplastic chemotherapy medicine is dissolved in organic solvent as oil phase;
B. with described TNFSF protein solution as water;
C. described oil phase and described water are made oil/water colostric fluid;
D. oil/water colostric fluid is carried out film emulsifying, get the film emulsion;
E. the curing with the film emulsion makes micro-nano ball;
F. with the micro-nano ball lyophilization.
8. the method for making of albumen micro-nano ball as claimed in claim 6 is characterized in that, described albumen is TNFSF albumen, comprises the steps:
A. described antineoplastic chemotherapy medicine is dissolved in organic solvent as oil phase;
B. with the TNFSF protein solution as water;
C. with described oil phase as decentralized photo, described water is carried out film emulsifying as continuous phase, get the film emulsion;
D. the curing with the film emulsion makes micro-nano ball;
E. with the micro-nano ball lyophilization.
9. the method for making of albumen micro-nano ball as claimed in claim 6 is characterized in that, described albumen is HSA albumen, and described antineoplastic chemotherapy medicine is paclitaxel, comprises the steps:
A. paclitaxel is dissolved in organic solvent as oil phase;
B. HAS albumen is water-soluble as water;
C. described oil phase and described water are made oil/water colostric fluid;
D. oil/water colostric fluid is carried out film emulsifying, get the film emulsion;
E. the curing with the film emulsion makes micro-nano ball;
F. with the micro-nano ball lyophilization.
10. the method for making of albumen micro-nano ball as claimed in claim 7 is characterized in that, described antitumor drug is paclitaxel, and described TNFSF albumen is TRAIL, comprises the steps:
A. the paclitaxel with the 20-40 weight portion is dissolved in ethyl acetate as oil phase;
B. with 50 weight portion TRAIL aqueous solutions as water;
C. under the ice-water bath condition, adopt ultrasonication that oil phase is scattered in aqueous phase and prepare oil/water colostric fluid;
D. described colostric fluid is poured in the quick film emulsifier unit, under room temperature, under the 0.5-1.0MPa nitrogen pressure, pressed fenestra to carry out film emulsifying repeatedly described colostric fluid, get the film emulsion;
E. organic solvent being removed in the magnetic agitation volatilization at normal temperatures of described film emulsion makes its curing make micro-nano ball;
F. described micro-nano ball deionized water centrifuge washing 5 times after will solidifying, and described compositions is made in lyophilization.
11. the method for preparing micro-nano ball as claimed in claim 7 is characterized in that, the weight portion proportioning of described ethyl acetate and water is 20: 50.
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| CN103432072A (en) * | 2013-09-11 | 2013-12-11 | 中国药科大学 | In-dissolvable drug food protein stabilizing suspension for injection and preparation method thereof |
| CN112089704A (en) * | 2020-09-27 | 2020-12-18 | 中国药科大学 | Bionic nano-carrier and preparation method and application thereof |
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| 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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| 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 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103432072A (en) * | 2013-09-11 | 2013-12-11 | 中国药科大学 | In-dissolvable drug food protein stabilizing suspension for injection and preparation method thereof |
| CN112089704A (en) * | 2020-09-27 | 2020-12-18 | 中国药科大学 | Bionic nano-carrier and preparation method and application thereof |
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