CN109893664B

CN109893664B - Cationic lipid nanoparticle/DNA compound and preparation method thereof

Info

Publication number: CN109893664B
Application number: CN201810038987.4A
Authority: CN
Inventors: 彭飞; 夏言富; 靳开远
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2017-12-11
Filing date: 2018-01-16
Publication date: 2022-03-11
Anticipated expiration: 2038-01-16
Also published as: CN109893664A

Abstract

The invention provides a cationic lipid nanoparticle/DNA compound and a preparation process thereof, wherein the preparation process comprises the following steps: (1) dissolving a cationic lipid material in anhydrous ethanol by heating; (2) dropwise adding the ethanol solution prepared in the step (1) into the water phase solution, and self-assembling to form cationic lipid nanoparticles; (3) removing residual ethanol in the cationic lipid nanoparticles obtained in the step (2); (4) and (5) filtering. (5) Preparing a DNA solution; (6) mixing the cationic lipid nanoparticles prepared in the step (4) and the DNA solution prepared in the step (5) according to a certain mass ratio to form a cationic lipid nanoparticle/DNA complex; (7) and (5) filtering. The preparation method is simple and rapid to operate, the particle size of the prepared cationic lipid nanoparticle/DNA compound is 50-150 nm, PDI is less than 0.3, the compound is in monodispersity distribution and stable in structure, and the compound is subjected to terminal filtration sterilization, so that the safety of the compound in clinical application of preparing and treating tumor medicines is effectively guaranteed.

Description

Cationic lipid nanoparticle/DNA compound and preparation method thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a cationic lipid nanoparticle/DNA compound and a preparation method thereof.

Background

The key to the success of gene therapy is whether it is possible to deliver the therapeutic agent safely and effectively into the target cells via the vector in vivo. Gene therapy vectors are divided into viral vectors and non-viral vectors. Although the viral vector is used as a highly efficient delivery system to achieve the purpose of target gene transfection and treatment, the Lipid Nanoparticles (LNPs) as non-viral vectors are widely concerned due to the problems of containing immunogenic viral proteins, limited target gene loading, high price and the like, and are widely applied to gene therapy research of congenital and acquired genetic defects. LNPs refer to vesicles formed from one or more lipid components that are effective in compressing and delivering a variety of nucleic acid molecules, from DNA, RNA, to chromosomes and even cells; LNPs are beneficial to large-scale production due to the characteristics of determined construction scheme, easy modification of targeting ligand and the like.

The cationic lipid is an amphiphilic molecule, is mainly connected with a hydrophobic long chain by a positively charged polar head through a chemical bond, and has the characteristics of easy synthesis and convenient modification in each component domain. The complex formed by the cationic lipid nanoparticle and the nucleic acid through electrostatic adsorption can not only compress the nucleic acid to increase the density of the nucleic acid, but also protect the nucleic acid from degradation of nuclease, and effectively bind to a target cell through electrostatic interaction with a negatively charged cell membrane, so that the nucleic acid is delivered into the target cell and mediates high-level gene expression.

The particle size of the complex formed by the cationic lipid nanoparticles and the DNA, namely the cationic lipid nanoparticle/DNA complex, is a main factor influencing the transfection efficiency of cells in vitro; cationic lipid nanoparticles with larger particle sizes are easier to be cleared by the blood circulation system in vivo than those with smaller particle sizes, thereby affecting the amount of DNA drug reaching the target tissue or target cell. The main factors influencing the particle size of the cationic lipid nanoparticle/DNA complex include the composition of the lipid, the ratio of the lipid to the nucleic acid, the preparation method thereof and the like.

Currently, methods for preparing LNPs include thin film dispersion, reverse phase evaporation, ethanol injection, microemulsion cooling, freeze-thaw, and freeze-drying. Wherein, the ethanol injection method is to inject the ethanol solution of the lipid and/or the drug into the water phase solution which is stirred at a high speed at a proper temperature, and the ethanol is diffused rapidly, thereby leading the lipid molecules to be aggregated to form LNPs due to thermodynamic reasons. The method has the following advantages: (1) ethanol is used for replacing the conventional organic solvent chloroform, so that the safety coefficient of the application of LNPs is increased, and the quality of the product is ensured; (2) the method can form LNPs only by directly injecting the oil phase containing lipid into the water phase, is simple and convenient, and is easy for large-scale production; (3) the method has mild conditions, is not easy to degrade or oxidize and denature the lipid, and reduces the damage to the LNPs structure; (4) the particle size of LNPs prepared by ethanol injection method is less than 100nm, which is beneficial to phagocytosis and transfection of cells; (5) by removing the injected ethanol after the formation of LNPs, the potential drug toxicity of the organic residual solvent can be avoided. Therefore, the ethanol injection method is gradually becoming a scale production method of great interest.

However, there are many limiting factors in the current ethanol injection method commonly used in industry, for example, the particle size and uniformity of the formed LNPs are greatly related to the concentration of the lipid solution, the stirring speed, the lipid composition, and the ratio of the oil phase and the water phase; in addition, the ethanol injection method usually injects an oil phase into a corresponding volume of an aqueous phase by mechanical force according to a certain volume ratio to form LNPs, and continuous production cannot be realized due to the volume limitation of the aqueous phase storage tank, thereby increasing batch-to-batch variation. The above factors all limit the scale production of LNPs.

LNPs or liposomes prepared by ethanol injection methods often require a homogenization step (such as high pressure homogenization, sonication, or extrusion) to improve the uniformity of the nanoparticles, but there are limitations in using an extrusion method most suitable for large-scale production:

(1) in the vertical extrusion process of the conventional liposome extrusion instrument with the size of 47mm, a filter membrane is easy to block, and even the quality of the whole batch of liposome or LNPs is influenced, so that the large-scale production of the liposome or LNPs is severely limited.

(2) In order to increase the throughput of samples, mass production is generally achieved by increasing the membrane area, but increasing the membrane area requires a larger extrusion device, which not only greatly increases the production cost, but also does not ensure the extrusion stability.

Therefore, in the mass production of cationic lipid nanoparticles, the problems of uneven particle size distribution, unstable structure, difficult quality control and the like of the nanoparticles still exist by adopting a homogenization step.

At present, an ethanol direct dissolution method is commonly adopted to prepare LNPs/nucleic acid complexes, namely, ethanol is dissolved in cationic lipid and then is directly mixed with buffer solution containing nucleic acid to prepare cationic lipid nanoparticles/nucleic acid complexes, and in the method, ethanol is not removed before the ethanol is mixed with the nucleic acid, so that certain influence is generated on the structural stability of the nucleic acid. Due to the existence of nucleic acid in the compound, ethanol cannot be removed by using the rotary evaporation method, and the ethanol in the sample needs to be removed by adopting mild methods such as ultrafiltration or dialysis, but the methods have long treatment time and complicated operation, the sample loss is large, and the residual amount of the ethanol in the sample is higher than that of the rotary evaporation method. The dilution method is adopted immediately after mixing to reduce the influence of ethanol on the stability of the compound, thereby being not beneficial to the quality control of the product. If the ethanol in the sample is not removed, the safety of the compound in clinical medication is affected.

Even if the homogenization step is used, the problem that the cationic nanoparticles are heterogeneous can not be solved, and the cationic lipid/nucleic acid composite obtained by mixing the cationic lipid/nucleic acid composite with nucleic acid has larger particle size and can not pass through a 0.22 mu m filter membrane to filter and sterilize a product, so that the risk of clinical medication is increased. Since the terminal filtration sterilization method cannot be used, it is necessary to perform an operation under aseptic conditions in the process of preparing the cationic lipid/nucleic acid complex, thereby increasing production costs and having poor quality controllability.

In the preparation of the cationic lipid nanoparticle/nucleic acid complex, nucleic acid is injected in a dropwise manner into the cationic lipid nanoparticle prepared by the ethanol injection method, usually by the action of a pump, and stirred without stopping to form the cationic lipid nanoparticle/nucleic acid complex. This method has the following problems: firstly, the cationic lipid nanoparticles and nucleic acid have shear force effect due to stirring in the mixing process, so that the mixing action time of the cationic lipid nanoparticles and the nucleic acid is short, and effective compounding cannot be achieved to form a stable compound; secondly, in order to prevent the complex from having high local concentration, large particle size and poor uniformity, the dripping speed of nucleic acid is slow, and the production time is long; thirdly, the method is limited by the size of the stirrer volume and the stirring capacity thereof, and the average particle size of the prepared cationic lipid nanoparticle/nucleic acid complex is often more than 220nm, so that the terminal filtration sterilization of the complex cannot be realized, and therefore, the method needs to be carried out in an ultra-clean bench or an isolator, and therefore, the method is not suitable for the large-scale production and preparation of the cationic lipid nanoparticle/nucleic acid complex.

Therefore, how to prepare the cationic lipid nanoparticle/nucleic acid composite with uniform particle size and monodispersity and the preparation process can realize experimental large-scale production, meet clinical requirements and ensure the safety of clinical medication is a problem to be solved urgently in the process research of the current cationic lipid nanoparticle/nucleic acid composite.

The invention content is as follows:

the invention aims to provide a method for preparing a cationic lipid nanoparticle/DNA complex by an improved ethanol injection method, which specifically comprises the following steps: after preparing cationic lipid nanoparticles with the concentration of not less than 6mg/ml by using a modified ethanol injection method, removing ethanol by a rotary evaporation method without homogenization (homogenization) step, mixing with a DNA solution, and filtering and sterilizing to obtain the cationic lipid nanoparticle/DNA compound. The preparation method is simple and rapid to operate, not only can ensure that the particle size of the cationic lipid nanoparticle is less than 100nm, the particle size is uniform, and the cationic lipid nanoparticle/DNA compound has monodispersity, but also has uniform particle size distribution and stable structure, the ethanol residual quantity meets the medicinal standard, and the safety of the compound in the clinical application of preparing and treating the tumor medicament is effectively ensured by filtering and sterilizing the product terminal.

The purpose of the invention is realized by the following technical scheme:

the invention firstly provides a preparation method of a cationic lipid nanoparticle/DNA complex, which comprises a preparation method of a cationic lipid nanoparticle and a preparation method of a cationic lipid nanoparticle/DNA complex.

The preparation method of the cationic lipid nanoparticle comprises the following steps:

(1) ethanol injection method: dissolving cationic lipid material in anhydrous ethanol, and heating to dissolve;

(2) dropwise adding the ethanol solution containing the cationic lipid material prepared in the step (1) into the aqueous phase solution, and self-assembling to form cationic lipid nanoparticles;

(3) removing residual ethanol in the cationic lipid nanoparticles obtained in the step (2);

(4) filtering;

the preparation method of the cationic lipid nanoparticle/DNA complex comprises the following steps:

(5) preparing a DNA solution;

(6) mixing the cationic lipid nanoparticles prepared in the step (4) and the DNA solution prepared in the step (5) according to a certain mass ratio to form a cationic lipid nanoparticle/DNA complex;

(7) filtering;

in the preparation method, the concentration of the cationic lipid material in the absolute ethyl alcohol in the step (1) is 30-100 mg/ml, preferably 50-60 mg/ml; the volume ratio of the ethanol solution to the water phase solution in the step (2) is 1: 3-1: 6, and the final concentration of the cationic lipid material in the mixed solution of ethanol and water phase is 6-25 mg/ml; the particle size of the formed cationic lipid nanoparticle/DNA compound is 50-150 nm, and the dispersion index PDI of the compound is less than 0.3.

When the concentration of the cationic lipid in the absolute ethyl alcohol is more than 100mg/ml, the cationic lipid nanoparticles with the particle size less than 150nm and PDI less than 0.3 cannot be formed; however, when the concentration of the cationic lipid material in the anhydrous ethanol is less than 30mg/ml, heterogeneous cationic lipid nanoparticles are easily generated and the polydispersity increases because the concentration value of the cationic lipid material is small.

In the preparation method, the cationic lipid nanoparticles with the particle size of 50-150 nm, which are monodisperse and uniform, can be obtained by using an ethanol injection method to prepare 6-25 mg/ml cationic lipid nanoparticles without further homogenization.

The cationic lipid nanoparticles with the particle size of less than 150nm and PDI of less than 0.3 prepared by the preparation method are suitable for being used as a DNA transmission carrier, can further improve the effects of compressing cationic lipid materials and stabilizing DNA transfection and transportation into cells, is convenient for filtration by a 0.22 mu m filter membrane, and has controllable quality.

Wherein the cationic lipid in the above preparation method is at least one selected from (2, 3-dioleoxypropyl) trimethylammonium chloride (DOTAP), N- [1- (2, 3-dioleoyl chloride) propyl ] -N, N-trimethylamine chloride (DOTMA), dimethyl-2, 3-dioleyloxypropyl-2- (2-sperminoylamido) ethylammonium (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB) and dimethyldioctadecylammonium bromide (DDAB), and the preferred cationic lipid is DOTAP.

The DOTAP is a commonly used cationic lipid, and can be used as a eukaryotic cell transfection reagent for transfection of eukaryotic cells and a vector of DNA vaccines because the DOTAP forms a stable transfection complex with DNA or RNA to enter cells and releases nucleic acid into the cells.

In the preparation method, the mass ratio of the cationic lipid nanoparticles to the DNA in the step (6) is 6: 1-125: 1, preferably 6: 1-20: 1.

When the mass ratio of the cationic lipid nanoparticles to the DNA is 6: 1-125: 1, the particle size of the formed cationic lipid nanoparticle/DNA compound is 50-150 nm, and the PDI is less than 0.3; particularly, when the mass ratio of the cationic lipid nanoparticles to the DNA is 6: 1-20: 1, the particle size of the formed cationic lipid nanoparticle/DNA compound is less than 100nm, the PDI is less than 0.3, the structure is more uniform and stable, and the effect of inhibiting the growth of tumor cells is better exerted. When the mass ratio of the two is less than 6:1, the DNA cannot be completely complexed with the cationic lipid nanoparticle. Because the maximum concentration of the cationic lipid nanoparticles prepared by the method is about 25mg/ml, and the DNA concentration of the cationic lipid nanoparticle/DNA complex is generally more than 0.1mg/ml in terms of drug formation, the DNA concentration before the cationic lipid nanoparticles are compounded with DNA in equal volume is more than 0.2 mg/ml. Thus, the mass ratio of cationic lipid nanoparticles to DNA is 125:1 at the maximum.

In the preparation method, the cationic lipid material is a mixture of the cationic lipid and a helper lipid, and the helper lipid is at least one selected from Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), cholesterol (Chol) or Dioleoylphosphatidylethanolamine (DOPE).

Further, the mass ratio of the cationic lipid to the helper lipid in the mixture of the cationic lipid and the helper lipid described in the above preparation method is greater than 1: 1.

The helper lipid may improve the stability of the cationic lipid nanoparticle. Common auxiliary lipid, such as cholesterol, has the solubility of about 33.6mg/ml at 50 ℃ in absolute ethyl alcohol, or DOPE has the solubility of about 36.5mg/ml at 50 ℃ in absolute ethyl alcohol, so when the auxiliary lipid is added into the cationic lipid, the cationic lipid nanoparticles with the particle size of 40-100 nm and monodispersity (PDI < 0.3) can be obtained when the mass ratio of the two is more than 1: 1; on the other hand, when the cationic lipid nanoparticle does not contain a helper lipid, the stability of the complex formed by the cationic lipid nanoparticle and the DNA can be maintained as well. However, when the mass ratio of the cationic lipid to the helper lipid is 1:1 or less, the prepared cationic lipid nanoparticle cannot simultaneously satisfy the particle size of less than 150nm and the PDI < 0.3.

The cationic lipid nanoparticle/DNA complex described in step (6) of the above preparation method may be optionally prepared in a semi-automatic apparatus comprising a T-type connector. The semi-automatic device of the T-shaped connector is selected from a combined device shown in fig. 1, fig. 2a or fig. 2b, wherein the device shown in fig. 1 is formed by sequentially connecting a constant flow pump, a sterile filter, a liquid storage bottle, a constant flow pump and the T-shaped connector according to the arrow direction; the device shown in fig. 2a is composed of a constant flow pump, a T-shaped connector, a liquid storage bottle containing an air filter and a connecting pipeline thereof in sequence according to the arrow direction; the device shown in fig. 2b consists of a syringe pump, a syringe, a T-shaped connector and a liquid storage bottle from right to left in sequence; the preferred apparatus is that shown in figure 1.

The preparation method of the cationic lipid nanoparticle/DNA complex in the step (6) comprises the steps of firstly assembling a semi-automatic device containing a T-shaped connector as shown in figure 1, figure 2a or figure 2b, then respectively placing the cationic lipid nanoparticle prepared in the step (4) and the DNA solution prepared in the step (5) into containers of the device according to the mass ratio of 6: 1-125: 1, mixing the cationic lipid nanoparticle and the DNA solution in the T-shaped connector at the speed of 20-100 ml/min, and then feeding the mixture into a new container to form the cationic lipid nanoparticle/DNA complex.

When the mixing speed is 20-100 ml/min, the cationic lipid nanoparticle/DNA compound with the particle size less than 150nm and uniform size (PDI less than 0.3) can be obtained, and the higher the mixing speed is in the range, the smaller the particle size of the compound is, the higher the uniformity is; when the mixing speed is less than 20ml/min, the cationic lipid nanoparticles and the DNA solution are easy to aggregate when being mixed in the T-shaped connector, thereby influencing the particle size and uniformity of the compound and being not beneficial to the quality control of the compound; when the mixing speed is more than 100ml/min, the mixing of the cationic lipid nanoparticles and the DNA solution in the T-shaped connector cannot be realized due to the limitation of a constant flow pump and a connecting pipeline in the device.

On one hand, the semi-automatic device containing the T-shaped connector is simple in structure and easy to assemble; on the other hand, the cationic lipid nanoparticle/DNA complex having a particle size of <150nm and a uniform size (PDI < 0.3) can be stably produced by a simple process repeatedly regardless of the order of the dropping of the cationic lipid nanoparticle and the DNA solution. Further, the device shown in fig. 1 can perform integral high-temperature sterilization before the cationic lipid nanoparticles and the DNA solution are compounded, and even in an unclean production environment without expensive instruments and equipment such as an isolator, a super clean bench and the like, the cationic lipid nanoparticles and the DNA solution can be compounded in a simple closed sterile device environment, so that the production cost is saved, large-scale production is easy to realize, and the prepared cationic lipid nanoparticles/DNA compound has a particle size of less than 150nm and a uniform particle size (PDI of less than 0.3).

In the preparation method, the heating in the step (1) is preferably water bath heating, and the heating temperature is 40-60 ℃, preferably 50-60 ℃; when the water bath temperature is room temperature, the cationic lipid nanoparticles are distributed disorderly, but the particle size and the distribution of the cationic lipid nanoparticles tend to be stable along with the increase of the water bath temperature, and the optimal particle size and the distribution of the cationic lipid nanoparticles are achieved particularly at 50-60 ℃.

In the above-mentioned production method, the aqueous solution in the step (2) is an aqueous solution or an aqueous solution containing a saccharide selected from lactose, maltose, sucrose, glucose or trehalose, and the concentration of the aqueous solution of the saccharide is 2 to 20%, preferably 4 to 10%.

The method for removing ethanol in step (3) in the above preparation method is spin-steaming, dialysis, ultrafiltration, spray drying or lyophilization, etc., preferably spin-steaming.

The residual quantity of ethanol in the spirally steamed cationic nano-lipid particles meets the medicinal standard of 0.5 percent.

In the preparation method, the filtration in the step (4) and the step (7) adopts a 0.22 mu m filter membrane for filtration, and the filtration can simultaneously play a role in sterilization.

The length of the DNA is 100-2500 bp.

The DNA solution in the step (5) in the above production method is an aqueous DNA solution.

The cationic lipid nanoparticles in the step (6) in the preparation method are obtained by diluting the cationic lipid nanoparticles prepared in the step (4) with water, and the concentration of the diluted cationic lipid nanoparticles is 1-4 mg/ml.

The cationic lipid nanoparticles prepared by the method are suitable for forming a compound with uniform particle size and monodispersity with DNA. Before compounding, the cationic lipid nanoparticles are diluted to the concentration of 1-4 mg/ml, the preparation method can be realized through a simple preparation method, different auxiliary materials can be added according to different preparation requirements, and the subsequent preparation process and large-scale production are facilitated.

The DNA described in the step (5) in the above production method is a plasmid or a variant thereof, or a plasmid loaded with another DNA; either mitochondrial DNA or fragments of mitochondrial DNA.

Further, the DNA described in the above preparation method in the step (5) is a plasmid selected from at least one of pVAX1, pcDNA3.1, pBR322, or pUC 18;

the DNA described in the step (5) of the above preparation method may also be a variant of pVAX1, i.e., a replicable plasmid having the most basic structural unit formed after reconstitution and comprising the kanamycin resistance gene, the pUC origin sequence and the plasmid backbone sequence, the nucleotide sequence of the pMVA plasmid is shown in SEQ ID NO: 1;

the DNA described in the step (5) in the above preparation method may also be subjected to base mutation or deletion at a specific nucleotide site of the pMVA plasmid to obtain a pMVA-1 plasmid, the nucleotide sequence of which is shown in SEQ ID NO: 2.

The DNA in the step (5) in the above preparation method is a plasmid loaded with other DNA, and is selected from at least one of plasmids having a nucleotide sequence shown in SEQ ID NO. 6-11 or a nucleotide sequence having more than 90% homology with the sequence shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.11,

preferably, the DNA is mitochondrial DNA or a fragment of mitochondrial DNA. The nucleotide sequence of the mitochondrial DNA segment is shown as SEQ ID NO. 3-5, or the nucleotide sequence has more than 90% homology with the sequence shown as SEQ ID NO. 3-5. The invention also aims to provide a cationic lipid nanoparticle/DNA compound, which is prepared by the preparation method, wherein the particle size of the formed cationic lipid nanoparticle/DNA compound is 50-150 nm, and the dispersion index PDI of the compound is less than 0.3.

Wherein the cationic lipid nanoparticle/DNA complex is preferably a DOTAP nanoparticle/pMVA-1 complex.

The cationic lipid nanoparticle/DNA compound prepared by the preparation method has a uniform and stable structure, is suitable for filtration sterilization at a terminal of process preparation, is not only beneficial to product quality control, but also can improve the inhibitory activity of the compound on the growth of tumor cells.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the preparation method of the cationic lipid nanoparticle/DNA compound is provided, and the cationic lipid nanoparticle prepared by the method has the advantages of uniform distribution, monodispersity, high activity of inhibiting tumor cells and the like, and has the particle size of less than 150 nm.

2. The method for preparing the cationic lipid nanoparticle/DNA compound has the advantages that the particle size of the cationic lipid nanoparticle/DNA compound prepared by the method is less than 150nm, and the compound is stable in structure and controllable in quality.

3. The preparation method of the cationic lipid nanoparticle/DNA compound is provided, the organic solvent residue of the cationic lipid nanoparticle/DNA compound prepared by the method meets the medicinal standard, and the safety of the compound serving as a tumor medicament in clinical medication can be guaranteed.

4. Provided is a method for preparing a cationic lipid nanoparticle/DNA complex, which can prepare the cationic lipid nanoparticle/DNA complex in a semi-automatic apparatus having a T-shaped connector, which has the characteristics of simple structure and easy assembly, and can provide a simple, sealed and sterile apparatus environment, and can repeatedly and stably produce the cationic lipid nanoparticle/DNA complex having a particle size of <150nm and a uniform size (PDI < 0.3) through simple steps, regardless of the sequential order of the dropping of the cationic lipid nanoparticle and the DNA solution. The method can realize the large-scale production of the compound as an anti-tumor medicament in the device so as to meet the requirement of clinical medication.

5. The method for preparing the cationic lipid nanoparticles by adopting the ethanol injection method is provided, the homogenization step is not needed, the method is simple and easy to operate, and the production cost is saved; the ethanol is removed by rotary evaporation in the process of preparing the cationic lipid nanoparticles, and compared with the method for removing the ethanol after the preparation of the cationic lipid nanoparticle/DNA compound is finished, the method is more favorable for saving the production time and ensuring the quality control of the compound, and is favorable for preparing the compound into tumor medicaments for clinical application.

6. The method for preparing the cationic lipid nanoparticle/DNA complex enables the complex product to realize terminal filtration sterilization, so that the quality of clinical medication of the complex is strongly guaranteed.

7. Provides a preparation method of cationic lipid nanoparticle/DNA compound, the particle size of the compound prepared by the method is less than 150nm, and the compound is suitable for gene therapy of systemic administration.

Description of the drawings:

FIG. 1 is a schematic view showing the preparation of a cationic lipid nanoparticle/DNA complex using the preparation method of the present invention using an apparatus 1 composed of a constant flow pump, a sterile filter,

liquid storage bottles

1 and 2 containing an air filter, a constant flow pump, a T-shaped connector, and a liquid storage bottle 3 in the order of arrow; wherein the components of the box-line labeled parts indicate that assembly and bulk autoclaving can be performed prior to preparation of the cationic lipid nanoparticle/DNA complex to maintain a sterile environment;

FIG. 2 is a schematic diagram of the preparation of cationic lipid nanoparticle/DNA complex using the preparation method of the present invention using apparatus 2 (FIG. 2a) and apparatus 3 (FIG. 2b), wherein apparatus 2 shown in FIG. 2a is composed of a constant flow pump, a T-shaped connector, and a liquid storage bottle containing an air filter and its connecting pipe in the order of arrow; the device shown in fig. 2b consists of a syringe pump, a syringe, a T-shaped connector and a liquid storage bottle from right to left in sequence;

FIG. 3, agarose gel electrophoresis detection images of DOTAP nanoparticle/DNA complexes of different mass ratios;

FIG. 4 shows the particle size and distribution of the DOTAP nanoparticle/pMVA-1 complex (mass ratio of DOTAP nanoparticle to pMVA-1 is 10:1) obtained by the preparation method of the present invention;

fig. 5 is a graph showing the inhibition of a549 cell activity by the cationic lipid nanoparticle/DNA complex prepared by the preparation method of the present invention and the cationic lipid nanoparticle/DNA complex prepared by the high-pressure homogenization step after the ethanol injection method, wherein ■ represents the preparation method of the present invention (referred to as a new process), ● represents the high-pressure homogenization step after the ethanol injection method (referred to as an old process);

FIG. 6, cationic lipid nanoparticle/DNA complex inhibits tumor growth in mice model cervical carcinoma subcutaneous tumor, wherein, t represents the preparation method of the present invention (referred to as new process), and ■ represents the use of high pressure homogenization step after ethanol injection (referred to as old process);

FIG. 7, particle size and distribution of cationic lipid nanoparticle/DNA complexes prepared using a high pressure homogenization step after ethanol injection;

fig. 8 is a graph showing the detection results of inhibition of a549 cell activity by the cationic lipid nanoparticle/DNA complex prepared by the preparation method of the present invention and the cationic lipid nanoparticle/DNA complex prepared by the ethanol direct dissolution method, wherein a is the preparation method of the present invention (referred to as new process) and a is the ethanol direct dissolution method.

FIG. 9 is a graph showing the inhibition of A549 cell activity by complexes of DOTAP nanoparticles and DNA prepared by the preparation method of the present invention at different final concentrations in an aqueous ethanol solution, wherein ● represents the complex (sample) of DOTAP nanoparticles and DNA prepared at a concentration of 50mg/ml in absolute ethanol and a volume ratio of ethanol to water of 1:5 (final concentration of DOTAP in an aqueous ethanol solution is about 8.3 mg/ml); ■ shows a complex of DOTAP nanoparticles and DNA prepared with a volume ratio of ethanol to water of 20mg/ml in absolute ethanol and a final concentration of DOTAP in aqueous ethanol of about 4mg/ml (control 1); tangle-solidup indicates a complex of DOTAP nanoparticles and DNA prepared at a DOTAP concentration of 100mg/ml in absolute ethanol and a ethanol to water volume ratio of 1:2 (final DOTAP concentration in aqueous ethanol solution of about 33.3mg/ml) (control 2).

Detailed Description

The technical means of the present invention will be further described below by way of specific embodiments. It should be noted that the present invention relates to some common molecular biology procedures and common procedures for preparing pharmaceutical preparations, and those skilled in the art can be implemented by combining textbooks, handbooks and instructions for using relevant equipment and reagents in the field on the basis of the present specification.

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the invention are not limited thereto.

Example 1 preparation of DOTAP nanoparticles and characterization thereof

1. Preparation of DOTAP nanoparticles

(1) As shown in Table 1, different masses of DOTAP were weighed into 250ml PETG (polyethylene terephthalate-1, 4-cyclohexanedimethanol) bottles, added to an absolute ethanol solution, heated in a water bath at 50 ℃ and gently shaken to accelerate complete dissolution of DOTAP.

(2) And (2) erecting a peristaltic pump, setting the rotating speed to be 6-8 rpm, and dropwise adding the ethanol solution containing the DOTAP prepared in the step (1) into the aqueous phase solution at the speed of 30-120 ml/min, wherein the total volume of the ethanol solution and the aqueous phase solution is 60ml, and the volume ratio of the ethanol solution to the aqueous phase solution is shown in Table 1. The distance between the droplet outlet and the liquid surface is kept about 5cm, and the DOTAP can be self-assembled in an ethanol aqueous solution to form the DOTAP nano-particles.

(3) And (3) after the DOTAP ethanol solution is dropwise added into the water-phase solution in the step (2), washing the pipeline and the PETG bottle by using absolute ethanol. After the rinsing was complete, the dispersion was stirred at 140rpm for a further 10 min.

(4) And (3) removing ethanol by reduced pressure distillation:

starting a rotary evaporator, and setting the water bath temperature to be 40 ℃; and starting a cooling circulating pump, and setting the refrigeration temperature to be 5 ℃. Transferring the DOTAP nanoparticle dispersed solution prepared in the step (3) into a rotary evaporation bottle, and carrying out reduced pressure distillation; and turning on a vacuum pump, and turning off all rotary ports of the rotary evaporator. And stopping distillation after the waste liquid collected in the distillation flask is more than 120 ml. Transferring the liquid in the rotary evaporation bottle into a new PETG bottle, and fixing the volume to 500ml by using pure water.

(5) Filtering and storing the DOTAP nanoparticles subjected to rotary evaporation and ethanol removal in the step (4) through a 0.22-micrometer filter membrane for later use.

2. Characterization of DOTAP nanoparticles:

(1) preparing a detection sample of the DOTAP nanoparticles:

and (3) adding sterilized distilled water into the DOTAP nano-particles with different concentrations prepared in the steps, oscillating at a high speed to fully dissolve the DOTAP nano-particles, and standing at room temperature.

(2) Detection of particle size of DOTAP nanoparticles

And (2) adding the DOTAP Nano-particle detection sample prepared in the step (1) into a sample vessel of a Nano-particle size potential analyzer (Malvern Zetasizer Nano ZS), placing the sample vessel into a test slot, setting the balance time to be 1min, and testing 3 groups of data in parallel on each sample. The average particle size of the composite sample was obtained.

The detection results are shown in Table 1, and the underlined data meet the conditions that the particle size is less than 150nm and PDI is less than 0.3, so that the solubility of DOTAP in ethanol is 30-100 mg/ml (particularly 50-60 mg/ml), the volume ratio of ethanol to an aqueous phase solution is 1: 3-1: 6, the final concentration of DOTAP in a mixed solution of ethanol and an aqueous phase is 6-25 mg/ml, the particle size of the prepared DOTAP nanoparticles is 50-150 nm, and PDI is less than 0.3.

TABLE 1 particle size and PDI values of nanoparticles formed in different concentrations of DOTAP in absolute ethanol and in different volume ratios of aqueous ethanol solutions

"-" indicates that the requirements that the particle diameter of the cationic lipid nanoparticle/DNA compound is 50-150 nm and PDI is less than 0.3 are obviously not met.

Example 2 preparation of DOTAP/Cholesterol nanoparticles and characterization thereof

1. Preparation of DOTAP/Chol nanoparticles:

(1) 33.6mg of DOTAP and 33.6mg of cholesterol (in a mass ratio of 1:1) which are weighed respectively are respectively put into a 250ml PETG bottle, added into an absolute ethyl alcohol solution, heated in a water bath at 50 ℃, and gently shaken to accelerate complete dissolution of the DOTAP and the cholesterol.

(2) And (2) erecting a peristaltic pump, setting the rotation speed to be 6-8 rpm, and dropwise adding the ethanol solution containing the DOTAP prepared in the step (1) into the aqueous phase solution at the speed of 30-120 ml/min, wherein the volume ratio of the ethanol solution to the aqueous phase solution is 1:3 or 1:4, the distance between a droplet outlet and a liquid level is kept at about 5cm, and the DOTAP and cholesterol can be self-assembled in the ethanol aqueous solution to form the DOTAP/Chol nano-particles.

(3) Same as example 1, step (3).

(4) Same as example 1, step (4).

2. Characterization of DOTAP/Chol nanoparticles:

the procedure was the same as for the characterization of DOTAP nanoparticles in example 1.

The detection result is shown in Table 2, when the mass ratio of the DOTAP to the cholesterol is more than 1:1, and the volume ratio of the ethanol to the aqueous phase solution is 1: 3-1: 6, the particle size of the prepared DOTAP/Chol nano-particles is less than 150nm, and the PDI is less than 0.3.

Example 3 preparation and characterization of DOTAP/DOPE nanoparticles

1. Preparation of DOTAP/DOPE nanoparticles:

(1) respectively weighing 36.5mg of DOTAP and 36.5mg of DOPE (mass ratio of 1:1) into a 250ml PETG bottle, adding into an absolute ethanol solution, heating in a water bath at 50 ℃, and slightly shaking to accelerate complete dissolution of DOTAP and DOPE.

(2) Erecting a peristaltic pump, setting the rotation speed to be 6-8 rpm, and dropwise adding the ethanol solution containing the DOTAP prepared in the step (1) into the water phase solution at the speed of 30-120 ml/min, wherein the volume ratio of the ethanol solution to the water phase solution is 1:3 or 1:4, the distance between a droplet outlet and the liquid level is kept at about 5cm, and the DOTAP and DOPE can be self-assembled in the ethanol water solution to form the DOTAP/DOPE nanoparticles.

(3) Same as example 1, step (3).

(4) Same as example 1, step (4).

2. Characterization of DOTAP/DOPE nanoparticles:

The detection result is shown in Table 2, when the mass ratio of DOTAP to DOPE is more than 1:1 and the volume ratio of ethanol to aqueous phase solution is 1: 3-1: 6, the particle size of the prepared DOTAP/DOPE nano-particles is less than 150nm, and the PDI is less than 0.3.

TABLE 2 particle size and PDI values of DOTAP/helper lipid nanoparticles in different ethanol aqueous solution volume ratios

The helper lipid may improve the stability of the cationic lipid nanoparticle. The solubility of common auxiliary lipid, such as cholesterol in absolute ethyl alcohol at 50 ℃ is about 33.6mg/ml, or the solubility of DOPE in absolute ethyl alcohol at 50 ℃ is about 36.5mg/ml, so when the auxiliary lipid is added into the cationic lipid, the mass ratio of the two is more than 1:1, and the cationic lipid nanoparticles with the particle size of 40-100 nm and monodispersity (PDI < 0.3) can be obtained.

And when the mass ratio of the cationic lipid to the auxiliary lipid is less than or equal to 1:1, the prepared cationic lipid nanoparticles cannot simultaneously meet the requirements that the particle size is less than 150nm and the PDI is less than 0.3.

Example 4 preparation of cationic lipid nanoparticle/DNA complexes and characterization thereof at different mixing speeds using device 1

1. Preparation of cationic lipid nanoparticle/DNA complexes Using apparatus 1 at different mixing speeds

(1) Solutions of pMVA-1(SEQ ID NO:2) and other plasmids, such as pMVA-2(SEQ ID NO:6), pMVA-3(SEQ ID NO:7), pMVA-4(SEQ ID NO:8), pMVA-5(SEQ ID NO:9), pMVA-6(SEQ ID NO:10), and pMVA-7(SEQ ID NO:11), were prepared.

(2) After the

liquid storage bottles

1, 2 and 3, the sterile filter and the pipelines among the sterile filter are connected according to the figure 1, the whole high-temperature sterilization is carried out, and the sterile filter and the constant flow pump are sequentially connected to form a closed sterile environment.

(3) As shown in fig. 1, the cationic lipid nanoparticles prepared in examples 1 to 3 and the DNA solution prepared in step (1) are respectively placed in a beaker at a mass ratio of 10:1, sterile-filtered by a constant flow pump and a sterile filter into a liquid storage bottle 1 and a liquid storage bottle 2, and after the sterile-filtering is completed, mixed in a T-shaped connector at a speed of 5 to 100ml/min by the constant flow pump according to table 3, and dripped into the liquid storage bottle 3, and left to stand for 30min, so that the cationic lipid nanoparticle/DNA complex can be formed.

(4)0.22 μm filter membrane was sterilized by filtration.

2. Characterization of cationic lipid nanoparticle/DNA complexes prepared at different mixing speeds

(1) Preparing a cationic lipid nanoparticle/DNA complex detection sample:

adding sterilized distilled water into the cationic lipid nanoparticle/DNA complex prepared in the step 1 at different mixing speeds, oscillating at a high speed to dissolve the complex, and standing at room temperature.

(2) Detection of particle size of cationic lipid nanoparticle/DNA complexes

Adding the cationic lipid nanoparticle/DNA complex detection sample prepared in the step (1) into a sample dish of a nanoparticle size potential analyzer (Malvern Zetasizer Nano ZS), placing the sample dish into a test slot, setting the balance time to be 1min, and testing 3 groups of data in parallel on each sample.

The detection results are shown in Table 3, when the mixing speed is 20-100 ml/min, the cationic lipid nanoparticle/DNA compound with the particle size less than 150nm and uniform size (PDI less than 0.3) can be obtained, and the higher the mixing speed is, the smaller the particle size of the compound is, and the higher the uniformity is; when the mixing speed is less than 20ml/min, the cationic lipid nanoparticles and the DNA solution are easy to aggregate when being mixed in a T-shaped connector, the particle size of the composite is more than 150nm, and the homogeneity is poor (PDI is more than 0.3); when the mixing speed is more than 100ml/min, the mixing of the cationic lipid nanoparticles and the DNA solution in the T-shaped connector cannot be realized due to the limitation of a constant flow pump and a connecting pipeline in the device.

Similarly, when the device 2 or the device 3 is used and the mixing speed of the cationic lipid nanoparticles and the DNA solution in the T-shaped connector is set to be 20-100 ml/min, the particle size of the cationic lipid nanoparticle/DNA compound is less than 150nm, and the PDI is less than 0.3.

TABLE 3 particle size and PDI values of cationic lipid nanoparticle/DNA complexes prepared at different mixing speeds using apparatus 1

Example 5 preparation of cationic lipid nanoparticle/DNA complexes of different mass ratios and characterization thereof using device 1:

1. preparation of cationic lipid nanoparticle/DNA complexes Using device 1

(2) After the

liquid storage bottles

(3) Respectively placing the cationic lipid nanoparticles prepared in the examples 1-3 and the DNA solution prepared in the step (1) in a mass ratio of 1: 1-125.1: 1 in a beaker as shown in figure 1, performing sterile filtration by a constant flow pump and a sterile filter in a liquid storage bottle 1 and a liquid storage bottle 2, after the sterile filtration is completed, respectively mixing the cationic lipid nanoparticles and the DNA solution in a T-shaped connector at a speed of 50ml/min by the constant flow pump, dropwise adding the mixture into the liquid storage bottle 3, and standing for 30min to obtain the cationic lipid nanoparticle/DNA complex.

(4)0.22 μm filter membrane was sterilized by filtration.

2. Agarose gel electrophoresis detection of cationic lipid nanoparticle/DNA complexes of different mass ratios

(1) Preparation of a 1% agarose gel: weighing agarose, placing the agarose in a conical flask, adding 1 XTAE, heating and boiling the agarose in a microwave oven until the agarose is completely melted, adding a DNA dye Golden View, and shaking the mixture uniformly to prepare a 1.0% agarose gel solution.

(2) Preparing a gel plate: and (3) after preparing the gel plate, cooling the agarose gel prepared in the step (1) to 65 ℃, and pouring the agarose gel onto the glass plate with the inner groove to form a homogeneous gel layer. Standing at room temperature until the gel is completely solidified, and placing the gel and the inner groove into an electrophoresis groove. Add 1 XTAE electrophoresis buffer until the gel plate is 1-2mm submerged.

(3) Sample adding: and (3) respectively mixing the cationic lipid nanoparticle/DNA compound detection samples with the cationic lipid nanoparticle to DNA mass ratios of 1:1, 6:1, 10:1, 15:1 and 20:1 with the loading buffer solution, and adding the mixture into the gel pores prepared in the step (2).

(4) Electrophoresis: the gel plate after sample application was immediately subjected to electrophoresis by energization. When bromophenol blue moved about 1cm from the lower edge of the gel plate, the electrophoresis was stopped.

(5) And (5) taking a picture and storing by using a gel imaging system.

Agarose gel electrophoresis detection of cationic lipid nanoparticle/DNA complexes of different mass ratios, as shown in fig. 3, shows that DNA can be effectively retained under the action of agarose gel electrophoresis when the mass ratios of DOTAP nanoparticles to DNA in the cationic lipid nanoparticle/DNA complexes are 6:1, 10:1, 15:1 and 20:1, respectively. And the ratio 1:1, DNA is free, and the formation of a cationic lipid nanoparticle/DNA complex meeting the requirement is difficult.

3. Characterization of cationic lipid nanoparticle/DNA complexes of different mass ratios:

the method was the same as the characterization method of the cationic lipid nanoparticle/DNA complex prepared in example 4 at different mixing speeds.

The detection results are shown in Table 4 and FIG. 4, and the average particle size of the obtained composite sample is 50-150 nm, PDI is less than 0.3, the proportion of the composite with the particle size of not less than 220nm is less than 0.01%, and the proportion of the composite with the particle size of less than 150nm is about 60.12%.

TABLE 4 particle size and PDI values for cationic lipid nanoparticle/DNA complexes of different mass ratios

Example 6 preparation of cationic lipid nanoparticle/DNA complexes and characterization thereof using device 2:

1. preparation of cationic lipid nanoparticle/DNA complexes using device 2

(2) According to the scheme shown in FIG. 2a, the cationic lipid nanoparticles prepared in examples 1-3 are diluted with water to a concentration of 1-4 mg/ml, and then placed in liquid storage bottles labeled with "cationic lipid nanoparticles" and "DNA" respectively at a mass ratio of 1: 1-125: 1 with the plasmid prepared in step (1), the cationic lipid nanoparticles and the DNA solution in the two liquid storage bottles are simultaneously pumped into a T-shaped connector at a speed of 50ml/min by a constant flow pump for mixing, and are dripped into the liquid storage bottles, and the mixture is left standing for 30min, so as to obtain the cationic lipid nanoparticle/DNA complex.

(3)0.22 μm filter membrane was sterilized by filtration.

2. Characterization of the resulting cationic lipid nanoparticle/DNA complexes prepared using device 2

The average particle size of the cationic lipid nanoparticle/DNA complex prepared by using the device 2 is 50-150 nm, PDI is less than 0.3, and the proportion of the complex with the particle size less than 150nm is about 24.82%.

Example 7 preparation of cationic lipid nanoparticle/DNA complexes and characterization thereof using device 3:

1. preparation of cationic lipid nanoparticle/DNA complexes Using device 3

(2) According to the scheme shown in FIG. 2b, after the cationic lipid nanoparticles prepared in examples 1-3 are diluted with water to a concentration of 1-4 mg/ml, the diluted cationic lipid nanoparticles and the plasmid prepared in step (1) are respectively placed in syringes labeled with "cationic lipid nanoparticles" and "DNA" according to a mass ratio of 1: 1-125: 1, two syringe pumps are adjusted to the same horizontal position, the syringe pumps are pushed, the cationic lipid nanoparticles and the DNA solution in the two syringes are simultaneously injected into a T-shaped connector at a speed of 50ml/min for mixing, and are dripped into a liquid storage bottle, and the cationic lipid nanoparticle/DNA complex is obtained after standing for 30 min.

(3)0.22 μm filter membrane was sterilized by filtration.

2. Characterization of the resulting cationic lipid nanoparticle/DNA complexes prepared using device 3

The average particle size of the cationic lipid nanoparticle/DNA complex prepared by the device 3 is 50-150 nm, and PDI is less than 0.3.

Example 8 Effect of cationic lipid nanoparticle/DNA complexes on A549 cell Activity

(1) Cell plating

A549 cells in logarithmic growth phase were prepared as cell suspension and diluted to 5X 10 with 10% FBS-1640⁴cells/ml, 100. mu.l/well into 96-well cell culture plates, 37.0 ℃ 5% CO₂Culturing for 24h under the condition. After the cells are attached to the wall, the culture medium without serum 1640 is replaced for starvation culture for 24 hours.

(2) Preparation of samples to be tested

The DOTAP nanoparticle/pMVA-1 complex prepared by mixing in the mass ratio of 10:1 using the device 1 in example 5 was diluted to 200 μ g/ml with 1640 medium, and then diluted 3-fold for a total of 9 dilution gradients, and prepared into assay samples containing different DOTAP concentrations.

(3) Sample application

The medium 1640 in the 96-well plate was discarded and the assay sample from step (2) was added, 3 replicates per gradient for a total of 9 gradients, with the last row serving as the cell blank, and the blank. Culturing at 37.0 deg.C and 5% CO2 for 48 h.

(4) CCK-8 detection of cell Activity

And (3) uniformly mixing CCK-8 and 1640 culture medium according to a ratio of 1:1, adding 20 mu l of the mixture into the 96-well plate in the step (3), culturing at 37.0 ℃ and 5% CO2 for 2h, and reading the light absorption value of OD450nm by using an enzyme labeling instrument.

(5) Data analysis

And (4) fitting a 4-parameter curve to the concentration gradient of the sample to be detected and the light absorption value obtained by detection in the step (4) to form an inverted S curve, and calculating the half effective concentration (EC50) according to the fitted curve.

As shown in FIG. 5, the DOTAP nanoparticle/pMVA-1 complex prepared by the invention can effectively inhibit the growth activity of A549 tumor cells.

Likewise, the cationic lipid nanoparticle/DNA complexes prepared in examples 6 and 7 also have the activity of inhibiting the growth of a549 tumor cells.

Example 9 stability test of cationic lipid nanoparticle/DNA Complex

1. The method of the invention prepares the cationic lipid nanoparticle/DNA complex

(1) And preparing DOTAP nanoparticles:

the procedure is as in example 1, wherein the concentrations of DOTAP and water in ethanol are 30mg/ml and the volume ratio of ethanol to water is 1: 4.

(2) Preparation of DOTAP nanoparticle/pMVA-1 complexes:

the procedure was the same as in example 5 for the preparation of cationic lipid nanoparticle/DNA complex using the apparatus 1, wherein the concentration of DOTAP nanoparticles was 4mg/ml and the concentration of pMVA-1 plasmid was 0.4mg/ml (mass ratio of cationic lipid to plasmid was 10: 1).

2. The method of the invention prepares the cationic lipid nanoparticle/DNA complex

(1) Preparation of DOTAP/cholesterol (DOTAP/Chol) nanoparticles:

the method is the same as example 2, wherein the concentrations of DOTAP and cholesterol in ethanol are both 30mg/ml (the mass ratio of the DOTAP and the cholesterol is 1:1), and the volume ratio of ethanol to water is 1: 4.

(2) Preparation of DOTAP-Chol/pMVA-1 Complex:

the procedure was the same as in example 5 using the apparatus 1 for preparing cationic lipid nanoparticle/DNA complexes, wherein the concentration of DOTAP/Chol nanoparticles was 4mg/ml and the concentration of pMVA-1 plasmid was 0.4mg/ml (mass ratio of cationic lipid to plasmid was 10: 1).

3. Preparation of DOTAP nanoparticle/DNA complex by high-pressure homogenization after ethanol injection

The method is the same as that of comparative example 1, wherein the concentrations of DOTAP and cholesterol in ethanol are both 10mg/ml (the mass ratio of the DOTAP to the cholesterol is 1:1), and the volume ratio of ethanol to water is 1: 4;

in the formed DOTAP/Chol-pMVA-1 complex, the concentration of the DOTAP/Chol nano-particles is 4mg/ml, and the concentration of the pMVA-1 plasmid is 0.4mg/ml (the mass ratio of the cationic lipid to the plasmid is 10: 1).

3. The cationic lipid nanoparticle/DNA complex (including DOTAP nanoparticle/pMVA-1 complex and DOTAP-Chol/pMVA-1 complex) prepared by the 2 different methods was subjected to particle size detection and PDI detection at 10d after acceleration at 0 day and 37 ℃.

As shown in Table 5, the particle size of the cationic lipid nanoparticle/DNA complex prepared by the method of the present invention is smaller than that of the cationic lipid nanoparticle/DNA complex prepared by the high pressure homogenization step after the ethanol injection method, the former has better uniformity and the former has better stability after acceleration.

In addition, the stability of the DOTAP nanoparticle/pMVA-1 complex prepared by the method is not significantly different from that of the DOTAP-Chol/pMVA-1 complex, which indicates that the stability of the complex can be maintained when the cationic lipid nanoparticle in the cationic lipid nanoparticle/DNA complex prepared by the method does not contain auxiliary lipid.

TABLE 5 comparison of particle size and PDI values of cationic lipid nanoparticle/DNA complexes prepared by the inventive preparation method and ethanol injection followed by high pressure homogenization procedure at 0d and 10d after acceleration

Also, the cationic lipid nanoparticle/DNA complexes prepared in examples 6 and 7 have good stability.

Example 10 inhibition of tumor growth in cervical carcinoma subcutaneous tumor model mice by cationic lipid nanoparticle/DNA complexes

1. Breeding female Kunming mice of 6-8 weeks old.

2. Mouse cervical carcinoma U14 cell culture: using DMEM medium containing inactivated 10% fetal calf serum, 100U/ml penicillin and 100. mu.g/ml streptomycin and 2mM glutamine at 37 ℃ with 5% CO₂The culture box is used for culturing the tumor cells, bottle-dividing passage is carried out after the cells grow full every 2 to 3 days, the passage is controlled between 2 to 10 generations, and the tumor cells in logarithmic growth phase are used for in vivo tumor inoculation.

3. Tumor cell inoculation and experimental animal grouping

Washing tumor cells twice with antibiotic-free and serum-free medium, removing serum existing in cells, and then resuspending tumor cells with DMEM (DMEM) without double antibody at concentration of 2 × 10⁷And/ml, performing subcutaneous injection on the right back of the experimental animal, injecting 100 μ l of each mouse, and performing group administration 5 days after tumor cell inoculation, wherein the group consists of 6 groups, as shown in table 6, wherein the new process group refers to the DOTAP nanoparticle/pMVA-1 complex prepared in the example 5 (wherein the mass of the DOTAP nanoparticle is 100 μ g, and the mass of the pMVA-1 plasmid is 10 μ g), the old process group refers to the DOTAP nanoparticle/pMVA-1 complex prepared in the comparative example 1 (wherein the mass of the DOTAP nanoparticle is 100 μ g, and the mass of the pMVA-1 plasmid is 10 μ g), and the vehicle control group is physiological saline.

4. Closely observing the growth of tumor after the tumor is inoculated until the tumor grows to 80-100mm³In the meantime, each group of experimental mice was administered with the dose volume of100 μ l/tube. The administration was performed every three days later, and all mice were sacrificed by day 21 after tumor inoculation, 5 times in total, and the tumor volume of each group of mice was measured.

TABLE 6 grouping situation and administration dose of DOTAP/pMVA-1 complex for treating cervical carcinoma subcutaneous tumor model mice

The experimental result is shown in fig. 6, the volume of the mice in the new process group is significantly lower than that of other groups, which indicates that the DOTAP nanoparticle/pMVA-1 complex prepared in the embodiment 5 of the present invention can significantly inhibit the tumor growth of tumor-bearing mice, and the inhibition effect is significantly better than that of the DOTAP nanoparticle/pMVA-1 complex prepared in the old process.

Similarly, the cationic lipid nanoparticle/DNA complexes prepared in examples 6 and 7 also have the activity of inhibiting tumor growth in tumor-bearing mice.

Comparative example 1 ethanol injection followed by high pressure homogenization procedure for preparation of DOTAP nanoparticle/DNA complexes

1. Preparation of cationic lipid nanoparticles by ethanol injection method

(1) Preparation of DOTAP ethanol solution:

weighing 3g of DOTAP into a 250ml PETG bottle, adding 100ml of absolute ethyl alcohol, heating in a water bath at 50 ℃, and slightly shaking to accelerate dissolution to prepare a DOTAP ethanol solution, wherein the concentration of the DOTAP in the ethanol is 30 mg/ml.

(2) Injecting DOTAP ethanol solution into the water phase solution

And (2) setting a peristaltic pump at a set rotating speed of 6-8 rpm, slowly dripping the DOTAP ethanol solution prepared in the step (1) into 400ml of an aqueous solution (the volume ratio of ethanol to water is 1:4), and keeping the distance between a droplet outlet and the liquid level to be about 5 cm.

(3) And (3) after the DOTAP ethanol solution is dropwise added into the aqueous solution in the step (2), washing the pipeline and the PETG bottle by using absolute ethanol. After the rinsing was complete, the dispersion was stirred at 140rpm for a further 10 min.

2. And (3) removing ethanol by reduced pressure distillation:

the procedure was as in (4) of example 1

3. High pressure homogenizing and granulating

And starting a low-temperature cooling circulating pump, and setting the refrigerating temperature to be 5 ℃. The high pressure homogenizer was rinsed with pure water for at least 3 times. Adding 500ml of DOTAP nano-particles prepared in the step 2, setting the homogenization pressure to be 500-800bar, and repeatedly homogenizing for 3-10 times. After completion of homogenization, the liquid was transferred to a 1L PETG bottle. Washing the homogenizer for 3 times with pure water, and storing with anhydrous ethanol.

4. And (3) filtering and sterilizing: filtration through a 0.22 μm filter.

5. Preparation of DOTAP nanoparticle/pMVA-1 Complex

(1) Filtering and sterilizing a DNA solution: the pMVA-1 plasmid solution was filtered using a 0.22 μm filter

(2) Preparation of DOTAP/pMVA-1 Complex:

and (3) mixing the DOTAP nanoparticles prepared in the step (4) with the pMVA-1 plasmid solution obtained by filtration sterilization in a mass ratio of 10:1 in a sterile environment, and standing for 30min to form a DOTAP nanoparticle/pMVA-1 complex.

6. Characterization of DOTAP nanoparticle/DNA complexes prepared using a high pressure homogenization step after ethanol injection

The detection result is shown in fig. 7, the average particle size of DOTAP nanoparticle/DNA complex prepared by a high-pressure homogenization step after an ethanol injection method is 150.4nm, PDI is 0.238, although the PDI of the complex prepared by the method is less than 0.3, the complex has polydispersity, and the proportion of the complex with the particle size of more than 220nm reaches 27% (obviously exceeding the requirement that the particle size of the complex prepared by the invention is less than 150 nm), so that when a 0.22 μm filter membrane is used for filtration, the resistance is large, the effect of filtration sterilization cannot be effectively achieved, and further the method cannot realize large-scale production.

7. Effect of DOTAP nanoparticle/DNA complexes prepared by high-pressure homogenization after ethanol injection on A549 cell activity

The procedure is as in example 8.

As shown in fig. 5 and table 7, although the DOTAP nanoparticle/DNA complex prepared by the high-pressure homogenization step (i.e., ethanol injection + high-pressure homogenization) after the ethanol injection method can inhibit the growth activity of a549 cells, the inhibition effect is about 15.2% lower than that of the DOTAP nanoparticle/DNA complex prepared by the present invention.

TABLE 7 comparison of the inhibition of A549 cells by DOTAP nanoparticle/DNA complexes prepared according to the methods of the present invention and ethanol injection followed by a high pressure homogenization step

	Ethanol injection + high pressure homogenization	Inventive method (example 5)
			EC50(μg/ml)	8.879	7.709

Comparative example 2 preparation of DOTAP nanoparticle/DNA Complex by ethanol direct dissolution method

1. Preparation of DOTAP ethanol solution

DOTAP was directly dissolved in absolute ethanol to prepare an ethanol solution containing 4mg/ml DOTAP.

2. Preparation of DOTAP nanoparticle/DNA Complex

And (2) mixing the DOTAP ethanol solution prepared in the step (1) with a pMVA-1 plasmid solution with the concentration of 0.4mg/ml in an equal volume (the mass ratio of the DOTAP to the pMVA-1 is 10:1), and standing for 30min to form the DOTAP nanoparticle/pMVA-1 complex.

3. Characterization of DOTAP nanoparticle/DNA complexes prepared by ethanol direct dissolution method

The average particle diameter of the DOTAP nanoparticle/DNA compound prepared by the ethanol direct dissolution method is 240.4nm, and the PDI is 0.155.

Therefore, the average particle size of the DOTAP nano-particle/DNA compound prepared by the ethanol direct dissolution method is far larger than that of the compound prepared by the method.

4. Comparing the effect of the ethanol direct dissolution method and the DOTAP nanoparticle/DNA compound prepared by the method of the invention on the activity of A549 cells

The procedure is as in example 8.

The detection results are shown in fig. 8 and table 8, and the inhibition effect of the DOTAP nanoparticle/DNA complex prepared by the method of the present invention on the growth activity of a549 cells is significantly higher than that of the complex prepared by the ethanol direct dissolution method.

TABLE 8 comparison of the inhibitory effects of DOTAP nanoparticle/DNA complexes prepared by the method of the present invention and ethanol direct dissolution on A549 cells

5. Characterization of DOTAP nanoparticle/DNA complex prepared by the method and the ethanol direct dissolution method after being placed at room temperature for N days

After the DOTAP nanoparticle/DNA complex prepared in example 5 and the DOTAP nanoparticle/DNA complex prepared in comparative example 1 were left at room temperature for 1d and 4d, the particle size and PDI of the complexes prepared by the two methods were examined.

The detection results are shown in table 9, and after the compound prepared by the method is placed for 1d and 4d, the particle size and PDI of the compound are not obviously changed; and after the compound prepared by the ethanol direct dissolution method is placed for 4 days, the particle size of the compound is obviously increased compared with 0 d. The cationic lipid nanoparticle/DNA compound prepared by the method is shown to be in single distribution, the particle size is less than 150nm, and the structure is stable and can be stored at room temperature.

TABLE 9 characterization of DOTAP nanoparticle/DNA complexes prepared by the method of the present invention and direct ethanol dissolution after 1 and 4 days at room temperature

Comparative example 3. complexes of DOTAP nanoparticles with DNA at different final concentrations in aqueous ethanol prepared by the method of the present invention

1. Preparation of DOTAP nanoparticles

According to the preparation method of example 1, DOTAP nanoparticles (DOTAP nanoparticle sample) prepared with DOTAP at a concentration of 50mg/ml in absolute ethanol and a volume ratio of ethanol to water of 1:5(DOTAP at a final concentration of about 8.3mg/ml in aqueous ethanol) were selected, DOTAP nanoparticles (DOTAP nanoparticle control 1) prepared with DOTAP at a concentration of 20mg/ml in absolute ethanol and a volume ratio of ethanol to water of 1:4(DOTAP at a final concentration of about 4mg/ml in aqueous ethanol), and DOTAP nanoparticles (DOTAP nanoparticle control 2) prepared with DOTAP at a concentration of 100mg/ml in absolute ethanol and a volume ratio of ethanol to water of 1:2(DOTAP at a final concentration of about 33.3mg/ml in aqueous ethanol).

2. Preparation of DOTAP/pMVA-1 Complex

According to the preparation method of example 5, the DOTAP nanoparticles (DOTAP nanoparticle sample, DOTAP nanoparticle reference 1, and DOTAP nanoparticle reference 2) with different concentrations prepared in step 1 are diluted, mixed with the pMVA-1 plasmid according to the mass ratio of 10:1, and left standing for 30min to form 3 different DOTAP nanoparticle/pMVA-1 complexes (sample, reference 1, and reference 2), respectively.

3. Effect of DOTAP nanoparticle/pMVA-1 Complex on A549 cell Activity

The effect of the 3 different DOTAP nanoparticle/pMVA-1 complexes prepared in step 2 on the activity of a549 cells was examined in the same manner as in example 8.

As shown in fig. 9 and table 10, the inhibition effect of control 1 and control 2 on a549 cells was about 53.3% and 44.6% lower than that of the sample, respectively.

Therefore, when the final concentration of the DOTAP in an ethanol water solution is 6-25 mg/ml and the volume ratio of ethanol to water is 1: 3-1: 6, the compound formed by compounding the DOTAP nanoparticles prepared by the method and the DNA has uniform particle size and monodispersity, and has good biological activity of inhibiting the growth of tumor cells.

TABLE 10 comparison of the inhibitory effects of DOTAP nanoparticles/pMVA-1 complexes formed with different concentrations of DOTAP nanoparticles and pMVA-1 plasmid on A549 cells

	Sample (I)	Reference 1	Control 2
				EC50(μg/ml)	10.25	15.72	14.82

Comparative example 4 preparation of cationic lipid nanoparticle/DNA Complex in dropwise addition

1.DOTAP nanoparticles were prepared according to the preparation method of example 1, diluted to 4mg/ml and placed in a beaker with constant stirring.

2. pMVA-1 plasmid solution was prepared according to the method of step 1 of example 4 and diluted to 0.4 mg/ml.

3. Slowly dripping the pMVA-1 plasmid solution prepared in the step 2 into the DOTAP nanoparticle solution prepared in the step 1 at the speed of 10ml/min, and standing for 30min after dripping is finished to form the DOTAP nanoparticle/pMVA-1 complex.

4. Characterization of cationic lipid nanoparticle/DNA complexes prepared in a drop-wise manner:

by using the detection method of example 4 for the characterization of the cationic lipid nanoparticle/DNA complex, the DOTAP nanoparticle/pMVA-1 complex prepared in a dropwise manner had a particle size of 325.4nm and a PDI of 0.359.

Therefore, the average particle diameter of the cationic lipid nanoparticle/DNA complex prepared by the dripping mode is far larger than that of the complex prepared by the method, and the homogeneity of the complex is poor (PDI > 0.3).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> Chengdu King Biotechnology Ltd

<120> cationic lipid nanoparticle/DNA complex and preparation method thereof

<130>

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 1978

<212> DNA

<213> Artificial Sequence

<220>

<221> variation

<223> nucleotide sequence of pMVA

<400> 1

gactcttcgc gatgtacggg ccagatatac gccttctact gggcggtttt atggacagca 60

agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta 120

aactggatgg ctttctcgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa 180

gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 240

gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 300

gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 360

ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg 420

acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 480

ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 540

gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 600

ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 660

gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 720

aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 780

ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 840

ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 900

ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 960

cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc 1020

ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acaggtggca 1080

cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 1140

tgtatccgct catgagacaa taaccctgat aaatgcttca ataatagcac gtgctaaaac 1200

ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 1260

tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 1320

cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 1380

taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 1440

gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 1500

acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 1560

ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 1620

ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 1680

cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 1740

aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 1800

gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 1860

gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 1920

gcaacgcggc ctttttacgg ttcctgggct tttgctggcc ttttgctcac atgttctt 1978

<210> 2

<211> 1977

<212> DNA

<213> Artificial Sequence

<220>

<221> variation

<223> nucleotide sequence of pMVA-1

<400> 2

gctgcttcgc gatgtacggg ccagatatac gccttctact gggcggtttt atggacagca 60

agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta 120

aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa 180

gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 240

gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 300

gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 360

ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg 420

acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 480

ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 540

gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 600

ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 660

gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 720

aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 780

ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 840

ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 900

ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 960

cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc 1020

ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat caggtggcac 1080

ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 1140

gtatccgctc atgagacaat aaccctgata aatgcttcaa taatagcacg tgctaaaact 1200

tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 1260

cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 1320

ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 1380

accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 1440

cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 1500

cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 1560

tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 1620

taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 1680

gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 1740

agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 1800

ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 1860

acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 1920

caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctt 1977

<210> 3

<211> 120

<212> DNA

<213> Artificial Sequence

<220>

<221> gene

<223> nucleotide sequence 1 of mtDNA

<400> 3

cccattattc ctagaaccag gcgacctgcg actccttgac gttgacaatc gagtagtact 60

cccgattgaa gcccccattc gtataataat tacatcacaa gacgtcttgc actcatgagc 120

<210> 4

<211> 600

<212> DNA

<213> Artificial Sequence

<220>

<221> gene

<223> nucleotide sequence 2 of mtDNA

<400> 4

ctgaactatc ctgcccgcca tcatcctagt cctcatcgcc ctcccatccc tacgcatcct 60

ttacataaca gacgaggtca acgatccctc ccttaccatc aaatcaattg gccaccaatg 120

gtactgaacc tacgagtaca ccgactacgg cggactaatc ttcaactcct acatacttcc 180

cccattattc ctagaaccag gcgacctgcg actccttgac gttgacaatc gagtagtact 240

cccgattgaa gcccccattc gtataataat tacatcacaa gacgtcttgc actcatgagc 300

tgtccccaca ttaggcttaa aaacagatgc aattcccgga cgtctaaacc aaaccacttt 360

caccgctaca cgaccggggg tatactacgg tcaatgctct gaaatctgtg gagcaaacca 420

cagtttcatg cccatcgtcc tagaattaat tcccctaaaa atctttgaaa tagggcccgt 480

atttacccta tagcaccccc tctaccccct ctagagccca ctgtaaagct aacttagcat 540

taacctttta agttaaagat taagagaacc aacacctctt tacagtgaaa tgccccaact 600

<210> 5

<211> 2000

<212> DNA

<213> Artificial Sequence

<220>

<221> gene

<223> nucleotide sequence 3 of mtDNA

<400> 5

tacgttgtag ctcacttcca ctatgtccta tcaataggag ctgtatttgc catcatagga 60

ggcttcattc actgatttcc cctattctca ggctacaccc tagaccaaac ctacgccaaa 120

atccatttca ctatcatatt catcggcgta aatctaactt tcttcccaca acactttctc 180

ggcctatccg gaatgccccg acgttactcg gactaccccg atgcatacac cacatgaaac 240

atcctatcat ctgtaggctc attcatttct ctaacagcag taatattaat aattttcatg 300

atttgagaag ccttcgcttc gaagcgaaaa gtcctaatag tagaagaacc ctccataaac 360

ctggagtgac tatatggatg ccccccaccc taccacacat tcgaagaacc cgtatacata 420

aaatctagac aaaaaaggaa ggaatcgaac cccccaaagc tggtttcaag ccaaccccat 480

ggcctccatg actttttcaa aaaggtatta gaaaaaccat ttcataactt tgtcaaagtt 540

aaattatagg ctaaatccta tatatcttaa tggcacatgc agcgcaagta ggtctacaag 600

acgctacttc ccctatcata gaagagctta tcacctttca tgatcacgcc ctcataatca 660

ttttccttat ctgcttccta gtcctgtatg cccttttcct aacactcaca acaaaactaa 720

ctaatactaa catctcagac gctcaggaaa tagaaaccgt ctgaactatc ctgcccgcca 780

tcatcctagt cctcatcgcc ctcccatccc tacgcatcct ttacataaca gacgaggtca 840

acgatccctc ccttaccatc aaatcaattg gccaccaatg gtactgaacc tacgagtaca 900

ccgactacgg cggactaatc ttcaactcct acatacttcc cccattattc ctagaaccag 960

gcgacctgcg actccttgac gttgacaatc gagtagtact cccgattgaa gcccccattc 1020

gtataataat tacatcacaa gacgtcttgc actcatgagc tgtccccaca ttaggcttaa 1080

aaacagatgc aattcccgga cgtctaaacc aaaccacttt caccgctaca cgaccggggg 1140

tatactacgg tcaatgctct gaaatctgtg gagcaaacca cagtttcatg cccatcgtcc 1200

tagaattaat tcccctaaaa atctttgaaa tagggcccgt atttacccta tagcaccccc 1260

tctaccccct ctagagccca ctgtaaagct aacttagcat taacctttta agttaaagat 1320

taagagaacc aacacctctt tacagtgaaa tgccccaact aaatactacc gtatggccca 1380

ccataattac ccccatactc cttacactat tcctcatcac ccaactaaaa atattaaaca 1440

caaactacca cctacctccc tcaccaaagc ccataaaaat aaaaaattat aacaaaccct 1500

gagaaccaaa atgaacgaaa atctgttcgc ttcattcatt gcccccacaa tcctaggcct 1560

acccgccgca gtactgatca ttctatttcc ccctctattg atccccacct ccaaatatct 1620

catcaacaac cgactaatca ccacccaaca atgactaatc aaactaacct caaaacaaat 1680

gataaccata cacaacacta aaggacgaac ctgatctctt atactagtat ccttaatcat 1740

ttttattgcc acaactaacc tcctcggact cctgcctcac tcatttacac caaccaccca 1800

actatctata aacctagcca tggccatccc cttatgagcg ggcgcagtga ttataggctt 1860

tcgctctaag attaaaaatg ccctagccca cttcttacca caaggcacac ctacacccct 1920

tatccccata ctagttatta tcgaaaccat cagcctactc attcaaccaa tagccctggc 1980

cgtacgccta accgctaaca 2000

<210> 6

<211> 2098

<212> DNA

<213> Artificial Sequence

<220>

<221> variation

<223> nucleotide sequence of pMVA-2

<400> 6

gactcttcgc gatgtacggg ccagatatac gccccattat tcctagaacc aggcgacctg 60

cgactccttg acgttgacaa tcgagtagta ctcccgattg aagcccccat tcgtataata 120

attacatcac aagacgtctt gcactcatga gccttctact gggcggtttt atggacagca 180

agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta 240

aactggatgg ctttctcgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa 300

gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 360

gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 420

gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 480

ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg 540

acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 600

ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 660

gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 720

ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 780

gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 840

aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 900

ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 960

ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1020

ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1080

cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc 1140

ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acaggtggca 1200

cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 1260

tgtatccgct catgagacaa taaccctgat aaatgcttca ataatagcac gtgctaaaac 1320

ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 1380

tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 1440

cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 1500

taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 1560

gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 1620

acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 1680

ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 1740

ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 1800

cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 1860

aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 1920

gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 1980

gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 2040

gcaacgcggc ctttttacgg ttcctgggct tttgctggcc ttttgctcac atgttctt 2098

<210> 7

<211> 2578

<212> DNA

<213> Artificial Sequence

<220>

<221> variation

<223> nucleotide sequence of pMVA-3

<400> 7

gactcttcgc gatgtacggg ccagatatac gcctgaacta tcctgcccgc catcatccta 60

gtcctcatcg ccctcccatc cctacgcatc ctttacataa cagacgaggt caacgatccc 120

tcccttacca tcaaatcaat tggccaccaa tggtactgaa cctacgagta caccgactac 180

ggcggactaa tcttcaactc ctacatactt cccccattat tcctagaacc aggcgacctg 240

cgactccttg acgttgacaa tcgagtagta ctcccgattg aagcccccat tcgtataata 300

attacatcac aagacgtctt gcactcatga gctgtcccca cattaggctt aaaaacagat 360

gcaattcccg gacgtctaaa ccaaaccact ttcaccgcta cacgaccggg ggtatactac 420

ggtcaatgct ctgaaatctg tggagcaaac cacagtttca tgcccatcgt cctagaatta 480

attcccctaa aaatctttga aatagggccc gtatttaccc tatagcaccc cctctacccc 540

ctctagagcc cactgtaaag ctaacttagc attaaccttt taagttaaag attaagagaa 600

ccaacacctc tttacagtga aatgccccaa ctcttctact gggcggtttt atggacagca 660

agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta 720

aactggatgg ctttctcgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa 780

gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 840

gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 900

gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 960

ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg 1020

acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 1080

ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 1140

gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 1200

ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 1260

gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 1320

aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 1380

ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1440

ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1500

ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1560

cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc 1620

ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acaggtggca 1680

cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 1740

tgtatccgct catgagacaa taaccctgat aaatgcttca ataatagcac gtgctaaaac 1800

ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 1860

tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 1920

cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 1980

taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 2040

gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 2100

acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 2160

ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 2220

ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 2280

cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 2340

aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 2400

gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 2460

gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 2520

gcaacgcggc ctttttacgg ttcctgggct tttgctggcc ttttgctcac atgttctt 2578

<210> 8

<211> 3978

<212> DNA

<213> Artificial Sequence

<220>

<221> variation

<223> nucleotide sequence of pMVA-4

<400> 8

gactcttcgc gatgtacggg ccagatatac gctacgttgt agctcacttc cactatgtcc 60

tatcaatagg agctgtattt gccatcatag gaggcttcat tcactgattt cccctattct 120

caggctacac cctagaccaa acctacgcca aaatccattt cactatcata ttcatcggcg 180

taaatctaac tttcttccca caacactttc tcggcctatc cggaatgccc cgacgttact 240

cggactaccc cgatgcatac accacatgaa acatcctatc atctgtaggc tcattcattt 300

ctctaacagc agtaatatta ataattttca tgatttgaga agccttcgct tcgaagcgaa 360

aagtcctaat agtagaagaa ccctccataa acctggagtg actatatgga tgccccccac 420

cctaccacac attcgaagaa cccgtataca taaaatctag acaaaaaagg aaggaatcga 480

accccccaaa gctggtttca agccaacccc atggcctcca tgactttttc aaaaaggtat 540

tagaaaaacc atttcataac tttgtcaaag ttaaattata ggctaaatcc tatatatctt 600

aatggcacat gcagcgcaag taggtctaca agacgctact tcccctatca tagaagagct 660

tatcaccttt catgatcacg ccctcataat cattttcctt atctgcttcc tagtcctgta 720

tgcccttttc ctaacactca caacaaaact aactaatact aacatctcag acgctcagga 780

aatagaaacc gtctgaacta tcctgcccgc catcatccta gtcctcatcg ccctcccatc 840

cctacgcatc ctttacataa cagacgaggt caacgatccc tcccttacca tcaaatcaat 900

tggccaccaa tggtactgaa cctacgagta caccgactac ggcggactaa tcttcaactc 960

ctacatactt cccccattat tcctagaacc aggcgacctg cgactccttg acgttgacaa 1020

tcgagtagta ctcccgattg aagcccccat tcgtataata attacatcac aagacgtctt 1080

gcactcatga gctgtcccca cattaggctt aaaaacagat gcaattcccg gacgtctaaa 1140

ccaaaccact ttcaccgcta cacgaccggg ggtatactac ggtcaatgct ctgaaatctg 1200

tggagcaaac cacagtttca tgcccatcgt cctagaatta attcccctaa aaatctttga 1260

aatagggccc gtatttaccc tatagcaccc cctctacccc ctctagagcc cactgtaaag 1320

ctaacttagc attaaccttt taagttaaag attaagagaa ccaacacctc tttacagtga 1380

aatgccccaa ctaaatacta ccgtatggcc caccataatt acccccatac tccttacact 1440

attcctcatc acccaactaa aaatattaaa cacaaactac cacctacctc cctcaccaaa 1500

gcccataaaa ataaaaaatt ataacaaacc ctgagaacca aaatgaacga aaatctgttc 1560

gcttcattca ttgcccccac aatcctaggc ctacccgccg cagtactgat cattctattt 1620

ccccctctat tgatccccac ctccaaatat ctcatcaaca accgactaat caccacccaa 1680

caatgactaa tcaaactaac ctcaaaacaa atgataacca tacacaacac taaaggacga 1740

acctgatctc ttatactagt atccttaatc atttttattg ccacaactaa cctcctcgga 1800

ctcctgcctc actcatttac accaaccacc caactatcta taaacctagc catggccatc 1860

cccttatgag cgggcgcagt gattataggc tttcgctcta agattaaaaa tgccctagcc 1920

cacttcttac cacaaggcac acctacaccc cttatcccca tactagttat tatcgaaacc 1980

atcagcctac tcattcaacc aatagccctg gccgtacgcc taaccgctaa cacttctact 2040

gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg 2100

ttgggaagcc ctgcaaagta aactggatgg ctttctcgcc gccaaggatc tgatggcgca 2160

ggggatcaag ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg 2220

gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac 2280

aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg 2340

ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc 2400

ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg 2460

aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc 2520

accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc 2580

ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta 2640

ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg 2700

cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg 2760

tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat 2820

tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc 2880

gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta 2940

tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa 3000

ttattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt 3060

cacaccgcat acaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 3120

tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 3180

ataatagcac gtgctaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 3240

tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 3300

cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 3360

gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 3420

tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 3480

gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 3540

gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 3600

ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 3660

acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 3720

agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 3780

cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 3840

tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 3900

gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctgggct tttgctggcc 3960

ttttgctcac atgttctt 3978

<210> 9

<211> 2097

<212> DNA

<213> Artificial Sequence

<220>

<221> variation

<223> nucleotide sequence of pMVA-5

<400> 9

gctgcttcgc gatgtacggg ccagatatac gccccattat tcctagaacc aggcgacctg 60

cgactccttg acgttgacaa tcgagtagta ctcccgattg aagcccccat tcgtataata 120

attacatcac aagacgtctt gcactcatga gccttctact gggcggtttt atggacagca 180

agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta 240

aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa 300

gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 360

gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 420

gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 480

ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg 540

acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 600

ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 660

gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 720

ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 780

gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 840

aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 900

ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 960

ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1020

ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1080

cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc 1140

ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat caggtggcac 1200

ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 1260

gtatccgctc atgagacaat aaccctgata aatgcttcaa taatagcacg tgctaaaact 1320

tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 1380

cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 1440

ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 1500

accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 1560

cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 1620

cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 1680

tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 1740

taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 1800

gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 1860

agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 1920

ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 1980

acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 2040

caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctt 2097

<210> 10

<211> 2577

<212> DNA

<213> Artificial Sequence

<220>

<221> variation

<223> nucleotide sequence of pMVA-6

<400> 10

gctgcttcgc gatgtacggg ccagatatac gcctgaacta tcctgcccgc catcatccta 60

gtcctcatcg ccctcccatc cctacgcatc ctttacataa cagacgaggt caacgatccc 120

tcccttacca tcaaatcaat tggccaccaa tggtactgaa cctacgagta caccgactac 180

ggcggactaa tcttcaactc ctacatactt cccccattat tcctagaacc aggcgacctg 240

cgactccttg acgttgacaa tcgagtagta ctcccgattg aagcccccat tcgtataata 300

attacatcac aagacgtctt gcactcatga gctgtcccca cattaggctt aaaaacagat 360

gcaattcccg gacgtctaaa ccaaaccact ttcaccgcta cacgaccggg ggtatactac 420

ggtcaatgct ctgaaatctg tggagcaaac cacagtttca tgcccatcgt cctagaatta 480

attcccctaa aaatctttga aatagggccc gtatttaccc tatagcaccc cctctacccc 540

ctctagagcc cactgtaaag ctaacttagc attaaccttt taagttaaag attaagagaa 600

ccaacacctc tttacagtga aatgccccaa ctcttctact gggcggtttt atggacagca 660

agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta 720

aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa 780

gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 840

gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 900

gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 960

ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg 1020

acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 1080

ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 1140

gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 1200

ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 1260

gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 1320

aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 1380

ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1440

ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1500

ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1560

cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc 1620

ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat caggtggcac 1680

ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 1740

gtatccgctc atgagacaat aaccctgata aatgcttcaa taatagcacg tgctaaaact 1800

tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 1860

cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 1920

ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 1980

accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 2040

cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 2100

cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 2160

tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 2220

taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 2280

gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 2340

agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 2400

ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 2460

acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 2520

caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctt 2577

<210> 11

<211> 3977

<212> DNA

<213> Artificial Sequence

<220>

<221> variation

<223> nucleotide sequence of pMVA-7

<400> 11

gctgcttcgc gatgtacggg ccagatatac gctacgttgt agctcacttc cactatgtcc 60

tatcaatagg agctgtattt gccatcatag gaggcttcat tcactgattt cccctattct 120

caggctacac cctagaccaa acctacgcca aaatccattt cactatcata ttcatcggcg 180

taaatctaac tttcttccca caacactttc tcggcctatc cggaatgccc cgacgttact 240

cggactaccc cgatgcatac accacatgaa acatcctatc atctgtaggc tcattcattt 300

ctctaacagc agtaatatta ataattttca tgatttgaga agccttcgct tcgaagcgaa 360

aagtcctaat agtagaagaa ccctccataa acctggagtg actatatgga tgccccccac 420

cctaccacac attcgaagaa cccgtataca taaaatctag acaaaaaagg aaggaatcga 480

accccccaaa gctggtttca agccaacccc atggcctcca tgactttttc aaaaaggtat 540

tagaaaaacc atttcataac tttgtcaaag ttaaattata ggctaaatcc tatatatctt 600

aatggcacat gcagcgcaag taggtctaca agacgctact tcccctatca tagaagagct 660

tatcaccttt catgatcacg ccctcataat cattttcctt atctgcttcc tagtcctgta 720

tgcccttttc ctaacactca caacaaaact aactaatact aacatctcag acgctcagga 780

aatagaaacc gtctgaacta tcctgcccgc catcatccta gtcctcatcg ccctcccatc 840

cctacgcatc ctttacataa cagacgaggt caacgatccc tcccttacca tcaaatcaat 900

tggccaccaa tggtactgaa cctacgagta caccgactac ggcggactaa tcttcaactc 960

ctacatactt cccccattat tcctagaacc aggcgacctg cgactccttg acgttgacaa 1020

tcgagtagta ctcccgattg aagcccccat tcgtataata attacatcac aagacgtctt 1080

gcactcatga gctgtcccca cattaggctt aaaaacagat gcaattcccg gacgtctaaa 1140

ccaaaccact ttcaccgcta cacgaccggg ggtatactac ggtcaatgct ctgaaatctg 1200

tggagcaaac cacagtttca tgcccatcgt cctagaatta attcccctaa aaatctttga 1260

aatagggccc gtatttaccc tatagcaccc cctctacccc ctctagagcc cactgtaaag 1320

ctaacttagc attaaccttt taagttaaag attaagagaa ccaacacctc tttacagtga 1380

aatgccccaa ctaaatacta ccgtatggcc caccataatt acccccatac tccttacact 1440

attcctcatc acccaactaa aaatattaaa cacaaactac cacctacctc cctcaccaaa 1500

gcccataaaa ataaaaaatt ataacaaacc ctgagaacca aaatgaacga aaatctgttc 1560

gcttcattca ttgcccccac aatcctaggc ctacccgccg cagtactgat cattctattt 1620

ccccctctat tgatccccac ctccaaatat ctcatcaaca accgactaat caccacccaa 1680

caatgactaa tcaaactaac ctcaaaacaa atgataacca tacacaacac taaaggacga 1740

acctgatctc ttatactagt atccttaatc atttttattg ccacaactaa cctcctcgga 1800

ctcctgcctc actcatttac accaaccacc caactatcta taaacctagc catggccatc 1860

cccttatgag cgggcgcagt gattataggc tttcgctcta agattaaaaa tgccctagcc 1920

cacttcttac cacaaggcac acctacaccc cttatcccca tactagttat tatcgaaacc 1980

atcagcctac tcattcaacc aatagccctg gccgtacgcc taaccgctaa cacttctact 2040

gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg 2100

ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca 2160

ggggatcaag ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg 2220

gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac 2280

aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg 2340

ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc 2400

ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg 2460

aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc 2520

accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc 2580

ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta 2640

ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg 2700

cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg 2760

tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat 2820

tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc 2880

gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta 2940

tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa 3000

ttattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt 3060

cacaccgcat caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 3120

ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 3180

taatagcacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 3240

gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 3300

gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 3360

caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 3420

ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 3480

tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 3540

ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 3600

tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 3660

cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 3720

gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 3780

ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 3840

gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 3900

agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 3960

tttgctcaca tgttctt 3977

Claims

1. A method for preparing a cationic lipid nanoparticle/DNA complex, the method comprising the steps of:

(1) dissolving cationic lipid material in anhydrous ethanol, and heating to dissolve;

(4) filtering;

(5) preparing a DNA solution;

(7) filtering;

wherein the cationic lipid material in the step (1) is (2, 3-dioleoxypropyl) trimethyl ammonium chloride (DOTAP), and the concentration of the cationic lipid material in ethanol is 30-100 mg/ml; the volume ratio of the ethanol solution to the water phase solution in the step (2) is 1: 3-1: 6, and the final concentration of the cationic lipid material in the mixed solution of ethanol and water phase is 6-25 mg/ml; the mass ratio of the cationic lipid nanoparticles to the DNA in the step (6) is 6: 1-125: 1;

preparing the cationic lipid nanoparticle and the DNA solution in the step (6) into the cationic lipid nanoparticle/DNA complex in a semi-automatic device containing a T-shaped connector; the semi-automatic device of the T-shaped connector is formed by sequentially connecting a constant flow pump, a sterile filter, a liquid storage bottle, a constant flow pump and the T-shaped connector in an arrow direction, firstly assembling the semi-automatic device containing the T-shaped connector, then respectively placing the cationic lipid nanoparticles prepared in the step (4) and the DNA solution prepared in the step (5) into containers of the device according to the mass ratio of 6: 1-125: 1, mixing in the T-shaped connector at the speed of 20-100 ml/min, and then adding the mixture into a new container;

the particle size of the formed cationic lipid nanoparticle/DNA compound is 50-150 nm, and PDI is less than 0.3;

the filtration in step (4) and step (6) was filtration using a 0.22 μm filter.

2. The method according to claim 1, wherein the cationic lipid material is a mixture of the cationic lipid and a helper lipid, wherein the mass ratio of the cationic lipid to the helper lipid is > 1:1, and the helper lipid is at least one selected from cholesterol (Chol) and Dioleoylphosphatidylethanolamine (DOPE).

3. The method according to claim 1, wherein the concentration of the cationic lipid material in ethanol in step (1) is 50 to 60 mg/ml.

4. The method according to claim 1, wherein the cationic lipid nanoparticle in step (6) is obtained by diluting the cationic lipid nanoparticle prepared in step (4) with water, and the concentration of the diluted cationic lipid nanoparticle is 1 to 4 mg/ml.

5. The method according to claim 1, wherein the heating in step (1) is water bath heating at a temperature of 40 to 60 ℃.

6. The preparation method according to claim 5, wherein the temperature of the water bath heating is 50-60 ℃.

7. The process according to claim 1, wherein the aqueous solution in the step (2) is an aqueous solution or an aqueous solution containing a saccharide selected from lactose, maltose, sucrose, glucose and trehalose, and the saccharide is present at a concentration of 2 to 20% by mass.

8. The production method according to claim 7, wherein the mass concentration of the saccharide is 4 to 10%.

9. The preparation method according to claim 1, wherein the dropping speed in the step (2) is 30 to 120 ml/min; the method for removing the ethanol in the step (3) is one of spin steaming, dialysis, ultrafiltration, spray drying or freeze drying.

10. The method according to claim 1, wherein the DNA in the step (5) is a plasmid selected from at least one of pVAX1 and pUC 18; or the plasmid is pMVA plasmid, the nucleotide sequence of the pMVA plasmid is shown as SEQ ID NO.1, or the nucleotide sequence of the pMVA plasmid has more than 90 percent of homology with the sequence shown as SEQ ID NO. 1; or the plasmid is a pMVA-1 plasmid, the nucleotide sequence of the pMVA-1 plasmid is shown in SEQ ID NO.2, or the nucleotide sequence of the pMVA-1 plasmid has more than 90 percent of homology with the sequence shown in SEQ ID NO. 2; or the plasmid is selected from at least one of the plasmids with the nucleotide sequence shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.11, or the nucleotide sequence with more than 90 percent of homology with the sequence shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO. 11;

or the DNA in the step (5) is mitochondrial DNA or mitochondrial DNA fragments, and the mitochondrial DNA fragments are at least one of DNA fragments with nucleotide sequences shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5, or DNA fragments with nucleotide sequences more than 90% homologous with the sequences shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5.

11. The method according to claim 1, wherein the DNA solution in the step (5) is an aqueous DNA solution, and the length of the DNA is 100 to 2500 bp.

12. A cationic lipid nanoparticle/DNA complex, wherein the complex is prepared by the method of any one of claims 1 to 11.