Novel targeting sequence in nanoparticle preparation for treating HPV infection and preparation method of novel PBAE

文档序号：1485953 发布日期：2020-02-28 浏览：7次中文

阅读说明：本技术 治疗hpv感染的纳米粒制剂中新的靶向序列以及新pbae的制备方法 (Novel targeting sequence in nanoparticle preparation for treating HPV infection and preparation method of novel PBAE ) 是由马丁汪辉祝达沈慧谭松巍熊劲风胡争程静于 2019-11-29 设计创作，主要内容包括：本发明提供了一种用于敲降或敲除HPV的靶向序列,还提供了用于靶向敲降或敲除HPV的重组质粒,还提供了一种用于治疗HPV感染的纳米粒制剂,还提供了一种制备聚(β氨基酯)聚合物的方法。本发明提供的靶向序列针对九种HVP亚型,并使用这些靶向序列构建了靶向敲降/敲除质粒,可有效地敲降/敲除被转染的细胞中的相应HPV。本发明制备的新PBAE可高效地将DNA转染至目标细胞内,并且具有更低的细胞毒性。(The invention provides a targeting sequence for knocking down or knocking out HPV, a recombinant plasmid for knocking down or knocking out HPV, a nanoparticle preparation for treating HPV infection and a method for preparing a poly (β amino ester) polymer.)

1. A targeting sequence for knocking down or knocking out HPV is characterized in that the sequence is shown as one of SEQ ID NO 1-45.

2. A recombinant plasmid for targeting knockdown or knockout of HPV, wherein said recombinant plasmid comprises the targeting sequence of claim 1.

3. The recombinant plasmid of claim 2, wherein the recombinant plasmid is a CRISPR plasmid, each of which comprises a sequence as set forth in one of SEQ ID NOs 1-27 as a targeting sequence.

4. The recombinant plasmid according to claim 2, wherein the recombinant plasmid is an shRNA plasmid, and each shRNA plasmid comprises a sequence shown in one of SEQ ID NO 28-45 as a targeting sequence.

5. A nanoparticle formulation for use in the treatment of HPV infection, comprising the recombinant plasmid of any one of claims 2-4 and a poly (β amino ester) polymer.

6. The nanoformulation according to claim 5, wherein the mass ratio of the poly (β amino ester) polymer to the recombinant plasmid is 20:1-40: 1.

7. A method of making a poly (β amino ester) polymer comprising the steps of:

s1: dissolving 1, 4-butanediol diacrylate and 4-amino-1-butanol in a solvent to react to obtain a reaction system;

s2: reacting the reaction system at 65-75 ℃ under the condition of keeping out of the sun to obtain a basic polymer;

s3 reacting the base polymer with a solution of 1, 4-butanediol diacrylate in DMF to give the poly (β amino ester) polymer.

8. The method of claim 7, wherein in S1, 1, 4-butanediol diacrylate and 4-amino-1-butanol are dissolved in N, N-dimethylformamide as a solvent to obtain the reaction system.

9. The method according to claim 7, wherein in S2, the temperature of the reaction system is slowly increased to 65-75 ℃, and the reaction is continuously stirred for 42-56 hours.

10. The method of claim 7, wherein in S3, the molar ratio of 1- (3-aminopropyl) -4-methylpiperazine in the base polymer is 1: 4 to 1: 6.

Technical Field

The invention relates to the field of biological pharmaceutical preparations, in particular to a targeting sequence for knocking down or knocking out HPV, a recombinant plasmid for knocking down or knocking out HPV, a nanoparticle preparation for treating HPV infection and a method for preparing a poly (β amino ester) polymer.

Background

Cervical cancer seriously harms women's health, accounting for the third place of female malignant tumor diseases worldwide. More than 85% of new cases of cervical Cancer and cases fatal to Cancer occur in developing countries (Jemal A, Bray F, Center MM, oral. Global Cancer statistics. CA Cancer J Clin,2011,61(2): 69-90.).

In recent years, with the continuous progress of basic medicine, a series of new recombinant plasmid DNAs with target knockdown or knockout HPV appear, including short hairpin RNA (shRNA) recombinant plasmid DNA with target knockdown HPV, regularly clustered short palindromic repeats (CRISPR) recombinant plasmid DNA and the like.

However, this knock-down/knock-out technique suffers from two problems, one is the requirement for efficient and accurate targeting, and the other is the requirement for a carrier to be able to penetrate tissue and cellular barriers effectively.

In the patent with application number 2015109993917 filed in 2015, a nanoparticle preparation for treating HPV infection is disclosed, which has the advantages of high transfection efficiency and high specificity. On the basis, the preparation methods of the targeting sequence and the polymer are improved through further research, and a novel nanoparticle preparation for treating HPV infection is obtained.

Disclosure of Invention

Based on the research, the invention provides a targeting sequence for knocking down or knocking out HPV, and the sequence is shown in one of SEQ ID NO 1-45.

The invention also provides a recombinant plasmid for targeted knock-down or knockout of HPV, wherein the recombinant plasmid comprises the targeting sequence.

In a specific embodiment, the recombinant plasmids are CRISPR plasmids, each of which comprises the sequence shown in one of SEQ ID NOs 1-27 as a targeting sequence.

In a specific embodiment, the recombinant plasmids are shRNA plasmids, and each shRNA plasmid comprises a sequence shown in one of SEQ ID NO. 28-45 as a targeting sequence.

The invention also provides a nano-particle preparation for treating HPV infection, which comprises the recombinant plasmid and the poly (β amino ester) polymer.

In a specific embodiment, the mass ratio of the poly (β amino ester) polymer to the recombinant plasmid is 20:1 to 40: 1.

The present invention also provides a method of preparing a poly (β amino ester) polymer comprising the steps of:

s1: dissolving 1, 4-butanediol diacrylate and 4-amino-1-butanol in a solvent to react to obtain a reaction system;

s2: reacting the reaction system at 65-75 ℃ under the condition of keeping out of the sun to obtain a basic polymer;

s3 reacting the base polymer with a solution of 1, 4-butanediol diacrylate in DMF to give the poly (β amino ester) polymer.

In a preferred embodiment, in S1, 1, 4-butanediol diacrylate and 4-amino-1-butanol are dissolved in N, N-dimethylformamide as a solvent to obtain the reaction system.

In a preferred embodiment, in S2, the reaction system is slowly warmed to 65-75 ℃ and reacted for 48 hours with continuous stirring.

In a preferred embodiment, in S3, the molar ratio of 1- (3-aminopropyl) -4-methylpiperazine of the base polymer is 1: 4 to 1: 6.

The invention provides targeting sequences aiming at nine HVP subtypes, and constructs targeting knockdown/knockout plasmids by using the targeting sequences, so that the corresponding HPV in transfected cells can be knocked down/knocked out effectively. The novel PBAE prepared by the invention can transfect DNA into target cells with high efficiency and has lower cytotoxicity.

Drawings

FIG. 1 is a statistical plot of cytotoxicity of the new PBAE of the invention at different PBAE-DNA mass ratios (20:1, 30:1, 40:1), with blank and previous PBAE (60:1) as controls;

FIG. 2 is a statistical plot of the transfection efficiency of the new PBAE of the invention at different PBAE-DNA mass ratios (20:1, 30:1, 40:1), with blank and previous PBAE (60:1) as controls;

FIG. 3 is a graph of the effect of transfection of HPV16-CRISPR-1 and HPV16-shRNA-1 on SiHa cell proliferation;

FIG. 4 shows the effect of HPV18-CRISPR-1 transfection and HPV18-shRNA-1 transfection on Hela cell proliferation;

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

1. Preparation of Poly (β amino ester) Polymer

Step 1: 2.18 g (11mmol) of 1, 4-butanediol diacrylate is weighed by an electronic balance and put into a beaker, 2mL of N, N-Dimethylformamide (DMF) is added, dissolved by ultrasonic, and stirred until the mixture is dispersed evenly.

Step 2: 0.89g (10mmol) of 4-amino-1-butanol is weighed in a beaker, added with a proper amount of DMF, dissolved by ultrasonic, and stirred until the mixture is dispersed evenly. The obtained solution was mixed with the solution of step 1, and DMF was added to make up to a total volume of 18.38mL, so that the molar ratio of the weighed 1, 4-butanediol diacrylate to 4-amino-1-butanol was 1.1:1 and the density was 167 mg/mL. It was surprisingly found in the study that this molar ratio gives better results.

And step 3: and (3) adding the solutions in the steps (1) and (2) into a light-proof glass flask, putting the flask into a constant-temperature magnetic stirring oil bath kettle, putting the liquid in the flask into magnetic beads, and installing a tap water condensing device and a balloon filled with nitrogen above the flask. Stirring is started, the temperature is slowly increased from room temperature to 65-75 ℃, the heating rate is 1 ℃/minute, and stirring is continuously carried out for 42-56 hours (the time is started when the temperature is increased to 40 ℃). Diluting the reaction solution with a proper amount of DMF, dripping the diluted reaction solution into diethyl ether with the volume of 6-10 times of the volume of the diluted reaction solution for precipitation, washing twice to remove unreacted monomers, centrifuging the precipitate by using a low-speed centrifuge, blowing nitrogen, and drying the precipitate in a vacuum drying oven to obtain the base polymer.

And 4, preparing 0.5M 1- (3-aminopropyl) -4-methylpiperazine solution by using DMF, mixing the base polymer synthesized in the step 3 with 1- (3-aminopropyl) -4-methylpiperazine solution with the molar ratio of 4-6 times, stirring at room temperature for 10min, standing for 24 hours, stopping the reaction, dripping the reaction solution into diethyl ether with the corresponding volume for precipitation, washing twice, centrifuging by using a low-speed centrifuge to obtain precipitate, blowing nitrogen, and drying in a vacuum drying oven to obtain a final product poly (β amino ester) polymer, namely the new PBAE of the invention, wherein the cytotoxicity and transfection efficiency of the new PBAE of the invention are compared with those of PBAE in the prior granted patent 2015109993917.

2. Sequences designed for targeted knockdown or knockdown hpv

The invention designs corresponding targeting sequences aiming at HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58, and is used for targeting knock-down or knocking out corresponding HPV. SEQ ID NO 1-27 are designed as CRISPR targeting sequences, and SEQ ID NO 28-45 are designed as short hairpin RNA gene sequences. Wherein:

1-3 are CRISPR targeting sequences (corresponding to targeting recombinant plasmids HPV6-CRISPR-1, 2, 3) aiming at HVP 6;

4-6 are CRISPR targeting sequences (corresponding to targeting recombinant plasmids HPV11-CRISPR-1, 2, 3) aiming at HVP 11;

7-9 are CRISPR targeting sequences (corresponding to targeting recombinant plasmids HPV16-CRISPR-1, 2 and 3) aiming at HVP 16;

10-12 are CRISPR targeting sequences against HVP18 (corresponding to targeting recombinant plasmid HPV18-CRISPR-1, 2, 3);

13-15 are CRISPR targeting sequences against HVP31 (corresponding to targeting recombinant plasmid HPV31-CRISPR-1, 2, 3);

16-18 are CRISPR targeting sequences against HVP33 (corresponding to targeting recombinant plasmid HPV33-CRISPR-1, 2, 3);

19-21 are CRISPR targeting sequences against HVP45 (corresponding to targeting recombinant plasmid HPV45-CRISPR-1, 2, 3);

22-24 are CRISPR targeting sequences against HVP52 (corresponding to targeting recombinant plasmid HPV52-CRISPR-1, 2, 3);

25-27 are CRISPR targeting sequences against HVP58 (corresponding to targeting recombinant plasmid HPV58-CRISPR-1, 2, 3);

28-29 are shRNA sequences (corresponding to targeting recombinant plasmids HPV6-shRNA-1, 2) aiming at HVP 6;

30-31 are shRNA sequences aiming at HVP11 (corresponding to targeting recombinant plasmids HPV11-shRNA-1, 2);

SEQ ID NO 32-33 is shRNA sequence aiming at HVP16 (corresponding to targeting recombinant plasmid HPV16-shRNA-1, 2);

34 is shRNA sequence aiming at HVP18 (corresponding to targeting recombinant plasmid HPV 34-shRNA-1);

35-36 are shRNA sequences (corresponding to targeting recombinant plasmids HPV31-shRNA-1, 2) aiming at HVP 31;

SEQ ID NO 37-38 is shRNA sequence aiming at HVP33 (corresponding to targeting recombinant plasmid HPV33-shRNA-1, 2);

39-40 are shRNA sequences (corresponding to targeting recombinant plasmids HPV45-shRNA-1, 2) aiming at HVP 45;

41-42 are shRNA sequences (corresponding to targeting recombinant plasmids HPV52-shRNA-1, 2) aiming at HVP 52;

SEQ ID NOS 43-45 are shRNA sequences (corresponding to targeting recombinant plasmids HPV58-shRNA-1, 2, 3) against HVP 58.

3. Construction of recombinant plasmid

Respectively cloning the sequences shown in SEQ ID NO 1-27 into CRISPER vectors to obtain CRISPR targeted knockout/knockdown plasmids aiming at corresponding HPV; the sequences shown in SEQ ID NO 28-45 are respectively cloned into shRNA plasmids to obtain shRNA targeted knockout/knockdown recombinant plasmids aiming at the corresponding HPV.

After the construction of the target recombinant plasmid is completed, the bacterial liquid containing the successfully recombined plasmid is respectively amplified, and the plasmid with endotoxin removed is extracted. For convenience of description, the "targeted knockdown or knock-out recombinant plasmid" will be referred to below simply as a targeted recombinant plasmid.

The extraction method for extracting the plasmid DNA is not particularly limited, as long as the integrity of the extracted plasmid DNA sample is ensured and endotoxin is removed, a commercial endotoxin-removing plasmid DNA extraction kit can be purchased, and the omega endotoxin-free plasmid large-scale extraction kit is adopted in the experiment and is operated according to the specification to remove the endotoxin. The extracted plasmid was stored at-20 ℃ separately.

The bacterial liquid containing the successfully recombined targeted recombinant plasmid can be frozen and preserved in a refrigerator at the temperature of minus 80 ℃ after being added with 10 percent of glycerol, and only the bacterial liquid for preserving the seeds needs to be amplified.

4. Preparation of nanoparticle suspensions

According to the test of the inventor, the solution with the mass ratio of the poly (β amino ester) polymer to the target recombinant plasmid DNA being 20:1 to 40:1 is prepared by properly diluting the poly (β amino ester) polymer and the target recombinant plasmid DNA respectively with 25mM sodium acetate solution, and according to the test of the inventor, the solution with the mass ratio of the poly (β amino ester) polymer to the target recombinant plasmid DNA being 20:1 to 40:1 is unexpectedly found to have better effect, particularly when the mass ratio is 40:1, for example, when the mass ratio is 40:1, 4000 mu g of poly (β amino ester) polymer is diluted to 40 mu L with 25mM sodium acetate solution, 100 mu g of plasmid DNA is diluted to 40 mu L with 25mM sodium acetate solution, the two solutions are rapidly mixed and vibrated, and are incubated for 15-20 minutes at room temperature, and 80 mu L of nanoparticle suspension for treating HPV infection is prepared.

According to the same method, the poly (β amino ester) polymer and the targeted recombinant plasmid prepared above are respectively set to three different mass ratios, namely, the mass ratio of the poly (β amino ester) polymer to the targeted recombinant plasmid DNA is 20:1, the mass ratio of the poly (β amino ester) polymer to the targeted recombinant plasmid DNA is 30:1, and the mass ratio of the poly (β amino ester) polymer to the targeted recombinant plasmid DNA is 40: 1.

5. Preparation of different dosage forms

The nanoparticle suspension can be added with pharmaceutical adjuvants or adjuvants to further make into different dosage forms. For example, the obtained nanoparticle suspension for treating HPV infection may be formulated with various suitable excipients such as chemicals, traditional Chinese medicines, etc. into clinically acceptable dosage forms, such as vaginal spray, vaginal lotion, vaginal gel, vaginal soft and hard capsules, vaginal suppository, vaginal membrane, vaginal tablet, vaginal effervescent tablet, male spray, ointment, cream, etc., buccal tablet, chewable tablet, chewing gum, etc. The lotion containing the nanoparticle suspension can be used for daily pudendum cleaning for women, and other dosage forms can be directly put into vagina to eliminate Human Papilloma Virus (HPV) infection of vagina and cervix. The following are exemplified by gel, lotion, pessary, and the like, but are not limited to the above. The amount of the compound to be used in each dosage form can be appropriately adjusted.

1) Preparation of vaginal gel

Vaginal gel preparation is represented by adding Pluronic F127 to nanoparticle suspension for treating HPV infection.

Adding 40g Pluronic F127 into 60mL water at 4 deg.C, stirring to obtain gel matrix; adding the nanoparticle suspension into the gel matrix on ice or at 4 deg.C, stirring, and making into homogeneous gel. The ratio of gel matrix to nanoparticle suspension can be optimized as desired, for example, 80 μ L of gel matrix is mixed with 120 μ L of nanoparticle suspension.

2) Preparation of vulva lotion

Adding the poly (β amino ester) plasmid nanoparticles into distilled water, adding 20g of glycerol, mixing, adding 0.3g of essence into 10g of ethanol, dissolving, adding the solution, mixing, adding distilled water to 1000mL, and subpackaging at 4 ℃ for storage.

3) Preparation of vaginal suppository

Heating and dissolving 9g of gelatin in distilled water, adding 7.2g of glycerol, fixing the volume to 5.6mL, stirring and uniformly mixing, adding the poly (β amino ester) plasmid nanoparticle suspension when the temperature of the glycerol gelatin is reduced to about 50 ℃, uniformly mixing, and subpackaging at 4 ℃ for storage.

6. Cytotoxicity determination and comparison of old and new PBAE

Toxicity tests are carried out on a lesion cell line SiHa before cervical cancer, and the specific method comprises the steps of mixing 100ng of green fluorescent plasmid with 10 mu L of 25mM sodium acetate, mixing poly (β amino ester) with 10 mu L of 25mM sodium acetate solution according to the PBAE-DNA mass ratio of 20:1, 30:1 and 40:1, 3, mixing the mixed poly (β amino ester) sodium acetate solution with the plasmid sodium acetate solution, standing for 15-20min, and adding the poly (β amino ester) sodium acetate solution into a 96-well plate, wherein the results of the original PBAE serving as a positive control (mass ratio of 60 to 1) are shown in figure 1, and the cell activity of cells transfected by using the new PBAE is higher than that of the original PBAE (60:1) after 72 hours of transfection, so that the cytotoxicity of the new PBAE is obviously lower than that of PBAE in a patent ZL 2015993917.

7. Determination and comparison of transfection efficiency of New and old PBAE

The PBAE-DNA composition described above was used to transfect HEK293 cells. As shown in FIG. 2, the transfection efficiency of the new PBAE of the present invention was as high as 80% or more, which is higher than the highest transfection efficiency (about 60:1, 70%) of PBAE in patent ZL2015109993917, with a PBAE-DNA mass ratio of 30: 1. At a mass ratio of 30:1, the PBAE of the invention have less cytotoxicity and higher transfection efficiency, superior to that of the PBAE in patent ZL 2015109993917.

Cervical cancer cell inhibition experiment using PBAE-targeted recombinant plasmid

The above targeted recombinant plasmids were used to combine with the new PBAE of the invention to corresponding plasmid nanoparticles, respectively, for HPV inhibition experiments. The results show that the plasmid nanoparticles of the present invention can inhibit the proliferation of the corresponding HPV-infected cells. Some examples of which are listed below.

1) Experiments for inhibiting siha cells by HPV16-CRISPR-1 and HPV16-shRNA-1 are shown in figure 3, and plasmid nanoparticles respectively consisting of HPV16-CRISPR-1 and HPV16-shRNA-1 can inhibit the proliferation of siha cells;

2) experiments on the inhibition of the HPV18-CRISPR-1 and the HPV18-shRNA-1 on Hela cells are shown in figure 4, and plasmid nanoparticles respectively consisting of the HPV18-CRISPR-1 and the HPV18-shRNA-1 can inhibit the proliferation of the Hela cells.

The plasmid nanoparticles formed by combining other targeted recombinant plasmids and the new PBAE can also inhibit the proliferation of corresponding uterine cancer cells, and are limited to space and not shown in a unified way.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> affiliated Tongji hospital of Tongji medical college of Huazhong university of science and technology

<120> novel targeting sequence in nanoparticle formulation for treating HPV infection and method for preparing novel PBAE

<141>2019-11-29

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