CN102283810A - Antibody targeting nano preparation and preparation method thereof - Google Patents
Antibody targeting nano preparation and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of an antibody targeting nano preparation. The antibody targeting nano preparation is characterized by comprising an anti-vascular endothelial growth factor antibody, polylactic acid nanoparticles and a coupling agent, wherein the mass ratio of the polylactic acid nanoparticles to the anti-vascular endothelial growth factor antibody is 10:1-8:1; and the mass ratio of the coupling agent to the anti-vascular endothelial growth factor antibody is 2:1. The 5-FU, which combines the anti-vascular endothelial growth factor antibody and the nanoparticles, can be combined with tumor cells in a targeting mode, so that the antitumor activity is greatly enhanced, the consumption of the 5-FU is reduced, the side effect of the 5-FU is lowered, and the tolerance of the patients is enhanced.
Description
Technical field
The present invention relates to a kind of preparation method of antibody target nanometer formulation.
Background technology
5-fluorouracil (5-FU) is the fluoride of miazines, belongs to the anti-metabolism antitumor drug, can suppress the thymidylic acid synzyme, and blocking-up deoxidation pyrimidine nucleotide converts thymic acid to, disturbs synthesizing of DNA.Still can suppress the formation of RNA in addition, thereby suppress the propagation of tumor cell, be the anti-metabolism antitumor drug that extensively uses clinically, but 5-FU might cause untoward reaction such as serious digestive tract reaction and bone marrow depression.
The antibody target nanometer formulation has wide application prospect as a kind of nano-grade medicine carrier at aspects such as improving drug solubility, increase medicine stability, slow controlled release drug administration, target administration.In present tumor research, the link coupled research report of existing medicine and monoclonal antibody.Therefore there are some researches show that the overexpression of vegf protein and tumor vascular formation and transfer have substantial connection, is that the antitumor drug of target spot just becomes one of method of treatment tumor with VEGF and receptor thereof.
Because the toxic and side effects of 5-fluorouracil has limited its application, if can make its directly effect tumor cell by targeting, and lack effect or do not act on normal cell, can reduce its toxic and side effects so, and minimizing dosage has reduced patient's chemical sproof risk.
The invention provides the antibody target nanometer formulation, it comprises anti-vascular endothelial growth factor antibody and polylactic acid nano microgranule, can effectively solve a prior art difficult problem.
Summary of the invention
Goal of the invention
The present invention adopts ultrasonic emulsification-solvent evaporated method to prepare the nanometer formulation medicine, prepared a kind ofly have slow releasing function, increase medicine in vivo the holdup time, have the antibody target nanometer formulation of targeting.
Technical scheme
The antibody target nanometer formulation is characterized in that it comprises anti-vascular endothelial growth factor antibody, polylactic acid nano microgranule, coupling agent; Wherein the mass ratio of polylactic acid nano microgranule and anti-vascular endothelial growth factor antibody is 10: 1～8: 1; The mass ratio of coupling agent and anti-vascular endothelial growth factor antibody is 2: 1.
Coupling agent is a carbodiimide.
The polylactic acid nano microgranule is made of polylactic acid, 5-fluorouracil, serum albumin, and its mass ratio is 4: 2: 1.
The mean diameter of described antibody target nanometer formulation is less than 300 nanometers.
The mean diameter of described antibody target nanometer formulation is the 230-280 nanometer.
Described antibody target nanometer formulation is a freeze-dried powder preparation.
Described serum albumin is bovine serum albumin or human serum albumin.
A kind of preparation method of antibody target nanometer formulation is characterized in that the following step composition:
A. prepare the 5-fluorouracil aqueous solution, standby as interior water;
B. take by weighing polylactic acid and be dissolved in the organic solvent, as oil phase, at 300w, the 3min ultrasound condition drips in the oil phase down, forms milky water/oily colostrum solution with interior water;
C. take by weighing serum albumin and be dissolved in the saturated 5-fluorouracil aqueous solution, be mixed with the serum albumin aqueous solution and make outer water, water/oily colostrum solution at 300W, is added drop-wise to outer aqueous phase under the 6min ultra-sonic dispersion condition, form the W/0/W compound system;
D. electromagnetic agitation W/O/W compound system makes organic solvent wherein slowly evaporate into volatilization fully; Obtain the polylactic acid nano microgranule, constituting of polylactic acid nano microgranule wherein by polylactic acid, 5-fluorouracil, serum albumin, its mass ratio is 4: 2: 1;
E. take by weighing mass ratio and be 10: 1 polylactic acid nano microgranule and anti-vascular endothelial growth factor antibody, be dissolved in pH and be in 6.86 the buffer solution, under stirring, add carbodiimide, wherein the mass ratio of coupling agent and anti-vascular endothelial growth factor antibody is 2: 1,4 ℃ are stirred 2h down, make the antibody target nanoparticle.
Organic solvent described in step b and the d is an acetone.
Coupling agent is a carbodiimide among the step e.
Preparation process schematic flow sheet of the present invention is seen Fig. 1.
The encoding amino acid sequence of anti-vascular endothelial growth factor antibody (heavy chain):
MDWTWILFLVAAATRVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQA
PGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDH
GPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH
TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKZ
The encoding amino acid sequence of anti-vascular endothelial growth factor antibody (light chain):
MDWTWILFLVAAATRVHSQSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPG
KAPKLMIYEVSKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGSNNLVFGGG
TKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT
TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSZ
Beneficial effect
The principle of the invention is explained: the present invention adopts anti-vascular endothelial growth factor antibodies nanoparticle, wherein includes 5-FU in the nanoparticle.Wherein the anti-VEGF monoclonal antibody in total man source specificity is in conjunction with VEGF; And angiogenesis is the basis of reproduction, growth promoter and reparation, and most malignant entity tumors such as ovarian cancer, hepatocarcinoma, cervical cancer and breast carcinoma etc. all are the blood vessel dependent tumors.In tumor vascular endothelial cell, VEGF shows as rise, and the vegf expression amount is very low in the normal structure; Therefore adopt the anti-vascular endothelial growth factor antibody specificity in conjunction with the VEGF that raises in the tumor cell, realize the targeting purpose; This human vessel endothelium growth factor resisting antibody, be that China Medicine University cooperates with Shanghai Research Center of Genetically Modified, utilization changes whole human source anti-vascular endothelial cell growth factor (ECGF) (VEGF) monoclonal antibody that human immunoglobulin gene's five feature transgenic mices successfully prepare over to, itself has significant inside and outside anti-tumor activity; The 5-FU of anti-vascular endothelial growth factor antibodies nanoparticle of the present invention, not only targeting is in conjunction with tumor cell, and makes anti-tumor activity strengthen greatly, and reduced the consumption of 5-FU, reduced the side effect of 5-FU, increased patient's toleration.Following is detailed explanation:
1, as can be seen from Figure 3, the anti-VEGF monoclonal antibody in total man source can be in blood, heart, liver, spleen, lung and bone, and behind the intravenously administrable, its radioactivity reaches maximum rapidly, and the prolongation radioactivity along with the time progressively reduces then; And in stomach, muscle, gonad and small intestinal, it is maximum that its radioactivity 1h after administration reaches, and reducing along with the prolongation of time then; Radioactivity in the brain is very low all the time, shows that the anti-VEGF monoclonal antibody in total man source can't pass through blood brain barrier.From table one as can be known, the antibody activity of antibody target nanoparticle can be preserved more than 80%, and therefore, the targeted nano preparation can be assembled at some position, thereby effectively reduces general toxicity.
2,5-fluorouracil low price and kinds of tumors such as digestive tract tumor, breast carcinoma, ovarian cancer, chorionic epithelioma, cervical cancer, hepatocarcinoma, bladder cancer, skin carcinoma (partial smearing) leukoplakia vulvae (partial smearing) etc. all had certain curative effect.Separately or with the other drug use in conjunction in breast carcinoma and gastroenteric tumor operation auxiliary treatment, also be used for the palliative treatment of some non-operation malignant tumor, especially the malignant tumor of those gastrointestinal tract, mammary gland, incidence, liver, urinary system and pancreas.But because 5-fluorouracil has strong general toxicity,, influenced its clinical use value, so we prepare 5-fluorouracil polylactic acid targeted nano preparation, wash and to reduce its 5-fluorouracil general toxicity as gastrointestinal reaction and bone marrow depression.By the result of Fig. 3 as can be known, the slow release effect of targeted nano preparation is 4-5 days, and targeting preparation can slowly discharge 5-fluorouracil in the target site enrichment, thereby reaches the effect of slow release.Because it discharges 5-fluorouracil at targeting moiety, targeting preparation can significantly reduce the general toxicity of 5-fluorouracil.
3, preparation 5-fluorouracil polylactic acid targeted nano preparation, its significant advantage can be utilized carbodiimide (EDC) coupling agent and the coupling of anti-vascular endothelial growth factor monoclonal antibody exactly, thereby improve the tumor-targeting of 5-fluorouracil polylactic acid targeted nano preparation, reduce toxicity.From the interpretation of result of Fig. 4, Fig. 5 and Fig. 6 as can be known, total man's resource monoclonal antibody has significant inhibitory effect to the expression of angiogenesis factor VEGF, and assembles explanation antibody in particular organization VEGF is had good targeting.Therefore from table two as can be known, the antibody activity of antibody 2. targeted nano microgranules can be preserved about 80%, can improve nanometer formulation after the coupling significantly to the targeting of cancerous cell.
4, as can be known, preparation 5-fluorouracil polylactic acid targeted nano preparation is under identical 5-FU dosage, and its general toxicity to the tumor model mouse is less, and losing weight of model mouse is less, proves that targeting preparation has effectively reduced the general toxicity of 5-FU from table three; And from the tumour inhibiting rate result, the targeting preparation of same dose is better than the tumor killing effect of the preparation of independent 5-FU, and the drug effect of 5-FU is significantly improved.
5, the present invention adopts ultrasonic emulsification-solvent evaporated method to prepare the nanometer formulation medicine, and operational approach is simple.Utilize the nanometer formulation medicine of this method preparation, measure its particle diameter and distribution, the results are shown in Figure 7 by photon correlation spectroscopy.As seen, mean diameter is 280nm among the figure, and polydispersity index is 0.07, and the narrower and rare macroparticle of particle size distribution range illustrates that the nanometer formulation dairy industry that adopts this method to make is the stabilizing solution system.
Description of drawings
Fig. 1 is a preparation process schematic flow sheet of the present invention.
Fig. 2 is a targeted nano preparation release curve.
Fig. 3 is the distribution of the anti-VEGF monoclonal antibody in total man source in mouse tissue behind the intravenously administrable
Fig. 4 is the influence of the anti-VEGF monoclonal antibody in total man source to angiogenesis.(a. normal group, b. model group, c. low dose group, d. high dose group, e. positive control)
Fig. 5 is the influence of the anti-VEGF monoclonal antibody in total man source to vegf expression.(a. normal group, b. model group, c. low dose group, d. high dose group, e. positive control)
Fig. 6 is the particle size distribution figure of nanometer formulation.
Fig. 7 is the particle size distribution figure of targeted nano preparation.
The specific embodiment
Following examples material therefor and instrument and equipment are:
Implement material: bovine serum albumin (Nanjing Ao Duofuni bio tech ltd), human serum albumin's (Beijing whole world development in science and technology company limited), 5-fluorouracil crude drug (Nanjing Ao Duofuni bio tech ltd), polylactic acid (Mw=30000) (Mount Tai, Jinan, Shandong handle of the Big Dipper Science and Technology Ltd.), anti-vascular endothelial growth factor antibody (prepares Yu Liu by literature method, *, Dong-jie Yun, Jian-quan Chen, Jian-yang Zhao, Si-guo Liu, Guo-xiang Cheng.Isolation and characterization of human anti-VEGF 165monoclonal antibody with antitumor efficacy from transgenic mice expressing human immunoglobulin loci.Cancer letters, 2009), carbodiimide (Sigma company).
Instrument and equipment: ultrasonic cell disintegration instrument (the new sesame bio tech ltd in Ningbo), laser particle analyzer (U.S.'s Brooker-Hai Wen company), refrigerated centrifuger (U.S. Thermo company), magnetic force heating stirrer (Shanghai Si Le Instr Ltd.), electronic balance (Satourius company), ultraviolet-visible spectrophotometer (Shanghai essence instrument Science and Technology Ltd.).
The employing method is with reference to 989～999 pages of " pharmaceutics " third editions (Tu Xide, Zhang Jun longevity, Zhu Jiabi chief editor) in the present embodiment; 337～348 pages of " medicine controlled releasing novel form " (Jia Wei, Gao Wenyuan chief editors).
Embodiment 1
The antibody target nanometer formulation is characterized in that it comprises anti-vascular endothelial growth factor antibody, polylactic acid nano microgranule, coupling agent; Wherein the mass ratio of polylactic acid nano microgranule and anti-vascular endothelial growth factor antibody is 10: 1; The mass ratio of coupling agent and anti-vascular endothelial growth factor antibody is 2: 1.
Coupling agent is a carbodiimide.
The polylactic acid nano microgranule is made of polylactic acid, 5-fluorouracil, bovine serum albumin, and its mass ratio is 4: 2: 1.
The mean diameter of described antibody target nanometer formulation is less than 300 nanometers.
A kind of preparation method of antibody target nanometer formulation is characterized in that the following step composition:
A. prepare mass volume ratio and be 5%～10% 5-fluorouracil aqueous solution, gained solution is standby as interior water;
B. polylactic acid is dissolved in the organic solvent, is mixed with mass volume ratio and is 5%～6% solution and make oil phase, at 300w, the 3min ultrasound condition drips in the oil phase down, forms milky water/oily colostrum solution with the interior water of 2.5ml;
C. taking by weighing bovine serum albumin is dissolved in the saturated 5-fluorouracil aqueous solution, be mixed with mass volume ratio and be 0.3%～0.5% Bovine Serum Albumin in Aqueous Solution and make outer water, colostrum solution at 300W, is added drop-wise to outer aqueous phase under the 6min ultra-sonic dispersion condition, forms the W/O/W compound system;
D. electromagnetic agitation W/0/W compound system makes organic solvent wherein slowly evaporate into volatilization fully; Obtain the polylactic acid nano microgranule, it is made up of polylactic acid, 5-fluorouracil and bovine serum albumin, and its mass ratio is 4: 2: 1;
E.16000 the polylactic acid nano microgranule that makes of rev/min high speed centrifuge separated and collected, with deionized water wash three to four times, lyophilization is preserved standby in-20 ℃ of refrigerators;
F. take by weighing mass ratio and be 10: 1 polylactic acid nano microgranule and anti-vascular endothelial growth factor antibody, be dissolved in pH and be in 6.86 the buffer solution, under stirring, add carbodiimide, wherein the mass ratio of carbodiimide and monoclonal antibody is 2: 1, and 4 ℃ are stirred 2h down, with gained solution 16000r/min, the 5min centrifugalize, use deionized water wash three times behind the ultra-sonic dispersion, lyophilization, gained white powdery solid is the antibody target nanoparticle.
Embodiment 2
The antibody target nanometer formulation is characterized in that it comprises anti-vascular endothelial growth factor antibody, polylactic acid nano microgranule, coupling agent; Wherein the mass ratio of polylactic acid nano microgranule and anti-vascular endothelial growth factor antibody is 8: 1; The mass ratio of coupling agent and anti-vascular endothelial growth factor antibody is 2: 1.
Coupling agent is a carbodiimide.
The polylactic acid nano microgranule is made of polylactic acid, 5-fluorouracil, bovine serum albumin, and its mass ratio is 4: 2: 1.
The mean diameter of described antibody target nanometer formulation is the 230-280 nanometer.
A kind of preparation method of antibody target nanometer formulation is characterized in that the following step composition:
A. prepare mass volume ratio and be 5%～10% 5-fluorouracil aqueous solution, gained solution is standby as interior water;
B. polylactic acid is dissolved in the organic solvent, is mixed with mass volume ratio and is 5%～6% solution and make oil phase, at 300w, the 3min ultrasound condition drips in the oil phase down, forms milky water/oily colostrum solution with the interior water of 2.5ml;
C. taking by weighing bovine serum albumin is dissolved in the saturated 5-fluorouracil aqueous solution, be mixed with mass volume ratio and be 0.3%～0.5% Bovine Serum Albumin in Aqueous Solution and make outer water, colostrum solution at 300W, is added drop-wise to outer aqueous phase under the 6min ultra-sonic dispersion condition, forms the W/O/W compound system;
D. electromagnetic agitation W/O/W compound system makes organic solvent wherein slowly evaporate into volatilization fully; Obtain the polylactic acid nano microgranule, it is made up of polylactic acid, 5-fluorouracil and bovine serum albumin, and its mass ratio is 4: 2: 1;
E.16000 the polylactic acid nano microgranule that makes of rev/min high speed centrifuge separated and collected, with deionized water wash three to four times, lyophilization is preserved standby in-20 ℃ of refrigerators;
F. take by weighing mass ratio and be 8: 1 polylactic acid nano microgranule and anti-vascular endothelial growth factor antibody, be dissolved in pH and be in 6.86 the buffer solution, under stirring, add carbon: imines, wherein the mass ratio of carbodiimide and monoclonal antibody is 2: 1, and 4 ℃ are stirred 2h down, with gained solution 16000r/min, the 5min centrifugalize, use deionized water wash three times behind the ultra-sonic dispersion, lyophilization, gained white powdery solid is the antibody target nanoparticle.
Embodiment 3
The antibody target nanometer formulation is characterized in that it comprises anti-vascular endothelial growth factor antibody, polylactic acid nano microgranule, coupling agent; Wherein the mass ratio of polylactic acid nano microgranule and anti-vascular endothelial growth factor antibody is 8: 1; The mass ratio of coupling agent and anti-vascular endothelial growth factor antibody is 2: 1.
Coupling agent is a carbodiimide.
The polylactic acid nano microgranule is made of polylactic acid, 5-fluorouracil, human serum albumin, and its mass ratio is 4: 2: 1.
The mean diameter of described antibody target nanometer formulation is the 230-280 nanometer.
A kind of preparation method of antibody target nanometer formulation is characterized in that the following step composition:
A. prepare mass volume ratio and be 5%～10% 5-fluorouracil aqueous solution, gained solution is standby as interior water;
B. polylactic acid is dissolved in the organic solvent, is mixed with mass volume ratio and is 5%～6% solution and make oil phase, at 300w, the 3min ultrasound condition drips in the oil phase down, forms milky water/oily colostrum solution with the interior water of 2.5ml;
C. taking by weighing the human serum albumin is dissolved in the saturated 5-fluorouracil aqueous solution, be mixed with mass volume ratio and be human serum albumin's aqueous solution of 0.3%～0.5% and make outer water, colostrum solution at 300W, is added drop-wise to outer aqueous phase under the 6min ultra-sonic dispersion condition, forms the W/0/W compound system;
D. electromagnetic agitation W/0/W compound system makes organic solvent wherein slowly evaporate into volatilization fully; Obtain the polylactic acid nano microgranule, it is made up of polylactic acid, 5-fluorouracil and bovine serum albumin, and its mass ratio is 4: 2: 1;
E.16000 the polylactic acid nano microgranule that makes of rev/min high speed centrifuge separated and collected, with deionized water wash three to four times, lyophilization is preserved standby in-20 ℃ of refrigerators;
F. take by weighing mass ratio and be 8: 1 polylactic acid nano microgranule and anti-vascular endothelial growth factor antibody, be dissolved in pH and be in 6.86 the buffer solution, under stirring, add carbodiimide, wherein the mass ratio of carbodiimide and monoclonal antibody is 2: 1, and 4 ℃ are stirred 2h down, with gained solution 16000r/min, the 5min centrifugalize, use deionized water wash three times behind the ultra-sonic dispersion, lyophilization, gained white powdery solid is the antibody target nanoparticle.
Embodiment 4
The antibody target nanometer formulation is characterized in that it comprises anti-vascular endothelial growth factor antibody, polylactic acid nano microgranule, coupling agent; Wherein the mass ratio of polylactic acid nano microgranule and anti-vascular endothelial growth factor antibody is 10: 1; The mass ratio of coupling agent and anti-vascular endothelial growth factor antibody is 2: 1.
Coupling agent is a carbodiimide.
The polylactic acid nano microgranule is made of polylactic acid, 5-fluorouracil, human serum albumin, and its mass ratio is 4: 2: 1.
The mean diameter of described antibody target nanometer formulation is less than 300 nanometers.
A kind of preparation method of antibody target nanometer formulation is characterized in that the following step composition:
A. prepare mass volume ratio and be 5%～10% 5-fluorouracil aqueous solution, gained solution is standby as interior water;
B. polylactic acid is dissolved in the organic solvent, is mixed with mass volume ratio and is 5%～6% solution and make oil phase, at 300w, the 3min ultrasound condition drips in the oil phase down, forms milky water/oily colostrum solution with the interior water of 2.5ml;
C. taking by weighing the human serum albumin is dissolved in the saturated 5-fluorouracil aqueous solution, be mixed with mass volume ratio and be 0.3%～0.5% Bovine Serum Albumin in Aqueous Solution and make outer water, colostrum solution at 300W, is added drop-wise to outer aqueous phase under the 6min ultra-sonic dispersion condition, forms the W/0/W compound system;
D. electromagnetic agitation W/0/W compound system makes organic solvent wherein slowly evaporate into volatilization fully; Obtain the polylactic acid nano microgranule, it is made up of polylactic acid, 5-fluorouracil and human serum albumin, and its mass ratio is 4: 2: 1;
E.16000 the polylactic acid nano microgranule that makes of rev/min high speed centrifuge separated and collected, with deionized water wash three to four times, lyophilization is preserved standby in-20 ℃ of refrigerators;
F. take by weighing mass ratio and be 10: 1 polylactic acid nano microgranule and anti-vascular endothelial growth factor antibody, be dissolved in pH and be in 6.86 the buffer solution, under stirring, add carbodiimide, wherein the mass ratio of carbodiimide and monoclonal antibody is 2: 1, and 4 ℃ are stirred 2h down, with gained solution 16000r/min, the 5min centrifugalize, use deionized water wash three times behind the ultra-sonic dispersion, lyophilization, gained white powdery solid is the antibody target nanoparticle.
A. detect the antibody activity of antibody antibody 2. antibody target nanoparticles with the Elisa detection method
1. use the vegf protein bag by 96 orifice plates.
2. every hole adds 100ul, 1%BSA one buffer solution, and 4 ℃ were sealed 24 hours, washed the exhaustion residual liquid 2 times.
3. dilution buffer liquid is pressed monoclonal anti scale of construction gradient dilution with monoclonal antibody and antibody target nanoparticle, and every hole adds 100ul, hatches 2 hours for 37 ℃.
4. wash 5 times, add two anti-(dilution in 1: 2000) every hole of 100ul, hatched 2 hours for 37 ℃.
5. wash 5 times, add the every hole of substrate solution 100ul, hatched 0.5 hour, and added stop buffer for 37 ℃.
6. microplate reader detects the 492nm wavelength OD of place value, and the corresponding monoclonal anti bulk concentration mapping of absorbance with monoclonal antibody and antibody target nanoparticle the results are shown in Table 1;
Table one
The b.MTT colorimetry is estimated the inhibitory action of antibody target nanoparticle to tumor cell
MTT is a kind of water solublity tetrazolium, and competent cell is learned the dehydrogenase of case body internal respiration chain, yellowish green MTT degraded can be formed water-fast bluish violet crystallization.This crystallization dissolves in DMSO and au bleu, and its shade is directly proportional with viable count.But the suppression ratio of the survival rate of quantitative assay cell and medicine pair cell thus.
Suppression ratio=(matched group OD value-experimental group OD value)/matched group OD value * 100%
Experimental procedure
With the SGC7901 cell inoculation in 96 orifice plates, in 37 ℃, 5%CO2 incubator, cultivate 48h, adherent.
2. the nanoparticle with variable concentrations adds in 96 orifice plates, the every hole of 100ul, 6 every group multiple holes.Setting the free auxocyte that only adds culture fluid simultaneously is matched group.Cultivate 48h in 37 ℃, 5%CO2 incubator.
3. add 5ml/mgMTT, the every hole of 20ul continues to cultivate 4h, abandons supernatant, adds 100ulDMSO, and concussion 10min all dissolves crystallization.Microplate reader 490nm surveys the OD value.
4. calculating suppression ratio.
Tumor control rate the results are shown in Table two
Table two
Wherein said antibody target nanoparticle obtains the antibody target nanoparticle for embodiment 1-4.
Embodiment 6
The foundation of a.ICR mouse tumor model
1. cultured S180 tumor cell is prepared into the cancerous cell suspension with PBS after trypsinization, the adjustment cell concentration is 1*10
6, 0.5ml cell suspension is advanced in the ICR mouse peritoneal by lumbar injection, collect ascites after the week, ascites is with 4 times of PBS dilutions, stand-by.
2. choose the ICR mice of body weight 18g-22g, the good ascites 0.2ml of dilution that oxter injection dilution is good, one week the back observe the tumor growth situation, choose ICR mice about the about 1cm of big or small diameter of tumor as experimental model.
B. the antineoplaston of antibody target nanoparticle
S180 tumor model ICR mice is divided into 4 groups at random, that is: 5-fluorouracil treatment group, antibody 2. targeted nano microgranule high dose group, antibody 2. targeted nano microgranule low dose group, model group.Concrete Therapeutic Method is as follows:
1.5-fluorouracil in treatment group (6): with the 5-fluorouracil injection of 10mg/ml according to the dosage of 20mg/kg from mouse tail vein injection.Be administered once in per 5 days.
2. antibody 2. antibody 2. targeted nano microgranule high dose group (6): with an amount of antibody 2. targeted nano microparticulate in the PBS isosmotic solution, ultra-sonic dispersion, injected dose is equivalent to the 5-fluorouracil dosage of 5-fluorouracil treatment group, from mouse tail vein injection.Be administered once in per 5 days.
3. antibody 2. targeted nano microgranule low dose group (6): an amount of antibody 2. antibody target nanoparticles are scattered in the PBS isosmotic solution, ultra-sonic dispersion, injected dose is equivalent to half of 5-fluorouracil dosage of 5-fluorouracil treatment group, from mouse tail vein injection.Be administered once in per 5 days.
4. model group (6): tail vein injection saline.
C. the result measures
Put to death mice after 15 days, peel off the tumor body and weigh, calculate and press down tumor efficient.Press down tumor efficient=(the average tumor of the average tumor weight/model group of the average tumor weight-experimental group of model group is heavy) * 100%.Measure diameter of tumor, relatively tumor killing effect.The results are shown in Table three
Table three
Wherein said antibody target nanoparticle obtains the antibody target nanoparticle for embodiment 1-4.
Claims (10)
1. the antibody target nanometer formulation is characterized in that it comprises anti-vascular endothelial growth factor antibody, polylactic acid nano microgranule, coupling agent; Wherein the mass ratio of polylactic acid nano microgranule and anti-vascular endothelial growth factor antibody is 10: 1～8: 1; The mass ratio of coupling agent and anti-vascular endothelial growth factor antibody is 2: 1.
2. antibody target nanometer formulation according to claim 1 is characterized in that coupling agent is a carbodiimide.
3. antibody target nanometer formulation according to claim 1 is characterized in that the polylactic acid nano microgranule is made of polylactic acid, 5-fluorouracil, serum albumin, and its mass ratio is 4: 2: 1.
4. antibody target nanometer formulation according to claim 3 is characterized in that described serum albumin is bovine serum albumin or human serum albumin.
5. antibody target nanometer formulation according to claim 1, the mean diameter that it is characterized in that described antibody target nanometer formulation is less than 300 nanometers.
6. according to the described antibody target nanometer formulation of claim 5, it is characterized in that: the mean diameter of described antibody target nanometer formulation is the 230-280 nanometer.
7. according to the described antibody target nanometer formulation of claim 1, it is characterized in that: described antibody target nanometer formulation is a freeze-dried powder preparation.
8. the preparation method of an antibody target nanometer formulation is characterized in that the following step forms:
A. be to take by weighing raw material at 4: 2: 1 by polylactic acid, 5-fluorouracil, sero-abluminous mass ratio; Preparation 5-fluorouracil aqueous solution, standby as interior water;
B. take by weighing polylactic acid and be dissolved in the organic solvent, as oil phase, at 300w, the 3min ultrasound condition drips in the oil phase down, forms milky water/oily colostrum solution with interior water;
C. take by weighing serum albumin and be dissolved in the saturated 5-fluorouracil aqueous solution, be mixed with Bovine Serum Albumin in Aqueous Solution and make outer water, water/oily colostrum solution at 300W, is added drop-wise to outer aqueous phase under the 6min ultra-sonic dispersion condition, form the W/0/W compound system;
D. electromagnetic agitation W/O/W compound system makes organic solvent wherein slowly evaporate into volatilization fully; Obtain the polylactic acid nano microgranule;
E. take by weighing mass ratio and be 10: 1～8: 1 polylactic acid nano microgranule and anti-vascular endothelial growth factor antibody, be dissolved in pH and be in 6.86 the buffer solution, under stirring, add carbodiimide, wherein the mass ratio of coupling agent and anti-vascular endothelial growth factor antibody is 2: 1,4 ℃ are stirred 2h down, make the antibody target nanoparticle.
9. according to the preparation method of right 8 described a kind of antibody target nanometer formulations, it is characterized in that the organic solvent described in step b and the d is an acetone.
10. according to the preparation method of right 8 described a kind of antibody target nanometer formulations, it is characterized in that coupling agent is a carbodiimide among the step e.
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| JP2018048192A (en) * | 2013-03-28 | 2018-03-29 | 三星電子株式会社Ｓａｍｓｕｎｇ Ｅｌｅｃｔｒｏｎｉｃｓ Ｃｏ．，Ｌｔｄ． | Fusion protein formed by coupling of c-met antibody and vegf-binding fragment |
| WO2021231237A3 (en) * | 2020-05-11 | 2021-12-16 | Augmenta Bioworks, Inc. | Antibodies for sars-cov-2 and uses thereof |
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| CN101045160A (en) * | 2006-03-31 | 2007-10-03 | 国家纳米科学中心 | Hydrophobic agent used for target treatment to tumor blood vessel, its preparing method and use |
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| CN101045160A (en) * | 2006-03-31 | 2007-10-03 | 国家纳米科学中心 | Hydrophobic agent used for target treatment to tumor blood vessel, its preparing method and use |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2018048192A (en) * | 2013-03-28 | 2018-03-29 | 三星電子株式会社Ｓａｍｓｕｎｇ Ｅｌｅｃｔｒｏｎｉｃｓ Ｃｏ．，Ｌｔｄ． | Fusion protein formed by coupling of c-met antibody and vegf-binding fragment |
| WO2021231237A3 (en) * | 2020-05-11 | 2021-12-16 | Augmenta Bioworks, Inc. | Antibodies for sars-cov-2 and uses thereof |
| US11919944B2 (en) | 2020-05-11 | 2024-03-05 | Augmenta Biosciences, Inc. | Antibodies for SARS-CoV-2 and uses thereof |
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