WO2024083221A1

WO2024083221A1 - Mucosal administration formulation, and preparation and use therefor

Info

Publication number: WO2024083221A1
Application number: PCT/CN2023/125644
Authority: WO
Inventors: 章文羿; 陈瀚; 曹禹琦; 李囡; 赵长有; 黄清瑞; 刘硕; 李小芳
Original assignee: 北京安必奇生物科技有限公司; 章文医药(北京)有限公司
Priority date: 2022-10-21
Filing date: 2023-10-20
Publication date: 2024-04-25

Abstract

A mucosal administration formulation, and a preparation method and use therefor. The preparation method for the mucosal administration formulation comprises the following steps: (1) mixing a nucleating agent solution, a drug protein solution and an inorganic salt buffer solution to obtain a mixed solution; (2) while stirring, spraying the mixed solution obtained in step (1) into a coating agent solution, adding a penetration enhancer, and freeze-drying to obtain the formulation. Nanoparticles prepared by ion crosslinking and using the nanoparticles to achieve mucosal administration can avoid the use of an industrial synthetic amphiphilic polymer reagent, providing better safety.

Description

A mucosal drug delivery preparation and its preparation method and application

Technical Field

The present invention relates to the field of medicines, and in particular to a mucosal drug delivery preparation and a preparation method and application thereof.

Background technique

The mucosal system, including the mucosa of the oral cavity, nasal cavity, digestive tract, urethra, vagina, etc., is an important protective barrier on the human body surface and an important way for exogenous microorganisms to infect and for drug molecules to enter the human body. Compared with the normal skin environment, the thickness of the mucosal layer is between 10-1000 microns. It does not have a keratinized surface and only contains a single layer or several layers of epithelial cells, which is conducive to the transmembrane transport of drug molecules. In addition, most mucosal surfaces contain microvilli structures, which further increase the surface area of the mucosa. Therefore, mucosa with high permeability and large surface area can be used as an important route for drug delivery. At present, there are many chemical small molecule drugs that can be delivered through mucosa.

On the other hand, according to the principles of immunology, mucosal immunity can activate the expression of secretory antibodies sIgA on the mucosal surface, directly blocking pathogens outside the body, fundamentally eliminating the occurrence of infection, and thus overcoming the formation of asymptomatic infections under ordinary humoral/cellular immunity.

However, in the field of biomacromolecule drugs and nucleic acid drugs, there is currently no mature mucosal drug delivery technology. The main obstacle is that biomacromolecules and negatively charged nucleic acids are difficult to penetrate cell membranes or intercellular spaces. Studies have found that mucosal cells, especially micro-wrinkled cells with a large number of microvilli on the surface, tend to engulf a large number of positively charged particles with a particle size of 100 nanometers to 1 micron. Therefore, using such particles to wrap biomacromolecules can achieve transmucosal biological delivery; regulating the electrical properties of nucleic acids through ion reactions and wrapping them with biocompatible materials to form nucleic acid nanoparticles of a specific size can also achieve transmucosal biological delivery. Another obstacle to mucosal drug delivery technology is the presence of a variety of nucleases on the mucosal surface, which poses a serious challenge to the stability of nucleic acid drugs.

Patent CN1812765B discloses a film-shaped dosage form for administering active ingredients to the human or animal body through the mucosa. During its preparation process, the pH value of the matrix used to prepare the dosage form, which includes a solvent or a mixture of solvents, at least one polymer forming a matrix, and at least one component selected from the group consisting of active pharmaceutical ingredients and aromatic substances, is close to or adapted to the physiological pH value of the mucosa to which the dosage form is applied. The use of this dosage form reduces or even prevents mucosal irritation during administration. The focus of this patent is on the "rapid release of active substances", but for polypeptide or protein drugs, their absorption process is generally slower than that of small molecule drugs, and the stability of the released polypeptide or protein drugs in the physiological environment of the mucosal surface is poor. Therefore, for polypeptide or protein drugs, sustained-release administration of nanoparticles has greater advantages than rapid-release administration of film preparations. The advantages.

Patent CN1720024 discloses a polymer foam or film composition for locally delivering pharmacologically effective agents to the nasal cavity, oral cavity, vagina, labia or scrotal epithelium or entering the systemic circulation through the nasal cavity, oral cavity, vagina, labia or scrotal epithelium, wherein the composition contains at least one matrix polymer or a mixture of a matrix polymer and a pharmacologically effective agent. The composition further contains a penetration enhancer, an absorption enhancer, a mucoadhesive, a hydrophilic or hydrophobic release modifier, or a mixture thereof, and can be applied to the surface of a complex drug delivery system. The dosage form of the patent is a foam or film, which does not have a nanoparticle structure. According to the literature, the mucosal layer has an efficient absorption capacity for nanoparticles of 100-1000nm, so the nano-preparation delivery system can be better applied to mucosal administration and absorption. In addition, the penetration enhancer of the patent depends on "diol derivatives" (page 42, section 15), while the preferred penetration enhancer of the patent does not contain diol derivatives, and the control example shows that the effect of the penetration enhancer selected by the patent is better than PEG400 (diol derivative used in Example 1 of the patent).

Patent CN1068585C and other similar patents disclose several cationic lipids for gene therapy. These lipids can encapsulate nucleic acid drugs through ion reactions, change their electrical properties, and finally form nanoliposomes/lipid particles to allow nucleic acid drugs to enter cells. However, such cationic materials often have certain cytotoxicity, and their pharmaceutical applications have many limitations. In addition, liposomes/lipid particles themselves have the problem of aggregation during long-term storage, and their adsorption/permeability on the mucosal surface is insufficient.

Patent CN108697803A discloses a transmucosal drug composition, which includes a lipophilic active compound, a polymer matrix formed by two or more water-soluble polymers, and a fast dissolving agent. At least one of the water-soluble polymers is an amphiphilic polymer, and at least one is a hydrophilic polymer or an amphiphilic polymer, and the hydrophilic-hydrophobic balance of the two new polymers is different from that of the first amphiphilic polymer. The applicable active ingredients of this patent are mainly "lipophilic active compounds", but most protein and polypeptide drugs are not lipophilic, and therefore are not applicable to the method of this patent.

In summary, the development of mucosal delivery technology for active protein drugs and nucleic acid drugs is still inadequate, and more necessary research is needed on mucosal delivery preparations for nucleic acid drugs.

In view of this, this patent focuses on the administration of proteins, peptide drugs, and nucleic acid drugs, so it is not necessary to contain amphiphilic polymers. This patent uses hydrophilic polymers from pure natural sources to achieve efficient mucosal administration of proteins, peptide drugs, and nucleic acid drugs, which can avoid the use of industrially synthesized amphiphilic polymer reagents and has better safety.

Summary of the invention

In view of the problems existing in the prior art, the present invention provides a mucosal drug delivery preparation and a preparation method and application thereof, nanoparticles prepared by ionic crosslinking, and the application of mucosal drug delivery using the nanoparticles.

The components of the nanoparticles include a nucleating agent, an active ingredient, a penetration enhancer, and a coating agent. The active ingredient is preferably a drug protein or a nucleic acid drug. Except for the active ingredient, the remaining components are from the Chinese Pharmacopoeia (2020 Edition) The safety of the included pharmaceutical excipients has been fully verified.

The preparation principle is that the active ingredient forms agglutination nuclei through electrostatic adsorption with a nucleating agent under specific pH conditions. A low-concentration solution containing agglutination nuclei is dispersed in a solution containing a high-concentration coating agent, and a nanoparticle solution containing the active ingredient can be quickly formed through ionic crosslinking between the nucleating agent and the coating agent. The size of the formed nanoparticles can be controlled by regulating the concentration and ionic conditions of the nucleating agent and the coating agent. A solid mucosal drug delivery preparation containing nanoparticles can be obtained by selecting a suitable nanoparticle solution, adding a permeation enhancer, and then freeze-drying. The transmembrane absorption of the active ingredient on the mucous membranes of the oral cavity, nasal cavity, digestive tract, urethra, vagina, etc. can be achieved.

To achieve the above purpose, the technical solution adopted by the present invention is as follows:

The present invention provides a method for preparing a mucosal drug delivery preparation, comprising the following steps:

(1) mixing a nucleating agent solution, an active ingredient solution, and an inorganic salt buffer solution to obtain a mixed solution;

(2) stirring the coating agent solution and then spraying it into the mixed solution obtained in step (1), adding a penetration enhancer, and freeze-drying to obtain;

The active ingredient is selected from at least one of a pharmaceutical protein and a nucleic acid drug;

When the active ingredient is a pharmaceutical protein, the nucleating agent or coating agent includes one or more of poloxamer 188, poloxamer 407, beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, polyoxyl stearate, chitosan, carboxymethyl chitosan, carbomer 940p, alginic acid and its sodium salt, hyaluronic acid and its sodium salt, polyvinyl alcohol 2000, polyvinyl alcohol 4000, polyvinyl alcohol 6000, polyvinyl alcohol 8000, polysorbate 80, sodium carboxymethyl cellulose, sodium carboxymethyl starch, polycarbophil, and povidone;

When the active ingredient is a nucleic acid drug, the nucleating agent or coating agent is selected from: poloxamer 188, poloxamer 407, beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, polyoxyl stearate, polyglutamic acid, polyaspartic acid, polyarginine, polylysine, chitosan, carboxymethyl chitosan, alginic acid and its sodium salt, hyaluronic acid and its sodium salt, polyvinyl alcohol 2000, polyvinyl alcohol 4000, polyvinyl alcohol 6000, polyvinyl alcohol 8000, polysorbate 80, sodium carboxymethyl cellulose, sodium carboxymethyl starch, and one or more of povidone.

Preferably, when the active ingredient is a nucleic acid drug, the nucleating agent and the capping agent are not the same substance.

Preferably, when the active ingredient is a pharmaceutical protein, the nucleating agent is alginate; when the active ingredient is a nucleic acid drug, the nucleating agent is chitosan or polylysine.

Preferably, when the active ingredient is a pharmaceutical protein, the raw material of the coating agent is chitosan; when the active ingredient is a nucleic acid drug, the raw material of the coating agent is alginate or dextran.

Furthermore, the penetration enhancer includes one or more of glucose, sucrose, maltose, trehalose, mannitol, sorbitol, EDTA and EGTA.

Preferably, when the active ingredient is a drug protein, the penetration enhancer is sucrose; when the active ingredient is a nucleic acid drug, the penetration enhancer is EDTA.

Preferably, the inorganic salt buffer solution is a phosphate buffer.

In the above steps, a stirring step may be included under some conditions. For example, a stirring step may be selectively included when a mixing operation is involved. The mixing operation may be the mixing of two solutions or the mixing of a solution and a solid. According to general understanding, the preparation of a solution should also include a stirring step.

The above steps may also include a filtration and sterilization step known in the art, which should be understood by those skilled in the art even if it is not limited. The solution is generally prepared with sterile water, and other standard water may also be used for preparation.

Furthermore, when the active ingredient is a pharmaceutical protein:

The concentration of the nucleating agent solution is a solution with a mass fraction of 0.01-5%; the pH range is 3-10. The ion concentration of the nucleating agent is 1-500mM. Sterilization is performed by filtration during the preparation process.

In some specific embodiments, the nucleating agent solution is prepared by preparing phosphate buffer, sterile water and a nucleating agent.

The concentration of the coating agent solution is 0.1-30% by mass, and the pH range is 3-10. The ion concentration of the coating agent is 1-500mM. Sterilization is performed by filtration during the preparation process.

In some specific embodiments, the coating agent solution is prepared by using acetic acid and a coating agent.

In some specific embodiments, the penetration enhancer solution is prepared using a penetration enhancer and sterile water.

The concentration range of the active ingredient solution is 1-100 mg/mL. The drug protein solvent is selected from a suitable phosphate buffer according to the properties of the active protein, and sterilized by filtration during the preparation process.

Furthermore, the drug protein solution is a new coronavirus antigen solution.

In some specific embodiments, the method for preparing the mucosal administration preparation comprises the following steps:

Inject the nucleating agent solution, new coronavirus antigen solution and phosphate buffer into the preparation tank in sequence, control the temperature at 4 degrees, and mix for 15 minutes.

Inject the coating agent solution into the preparation tank, and spray the liquid in the preparation tank into the preparation tank through a sprayer.

After spraying is completed, continue stirring, then drop the penetration enhancer solution, and continue stirring after the dropwise addition is completed.

Use an automatic filling machine to dispense the mixed solution in the preparation tank into vials, 0.5 mL/vial. After dispensing, transfer to the freeze dryer for pre-freezing. Start the freeze drying process. After freeze drying, cap.

After freeze-drying, samples are taken and reconstituted to test protein content, turbidity, viscosity, osmotic pressure, particle size and surface potential. The remaining samples are stored or tested for stability.

Furthermore, when the active ingredient is a nucleic acid drug:

The concentration of the nucleating agent solution is a solution with a mass fraction of 0.01%-5%, preferably 1%-3%; the pH range is 3-10, and the ion concentration of the nucleating agent is 1-500mM.

In some specific embodiments, the solvent of the nucleating agent solution is one or both of phosphate buffer and water.

The concentration of the coating agent solution is a solution with a mass fraction of 0.1%-20%, preferably 0.2%-10%, a pH range of 3-10, and an ion concentration of the coating agent of 1-500mM.

In some specific embodiments, the solvent of the coating agent solution is water.

The penetration enhancer is added in the form of a penetration enhancer solution. In some embodiments, the solvent of the penetration enhancer solution is water. In some specific embodiments, the penetration enhancer solution is an EDTA buffer with a pH of 7-9, preferably 8-9, and a concentration of 400-600 mM, preferably 500-600 mM.

The concentration of the active ingredient solution is in the range of 0.1-20 mg/mL, preferably 10-20 mg/mL.

The inorganic salt buffer is a phosphate buffer with a pH of 4-7.

In the step (2), a lyoprotectant is added at the same time as the penetration enhancer. The lyoprotectant is added in the form of a solution. Preferably, the lyoprotectant solution is a trehalose solution with a mass fraction of 20%-40%, more preferably 30%-40%, and even more preferably 30% trehalose solution.

Preferably, the nucleic acid drug is antigen mRNA, and more preferably SARS-CoV-2 antigen mRNA.

In the present invention, the SARS-CoV-2 is sometimes also referred to as the new coronavirus.

1) Inject phosphate buffer, nucleating agent solution and antigen mRNA solution into a low-temperature liquid preparation tank in sequence, control the temperature at 4°C, and mix for 15 minutes;

2) injecting the coating agent solution into the preparation tank, and spraying the liquid in the preparation tank into the preparation tank through a sprayer at a speed of 500-3000 rpm;

3) After spraying is completed, continue stirring, then drop the penetration enhancer solution and freeze-drying protective agent, and continue stirring after the dropwise addition is completed;

4) Use an automatic filling machine to dispense the mixed solution in the preparation tank into vials, 0.5 mL/vial; after dispensing, transfer to a freeze dryer; pre-freeze; start the freeze drying process; and cap after freeze drying;

5) After freeze-drying, samples are taken and re-dissolved to test nucleic acid content, turbidity, viscosity, osmotic pressure, particle size and surface potential; the remaining samples are stored or subjected to stability testing.

Preferably, the method for preparing the mucosal administration preparation comprises the following steps:

(1) mixing a nucleating agent solution, a SARS-CoV-2 antigen mRNA solution, and an inorganic salt buffer solution to obtain a mixed solution;

(2) spraying the mixed solution obtained in step (1) into the coating agent solution, adding the penetration enhancer solution and the protective agent solution, stirring again, and freeze-drying to obtain;

In the step (1):

The volume ratio of the nucleating agent solution, the SARS-CoV-2 antigen mRNA solution and the inorganic salt buffer is 4-6:1-3:0.5-1.5, preferably 5:2:1;

In the step (2):

The volume ratio of the coating agent solution, the penetration enhancer solution and the protective agent solution is 8-10:1:1, preferably 10:1:1; and the volume ratio of the coating agent solution to the SARS-CoV-2 antigen mRNA solution in step (1) is 4-6:1, preferably 5:1.

In some specific embodiments, the preparation method comprises the following steps:

(1) mixing a chitosan solution, a SARS-CoV-2 antigen mRNA solution, and a phosphate buffer solution to obtain a mixed solution;

(2) spraying the mixed solution obtained in step (1) into the alginate solution, adding the EDTA solution and the trehalose solution, stirring again, and freeze-drying to obtain;

In the step (1):

The volume ratio of the nucleating agent solution, the SARS-CoV-2 antigen mRNA solution and the phosphate buffer is 4-6:1-3:0.5-1.5, preferably 5:2:1;

In the step (2):

The volume ratio of the alginate solution, the EDTA solution and the trehalose solution is 8-10:1:1, preferably 10:1:1; and the volume ratio of the alginate solution to the SARS-CoV-2 antigen mRNA solution in step (1) is 4-6:1, preferably 5:1.

Furthermore, in step (2), the freeze-drying includes pre-freezing, main drying and analysis; the pre-freezing time is 4-24 hours, the pre-freezing temperature is -80°C, the main drying time is 4-72 hours, the main drying temperature is -80°C to -10°C, and the gradient setting is; the analysis time is 2-24 hours, and the analysis temperature is -10°C to 50°C, and the gradient setting is.

Furthermore, the preparation of the mucosal drug delivery preparation is completed in a Class A clean environment. A low-temperature liquid preparation tank and a preparation tank with 1-1000 spray devices and a mechanical stirrer are used. The spray speed of each nozzle is 0.01-100 mL/min. The preparation temperature selection range is 0-50°C.

On the other hand, the present invention also provides a mucosal administration preparation prepared by the above-mentioned preparation method.

Furthermore, the mucosal administration preparation is in the form of a solid dry powder with a water content of 0.5-10%, and the obtained preparation is redissolved in water to form a nanoparticle solution, wherein the particle size of the active component particles is 0.1-10 μM. The stability of the dry powder preparation is more than 1 year.

Furthermore, when the active ingredient is a drug protein, the water content of the mucosal administration preparation is between 1-10%, and when the active ingredient is a nucleic acid drug, the water content of the mucosal administration preparation is between 0.5%-5%.

The technical effects achieved by the present invention are:

The present invention designs a mucosal drug delivery technology. The key point of this technology is to achieve transmembrane absorption of proteins, peptide drugs, and nucleic acid drugs on the mucosa through drug loading with nanoparticles. By optimizing the protein, the ability of the protein itself to target mucosal immune cells is enhanced. Polymer materials that comply with the pharmaceutical excipients of the "Chinese Pharmacopoeia" are used to form complexes with proteins, peptides, and nucleic acid drugs through electrostatic adsorption to optimize the structure, adhesion, and stability of the preparation. On the other hand, the activity of the loaded antigen and the mucosal absorption effect are improved. This vaccine program integrates nanotechnology with protein recombination technology/nucleic acid drug technology. Through oral/nasal spray vaccination, it can innovatively and effectively induce the triple immune pathways of mucosa, humor, and cells at the same time, and has the characteristics of high neutralizing antibody production efficiency, high safety, and room temperature stability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the particle size distribution of the preparations of Example 1, Example 2 and Comparative Example 1.

FIG. 2 is a graph showing the stability of the protein in the formulation of Example 1.

FIG3 is a comparison of cell absorption of the positive control, Examples 1-3, Control Example 1 and the negative control.

FIG. 4 shows the neutralizing antibody titer on the mucosa after intramuscular injection, as well as mucosal administration of Examples 1-3 and Control Example 1 to mice.

FIG. 5 is a particle size distribution diagram of drug-loaded nanoparticles of Examples 4-6 and Comparative Example 6.

FIG6 is a graph showing the surface potential distribution of drug-loaded nanoparticles of Examples 4-6 and Comparative Example 6.

FIG. 7 is a graph showing the drug-loaded cell absorption of nanoparticles of Examples 4-6 and Comparative Example 6.

FIG8 shows the experimental results of the nanoparticle drug delivery of Examples 4-6 and Control Example 6 for mouse immunization.

Detailed ways

The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

It is worth noting that the raw materials used in the present invention are all common commercially available products, so their sources are not specifically limited.

The sources of some raw materials in the present invention are as follows:

1. Preparation of active protein mucosal drug delivery preparations

Example 1

All preparations are made with sterile water in an isolator to ensure the sterility of the solution. All reagents are placed on ice as much as possible to prevent degradation. The obtained working solution is divided into appropriate specifications and frozen at -20℃. Reagents at room temperature are only used on the same day.

Prepare phosphate buffer at pH 8, concentration 100 mM, and filter sterilize or autoclave.

Prepare a core agent solution by using a pH=8 phosphate buffer and sterile water to prepare a 0.5% solution of alginic acid, a salt concentration of 100 mM, and a volume of 200 mL. Add 100 mL of EDTA solution (0.5 M, pH=8), mix well, and filter to sterilize.

Prepare the coating agent solution by dissolving chitosan in 1% acetic acid to a final concentration of 1%, prepare about 1 L, and sterilize by autoclave.

The SARS-CoV-2 antigen solution has a concentration of 5 mg/mL and is filtered and sterilized.

To prepare the penetration enhancer solution, weigh 100 g of sucrose, dissolve it in sterile water to prepare a 20% solution, and filter and sterilize.

Nanoparticle formulation:

All operations are completed in the clean bench. The low-temperature liquid preparation tank, as well as the preparation tank with a spray device and a mechanical stirrer are transferred to the isolator. The freeze dryer is turned on and the pre-freezing temperature is set to -50 degrees.

Inject 300mL of nucleating agent solution, 600mL of new coronavirus antigen solution, and 100mL of phosphate buffer into the preparation tank in sequence, control the temperature at 4 degrees, and mix for 15 minutes.

1L of coating agent solution was injected into the preparation tank, and the speed was set to 800rpm. The liquid in the preparation tank was sprayed into the preparation tank through a sprayer at a spray rate of 50mL/min. The nozzle height was set to 30cm.

After spraying, continue stirring for 10 minutes, then drip 400 mL of permeation enhancer at a dripping rate of 40 mL/min, and continue stirring for 10 minutes after dripping.

Use an automatic filling machine to dispense the mixed solution in the preparation tank into vials, 0.5 mL/vial. After dispensing, transfer to the freeze dryer and pre-freeze for 4 hours. Start the freeze drying process. After freeze drying, cap.

Example 2

The method is similar to that of Example 1, except that the nucleating agent is replaced with an equal amount of sodium hyaluronate.

Example 3

The method is similar to that of Example 1, except that the coating agent is replaced with an equal amount of dextran.

Comparative Example 1

The method is similar to that of Example 1, except that the coating agent is replaced with an equal amount of polyvinyl alcohol 400.

2. Preparation of Nucleic Acid Drug Mucosal Delivery Preparations

Example 4 Preparation experiment of nanoparticles

In this embodiment, unless otherwise specified, all preparations are made using sterile water as a solvent in an isolator to ensure that the solution is sterile. All reagents are placed on ice as much as possible to prevent degradation. The resulting working solution is packaged into appropriate specifications and frozen at -20°C. Reagents at room temperature are limited to use on the same day.

(1) Preparation of basic solution:

Prepare phosphate buffer at pH 5, concentration 100 mM, and filter sterilize or autoclave.

Prepare a core agent solution, use pH=5 phosphate buffer and sterile water to prepare a chitosan solution with a mass fraction of 1%, a phosphate concentration of 10mM, and a volume of 200mL. Dissolve and mix thoroughly, then filter and sterilize.

Prepare the coating solution by dissolving alginate in sterile water to a final concentration of 0.2%, prepare about 1 L, and sterilize by autoclave.

The SARS-CoV-2 antigen mRNA solution has a concentration of 20 mg/mL and is filtered and sterilized.

Prepare EDTA buffer with pH=8 and concentration of 500 mM, and sterilize by filtration or autoclaving.

Prepare freeze-dried protective agent solution: weigh 100g trehalose, dissolve it in sterile water to prepare a solution with a mass fraction of 30%, and filter and sterilize.

(2) Nanoparticle preparation:

All operations were completed in a clean bench. The low-temperature liquid preparation tank, as well as the preparation tank with a spray device and a mechanical stirrer were transferred to the isolator. The freeze dryer was turned on and the pre-freezing temperature was set to -50°C.

Inject 500 mL of nucleating agent solution, 200 mL of new coronavirus antigen solution, and 100 mL of phosphate buffer into the preparation tank in sequence, control the temperature at 4°C, and mix for 15 minutes.

Inject 1L of coating agent solution into the preparation tank, set the speed to 1500rpm. Spray the liquid in the preparation tank into the preparation tank through a sprayer at a spray rate of 120mL/min. Set the nozzle height to 30cm.

After spraying, continue stirring for 10 minutes, then drip 100 mL of permeation enhancer at a rate of 10 mL/min, continue dripping 100 mL of freeze-dried protective agent at a rate of 50 mL/min, and continue stirring for 10 minutes after dripping.

After freeze-drying, samples are taken and reconstituted to test nucleic acid content, turbidity, viscosity, osmotic pressure, particle size and surface potential. The remaining samples are stored or tested for stability.

Example 5

Referring to Example 1, the coating agent was replaced by dextran instead of alginate.

Example 6

Referring to the arrangement of Example 4, the nucleating agent was replaced by polylysine instead of chitosan.

Comparative Example 2

The arrangement is similar to that of Example 4, except that both the coating agent and the nucleating agent are alginic acid.

Comparative Example 3

The arrangement is similar to that of Example 4, except that both the coating agent and the nucleating agent are chitosan.

Comparative Example 4

The arrangement is similar to that of Example 4, except that both the coating agent and the nucleating agent are dextran.

Comparative Example 5

The arrangement is similar to that of Example 4, except that both the coating agent and the nucleating agent are polylysine.

Comparative Example 6

Prepared according to the method of Example 17 of patent CN102625696B, using the new coronavirus antigen solution instead of the siRNA in this example.

Comparative Example 7

The drug preparation is prepared with reference to the preparation method of Example 1 in the prior art CN115590826A, with the difference that the new coronavirus antigen solution (protein drug) is replaced by the new coronavirus antigen mRNA solution (nucleic acid drug) of Example 1 of the present application.

3. Characterization of Nanoparticles:

Preferably, the particle size and surface potential of the nanoparticles are evaluated using a Zetasizer particle size analyzer from Malvarn. Other instruments with similar testing principles and testing capabilities may also be used to complete the test.

Randomly take one preparation product and inject 0.5 mL of water for injection. Invert and shake 10 times. Draw out the solution and mix it with 0.5 mL of phosphate buffer of a specific pH, preferably 20 mM phosphate buffer of pH=7.4 in the present invention. Put it into the sample pool and measure the particle size. Measure three times in a row and take the average value. The particle size of the nanoparticles in Example 1 is between 0.3-1 μM. Simultaneously carry out relevant experiments on the products of Examples 1-2 and Comparative Example 1, and the obtained nanoparticle drug-loaded particle size distribution diagram is shown in Figure 1.

The particle size of the nanoparticles in Examples 4-6 is between 255.97-585.97 nm, which is consistent with the particle size distribution of 100 nm to 600 nm. FIG5 shows the particle size distribution of the drug-loaded nanoparticles in Examples 4-6 and Comparative Example 6.

Randomly take 10 preparation products, each of which is injected with 0.5mL of water for injection. Shake upside down 10 times. The solution is extracted and mixed with 0.5mL of phosphate buffer of continuously changing pH, preferably 20mM phosphate buffer of pH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8 in this embodiment. According to the pH order, each sample is placed in the sample cell in turn to measure the surface potential. Three consecutive measurements are taken and the average value is taken. The obtained surface potential is plotted against pH, and the isoelectric point of the nanoparticles and the surface potential under physiological conditions are read from the figure. Figure 6 shows the surface potential distribution diagram of the nanoparticle drug loading of Examples 4-6 and Comparative Example 5.

The specific results of Examples 4-6 and Comparative Examples 2-7 are as follows:

2. Evaluation of Nanoparticle Protein Drug Delivery:

Randomly take one preparation product and inject 0.5mL of water for injection. Invert and shake 10 times. Draw out the solution and centrifuge the solution using an ultra-high speed centrifuge at 100,000rpm, 4 degrees Celsius, for 24 hours. Take the supernatant. Determine the protein content using ultraviolet spectrophotometry. For the preparation in Example 1, the protein content in the supernatant is not higher than 0.025mg/mL. That is, the protein encapsulation rate is greater than 96%. The specific results of Examples 4-6 and Control Examples 2-7 are as follows:

3. Nanoparticle-protein stability assessment:

This experiment was completed using commercial ELISA detection kits and SDS-PAGE kits.

10 preparations of Example 1 were randomly taken and stored at 4°C. They were taken out at 0 days, 10 days, 20 days, 30 days, 50 days, 100 days, 150 days, 200 days, 300 days, and 365 days, respectively. 0.5 mL of water for injection was then immediately injected. Shake upside down 10 times. The solution was extracted and tested using PAGE and ELISA under non-reducing conditions. The nucleic acid in the sample was extracted using a commercial nucleic acid extraction kit (Suzhou Beaver #70410), and the nucleic acid integrity was determined using a capillary electrophoresis kit (Hangzhou Houze Qsep100). It was found by ELISA that the total preservation rate of the freeze-dried and re-dissolved products of Example 1-3 was always above 80% compared with that of Control Example 1. Further analysis of the total stored protein using non-reducing PAGE found that the protein with a complete molecular weight was always above 95%, indicating that the solid preparation had good stability at 4°C within a shelf life of 1 year. The results of nucleic acid integrity showed that the total preservation rate (%) of the freeze-dried and reconstituted products was always at a higher level than that of the control group, indicating that the solid preparation had good stability at 4°C within the shelf life of 1 year.

The drug-carrying stability diagram of the nanoparticles of the present invention is shown in Figure 2. The specific results of the preservation rate (%) of Examples 4-6 and Comparative Examples 2-7 are as follows:

IV. Evaluation of Cellular Uptake of Nanoparticle-Delivered Drugs:

Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Transwell polycarbonate filter inserts (12-port, 3 mm pore size) were purchased from Corning Costar (New York, NY).

(1) Determination of active protein mucosal delivery preparations

Caco-2 cells were cultured in DMEM (25 mM glucose) with 10% (v/v) FBS (Hyclone Laboratories, Logan, UT), and Raji cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS.

0.1 and 0.5 mL DMEM supplemented with 10% FBS were added to the apical and basolateral sides of the transverse wells, respectively, and the transverse wells were pre-incubated in a CO2 incubator for 30 minutes. Caco-2 cells were then spread to the top of the transwells. After 3 hours of incubation, the culture medium on the apical side was replaced, and the upper culture medium was replaced every other day for the next 14 days of culture. Raji cells suspended in a mixture of RPMI1640/DMEM (1:2) were then added to the basolateral chamber of the transverse wells and co-cultured for 7 days.

Randomly take one preparation product, inject 0.5mL of water for injection, and shake it upside down 10 times. Draw out the solution, take 100μL and incubate it on the top of the transfer well, add 100μL of saline sample containing 125μg protein to the control well, and add 100μL of saline to the negative control well. Then pre-incubate the cross well in a _CO2 incubator for 2h. Collect the culture medium and caco2 cells and measure them. Testing SDS-PAGE, it can be observed that in the caco2 cell recovery sample, only the preparation group has obvious target protein signals. Compared with the standard concentration sample, it is judged that its absorption effect is greater than 50%.

Relevant experiments of the products of Examples 1-3 and Comparative Example 1 were carried out simultaneously, and the obtained nanoparticle drug-loaded cell absorption diagram is shown in Figure 3.

(2) Determination of nucleic acid drug mucosal delivery preparations

Caco-2 cells (ATCC #HTB-37) were cultured in DMEM (25 mM glucose) with 10% (v/v) FBS (Hyclone Laboratories, Logan, UT), and Raji cells (ATCC #CCL-86) were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS.

0.1mL and 0.5mL DMEM supplemented with 10% FBS were added to the top and basolateral sides of the transverse wells, respectively, and the transverse wells were pre-incubated in a CO ₂ incubator for 30 minutes. Caco-2 cells were then spread to the top of the transwell. After 3 hours of incubation, the medium on the top side was replaced, and the upper medium was replaced every other day for the next 14d culture. Raji cells suspended in a mixture of RPMI1640/DMEM (1:2) were then added to the basolateral chamber of the transverse wells and maintained in co-culture for 7 days.

Randomly take 1 preparation product, inject 0.5mL of injection water, and shake it upside down 10 times. Draw out the solution, take 100μL and incubate it at the top of the transwell, add 200μg of new crown antigen mRNA and appropriate liposome transfection reagent, such as lipofectine, to the positive control well, and dilute it to 100μL with injection water; add 100μL of normal saline to the negative control well. Then pre-incubate the horizontal well in a _CO2 incubator for 2h. Replace the reagent in the well with ordinary culture medium and continue to culture for 48 hours. Harvest the upper Caco-2 cells and the lower Raji cells and measure them. Testing SDS-PAGE, it can be observed that in the Caco-2 cell recovery sample, obvious target protein signals were observed in the preparation group and the positive group. In Raji cells, only the target protein signal was observed in the preparation group. Compared with the positive group standard nucleic acid-lipofectine group sample, it is judged that its absorption effect is significantly better than the liposome transfection scheme. Figure 7 shows the absorption diagram of nanoparticle-loaded cells in Examples 4-6 and Control Example 6.

5. Animal Model Evaluation of Nanoparticle Drug Delivery:

(1) Determination of active protein mucosal delivery preparations

SPF BALB/c mice, aged 6-8 weeks and weighing about 20 g, were selected and kept in a clean environment.

The mice were randomly divided into 3 groups, 10 mice in each group, half of them were male and half were female. After enrollment, 10 μL of blood was collected every week to measure blood routine, and the test was performed for 3 weeks to confirm that there was no abnormality.

The dosing experiment was conducted at the 1st, 3rd and 7th weeks. Each time the drug was administered, one preparation product was randomly selected, injected with 0.5 mL of water for injection, inverted and shaken 10 times, and the solution was drawn out. In the preparation group, 25 μL of the preparation solution was sprayed into each nostril of each mouse. In the protein control group, 25 μL of saline solution containing 0.625 mg/mL of antigen protein was sprayed into each nostril of each mouse. In the negative control group, 25 μL of saline was sprayed into each nostril of each mouse.

Observe the respiratory rate of mice before and during each spraying, count with a stopwatch, and record the respiratory rate. Weigh the mice before and after each spraying, record the weight data, and measure the body temperature.

After enrollment, before each dosing experiment, and 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, and 6 months after the third dosing, mouse mucosal sampling and venous blood collection were performed. Each time, 100 μL of mouse venous blood was drawn, anticoagulated with 0.15 mg of EDTA-K2, and 10 μL was taken for analysis by a routine blood analyzer, and the data was recorded. The remaining blood sample was centrifuged at 4°C, 3000 rpm for 20 minutes, and 20 μL of supernatant plasma was immediately used for ELISA activity test to test the antibody level against the administered protein in the mouse. The remaining plasma was directly frozen in liquid nitrogen. Oral swabs were used to collect mouse mucosal samples, and the resulting samples were immediately placed in protein preservation solution. 20 μL was immediately used for ELISA activity test to test the antibody level against the antigen protein on the mouse mucosa.

The day after the third administration of the mice, half of each group was killed, and the main organs were collected and fixed with formaldehyde. Paraffin sections were made from the nasal cavity and lung tissues of the mice, routinely dewaxed and hydrated, stained with hematoxylin and eosin, dehydrated with gradient alcohol, transparentized with xylene, and sealed with neutral gum. Photos were taken under an upright high-power microscope, and acute injury scores were performed using the Smith method.

The results obtained show that the preparation of the present invention can effectively induce immune activity against protein antigens and has no obvious irritation to mice.

Relevant experiments of the products of Examples 1-3 and Comparative Example 1 were carried out simultaneously, and the experimental effect of the obtained nanoparticles loaded with drugs for mouse immunization is shown in Figure 4.

(2) Determination of nucleic acid drug mucosal delivery preparations

The pharmaceutical stability of control examples 2-5 and control example 7 is not sufficient, and it is not meaningful to carry out animal level antibody evaluation. This evaluation experiment only verifies examples 4-6 and control example 6.

The dosing experiment was conducted at weeks 1, 3, and 7. Each time the drug was administered, one preparation product was randomly taken, injected with 0.5 mL of water for injection, inverted and shaken 10 times, and the solution was drawn out. In the preparation group, 25 μL of the preparation solution was sprayed into each nostril of each mouse. The antigen control group used a saline solution containing 2 mg/mL of the new crown mRNA, and 25 μL was sprayed into each nostril of each mouse. In the negative control group, 25 μL of saline was sprayed into each nostril of each mouse.

After enrollment, before each dosing experiment, and 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, and 6 months after the third dosing, mouse mucosal sampling and venous blood sampling were performed. Each time, 100 μL of mouse venous blood was drawn, anticoagulated with 0.15 mg of EDTA-K2, and 10 μL was taken for flow cytometry analysis to determine the proportion of CD8+ cells. The remaining blood samples were centrifuged at 4°C, 3000 rpm for 20 minutes, and 20 μL of supernatant plasma was immediately used for ELISA activity test (Anbiqi Biological, New Coronavirus IgG Antibody Detection Kit (Enzyme-Linked Immunoassay)) to test the antibody level against the administered protein in mice. The remaining plasma was directly frozen in liquid nitrogen. Oral swabs were used to collect mouse mucosal samples, and the resulting samples were immediately placed in protein preservation solution, and 20 μL was immediately used for ELISA activity test (Anbiqi Biological, New Coronavirus IgA Antibody Detection Kit (Enzyme-Linked Immunoassay)) to test the antibody level against the antigen protein on the mouse mucosa. The geometric mean titer (GMT) is a commonly used indicator for measuring viral antibody titers. It is the geometric mean of viral antibody titers in all tested samples. The results are as follows:

The day after the third administration of the mice, half of each group was killed, and the main organs were collected and fixed with formaldehyde. Paraffin sections were made from the mouse nasal cavity and lung tissues, routinely dewaxed and hydrated, stained with hematoxylin and eosin in turn, dehydrated with gradient alcohol, transparentized with xylene, and sealed with neutral gum. Photos were taken under an upright high-power microscope, and acute injury scores were performed using the Smith method. The results are as follows:

Figure 8 shows the experimental effects of nanoparticle drug delivery in Examples 4-6 and Control Example 6 for mouse immunization (titer G.M.T of each antibody).

Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

A method for preparing a mucosal drug delivery preparation, characterized in that it comprises the following steps:

(1) mixing a nucleating agent solution, an active ingredient solution, and an inorganic salt buffer solution to obtain a mixed solution;

(2) stirring the coating agent solution and then spraying it into the mixed solution obtained in step (1), adding a penetration enhancer, and freeze-drying to obtain;

The active ingredient is selected from at least one of a pharmaceutical protein and a nucleic acid drug;

When the active ingredient is a pharmaceutical protein, the nucleating agent or coating agent includes one or more of poloxamer 188, poloxamer 407, beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, polyoxyl stearate, chitosan, carboxymethyl chitosan, carbomer 940p, alginic acid and its sodium salt, hyaluronic acid and its sodium salt, polyvinyl alcohol 2000, polyvinyl alcohol 4000, polyvinyl alcohol 6000, polyvinyl alcohol 8000, polysorbate 80, sodium carboxymethyl cellulose, sodium carboxymethyl starch, polycarbophil, and povidone;

When the active ingredient is a nucleic acid drug, the nucleating agent or coating agent is selected from: poloxamer 188, poloxamer 407, beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, polyoxyl stearate, polyglutamic acid, polyaspartic acid, polyarginine, polylysine, chitosan, carboxymethyl chitosan, alginic acid and its sodium salt, hyaluronic acid and its sodium salt, polyvinyl alcohol 2000, polyvinyl alcohol 4000, polyvinyl alcohol 6000, polyvinyl alcohol 8000, polysorbate 80, sodium carboxymethyl cellulose, sodium carboxymethyl starch, and one or more of povidone.
The preparation method according to claim 1 is characterized in that, when the active ingredient is a nucleic acid drug, the nucleating agent and the coating agent are not the same substance.
The preparation method according to claim 1 is characterized in that when the active ingredient is a drug protein, the nucleating agent is alginate; when the active ingredient is a nucleic acid drug, the nucleating agent is chitosan or polylysine.
The preparation method according to claim 1 is characterized in that when the active ingredient is a drug protein, the raw material of the coating agent is chitosan; when the active ingredient is a nucleic acid drug, the coating agent is alginate or dextran.
The preparation method according to claim 1, characterized in that the penetration enhancer comprises one or more of glucose, sucrose, maltose, trehalose, mannitol, sorbitol, EDTA and EGTA.
The preparation method according to claim 5, characterized in that when the active ingredient is a drug protein, the penetration enhancer is sucrose; when the active ingredient is a nucleic acid drug, the penetration enhancer is EDTA.
The preparation method according to claim 1, characterized in that the inorganic salt buffer solution is a phosphate buffer.
The preparation method according to claim 1, characterized in that when the active ingredient is a pharmaceutical protein:

The concentration of the nucleating agent solution is a solution with a mass fraction of 0.01-5%; the pH range is 3-10, and the ion concentration of the nucleating agent is 1-500mM.
The preparation method according to claim 1, characterized in that when the active ingredient is a pharmaceutical protein: The concentration of the coating agent solution is a solution with a mass fraction of 0.1-30%, a pH range of 3-10, and an ion concentration of the coating agent of 1-500mM.
The preparation method according to claim 1 is characterized in that, when the active ingredient is a pharmaceutical protein: the concentration range of the active ingredient solution is 1-100 mg/mL.
The preparation method according to claim 1 is characterized in that when the active ingredient is a drug protein: the drug protein solution is a new coronavirus antigen solution.

In some specific embodiments, the method for preparing the mucosal administration preparation comprises the following steps:
The preparation method according to claim 1, characterized in that when the active ingredient is a nucleic acid drug:

The concentration of the nucleating agent solution is 0.01%-5% by mass, preferably 1%-3%; the pH range is 3-10, the ion concentration of the nucleating agent is 1-500mM, and the solvent of the nucleating agent solution is one or both of phosphate buffer and water.
The preparation method according to claim 1, characterized in that when the active ingredient is a nucleic acid drug:

The concentration of the coating agent solution is a solution with a mass fraction of 0.1%-20%, preferably 0.2%-10%, a pH range of 3-10, an ion concentration of the coating agent of 1-500mM, and a solvent of the coating agent solution is water.
The preparation method according to claim 1, characterized in that when the active ingredient is a nucleic acid drug:

The penetration enhancer is added in the form of a penetration enhancer solution, which is an EDTA buffer solution with a pH of 7-9 and a concentration of 400-600 mM.
The preparation method according to claim 1, characterized in that when the active ingredient is a nucleic acid drug:

The concentration of the active ingredient solution is in the range of 0.1-20 mg/mL.
The preparation method according to claim 1, characterized in that when the active ingredient is a nucleic acid drug:

The inorganic salt buffer is a phosphate buffer with a pH of 4-7.
The preparation method according to claim 1, characterized in that when the active ingredient is a nucleic acid drug:

In the step (2), a lyophilization protectant is added at the same time as the penetration enhancer, and the lyophilization protectant is added in the form of a solution.
The preparation method according to claim 17, characterized in that the lyophilization protectant solution is a trehalose solution with a mass fraction of 20%-40%.
The preparation method according to claim 1 is characterized in that when the active ingredient is a nucleic acid drug: the nucleic acid drug is antigen mRNA.
The preparation method according to any one of claims 1, 12-19, characterized in that the preparation method of the mucosal administration preparation comprises the following steps:

(1) mixing a nucleating agent solution, a SARS-CoV-2 antigen mRNA solution, and an inorganic salt buffer solution to obtain a mixed solution;

(2) spraying the mixed solution obtained in step (1) into the coating agent solution, adding the penetration enhancer solution and the protective agent solution, stirring again, and freeze-drying to obtain the product.
The preparation method according to claim 20 is characterized in that in step (1): the volume ratio of the nucleating agent solution, the SARS-CoV-2 antigen mRNA solution and the inorganic salt buffer is 4-6:1-3:0.5-1.5; in the step (2): the volume ratio of the coating agent solution, the penetration enhancer solution and the protective agent solution is 8-10:1:1, and the volume ratio of the coating agent solution to the SARS-CoV-2 antigen mRNA solution in step (1) is 4-6:1.
The preparation method according to claim 20 is characterized in that in step (1): the volume ratio of the nucleating agent solution, the SARS-CoV-2 antigen mRNA solution and the inorganic salt buffer is 5:2:1; in the step (2): the volume ratio of the coating agent solution, the penetration enhancer solution and the protective agent solution is 10:1:1, and the volume ratio of the coating agent solution to the SARS-CoV-2 antigen mRNA solution in step (1) is 5:1.
The preparation method according to claim 1 is characterized in that in step (2), the freeze-drying includes pre-freezing, main drying and analysis; the pre-freezing time is 4-24 hours, the pre-freezing temperature is -80°C, the main drying time is 4-72 hours, the main drying temperature is -80°C to -10°C, and the gradient setting is; the analysis time is 2-24 hours, and the analysis temperature is -10°C to 50°C, and the gradient setting is.
A mucosal administration preparation prepared by the preparation method according to any one of claims 1 to 23.