CN102266291B - Preparation method of strychnine immune nanoparticles - Google Patents
Preparation method of strychnine immune nanoparticles Download PDFInfo
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Abstract
The invention relates to a preparation method of strychnine immune nanoparticles, and the preparation method comprises the following specific steps: step 1, dissolving carboxylated poly(ethylene glycol)-polylactic acid block copolymer and strychnine in an organic solvent, and then mixing with a polyvinyl alcohol aqueous solution to form a primary emulsion; step 2, removing the organic solvent andimpurities to obtain a strychnine nanoparticle concentrate; and step 3, sequentially adding a carbodiimide salt used as a carboxyl activation reagent and an anti-human-AFP (alpha-fetoprotein) monoclonal antibody to the strychnine nanoparticle concentrate, thus linking the carboxyl group on polyethylene glycol and the amino group of the anti-human-AFP monoclonal antibody by chemical coupling to obtain the strychnine immune nanoparticles. The preparation method provided by the invention has the advantages of simple process, high encapsulation rate, stable drug release and good drug ballability;and the obtained strychnine immune nanoparticles are used for preparing anti-tumor immune targeting drugs, and have the advantages of precise targeting drug accumulation in tumor tissue cells, stabledrug release, good anti-cancer effect, safety and the like.
Description
Technical field
The present invention relates to a kind of preparation method of Brucine immune nanoparticles, particularly, relating to a kind of liver cancer-specific target therapeutic agent-anti-human AFP McAb(is anti-human alpha-fetoprotein monoclonal antibody)-preparation method of polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles.
Background technology
Strychnine (Brucine) is one of Semen Strychni main component, and its chemical constitution is as follows:
This strychnine is a kind of alkalescence alkaloid, poorly water-soluble, represent medicine as promoting blood circulation to remove obstruction in the collateral and " treating the poisonous disease with poisonous drugs ", the T lymphopoiesis there is facilitation, the Function Regulation that Lymphocyte Function is had dose dependent, have obvious tumor-inhibiting action and very strong analgesic activity, Deng Xukun etc. estimate strychnine to the tumor inhibition effect of transplanted tumor model tumor-bearing mice and the impact of life span, found that strychnine all is not less than 30% at 1.61-6.46 mg to the tumour inhibiting rate of solid tumor Heps model mice and S180 model mice, strychnine can also improve weight and the index thereof of the immune organ of mice, but it is to ascites tumor model (EAC, Heps) life span of tumor-bearing mice is without obviously prolongation effect, thereby confirmed that strychnine can suppress the growth of solid tumor models mouse interior tumor to a certain extent.Short application use does not have obvious toxicity to hemopoietic, immune system and the Liver and kidney of animal, can also stimulate and promote hemopoietic system and immune function, recover the damage of the hepatic and renal function that mice causes because of the strain of inoculation hepatocarcinoma Heps tumor, strychnine may become new promising antitumor drug.
Prior art research: (1) preparation vauqueline liposome, contrast with strychnine solution, carrying out pharmacokinetic with White Rabbit finds, the administration of strychnine solution has not measured Brucine in the blood after 2 hours, slow distribution half-life 7.5 minutes, and after strychnine is encapsulated in liposome, administration still keeps certain CONCENTRATION DISTRIBUTION in the blood after 12 hours, the elimination half-life has prolonged 6.6 than the former, but drug release is still too fast, and liver targeting is not strong, is difficult to reach in the part effective drug level; (2) strychnine and liposome thereof all show obvious inhibitory action to the growth of mouse bearing liver cancer (Heps) and Transplanted murine tumors sarcoma S180 cell, but both to the life span of ascites tumor model (EAC and Heps) tumor-bearing mice without the prolongation effect; (3) strychnine polylactic acid nano particle (Bru-PLA-NPs) intravenous administration is used for the pharmacokinetics observation in the rabbit body, the medicine dynamics data of strychnine in the rabbit body meets three-compartment model after found that single dose intravenous injection Bru-PLA-NPs (4 mg/ kg), meet two-compartment model behind the quiet notes strychnine of the single dose solution (4 mg/ kg), with strychnine solution phase ratio, after the Bru-PLA-NPs administration, the elimination half-life of strychnine (t1/ 2 β) is improved 6.6 times; Bioavailability improves 8.7 times.With strychnine solution phase ratio, significant change has occured in the pharmacokinetics behavior of strychnine in the rabbit body behind the Bru-PLA-NPs intravenous administration, but strychnine is made nanoparticle prolong drug circulation and action time in animal body, improve bioavailability; (4) strychnine solution is used in the abdominal cavity, strychnine conventional liposome and strychnine hidden liposome treatment transplanted hepatoma H22 mice, strychnine dosage is 3.23mgkg-1, be used in conjunction 8d, found that the strychnine solution with dosage, the strychnine conventional liposome, the tumour inhibiting rate of strychnine hidden liposome is respectively 28.64%, 57.96% and 71.36%, strychnine conventional liposome and hidden liposome to tumor-bearing mice all without immunosuppressive action, compare with conventional liposome, hidden liposome can further strengthen its antitumous effect as the carrier of strychnine.
Our recent research finds that strychnine and strychnine-polyethylene glycol-polylactic acid block copolymer nano microgranule significantly suppress the SMMC-7721 human liver cancer cell and enter G2 from the S phase, the M phase, liver cancer apoptosis reducing, and strychnine nanoparticle inhibition tumor cell cultivation effect significantly is better than the strychnine monomer, strychnine can suppress the matrix adhesion of people's SMMC-7721 liver cancer cells, growth along with drug dose, the matrix adhesion inhibitory action is strengthened gradually, observation of cell motion migration rate was 17.46% when the strychnine consumption was 320 μ g/ml, the invasion and attack rate is 2.52%, compares migration rate with 40 μ g/ml and all there is significant difference in the invasion and attack rate.Show that strychnine can the establishment hepatoma carcinoma cell adhere to, stop the hepatoma carcinoma cell mobility and invasiveness, theoretical basis has been established in the further investigation that is used for the targeted therapy hepatocellular carcinoma for strychnine.
But strychnine is hypertoxic Chinese medicine, and its therapeutic dose and toxic dose are very approaching, and systemic administration dosage is large, and toxic and side effects is strong.How further improving targeting and the tumor by local medicine of strychnine and assemble, continue the performance Graft Versus Tumor, reduce the whole body toxicity, is the key point that improves the anti-hepatocarcinoma effect of strychnine.
Summary of the invention
The preparation method that the purpose of this invention is to provide a kind of liver neoplasm immunity targeted nano medicine-anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles, the method simple process, envelop rate is high, drug release is stable, balling-up is good, and prepared immune targeted nano microgranule meets the needs of neoplasm targeted therapy at aspects such as physicochemical properties.
For realizing above purpose, the invention provides a kind of preparation method of Brucine immune nanoparticles, take strychnine as active component, with PLA-PEG-CH2-CH2-COOH as pharmaceutical pack by adjuvant, the anti-human AFP McAb(of outer link is anti-human alpha-fetoprotein monoclonal antibody) make immune targeted nano microgranule, the method comprises following concrete steps:
Step 2 is removed organic solvent and impurity, makes strychnine nanoparticle concentrated solution;
Step 3, in the strychnine nanoparticle concentrated solution of step 2 gained, add successively activated carboxylic reagent carbodiimide salt and anti-human AFP monoclonal antibody, so that the amino of Polyethylene Glycol carboxyl and anti-human AFP monoclonal antibody links by chemical coupling, obtain Brucine immune nanoparticles.
The preparation method of above-mentioned Brucine immune nanoparticles, wherein, in step 2, the method for removing organic solvent and impurity comprises: at first, to the described just Ruzhong of step 1, add the pure water dilution, fluid drying is removed organic solvent, then, centrifugal treating, remove possible aggregation impurity, get the supernatant, be i.e. the strychnine nanoemulsions; At last, by ultra-filtration centrifuge tube, remove free strychnine, and washing, strychnine nanoparticle concentrated solution obtained.
The preparation method of above-mentioned Brucine immune nanoparticles, wherein, in step 1, the general formula of described carboxylated polyethylene glycol-polylactic acid block copolymer is PLA-PEG-CH2-CH2-COOH, the scope of its molecular weight is 40-50kD, be carboxylated Polyethylene Glycol and polylactic acid take part by weight as 1:1.5 ~ 3 ratio copolymerization forms.
The preparation method of above-mentioned Brucine immune nanoparticles, wherein, in step 1, described carboxylated polyethylene glycol-polylactic acid block copolymer is that carboxylated Polyethylene Glycol and the ratio copolymerization of polylactic acid take part by weight as 1.5:2.5 form.
The preparation method of above-mentioned Brucine immune nanoparticles, wherein, in step 1, the weight ratio of described strychnine and carboxylated polyethylene glycol-polylactic acid block copolymer is 1:15 ~ 30.
The preparation method of above-mentioned Brucine immune nanoparticles, wherein, in step 1, the weight ratio of described strychnine and carboxylated polyethylene glycol-polylactic acid block copolymer is 1:20.
The preparation method of above-mentioned Brucine immune nanoparticles, wherein, in step 3, described activated carboxylic reagent is carbodiimide hydrochloride.
The preparation method of above-mentioned Brucine immune nanoparticles, wherein, it is 5 ~ 7% that described Brucine immune nanoparticles carries strychnine content, envelop rate is 70 ~ 85%.
The preparation method of above-mentioned Brucine immune nanoparticles, wherein, described Brucine immune nanoparticles carries strychnine content 5.6 ± 0.2%, and envelop rate is 76.0 ± 2.3%.
The preparation method of above-mentioned Brucine immune nanoparticles, wherein, in the described Brucine immune nanoparticles, the concentration range of anti-human AFP monoclonal antibody is 10 ~ 20 μ g antibody/mg nanoparticles.
The preparation method of above-mentioned Brucine immune nanoparticles, wherein, in the described Brucine immune nanoparticles, the concentration of anti-human AFP monoclonal antibody is 15 μ g antibody/mg nanoparticles.
The present invention utilizes the Release Properties of carboxylated Polyethylene Glycol and polylactic acid, prepare nanoparticle take strychnine, carboxylated Polyethylene Glycol and polylactic acid block cosslinking as framing structure, carboxylated Polyethylene Glycol and polylactic acid form block copolymer, the copolymer end contains the water-soluble polymer Polyethylene Glycol to be inlayed, significantly improved long circulation time in the body, avoided independent with Polyethylene Glycol Brucine immune nanoparticles in vivo release excessive velocities, in the body holdup time short, be difficult to reach at tumor by local the defectives such as effective antitumor drug level.
Utilization of the present invention is difficult to reach in the part defectives such as effective antitumor drug level.Utilize Polyethylene Glycol to expose carboxyl, successfully link by the chemical coupling technology with the amino of anti-human AFP McAb, significantly improved the link efficiency of anti-human AFP McAb and carboxylated polyethylene glycol/polylactic acid block copolymer strychnine nanoparticle, not only can control Nano medication discharges and degradation speed in vivo, and reach accurate targeting, improve curative effect of medication, greatly reduced the strychnine drug toxicity.
The strychnine immunity targeted nano particulate starting material that the present invention adopts is selected good biocompatibility, degradable polymer---polylactic acid is as skeleton carrier, and polymer ends is inlayed by containing water-soluble polymer PEG, reaches macrocyclic controlled-release effect in the body.
The present invention is take strychnine as active component, with the carboxylated Polyethylene Glycol of biodegradation material (carboxylation polyethylene glycol, CPEG) and polylactic acid (polylactic acid, PLA) as excipient substance, by chemical modification technique development PLA-PEG-CH2-CH2-COOH, adopt ultrasonic emulsification-chemical crosslink technique fabricating technology, preparation strychnine-PLA-PEG-CH2-CH2-COOH nanoparticle (Brucine/CPEG/PLA Nanoparticles, BCPNS), by chemical coupling method anti-human AFP McAb is linked with PLA-PEG-CH2-CH2-COOH strychnine nanoparticle, anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles (Anti-hAFP McAb-Brucine/CPEG/PLA Nanoparticles, AHMBCPNS) is developed in success.Particle diameter 274.5 ± 64.3 nm, the Zeta current potential is-4.73 ± 4.73 mV, carrying drug ratio: 5.6 ± 0.2%, and envelop rate: 76 ± 2.3%, carrying strychnine concentration is 800 μ g/ml, and the concentration of anti-human AFP McAb on the strychnine nanoparticle is 15 μ g antibody/mg nanoparticles.Vitro drug release result: 24 hours cumulative release of strychnine surpass 80%, 48 hour and discharge fully.
The anti-human AFP McAb-of the present invention polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles internal metabolism, tissue distribution and antitumor action:
Strychnine is at the half-life (t of strychnine group, strychnine nanoparticle group and Brucine immune nanoparticles group in the rat body
1/2) be respectively 6.98 ± 0.65 h, 16.88 ± 5.67 h and 15.69 ± 3.76 h.Strychnine nanoparticle group and Brucine immune nanoparticles group half-life obviously are longer than the strychnine group, and both in vivo metabolic process be close, namely initially be the process of a tachymetabolism, eliminate slowly to the later stage, show obvious slow-releasing.And present a tachymetabolism process in the strychnine body.
All do not measure strychnine in each tissue in the strychnine group tumor bearing nude mice body behind the administration 3d, strychnine nanoparticle group strychnine concentration in the other hepatic tissue of spleen, lung, kidney, muscle, hepatocarcinoma and cancer is respectively 341.00 ng/ mg, 9.09 ng/mg, 7.90 ng/ mg, 14.41 ng/ mg, 24.00 ng/ mg and 246.50 ng/ mg, and remaining tissue is not measured; The Brucine immune nanoparticles group only in spleen, hepatocarcinoma and the other hepatic tissue of cancer strychnine concentration be respectively 341.50 ng/ mg, 57.13 ng/ mg and 135.93 ng/ mg.The drug level of Brucine immune nanoparticles group strychnine in liver cancer tissue is higher than strychnine nanoparticle group, and the strychnine drug level is starkly lower than strychnine nanoparticle group in the other hepatic tissue of cancer, difference have statistical significance (
F=445.85,
P＜0.05).
Tumour inhibiting rate is respectively 36.73%, 54.77%, 69.48%, 75.96% when Brucine immune nanoparticles group administration 7d, 14d, 21d and 30d, is significantly higher than other positive administration groups.The 5-Fu(5-fluorouracil) group, strychnine group, strychnine nanoparticle group and Brucine immune nanoparticles group tumor animal life cycle (d) are respectively 63.60 ± 16.26,50.10 ± 11.19%, 60.50 ± 12.54,81.50 ± 14.25, especially the longest with Brucine immune nanoparticles group life span, compare with other experimental grouies significant difference (
F=9.010,
P＜0.05); 5-Fu group, strychnine group, strychnine nanoparticle group and Brucine immune nanoparticles group increase in life span are respectively 25.20% ,-1.38%, 19.09% and 60.43%, and wherein Brucine immune nanoparticles group tumor animal life span obviously prolongs.
The preparation method of anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles provided by the invention, preparation technology is easy, envelop rate is high, drug release is stable, balling-up is good, and prepared immune targeted nano microgranule meets the needs of neoplasm targeted therapy at aspects such as physicochemical properties.The anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles of the present invention's preparation, be used for the hepatocarcinoma treatment, make its medicine carrying accumulate in tumor tissues more " accurately ", greatly bring into play the strychnine antitumor action, reduce to greatest extent the toxic action of strychnine, the treatment that is used for hepatocarcinoma has good potential applicability in clinical practice.
Description of drawings
Fig. 1 is PLA-PEG-CH2-CH2-COOH (PLA-PEG-CH2-CH2-COOH)
1H-NMR measures collection of illustrative plates.
Fig. 2 a is anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer strychnine immune nano
The microgranule transmission electron microscope picture, transmission electron microscope * 10,000.
Fig. 2 b is anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer strychnine immune nano
The microgranule scanning electron microscope (SEM) photograph, scanning electron microscope * 50,000.
Fig. 3 is anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles particle size distribution figure.
Fig. 4 is anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles vitro drug release.
Fig. 5 is that anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles targeting is measured.
Fig. 6 is that anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles is to the hepatoma cell growth inhibitory action.
Fig. 7 is anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles pharmacokinetics.
Fig. 8 is that anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles is at the tumor bearing nude mice distribution.
Fig. 9 is anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles antitumor action.
Figure 10 is that anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles is on tumor animal impact life cycle.
The specific embodiment
Describe the specific embodiment of the present invention in detail below in conjunction with accompanying drawing.
The preparation method of anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles
Composition of raw materials is as follows:
Wherein, polymeric material (PLA-PEG-CH2-CH2-COOH) be carboxylated Polyethylene Glycol and polylactic acid take part by weight as 1:1.5 ~ carboxylated polyethylene glycol-polylactic acid block copolymer that 3 ratio (preferred 1.5:2.5) copolymerization forms; Its general formula is PLA-PEG-CH2-CH2-COOH, and the scope of its molecular weight is 40-50kD, and its structure fragment reaches
1H-NMR measures collection of illustrative plates as shown in Figure 1.
Above-mentioned oil phase and water are mixed, disperse with the high shear dispersing emulsification machine, 12000rpm rotating speed down cut 5min gets dispersion liquid, again this dispersion liquid is continued emulsifying under 300w power ultrasonic condition, and ultrasonic 5 times, each 30s gets colostric fluid.Colostric fluid is poured in the diluent of recipe quantity, the limit edged stirs, and mixing speed 700rpm continues to stir 6-8h and carries out fluid drying under normal temperature condition, fling to CH
2Cl
2, with the centrifugal 10min under the 5000rpm rotating speed of the emulsion behind the fluid drying, remove possible aggregation (comprising carboxylated polyethylene glycol-polylactic acid block copolymer, polyvinyl alcohol), collect supernatant and namely get the strychnine nanoemulsions.With strychnine nanoemulsions (the Millipore Amicon100 in the ultra-filtration centrifuge tube that packs into, 000), at centrifugal force 3, centrifugal 30min under the 000g condition, concentrate and remove free strychnine, use the purified water concentrated solution for washing after concentrating, ultrafiltration is once centrifugal again, obtain strychnine nanoparticle concentrated solution
60The Co irradiation sterilization, 4 ℃ of Refrigerator stores are for subsequent use.
30ml strychnine medicine-carried nano particles concentrated solution is divided into 10 parts, be every part of 3ml, in every part of medicine-carried nano particles concentrated solution, add 5mg EDAC(carbodiimide hydrochloride), shaking table vibration 15min(200rpm/min), then after adding AFP antibody (1mg/ml), 4 ℃ of shaking tables vibration 3h(200rpm/min), centrifugal 5 minutes 10000 rpm again, 0.01M PBS(pH 7.4) washing particle 3 times.By the centrifugation of centrifugal force, coagulation was at the pipe end under the effect of centrifugal force after nanoparticle was connected with antibody, and the antibody that connects can not avale to be suspended in the upper strata under the rotating speed of 10000rpm.Obtain the Brucine immune nanoparticles concentrated solution through the ultrafiltration centrifugalize, in 4 ℃ of refrigerators, save backup.
The Brucine immune nanoparticles testing result shows, particle size is even, the profile rounding, particle diameter 274.5 ± 64.3 nm, such as Fig. 2 a, Fig. 2 b and shown in Figure 3, the Zeta current potential is-4.73 ± 4.73 mV, carrying drug ratio: 5.6 ± 0.2%, envelop rate: 76 ± 2.3%, carrying strychnine concentration is 800 μ g/ml, the concentration of anti-human AFP McAb on the strychnine nanoparticle is 15 μ g antibody/mg nanoparticles.Vitro drug release result: 24 hours cumulative release of strychnine surpass 80%, 48 hour and discharge fully.Anticancer experiment in vitro: Brucine immune nanoparticles has obvious inhibitory action to hepatoma carcinoma cell SMMC-7721 growth, and is with the increase inhibitory action of drug level and increases progressively trend, has concentration dependent.Compare with the strychnine nanoparticle with strychnine, Brucine immune nanoparticles approaches IC to inhibitory action and the 5-FU of hepatoma carcinoma cell SMMC-7721
50(5-FU organizes IC to differ minimum
50Be 16.7 μ g/ml; Brucine immune nanoparticles group IC
50Be 28.2 μ g/ml).
Embodiment 2
Anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles vitro drug release test
Precision pipettes Brucine immune nanoparticles concentrated solution 2.0ml, placing molecular cut off is 3, in the 000 daltonian bag filter, tighten at two ends, 3 parts of operation repetitives, put in the PBS medium of 50ml pH7.4, in 37 ℃, carry out release in vitro under the 100rpm speed conditions, discharge after the beginning the 0.5th, 1,2,4,8,12,16,18,24,36,48h takes a sample 4ml, fluid infusion 4ml, sample measure the UV absorption value under the 263nm wavelength, calculate Bru(strychnine in the release medium) concentration and cumulative release percentage rate.
Compound concentration is 0.427mg/mlBru-PBS solution in addition, precision pipettes this solution 2.0ml, placing molecular cut off is 3, in the 000 daltonian bag filter, tighten at two ends, 3 parts of operation repetitives, put in the PBS medium of 50mlpH7.4, in 37 ℃, carry out release in vitro under the 120rpm speed conditions, discharge beginning the rear the 10th, 20,30,60,120,210, the 300min 4ml that takes a sample respectively, fluid infusion 4ml, sample is measured the UV absorption value under the 263nm wavelength, calculate concentration and the cumulative release percentage rate of strychnine in the release medium, is used for investigating the retardation that bag filter discharges strychnine.
Release in vitro result shows: 24 hours cumulative release of strychnine surpass 80%, 48 hour and discharge fully, as shown in Figure 4.
Embodiment 3
Anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles targeting is measured
Anti-human AFP McAb strychnine immune nano particle and hepatoma carcinoma cell SMMC-7721 are hatched 4h, use 0.01M PBS washed cell 3 times; Two anti-addings with the FITC labelling continue to hatch 2h, after PBS washing 3 times; Under Laser Scanning Confocal Microscope, observe and replenish immunofluorescence method.Show under 200 times of Laser Scanning Confocal Microscopes, Brucine immune nanoparticles comparatively is evenly distributed near the hepatoma cell membrane, presents approximate " refer to guard against " shape, shows good targeting location, as shown in Figure 5 (photo under the Laser Scanning Confocal Microscope * 200 times).
Embodiment 4
Anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles is to the liver cancer cell growth inhibitory action
Need to be divided into RPMI-1640 group, 5-FU group, strychnine group, strychnine nanoparticle group, PLA-PEG-CH2-CH2-COOH block copolymer nano microgranule group, anti-human AFP McAb group, the blank Immunological nanometer particles group of anti-human AFP McAb and Brucine immune nanoparticles group according to experiment.Strychnine group, strychnine nanoparticle group and Brucine immune nanoparticles group are chosen respectively the different strychnine concentration of 10,20,40,80,160,240 μ g/ml, the molar dose of contained blank nanoparticle under blank nanoparticle matched group consumption and the corresponding equal drug level of nanoparticle that contains strychnine, 5-FU chooses respectively 10,20,40 μ g/ml.External act on respectively SMMC-7721 hepatoma carcinoma cell 72h after, adopt mtt assay to measure each group to hepatoma carcinoma cell SMMC-7721 growth inhibition ratio.The result shows: the drug effect of (1) negative control group (RPMI-1640 group, PLA-PEG-CH2-CH2-COOH block copolymer nano microgranule group, anti-human AFP McAb group, anti-human AFP McAb-PEG-PLA block copolymer group) variable concentrations 72 hours to people's SMMC-7721 liver cancer cells all without obvious inhibitory action, increase along with drug level, each organize medicine to the growth of people's SMMC-7721 liver cancer cells all without obvious inhibitory action, equal not statistically significants (P〉0.05).(2) medicine of each group (strychnine group, strychnine nanoparticle group, Brucine immune nanoparticles group) variable concentrations of 5-FU group and experiment all has obvious inhibitory action to people's SMMC-7721 liver cancer cells, the drug effect same time, increase along with drug level, each organizes medicine all has obvious inhibitory action to the growth of people's SMMC-7721 liver cancer cells, between group in twos comparing difference statistical significance (P＜0.05) is arranged, as shown in Figure 6.
Anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles pharmacokinetics
Choose 6 of healthy male Wistar rats, dosage with 3.23mg/kg injects strychnine, strychnine nanoparticle and Brucine immune nanoparticles through the tail vein, injection rear 5min, 10min, 15min, 30min, 45min, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h respectively tail venous blood sampling 0.5ml place the anticoagulant test tube, 1500rpm * 5min draws blood plasma for subsequent use in 4 ℃ of cryopreservation tubes.Adopt the 3200Q-Trap tandem mass spectrometer to measure a time point blood plasma strychnine concentration.Strychnine t1/2 is 6.98 ± 0.65 h, strychnine nanoparticle t
1/2Be 16.88 ± 5.67 h, Brucine immune nanoparticles t
1/2Be 15.69 ± 3.76 h, the result shows, relative strychnine, and Brucine immune nanoparticles significant prolongation strychnine in vivo circulation time (sees Table 1, Fig. 7).
Table 1: pharmacokinetics behind the Brucine immune nanoparticles intravenously administrable (n=6)
Embodiment 6
Anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles is at lotus Liver Cancer Bearing Nude Mice distribution
Set up the Orthotopic implantation in nude mice liver cancer model, 2 weeks after the modeling, by nude mice tail intravenously administrable, dosage with 3.23mg/kg injects strychnine through the tail vein, strychnine nanoparticle and Brucine immune nanoparticles, 72hr after the administration, leave and take the tumor bearing nude mice heart, liver, spleen, lung, kidney, brain, stomach, muscle, fat, cancerous tissue, volume ratio 1:2 adding distil water by weight, tissue homogenate, get homogenate 100 μ l, add mark (loratadine 100ng/ml) in the 10 μ l, add 400 μ l acetonitriles, vortex vibration, the centrifugal 3min of 15000rpm, get supernatant 100 μ l and be transferred to sample introduction in the sample feeding pipe, adopt the 3200Q-Trap tandem mass spectrometer to measure in vivo tissue distribution of strychnine nanoparticle.The result shows 72hr after the administration, strychnine is not all measured at above-mentioned each tissue concentration, and the concentration of strychnine nanoparticle group in liver, spleen and tumor all is higher than lung, kidney, muscle, remaining tissue is not all measured, and the drug level in the tumor tissues is 23.96ng/ml, liver 246.5 ng/ml, spleen 341 ng/ml, lung 9.09 ng/ml, kidney 7.89 ng/ml, muscle 14.4 ng/ml.72hr liver tumor targeting index is 71.42 after the administration; Kidney, lung, muscle etc. organize Chinese medicine concentration obviously to reduce, in vivo metabolism time lengthening of medicine, and tumor target tissue concentration is higher.The Brucine immune nanoparticles group only in spleen, tumor and the other hepatic tissue of cancer strychnine concentration be respectively 341.50 ng/ mg, 57.13 ng/ mg and 135.93 ng/ mg.The drug level of strychnine in liver cancer tissue is higher than strychnine nanoparticle group, and the strychnine drug level is starkly lower than strychnine nanoparticle group in the other hepatic tissue of cancer, difference have statistical significance (
F=445.85,
P＜0.05), as shown in Figure 8.
Embodiment 7
The effect of anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles treatment hepatocellular carcinoma in nude mice
Set up the Orthotopic implantation in nude mice liver cancer model, be divided at random normal saline group (NS), blank nanoparticle group (being PLA-PEG-CH2-CH2-COOH block copolymer nano microgranule group), anti-human AFP McAb group, the blank Immunological nanometer particles group of anti-human AFP McAb, 5-FU group, strychnine group, strychnine nanoparticle group and Brucine immune nanoparticles group.Be 20mg/kg through tail vein saline injection, 5-FU(consumption respectively), strychnine (consumption is 3.23mg/kg), blank nanoparticle consumption is the corresponding molar dose that contains contained blank nanoparticle under the equal drug level of strychnine nanoparticle.Put to death nude mice behind each group treatment 30d, respectively organize gross tumor volume, neoplasm necrosis degree; Adopt the TUNEL labelling method to detect apoptosis rate, Use immunohistochemistrySP SP detects the expression of Ki-67.The result shows: compare with 5-FU group (tumor control rate is 58.87 ± 1.67%), strychnine group (tumor control rate is 47.08 ± 2.69%), strychnine nanoparticle group (tumor control rate is 51.38 ± 3.18%) function of tumor inhibition is similar to 5-FU, and difference has statistical significance (P＜0.05); Tumour inhibiting rate is respectively 36.73%, 54.77%, 69.48%, 75.96% when Brucine immune nanoparticles group administration 7d, 14d, 21d and 30d, is significantly higher than 5-Fu group, strychnine group, strychnine nanoparticle group, as shown in Figure 9.
Anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles is on tumor animal impact life cycle
Set up the Orthotopic implantation in nude mice liver cancer model, be divided at random normal saline group, blank nanoparticle group, anti-human AFP McAb group, the blank Immunological nanometer particles group of anti-human AFP McAb, 5-FU group, strychnine group, strychnine nanoparticle group and Brucine immune nanoparticles group.Be 20mg/kg through tail vein saline injection, 5-FU(consumption respectively), strychnine (consumption is 3.23mg/kg), blank nanoparticle consumption is the corresponding molar dose that contains contained blank nanoparticle under the equal drug level of strychnine nanoparticle.Therapeutic outcome shows, 5-Fu group, strychnine group, strychnine nanoparticle group and Brucine immune nanoparticles group tumor animal life cycle (d) are respectively 63.60 ± 16.26,50.10 ± 11.19,60.50 ± 12.54,81.50 ± 14.25, especially the longest with Brucine immune nanoparticles group life span, compare with other experimental grouies significant difference (
F=9.010,
P＜0.05), shows that Brucine immune nanoparticles has good targeting, significantly improve the strychnine Graft Versus Tumor, prolong tumor animal life cycle, as shown in figure 10.
The present invention utilizes anionic polymerisation and chemical modification technique to prepare carboxylated Polyethylene Glycol and polylactic-acid block copolymer material, by ultrasonic emulsification and chemical crosslinking technology development PLA-PEG-CH2-CH2-COOH strychnine nanoparticle, prepare anti-human AFP McAb-polyethylene glycol-polylactic acid block copolymer Brucine immune nanoparticles by chemical coupling method, be used for the hepatocarcinoma treatment, make its medicine carrying accumulate in tumor tissues more " accurately ", greatly bring into play the strychnine antitumor action, reduce to greatest extent the toxic action of strychnine.
Although content of the present invention has been done detailed introduction by above preferred embodiment, will be appreciated that above-mentioned description should not be considered to limitation of the present invention.After those skilled in the art have read foregoing, for multiple modification of the present invention with to substitute all will be apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Claims (7)
1. the preparation method of a Brucine immune nanoparticles is characterized in that, the method comprises following concrete steps:
Step 1 is dissolved in PLA-PEG-CH2-CH2-COOH and strychnine in the organic solvent, mixes with polyvinyl alcohol water solution to form colostrum again;
Step 2 is removed organic solvent and impurity, makes strychnine nanoparticle concentrated solution;
Step 3, in the strychnine nanoparticle concentrated solution of step 2 gained, add successively activated carboxylic reagent carbodiimide salt and anti-human AFP monoclonal antibody, so that the amino of Polyethylene Glycol carboxyl and anti-human AFP monoclonal antibody links by chemical coupling, obtain Brucine immune nanoparticles;
The general formula of described carboxylated polyethylene glycol-polylactic acid block copolymer is PLA-PEG-CH2-CH2-COOH, and the scope of its molecular weight is 40-50kD, be carboxylated Polyethylene Glycol and polylactic acid take part by weight as 1:1.5 ~ 3 ratio copolymerization forms;
Described carboxylated polyethylene glycol-polylactic acid block copolymer is that carboxylated Polyethylene Glycol and the ratio copolymerization of polylactic acid take part by weight as 1.5:2.5 form;
The weight ratio of described strychnine and carboxylated polyethylene glycol-polylactic acid block copolymer is 1:15 ~ 30.
2. the preparation method of Brucine immune nanoparticles as claimed in claim 1, it is characterized in that, in step 2, the method for removing organic solvent and impurity comprises: at first, to the described just Ruzhong of step 1, add the pure water dilution, fluid drying is removed organic solvent, then, centrifugal treating, remove aggregation impurity, get the supernatant, be i.e. the strychnine nanoemulsions; At last, by ultra-filtration centrifuge tube, remove free strychnine, and washing, strychnine nanoparticle concentrated solution obtained.
3. the preparation method of Brucine immune nanoparticles as claimed in claim 1 is characterized in that, in step 1, the weight ratio of described strychnine and carboxylated polyethylene glycol-polylactic acid block copolymer is 1:20.
4. such as the preparation method of the described Brucine immune nanoparticles of any one among the claim 1-3, it is characterized in that, in step 3, described activated carboxylic reagent is carbodiimide hydrochloride.
5. such as the preparation method of the described Brucine immune nanoparticles of any one among the claim 1-3, it is characterized in that, it is 5 ~ 7% that described Brucine immune nanoparticles carries strychnine content, and envelop rate is 70 ~ 85%.
6. the preparation method of Brucine immune nanoparticles as claimed in claim 5 is characterized in that, described Brucine immune nanoparticles carries strychnine content 5.6 ± 0.2%, and envelop rate is 76.0 ± 2.3%.
7. the preparation method of Brucine immune nanoparticles as claimed in claim 6 is characterized in that, in the described Brucine immune nanoparticles, the concentration range of anti-human AFP monoclonal antibody is 10 ~ 20 μ g antibody/mg nanoparticles.
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