Lipid nanoparticle composition comprising modified resiquimod and use thereof

Abstract

The present invention relates to a novel compound modified with resiquimod, a lipid nanoparticle composition comprising same, and a vaccine composition comprising same. More specifically, the vaccine composition comprising the lipid nanoparticles of the present invention has an excellent immune-enhancing effect and can effectively induce an antigen-specific immune response in nasal mucosa.

Description

Lipid nanoparticle composition comprising modified resiquimod and use thereof

[0001] The present invention relates to a lipid nanoparticle composition comprising a modified resiquimod and a vaccine composition comprising the same.

[0002] Nucleic acid-based medicines, which began about 40 years ago by injecting plasmid DNA into the body to help produce deficient proteins, have since been reported to include various types, including antigene, decoy, antisense, siRNA, and miRNA, which inhibit gene transcription and translation. Nucleic acid-based medicines target DNA or RNA, rather than proteins, and have garnered attention as personalized treatments through complementary binding with specific DNA or RNA sequences. Nucleic acid-based medicines are utilized not only as therapeutic agents but also as preventive agents that protect against diseases by injecting genes that can express antigens for specific diseases. Gene-based vaccines are categorized as DNA vaccines, RNA vaccines, and viral vector vaccines. Among them, RNA vaccines inject mRNA encoding antigens into the body, causing the antigens to be expressed in the body and inducing antibody formation. RNA vaccines do not carry the potential risks of viral vector-based vaccines, such as infection, or the genetic mutations of DNA vaccines. They also offer the advantage of rapid development, garnering attention as an effective response to the COVID-19 outbreak in 2019.

[0003] Due to the instability of these mRNA vaccines, there is a need for delivery materials that remain stable throughout the manufacturing, distribution, and in vivo delivery processes, while also being degraded without adverse effects. Currently, lipid nanoparticles (LNPs) perform this role, and are typically composed of a specific ratio of four components: ionized lipids, phospholipids (helper lipids), cholesterol (structural lipids), and PEGylated lipids. However, hypersensitivity reactions, including anaphylaxis, and unexpected immune responses have been reported with many PEG-containing formulations. Therefore, the development of novel formulations with fewer side effects and greater stability in the body is needed.

[0004] Resiquimod is a drug known to act as an immune response modulator and possesses antiviral and antitumor activity. Hua Zhang et al., J Control Release. 2021 Feb 10:330:1080-1094.

[0005] In , it is disclosed that liposomes containing resiquimod can be used as drug delivery vehicles.

[0006] The technical problem to be achieved by the present invention is to provide a lipid nanoparticle composition comprising a modified resiquimod.

[0007] Another technical task of the present invention is to provide a vaccine composition comprising the lipid nanoparticles.

[0008] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0009] To solve the above problem, the present invention provides a lipid nanoparticle composition comprising a compound represented by the following chemical formula 1, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof:

[0010]

[0011] The above R1 and R2 are each independently a saturated or unsaturated hydrocarbon having 6 to 22 carbon atoms.

[0012] According to one aspect, R1 and R2 are each independently an unsaturated hydrocarbon and may have 1 to 3 double bonds.

[0013] According to one side, the above R1 and R2 are identical to each other and can be expressed by the following chemical formula 2:

[0014]

[0015] According to one side, the compound may be an immunomodulator.

[0016] According to one aspect, the composition may further comprise one or more of oleate or excipients.

[0017] According to one aspect, the oleate may be a sodium salt, and the excipient may be glyceryl monooleate.

[0018] According to one aspect, the composition comprises the compound of the above chemical formula 1, an oleate salt and an excipient, and may comprise the compound in an amount of 10 to 60 ug / mL, the oleate salt in an amount of 0.5 to 1.0 mg / mL and the excipient in an amount of 60 to 150 ug / mL, and most preferably, the compound in an amount of 20 ug / mL, the oleate salt in an amount of 0.5 mg / mL and the excipient in an amount of 150 ug / mL.

[0019] According to one aspect, the lipid nanoparticle composition may contain a therapeutic or prophylactic agent therein.

[0020] According to one aspect, the therapeutic or prophylactic agent may be selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.

[0021] According to one aspect, the therapeutic or preventive agent is messenger RNA, and the lipid nanoparticle may have a diameter of 100 to 150 nm and an internal zeta potential of -60 to -40 mV.

[0022] According to another embodiment of the present invention, a vaccine composition comprising the lipid nanoparticle composition is provided. In one aspect, the vaccine composition may be administered nasally.

[0023] The novel compound modified with the resiquimod of the present invention can form lipid nanoparticles, stably maintain their structure, and has excellent physical stability.

[0024] In addition, a vaccine composition comprising the lipid nanoparticles can effectively induce an antigen-selective immune response in the nasal mucosa.

[0025] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0026] Figure 1 is ResiOA's 1 This is the H-NMR result.

[0027] Figure 2 shows the results of flow cytometry analysis of the degree of dendritic cell activation by Resiquimod and ResiOA.

[0028] Figure 3 shows an ultra-low temperature electron microscope image of the manufactured lipid nanoparticles and the results of dynamic light scattering particle size analysis.

[0029] Figure 4 shows the measurement of the zeta potential of the manufactured lipid nanoparticles.

[0030] Figure 5 shows the results of NanoFACS analysis of lipid nanoparticles.

[0031] Figure 6 is a fluorescence imaging result showing that an oleic acid-based transporter containing ResiOA is expressed nasally-specifically.

[0032] Figure 7 shows the results of an experiment to determine the optimal sodium oleate ratio that maximizes mRNA expression.

[0033] Figure 8 shows the results of an experiment to determine the optimal amount of ResiOA to maximize mRNA expression.

[0034] Figure 9 shows the results of an experiment to determine the amount of glyceryl monooleate that maximizes mRNA expression.

[0035] Figure 10 shows the results of a comparative experiment to determine the immune enhancement effect through the degree of activation (CD80+ / CD86+) of dendritic cells (CD11c+).

[0036] Figure 11 shows the results showing the concentration of IgA in response to ovalbumin in the nasal cavity.

[0037] In order to solve the above-described problem, the present inventors synthesized a novel immunomodulator by modifying the known immunomodulator Resiquimod, and confirmed that this acts as a lipid adjuvant in the production of lipid nanoparticles, thereby maintaining the spherical membrane shape of the lipid nanoparticles, and has a considerably excellent immune-enhancing effect and an effect of inducing a nasal mucosa-specific immune response, especially when composed of a vaccine composition to be sprayed into the nasal cavity. The present inventors intend to complete and provide this invention.

[0038] More specifically, the present invention provides a lipid nanoparticle composition comprising a compound represented by the following chemical formula 1, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof to solve the above problem:

[0039]

[0040] The above R1 and R2 are each independently a saturated or unsaturated hydrocarbon having 6 to 22 carbon atoms.

[0041] The compound of the above chemical formula 1 may preferably be synthesized from a resiquimod represented by the following chemical formula 1-1, and may be synthesized under the reaction conditions shown in Example 1 below.

[0042]

[0043] According to one aspect, R1 and R2 are each independently an unsaturated hydrocarbon and may have 1 to 3 double bonds.

[0044] The term “saturated” used in the present invention means that the hydrocarbon is composed of only single bonds, and the term “unsaturated” used in the present invention means that the hydrocarbon contains at least one double bond or triple bond among carbon-carbon bonds.

[0045] The above R1 and R2 are preferably C each independently having one double bond. 18 It may be a hydrocarbon, and the double bond may be formed between the 9th carbon and the 10th carbon of the hydrocarbon, and most preferably may be expressed by the following chemical formula 2:

[0046]

[0047] According to one side, the compound may be an immunomodulator.

[0048] The terms "immunomodulator," "adjuvant," or "immunopotentiator" as used herein refer to a substance that enhances the immune response induced by an antigen. An immunopotentiator can enable a vaccine to exhibit the same efficacy with a small amount of antigen, or can induce a favorable immune response through a mechanism known or unknown in the art. In particular, the immunomodulator in the present invention not only enhances the immune response in the body, but also enables a vaccine action specific to the nasal mucosa and performs a role similar to lipids, thereby forming lipid nanoparticles.

[0049] According to one aspect, the composition may further comprise one or more of an oleate salt or an excipient. Preferably, the oleate salt may be a sodium salt, and the excipient may be glyceryl monooleate. As an excipient, glyceryl monooleate may increase the physical stability of lipid nanoparticles, and the effects thereof are as disclosed in Example 5, etc.

[0050] According to one aspect, the composition comprises the compound of the above chemical formula 1, an oleate, and an excipient, and may comprise the compound in an amount of 10 to 60 ug / mL, the oleate in an amount of 0.5 to 1.0 mg / mL, and the excipient in an amount of 60 to 150 ug / mL, and most preferably the compound in an amount of 20 ug / mL, the oleate in an amount of 0.5 mg / mL, and the excipient in an amount of 150 ug / mL. In the above concentration range, the nasal-specific antigen expression effect was excellent, as shown in Examples 5 and 6, etc.

[0051] According to one aspect, the lipid nanoparticle composition may contain a therapeutic or prophylactic agent therein. According to one aspect, the therapeutic or prophylactic agent may be selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0052] According to one aspect, the therapeutic or prophylactic agent may most preferably be messenger RNA, and the lipid nanoparticle may have a diameter of 100 to 150 nm and an internal zeta potential of -60 to -40 mV.

[0053] According to another embodiment of the present invention, a vaccine composition comprising the lipid nanoparticle composition is provided. The vaccine composition may preferably be an mRNA vaccine and may further comprise a pharmaceutically acceptable salt or adjuvant.

[0054] According to one aspect, the route of administration of the vaccine composition of the present invention may be, for example, oral or parenteral. Here, parenteral refers to a broad route of administration, and includes, for example, intranasal, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intranasal, sublingual, intrathecal (intrathecal), inhalation, ocular, rectal, vaginal, and ventricular administration, but most preferably, it may be administered by spraying into the nasal cavity. The lipid nanoparticles containing the compound represented by the chemical formula 1 of the present invention can be fixed to the nasal mucosa when administered by spraying into the nasal cavity, and can effectively induce an immune response specific to the nasal mucosa. This can be helpful for effective vaccine action by inducing an immune response in the nasal mucosa, which is the primary antigen introduction route in respiratory infectious diseases, and in particular, can increase the amount of antigen-specific IgA expression in the nasal mucosa.

[0055] When formulating the above composition, it is manufactured using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0056] Solid preparations for oral administration include tablets, tablets, powders, granules, capsules, troches, etc., and these solid preparations are prepared by mixing one or more compounds according to the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included.

[0057] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, etc.

[0058] Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0059] The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to administer an amount that achieves the maximum effect with the minimum amount without causing side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0060]

[0061] The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0062] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0063] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0064]

[0065] Example 1. Synthesis of ResiOA

[0066] ResiOA of the present invention was synthesized as follows. 1.00 g of oleyl alcohol (3.725 mmol) was dissolved in 10 mL of dichloromethane in a 50 mL round-bottom flask and the mixture was kept on ice. A 15 wt% phosgene solution (2.66 mL, 3.725 mmol) was carefully added to the flask, followed by the gradual addition of 0.519 mL of triethylamine (3.725 mmol) to the precooled solution. The reaction mixture was stirred at room temperature for an additional 2 hours. After the reaction, the mixture was rapidly extracted with ice water, the organic layer was separated, and then dried over magnesium sulfate. The solvent was evaporated from the organic layer to obtain oleyl chloroformate.

[0067] In a 20 mL brown vial, 20 mg of resiquimod (63.6 μmol), 20 mg of potassium carbonate (140 μmol), and 5 mL of chloroform (CHCl3) were added and reacted for 3 hours under an argon atmosphere. Oleyl chloroformate (42.1 mg, 127 μmol) was added to the solution and stirred at room temperature for 24 hours. Afterwards, the reaction solvent was evaporated under reduced pressure to obtain a residue. This residue was subjected to column chromatography with a ratio of 5:1 of methylene chloride and ethyl acetate to finally obtain ResiOA. The resulting ResiOA 1 The H-NMR results are shown in Figure 1.

[0068]

[0069] Example 2. Comparison of the efficacy equivalence of Resiquimod and ResiOA

[0070] To compare the efficacy equivalence of Resiquimod and ResiOA of the present invention, the immune-enhancing effect was compared through the degree of dendritic cell activation as follows. Bone marrow cells were isolated from the femurs of 10-week-old Balb / C male mice and cultured in RPMI1640 medium supplemented with 10% FBS, 10 mM HEPES, 50 μM β-mercaptoethanol, 20 ng / mL GM-CSF, and 20 ng / mL IL-4. The medium was changed once every two days. After culturing for 7 days, dendritic cells were selected and cultured in a 24-well plate with 1 νg / mL of LPS as a positive control, 5, 10, and 20 νM ResiOA, and 5, 10, and 20 νN for 24 hours. Afterwards, it was stained with FITC-CD11c antibody, PE-CD86, and PE-Cy5-CD80 antibody for 20 minutes at 4°C, and the results were measured by flow cytometry. The results are shown in Fig. 2. It was confirmed that activated dendritic cells positive for CD80 and CD86 increased in ResiOA to a similar degree as that of Resiquimod, confirming that ResiOA has an immune-enhancing effect at a similar level as that of Resiquimod.

[0071]

[0072] Example 3. Preparation of mRNA lipid nanoparticles

[0073] The nasal vaccine containing ResiOA of the present invention was prepared as follows. Sodium oleate (0.3 mg / mL) was dissolved in NFW, and ResiOA was dissolved in ethanol. The ratio of water to ethanol was maintained at 99:1 (v / v%). The final concentration of mRNA was set at 0.3 mg / mL. The final concentrations of ResiOA and glyceryl monooleate were prepared at 300 νη / νM and 150 νη / νM, respectively. Glyceryl monooleate was included to improve the stability of lipid nanoparticles. OA-NPs were prepared by vortexing sodium oleate at 25°C (room temperature) for 10 minutes.

[0074] OA-mRNA NPs were prepared by mixing sodium oleate and mRNA at a weight ratio of 5:1 and vortexing at 25°C (room temperature) for 10 minutes. OA-mRNA-ResiOA NPs were prepared by mixing sodium oleate and mRNA at a weight ratio of 5:1 and ResiOA dissolved in ethanol and vortexing at 25°C (room temperature) for 10 minutes.

[0075] OA-mRNA-ResiOA-GMO NPs were prepared by mixing sodium oleate and mRNA in a weight ratio of 5:1 and ResiOA and glyceryl monooleate dissolved in ethanol, and vortexing at 25 degrees (room temperature) for 10 minutes.

[0076]

[0077] Example 4. Characterization of manufactured lipid nanoparticles

[0078] The cryo-TEM (Cryo-TEM, Talos L120C, FEI, NL) image of the manufactured lipid nanoparticles is shown in Fig. 3a, and the size was analyzed using dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern, UK) and shown in Fig. 3b. From the left, Figs. 3a and 3b show nanoparticles manufactured only with oleic acid, nanoparticles manufactured with oleic acid and mRNA, and nanoparticles composed of oleic acid, mRNA, and ResiOA. In the case of OA NP and OA-mRNA NP, the sizes were 79.6 ± 4.3 nm and 79.5 ± 9.3 nm, respectively, and were observed in the form of a multivesicular vesicle (MVV) type, while OA-mRNA-ResiOA NP was observed in the form of a solid-lipid nanoparticle type with a size of 122.7 ± 12.2 nm.

[0079] The manufactured lipid nanoparticles were diluted to 1 / 10 and the zeta potential was measured using a zeta potential meter (Zetasizer Nano ZS, Malvern, UK) at 25 degrees (room temperature), and the results are shown in Fig. 4.

[0080] To confirm the mRNA encapsulation efficiency in nanoparticles, the NanoFACS analysis results of lipid nanoparticles manufactured using fluorescently labeled mRNA and lipid, respectively, are shown in Figure 5.

[0081] Specifically, OA-NP rhodamine was prepared by the same method as described in Example 3, including 5% of the total lipid as rhodamine-labeled lipid. Free mRNA-Cy5 is Cy5-labeled mRNA, and OA-mRNA NP is a nanoparticle prepared by the same method as described in Example 3 using both rhodamine-labeled lipid and Cy5-labeled mRNA. All samples were identified as nano-sized particles by detecting scattered light at 405 nm using NanoFACS (Cytoflex, Beckman Coulter, USA), and the fluorescence of the nanoparticles was analyzed through the rhodamine and Cy5 fluorescence channels. OA-NP rhodamine was a nanoparticle, and only the rhodamine fluorescence signal was detected, while the Cy5 fluorescence signal was not detected. Free mRNA Cy5 was theoretically less than several nanometers in size, so it was not identified as a nanoparticle based on the specifications of the NanoFACS used, and thus no fluorescence signal was detected. OA-mRNA NPs are nanoparticles, and 74.21% of particles were detected to exhibit simultaneous rhodamine and Cy5 fluorescence signals. This confirmed that 74.21% of mRNA was encapsulated in oleic acid-based nanoparticles.

[0082]

[0083] Example 5. Animal testing

[0084] To confirm the nasal-specific protein expression level of the lipid nanoparticles of the present invention and to optimize the protein expression amount in the nasal cavity, luciferase mRNA (CleanCap FLuc mRNA, Trilnk Biotechnology, US) was used, and D-luciferin was used as a substrate for the expressed luciferase to compare the protein expression amount through bioluminescence imaging.

[0085] Two lipid nanoparticle formulations used in animal experiments were prepared as OA-mRNA NP and OA-mRNA-ResiOA NP in Example 3. As controls, commercially available in vivo jetRNA and lipofectamine were prepared using the provided method, and LNP particles for Moderna vaccine composition (50% SM-102, 10% DSPC, 1.5% DMG-PEG2000, 38.5% cholesterol) were prepared using Microfluidics (the NanoAssembler Spark TM , PNI, US) equipment was used. The above five compositions were administered intranasally and intramuscularly to 10-week-old Balb / c male mice. 1 ug of mRNA was administered intranasally, and 10 ug of mRNA was administered intramuscularly. 3 hours after administration, 0.9 mg of D-luciferin was injected intranasally, and 3 mg was injected intravenously. The bioluminescence signal level was measured and shown in Fig. 6. It was confirmed that the oleic acid-based delivery system of the present invention, particularly the delivery formulation containing ResiOA, was expressed specifically in the nasal cavity.

[0086] Next, in order to optimize the antigen expression efficiency in the in vivo nasal cavity, the optimal sodium oleate ratio that maximizes mRNA expression was found during the LNP manufacturing step, and experiments were conducted with the ratio of sodium oleate to mRNA ranging from 1:1 to 20:1. The experimental results are shown in Fig. 7, and the best results were obtained when the sodium oleate to mRNA ratio was 5:1 (wt / wt%).

[0087] Likewise, in order to maximize antigen expression efficiency within the nasal cavity while incorporating the maximum amount of ResiOA, we sought to find the optimal ResiOA composition during the manufacturing stage of LNPs. The sodium oleate to mRNA ratio was fixed at 5:1 (wt / wt%), and particles were manufactured by varying the amount of ResiOA from 0 to 600 ug / mL, and the protein expression level in the nasal cavity of a small animal model was observed. The experimental results are shown in Fig. 8, and it was shown that when 10 ug / mL of ResiOA was included in the particle, the expression level of the antigen in the nasal cavity increased by more than 3-fold compared to when ResiOA was not included in the particle. However, there was no significant difference in the expression level of luciferase in the nasal cavity in the range of 10 to 300 ug / mL of ResiOA.

[0088] In addition, to maximize antigen expression efficiency in the nasal cavity in vivo, we sought to find the optimal composition of glyceryl monooleate for increased particle stability. The sodium oleate and mRNA ratio was fixed at 5:1 (wt / wt%), ResiOA 60 ug / mL, and glyceryl monooleate was varied from 0 to 600 ug / mL to find the optimal composition. The experimental results are shown in Fig. 9, and it was confirmed that the antigen expression efficiency in the nasal cavity was maximized when 150 ug / mL of glyceryl monooleate was included.

[0089]

[0090] Example 6. Confirmation of in vivo effects

[0091] To induce a nasal mucosal-specific immune response, Ova-mRNA (CleanCap OVA mRNA, Trilnk Biotechnology, US) for expression of the model antigen ovalbumin was used. The immune enhancement effect of the optimized nasal vaccine was compared through the degree of dendritic cell (CD11c+) activation (CD80+ / CD86+), and the results are shown in Figure 10. Bone marrow cells were isolated from the femurs of 10-week-old Balb / C male mice and cultured in RPMI 1640 medium supplemented with 10% FBS, 10 mM HEPES, 50 μM β-mercaptoethanol, 20 ng / mL GM-CSF, and 20 ng / mL IL-4. The medium was changed once every two days. After 7 days of culture, dendritic cells were selected and cultured in 24-well plates with positive control groups (LPS 1 ug / mL, ResiOA 1 ug / mL, OTP LNP 1 ug / mL, Free OVA mRNA, and OA-mRNA-GMO NP) for 24 hours. Afterwards, they were stained with FITC-CD11c antibody, PE-CD86, and PE-Cy5-CD80 antibodies for 20 minutes at 4°C, and the results were measured by flow cytometry.

[0092] As a result, the optimized intranasal vaccine platform of the present invention (4. OPT LNP in FIG. 10) showed an immune-enhancing effect similar to that of the positive control group (No. 3, chemically modified resiquimod (ResiOA)) and an immune-enhancing effect of more than 50% compared to the negative control groups (Nos. 5 and 6). In addition, it was confirmed that the immune-enhancing effect of ResiOA was maintained even after particle formation.

[0093] Afterwards, to confirm whether antigen-selective antibodies were produced in the body, the following experiment was conducted. The optimized nasal vaccine of the present invention was inoculated according to the inoculation schedule (first inoculation on day 0, boost inoculation on days 7 and 14, and sacrifice on day 21), and 100 ug of mRNA was administered intranasally to the animal model at 33.3 uL / day. On day 21, the concentration of IgA against ovalbumin in the nasal cavity was measured to confirm the production of antigen-selective antibodies in the nasal cavity, and the results are shown in Fig. 11. As a result, the optimized nasal vaccine of the present invention showed a very high nasal expression amount of selective IgA against the antigen (ovalbumin) by more than twice that of the control group.

[0094]

[0095] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0096] Therefore, other implementations, other manufacturing examples and equivalents to the patent claims also fall within the scope of the claims described below.

[0097]

[0098] This invention was completed with the support of the Future Source Medical Technology Development Project - Development of Theragnosis Technology for Controlling Intractable Diseases (2E32330:20230101~20231231:) and Bridge Program=>(BP) Development of Novel Gene Therapy Delivery System (2V09510:20230101~20230831: Main Invention Project), and the National Research Project of the Ministry of Science and ICT, Nano Future Material Source Technology Development Project - Development of Next-Generation Anticancer Technology Based on Bacterial Shuttle-Induced Nanomaterial and Peptide Light Source (2N72040:20230101~20231231:).

Claims

1. A lipid nanoparticle composition comprising a compound represented by the following chemical formula 1, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof: The above R1 and R2 are each independently a saturated or unsaturated hydrocarbon having 6 to 22 carbon atoms.

2. In paragraph 1, A lipid nanoparticle composition, wherein R1 and R2 are each independently an unsaturated hydrocarbon having 1 to 3 double bonds.

3. In paragraph 1, The above R1 and R2 are the same, and the lipid nanoparticle composition is represented by the following chemical formula 2:

4. In paragraph 1, The above compound is an immunomodulatory agent, a lipid nanoparticle composition.

5. In paragraph 1, A lipid nanoparticle composition, characterized in that the composition further comprises at least one of oleate and excipients.

6. In paragraph 5, A lipid nanoparticle composition wherein the above oleate is a sodium salt.

7. In paragraph 5, A lipid nanoparticle composition wherein the excipient is glyceryl monooleate.

8. In any one of paragraphs 1 to 7, A lipid nanoparticle composition comprising the compound of the above chemical formula 1, an oleate, and an excipient, wherein the compound is contained in an amount of 10 to 60 ug / mL, the oleate is contained in an amount of 0.5 to 1.0 mg / mL, and the excipient is contained in an amount of 60 to 150 ug / mL.

9. In paragraph 8, A lipid nanoparticle composition comprising the compound of the above chemical formula 1, an oleate, and an excipient, wherein the compound is contained in an amount of 20 ug / mL, the oleate is contained in an amount of 0.5 mg / mL, and the excipient is contained in an amount of 150 ug / mL.

10. In paragraph 8, A lipid nanoparticle composition comprising a therapeutic or preventive agent therein.

11. In paragraph 10, A lipid nanoparticle composition, characterized in that the therapeutic or prophylactic agent is selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.

12. In paragraph 11, The above therapeutic or prophylactic agent is messenger RNA, A lipid nanoparticle composition, characterized in that the lipid nanoparticle has a diameter of 100 to 150 nm and an internal zeta potential of -60 to -40 mV.

13. A vaccine composition comprising the lipid nanoparticle composition of Article 12.

14. A vaccine composition according to claim 13, wherein the vaccine composition is administered nasally.

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