CN101953796B - Method for preparing recombinant human endostatin chitosan nanoparticles for injection - Google Patents

Abstract

The invention relates to a preparation method of recombinant human vascular endostatin chitosan nanoparticles for injection, in particular to a method using biodegradable chitosan as a carrier, selecting sodium tripolyphosphate as an ion cross-linking agent, and continuously stirring Under the conditions, the chitosan solution containing the recombinant human endostatin and the sodium tripolyphosphate solution undergo an ion cross-linking reaction to prepare the recombinant human endostatin chitosan nanoparticles.

Description

A preparation method of recombinant human endostatin chitosan nanoparticles for injection

technical field

The invention relates to a preparation method of recombinant human endostatin chitosan nanoparticle for injection, in particular to a preparation method using biodegradable chitosan as a matrix. The particle size range of the nanoparticles prepared by the method is controllable, the drug loading capacity is high, and the nanoparticles are safe, non-toxic and easy to operate.

Background technique

Recombinant human endostatin for injection (Endostar, Endostar) was developed by Shandong Simcere Maidejin Biopharmaceutical Co., Ltd. (a holding subsidiary of Simcere Pharmaceuticals), and obtained the new drug certificate issued by the State Food and Drug Administration in 2005. Obtained the production approval in 2006, and is currently the world's first recombinant human endostatin approved by the national food and drug regulatory agency for marketing.

Recombinant human endostatin is produced by modifying the nucleotide coding sequence of human endostatin to produce recombinant human endostatin (ZL 00107569.1) with 9 additional amino acid sequences at the N-terminal, and its amino acid sequence is:

(M) GGSHHHHHHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLGGRLLGQSAASCHHAYIVLCIENS when expressed by the end part of E.

The anti-tumor mechanism of recombinant human endostatin is based on the theory of "starving tumor therapy" proposed by Professor Folkman of Harvard Medical School. Studies have found that some endogenous angiogenesis inhibitors such as Angiostatin and Endostatin can hinder the growth of blood vessels in tumor tissue, and stagnate tumor growth and metastasis (O'Relly, M.S., et al.Cell.79:315-328, 1994; O'Relly, M.S., et al. Cell. 88:277-285, 1997). However, due to the problems of easy precipitation and difficult renaturation of Endostatin in the process of expression and preparation, its large-scale application in tumor patients is limited. The recombinant human endostatin Endostar maintains all the biological activities of endogenous Endostatin, and at the same time solves the difficulties in the production process of Endostatin, with higher expression level and stronger curative effect, and does not cause immunity in vivo due to the addition of N-terminal sequence Originality. However, as an exogenous protein, recombinant human endostatin is easily recognized by the immune system in the body and then degraded. Therefore, the half-life in the body is very short. In clinical use, patients need to inject once a day for 14 consecutive days as a course of treatment. After one week, proceed to the next course of treatment. Clinical compliance is poor due to frequent injection administration. Recent studies have found that continuous administration of small doses of Endostatin in animals is more effective than intermittent administration (Minoru Kuroiwa, et al.J.Pediatr.Surg.38:1499-1505, 2003; Oliver Kisker, et al.Cancer Res .61:7669-7674, 2001). Therefore, it is very necessary to develop it into a long-acting preparation that can be injected once and can release medicine continuously.

Sustained and controlled release targeted drug delivery systems mainly include liposomes, microspheres, nanoparticles, etc. Among them, biodegradable polymer nanoparticles have attracted extensive attention because of their good biocompatibility, ultrafine particle size, reasonable in vivo distribution and high drug utilization. Currently used drug sustained-release materials are some synthetic (such as PEG, PVA) or natural (such as sodium alginate, collagen chitosan, etc.) biodegradable materials. Among them, chitosan has good biocompatibility and biodegradability, and its positive charge enables it to interact with negatively charged polymers, macromolecules and even some polyanions in liquid media, thus The resulting sol-gel transition process can be conveniently used in the preparation of drug-loaded nanoparticles. Chitosan nanoparticles have been considered as a kind of drug controlled release carrier with great application prospects, especially suitable for the embedding of bioactive macromolecular drugs.

Contents of the invention

The purpose of the present invention is to provide a preparation method of recombinant human endostatin chitosan nanoparticles for injection, which can avoid the use of organic solvents, and simultaneously obtain nanoparticles with smaller particle size, uniform distribution and high drug loading capacity .

The nano particle system of the present invention is prepared by ion cross-linking reaction between chitosan and sodium tripolyphosphate, the system can uniformly control the particle size of the product, and simultaneously obtain higher drug loading capacity.

The preparation of recombinant human endostatin chitosan nanoparticle for injection of the present invention comprises the following steps:

(1) Chitosan is added in the acetic acid solution, stirred until completely dissolved;

(2) adding the buffer containing recombinant human endostatin into the chitosan acetic acid solution prepared in step (1), adding sodium tripolyphosphate under the condition of constant stirring to make an emulsion;

(3) ultrasonically disperse the above obtained emulsion, centrifuge, filter off the supernatant to obtain a concentrated solution or a precipitate;

(4) Redisperse the above-mentioned concentrated solution or precipitate in water, add a lyoprotectant, and freeze-dry to obtain recombinant human endostatin chitosan nanoparticles.

The molecular weight range of chitosan in the present invention is 10,000-500,000 Daltons, preferably 50,000-200,000 Daltons.

In the present invention, the concentration of the acetic acid solution is 0.5-2 mg/mL, preferably 1 mg/mL, the concentration of chitosan in the chitosan acetic acid solution is 0.5-10 mg/mL, and the concentration of the sodium tripolyphosphate aqueous solution is 0.1-10 mg/mL.

In the present invention, recombinant human endostatin is dissolved in a buffer, wherein the buffer can be one of acetate buffer, phosphate buffer, Tris buffer, glycine-hydrochloric acid buffer, preferably acetate buffer liquid. Since the recombinant human endostatin is only stable at a certain pH value, the pH value of the buffer solution is between 2-9, preferably between 4-7.

The formation of nano particles in the present invention is related to the addition ratio of chitosan and sodium tripolyphosphate, and the mass ratio of chitosan and sodium tripolyphosphate is 1:1-10:1.

In the present invention, sodium tripolyphosphate is an ion cross-linking agent. During the stirring process, chitosan and sodium tripolyphosphate are cross-linked to form nanoparticles. Magnetic stirring and propeller stirring can be used for stirring, and the stirring speed is 100 to 600 rpm. Minutes, stirring time in 1 ~ 120 minutes.

In the present invention, in order to control the particle size of the nanoparticles, ultrasound with a power of 50W-200W can be used, and the ultrasound time is 0-10min.

In the present invention, in order to separate free drug and nanoparticle, can adopt ultrafiltration centrifugation mode, promptly use the molecular weight cut off to be the ultrafiltration membrane of 30000～100000 Daltons, the centrifugal speed is controlled at 3000～5000 rpm, and the centrifugation time is 5 ~30 minutes, or adopt refrigerated centrifugation, that is, the temperature is controlled at 0-10 degrees Celsius, the centrifugal speed is controlled at 6000-16000 rpm, and the centrifugation time is 10-60 minutes.

In the present invention, necessary drying treatment is required after the nanoparticle precipitation is obtained. The drying step adopts freeze-drying, and the freeze-drying protective agent used includes one or more of mannitol, lactose, trehalose, glucose, and sucrose, preferably trehalose. The mass ratio of the lyoprotectant to the recombinant human endostatin chitosan nanoparticle is 0.1:1-10:1.

In the present invention, the recombinant human endostatin chitosan nanoparticle has a weight percentage of recombinant human endostatin, that is, a drug loading amount of 1%wt-50%wt, and the average particle diameter of the nanoparticle is between 100nm and 600nm.

The recombinant human endostatin described in the present invention is the recombinant human endostatin Endostar.

Advantage of the present invention has:

1. Chitosan has good biocompatibility, biodegradability, non-toxic and economical source;

2. No organic solvent is used in the preparation method, which can avoid affecting the activity of the protein, and the method is easy to operate;

3. The particle size range of nanoparticles is controllable, the shape is spherical, the distribution is uniform, and the drug loading capacity is high.

4. Nanoparticles can achieve the purpose of sustained drug release.

Description of drawings

Fig. 1 is the morphological diagram of nanoparticles in Example 1.

The particle size distribution diagram of nanoparticles in Fig. 2 embodiment two.

The particle size distribution diagram of nanoparticles in Fig. 3 embodiment three.

The cumulative release curve of nanoparticles in Fig. 4 Example 4.

Fig. 5 The proliferative activity of recombinant human endostatin on HUVEC cells before and after making nanoparticles.

Detailed ways

The present invention is described in more detail through the following examples, but they cannot be construed as limiting the protection scope of the present invention.

Embodiment one

Take by weighing the chitosan 500mg that molecular weight is 100000 Daltons, be dissolved in the acetic acid solution of 50mL 1%, obtain the chitosan solution of 10mg/mL. Prepare 20 mL of 5 mg/mL sodium polyphosphate solution with distilled water. Add 200 mg of Endostar acetate buffer solution (pH 5.5) into the chitosan solution, slowly add sodium polyphosphate solution dropwise under magnetic stirring, and react for 30 minutes to obtain recombinant human endostatin nanoparticles. The above nanoparticle solution was centrifuged at 4°C and 10,000 rpm for 30 minutes to separate the nanoparticles. The obtained nanoparticles were redispersed in 50 mL of water, 1 g of trehalose was added, and freeze-dried to obtain recombinant human endostatin nanoparticles with uniform particle size distribution.

Appropriately dilute the supernatant solution after centrifugation, and use the BCA method (the kit is from ThermoScientific, named BCA ^TM Protein Assay Kit) to measure the protein concentration, that is, the free protein concentration, and calculate the weight of recombinant human endostatin in the nanoparticles The percentage is the drug loading. Wherein drug loading=(added protein weight-free protein weight)÷the total weight of drug-loaded nanoparticles. The freeze-dried nanoparticles were redissolved in distilled water, and the morphology of the nanoparticles was observed with a transmission electron microscope. The obtained nanoparticles have a drug loading capacity of 16.3% and a spherical shape, as shown in FIG. 1 .

Embodiment two

Take by weighing the chitosan 500mg that molecular weight is 100000 Daltons, be dissolved in the acetic acid solution of 50mL 1%, obtain the chitosan solution of 10mg/mL. Prepare 20 mL of 5 mg/mL sodium polyphosphate solution with distilled water. Take 200mg of Endostar acetate buffer solution (pH5.5) and add it to the chitosan solution, slowly add the sodium polyphosphate solution dropwise under magnetic stirring, react for 5 minutes, and ultrasonicate the probe at 100W for 2 minutes to obtain the recombinant human blood vessel Endostatin nanoparticles. The above nanoparticle solution was centrifuged at 4°C and 10,000 rpm for 30 minutes to separate the nanoparticles. The obtained nanoparticles were redispersed in 50 mL of water, 1 g of trehalose was added, and freeze-dried to obtain recombinant human endostatin nanoparticles with uniform particle size distribution.

The determination of drug loading is the same as in Example 1. The freeze-dried nanoparticles were redissolved in distilled water, and the particle size of the nanoparticles was measured with a nano-ZS90 Malvern particle size analyzer. The obtained nanoparticles have a drug loading capacity of 20.8%, an average particle size of 241 nm, and the particle size distribution is shown in FIG. 2 .

Embodiment Three

Take by weighing the chitosan 250mg that molecular weight is 100000 Daltons, be dissolved in the acetic acid solution of 50mL 1%, obtain the chitosan solution of 5mg/mL. Prepare 15 mL of 5 mg/mL sodium polyphosphate solution with distilled water. Add 200 mg of Endostar phosphate buffer solution (pH5.0) into the chitosan solution, slowly add sodium polyphosphate solution dropwise under propeller stirring (300 rpm), react for 10 minutes, and ultrasonically probe for 2 minutes under 100W power, that is Recombinant human endostatin nanoparticles were obtained. The nanoparticle solution was centrifuged at 4000 rpm for 10 minutes with an ultrafiltration membrane with a molecular weight cut-off of 50,000 Daltons to separate the nanoparticles. The obtained nanoparticle concentrate was dispersed in 30 mL of water, 1.5 g of trehalose was added, and freeze-dried to obtain recombinant human endostatin nanoparticles with uniform particle size distribution.

Appropriately dilute the ultrafiltrate after centrifugation, and use the BCA method (the kit is from Thermo Scientific, named BCA ^TM Protein Assay Kit) to measure the protein concentration, that is, the free protein concentration, and calculate the recombinant human endostatin in the nanoparticles. The weight percentage is the drug loading. Wherein drug loading=(added protein weight-free protein weight)÷the total weight of drug-loaded nanoparticles. The freeze-dried nanoparticles were redissolved in distilled water, and the particle size of the nanoparticles was measured with a nano-ZS90 Malvern particle size analyzer. The obtained nanoparticles have a drug loading capacity of 34.7%, an average particle size of 178 nm, and the particle size distribution is shown in FIG. 3 .

Embodiment Four

Take by weighing the chitosan 250mg that molecular weight is 50000 Daltons, be dissolved in the acetic acid solution of 50mL 1%, obtain the chitosan solution of 5mg/mL. Prepare 15 mL of 5 mg/mL sodium polyphosphate solution with distilled water. Take 200mg of Endostar acetate buffer solution (pH5.5) and add it to the chitosan solution, slowly add sodium polyphosphate solution dropwise under magnetic stirring, react for 5 minutes, and use the probe at 100W power for 2 minutes to obtain recombinant human vascular endothelium Inhibin nanoparticles. The above nanoparticle solution was centrifuged at 4°C and 10,000 rpm for 30 minutes to separate the nanoparticles. The obtained nanoparticles were redispersed in 50 mL of water, 1.5 g of sucrose was added, and freeze-dried to obtain recombinant human endostatin nanoparticles with uniform particle size distribution.

The determination of drug loading is the same as in Example 1. The freeze-dried nanoparticles were redissolved in distilled water, and the particle size of the nanoparticles was measured with a nano-ZS90 Malvern particle size analyzer. The drug-loading capacity of the obtained nanoparticles is 32.1%, and the average particle diameter is 143nm.

Take 50 mg of freeze-dried nanoparticles and add 5 mL of phosphate buffer (pH7.4), shake and release at 37 degrees at 100 rpm for 10 days, take samples on 1d, 2d, 4d, 6d, 8d, and 10d respectively, and use the BCA method ( The kit is from Thermo Scientific (named BCA ^TM Protein Assay Kit) to measure the concentration of protein in it, that is, the concentration of released protein, and calculate the cumulative release rate. The cumulative release reached 90% in 10 days, and the cumulative release curve is shown in Figure 4.

Embodiment five

Take by weighing the chitosan 100mg that molecular weight is 200000 Daltons, be dissolved in the acetic acid solution of 50mL 1%, obtain the chitosan solution of 2mg/mL. Prepare 10 mL of 2 mg/mL sodium polyphosphate solution with distilled water. Add 100mg of Endostar acetate buffer (pH5.5) into the chitosan solution, slowly add sodium polyphosphate solution dropwise under propeller stirring (300 rpm), react for 30 minutes, and ultrasonicate for 5 minutes under 100W power, that is Recombinant human endostatin nanoparticles were obtained. The nanoparticle solution was centrifuged at 4000 rpm for 10 minutes with an ultrafiltration membrane with a molecular weight cut-off of 50,000 Daltons to separate the nanoparticles. The obtained nanoparticle concentrate was dispersed in 20 mL of water, 1 g of sucrose was added, and freeze-dried to obtain recombinant human endostatin nanoparticles with uniform particle size distribution.

The determination of drug loading and particle size is the same as in Example 3. The drug-loading capacity of the obtained nanoparticles is 41.7%, and the average particle diameter is 349nm.

Take the reconstituted nanoparticles for Endostar bioactivity assay:

1. HUVEC cells were cultured with ECM supplemented with FBS, ECGS, and P/O Solution in an incubator at 37°C and 5% CO ₂ , and inoculated after the cells were in good condition and entered the logarithmic growth phase.

2. Digest the cells with 0.25% trypsin, centrifuge at 1000rpm for 5min, discard the supernatant, resuspend with the medium, and count live cells with a hemocytometer under a microscope. Adjust the cell density to 5000 cells/mL, add 160 μL of cell suspension to each well, and culture in a 5% CO ₂ incubator at 37°C.

3. Pre-dilute the Endostar stock solution and the reconstituted nanoparticles to 2.5 mg/mL with pH 7.4 phosphate buffer solution, and the final concentration in the well is 500, 250, 125, 62.5, 31.25, 15.625 μg/mL, A volume of 40 μl of drug was added to each well, and three parallels were set up for each gradient, and cultured in a 5% CO ₂ incubator at 37° C. for 96 hours.

4. Add 5 mg/mL MTT working solution, 20 μL per well, culture in a 5% CO ₂ incubator at 37°C for 4 hours, discard the cell supernatant, and add 200 μL of DMSO to each well. Leave it for 10 min, and measure the OD value with a microplate reader at a wavelength of 490 nm.

5. Calculate the cell inhibition rate according to the OD value, and the calculation formula is inhibition rate (IR)=(the average OD value of the control group-the average OD value of the experimental group)/(the average OD value of the control group-the average OD value of the blank) . (Figure 5)

It can be seen from the figure that the Endostar in the nanoparticle and the stock solution have the same degree of inhibition on cell proliferation.

Claims (10)

1. a preparation method of recombinant human endostatin chitosan nanoparticle for injection, characterized in that: (1) Add chitosan to the acetic acid solution and stir until completely dissolved; (2) Add the buffer solution containing recombinant human endostatin into the chitosan acetic acid solution prepared in step (1), add sodium tripolyphosphate under the condition of constant stirring to make an emulsion; (3) ultrasonically disperse the above obtained emulsion, centrifuge, filter off the supernatant to obtain a concentrate or precipitate; (4) Redisperse the above-mentioned concentrated solution or precipitate in water, add a lyoprotectant, and freeze-dry to obtain recombinant human endostatin chitosan nanoparticles; the lyoprotectant is selected from lactose, glucose, sucrose one or more of them. 2. preparation method according to claim 1 is characterized in that described chitosan molecular weight scope is at 10000～～500000 Daltons, and chitosan concentration is 0.5～10mg/mL in chitosan acetic acid solution. 3. preparation method according to claim 1 is characterized in that described acetic acid solution concentration is 0.5～～2mg/mL. 4. The preparation method according to claim 1, characterized in that the sodium tripolyphosphate in step (2) can be directly added or added as an aqueous solution, and the prepared sodium tripolyphosphate aqueous solution has a concentration of 0.1 to 10 mg/mL. 5. preparation method according to claim 1, it is characterized in that buffer described in step (2) can be one of acetate buffer, phosphate buffer, Tris buffer, glycine～hydrochloric acid buffer species; wherein the pH value of the buffer is between 2 and 9. 6. preparation method according to claim 1 is characterized in that the weight ratio of chitosan and sodium tripolyphosphate is 1: 1～～10: 1 in the step (2). 7. The preparation method according to claim 1, characterized in that the ultrasonic time in step (3) is 0-10min, and the centrifugation can adopt ultrafiltration centrifugation or refrigerated centrifugation; the centrifugal speed of refrigerated centrifugation is 6000-16000 rpm The centrifugation time is 10 to 60 minutes, and the temperature is controlled at 0 to 10 degrees Celsius; the ultrafiltration centrifugation adopts an ultrafiltration membrane with a molecular weight cut-off of 30,000 to 100,000 Daltons, the centrifugal speed is 3,000 to 5,000 rpm, and the centrifugation time is 5 to 5 30 minutes. 8. The preparation method according to claim 1, characterized in that the mass ratio of the lyoprotectant to recombinant human endostatin chitosan nanoparticles in step (4) is 0.1:1 to 10:1. 9. The preparation method according to claim 1, characterized in that the weight percent of recombinant human endostatin in the recombinant human endostatin chitosan nanoparticles accounts for 1%wt～50%wt, and the nanoparticle average The particle size is between 100nm and 600nm. 10. The method according to any one of claims 1-9, characterized in that the recombinant human endostatin is recombinant human endostatin Endostar.

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