Optogenetic Anti-inflammatory composition containing nanoparticles including agent for inhibiting formation of NLRP3 inflammasome as active ingredient

Abstract

The present invention relates to an optogenetic anti-inflammatory composition containing, as an active ingredient, nanoparticles including an agent for inhibiting the formation of the nod-like receptor protein 3 (NLRP3) inflammasome, wherein the nanoparticles contain: a vector including a polynucleotide encoding a truncated version of cryptochrome-interacting basic-helix-loop-helix 1 (CIBN)-NLRP3 fusion protein; and a vector including a polynucleotide encoding the cryptochrome 2 (CRY2) protein or a variant thereof. The optogenetic anti-inflammatory composition has excellent anti-inflammatory activity due to non-invasive light stimulation and can thus be highly effectively used in related fields.

Description

Optogenetic anti-inflammatory composition containing nanoparticles as an active ingredient, which include an agent that inhibits the formation of NLRP3 inflammasome

[0001] The present invention relates to an optogenetic anti-inflammatory composition containing nanoparticles as an active ingredient, which include an agent that inhibits the formation of NLRP3 (nod-like receptor protein 3) inflammasome.

[0002]

[0003] This work was supported by the National Research Foundation of Korea (NRF) under the Ministry of Science and ICT's Individual Basic Research Program (Project No. 2022R1A2B5B02001886) and the National Institute for Basic Science Research Operation Support Program (Project No. IBS-R001-D2-2023-A00) under the Ministry of Science and ICT's Basic Science Research Program (Project No. 2022R1A2B5B02001886).

[0004] Inflammation is one of the most common immune responses that occurs when the body is abnormal. It functions to minimize tissue damage and eliminate infectious agents through the body's immune cells when exposed to external stimuli such as bacteria and viruses or internal stimuli such as increased toxins. Various studies have shown that microglia and macrophages are activated in various inflammatory situations in the body, including brain inflammation, and are known to induce inflammatory responses. Recently, it has been reported that peripheral nerve injury activates microglia in the spinal cord, and that inflammatory responses such as the secretion of inflammatory cytokines play a key role in the development of neuropathic pain.

[0005] Inflammasome is a protein complex that mediates inflammatory responses and is activated by stimulation from external PAMPs (Pathogen-associated molecular patterns) or internal DAMPs (Damage-associated molecular patterns). In particular, the NLRP3 (nod-like receptor protein 3) inflammasome is currently the most fully characterized inflammasome and is the most widely studied as a major mechanism of inflammatory factor secretion by microglia.

[0006] Recently, various optogenetic control technologies have been developed. Existing optogenetic technologies have been studied in the direction of increasing or decreasing the excitability of neurons by activating ion channels with light. Recently, various studies are being conducted to graft optogenetic technologies to microglia or astrocytes in addition to neurons. However, the development of practically applicable technologies has been hindered due to difficulties in producing vectors sensitive to photostimulation, difficulties in developing implant technologies for in vivo gene delivery for photostimulation, and the lack of methods for delivering vectors to specific target cells. The present invention aims to overcome the shortcomings of general anti-inflammatory agents that act throughout the body by developing a technology that can alleviate inflammatory responses that occur in various body parts such as the brain, muscles, knees, and waist by inducing photostimulation at a desired site and time using such optogenetic technology.

[0007] Meanwhile, Korean Patent Registration No. 2212997 discloses a 'composition for improving, preventing, or treating chronic inflammatory diseases caused by overactivation of NLRP3 inflammasome containing burdock extract as an active ingredient', and Korean Patent Publication No. 2022-0144908 discloses an 'NLRP3 inflammasome inhibitory peptide for treating inflammatory diseases', but there is no disclosure regarding the 'optogenetic anti-inflammatory composition containing nanoparticles containing an agent that inhibits the formation of NLRP3 inflammasome as an active ingredient' of the present invention.

[0008] The present invention was derived from the above-mentioned needs, and the present inventors treated microglial cells with a vector including a polynucleotide encoding a CIBN (truncated version of cryptochrome-interacting basic-helix-loop-helix 1) -NLRP3 (nod-like receptor protein 3) fusion protein; and a vector including a polynucleotide encoding a CRY2 (cryptochrome 2) protein or a variant thereof, and after treating with LPS (Lipopolysaccharide) and inducing light stimulation, they confirmed that the expression of inflammatory cytokines was reduced compared to a control group that was not inducible by light stimulation. In addition, the vector including a polynucleotide encoding a CIBN-NLRP3 fusion protein; And a vector including a polynucleotide encoding a CRY2 protein or a variant thereof; After manufacturing a nanoparticle containing the vector, the nanoparticle was injected into a neuropathic pain animal model, and then photo-stimulation was induced. As a result, a significant analgesic effect was confirmed compared to a control group that did not induce photo-stimulation, thereby completing the present invention.

[0009] To solve the above problem, the present invention provides an optogenetic anti-inflammatory pharmaceutical composition comprising nanoparticles as an active ingredient, the nanoparticles comprising a vector comprising a polynucleotide encoding a CIBN (truncated version of cryptochrome-interacting basic-helix-loop-helix 1)-NLRP3 (nod-like receptor protein 3) fusion protein; and a vector comprising a polynucleotide encoding a CRY2 (cryptochrome 2) protein or a variant thereof.

[0010] In addition, the present invention provides a method for suppressing inflammation through optogenetic stimulation, comprising the steps of: injecting the pharmaceutical composition into a subject other than a human; and irradiating a photo-stimulation.

[0011] In addition, the present invention provides an optogenetic anti-inflammatory medical device using the pharmaceutical composition.

[0012] In addition, the present invention provides an optogenetic anti-inflammatory veterinary composition comprising nanoparticles as an active ingredient, wherein the nanoparticles comprise a vector comprising a polynucleotide encoding a CIBN-NLRP3 fusion protein; and a vector comprising a polynucleotide encoding a CRY2 protein or a variant thereof.

[0013] In addition, the present invention provides a nanoparticle comprising a vector comprising a polynucleotide encoding a CIBN-NLRP3 fusion protein; and a vector comprising a polynucleotide encoding a CRY2 protein or a variant thereof.

[0014] The present invention relates to an optogenetic anti-inflammatory composition comprising, as an active ingredient, nanoparticles including an agent that inhibits the formation of NLRP3 inflammasome, wherein the nanoparticles include a vector including a polynucleotide encoding a CIBN (truncated version of cryptochrome-interacting basic-helix-loop-helix 1)-NLRP3 (nod-like receptor protein 3) fusion protein; and a vector including a polynucleotide encoding a CRY2 (cryptochrome 2) protein or a variant thereof, and since the nanoparticles have excellent anti-inflammatory activity by non-invasive photo-stimulation, they can be very usefully used in related fields.

[0015] Figure 1 shows the results of confirming CIBN-CRY2 binding by mixing CIBN-NLRP3 vector and CRY2-mRuby3 vector (A) at various ratios and treating HEK293T cells, followed by confirmation of mRuby3 fluorescence signal through fluorescence microscopy (C, D), 3D image analysis through IMARIS software (E), and video analysis through confocal microscopy (F).

[0016] Figure 2 shows the results of analyzing the anti-inflammatory effect after treating BV2 cells with the CIBN-NLRP3 vector and the CRY2-mRuby3 vector at a ratio of 1:3, measuring the expression level of the IL-1β gene under conditions of LPS (Lipopolysaccharide) treatment after light stimulation (A, B) and measuring the expression level of the IL-1β gene under conditions of light stimulation after pretreatment with LPS (C, D). **:p<0.01, ***:p<0.001.

[0017] Figure 3 shows the results of analyzing the anti-inflammatory effect after treating RAW264.7 cells with the CIBN-NLRP3 vector and the CRY2-mRuby3 vector at a ratio of 1:3. The results are the results of measuring the expression level of the IL-1β gene under light-stimulated conditions after pretreatment with LPS. CTL is the untreated control group. *:p<0.05.

[0018] Figure 4 shows the results of measuring the size (A) and zeta potential value (B) of PLGA nanoparticles loaded with CIBN-NLRP3 vector and CRY2-mRuby3 vector at a ratio of 1:3, observing the shape through scanning electron microscopy (SEM) (C), analyzing the toxicity in BV2 cells (D), and measuring the amount of CIBN-CRY2 DNA released from the nanoparticles (E).

[0019] Figure 5 shows the results of measuring the pain threshold using von Frey filaments after performing SNI (spared nerve injury) surgery on mice to produce a neuropathic pain animal model (A), and analyzing the expression of Iba1 protein (B), inflammatory cytokine expression (C), anti-inflammatory cytokine expression (D), and microglial morphology (E to J) using Sholl analysis in microglia isolated from the spinal cord of mice 14 days after surgery. Sham: Control group that underwent surgery in the same way as the experimental group but did not induce SNI, POD: postoperative day, it: intrathecal. *:p<0.05, **:p<0.01, ***:p<0.001.

[0020] Figure 6 shows the evaluation of the analgesic efficacy according to the injection of CIBN-NLRP3 / CRY2-mRuby3 PLGA nanoparticles and invasive optical stimulation in a neuropathic pain animal model. 5A is a schematic diagram of the experimental plan, and 5B and 5C are the results of measuring the pain threshold using von Frey filament. POD: postoperative day, it: intrathecal.

[0021] Figure 7A is a photograph showing a blue light LED plate attached to the cage ceiling used to induce non-invasive photostimulation, 7B is a schematic diagram of an experimental plan to evaluate the analgesic efficacy according to nanoparticle injection and non-invasive photostimulation in a neuropathic pain animal model, 7C is a result of measuring the pain threshold value using von Frey filament by CIBN-NLRP3 / CRY2-mRuby3 PLGA nanoparticle injection and invasive photostimulation and CIBN-NLRP3 / CRY2 (E281A, A9) PLGA nanoparticle injection and non-invasive photostimulation, 7D is a schematic diagram of an experimental plan to compare the analgesic efficacy according to the nanoparticle injection frequency and photostimulation time in a neuropathic pain animal model, 7E and 7F are the results of measuring the pain threshold value according to the nanoparticle injection frequency and photostimulation time in a neuropathic pain animal model, and 7G is This is the result of confirming the expression of Iba1 protein in microglia. POD: postoperative day, it: intrathecal.

[0022] Figure 8 is a schematic diagram showing the process in which inflammasome formation is inhibited and the formation of inflammatory cytokines (IL-1β) is inhibited when photo-stimulation is applied to nanoparticles containing the CIBN-NLRP3 protein and CRY2 protein of the present invention.

[0023] The present invention provides an optogenetic anti-inflammatory pharmaceutical composition comprising nanoparticles as an active ingredient, wherein the nanoparticles comprise a vector comprising a polynucleotide encoding a CIBN (truncated version of cryptochrome-interacting basic-helix-loop-helix 1)-NLRP3 (nod-like receptor protein 3) fusion protein; and a vector comprising a polynucleotide encoding a CRY2 (cryptochrome 2) protein or a variant thereof.

[0024] In the optogenetic anti-inflammatory pharmaceutical composition of the present invention, the CIBN-NLRP3 fusion protein may be composed of an amino acid sequence of SEQ ID NO: 1, and the CRY2 protein or variant thereof may be composed of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, respectively, but is not limited thereto.

[0025] The above CRY2 variant is a mutant CRY2 (E281A, A9) protein in which glutamic acid, the 281st amino acid of the CRY2 protein, is replaced with alanine and 9 amino acids are added to the C-terminus, and is characterized by improved light sensitivity.

[0026] In addition, in the optogenetic anti-inflammatory pharmaceutical composition of the present invention, the nanoparticles may be PLGA (poly(lactic-co-glycolic acid)) nanoparticles, and a vector including a polynucleotide encoding the CIBN-NLRP3 fusion protein and a vector including a polynucleotide encoding the CRY2 protein or a variant thereof may be included in a weight ratio of 1:2.5 to 3.5, preferably 1:3, but is not limited thereto.

[0027] In the optogenetic anti-inflammatory pharmaceutical composition of the present invention, the anti-inflammation may be general anti-inflammation, and preferably may be inflammation caused by a chronic pain disease, but is not limited thereto.

[0028] The above "chronic pain disease" means a rather severe pain affecting one or more parts of the body and lasting for more than three months, and includes a pain syndrome whose intensity may change over time, and is preferably at least one disease selected from the group consisting of neuropathic pain, chronic pain due to arthritis, complex regional pain syndrome, post-herpetic neuralgia, and pain syndrome after spinal surgery, but is not limited thereto.

[0029] The above "neuropathic pain" refers to a chronic neurological disease caused when the nervous system is damaged by various causes such as inflammation, trauma, ischemic damage, or metabolic products, and may be induced by peripheral nerve damage or central nerve damage, and preferably includes at least one disease selected from the group consisting of peripheral nervous system abnormalities or damage, multiple sclerosis including spinal nerve damage, spinal cord injury, hyperalgesia, hyperesthesia, neuropathy, diabetic neuropathy, neuritis, neuralgia, causalgia, allodynia, postherpetic neuralgia, lumbar nerve root compression, pain associated with cancer, alcoholism, atypical facial pain, herpetic neuralgia, post-stroke pain, HIV-associated neuropathy, osteoarthritis, and phantom limb pain, but is not limited thereto.

[0030] In addition, the nanoparticles containing the vector including the polynucleotide encoding the CIBN-NLRP3 fusion protein and the vector including the polynucleotide encoding the CRY2 protein or a variant thereof may target one or more species selected from the group consisting of microglia cells, neuronal cells, and astrocytic cells, but are not limited thereto.

[0031] In the optogenetic anti-inflammatory pharmaceutical composition of the present invention, the term “optogenetic” preferably means activation by non-invasive optical stimulation, and the optical stimulation may be, but is not limited to, blue light of 470 nm.

[0032] The pharmaceutical composition according to the present invention is preferably in the form of any one selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols, but is not limited thereto. In addition to the above-described active ingredients, the composition may further include a pharmaceutically acceptable carrier, excipient, or diluent. Examples of the carrier, excipient, or diluent that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0033] The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The pharmaceutical composition of the present invention may be administered via any generally accepted route as long as it can reach the target tissue.

[0034] The present invention also provides a method for suppressing inflammation through optogenetic stimulation, comprising the steps of: injecting the pharmaceutical composition into a non-human subject; and irradiating the subject with light stimulation.

[0035] In the method of suppressing inflammation through optogenetic stimulation of the present invention, the optogenetic stimulation may be, but is not limited to, irradiation with blue light of 470 nm.

[0036] The method of suppressing inflammation through optogenetic stimulation of the present invention is characterized in that it can treat chronic pain diseases by reducing pain due to suppression of inflammation.

[0037] In the method of suppressing inflammation through optogenetic stimulation of the present invention, the injection may preferably be an injection into an inflamed area, and more preferably an intrathecal or intravenous injection, but is not limited thereto.

[0038] The present invention also provides an optogenetic anti-inflammatory medical device using the pharmaceutical composition.

[0039] The optogenetic anti-inflammatory medical device of the present invention may induce an anti-inflammatory effect by injecting a pharmaceutical composition according to the present invention and then irradiating it with light stimulation. The light stimulation may be irradiating blue light of 470 nm, but is not limited thereto.

[0040] The present invention also provides an optogenetic anti-inflammatory veterinary composition comprising nanoparticles as an active ingredient, wherein the nanoparticles comprise a vector comprising a polynucleotide encoding a CIBN (truncated version of cryptochrome-interacting basic-helix-loop-helix 1)-NLRP3 (nod-like receptor protein 3) fusion protein; and a vector comprising a polynucleotide encoding a CRY2 (cryptochrome 2) protein or a variant thereof.

[0041] In the optogenetic anti-inflammatory veterinary composition of the present invention, the CIBN-NLRP3 fusion protein and the CRY2 protein or a variant thereof are as described above.

[0042] The veterinary composition of the present invention may further comprise suitable excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil. The veterinary composition according to the present invention may further include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc., and the veterinary composition according to the present invention may be formulated using methods well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile topical preparations, etc.

[0043] The effective amount of the veterinary composition according to the present invention can be appropriately selected depending on the individual animal. This may be determined based on factors including the severity of the disease or condition, the individual's age, weight, health status, or sex, sensitivity to the active ingredient of the present invention, the route of administration, the duration of administration, other compositions combined with or used concurrently with the composition, and other factors well known in the fields of physiology and veterinary medicine.

[0044] The present invention also provides a nanoparticle comprising a vector comprising a polynucleotide encoding a CIBN (truncated version of cryptochrome-interacting basic-helix-loop-helix 1)-NLRP3 (nod-like receptor protein 3) fusion protein; and a vector comprising a polynucleotide encoding a CRY2 (cryptochrome 2) protein or a variant thereof.

[0045]

[0046] Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0047]

[0048] Example 1. Establishment of optimal conditions for CIBN-CRY2 bond formation induced by photostimulation.

[0049] In order to determine the conditions under which the binding of CIBN protein and CRY2 protein is most effectively formed when there is a photo-stimulation, the CIBN-NLRP3 vector and the CRY2-mRuby3 vector (Fig. 1A; Nanoglia, Korea) were mixed at various ratios and treated into HEK293T cells, and then the binding of CIBN-CRY2 was confirmed. In a state where the CIBN-NLRP3 fusion protein and CRY2 protein were expressed by the vector according to the present invention, a photo-stimulation was applied so that the CIBN protein and CRY2 protein bind to form clusters of the CIBN-NLRP3-CRY2 protein, thereby suppressing the formation of the NLRP3 inflammasome (Fig. 1B).

[0050] Specifically, HEK293T cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (Fetal Bovine Serum) and 1% antibiotics at 37°C and 5% CO2, seeded in 6-well plates, and transfected with CIBN-NLRP3 vector and CRY2-mRuby3 vector at a weight ratio of 1:1, 1:2, 1:3, 2:1, or 3:1, respectively. Afterwards, while maintaining the cell culture conditions, photostimulation was performed for 30 minutes or 2 hours using a 470 nm blue light LED plate (10 cm, 40 mW, Thorlabs), and the mRuby3 fluorescence signal was confirmed through a fluorescence microscope (Axio scope 5, Carl Zeiss). 3D images were analyzed using IMARIS software, and video analysis was performed using a confocal microscope.

[0051] As a result, it was confirmed that the CIBN-CRY2 binding was formed the most and aggregated in the experimental group transfected with the CIBN-NLRP3 vector and CRY2-mRuby3 vector at a ratio of 1:3 under the light stimulation condition for 30 minutes (Fig. 1C). In addition, to confirm the persistence of the CIBN-CRY2 binding, after inducing binding with light stimulation for 2 hours and then turning off the light and re-observing 2 hours later, it was confirmed that the binding was still maintained under the condition of transfecting the CIBN-NLRP3 vector and CRY2-mRuby3 vector at a ratio of 1:3 (Fig. 1D). As a result of additionally confirming the cells transfected with the CIBN-NLRP3 vector and CRY2-mRuby3 vector at a ratio of 1:3 through 3D image analysis and video analysis, it was confirmed that the binding and aggregation of CIBN-CRY2 were induced to form puncta (Fig. 1E, 1F).

[0052]

[0053] Example 2. Analysis of the anti-inflammatory effect due to CIBN-CRY2 bond formation induced by photostimulation.

[0054] 2-1. Analysis of anti-inflammatory effects under LPS treatment conditions after photostimulation

[0055] Microglia cell line BV2 cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics at 37°C and 5% CO2, seeded in 6-well plates, and transfected with a mixture of CIBN-NLRP3 vector and CRY2-mRuby3 vector at a ratio of 1:3.

[0056] The day after transfection of CIBN-NLRP3 vector and CRY2-mRuby3 vector, inflammatory response was induced by photostimulation for 2 h using a 470 nm blue light LED plate, followed by treatment with 0.5 μg / ml LPS (Lipopolysaccharide) for 0.5, 2, or 12 h, respectively (Fig. 2A). Afterwards, each cell was harvested, mRNA was isolated, cDNA was synthesized, and the expression level of the inflammatory cytokine IL-1β gene was confirmed through qRT-PCR analysis using the primers in Table 1 below. The expression level of the IL-1β gene was normalized to the expression level of the GAPDH gene, and the value was 2 -ΔΔCt It was calculated and expressed by the method.

[0057] Information on primers used in the present invention Forward primer (sequence number) Reverse primer (sequence number) IL-1β 5'-TTGTGGCTGTGGAGAAGCTGT-3'(4) 5'-AACGTCACACACCAGCAGGTT-3'(5) TNF-α 5'-AGCAAACCACCAAGTGGAGGA-3'(6) 5'-GCTGGCACCACTAGTTGGTTGT-3'(7) iNOS 5'-AGGGAGTGTTGTTCCAGGTG-3'(8) 5'-GCACATCGCCACAAACATAG-3'(9) IL-6 5'-CCGGAGAGGAGACTTCACAG-3'(10) 5'-ACAGTGCATCATCGCTGTTC-3' (11)IL-185'-GGACTGGCTGTGACCCTATC-3'(12)5'-TGTCCTGGCACACGTTTCTG-3'(13)IL-105'-TGCTATGCTGCCTGCTCTCTTA-3'(14)5'-ATGTTGTCCAGCTGGTCCTT-3'( 15)Arg15'-CTTCAGAGAAGTGGCCCAAC-3'(16)5'-GGTGGTGGGTATCACAGGAC-3'(17)GAPDH5'-ACCCAGAAGACTGTGGATGG-3'(18)5'-CACATTGGGGGTAGGAACAC-3'(19)

[0058] As a result, it was confirmed that there was no significant difference in the expression level of the IL-1β gene under LPS treatment conditions after photostimulation (Fig. 2B).

[0059]

[0060] 2-2. Analysis of anti-inflammatory effects under light-stimulated conditions after pretreatment with LPS

[0061] Microglia cell line BV2 cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics at 37°C and 5% CO2, seeded in 6-well plates, and transfected with a mixture of CIBN-NLRP3 vector and CRY2-mRuby3 vector at a ratio of 1:3.

[0062] The day after transfection with CIBN-NLRP3 vector and CRY2-mRuby3 vector, inflammation was induced by pretreatment with 0.5 μg / ml LPS for 2, 2.5, 4, or 8 hours, respectively, followed by photostimulation using a 470 nm blue light LED plate for 30 minutes, 2 hours, or 6 hours, respectively (Fig. 2C). Afterwards, each cell was harvested, mRNA was isolated, cDNA was synthesized, and the expression level of the IL-1β gene, an inflammatory cytokine, was confirmed through qRT-PCR analysis using the primers in Table 1.

[0063] As a result, it was confirmed that after LPS pretreatment, the IL-1β expression level under 2-hour or 6-hour light stimulation conditions was significantly reduced compared to the non-light-stimulated control group (Fig. 2D).

[0064] Through the above results, it was found that the longer the exposure to light stimulation was made after inducing hyperplasia of intracellular NLRP3 inflammasomes through pretreatment with LPS, the longer the binding of CIBN-CRY2 was maintained, resulting in an anti-inflammatory effect. This means that the vector according to the present invention and intracellular NLRP3 must form NLRP3 inflammasomes together to be effective in inhibiting NLRP3 inflammasome formation through binding of CIBN-CRY2 due to light stimulation.

[0065]

[0066] 2-3. Analysis of the anti-inflammatory effect of CIBN-CRY2 binding induced by photostimulation in Raw264.7 macrophages.

[0067] Macrophage cell line RAW264.7 cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics at 37°C and 5% CO2, seeded in 6-well plates, and transfected with a mixture of CIBN-NLRP3 vector and CRY2-mRuby3 vector at a ratio of 1:3.

[0068] The day after transfection with CIBN-NLRP3 vector and CRY2-mRuby3 vector, inflammation was induced by pretreatment with 0.5 μg / ml LPS for 4 or 8 hours, respectively, followed by photostimulation using a 470 nm blue light LED plate for 2 or 6 hours, respectively (Fig. 3A). Afterwards, each cell was harvested, mRNA was isolated, and cDNA was synthesized. The expression level of the IL-1β gene, an inflammatory cytokine, was confirmed through qRT-PCR analysis using the primers in Table 1.

[0069] As a result, it was confirmed that after LPS pretreatment, the IL-1β expression level under 2-hour or 6-hour light stimulation conditions was significantly reduced compared to the non-light stimulation control group (Fig. 3B).

[0070] Through the above results, it was found that the longer the exposure to light stimulation was made after inducing hyperplasia of intracellular NLRP3 inflammasomes through pretreatment with LPS, the longer the binding of CIBN-CRY2 was maintained, resulting in an anti-inflammatory effect. This means that the vector according to the present invention and intracellular NLRP3 must form NLRP3 inflammasomes together to be effective in inhibiting NLRP3 inflammasome formation through binding of CIBN-CRY2 due to light stimulation.

[0071]

[0072] Example 3. Preparation and cytotoxicity analysis of PLGA nanoparticles containing CIBN-NLRP3 vector and CRY2-mRuby3 vector.

[0073] A primary W1 / O emulsion was prepared by adding 25 μg of CIBN-NLRP3 vector, 75 μg of CRY2-mRuby3 vector, and 25 mg of PLGA (poly(lactic-co-glycolic acid), Corbion, Netherlands) to 200 μl of TE7.5 buffer, and sonicating (UP100H sonicator, Germany). Then, 2 ml of 2% (w / v) PVA1500 (Thermo Fisher Scientific) was added, and sonicating and emulsifying for 1 min to prepare a W1 / O / W2 double emulsion. The prepared W1 / O / W2 double emulsion was diluted with 6 ml of 2% (w / v) PVA1500 and stirred in a hood at room temperature for 3 hours to evaporate dichloromethane (DCM), then centrifuged at 38,000 g for 10 minutes at 4°C to collect the pellet, which was then washed twice and lyophilized to obtain CIBN-NLRP3 / CRY2-mRuby3 PLGA nanoparticles. In the above method, CIBN-NLRP3 / CRY2(E281A, A9) PLGA nanoparticles were prepared using a vector containing a mutant CRY2(E281A, A9) instead of the CRY2-mRuby3 vector. The mutant CRY2(E281A, A9) is a form in which nine amino acids are added to the C-terminus of CRY2, and is characterized by improved photosensitivity.

[0074] Afterwards, the physical characteristics (size, zeta potential, integrity) of the nanoparticles were confirmed using a scanning electron microscope (SEM) and a Zetasizer Nano ZS (Malvern instrument, UK), and the amount of CIBN-CRY2 DNA released from the nanoparticles was measured for 48 hours using a NanoDrop (Thermo Fisher Scientific). In addition, 5 × 10 BV2 cells were seeded in a 96-well plate. 3Cells were seeded per well and treated with CIBN-NLRP3 / CRY2 PLGA nanoparticles at 0, 10, 50, 100, or 200 μg / ml for 24 hours, and cell viability was analyzed using a cytotoxicity assay kit (EZ-Cytox, DogenBIO).

[0075] As a result, the size of the PLGA nanoparticles loaded with the CIBN-NLRP3 vector and the CRY2-mRuby3 vector at a ratio of 1:3 was 242.3 nm, the zeta potential value was -44 mV, and it was confirmed that they had a spherical shape, did not show toxicity in BV2 cells, and approximately 96% of the CIBN-CRY2 DNA was released over 36 hours (Fig. 4).

[0076] Through the above results, it was found that the PLGA nanoparticles containing CIBN and CRY2 of the present invention have stable and safe characteristics.

[0077]

[0078] Example 4. Preparation and characterization of a mouse neuropathic pain animal model.

[0079] Seven-week-old male C57BL / 6 mice were housed in cages of five per cage under a 12-hour light / dark cycle, with free access to food and water. The animal experiments of the present invention were approved by the Animal Care and Use Committee of Chungnam National University (CNUH-2023-1A0009) and were conducted in accordance with the ethical guidelines for pain research of the Ministry of Food and Drug Safety.

[0080] To create a spared nerve injury (SNI) model, a neuropathic pain model, mice were anesthetized. The skin and muscles on the left thigh were incised, exposing the sciatic nerve and three peripheral nerve branches (sural, peroneal, and tibial). The peroneal and tibial nerves were isolated and tightly tied with 4-0 silk thread. After complete hemostasis, the muscles and skin were sutured and recovery was induced. The control group (Sham) was operated on in the same manner as the experimental group, but SNI was not induced.

[0081]

[0082] 4-1. Pain behavior test

[0083] Mechanical thresholds were measured using 0.008–2 g von Frey filaments on days 3, 5, 7, 10, and 14 postoperatively. Specifically, von Frey filaments were applied three times at 10-minute intervals to the plantar surface of the hind paws of mice, and the thresholds of each paw were measured using an up-down test paradigm.

[0084] As a result, it was confirmed that the pain threshold began to decrease from the 3rd day after SNI surgery and the decreased pain threshold was maintained until the 14th day after surgery (Fig. 5A).

[0085]

[0086] 4-2. Analysis of microglia activation and inflammatory factor expression

[0087] On the 14th day after surgery, the L5 spinal cord of the mouse was isolated and stained using an antibody against Iba1, a marker of microglia. mRNA was isolated from microglia isolated from the spinal cord, cDNA was synthesized, and the expression levels of inflammatory cytokines TNF-α, IL-1β, iNOS, IL-6, IL-18 genes and anti-inflammatory cytokines IL-10 and Arg1 genes were confirmed through qRT-PCR analysis using the primers in Table 1. The expression levels of each gene were normalized to the expression levels of the GAPDH gene, and the values ​​were 2 -ΔΔCt It was calculated and expressed by the method.

[0088] As a result, it was confirmed that microglia were activated by SNI surgery by confirming that the expression of Iba1 protein increased in the dorsal horn of the spinal cord of the operated group compared to the control group (Sham) (Fig. 5B). It was also confirmed that the expression of inflammatory cytokines increased in the operated group compared to the control group (Sham), while the expression of anti-inflammatory cytokines decreased or showed no significant difference (Fig. 5C, 5D).

[0089]

[0090] 4-3. Morphological analysis of microglia

[0091] To further analyze the morphology of microglia isolated from the spinal cord of mice 14 days after surgery, Sholl analysis was performed. As a result, it was confirmed that the Sholl intersections of microglia were reduced, and the length, thickness, number of branches, and segment size of processes were reduced (Figures 5E to 5J).

[0092]

[0093] Example 5. Evaluation of analgesic efficacy by injection of CIBN-NLRP3 / CRY2-mRuby3 PLGA nanoparticles and invasive photostimulation.

[0094] One vial of CIBN-NLRP3 / CRY2-mRuby3 PLGA nanoparticles prepared in Example 3 was diluted in 250 μl of PBS, and 20 μl of the prepared CIBN-NLRP3 / CRY2-mRuby3 PLGA nanoparticles were intrathecally injected 7 days and 4 days before SNI surgery using the method of Example 4. Thereafter, a ferrule, which is an optical fiber connector for invasive photostimulation delivery, was implanted into the spinal cord, and on the 3rd and 5th days after SNI surgery, photostimulation was performed using a 470 nm blue light LED plate (26.3 mW) for 2 hours, and then the mechanical threshold was measured using a von Frey filament (Fig. 6A).

[0095] As a result, it was confirmed that the pain threshold of the photostimulated experimental group increased compared to the non-photostimulated control group, indicating that CIBN-NLRP3 / CRY2-mRuby3 PLGA nanoparticle injection and invasive photostimulation had analgesic efficacy in a neuropathic pain animal model (Fig. 6B). However, it was confirmed that the baseline, which is most important in pain behavioral experiments, was affected due to the ferrule implanted in the spinal cord to deliver invasive photostimulation (Fig. 6C).

[0096]

[0097] Example 6. Evaluation of analgesic efficacy by injection of CIBN-NLRP3 / CRY2 (E281A, A9) PLGA nanoparticles and noninvasive optical stimulation.

[0098] To overcome the shortcomings of invasive photostimulation using ferrules and to induce non-invasive photostimulation without the need for ferrule insertion, CIBN-NLRP3 / CRY2(E281A, A9) PLGA nanoparticles containing mutant CRY2(E281A, A9) with improved light sensitivity and a 26.3 mW 470 nm blue light LED plate (Fig. 7A) were attached to the cage ceiling.

[0099] One vial of CIBN-NLRP3 / CRY2 (E281A, A9) PLGA nanoparticles prepared in Example 3 was diluted in 250 μl of PBS, and 20 μl of the prepared CIBN-NLRP3 / CRY2 (E281A, A9) PLGA nanoparticles were intrathecally injected 7 days and 4 days before SNI surgery according to the method of Example 4. Afterwards, on the 3rd and 5th days after SNI surgery, mechanical thresholds were measured using von Frey filaments after 2 hours of photostimulation. CIBN-NLRP3 / CRY2-mRuby3 PLGA nanoparticles were injected according to the method of Example 5, and invasive photostimulation was performed to serve as a control group (Fig. 7B).

[0100] As a result, it was confirmed that CIBN-NLRP3 / CRY2 (E281A, A9) PLGA nanoparticle injection and non-invasive photostimulation had analgesic efficacy, and unlike invasive photostimulation, it was confirmed that the baseline was not affected (Fig. 7C).

[0101] Additionally, the injection frequency of CIBN-NLRP3 / CRY2 (E281A, A9) PLGA nanoparticles (1 st only, 2 nd only or 1 st &2 nd ) and the analgesic efficacy according to the photostimulation time (0.5 hour or 2 hours) were compared and analyzed. When two nanoparticle injections and two hours of photostimulation were given, an immediate and sustained analgesic efficacy was observed (Figs. 7D to 7F), and a decrease in the expression of Iba1 protein in microglia was confirmed (Fig. 7G).

[0102] Based on the above results, it was found that when CIBN-NLRP3 / CRY2 (E281A, A9) PLGA nanoparticles were injected and photostimulated, analgesic efficacy was effectively exhibited and microglial activation was reduced (Fig. 8).

Claims

1. An optogenetic anti-inflammatory pharmaceutical composition comprising nanoparticles as an active ingredient, the nanoparticles comprising a vector comprising a polynucleotide encoding a CIBN (truncated version of cryptochrome-interacting basic-helix-loop-helix 1)-NLRP3 (nod-like receptor protein 3) fusion protein; and a vector comprising a polynucleotide encoding a CRY2 (cryptochrome 2) protein or a variant thereof.

2. An optogenetic anti-inflammatory pharmaceutical composition according to claim 1, characterized in that the CIBN-NLRP3 fusion protein consists of an amino acid sequence of sequence number 1.

3. An optogenetic anti-inflammatory pharmaceutical composition according to claim 1, wherein the CRY2 protein or its variant is each composed of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

4. An optogenetic anti-inflammatory pharmaceutical composition according to claim 1, characterized in that the nanoparticles are PLGA (poly(lactic-co-glycolic acid)) nanoparticles.

5. An optogenetic anti-inflammatory pharmaceutical composition according to claim 1, characterized in that the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient.

6. An optogenetic anti-inflammatory pharmaceutical composition according to claim 1, characterized in that the composition is prepared in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols.

7. A method for suppressing inflammation through optogenetic stimulation, comprising the steps of: injecting a pharmaceutical composition of any one of claims 1 to 6 into a subject other than a human; and irradiating a photo-stimulation.

8. An optogenetic anti-inflammatory medical device using a pharmaceutical composition according to any one of claims 1 to 6.

9. A veterinary composition for optogenetic anti-inflammatory treatment comprising nanoparticles as an active ingredient, the nanoparticles comprising a vector comprising a polynucleotide encoding a CIBN (truncated version of cryptochrome-interacting basic-helix-loop-helix 1)-NLRP3 (nod-like receptor protein 3) fusion protein; and a vector comprising a polynucleotide encoding a CRY2 (cryptochrome 2) protein or a variant thereof.

10. A nanoparticle comprising a vector comprising a polynucleotide encoding a CIBN (truncated version of cryptochrome-interacting basic-helix-loop-helix 1)-NLRP3 (nod-like receptor protein 3) fusion protein; and a vector comprising a polynucleotide encoding a CRY2 (cryptochrome 2) protein or a variant thereof.

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