CN104814934A - Herceptin modified paclitaxel-carried targeting nanoparticle transfer system - Google Patents

Abstract

The invention discloses a herceptin modified paclitaxel-carried targeting nanoparticle transfer system and a preparation method thereof. Nanoparticles (PLNs) with PLGA/Lipid core-shell structures are prepared by an improved classical emulsion solvent evaporation method, paclitaxel-carried polymer lipid nanoparticles are prepared by a binary organic solvent method, and herceptin modified paclitaxel-carried targeting nanoparticles (T=PCNs) are built by a chemical modification method. The prepared T=PCNs release drugs in vitro, sudden release rate within 24h is 41.2%, cumulative release rate within 120h is 73.4%, and cancer therapy is facilitated by drug release level. Cell experiment results indicate that the T=PCNs are fine in targeting property for highly expressed HER2 cancer cells and have an obvious killing function for the cancer cells. Safety and the targeting property of paclitaxel are greatly improved.

Description

The targeted nano granule transmission system of carrying paclitaxel that the appropriate strain monoclonal antibody of one yeast inoculation is modified

Technical field

The present invention discloses the targeted nano granule transmission system of carrying paclitaxel that the appropriate strain monoclonal antibody of a yeast inoculation is modified, and provides the preparation method of this system simultaneously, belongs to cancer target and Atrigel technical field.

Background technology

Paclitaxel is the antitumor drug of extensive use clinically, can induce and promote tubulin polymerization, preventing depolymerization, makes tubulin keep stable, suppresses mitosis, play antitumor action.Paclitaxel has efficiently, low toxicity and broad spectrum anticancer active, especially has unique curative effect to breast carcinoma and ovarian cancer, and its injection is a line medication of the entity tumors such as treatment breast carcinoma, ovarian cancer, pulmonary carcinoma.But because taxol soluble is poor, making ejection preparation has certain difficulty.Current clinical conventional paclitaxel injection (Taxol) adopts polyoxyethylene castor oil etc. as solvent, can promote histamine release, cause serious anaphylaxis.In addition injection-type paclitaxel targeting is poor, easily causes general toxicity, therefore clinically in the urgent need to researching and developing the Novel Drug Delivery Systems of paclitaxel, to overcome one's shortcomings.

PLGA, biodegradability Poly(D,L-lactide-co-glycolide, has good biocompatibility and stability as pharmaceutical carrier, can wrap up hydrophobic paclitaxel, but PLGA nanoparticle particle diameter is general comparatively large, is unfavorable for intravenous injection.The liposome be made up of phospholipid material, similar biofilm structure, has particle diameter little, good biocompatibility, increases cellular uptake and endosome escape, delays the advantages such as drug release, but less stable.Polvmeric lipid nanoparticle is a kind of Novel Drug Delivery Systems with nucleocapsid structure combining the exploitation of both polymer and phospholipid advantage.Paclitaxel is loaded into polvmeric lipid nanoparticle, there is following advantage: particle diameter is little, entrapment efficiency is high, good biocompatibility, and release behavior is controlled, and after binding partner molecule, targeting strengthens.Thus improve active targeting, reduce paclitaxel toxic and side effects, reduce paclitaxel consumption, increase patient's compliance.

Some tumor cell surface overexpression receptor, can binding specificity part.Utilize this part guide effect, chemotherapeutics can be delivered to specific tumors tissue or cell, thus realize targeted therapy.Herceptin (Trastuzumab) is the monoclonal antibody of human epidermal growth factor receptor-2 (HER2), can identify and in conjunction with the tumor cell of HER2 process LAN, as breast carcinoma, and ovarian cancer, lung carcinoma cell etc.Herceptin has the features such as the large and space structure of protide macromole molecular weight is complicated, is vulnerable to the impact of operating environment, thus loses activity in nanoparticle preparation process.After drug-carrying nanometer particle basically forms, the Mal group introduced by nanoparticle surface, is reacted under mild conditions with antibody, thus antibody is connected to nanoparticle surface, can keep the natural activity of antibody, thus improves targeting effect.Therefore, by the receptor-mediated nanometer transmission system of HER2, the oncotherapy for HER2 process LAN provides the approach of potentiality.

Summary of the invention

The invention provides the targeted nano granule transmission system of carrying paclitaxel that the appropriate strain monoclonal antibody of a yeast inoculation is modified, the targeted nano granule transmission system of carrying paclitaxel that the appropriate strain monoclonal antibody of song for selectively targeted HER2 process LAN tumor cell is modified, solves defect that current formulation for paclitaxel exists and the single defect of nanoparticulate carriers material.

The present invention further provides the preparation method of carrying the targeted nano granule transmission system of paclitaxel that bent appropriate strain monoclonal antibody is modified.

The targeted nano granule transmission system of carrying paclitaxel that the appropriate strain monoclonal antibody of a yeast inoculation of the present invention is modified, is achieved through the following technical solutions:

By polymer P LGA and phospholipid be the polvmeric lipid nanoparticle of nucleocapsid structure prepared by carrier material, the bent appropriate strain monoclonal antibody of modified ligand that wraps the paclitaxel that is loaded in nanoparticle and nanoparticle surface forms.

The targeted nano granule transmission system of carrying paclitaxel that the appropriate strain monoclonal antibody of song of the present invention is modified, is characterized in that:

PLGA is carboxy blocking, model 5050 1A or 5050 1.5A, and viscosity is 0.16 ~ 0.20 dL/g.

The targeted nano granule transmission system of carrying paclitaxel that the appropriate strain monoclonal antibody of song of the present invention is modified, is characterized in that:

Phospholipid is wherein dipalmitoyl phosphatidyl choline and PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleimide.

The targeted nano granule transmission system of carrying paclitaxel that the appropriate strain monoclonal antibody of song of the present invention is modified, is characterized in that:

Paclitaxel: PLGA: dipalmitoyl phosphatidyl choline: the mass ratio of PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleimide is (10 ~ 15): 60:(15 ~ 20): 10.

The targeted nano granule transmission system of carrying paclitaxel that the appropriate strain monoclonal antibody of song of the present invention is modified, is characterized in that the mol ratio of the appropriate strain monoclonal antibody of song wherein and PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleimide is 1:5 ~ 1.

The preparation method of carrying the targeted nano granule transmission system of paclitaxel that the appropriate strain monoclonal antibody of song of the present invention is modified, comprises the following steps:

1) preparation of the emulsion-solvent evaporation method of improvement is adopted to wrap up and deliver PLGA/Lipid nanoparticle of paclitaxel;

2) adopt the method for chemical coupling, use activating reagent by certain amino mercapto of appropriate for song strain monoclonal antibody, with the Mal group generation additive reaction on nanoparticle surface, form thioether bond, and then aglucon is connected with nanoparticle.

Preparation method of the present invention, is characterized in that specifically comprising the following steps:

1) PLGA of the paclitaxel and 60 mg that take 10 ~ 15 mg is dissolved in the acetone of 2.4 mL, and the DPPC and the 10 mg DSPE-PEG2000-Mal that take 15 ~ 20 mg are dissolved in the ethanol of 1.6 mL; Mixing acetone ethanol solution (long-pending than being 3:2) forms binary organic facies (O phase), add appropriate 1.0 ~ 2.0% PLURONICS F87 aqueous solutions (W phase), be interrupted ultrasonic disperse at 0 ~ 4 DEG C and prepare O/W Emulsion, supplement 1.0 ~ 2.0% PLURONICS F87 aqueous solutions of 10 ~ 20 mL again, room temperature stirring at low speed, volatilization removing binary of spending the night organic facies, crosses film removing Large stone nanoparticle, and collected by centrifugation nanoparticle, lyophilization;

2) 20 DEG C, under pH 8.0 buffer incubation conditions, use activating reagent by a part of amino mercapto of Herceptin, with the Mal group generation additive reaction on nanoparticle surface, form thioether bond, and then aglucon is connected with nanoparticle, centrifuge washing removes unreacted monoclonal antibody, and collects nanoparticle, obtains the targeted nano granule transmission system of carrying paclitaxel that Herceptin is modified.

Preparation method of the present invention, is characterized in that:

Amino group activating reagents is 2 Iminothiolane hydrochloride, and working concentration is 6 ~ 7 mg/mL, and amount ranges is 20 ~ 100 μ L.

PLGA/Lipid nanoparticle transmission system prepared by the binary solvent emulsifying volatility process that the present invention introduces, the medicine of parcel is not limited only to paclitaxel, also has the hydrophobicity chemotherapeutics of good biocompatibility widenable to other and PLGA; The part of coupling is also not limited only to bent appropriate strain monoclonal antibody, also can comprise those and have the micromolecule polypeptide of targeting and the monoclonal antibody of other kind.

Targeted nano granule (T=PCNs) prepared by the present invention is lyophilized formulations, belongs to intravenously administrable system.For intravenously administrable system, solvent is generally aseptic aqueous solution.Suitable adjustment solution ph also makes solution isotonic (as adding sodium chloride or glucose), and be namely applicable to intravenously administrable, nanoparticle taked the mode sterilizing of 0.22 μm of sterilised membrane filter.

good effect of the present invention is:combine the advantage of PLGA and phospholipid and combine with monoclonal antibody, having prepared the targeted nano granule (T=PCNs) carrying paclitaxel that the appropriate strain monoclonal antibody of the song with nucleocapsid structure is modified, formed the nano_scale particle with nucleocapsid structure; Solve defect and the single deficiency of nanoparticulate carriers material that existing formulation for paclitaxel exists.There is particle diameter little (150 ~ 200 nm), more be conducive to cellular uptake (<200 nm), entrapment efficiency high (75 ~ 83%), good stability (current potential 0 about mV) in the solution, good biocompatibility, increase endosome to escape, release behavior can maintain paclitaxel treatment level, in conjunction with the active targeting more achieving medicine after bent appropriate strain monoclonal antibody.Improve the ingestion efficiency of the tumor cell of HER2 process LAN, greatly improve safety and the targeting of paclitaxel.The targeted nano granule transmission system of carrying paclitaxel that the appropriate strain monoclonal antibody of song that the present invention builds is modified is a kind of excellent delivery system, is expected to be applied to the treatment of the entity tumors such as the breast carcinoma of HER2 process LAN, ovarian cancer and pulmonary carcinoma.

Accompanying drawing explanation

Fig. 1 is the schematic diagram of nanoparticle;

Fig. 2 is the particle diameter potential image of nanoparticle: (A) PCNs particle size distribution; (B) T=PCNs particle size distribution; (C) PCNs Potential distribution; (D) T=PCNs Potential distribution;

Fig. 3 is the SEM figure of nanoparticle; (A) PCNs; (B) T=PCNs;

Fig. 4 is the tablets in vitro curve of nanoparticle;

Fig. 5 is that PTX, HER, PCNs, T=PCNs are to SKBR3(A) and the cytotoxicity of MCF7 cell (B);

Fig. 6 is the laser co-focusing result that SKBR3 and MCF7 cell absorbs T=PCNs;

Fig. 7 is the flow cytometer result that SKBR3 and MCF7 cell absorbs T=PCNs: (A) rectangular histogram (B) bar diagram.

Specific embodiments

Describe the present invention below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

embodiment 1 :

1, take paclitaxel 10 mg and PLGA 1A 60mg to be dissolved in 2.4 mL acetone, take DPPC 20 mg and DSPE-PEG2000-Mal 10 mg and be dissolved in the ethanol of 1.6 mL.Mixing acetone ethanol solution forms binary organic facies (O phase), add 15 mL 1.0 ~ 2.0% PLURONICS F87 aqueous solutions (W phase), ultrasonic (100 W, 150 s) dispersion preparation O/W Emulsions, then supplement 1.0 ~ 2.0% PLURONICS F87 aqueous solutions of 15 mL are interrupted at 0 ~ 4 DEG C, room temperature stirring at low speed (150 rpm), volatilization removing binary of spending the night organic facies, 0.22 μm of filtering with microporous membrane, and collected by centrifugation nanoparticle (18000 rpm, 15 min), lyophilization;

2,20 μ L, 6 mg/mL Traut ' s Reagent are got, add the bent appropriate strain monoclonal antibody solution (pH 8.0 PBS) of 1 mL, 1 mg/mL, 2 h are activated under 20 DEG C of conditions, add the above-mentioned nanoparticle of 5 mg again, hatch 18 h under 20 DEG C of conditions, centrifuge washing removes the monoclonal antibody (18000 rpm, 15 min) do not connected, lyophilization, namely obtains the targeted nano granule carrying paclitaxel that Herceptin is modified.

embodiment 2 :

1, take paclitaxel 15 mg and PLGA 1A 60mg to be dissolved in 2.4 mL acetone, take DPPC 15 mg and DSPE-PEG2000-Mal 10 mg and be dissolved in the ethanol of 1.6 mL.Mixing acetone ethanol solution forms binary organic facies (O phase), add 15 mL 1.0 ~ 2.0% PLURONICS F87 aqueous solutions (W phase), ultrasonic (100 W, 150 s) dispersion preparation O/W Emulsions, then supplement 1.0 ~ 2.0% PLURONICS F87 aqueous solutions of 15 mL are interrupted at 0 ~ 4 DEG C, room temperature stirring at low speed (150 rpm), volatilization removing binary of spending the night organic facies, 0.22 μm of filtering with microporous membrane, and collected by centrifugation nanoparticle (18000 rpm, 15 min), lyophilization;

2,20 μ L, 6 mg/mL Traut ' s Reagent are got, add the bent appropriate strain monoclonal antibody solution (pH 8.0 PBS) of 1 mL, 1 mg/mL, 2 h are activated under 20 DEG C of conditions, add the above-mentioned nanoparticle of 5 mg again, hatch 18 h under 20 DEG C of conditions, centrifuge washing removes the monoclonal antibody (18000 rpm, 15 min) do not connected, lyophilization, namely obtains the targeted nano granule carrying paclitaxel that Herceptin is modified.

embodiment 3 :

1, take paclitaxel 10 mg and PLGA 1.5A 60mg to be dissolved in 2.4 mL acetone, take DPPC 20 mg and DSPE-PEG2000-Mal 10 mg and be dissolved in the ethanol of 1.6 mL.Mixing acetone ethanol solution forms binary organic facies (O phase), add 15 mL 1.0 ~ 2.0% PLURONICS F87 aqueous solutions (W phase), ultrasonic (100 W, 150 s) dispersion preparation O/W Emulsions, then supplement 1.0 ~ 2.0% PLURONICS F87 aqueous solutions of 15 mL are interrupted at 0 ~ 4 DEG C, room temperature stirring at low speed (150 rpm), volatilization removing binary of spending the night organic facies, 0.22 μm of filtering with microporous membrane, and collected by centrifugation nanoparticle (18000 rpm, 15 min), lyophilization;

2,20 μ L, 6 mg/mL Traut ' s Reagent are got, add the bent appropriate strain monoclonal antibody solution (pH 8.0 PBS) of 1 mL, 1 mg/mL, 2 h are activated under 20 DEG C of conditions, add the above-mentioned nanoparticle of 5 mg again, hatch 18 h under 20 DEG C of conditions, centrifuge washing removes the monoclonal antibody (18000 rpm, 15 min) do not connected, lyophilization, namely obtains the targeted nano granule carrying paclitaxel that Herceptin is modified.

embodiment 4 :

1, take paclitaxel 15 mg and PLGA 1.5A 60mg to be dissolved in 2.4 mL acetone, take DPPC 15 mg and DSPE-PEG2000-Mal 10 mg and be dissolved in the ethanol of 1.6 mL.Mixing acetone ethanol solution forms binary organic facies (O phase), add 15 mL 1.0 ~ 2.0% PLURONICS F87 aqueous solutions (W phase), ultrasonic (100 W, 150 s) dispersion preparation O/W Emulsions, then supplement 1.0 ~ 2.0% PLURONICS F87 aqueous solutions of 15 mL are interrupted at 0 ~ 4 DEG C, room temperature stirring at low speed (150 rpm), volatilization removing binary of spending the night organic facies, 0.22 μm of filtering with microporous membrane, and collected by centrifugation nanoparticle (18000 rpm, 15 min), lyophilization;

2,20 μ L, 6 mg/mL Traut ' s Reagent are got, add the bent appropriate strain monoclonal antibody solution (pH 8.0 PBS) of 1 mL, 1 mg/mL, 2 h are activated under 20 DEG C of conditions, add the above-mentioned nanoparticle of 5 mg again, hatch 18 h under 20 DEG C of conditions, centrifuge washing removes the monoclonal antibody (18000 rpm, 15 min) do not connected, lyophilization, namely obtains the targeted nano granule carrying paclitaxel that Herceptin is modified.

embodiment 5 :

1, take paclitaxel 10 mg and PLGA 1A 60mg to be dissolved in 2.4 mL acetone, take DPPC 20 mg and DSPE-PEG2000-Mal 10 mg and be dissolved in the ethanol of 1.6 mL.Mixing acetone ethanol solution forms binary organic facies (O phase), add 15 mL 1.0 ~ 2.0% PLURONICS F87 aqueous solutions (W phase), ultrasonic (100 W, 150 s) dispersion preparation O/W Emulsions, then supplement 1.0 ~ 2.0% PLURONICS F87 aqueous solutions of 15 mL are interrupted at 0 ~ 4 DEG C, room temperature stirring at low speed (150 rpm), volatilization removing binary of spending the night organic facies, 0.22 μm of filtering with microporous membrane, and collected by centrifugation nanoparticle (18000 rpm, 15 min), lyophilization;

2,100 μ L, 6 mg/mL Traut ' s Reagent are got, add the bent appropriate strain monoclonal antibody solution (pH 8.0 PBS) of 1 mL, 5 mg/mL, 2 h are activated under 20 DEG C of conditions, add the above-mentioned nanoparticle of 5 mg again, hatch 18 h under 20 DEG C of conditions, centrifuge washing removes the monoclonal antibody (18000 rpm, 15 min) do not connected, lyophilization, namely obtains the targeted nano granule carrying paclitaxel that Herceptin is modified.

embodiment 6 :

1, take paclitaxel 15 mg and PLGA 1A 60mg to be dissolved in 2.4 mL acetone, take DPPC 15 mg and DSPE-PEG2000-Mal 10 mg and be dissolved in the ethanol of 1.6 mL.Mixing acetone ethanol solution forms binary organic facies (O phase), add 15 mL 1.0 ~ 2.0% PLURONICS F87 aqueous solutions (W phase), ultrasonic (100 W, 150 s) dispersion preparation O/W Emulsions, then supplement 1.0 ~ 2.0% PLURONICS F87 aqueous solutions of 15 mL are interrupted at 0 ~ 4 DEG C, room temperature stirring at low speed (150 rpm), volatilization removing binary of spending the night organic facies, 0.22 μm of filtering with microporous membrane, and collected by centrifugation nanoparticle (18000 rpm, 15 min), lyophilization;

2,100 μ L, 6 mg/mL Traut ' s Reagent are got, add the bent appropriate strain monoclonal antibody solution (pH 8.0 PBS) of 1 mL, 5 mg/mL, 2 h are activated under 20 DEG C of conditions, add the above-mentioned nanoparticle of 5 mg again, hatch 18 h under 20 DEG C of conditions, centrifuge washing removes the monoclonal antibody (18000 rpm, 15 min) do not connected, lyophilization, namely obtains the targeted nano granule carrying paclitaxel that Herceptin is modified.

embodiment 7 :

1, take paclitaxel 10 mg and PLGA 1.5A 60mg to be dissolved in 2.4 mL acetone, take DPPC 20 mg and DSPE-PEG2000-Mal 10 mg and be dissolved in the ethanol of 1.6 mL.Mixing acetone ethanol solution forms binary organic facies (O phase), add 15 mL 1.0 ~ 2.0% PLURONICS F87 aqueous solutions (W phase), ultrasonic (100 W, 150 s) dispersion preparation O/W Emulsions, then supplement 1.0 ~ 2.0% PLURONICS F87 aqueous solutions of 15 mL are interrupted at 0 ~ 4 DEG C, room temperature stirring at low speed (150 rpm), volatilization removing binary of spending the night organic facies, 0.22 μm of filtering with microporous membrane, and collected by centrifugation nanoparticle (18000 rpm, 15 min), lyophilization;

2,100 μ L, 6 mg/mL Traut ' s Reagent are got, add the bent appropriate strain monoclonal antibody solution (pH 8.0 PBS) of 1 mL, 5 mg/mL, 2 h are activated under 20 DEG C of conditions, add the above-mentioned nanoparticle of 5 mg again, hatch 18 h under 20 DEG C of conditions, centrifuge washing removes the monoclonal antibody (18000 rpm, 15 min) do not connected, lyophilization, namely obtains the targeted nano granule carrying paclitaxel that Herceptin is modified.

embodiment 8 :

1, take paclitaxel 15 mg and PLGA 1.5A 60mg to be dissolved in 2.4 mL acetone, take DPPC 15 mg and DSPE-PEG2000-Mal 10 mg and be dissolved in the ethanol of 1.6 mL.Mixing acetone ethanol solution forms binary organic facies (O phase), add 15 mL 1.0 ~ 2.0% PLURONICS F87 aqueous solutions (W phase), ultrasonic (100 W, 150 s) dispersion preparation O/W Emulsions, then supplement 1.0 ~ 2.0% PLURONICS F87 aqueous solutions of 15 mL are interrupted at 0 ~ 4 DEG C, room temperature stirring at low speed (150 rpm), volatilization removing binary of spending the night organic facies, 0.22 μm of filtering with microporous membrane, and collected by centrifugation nanoparticle (18000 rpm, 15 min), lyophilization;

2,100 μ L, 6 mg/mL Traut ' s Reagent are got, add the bent appropriate strain monoclonal antibody solution (pH 8.0 PBS) of 1 mL, 5 mg/mL, 2 h are activated under 20 DEG C of conditions, add the above-mentioned nanoparticle of 5 mg again, hatch 18 h under 20 DEG C of conditions, centrifuge washing removes the monoclonal antibody (18000 rpm, 15 min) do not connected, lyophilization, namely obtains the targeted nano granule carrying paclitaxel that Herceptin is modified.

embodiment 9 :

The sign of nanoparticle

Adopt laser particle analyzer to analyze particle diameter and the Potential distribution of PCNs and T=PCNs, its particle diameter and Potential distribution are as shown in Figure 2.Droplet measurement result shows, and the mean diameter of PCNs is between 130 ~ 160 nm, and current potential is at about-10 mV.The mean diameter of T=PCNs is between 150 ~ 200 nm, and current potential is at about 0 mV (table 1).

Adopt the pattern of scanning electronic microscope observation PCNs and T=PCNs, lyophilizing nanoparticle is placed on metal spraying process on silicon chip, and result as shown in Figure 3.Surface sweeping Electronic Speculum result shows, PCNs and T=PCNs is all in spheroidal, and size is homogeneous, between 100 ~ 200 nm, has a small amount of gathering between T=PCNs group microgranule.

Nanoparticle particle diameter, Potential distribution and entrapment efficiency obtained by table 1. embodiment 1 ~ 8

* ¹t:PCNs is the ratio of bent appropriate strain monoclonal antibody and nanoparticle; * ²eE is paclitaxel envelop rate.

embodiment 10 :

The drug loading of nanoparticle and release in vitro behavior are investigated

High performance liquid chromatography (HPLC) is adopted to measure the envelop rate of PCNs: take 10 mg lyophilizing PCNs, be dissolved in 1 mL acetonitrile, spiral dissolves 10 min, adds 75% methanol solution of 9 mL, and 0.22 μm of filtering with microporous membrane, filtrate carries out chromatography.HPLC condition, mobile phase methanol: water (75:25, v/v) solution, flow velocity 1 mL/min, determined wavelength 230 nm, sample size 20 μ L.According to quality × 100% of following formulae discovery envelop rate (EE, %)=the record quality/input paclitaxel of paclitaxel.Recording envelop rate is 75% ~ 83%(table 1).

Adopt the release in vitro behavior of methods analyst PCNs and T=PCNs of dialysis: the lyophilizing nanoparticle taking 20 mg, is dispersed in 1 mL pH 7.4 PBS(1% Tween-80, v/v) in solution, load bag filter (MWCO, 10 kD).Bag filter is inserted containing 40 mL pH 7.4 PBS(1% Tween-80, v/v) in solution, be placed in 37 DEG C of constant-temperature tables, 120 rpm continue to jolt.Respectively at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 1 d, 2 d, 3 d, it is for subsequent use that 4 d take out dialysis solution 1 mL, supplements the fresh PBS solution of 1 mL simultaneously.A μm microporous filter membrane crossed by dialysis solution, and filtrate HPLC detects content of taxol, and calculates the preparation of paclitaxel.With commercially available paclitaxel injection (Taxol) for contrast, compare the releasability of slow release nano-particle, result as shown in Figure 4.In 6 initial h, all to present in various degree prominent releases behavior (61.2%, 43.3%, 34.4%) for three kinds of preparations, and paclitaxel injection group is prominent, and to release phenomenon the most obvious, namely discharges complete, without slow release behavior in 24 h.There is slow releasing behavior in PCNs and T=PCNs group, T=PCNs group accumulative release rate in 96 h reaches 78.2% after prominent releasing.T=PCNs release behavior is first prominent releases slow release again, and slow release effect is obvious.This gives the credit to the nucleocapsid structure that its PLGA and phospholipid are formed, and the monomolecular phosphorus lipid layer on PLGA surface regulates the release of paclitaxel, after surperficial coupling monoclonal antibody, has further delayed the release of paclitaxel, has maintained the treatment concentration of paclitaxel within a certain period of time.

embodiment 11 :

The cellulotoxic experiment of nanoparticle

Mankind mastopathy cell SKBR3 and MCF7 is incubated in the DMEM cell culture fluid containing 10% hyclone, 1% Pen .-Strep respectively, and culture bottle is placed in 37 DEG C, containing 5% CO ₂in incubator.The cell of trophophase of taking the logarithm carries out following experiment.Wherein, SKBR3 cell transition expresses HER2 antigen, MCF7 cell low expression HER2 antigen.

With 1 × 10 ⁴the density in individual/hole will be in the cancer cell inoculation of logarithmic (log) phase in 96 microwell plates, cultivating 24 h for 37 DEG C makes it adherent, within second day, remove culture fluid, add 100 μ L pastille culture medium (PTX, HER respectively, PCNs and T=PCNs), 37 DEG C hatch 4 h after, remove pastille culture medium, change fresh culture, continue cultivation 24 h, 48 h and 72 h respectively.Remove culture medium, every hole adds 10 μ L, 5mg/mL MTT reagent, hatches 4 h for 37 DEG C, and DMSO dissolves the first a ceremonial jade-ladle, used in libation crystallization generated, microplate reader 492 nm analytical solution absorbance, and calculates cell survival rate.

Experimental result as shown in Figure 5, T=PCNs to the lethal effect (47.2%) after SKBR3 cell 72 h apparently higher than PTX(76.3%) and PCNs(85.5%).This gives the credit to the HER2 antigen of SKBR3 overexpression, illustrates that its tumor-targeting is good.Test as a control group with MCF7 cell, T=PCNs does not obviously distinguish after effect 72 h to the lethal effect (54.9%) of MCF7 cell and PCNs(56.6%).Cell toxicant result illustrates, T=PCNs extracorporeal anti-tumor successful, be unable to do without the targeting of the good biocompatibility of its carrier material and monoclonal antibody.

embodiment 12 :

The cellular uptake experiment of nanoparticle

Use the T=PCNs of double fluorescence labeling in cellular uptake experiment, rhodamine B labelling PCNs, the bent appropriate strain monoclonal antibody of FITC labelling, then carry out chemical coupling, concrete preparation technology is as follows:

(A) preparation of the PCNs of rhodamine B is carried: take rhodamine B 2 mg and PLGA 1A 60mg and be dissolved in 2.4 mL acetone, take DPPC 20 mg and DSPE-PEG2000-Mal 10 mg and be dissolved in the ethanol of 1.6 mL.Mixing acetone ethanol solution forms binary organic facies (O phase), add 15 mL 1.0 ~ 2.0% PLURONICS F87 aqueous solutions (W phase), ultrasonic (100 W, 150 s) dispersion preparation O/W Emulsions, then supplement 1.0 ~ 2.0% PLURONICS F87 aqueous solutions of 15 mL are interrupted at 0 ~ 4 DEG C, room temperature stirring at low speed (150 rpm), to spend the night volatilization removing binary organic facies, 0.22 μm of filtering with microporous membrane, and centrifuge washing collect nanoparticle (18000 rpm, 15 min), lyophilization;

(B) preparation of the appropriate strain monoclonal antibody of the song of FITC labelling: (1) uses crosslinked fluid (NaHCO3, the Na of pH 9.0 ₂cO ₃, NaCl) and prepare bent appropriate strain monoclonal antibody solution, concentration is 10 mg/mL.(2) FITC is dissolved in DMSO, and concentration is 1mg/mL.(3) FITC solution is slowly added drop-wise in antibody-solutions, rocks gently and make it mix homogeneously with antibody, 4 DEG C, dark place reaction, 6 h.(4) NH4Cl of 5 mol/L is added to final concentration 50 mmol/L, 4 DEG C of cessation reaction 1 h.(5) dialyse in bent appropriate strain monoclonal antibody crosslinked fluid pH 7.4 PBS of FITC-, remove the FITC do not connected.

(C) coupling of the appropriate strain monoclonal antibody of song of PCNs and the FITC labelling of rhodamine B is carried: get 20 μ L, 6 mg/mL Traut ' s Reagent, add the bent appropriate strain monoclonal antibody solution (pH 8.0 PBS) of 1 mL, 1 mg/mL FITC-, 2 h are activated under 20 DEG C of conditions, add the above-mentioned nanoparticle of 5 mg again, 18 h are hatched under 20 DEG C of conditions, centrifuge washing removes monoclonal antibody (18000 rpm do not connected, 15 min), lyophilization, namely the targeted nano granule carrying paclitaxel that the Herceptin obtaining double fluorescence labeling is modified, keeps in Dark Place.

Laser scanning confocal microscopy

With 2 × 10 ⁴the density in individual/hole will be in cancer cell inoculation culture dish at the bottom of glass of logarithmic (log) phase, cultivating 24 h for 37 DEG C makes it adherent, within second day, remove culture fluid, add 1 mL serum-free pastille culture medium (double fluorescence labeling T=PCNs) respectively, 37 DEG C hatch 2 h after, remove the culture medium containing fluorescence nano grain, pH 7.4 PBS cleans three times.4% formaldehyde fixed cell, DAPI solution carries out nuclear targeting, imaging under laser confocal microscope.Rhodamine B excitation wavelength Ex=540 nm, emission wavelength Em=625 nm.FITC excitation wavelength Ex=488 nm, Em=525 nm.

Flow cytometric analysis

With 1 ~ 2 × 10 ⁵the density in individual/hole will be in the cancer cell inoculation of logarithmic (log) phase in 24 orifice plates, cultivating 24 h for 37 DEG C makes it adherent, within second day, remove culture fluid, add 1 mL serum-free pastille culture medium (double fluorescence labeling T=PCNs) respectively, 37 DEG C hatch 3 h after, remove the culture medium containing fluorescence nano grain, pH 7.4 PBS cleans three times.0. 25% pancreatin (w/v) collecting cell, is resuspended in 4% formaldehyde fixative, carries out flow cytometry.Adopt laser channeling FL1 and FL3 respectively, detect the fluorescence of FITC and rhodamine B.

Cellular uptake experimental result as Fig. 6, shown in 7.The PCNs of rhodamine B labelling presents red fluorescence, and the appropriate strain monoclonal antibody of song of FITC labelling presents green fluorescence, and after the two combines, occur yellow fluorescence (T=PCNs), its bright-coloured degree depends on the content that tumor cell absorbs.This also indirectly shows, bent appropriate strain monoclonal antibody is successfully coupled at the surface of nanoparticle, and in cellular uptake process, the two is not separated.In SKBR3 cell, glassy yellow fluorescence appears at cytosolic domain, illustrates that tumor cell has higher uptake ratio to T=PCNs.Contrast MCF7 cell, yellow fluorescence is extremely not obvious, illustrates that MCF7 cell is lower to T=PCNs uptake ratio, and T=PCNs can not play its targeting (Fig. 6).In flow cytometry, the picked-up of tumor cell to T=PCNs is quantitatively analyzed.As shown in Fig. 7 B, SKBR3 cell to the picked-up of rhodamine B-PCNs and FITC-HER all higher than MCF7 cell.Cell experiment result describes the cancerous cell of T=PCNs to HER2 process LAN and has good targeting and cytotoxicity.

discuss

Present invention incorporates the advantage of PLGA and phospholipid and combine with monoclonal antibody, having prepared the targeted nano granule (T=PCNs) carrying paclitaxel that the appropriate strain monoclonal antibody of the song with nucleocapsid structure is modified.The core profile that phospholipid is coated on PLGA/ paclitaxel becomes monomolecular phosphorus lipid layer, phospholipid surface coupling monoclonal antibody molecule, thus forms the nano_scale particle with nucleocapsid structure.T=PCNs has possessed the advantage of PLGA and phospholipid: have particle diameter little (150 ~ 200 nm), more be conducive to cellular uptake (<200 nm), entrapment efficiency high (75 ~ 83%), good stability (current potential 0 about mV) in the solution, good biocompatibility, increase endosome to escape, release behavior can maintain paclitaxel treatment level, in conjunction with the active targeting more achieving medicine after bent appropriate strain monoclonal antibody.To the lethal effect of tumor cell apparently higher than PTX and PCNs, improve the ingestion efficiency of the tumor cell of HER2 process LAN, greatly improve safety and the targeting of paclitaxel.Especially extremely take effect in the breast carcinoma for the treatment of HER2 high expressed.

PLGA/Lipid nanoparticle transmission system prepared by the binary solvent emulsifying volatility process that the present invention introduces, the medicine of parcel is not limited only to paclitaxel, also has the hydrophobicity chemotherapeutics of good biocompatibility widenable to other and PLGA; The part of coupling is also not limited only to bent appropriate strain monoclonal antibody, also can comprise those and have the micromolecule polypeptide of targeting and the monoclonal antibody of other kind.

Targeted nano granule (T=PCNs) prepared by the present invention is lyophilized formulations, belongs to intravenously administrable system.For intravenously administrable system, solvent is generally aseptic aqueous solution.Suitable adjustment solution ph also makes solution isotonic (as adding sodium chloride or glucose), and be namely applicable to intravenously administrable, nanoparticle taked the mode sterilizing of 0.22 μm of sterilised membrane filter.

The targeted nano granule transmission system of carrying paclitaxel that the appropriate strain monoclonal antibody of song that the present invention builds is modified is a kind of excellent delivery system, is expected to be applied to the treatment of the entity tumors such as the breast carcinoma of HER2 process LAN, ovarian cancer and pulmonary carcinoma.

Claims (8)

1. the targeted nano granule transmission system of carrying paclitaxel of the appropriate strain monoclonal antibody modification of a yeast inoculation, is characterized in that:

By polymer P LGA and phospholipid be the polvmeric lipid nanoparticle of nucleocapsid structure prepared by carrier material, the bent appropriate strain monoclonal antibody of modified ligand that wraps the paclitaxel that is loaded in nanoparticle and nanoparticle surface forms.

2. the targeted nano granule transmission system of carrying paclitaxel of bent appropriate strain monoclonal antibody modification as claimed in claim 1, is characterized in that:

PLGA is carboxy blocking, model 5050 1A or 5050 1.5A, and viscosity is 0.16 ~ 0.20 dl/g.

3. the targeted nano granule transmission system of carrying paclitaxel of bent appropriate strain monoclonal antibody modification as claimed in claim 1, is characterized in that:

Phospholipid is wherein dipalmitoyl phosphatidyl choline and PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleimide.

4. the targeted nano granule transmission system of carrying paclitaxel of bent appropriate strain monoclonal antibody modification as claimed in claim 1, is characterized in that:

Paclitaxel: PLGA: dipalmitoyl phosphatidyl choline: the mass ratio of PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleimide is (10 ~ 15): 60:(15 ~ 20): 10.

5. the bent appropriate strain monoclonal antibody as claimed in claim 1 targeted nano granule transmission system of carrying paclitaxel of modifying, is characterized in that the mol ratio of the appropriate strain monoclonal antibody of song wherein and PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleimide is 1:5 ~ 1.

6. the preparation method of carrying the targeted nano granule transmission system of paclitaxel of bent appropriate strain monoclonal antibody modification as claimed in claim 1, comprises the following steps:

1) preparation of the emulsion-solvent evaporation method of improvement is adopted to wrap up and deliver PLGA/Lipid nanoparticle of paclitaxel;

2) adopt the method for chemical coupling, use activating reagent by certain amino mercapto of appropriate for song strain monoclonal antibody, with the Mal group generation additive reaction on nanoparticle surface, form thioether bond, and then aglucon is connected with nanoparticle.

7. preparation method as claimed in claim 6, is characterized in that specifically comprising the following steps:

1) PLGA of the paclitaxel and 60 mg that take 10 ~ 15 mg is dissolved in the acetone of 2.4 mL, and the DSPE-PEG2000-Mal of the DPPC and 10 mg that take 15 ~ 20 mg is dissolved in the ethanol of 1.6 mL; Mixing acetone ethanol solution (long-pending than being 3:2) forms binary organic facies (O phase), add appropriate 1.0 ~ 2.0% PLURONICS F87 aqueous solutions (W phase), be interrupted ultrasonic disperse at 0 ~ 4 DEG C and prepare O/W Emulsion, supplement 1.0 ~ 2.0% PLURONICS F87 aqueous solutions of 10 ~ 20 mL again, room temperature stirring at low speed, volatilization removing binary of spending the night organic facies, crosses film removing Large stone nanoparticle, and collected by centrifugation nanoparticle, lyophilization;

2) 20 DEG C, under pH 8.0 buffer incubation conditions, use activating reagent by a part of amino mercapto of Herceptin, with the Mal group generation additive reaction on nanoparticle surface, form thioether bond, and then aglucon is connected with nanoparticle, centrifuge washing removes unreacted monoclonal antibody, and collects nanoparticle, obtains the targeted nano granule transmission system of carrying paclitaxel that Herceptin is modified.

8. preparation method as claimed in claim 7, is characterized in that:

Amino group activating reagents is 2 Iminothiolane hydrochloride, and working concentration is 6 ~ 7 mg/mL, and amount ranges is 20 ~ 100 μ L.