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KR-102959299-B1 - Anti-inflammatory composition by optogenetic stimulation comprising nanoparticle containing agent for inhibiting formation of nod-like receptor protein 3 inflammasome as effective component

KR102959299B1KR 102959299 B1KR102959299 B1KR 102959299B1KR-102959299-B1

Abstract

The present invention relates to a optogenetic anti-inflammatory composition containing nanoparticles as an active ingredient that inhibit the formation of NLRP3 inflammasomes, wherein the nanoparticles comprise a vector containing 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 containing a polynucleotide encoding a CRY2 (cryptochrome 2) protein or a variant thereof, and since the anti-inflammatory activity by non-invasive photostimulation is excellent, it can be very usefully employed in related fields.

Inventors

  • 김동운
  • 이상규
  • 신주희

Assignees

  • 충남대학교산학협력단
  • 기초과학연구원

Dates

Publication Date
20260507
Application Date
20240206

Claims (10)

  1. A pharmaceutical composition for optogenetic anti-inflammatory drugs containing nanoparticles as an active ingredient, comprising: a vector containing a polynucleotide encoding a CIBN (truncated version of cryptochrome-interacting basic-helix-loop-helix 1)-NLRP3 (nod-like receptor protein 3) fusion protein consisting of the amino acid sequence of SEQ ID NO. 1; and a vector containing a polynucleotide encoding a CRY2 (cryptochrome 2) protein or a variant thereof.
  2. delete
  3. A pharmaceutical composition for optogenetic anti-inflammatory effects according to claim 1, wherein the CRY2 protein or a variant thereof is each composed of the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 3.
  4. A pharmaceutical composition for optogenetic anti-inflammatory effects according to claim 1, characterized in that the nanoparticles are PLGA (poly(lactic-co-glycolic acid)) nanoparticles.
  5. A pharmaceutical composition for optogenetic anti-inflammatory effects according to claim 1, characterized in that the composition further comprises, in addition to the active ingredient, a pharmaceutically acceptable carrier, excipient, or diluent.
  6. A pharmaceutical composition for optogenetic anti-inflammatory effects 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, 3 to 6 into an individual other than a human; and irradiating with photostimulation.
  8. A medical device for optogenetic anti-inflammatory use of a pharmaceutical composition according to any one of claims 1, 3 to 6.
  9. A veterinary composition for optogenetic anti-inflammatory treatment containing nanoparticles as an active ingredient, comprising: a vector containing a polynucleotide encoding a CIBN (truncated version of cryptochrome-interacting basic-helix-loop-helix 1)-NLRP3 (nod-like receptor protein 3) fusion protein consisting of the amino acid sequence of SEQ ID NO. 1; and a vector containing 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 consisting of the amino acid sequence of SEQ ID NO. 1; and a vector comprising a polynucleotide encoding a CRY2 (cryptochrome 2) protein or a variant thereof.

Description

Optogenetic anti-inflammatory composition comprising nanoparticle containing agent for inhibiting formation of NLRP3 inflammasome as effective component The present invention relates to an optogenetic anti-inflammatory composition containing nanoparticles as an active ingredient that contain a preparation that inhibits the formation of NLRP3 (nod-like receptor protein 3) inflammasome. Inflammation is one of the most common immune responses that occurs when there is an abnormality in the body. It plays a role in suppressing tissue damage and eliminating infectious agents through the body's immune cells in the presence of external stimuli, such as bacteria and viruses, or internal stimuli, such as an increase in toxins within the body. Various studies have shown that microglia or macrophages are activated in various inflammatory situations within the body, including brain inflammation, to induce inflammatory responses. Recently, it has been reported that microglia in the spinal cord are activated by peripheral nerve damage, and that inflammatory responses, such as the secretion of inflammatory cytokines, play a significant role in the development of neuropathic pain. An inflammasome refers to 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 studied as a major mechanism for the secretion of inflammatory factors by microglia. Recently, various optogenetic modulation technologies have been developed. However, existing optogenetic research has focused on increasing or decreasing neuronal excitability by activating ion channels with light. Although various studies are currently applying optogenetic technology to microglia or astrocytes in addition to neurons, the development of practically applicable technologies remains challenging due to difficulties in fabricating photostimulation-sensitive vectors, developing implant technologies to deliver in vivo genes for photostimulation, or the lack of methods to deliver vectors to specific target cells. This invention aims to overcome the limitations of conventional systemic anti-inflammatory drugs by developing a technology that can alleviate inflammatory responses in various body parts—such as the brain, muscles, knees, and lower back—by utilizing optogenetic technology to induce photostimulation at desired sites and times. Meanwhile, Korean Registered Patent No. 2212997 discloses a 'composition for the improvement, prevention, or treatment of chronic inflammatory diseases caused by the overactivity of NLRP3 inflammasomes containing burdock extract as an active ingredient,' and Korean Published Patent No. 2022-0144908 discloses an 'NLRP3 inflammasome inhibitory peptide for the treatment of inflammatory diseases,' but there is no disclosure regarding the 'optogenetic anti-inflammatory composition containing nanoparticles containing a preparation that inhibits the formation of NLRP3 inflammasomes' of the present invention as an active ingredient. Figure 1 shows the results of confirming CIBN-CRY2 binding by mixing CIBN-NLRP3 vector and CRY2-mRuby3 vector (A) in various ratios and treating HEK293T cells, then confirming mRuby3 fluorescence signals through fluorescence microscopy (C, D), 3D image analysis through IMARIS software (E), and video analysis through confocal microscopy (F). Figure 2 analyzes the anti-inflammatory effect after treating BV2 cells with CIBN-NLRP3 and CRY2-mRuby3 vectors in a 1:3 ratio. The results show the expression levels of the IL-1β gene measured under conditions of LPS (Lipopolysaccharide) treatment after photostimulation (A, B) and the expression levels of the IL-1β gene measured under conditions of photostimulation after pretreatment with LPS (C, D). **:p<0.01, ***:p<0.001. Figure 3 analyzes the anti-inflammatory effect after treating RAW264.7 cells with CIBN-NLRP3 and CRY2-mRuby3 vectors in a 1:3 ratio, showing the results of measuring the expression level of the IL-1β gene under photostimulation conditions after LPS pretreatment. CTL is the control group that received no treatment. *:p<0.05. 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 in a 1:3 ratio, observing the morphology 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). Figure 5 shows the results of measuring pain threshold values using von Frey filaments after performing SNI (spared nerve injury) surgery on mice to prepare an animal model of neuropathic pain (A); the expression of Iba1 protein (B), inflammatory cytokines (C), and anti-inflammatory cytokines (D) in mic