CN116036043A - Nucleic acid pharmaceutical preparation, freeze-dried powder and preparation method - Google Patents

Abstract

The application discloses a nucleic acid pharmaceutical preparation, freeze-dried powder and a preparation method thereof. The nucleic acid drug formulation of the present application self-assembles and encapsulates a nucleic acid in a trehalose solution from a cationic nucleic acid delivery polymer to form nucleic acid drug nanoparticles, thereby forming a nucleic acid drug formulation comprising the nucleic acid drug nanoparticles and the trehalose solution. Further, the nucleic acid drug preparation is subjected to freeze-drying treatment to obtain nucleic acid drug freeze-dried powder. The nucleic acid drug preparation and the nucleic acid drug freeze-dried powder prepared by the same have good sealing rate, uniform particle size distribution and good quality reproducibility after re-dissolution, can be stably stored at 4 ℃ for a long time, can be transported and stored for a long time by conventional cold chain transportation, reduce the difficulty and cost of nucleic acid drug storage and transportation, and lay a foundation for large-scale popularization and application of nucleic acid drugs.

Description

A nucleic acid drug preparation, lyophilized powder and preparation method

Technical Field

The present application relates to the technical field of nucleic acid drugs, and in particular to a nucleic acid drug preparation, a lyophilized powder and a preparation method thereof.

Background Art

Nucleic acid drugs, also known as nucleotide drugs, are various oligoribonucleotides (RNA) or oligodeoxyribonucleotides (DNA) with different functions. They mainly work at the genetic level, have specific targets and mechanisms of action, and are specific to pathogenic genes. Nucleic acids cannot directly enter the human body and reach the site of action, so a special delivery system is required. And because nucleic acid is an unstable substance, it faces huge challenges in formulation. At the same time, currently available nucleic acid drugs, such as a variety of mRNA vaccines, need to be stored and transported at strict low temperatures, which undoubtedly increases the difficulty and cost of storage and transportation of nucleic acid drugs, and significantly limits the large-scale promotion and popularization of nucleic acid drugs.

One of the most common methods to improve the stability of a preparation is to make the preparation into a freeze-dried dosage form, i.e., freeze-dried powder. However, there are many technical difficulties in making nucleic acid drugs into freeze-dried powder, such as difficulty in controlling particle size and poor quality reproducibility after reconstitution; and for nucleic acid drugs with different delivery systems, the quality and reconstitution effect of their freeze-dried powder are also different.

Therefore, how to prepare lyophilized powder of nucleic acid drugs with uniform particle size distribution and good quality reproducibility after reconstitution is still an important problem to be solved in the field of nucleic acid drug technology.

Summary of the invention

The purpose of this application is to provide an improved nucleic acid drug preparation, lyophilized powder and preparation method.

In order to achieve the above objectives, this application adopts the following technical solutions:

The first aspect of the present application discloses a nucleic acid drug preparation, which is formed by self-assembly of a cationic nucleic acid delivery polymer in a trehalose solution and encapsulation of nucleic acid to form nucleic acid drug nanoparticles, thereby forming a nucleic acid drug preparation comprising nucleic acid drug nanoparticles and a trehalose solution.

It should be noted that the nucleic acid drug preparation of the present application creatively performs self-assembly and nucleic acid encapsulation of cationic nucleic acid transfer polymers in trehalose solution, which can not only obtain nucleic acid drug nanoparticles with good encapsulation rate and uniform particle size distribution; moreover, the nucleic acid drug freeze-dried powder obtained by direct freeze-drying of the preparation also has a uniformly distributed particle size, and the quality reproducibility is good after reconstitution. More importantly, the nucleic acid drug freeze-dried powder obtained by freeze-drying the nucleic acid drug preparation of the present application can be stored stably for a long time at 4°C, -20°C, and -80°C; in one implementation of the present application, the nucleic acid drug freeze-dried powder is placed at 4°C for 5 months, and the particle size, Zeta potential, particle size distribution index (PDI) and encapsulation rate after reconstitution have good stability. The freeze-dried nucleic acid drug powder obtained by freeze-drying the nucleic acid drug preparation of the present application can meet the long-term stable storage requirements under conventional cold chain transportation, reduce the difficulty and cost of nucleic acid drug storage and transportation, and lay the foundation for the large-scale promotion and application of nucleic acid drugs.

In one implementation of the present application, the trehalose solution is an aqueous solution of trehalose, and the concentration thereof is 1%-10%.

Preferably, the concentration of the trehalose aqueous solution is 10%.

It should be noted that one of the key points of the present application is to use trehalose solution for self-assembly of cationic nucleic acid delivery polymers and nucleic acid encapsulation; it is understandable that as long as trehalose is contained therein, the technical effect of the present application can be achieved to varying degrees. However, in order to ensure the quality of the nucleic acid drug lyophilized powder, the present application preferably uses a trehalose solution with a concentration of 1%-10%.

In one implementation of the present application, the cationic nucleic acid delivery polymer is polyethyleneimine.

Preferably, the cationic nucleic acid delivery polymer is linear polyethyleneimine.

Preferably, the molecular weight of the linear polyethyleneimine is 10,000-70,000 Daltons, preferably 48,050 Daltons.

In one implementation of the present application, the nucleic acid is RNA.

Preferably, the RNA has 30-544 bases.

It should be noted that RNA is only a specific type of nucleic acid used in one implementation of the present application. Relatively speaking, DNA with better stability is also applicable to the present application. Moreover, for example, polyethyleneimine is specifically used as a nucleic acid delivery polymer in one implementation of the present application, and polyethyleneimine itself can also perform nucleic acid delivery on DNA. Therefore, the nucleic acid of the present application is not limited to various RNAs, but can also be applicable to various types of DNA.

In one implementation of the present application, the particle size of the nucleic acid drug nanoparticles is 95-195 nm.

Preferably, the Zeta potential of the nucleic acid pharmaceutical preparation is 34-46 mV.

Preferably, the mass ratio of the cationic nucleic acid delivery polymer to the nucleic acid is 1-2.5:1.5-3, preferably 1.5:2.

The second aspect of the present application discloses a nucleic acid drug lyophilized powder, which is obtained by lyophilizing the nucleic acid drug preparation of the present application.

It should be noted that the lyophilized powder of the nucleic acid drug of the present application has a good encapsulation rate, uniform particle size distribution, good quality reproducibility after reconstitution, and can be stored stably for a long time at 4°C, -20°C, and -80°C, respectively. In one implementation of the present application, the lyophilized powder of the nucleic acid drug is placed at 4°C for 5 months, and the particle size, Zeta potential, particle size distribution index (PDI) and encapsulation rate after reconstitution have good stability. The lyophilized powder of the nucleic acid drug of the present application can meet the long-term stable storage requirements under conventional cold chain transportation, reducing the difficulty and cost of storage and transportation of nucleic acid drugs, and laying the foundation for the large-scale promotion and application of nucleic acid drugs.

The third aspect of the present application discloses a method for preparing the lyophilized powder of the nucleic acid drug of the present application, comprising dissolving a cationic nucleic acid transfer polymer in a trehalose solution to prepare a solution A; dissolving a nucleic acid in a trehalose solution to prepare a solution B; mixing solution A and solution B evenly to obtain a nucleic acid drug preparation; and lyophilizing the nucleic acid drug preparation to obtain the lyophilized powder of the nucleic acid drug of the present application.

It should be noted that, in the present application, the cationic nucleic acid transfer polymer and the nucleic acid are dissolved in the trehalose solution respectively, and after the two are evenly dispersed in the trehalose solution, they are mixed in the liquid phase, which can ensure the uniformity of the contact between the cationic nucleic acid transfer polymer and the nucleic acid; after the two are evenly mixed, the positively charged groups of the cationic nucleic acid transfer polymer are utilized to self-assemble and encapsulate the nucleic acid through electrostatic interaction with the negatively charged nucleic acid.

In one implementation of the present application, the pH of solution A is 3.5-4, preferably 3.6.

In one implementation of the present application, solution A and solution B are mixed evenly, specifically comprising: dispensing equal amounts of solution A into two syringes, dispensing equal amounts of solution B into another two syringes, connecting the two syringes for dispensing solution A to pipelines 1 and 3 of the mixer, respectively, connecting the two syringes for dispensing solution B to pipelines 2 and 4 of the mixer, respectively, setting the total flow rate, and starting the injection pump of the mixer to mix solution A and solution B, thereby obtaining a nucleic acid drug preparation.

In one implementation of the present application, the total flow rate is 40-80 mL/min, preferably 80 mL/min.

In one implementation of the present application, the pressure of the freeze-drying process is 0.001-0.200 mbar, preferably 0.1 mbar.

Due to the adoption of the above technical solution, the beneficial effects of this application are:

The nucleic acid drug preparation of the present application and the nucleic acid drug lyophilized powder prepared therefrom have good sealing rate and uniform particle size distribution. The nucleic acid drug lyophilized powder has good quality reproducibility after reconstitution and can be stably stored for a long time at 4°C. It can be transported and stored over long distances for a long time through conventional cold chain transportation, which reduces the difficulty and cost of nucleic acid drug storage and transportation, and lays the foundation for the large-scale promotion and application of nucleic acid drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a transmission electron microscope observation image of the nucleic acid drug nanoparticles prepared in the example of the present application;

FIG2 is a particle size test result of the freeze-dried powder in the embodiment of the present application after being reconstituted at 4° C. for 5 months;

FIG3 is a potential test result of the freeze-dried powder in the embodiment of the present application after being stored at 4° C. for 5 months and then reconstituted;

FIG4 is a PDI test result of the lyophilized powder in the embodiment of the present application after being reconstituted within 5 months after being stored at 4° C.;

FIG5 is a test result of the encapsulation efficiency of the lyophilized powder in the embodiment of the present application after being reconstituted at 4° C. for 5 months;

FIG6 is a particle size test result of the freeze-dried powder in the embodiment of the present application after being reconstituted within 5 months of storage at -20°C;

FIG7 is a potential test result of the freeze-dried powder in the embodiment of the present application after being stored at -20°C for 5 months and then reconstituted;

FIG8 is a PDI test result of the freeze-dried powder in the embodiment of the present application after being stored at -20°C for 5 months and then reconstituted;

FIG9 is a test result of the encapsulation efficiency of the lyophilized powder in the embodiment of the present application after being reconstituted within 5 months after being stored at -20°C;

FIG10 is a particle size test result of the freeze-dried powder in the embodiment of the present application after being reconstituted within 5 months after being stored at -80°C;

FIG11 is a potential test result of the freeze-dried powder in the present application example after being stored at -80°C for 5 months and then reconstituted;

FIG12 is a PDI test result of the freeze-dried powder in the present application after being stored at -80°C for 5 months and then reconstituted;

FIG. 13 is a test result of the encapsulation efficiency of the lyophilized powder in the embodiment of the present application after being reconstituted within 5 months after being stored at -80°C.

DETAILED DESCRIPTION

This study found that the particle size of nucleic acid drug nanoparticles is affected by many factors. In order to prepare qualified nanoparticle preparations, it is necessary to optimize the formulation of raw materials, excipients, etc., and to perform quality control on indicators such as the particle size, potential, pH value, and encapsulation rate of the preparations, and ultimately determine the optimal formulation scheme and process.

Based on the above research and understanding, this application creatively proposes that the self-assembly of cationic nucleic acid delivery polymers and nucleic acid encapsulation in trehalose solution can not only obtain nucleic acid drug nanoparticles with good encapsulation rate and uniform particle size distribution; but also the nucleic acid drug lyophilized powder obtained thereby has the advantages of uniform particle size distribution and good quality reproducibility after reconstitution. More importantly, the nucleic acid drug lyophilized powder prepared thereby can be stored stably for at least 5 months at 4°C, reducing the difficulty and cost of nucleic acid drug storage and transportation.

It should be noted that trehalose is a commonly used lyoprotectant; however, the present application found that cationic nucleic acid delivery polymers can self-assemble and encapsulate nucleic acids in trehalose solutions to obtain nucleic acid drug nanoparticles with good encapsulation efficiency and uniform particle size distribution; and the nucleic acid drug lyophilized powder obtained by further freeze-drying has good quality reproducibility after reconstitution. For example, the nucleic acid drug lyophilized powder is placed at 4°C for 5 months, and the particle size, Zeta potential, particle size distribution index (PDI) and encapsulation efficiency after reconstitution are all stable.

In one implementation of the present application, a cationic polymer polyethyleneimine (PEI) is specifically used as a delivery system and excipient to carry RNA to the target site, which is combined with the nucleic acid RNA in a specific way through the action of charge, and the RNA is encapsulated, and a nano-scale particle solution is prepared through an efficient mixing process, that is, the nucleic acid drug preparation of the present application. Further, the solution is made into a lyophilized preparation, that is, the nucleic acid drug lyophilized powder of the present application, which is more convenient to store and transport, while increasing the stability of the preparation and reducing costs.

The application is further described in detail below by specific examples. The following examples only further illustrate the application and should not be construed as limiting the application. In the following embodiments, a lot of detailed descriptions are to enable the application to be better understood. However, those skilled in the art can recognize without difficulty that some features can be omitted in different situations, or can be replaced by other test kits, materials, methods. In some cases, some operations related to the application are not shown or described in the specification, and this is to avoid the core part of the application from being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and the related operations can be fully understood according to the description in the specification and the general technical knowledge in the art. In the following examples, reagents used or instruments are not indicated as manufacturers, and are conventional products that can be purchased and obtained on the market.

Example 1

The nucleic acid drug preparation of this example is formed by self-assembly of a cationic nucleic acid delivery polymer in a trehalose solution and encapsulation of nucleic acid to form nucleic acid drug nanoparticles, thereby forming a nucleic acid drug preparation comprising nucleic acid drug nanoparticles and a trehalose solution. This example respectively studies the effects of different RNA dosages on nucleic acid drug preparations. The RNA sequence is mGmUmGCCCAUUCGGGGGGGGCCGAAUCAGmCmA5meC, which was custom synthesized by LGC.

Specifically, as shown in Table 1, RNA of different masses was dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare RNA working solutions; in this example, 2.50 mg, 2.00 mg, 1.50 mg, and 1.00 mg of RNA were weighed and dissolved in 5 mL of 10% trehalose solution, i.e., solution B. 1.935 mg of PEI was dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare 0.387 mg/mL PEI working solution, and pH was adjusted to 3.80 with 0.5 M sodium hydroxide at room temperature, i.e., solution A. PEI is linear polyethyleneimine with a molecular weight of 48050 Daltons, purchased from Polyplus.

After the two working solutions are prepared, each working solution is divided equally into two syringes, that is, solution A is divided equally into two syringes, solution B is divided equally into two other syringes, and two injections containing RNA working solution are connected to the 1 and 3 pipelines of the mixer, and two injections containing PEI working solution are connected to the 2 and 4 pipelines of the mixer. After the connection is completed, the syringe is fixed on the syringe pump, and the total flow rate is 80mL/min. The syringe pump is started, and the RNA working solution and the PEI working solution are mixed, that is, the nucleic acid drug preparation of this example is prepared, that is, the preparation stock solution for preparing nucleic acid drug lyophilized powder. The particle size and PDI of the preparation stock solution are detected by Malvern particle size analyzer, and the results are shown in Table 2.

Table 1 Recipe information under different RNA quality conditions

Prescription/Group #1 #2 #3 #4 RNA 2.50mg 2.00mg 1.50mg 1.00mg PEI 1.935mg 1.935mg 1.935mg 1.935mg Trehalose 10% 10% 10% 10% Enzyme-free sterile water 10mL 10mL 10mL 10mL PEI working solution pH pH＝3.8 pH＝3.8 pH＝3.8 pH＝3.8

Table 2 Test results of nucleic acid drug preparations prepared with different quality RNA

The results in Table 2 show that the particle size of nucleic acid drug nanoparticles increases with the increase of RNA concentration; comparative analysis shows that when the RNA dosage is 1.50 mg, that is, when the RNA concentration in the reaction solution is 0.15 mg/mL, the particle size of nucleic acid drug nanoparticles is relatively small, and the PDI value is the smallest. Therefore, the preferred RNA concentration is determined to be 0.15 mg/mL.

Example 2

Based on Example 1, this example investigates the effect of PEI dosage on the preparation. All materials and methods in this example are the same as those in Example 1, with the only difference being the PEI dosage. Specifically, 1.5 mg RNA was dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare an RNA working solution. As shown in Table 3, different masses of PEI, specifically 1.935 mg, 2.15 mg, 2.365 mg, and 2.580 mg, were dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare a PEI working solution, and pH = 3.80 was adjusted with 0.5 M sodium hydroxide at room temperature.

After the two working solutions are prepared, each working solution is divided equally into two syringes, and the amount of liquid in the four syringes is kept the same. The two injections containing RNA working solution are connected to the 1 and 3 pipes of the mixer, and the two injections containing PEI working solution are connected to the 2 and 4 pipes of the mixer. After the connection is completed, the syringe is fixed on the syringe pump, the total flow rate is 80mL/min, and the syringe pump is started to prepare the preparation stock solution. The particle size and PDI of the preparation stock solution are tested using a Malvern particle size analyzer, and the results are shown in Table 4.

Table 3 Formulation information under different quality PEI conditions

Prescription/Group #1 #2 #3 #4 RNA 1.5mg 1.5mg 1.5mg 1.5mg PEI 1.935mg 2.15mg 2.365mg 2.580mg Trehalose 10% 10% 10% 10% Enzyme-free sterile water 10mL 10mL 10mL 10mL PEI working solution pH pH＝3.80 pH＝3.80 pH＝3.80 pH＝3.80

Table 4 Test results of nucleic acid drug preparations prepared with PEI of different qualities

Indicator/Group #1 #2 #3 #4 Particle size 132.80nm 124.90nm 126.67nm 123.17nm PDI 0.255 0.272 0.283 0.272

The results in Table 4 show that when the PEI dosage is 1.935 mg and 2.15 mg, that is, when the PEI concentration in the reaction solution is 0.1935 mg/mL and 0.2150 mg/mL, the particle size and PDI of the nucleic acid drug nanoparticles are almost the same, and the PDI is smaller than the two gradients of 0.2365 mg/mL and 0.2580 mg/mL. Therefore, in this experiment, the preferred PEI concentration is determined to be 0.2150 mg/mL.

Example 3

This example uses the RNA concentration determined in Example 1 and the PEI concentration determined in Example 2 to investigate the effect of the pH of the PEI working solution on the preparation. All materials and methods in this example are the same as those in Example 1. Specifically, 1.5 mg RNA was dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare an RNA working solution. 2.15 mg PEI was dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare a PEI working solution, and the pH was adjusted to different values shown in Table 5 with 0.5 M sodium hydroxide at room temperature, that is, the pH values were 3.5, 3.6, 3.8, and 4.0, respectively.

After the two working solutions are prepared, each working solution is divided equally into two syringes, keeping the amount of liquid in the four syringes the same, connecting the two injections containing RNA working solution to the 1 and 3 pipes of the mixer, and connecting the two injections containing PEI working solution to the 2 and 4 pipes of the mixer. After the connection is completed, the syringe is fixed on the syringe pump, the total flow rate is 80mL/min, the syringe pump is started, and the preparation stock solution is prepared. The particle size and PDI of the preparation stock solution are detected by Malvern particle size analyzer, and the pH value of the preparation stock solution is detected. The results are shown in Table 6.

Table 5 Formula information of PEI working solution with different pH values

Prescription/Group #1 #2 #3 #4 RNA 1.5mg 1.5mg 1.5mg 1.5mg PEI 2.15mg 2.15mg 2.15mg 2.15mg Trehalose 10% 10% 10% 10% Enzyme-free sterile water 10mL 10mL 10mL 10mL PEI working solution pH pH＝3.50 pH＝3.60 pH＝3.80 pH = 4.0

Table 6 Test results of nucleic acid drug preparations prepared by PEI at different pH values

Indicator/Group #1 #2 #3 #4 Particle size 133.20nm 132.77nm 124.9nm 133.00nm PDI 0.279 0.267 0.272 0.271 pH 3.78 3.88 4.09 4.30

The results in Table 6 show that the pH value of the PEI working solution is between 3.5 and 4.0, and nucleic acid drug nanoparticles with a particle size and PDI equivalent can be prepared, especially when the pH value of the PEI working solution is 3.6, the PDI of the nucleic acid drug nanoparticles is the smallest. Therefore, the preferred pH value of the PEI working solution is 3.6.

Example 4

This example uses the RNA concentration determined in Example 1, the PEI concentration determined in Example 2, and the pH value of the PEI working solution determined in Example 3 to investigate the effect of the total flow rate when the mixer mixes the RNA working solution and the PEI working solution on the preparation. Specifically, 1.5 mg RNA was dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare an RNA working solution. 2.15 mg PEI was dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare a PEI working solution, and the pH was adjusted to 3.6 with 0.5 M sodium hydroxide at room temperature.

After the two working solutions are prepared, each working solution is divided equally into two syringes, keeping the amount of liquid in the four syringes the same, connecting the two injections containing RNA working solution to the 1 and 3 lines of the mixer, and connecting the two injections containing PEI working solution to the 2 and 4 lines of the mixer. After the connection, the syringe is fixed on the syringe pump, and the total flow rate is set to 40, 60, and 80 mL/min according to Table 7, and the syringe pump is started to prepare the preparation stock solution. The particle size and PDI of the preparation stock solution are detected by Malvern particle size analyzer, and the results are shown in Table 8.

Table 7 Recipe information under different total flow rate conditions

Prescription/Group #1 #2 #3 RNA 1.5mg 1.5mg 1.5mg PEI 2.15mg 2.15mg 2.15mg Trehalose 10% 10% 10% Enzyme-free sterile water 10mL 10mL 10mL Total flow rate 40mL/min 60mL/min 80mL/min

Table 8 Test results of nucleic acid drug preparations prepared at different total flow rates

Indicator/Group #1 #2 #3 Particle size 122.1nm 116.1nm 95.8nm PDI 0.317 0.274 0.264

The results in Table 8 show that as the total flow rate increases, the particle size and PDI both decrease, indicating that increasing the total flow rate is beneficial to the preparation of nanomedicines with smaller and more uniform particle sizes, so the preferred total flow rate is 80 mL/min.

Example 5

In this example, 1.5 mg RNA was dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare RNA working solution. 2.15 mg PEI was dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare PEI working solution, and the pH was adjusted to 3.6 with 0.5 M sodium hydroxide at room temperature.

After the two working solutions are prepared, each working solution is divided equally into two syringes, and the liquid volume in the four syringes is kept the same. The two syringes containing RNA working solution are connected to the 1 and 3 pipes of the mixer, and the two syringes containing PEI working solution are connected to the 2 and 4 pipes of the mixer. After the connection is completed, the syringes are fixed on the syringe pump, the total flow rate is 80mL/min, and the syringe pump is started to prepare the preparation.

Take 10uL of the original solution and drop it into the carbon film copper mesh for transmission electron microscopy. After standing for 20 minutes, absorb the surface liquid. After the liquid evaporates overnight at room temperature, observe the morphology of the nanoparticles using a transmission electron microscope. The results are shown in Figure 1.

The results in Figure 1 show that most of the nucleic acid drug nanoparticles prepared in this example are spherical, which is consistent with the theory.

Example 6

This example uses the RNA concentration determined in Example 1, the PEI concentration determined in Example 2, and the pH value of the PEI working solution determined in Example 3 to study the effect of the trehalose ratio on the preparation. Specifically, 1.5 mg RNA was dissolved in 5 mL of enzyme-free sterile water containing different concentrations of trehalose to prepare an RNA working solution. 2.15 mg PEI was dissolved in 5 mL of enzyme-free sterile water containing different concentrations of trehalose to prepare a PEI working solution, and the pH was adjusted to 3.6 with 0.5M sodium hydroxide at room temperature. Among them, the different concentrations of trehalose are 0%, 1%, 2%, 4%, 6%, 8%, and 10% trehalose concentrations, respectively. The RNA working solution and the PEI working solution use the same concentration of trehalose aqueous solution, for example, RNA and PEI use 1% trehalose aqueous solution at the same time, and 2% trehalose aqueous solution at the same time, and so on. The specific formula is shown in Table 9, and the effects of different concentrations of trehalose on the preparation were tested respectively.

After the two working solutions are prepared, each working solution is divided equally into two syringes, keeping the amount of liquid in the four syringes the same, connecting the two injections containing RNA working solution to the 1 and 3 pipes of the mixer, and connecting the two injections containing PEI working solution to the 2 and 4 pipes of the mixer. After the connection, fix the syringe on the syringe pump, the total flow rate is 80mL/min, start the syringe pump, and prepare the preparation stock solution. The preparation stock solution is divided into vials, each with a volume of 2.5mL, placed in a -60℃ environment for about 4h, quickly transferred to the freeze dryer, turn on the vacuum pump, set the pressure to 0.1mbar, and freeze-dry for about 24 hours to obtain the freeze-dried powder of the nucleic acid drug in this example.

Take 2 mg of nucleic acid drug lyophilized powder sample and add 2.6 mL of enzyme-free sterile water for reconstitution.

The particle size, PDI and potential of the original solution and the reconstituted preparation were tested using a Malvern particle size analyzer. The RNA encapsulation efficiency in the original solution and the reconstituted preparation was tested using a Ribogreen kit. The test results are shown in Table 10.

Table 9 Formulation information under different concentrations of trehalose

Prescription/Group #0 #1 #2 #3 #4 #5 #6 RNA 1.5mg 1.5mg 1.5mg 1.5mg 1.5mg 1.5mg 1.5mg PEI 2.15mg 2.15mg 2.15mg 2.15mg 2.15mg 2.15mg 2.15mg Trehalose 0% 1% 2% 4% 6% 8% 10% Enzyme-free sterile water 10mL 10mL 10mL 10mL 10mL 10mL 10mL PEI working solution pH pH＝3.60 pH＝3.60 pH＝3.60 pH＝3.60 pH＝3.60 pH＝3.60 pH＝3.60

Table 10 Test results of nucleic acid drug preparations prepared with different concentrations of trehalose

Indicator/Group #0 #1 #2 #3 #4 #5 #6 Liquid particle size 115.9nm 99.88nm 100.83nm 96.42nm 100.04nm 105.30nm 96.43nm Original liquid PDI 0.248 0.277 0.268 0.277 0.265 0.285 0.274 Potential of stock solution 38.13mV 39.00mV 42.50mV 38.13mV 34.93mV 38.60mV 38.73mV Encapsulation efficiency of stock solution 100% 99.42% 99.34% 99.06% 99.67% 99.76% 99.60% Particle size of lyophilized reconstituted preparation Redissolution and precipitation 126.23nm 137.90nm 144.03nm 106.23nm 143.90nm 142.67nm Lyophilized reconstituted preparation PDI Redissolution and precipitation 0.270 0.234 0.261 0.242 0.241 0.253 Potential of lyophilized reconstituted preparations Redissolution and precipitation 43.23mV 45.33mV 36.43mV 38.03mV 40.63mV 37.30mV Encapsulation efficiency of freeze-dried reconstituted preparations Redissolution and precipitation 99.55% 99.47% 99.65% 99.98% 100.00% 98.83%

The results in Table 10 show that the trehalose concentration of 0%-10% can prepare nucleic acid drug nanoparticles with equivalent particle size, PDI, potential, and encapsulation efficiency. However, when 1%-10% trehalose is added, the particle size, PDI, potential, and encapsulation efficiency of the prepared nucleic acid drug lyophilized powder after reconstitution have good reproducibility.

Example 7

In this example, under the preferred conditions determined in the above examples, the stability of the finally prepared nucleic acid drug lyophilized powder at different temperatures was studied. Specifically, 1.5 mg RNA was dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare RNA working solution. 2.15 mg PEI was dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare PEI working solution, and the pH was adjusted to 3.6 with 0.5 M sodium hydroxide at room temperature.

After the two working solutions are prepared, each working solution is divided equally into two syringes, keeping the amount of liquid in the four syringes the same, connecting the two injections containing RNA working solution to the 1 and 3 lines of the mixer, and connecting the two injections containing PEI working solution to the 2 and 4 lines of the mixer. After the connection, fix the syringe on the syringe pump, the total flow rate is 80mL/min, start the syringe pump, and prepare the preparation stock solution. The preparation stock solution is divided into 7mL vials, each with a volume of 2.5mL, placed in a -60℃ environment for about 2h, quickly transferred to the freeze dryer, turn on the vacuum pump, set the pressure to 0.1mbar, and freeze-dry for about 24 hours to obtain the freeze-dried powder of the nucleic acid drug in this example.

The lyophilized powder of nucleic acid drugs was stored at 4°C, -20°C and -80°C, respectively. 0.5 mg of the lyophilized powder sample of nucleic acid drugs was taken each month and added with 1 mL of enzyme-free sterile water for reconstitution. The particle size, PDI and potential of the preparation after reconstitution were detected by Malvern particle size analyzer, and the RNA encapsulation rate in the preparation after reconstitution was detected by Ribogreen kit. The test results are shown in Figures 2 to 13; Figure 2 shows the particle size of the lyophilized powder of nucleic acid drugs after reconstitution tested monthly after being stored at 4°C for 5 months; Figure 3 shows the potential of the lyophilized powder of nucleic acid drugs after being stored at 4°C for 5 months; Figure 4 shows the PDI of the lyophilized powder of nucleic acid drugs after being stored at 4°C for 5 months; Figure 5 shows the encapsulation rate of the lyophilized powder of nucleic acid drugs after being reconstituted tested monthly after being stored at 4°C for 5 months; Figure 6 shows the particle size of the lyophilized powder of nucleic acid drugs after being reconstituted tested monthly after being stored at -20°C for 5 months; Figure 7 shows the particle size of the lyophilized powder of nucleic acid drugs after being reconstituted tested monthly after being stored at -20°C for 5 months Potential; Figure 8 is the PDI of the nucleic acid drug lyophilized powder after reconstitution tested monthly after being stored at -20°C for 5 months; Figure 9 is the encapsulation efficiency of the nucleic acid drug lyophilized powder after being stored at -20°C for 5 months and tested monthly; Figure 10 is the particle size of the nucleic acid drug lyophilized powder after reconstitution tested monthly after being stored at -80°C for 5 months; Figure 11 is the potential of the nucleic acid drug lyophilized powder after reconstitution tested monthly after being stored at -80°C for 5 months; Figure 12 is the PDI of the nucleic acid drug lyophilized powder after reconstitution tested monthly after being stored at -80°C for 5 months; Figure 13 is the encapsulation efficiency of the nucleic acid drug lyophilized powder after being stored at -80°C for 5 months and tested monthly.

After the freeze-dried powder preparations were stored at 4°C, -20°C, and -80°C, it can be seen from the particle size, PDI, and potential results tested monthly in May, that is, Figures 2 to 13, that the freeze-dried powder preparations can be stored at -20°C and -80°C for 5 months, and the fluctuation range of various indicators is small. At 4°C, except for the large fluctuations in the data in the 5th month, the data in the first 4 months were relatively stable. At the same time, regardless of the conditions of 4°C, -20°C, and -80°C, the encapsulation rate can be stabilized at nearly 100%, showing good stability.

Example 8

In this example, the effect of the number of RNA base pairs on the preparation was studied under the preferred conditions determined in the previous examples.

Specifically, 1.5 mg of RNA with different base numbers was dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare an RNA working solution. The RNA with different base numbers is shown in Table 11, wherein the 30 nt RNA was obtained by custom synthesis by LGC, and its sequence is as follows:

mGmUmGCCCAUUCGGGGGGGGCCGAAUCAGmCmA5meC

The 366nt RNA was obtained in-house, and its sequence is as follows:

ACCAGACAAGAGUUUAAGAGAUAUCCAUUCUUCCAAAUUUUCUUG

UCUCCCUGCAAGUUCCACUUACCAUUGUCAUAUGGACAAGUCCAAGAC

UUCCAGGUACCGCGGAGCUUCGAUCGUUCUGCACGACAGGGACUAAUU

AUUACGAGCUGUCAUAUGGCUCGAUAUCACCCAGUGAUCCAUCAUCAA

UCACGGUCGUGUAUUCAUUCUGCCUGGCCCCGAACAUCCUGACCGCCCC

UAAAAUCUUCAUCAAAAUCUUCAUUUCUUUGGUGAGUCUUGGACUUAU

CCACAUGACAACAGCAAGAAAAACUCACAAGAAGACAAGAAAAUUCAA

AAGAAUCAAUAUCUCCCAAACUCUUGUCUGGU

The 544nt RNA was obtained in-house, and its sequence is as follows:

ACCAGACAAGAGUUUAAGAGAUAUCCAUUCUUCCAAAUUUUCUUG

UCUCCCUGCAAGUUCCACUUACCAUUGUCAUAUGGACAAGUCCAAGAC

UUCCAGGUACCGCGGAGCUUCGAUCGUUCUGCACGACAGGGACUAAUU

AUUACGAGCUGUCAUAUGGCUCGAUAUCACCCAGUGAUCCAUCAUCAA

UCACGGUCGUGUAUUCAUUCUGCCUGGCCCCGAACAUCCUGACCGCCCC

UAAAAUCUUCAUCAAAAUCUUCAUUUCUUUGGGAGGAAUCCAUACGU

UAUACUAUGUAUAAUCCUCAAACCUGUCCAAUAAAGUUUUUGUGAUAA

CCCUCAGGUUCCUGAUCUCACGGGAUGACAAUGAAACCACUCCCAAUU

GAAGUCUUGCCUCAAACUUCUGGUCAGGGAAUGACCCAGCCACCAAUC

CUGUGGACAUAGAUAAAGAUAGUCUUGGACUUAUCCACAUGACAACAG

CAAGAAAAACUCACAAGAAGACAAGAAAAUUCAAAAGAAUCAAUAUCU

CCCAAACUCUUGUCUGGU

2.15 mg of PEI was dissolved in 5 mL of enzyme-free sterile water containing 10% trehalose to prepare a PEI working solution, and the pH was adjusted to 3.6 with 0.5 M sodium hydroxide at room temperature.

After the two working solutions are prepared, each working solution is divided equally into two syringes, keeping the amount of liquid in the four syringes the same, connecting the two injections containing RNA working solution to the 1 and 3 pipes of the mixer, and connecting the two injections containing PEI working solution to the 2 and 4 pipes of the mixer. After the connection, fix the syringe on the syringe pump, the total flow rate is 80mL/min, start the syringe pump, and prepare the preparation stock solution. The preparation stock solution is divided into vials, each with a volume of 2.5mL, placed in a -60℃ environment for about 4h, quickly transferred to the freeze dryer, turn on the vacuum pump, set the pressure to 0.1mbar, and freeze-dry for about 24 hours to obtain the freeze-dried powder of the nucleic acid drug in this example.

Take 2 mg of nucleic acid drug lyophilized powder sample and add 2.6 mL of enzyme-free sterile water for reconstitution.

The particle size, PDI and potential of the stock solution and the reconstituted preparation were tested using a Malvern particle size analyzer. The RNA encapsulation efficiency in the stock solution and the reconstituted preparation was tested using a Ribogreen kit. The test results are shown in Table 12.

Table 11 Prescription information under different base number RNA conditions

Table 12 Test results of nucleic acid drug preparations prepared from RNA with different base numbers

Indicator/Group #1 #2 #3 Liquid particle size 116.57nm 103.13nm 101.63nm Original liquid PDI 0.24 0.22 0.23 Potential of stock solution 34.50mV 42.27 24.87mV Encapsulation efficiency of stock solution 100% 100% 100% Particle size of lyophilized reconstituted preparation 123.80nm 107.77nm 163.63nm Lyophilized reconstituted preparation PDI 0.25 0.19 0.28 Potential of lyophilized reconstituted preparations 39.37mV 36.93mV 27.00mV Encapsulation efficiency of freeze-dried reconstituted preparations 100% 100% 100%

The results in Table 12 show that the particle size and PDI of the preparation stock solution are relatively small, and after the prepared lyophilized powder is dissolved, although the particle size fluctuates, the PDI, potential, and encapsulation efficiency remain within a relatively stable range. Therefore, RNA with a base number of 30-544 can prepare nucleic acid drug nanoparticles with equivalent PDI, potential, and encapsulation efficiency.

The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims (10)

1. A nucleic acid drug preparation, characterized in that: cationic nucleic acid delivery polymers self-assemble and encapsulate nucleic acids in a trehalose solution to form nucleic acid drug nanoparticles, thereby forming a nucleic acid drug preparation comprising nucleic acid drug nanoparticles and a trehalose solution. 2. The nucleic acid drug preparation according to claim 1, characterized in that: the trehalose solution is an aqueous solution of trehalose, and its concentration is 1%-10%; Preferably, the concentration of the trehalose aqueous solution is 10%. 3. The nucleic acid drug preparation according to claim 1 or 2, characterized in that: the cationic nucleic acid delivery polymer is polyethyleneimine; Preferably, the cationic nucleic acid delivery polymer is linear polyethyleneimine; Preferably, the molecular weight of the linear polyethyleneimine is 10,000-70,000 Daltons, preferably 48,050 Daltons. 4. The nucleic acid pharmaceutical preparation according to claim 1 or 2, characterized in that: the nucleic acid is RNA; Preferably, the RNA has a base number of 30-544. 5. The nucleic acid drug preparation according to claim 1 or 2, characterized in that: the particle size of the nucleic acid drug nanoparticles is 95-195 nm; Preferably, the Zeta potential of the nucleic acid pharmaceutical preparation is 34-46 mV; Preferably, the mass ratio of the cationic nucleic acid delivery polymer to the nucleic acid is 1-2.5:1.5-3, preferably 1.5:2. 6. A nucleic acid drug lyophilized powder, characterized in that it is lyophilized from the nucleic acid drug preparation according to any one of claims 1 to 5. 7. The method for preparing the lyophilized powder of nucleic acid drug according to claim 6 is characterized in that: it comprises dissolving a cationic nucleic acid transfer polymer in a trehalose solution to prepare a solution A; dissolving a nucleic acid in a trehalose solution to prepare a solution B; mixing solution A and solution B evenly to obtain the nucleic acid drug preparation; and lyophilizing the nucleic acid drug preparation to obtain the lyophilized powder of nucleic acid drug. 8. The preparation method according to claim 7, characterized in that the pH of the solution A is 3.5-4, preferably 3.6. 9. The preparation method according to claim 7 or 8, characterized in that: the mixing of solution A and solution B uniformly comprises: dispensing equal amounts of solution A into two syringes, dispensing equal amounts of solution B into another two syringes, connecting the two syringes for dispensing solution A to pipelines 1 and 3 of the mixer, respectively, connecting the two syringes for dispensing solution B to pipelines 2 and 4 of the mixer, respectively, setting the total flow rate, and starting the injection pump of the mixer to mix solution A and solution B, thereby obtaining the nucleic acid drug preparation. 10. The preparation method according to claim 9, characterized in that the total flow rate is 40-80 mL/min, preferably 80 mL/min. Preferably, the pressure of the freeze-drying process is 0.001-0.200 mbar, preferably 0.1 mbar.

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