Lipid nano-particles for realizing brain targeting through nasal cavity way as well as delivery method and application of lipid nano-particles

Abstract

The invention discloses lipid nanoparticles for realizing brain targeting through a nasal cavity way as well as a delivery method and application of the lipid nanoparticles. The lipid nanoparticles are prepared from the following raw materials: peptidyl ionizable lipid, auxiliary phospholipid, steroidal lipid and polyethylene glycol lipid, wherein the structural formula of the peptidyl ionizable lipid is as shown in formula I, and the peptidyl ionizable lipid accounts for 45-65% of the total lipid in mole percentage; the auxiliary phospholipid accounts for 10-20 mol% of the total lipid, the steroidal lipid accounts for 30-50 mol% of the total lipid, and the polyethylene glycol lipid accounts for 1-5 mol% of the total lipid. And wrapping nucleic acid molecules in the lipid nanoparticles to form the lipid nanoparticles entrapped with nucleic acid. According to the invention, a non-invasive and efficient brain-targeted mRNA delivery platform is established, and a solid foundation is laid for expanding the application of mRNA therapy in the field of nerves.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a lipid nanoparticle that achieves brain targeting via the nasal cavity, its delivery method, and its application. Background Technology

[0002] Central nervous system diseases, including common headaches, neurodegenerative diseases, and neurodevelopmental disorders, affect billions of people worldwide. The blood-brain barrier, a crucial physiological barrier of the central nervous system, strictly regulates the entry of substances into brain tissue through dense endothelial cell junctions and efficient efflux transport mechanisms. While maintaining brain homeostasis, this mechanism also makes it difficult for over 99% of macromolecular therapeutic drugs to be effectively delivered to the brain parenchyma, thus hindering their therapeutic efficacy against brain diseases. Although lipid nanoparticles have achieved success as effective mRNA delivery carriers in some areas (such as infectious vaccines), they naturally tend to accumulate in the liver and are difficult to effectively cross the blood-brain barrier after intravenous injection. To overcome this obstacle, current technologies mostly rely on invasive methods (such as intrathecal injection) or complex physical methods (such as focused ultrasound to open the blood-brain barrier) for brain parenchyma delivery. These methods are not only complex and costly, with low patient compliance, but also carry significant risks, making them unsuitable for the long-term treatment of chronic brain diseases.

[0003] In recent years, intranasal drug delivery has been widely used as a novel delivery method. Intranasal delivery utilizes the olfactory and trigeminal nerve pathways between the nasal cavity and the brain, allowing drugs to be taken up by neurons and directly transported to the cerebrospinal fluid and brain parenchyma, thus circumventing the blood-brain barrier. Because the delivered drug does not pass through the systemic bloodstream, or only a very small amount enters the bloodstream, its distribution in other tissues and organs (such as the liver) is greatly reduced, thereby lowering systemic toxicity. Simultaneously, this non-invasive delivery method can significantly improve patient compliance and effectively simplify the treatment process. Several clinical and preclinical studies have demonstrated the feasibility of intranasal delivery for treating target diseases, including brain tumors, metabolic diseases, and neurodegenerative diseases. Overall, these studies show that intranasal delivery exhibits higher bioavailability in the brain, avoids initial hepatic metabolism, and reduces systemic side effects. However, the complex enzymatic environment of the nasal cavity, mucus clearance mechanisms, and the low penetration efficiency of conventional LNP formulations into neural tissue mean that achieving efficient and stable intrabrain delivery of mRNA via this route still faces significant challenges. Summary of the Invention

[0004] Addressing the core challenge of low brain delivery efficiency of mRNA-LNPs in the treatment of central nervous system diseases using existing technologies, this invention systematically explores a solution for nasal administration. The inventors propose a core hypothesis: rationally designed mRNA-LNPs can efficiently enter the brain via the nasal route. Studies have successively confirmed: first, the surface charge of LNPs is a key parameter affecting delivery efficiency, with positively charged LNPs exhibiting optimal brain targeting ability; second, Cy5-labeled LNPs delivered via nasal drops are primarily transported to the brain through the olfactory nerve and trigeminal nerve pathways within the nasal cavity. Based on these findings, we constructed an INBT LNP (IntraNasal Brain Target LNP, INBT LNP) co-loaded with BDNF mRNA and IL-10 mRNA, and validated it in a traumatic brain injury (TBI) model: this therapy can improve the pathological process of TBI in multiple dimensions, including inhibiting acute neuroinflammation and cell death, intervening in glial scar formation, and promoting nerve regeneration, and can effectively improve cognition and control inflammation in the chronic phase. This invention establishes a non-invasive and efficient brain-targeting mRNA delivery platform, laying a solid foundation for expanding the application of mRNA therapy in the neurological field.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides lipid nanoparticles that achieve brain targeting via the nasal cavity.

[0006] The lipid nanoparticles are made from peptide-based ionizable lipids, cofactor phospholipids, steroidal lipids, and polyethylene glycol lipids. The structural formula of the peptide-based ionizable lipid is shown in Formula I:

[0007] in, n is an integer from 1 to 30, such as 2, 4, 6, 8, etc. m and o are integers from 0 to 30, such as 0, 1, 2, etc. p is an integer between 0 and 10, such as 2, 4, etc. X is either O or S independently each time it appears. A1, A2, and A3 are each independently represented by a hydrogen atom or an alkyl group when they appear. R2 and R3, each time they appear, independently represent the side groups contained in the amino acid building blocks of ionizable lipids, where R2 is any amino acid side group with a pKa greater than or equal to about 6.0, and R3 is any amino acid side group with a pKa less than about 6.0. A4 and A5 are hydrophobic tails and are independently substituted alkyl chains of 4 to 25 carbon atoms, either saturated or unsaturated, linear or branched, wherein the alkyl chains optionally contain one or more linking groups L, the linking groups L being selected from amide bonds, ester bonds, disulfide bonds, ketithiolide bonds, ether bonds, or combinations thereof. R1 represents the N-terminus of the peptide group that can ionize the lipid, and R1 is a hydrogen atom or an acetyl group. R4 represents the C-terminus of the peptide-ionizable lipid, and R4 is a hydroxyl group or has an amino group, amino acid group, and / or other functional group modification.

[0008] In some embodiments of the present invention, in the peptide-based ionizable lipids represented by Formula I, p is 2 or 4; A4 and A5 are independently selected from those having 8 carbon atoms. 14 alkyl chain.

[0009] In some embodiments of the present invention, in the peptide-based ionizable lipids represented by Formula I, p is 2 or 4; A4 and A5 are independently selected from alkyl chains having 12 carbon atoms.

[0010] In some embodiments of the present invention, the peptide-based ionizable lipid is any of the following lipids: a12Dab4, Am-Ka12K4, Ac-Ka12K4, Am-R3a12K4, Am-K3a12K4, a12K4, Ha12K4, Ca12K2, a12K4D-Ca, a12K4E2-Ca, a12K2E-Ca, a12K4E-Ca; preferably a12Dab4 or Am-Ka12K4; more preferably Am-Ka12K4.

[0011] Its structural formula is as follows:

[0012] The peptide-based ionizable lipids described in this invention can be prepared according to the method described in the patent (application publication number: CN118852328A).

[0013] In some embodiments of the present invention, the auxiliary phospholipids include, but are not limited to: DOPC (1,2) Dioleoyl sn glycerol 3 Phosphatidylcholine), DSPC (1,2) Distearate acyl sn glycerol 3 Phosphatidylcholine), POPE (1-palmitoyl-2-oleoyl-phosphatidylethanolamine), DPPC (1,2) Dipalmitoyl sn glycerol 3 Phosphatidylcholine), POPC (2 oleoyl 1 Palmitoyl sn glycerol 3 Phosphatidylcholine), DOPE (1,2) Dioleoyl sn glycerol 3 Phosphatidylethanolamine), DSPE (1,2) Distearate acyl sn glycerol 3 Phosphatidylethanolamine and DPPE (1,2) Dipalmitoyl sn glycerol 3 Phosphatidylethanolamine).

[0014] In a specific embodiment of the present invention, the auxiliary phospholipid is selected from DOPC, DSPC or POPE.

[0015] In some embodiments of the present invention, the steroidal lipids include, but are not limited to: cholesterol, cholesterolanol, 7α-hydroxyl group, etc. Hydroxycholesterol, β sitosterol, 7β Hydroxycholesterol, cholesterol, and cholesterolone.

[0016] In a specific embodiment of the present invention, the steroidal lipid is selected from cholesterol.

[0017] In some embodiments of the present invention, the polyethylene glycol lipid includes, but is not limited to: DMG-PEG2000 (1,2... Dimyristic acid rac glycerin 3 Methoxylated polyethylene glycol 2000), DSG PEG2000 (1,2) Distearate rac glycerin 3 Methoxylated polyethylene glycol 2000), DSPE mPEG2000 (distearylphosphatidylacetamide-methoxy polyethylene glycol 2000) and ALC 0159 (Methoxy polyethylene glycol bis(tetradecyl)acetamide).

[0018] In a specific embodiment of the present invention, the polyethylene glycol lipid is selected from DMG-PEG2000.

[0019] In some embodiments of the present invention, the peptide-based ionizable lipids account for 35-60% of the raw material (total lipids); the auxiliary phospholipids account for 5-25% of the raw material (total lipids); the steroidal lipids account for 30-50% of the raw material (total lipids); and the polyethylene glycol lipids account for 1-5% of the raw material (total lipids).

[0020] In a specific embodiment of the present invention, the peptide-based ionizable lipids account for 50% of the raw material (total lipids); the auxiliary phospholipids account for 10% of the raw material (total lipids); the steroidal lipids account for 38.5% of the raw material (total lipids); and the polyethylene glycol lipids account for 1.5% of the raw material (total lipids).

[0021] Secondly, the present invention provides a lipid nanoparticle loaded with nucleic acid that can achieve brain targeting via the nasal cavity.

[0022] The lipid nanoparticles carrying nucleic acids include the lipid nanoparticles described in the first aspect of the present invention and nucleic acid molecules encapsulated in the lipid nanoparticles.

[0023] In some embodiments of the present invention, the nucleic acid is mRNA.

[0024] In some embodiments of the present invention, the mRNA expresses a protein with therapeutic function or a protein with fluorescent reporter function.

[0025] In a specific embodiment of the present invention, the therapeutic function is to treat diseases of the central nervous system, such as traumatic brain injury (TBI), including acute TBI, subacute TBI and chronic TBI.

[0026] In a specific embodiment of the present invention, the protein with therapeutic function may be a protein with neuroprotective and immunomodulatory effects, such as BDNF, IL-10, etc.

[0027] In some embodiments of the present invention, the molar ratio of ionizable nitrogen atoms in the lipid molecules of the lipid nanoparticles to phosphate molecules in the nucleic acid molecules is fixed at (5-15):1, specifically 5:1, 10:1 or 15:1.

[0028] The lipid nanoparticles and nucleic acid-encapsulated lipid nanoparticles mentioned above are both administered via intranasal administration.

[0029] Thirdly, the present invention provides the application of the lipid nanoparticles described in the first aspect as carriers in drug delivery; wherein the drug is a nucleic acid. Fourthly, the present invention provides the use of the lipid nanoparticles described in the first aspect or the nucleic acid-loaded lipid nanoparticles described in the second aspect in the preparation of products having the functions described in A1) and / or A2) below: A1) This allows the drug to be delivered intranasally and targeted to the brain; A2) Treatment of central nervous system diseases.

[0030] Fifthly, the present invention provides a product having the functions described above (A1) and / or A2).

[0031] The product includes the lipid nanoparticles described in the first aspect of the present invention or the lipid nanoparticles loaded with nucleic acids described in the second aspect of the present invention.

[0032] The central nervous system diseases mentioned above can be traumatic brain injury (TBI), including acute TBI, subacute TBI, and chronic TBI.

[0033] In a sixth aspect, the present invention provides a method for preparing nucleic acid-loaded lipid nanoparticles that achieve brain targeting via the nasal cavity as described in the second aspect.

[0034] The preparation method provided by this invention includes the following steps: Step (a1): Dissolve peptide-based ionizable lipids, cofactor phospholipids, steroidal lipids, and polyethylene glycol lipids in an organic solvent at a specific molar percentage to obtain a lipid organic phase; Step (a2): Dissolve the nucleic acid molecules in an appropriate buffer solution to obtain a nucleic acid molecule solution; Step (a3): The lipid organic phase from step (a1) and the nucleic acid molecule solution from step (a) are mixed and incubated to obtain a solution of lipid nanoparticles loaded with nucleic acid.

[0035] In some embodiments of the present invention, the organic solvent includes any one or a combination of at least two of methanol, ethanol, propanol, tetrahydrofuran, and diethyl ether.

[0036] In some embodiments of the present invention, the buffer solution is a pH 3.0, 10 mM citrate-sodium citrate buffer solution.

[0037] In some embodiments of the present invention, the molar ratio of ionizable nitrogen atoms in the total lipids of the lipid organic phase to phosphate molecules in the nucleic acid molecules is fixed at (5-15):1.

[0038] In some embodiments of the present invention, the volume ratio of the lipid organic phase to the nucleic acid molecule solution is 1:(1-6).

[0039] In a specific embodiment of the present invention, the volume ratio of the lipid organic phase to the nucleic acid molecule solution is 1:3.

[0040] In some embodiments of the present invention, the incubation conditions are static incubation at room temperature for 10-30 minutes.

[0041] In a seventh aspect, the present invention provides a method for delivering brain-targeted lipid nanoparticles loaded with nucleic acids, comprising the following steps: 1) preparing the lipid nanoparticles loaded with nucleic acids as described in the second aspect of the present invention; 2) The nucleic acid-loaded lipid nanoparticles are administered intranasally to achieve brain-targeted delivery of nucleic acid-loaded nucleic acid lipid nanoparticles. The methods described above are all non-disease diagnostic and treatment methods. As non-disease diagnostic and treatment methods, they can be used for simple brain-targeting studies of nucleic acid-loaded lipid nanoparticles, or for studying the organ-targeted expression of nucleic acid-loaded lipid nanoparticles.

[0042] Eighthly, the present invention provides a method for delivering nucleic acid molecules to the brain or for preventing or treating diseases.

[0043] The method provided by the present invention includes applying an effective amount of the nucleic acid-loaded lipid nanoparticles described in the second aspect of the present invention to a desired object or tissue.

[0044] In some embodiments, the disease is any disease for which the nucleic acid molecules of the present invention can have a therapeutic or preventative effect. In some embodiments, the disease is a central nervous system disease and the nucleic acid molecule is a gene therapy agent. In some embodiments, the disease is a disease requiring brain-targeted therapy.

[0045] This invention utilizes mRNA-LNP via the nasal route for brain-targeted drug delivery and treatment. Its main innovations and key points are as follows: 1) Based on our independently developed peptide ionizable lipid (PIL) library (patent pending, publication number CN 118852328 A), this study prepared 12 LNPs with different surface charge properties. In vivo evaluation after nasal administration revealed that the surface charge of LNPs is closely related to their brain-targeted delivery efficiency and mRNA expression level. This study is the first to reveal the regulatory role of charge properties as a key design parameter on nasal-brain delivery efficiency, providing important design basis and theoretical guidance for developing efficient brain-targeted mRNA-LNP delivery systems.

[0046] 2) This study found that positively charged mRNA-LNPs are a key determinant for efficient nasal-brain delivery. Positively charged LNPs composed of PIL lipid Am-Ka12K4 were screened, exhibiting the highest efficiency in delivering mRNA to the brain. Based on this, the novel lipid nanoparticle INBT LNP was successfully developed. In vivo experiments confirmed that this carrier can efficiently deliver mRNA to the brain via the nasal route and achieve high levels of protein expression, demonstrating high brain-targeting specificity. This work confirms the feasibility of achieving brain targeting of mRNA-LNPs via nasal drops, laying a solid foundation for expanding its application in the treatment of central nervous system diseases.

[0047] 3) To elucidate the naso-brain transport pathway of LNP, this study administered Cy5-labeled LNPs via intranasal administration to TBI model mice. In vivo distribution studies showed that LNPs are mainly delivered to the brain via the olfactory nerve and trigeminal nerve pathways within the nasal cavity, through axoplasmic transport.

[0048] 4) This study presents a combined therapy based on the INBT LNP vector, delivering BDNF mRNA and IL-10 mRNA via the nasal cavity. This strategy synergistically exerts neuroprotective and immunomodulatory effects during the acute and subacute phases of TBI, effectively inhibiting neuroinflammation and neuronal apoptosis, and suppressing glial scar formation by regulating astrocyte activation, while promoting endogenous neurogenesis. In the chronic phase, this therapy can sustainably improve cognitive function and control chronic inflammation.

[0049] In summary, this invention has developed a novel, non-invasive TBI therapy that combines high targeting and convenience with potential therapeutic benefits. This delivery system is not only suitable for the treatment of TBI, but also offers a promising new treatment option for various brain diseases. Attached Figure Description

[0050] Figure 1 This diagram illustrates the preparation, in vivo nasal administration, tail vein administration, and imaging of mRNA-LNP.

[0051] Figure 2 Statistical analysis of in vitro brain imaging and fluorescence data of mice after intranasal and intravenous administration of mRNA-LNP.

[0052] Figure 3 To screen the physicochemical properties of LNP for nasal drops.

[0053] Figure 4 In vitro imaging of mouse brains after intranasal administration of mCherry mRNA-LNP with different charge properties and statistical graph showing the relationship between LNP surface charge and delivery efficiency.

[0054] Figure 5 In vitro imaging of major organs and nasal cavity in mice after intranasal administration of mCherry mRNA-LNP with different charge properties.

[0055] Figure 6 In vitro imaging and statistical diagrams of mouse brains after intranasal administration of different helper lipids mCherry mRNA-LNP.

[0056] Figure 7 In vitro imaging and statistical diagrams of mouse brains after intranasal administration of mCherry mRNA-LNP at different molar percentages.

[0057] Figure 8 In vitro brain imaging and statistical diagrams of TBI mice after intranasal administration of Cy5-Luc@INBT LNP.

[0058] Figure 9 Imaging images and statistical graphs of the nasal cavity, olfactory bulb, and trigeminal nerve in TBI mice after intranasal administration of Cy5-Luc@INBT LNP.

[0059] Figure 10 This is the primary route of delivery to the brain of TBI after Cy5-Luc@INBT LNP is administered via nasal drops.

[0060] Figure 11 In vitro brain imaging and statistical diagrams of TBI mice after intranasal administration of mCherry@INBT LNP.

[0061] Figure 12 In vitro imaging of mRNA expression in major organs and nasal cavity of TBI mice after administration of mCherry@INBT LNP via intranasal drops.

[0062] Figure 13 Immunofluorescence staining to show the colocalization of mCherry mRNA expression in the olfactory bulb of TBI mice with three types of brain cells after intranasal administration of mCherry@INBT LNP.

[0063] Figure 14Immunofluorescence staining to show the colocalization of mCherry mRNA expression in the cortex of TBI mice with three types of brain cells after intranasal administration of mCherry@INBT LNP.

[0064] Figure 15 Immunofluorescence staining to show the co-localization of mCherry mRNA expression in the hippocampus of TBI mice with three types of brain cells after intranasal administration of mCherry@INBT LNP.

[0065] Figure 16 Physicochemical properties of INBT LNP after encapsulating two types of mRNA.

[0066] Figure 17 To detect the in vitro expression and secretion of BDNF mRNA and IL-10 mRNA using Western blotting.

[0067] Figure 18 After delivering BDNF mRNA and IL-10 mRNA to INBT LNP, the levels of BDNF and IL-10 proteins in the cortex and hippocampus of the brain injury area of ​​TBI mice were detected by ELISA.

[0068] Figure 19 Immunofluorescence assays were performed to detect the levels of BDNF and IL-10 proteins in the cortex and hippocampus of the brain injury area in TBI mice after delivering BDNF mRNA and IL-10 mRNA to INBT LNP.

[0069] Figure 20 Experimental process and timeline diagram of treating acute and subacute TBI mice with mBDNF & mIL-10@INBT LNP.

[0070] Figure 21 After treating acute-phase TBI mice with mBDNF & mIL-10@INBT LNP, the expression of pro-inflammatory genes in the cortex and hippocampus of the brain injury area was detected by qPCR.

[0071] Figure 22 After treating acute-phase TBI mice with mBDNF & mIL-10@INBT LNP, the expression of anti-inflammatory genes in the cortex and hippocampus of the brain injury area was detected by qPCR.

[0072] Figure 23 After treating acute-phase TBI mice with mBDNF & mIL-10@INBT LNP, the expression of pro-apoptotic genes in the cortex and hippocampus of the brain injury area was detected by qPCR.

[0073] Figure 24After treating acute-phase TBI mice with mBDNF & mIL-10@INBT LNP, the expression of anti-apoptosis-related genes in the cortex and hippocampus of the brain injury area was detected by qPCR.

[0074] Figure 25 Bax / Bcl-2 values ​​in the cortex and hippocampus of mice with acute TBI after treatment with mBDNF & mIL-10@INBT LNP.

[0075] Figure 26 Immunofluorescence staining images and cell counts of activated microglia in the cortex and hippocampus of the brain injury area were obtained after treating subacute TBI mice with mBDNF & mIL-10@INBT LNP.

[0076] Figure 27 Immunofluorescence staining images and fluorescence intensity statistics of astrocytes in the cortex and hippocampus of the brain injury area after treatment of subacute TBI mice with mBDNF & mIL-10@INBT LNP.

[0077] Figure 28 Immunofluorescence staining and cell count of neurons in the cortex and hippocampus of the brain injury area were examined after treating subacute TBI mice with mBDNF & mIL-10@INBT LNP.

[0078] Figure 29 Immunofluorescence staining images and statistical graphs of neuronal regeneration in the hippocampus after mBDNF & mIL-10@INBT LNP treatment in subacute TBI mice.

[0079] Figure 30 Experimental process and timeline diagram of treating chronic TBI mice with mBDNF & mIL-10@INBT LNP.

[0080] Figure 31 Route map, average speed, and total distance traveled in open field experiments of mice with chronic TBI treated with mBDNF & mIL-10@INBT LNP.

[0081] Figure 32 The dwell time and number of times mice entered the center of the open field in open field experiments were statistically analyzed for mice with chronic TBI treated with mBDNF & mIL-10@INBT LNP.

[0082] Figure 33 A statistical chart showing the time required for mBDNF & mIL-10@INBT LNP to find a platform during the water maze training phase in chronic TBI mice.

[0083] Figure 34Statistical graphs showing the time required to find a platform, the number of times a platform was found, the total distance traveled, and the average speed in the water maze test phase for mice treated with mBDNF & mIL-10@INBT LNP in the chronic phase of TBI.

[0084] Figure 35 This is a flowchart of a conditioned fear experiment.

[0085] Figure 36 For situational fear memory freezing rate and cue-based fear memory freezing rate.

[0086] Figure 37 Immunofluorescence staining of CD45 cells at the location of cortical lesions in mice with chronic TBI treated with mBDNF & mIL-10@INBT LNP and statistical diagram of CD45+ cell count.

[0087] Figure 38 The levels of liver injury markers (AST, ALT) and kidney injury markers (CREA, BUN) in TBI mice 24 h after treatment with mBDNF & mIL-10@INBT LNP.

[0088] Figure 39 Serum liver function (AST, ALT) and kidney function (CREA, BUN) levels in TBI mice 48 h after treatment with mBDNF & mIL-10@INBT LNP.

[0089] Figure 40 H&E staining images of major organs in chronic TBI mice after treatment with mBDNF & mIL-10@INBT LNP.

[0090] Figure 41 H&E staining images of the nasal cavity of chronic TBI mice after treatment with mBDNF & mIL-10@INBT LNP. Detailed Implementation

[0091] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0092] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0093] The peptide-ionizable lipid a12Dab4 involved in the following examples has the following structural formula:

[0094] The chemical structural formulas of the 12 PIL lipids with different charges and the 2 control lipids involved in the following examples are shown below:

[0095] The aforementioned ionizable cationic lipids a12Dab4 and PIL lipids can be prepared according to the method described in the patent (application publication number: CN118852328A).

[0096] Example 1: Nasal drop administration enables the expression of mRNA-LNP in the mouse brain. To investigate whether mRNA-LNPs can achieve brain-targeted delivery, we first prepared LNPs loaded with mCherry mRNA.

[0097] The specific steps are as follows: LNPs were prepared using the peptide-based ionizable lipid a12Dab4, which was independently developed in our laboratory, along with three other auxiliary lipids: phospholipid DOPC, cholesterol, and polyethylene glycol lipid DMG-PEG2000. The molar ratios of each lipid were as follows: peptide-based ionizable lipid a12Dab4 accounted for 50% of the total lipids, phospholipid DOPC accounted for 10% of the total lipids, cholesterol accounted for 38.5% of the total lipids, and polyethylene glycol lipid DMG-PEG2000 accounted for 1.5% of the total lipids. The four lipids were dissolved in anhydrous ethanol according to the above molar ratios to form the ethanol phase; the mCherry mRNA molecule (whose coding sequence is sequence 1 shown in Table 1) was dissolved in 10 mM citrate-sodium citrate buffer at pH 3.0 to form the aqueous phase (for a dosage of 0.5 mg / kg, the concentration of the mRNA solution was 0.37 mg / mL). Subsequently, the aqueous and ethanol phases were rapidly mixed at a volume ratio of 3:1, maintaining a fixed molar ratio of ionizable nitrogen atoms in the total lipids to phosphate molecules in the nucleic acid molecules at 10:1. After mixing, the sample was incubated at room temperature for 15 minutes, then transferred to 1× phosphate-buffered saline (PBS) at pH 7.4 and dialyzed for 2–4 hours to remove organic solvents and non-encapsulated components. The final concentration (0.4 mg / mL for a mouse weighing approximately 20 g, based on a dose of 0.5 mg / kg (mRNA) and a volume of 25 μL) of mRNA-LNP solution was obtained (using PBS solution as the solvent).

[0098] The LNPs containing the prepared mCherry mRNA were administered intranasally to mice using a pipette and nasal dropper tip (IN), and intravenously via tail vein administration using a disposable syringe (IV). The expression of mRNA in the brain was detected using an IVIS imaging system (see [link to IV imaging]). Figure 1 ).

[0099] Each LNP formulation encapsulating mCherry mRNA was administered intranasally to mice using a pipette and nasal dropper tip. The specific administration method was as follows: Mice were held still, with their nasal cavity parallel to their neck and nearly perpendicular to their lower body. Droppers were administered alternately into both nostrils, with a volume of 25 μL per mouse. After complete administration, the mice were held in the dropper position for 30 seconds before being placed back in their cages. Three hours after the first administration, a second intranasal administration was given at a dose of 0.5 mg / kg, administered in the same manner as the first administration. Twelve hours after the second administration, the mouse brains were examined using an IVIS small animal in vivo imaging system. Results are as follows: Figure 2 As shown, a significant fluorescent signal was observed in the mouse brain via nasal administration alone, indicating that nasal administration can achieve mRNA-LNP expression in the mouse brain, while tail vein administration cannot achieve mRNA-LNP expression in the mouse brain.

[0100] Example 2: Screening LNP vectors suitable for nasal-brain delivery and with high brain-targeted delivery efficiency. To further improve the delivery efficiency of mRNA-LNP in the brain, we selected 12 PIL lipids with different charges from our laboratory-developed PIL lipid library to prepare LNPs, aiming to screen for LNPs with higher efficiency for intranasal administration to the brain. We also used the already approved ALC-0315 and SM-102 lipids to prepare LNPs as controls.

[0101] The preparation method of the following mRNA-LNP is basically the same as that in Example 1, except that the "peptide-based ionizable lipid a12Dab4" in Example 1 is replaced with equimolar amounts of PIL lipids with different charges, as well as ALC-0315 and SM-102 lipids. The molar ratios of each lipid in the LNP of ALC-0315 are as follows: ionizable cationic lipid ALC-0315 accounts for 46.3% of the total lipids, phospholipid DSPC accounts for 9.4% of the total lipids, cholesterol accounts for 42.7% of the total lipids, and polyethylene glycol lipid DMG-PEG accounts for 1.6% of the total lipids. The molar ratios of each lipid in the LNP of SM-102 are as follows: ionizable cationic lipid SM-102 accounts for 50% of the total lipids, phospholipid DSPC accounts for 10% of the total lipids, cholesterol accounts for 38.5% of the total lipids, and polyethylene glycol lipid DMG-PEG accounts for 1.5% of the total lipids. Both were formulated according to the optimal LNP formulations of the two marketed lipids.

[0102] Freshly prepared mRNA-LNP solutions were diluted with 1 × PBS to a mRNA concentration of 1 ng / mL, and then measured using a nanoparticle size and potential analyzer. Results are as follows: Figure 3 As shown in the left figure, the hydrodynamic diameters of all LNP samples were distributed between 150 and 200 nm, with a polydispersity index of approximately 0.2, indicating uniform particle size distribution and good monodispersity. Encapsulation efficiency was determined using the RiboGreen fluorescent dye assay. The encapsulation efficiency of LNP for mCherry mRNA was calculated by measuring the concentration of free mRNA in the mRNA-LNP solution and the concentration of all mRNA in the solution after complete lysis of mRNA-LNP with Triton X-100. 100 µL of mRNA-LNP solution diluted with TE buffer was added to a 96-well plate to determine the concentration of free mRNA. An equal volume of sample was added to TE buffer containing 0.1% Triton X-100, and the plate was incubated at room temperature with shaking for 30 minutes to completely lyse LNP and calculate the total mRNA concentration. Subsequently, 100 µL of RiboGreen dye diluted 500-fold with TE buffer was added to each well, and the fluorescence intensity was detected using a microplate reader. Encapsulation efficiency (%) = [(Actual concentration of lysed mRNA - Actual concentration of free mRNA) / Actual concentration of lysed mRNA] * 100%. Results are as follows: Figure 3 As shown in the right figure, the encapsulation efficiency of a series of LNPs is between 80% and 100%, which indicates that LNPs have a high encapsulation efficiency for mRNA and can be used as a vector for mRNA delivery.

[0103] To screen for suitable LNP vectors for nasal-brain delivery, we prepared mRNA-LNPs from the aforementioned 12 PIL lipids and FDA-approved SM-102. After intranasal administration to mice, the expression level of mCherry mRNA in their brains was analyzed using an IVIS imaging system. Results are as follows: Figure 4 As shown, negatively charged LNPs failed to elicit significant mCherry fluorescence signal expression in the brain; electrically neutral LNPs mediated some expression; while positively charged LNPs exhibited the strongest intrabrain fluorescence signal. This indicates that the surface charge of LNPs is closely related to their intrabrain delivery efficiency, and the positive charge may enhance the interaction between LNPs and nasal mucus and olfactory epithelial cells, thereby promoting their uptake and subsequent transport to the brain. Based on these screening results, we selected the Am-Ka12K4 lipid with the highest brain targeting efficiency for further in-depth research.

[0104] To evaluate the in vivo expression and distribution characteristics of LNPs, we prepared LNPs from the aforementioned 12 PIL lipids, as well as the FDA-approved SM-102 and ALC-0315, and administered them to mice via nasal drops (dose as in Example 1). Subsequently, the major organs (heart, liver, spleen, lung, and kidney) and nasal cavity of the mice were dissected, and the distribution of mRNA expression was observed using an IVIS imaging system. The results are as follows: Figure 5 As shown, no significant fluorescent signals were detected in any of the examined peripheral organs or the nasal cavity. These results indicate that nasal administration of LNPs effectively avoids expression in non-target organs, demonstrating high brain-targeting specificity and providing important evidence for its potential application as a precision delivery tool for the central nervous system.

[0105] To optimize the brain-targeted delivery efficiency of LNPs, we further screened their helper phospholipid components. LNPs were prepared using three helper phospholipids: DOPC, DSPC, and POPE, and administered to mice via nasal route. The LNPs prepared here were essentially the same as in Example 1, except that the "peptide-based ionizable lipid a12Dab4" in Example 1 was replaced with Am-Ka12K4 lipid, and the helper lipids were replaced with "DOPC, DSPC, or POPE," respectively. After administration, the expression of mCherry mRNA in the brain was detected using an IVIS in vivo imaging system. The results are as follows: Figure 6 As shown, the LNP prepared using DOPC as an assist phospholipid exhibited the highest fluorescence signal intensity in the brain, indicating its optimal nose-to-brain delivery efficiency. Therefore, we selected DOPC as the assist phospholipid component for LNPs in subsequent studies.

[0106] Subsequently, we optimized the molar percentages of each component of the LNP to further improve the naso-brain delivery efficiency. For example... Figure 7The left figure shows the lipid molar percentages used to optimize each component of the LNP. For example... Figure 7 As shown in the right figure, the molar percentage 1 (Am-Ka12K4 / DOPC / Chol / DMG-PEG2k = 50:10:38.5:1.5) exhibited the highest brain mRNA delivery efficiency compared to other ratios. Based on these results, we therefore named this platform INBT LNP (IntraNasal BrainTarget LNP, INBT LNP).

[0107] Example 3: Application of INBT LNP intranasal drops in a mouse model of traumatic brain injury To investigate the biodistribution of Cy5-Luc@INBT LNPs after intranasal administration, we established a mouse model of traumatic brain injury (TBI). The pathological characteristics mainly include the following: in the acute phase, microglia and astrocytes are rapidly activated, releasing pro-inflammatory cytokines, initiating an inflammatory cascade response, and causing cell death in the core injury area; in the subacute phase, widespread inflammatory damage, neuronal apoptosis, astrocyte hypertrophy and proliferation, and the initial formation of glial scars; in the chronic phase, persistent neuroinflammation and a lack of neurotrophic factors lead to abnormal synaptic structure and function, affecting learning, memory, and cognitive function. The TBI model used in this study was a repetitive mild traumatic brain injury (rmTBI) model. The main modeling method was as follows: mice were anesthetized by intraperitoneal injection of 2.5% tribromoethanol. Immediately after anesthesia, the mice were placed in a stereotaxic apparatus, and the scalp was exposed using surgical scissors. The surgical site was alternately cleaned with 70% ethanol and povidone-iodine. Using a stereotaxic apparatus, the left and right sides of the head, as well as the distribution of the coronal point (Bregma) and lambda point, were aligned to the same horizontal plane. The impact point was located on the craniofacial midline, approximately 2.0 mm anterior to the coronal point. The injury parameters of the impact device (Leica Impact One Stereotaxic Impactor) were: impact depth 2.0 mm, velocity 3.0 m / s, and residence time 100 ms, with the skull surface as the origin. Immediately after impact, the mice were removed from the stereotaxic frame and placed on a heated pad maintained at 37°C in a supine position until full consciousness was regained. To induce repeated injury, a second impact was performed 24 hours after the first, followed by a third impact 24 hours later, thus establishing a homogenized TBI mouse model. Six hours after successful model establishment, TBI mice were given Cy5-Luc@INBT LNP intranasally in two divided doses of 0.5 mg / kg each. One hour after the first nasal instillation, a second nasal instillation was administered. Two hours after the second nasal instillation, the mouse brain was examined using the IVIS small animal in vivo imaging system.

[0108] The Cy5-Luc@INBT LNP used in this embodiment has the following formulation (molar percentage of each component): 50% of the total lipids are peptide-ionizable lipid Am-Ka12K4, 10% of the total lipids are phospholipid DOPC, 38.5% of the total lipids are cholesterol, and 1.5% of the total lipids are polyethylene glycol lipid DMG-PEG. The mRNA used is Cy5-Luc mRNA (its coding sequence is shown in Sequence 2 in Table 1) or mCherry mRNA molecule (its coding sequence is shown in Sequence 1 in Table 1). The preparation method is the same as in Example 1.

[0109] The mCherry@INBT LNP used in this embodiment has the following formulation (molar percentage of each component): 50% of the total lipids are peptide-based ionizable lipid Am-Ka12K4, 10% of the total lipids are phospholipid DOPC, 38.5% of the total lipids are cholesterol, and 1.5% of the total lipids are polyethylene glycol lipid DMG-PEG. The mRNA used is the mCherry mRNA molecule (its coding sequence is shown in Sequence 1 in Table 1), and the preparation method is the same as in Example 1.

[0110] like Figure 8 As shown, a significant Cy5 fluorescence signal was observed in the brain of TBI mice by administering Cy5-Luc@INBT LNP via nasal instillation, indicating that LNP distribution and expression in the brain of TBI mice can be achieved through nasal instillation.

[0111] Cy5-Luc@INBT LNP was administered via nasal instillation to TBI mice. Two hours after instillation, the nasal cavity, olfactory bulb, and trigeminal nerve of the mice were examined using the IVIS small animal in vivo imaging system. Figure 9 As shown, significant Cy5 fluorescence signals were observed in the nasal cavity, olfactory bulb, and trigeminal nerve of TBI mice by administering Cy5-Luc@INBT LNP via nasal drops. These results confirm that LNP may enter the central nervous system through the olfactory nerve and trigeminal nerve in the nasal cavity.

[0112] Our in vivo distribution study of Cy5-labeled LNPs after intranasal administration in a TBI mouse model demonstrates that the optimized INBT LNPs can rapidly deliver mRNA to the central nervous system via the direct "nas-brain pathway," bypassing the blood-brain barrier. In summary, the in vivo distribution study confirms that the optimized INBT LNPs in this study, after intranasal administration, can efficiently utilize the "nas-brain pathway," primarily via the olfactory and trigeminal nerve pathways, through axonal retrograde transport mechanisms, to directly and rapidly deliver mRNA to the brain. Figure 10The main pathways from intranasal administration to mRNA-LNP reaching the brain of TBI mice are summarized.

[0113] Next, we observed mRNA expression in TBI mice. We administered mCherry@INBTLNP intranasally to TBI mice, and 12 hours after the instillation, we used the IVIS small animal in vivo imaging system to image and detect the mouse brain. Figure 11 As shown, by administering mCherry@INBT LNP via nasal instillation to TBI mice, a significant fluorescent signal of mCherry mRNA expression was observed in the mouse brain, indicating that mRNA expression in the brain of TBI mice can be achieved via nasal instillation.

[0114] TBI mice were intranasally instilled with mCherry@INBT LNP. Twelve hours after instillation, the major organs (heart, liver, spleen, lungs, and kidneys) and nasal cavity of the mice were examined using the IVIS small animal in vivo imaging system. Results are as follows: Figure 12 As shown, no significant mCherry fluorescence signal was observed in any of the tested peripheral tissues or nasal cavities. This result indicates that even under TBI pathological conditions, INBT LNP can still achieve precise brain-targeted delivery via nasal drops, demonstrating excellent peripheral tissue avoidance ability and high brain-targeting specificity.

[0115] Next, we collected brain tissue from TBI mice that had received intranasal mCherry@INBT LNP injections. After fixation with glycosylation, the tissue was embedded using OCT. The embedded brain tissue was then frozen sectioned, and immunofluorescence staining was used to observe the expression of mCherry mRNA in the olfactory bulb of the TBI mouse brain and its co-localization with microglia (Iba1), astrocytes (GFAP), and neurons (NeuN). Specifically, brain tissue sections were first fixed with 4% paraformaldehyde and infiltrated with 0.125% Triton X-100. The sections were then washed three times with PBS and incubated overnight at 4°C with primary antibodies against Iba1, GFAP, NeuN, and mCherry, respectively. Finally, the sections were washed three times with PBS and incubated with Alexa Fluor™ 568 and Alexa Fluor™ 647 conjugated secondary antibodies, respectively, followed by repeated washing with PBS. Images were collected using a laser confocal microscope (Leica Stellaris). Figure 13 As shown, mCherry mRNA can be expressed in the olfactory bulb of the brain of TBI mice via nasal drop, and it shows obvious colocalization with all three types of brain cells (indicated by arrows).

[0116] Subsequently, we used immunofluorescence staining to observe the expression of mCherry mRNA in the cortical area of ​​the brain injury zone in TBI mice and its co-localization with microglia (Iba1), astrocytes (GFAP), and neurons (NeuN). Figure 14 As shown, mCherry mRNA can be expressed in the brain cortex of TBI mice via nasal drop, and it shows obvious colocalization with all three types of brain cells (indicated by arrows).

[0117] We used immunofluorescence staining to observe the expression of mCherry mRNA in the hippocampus of TBI mice brain injury areas and its co-localization with microglia (Iba1), astrocytes (GFAP), and neurons (NeuN). Figure 15 As shown, although mCherry mRNA can reach and be expressed in the hippocampus of TBI mice via nasal drops, the signal is weak. This limitation may be due to the hippocampus being located deep in the brain, and its expression being mainly limited to microglia and astrocytes. Analysis of the immunofluorescence results above indicates that the optimized INBT LNP nasal drop delivery system in this study can not only achieve brain targeting via the naso-brain pathway, but also achieve efficient and specific expression of therapeutic proteins in key lesion areas (cortex and hippocampus) of TBI. This marks the completion of the validation of this technology from "vector delivery" to "functional protein expression."

[0118] Example 4: Therapeutic effect of INBT LNP on TBI mice We investigated the therapeutic effects of INBT LNP on TBI mice. TBI induces massive neuronal death, releasing damage-related molecules such as myelin fragments, leading to persistent neuroinflammation and ultimately hindering neurological function recovery. Furthermore, TBI is a well-established risk factor for various progressive neurodegenerative diseases and mental disorders. Brain-derived neurotrophic factor (BDNF) plays a central role in neurodegenerative diseases: it is crucial for the survival and functional maintenance of dopaminergic neurons by regulating synaptic plasticity and the neurotransmitter system. BDNF-based therapies not only have neuroprotective effects but also enhance neuroplasticity, potentially reversing functional deficits caused by brain injury. On the other hand, neuroimmunomodulation is also an important direction for TBI treatment. Recent studies have shown that intranasal administration of CD3 monoclonal antibodies can enhance the phagocytic capacity of microglia for damage-related debris through IL-10-mediated Treg-microglia interaction and effectively reduce neuroinflammation. Based on the above theory, this study innovatively uses INBT LNP as a delivery carrier to deliver BDNF mRNA and IL-10 mRNA together via nasal administration, aiming to exert a synergistic effect of neuroprotection and immune regulation, and to provide new experimental evidence for the combination therapy strategy of TBI.

[0119] The mBDNF & mIL-10@INBT LNP used in this embodiment has the following formulation: 50% of the total lipids are peptide-based ionizable lipid Am-Ka12K4, 10% of the total lipids are phospholipid DOPC, 38.5% of the total lipids are cholesterol, and 1.5% of the total lipids are polyethylene glycol lipid DMG-PEG. The mRNAs used are mBDNF (BDNF mRNA, whose coding sequence is sequence 3 shown in Table 1) and mIL-10 (IL-10 mRNA, whose coding sequence is sequence 4 shown in Table 1), with a mass ratio of 1:1. The preparation method is the same as in Example 1.

[0120] We diluted the freshly prepared mBDNF&mIL-10@INBT LNP solution with 1×PBS to a concentration of 1 ng / mL for mRNA. The particle size of the LNPs was then determined using a nanoparticle size and potential analyzer. The physicochemical properties of two different dosages (1 mg / kg and 1.4 mg / kg) of mBDNF&mIL-10@INBT LNP were tested. The results are as follows... Figure 16As shown in the left figure, all LNPs exhibit a relatively uniform size distribution, ranging from approximately 200 nm, with a polydispersity index (PDI) of around 0.2. Encapsulation efficiency was determined using the RiboGreen fluorescent dye assay for LNP mRNA encapsulation efficiency, and the results are shown below. Figure 16 As shown in the right figure, the encapsulation efficiency of the series of LNPs is 100%, which indicates that the INBT LNP pair can encapsulate two mRNAs simultaneously.

[0121] BDNF mRNA (coding sequence 3 in Table 1) and IL-10 mRNA (coding sequence 4 in Table 1) were synthesized via in vitro transcription and transfected into MDA-MB-231 cells. Total cell protein and culture supernatant total protein were extracted after 24 hours. The expression and secretion of BDNF and IL-10 were verified by Western blot. Figure 17 As shown, BDNF mRNA and IL-10 mRNA can be normally expressed inside cells and secreted outside cells.

[0122] TBI mice were administered 1 mg / kg of mBDNF & mIL-10@INBT LNP via intranasal drops. A control was administered the same volume (25 μL) of PBS. Twelve hours after administration, cortical and hippocampal tissues from the TBI lesion site were harvested for total protein extraction. Enzyme-linked immunosorbent assay (ELISA) was performed using a BDNF ELISA kit (Solarbio; product number: SEKM-0143) and an IL-10 ELISA kit (Solarbio; product number: SEKM-0010) to detect the protein levels of BDNF and IL-10 in the tissues. Results are as follows: Figure 18 As shown, compared with the PBS group, the levels of BDNF and IL-10 proteins in the cortex and hippocampus of the brain injury area after TBI were significantly increased after intranasal administration of LNP.

[0123] TBI mice were administered mBDNF & mIL-10@INBT LNP at a dose of 1 mg / kg via intranasal drip. Twelve hours after administration, the mice were anesthetized and subjected to cardiac perfusion. Brain tissue was collected, fixed with glycosylated material, and embedded in OCT. Immunofluorescence staining was used to detect the expression of BDNF mRNA and IL-10 mRNA in the brain. Results are as follows: Figure 19 As shown, BDNF mRNA and IL-10 mRNA are effectively expressed in the brains of TBI mice. These data demonstrate the successful in vivo expression of BDNF mRNA and IL-10 mRNA.

[0124] Next, we investigated the therapeutic effects of intranasal administration of mBDNF & mIL-10@INBT LNP on acute and subacute TBI mice. The experiment was divided into four groups (n=7): sham mice were administered PBS intranasally (G1), TBI mice were administered LNP only intranasally (G2), TBI mice were administered mBDNF & mIL-10@INBT LNP intranasally at a dose of 1 mg / kg (G3), and TBI mice were administered mBDNF & mIL-10@INBT LNP intranasally at a dose of 1.4 mg / kg (G4). Intranasal administration began 6 hours after the first TBI impact and was repeated every three days. Figure 20 ).

[0125] To evaluate the therapeutic effect of mBDNF & mIL-10@INBT LNP on acute-phase TBI, mouse brain tissue was collected on day 3, and the expression of pro-inflammatory genes in the cortex and hippocampus of the brain injury area was detected by qPCR. Figure 21 As shown, TBI modeling (G2 group) successfully induced a strong neuroinflammatory response, manifested by a significant upregulation of the mRNA levels of key pro-inflammatory factors IL-1β, IL-6, and TNF-α. In the G3 and G4 groups treated with mBDNF & mIL-10@INBT LNP, the expression of these factors was significantly inhibited, demonstrating that this therapy can effectively reduce early neuroinflammatory symptoms after TBI.

[0126] We used qPCR to detect the expression of anti-inflammatory genes in the cortex and hippocampus of TBI mice at the site of brain injury. The results are as follows: Figure 22 As shown, the expression of anti-inflammatory genes, including CD-206 and TGF-β, in the cortical and hippocampal brain injury areas of TBI mice (G3 and G4) treated with mBDNF&mIL-10@INBT LNP was significantly higher than that in the LNP-only group, indicating that mBDNF&mIL-10@INBT LNP treatment can significantly enhance the expression of anti-inflammatory genes in the brain injury areas of TBI mice.

[0127] We used qPCR to detect the expression of pro-apoptosis-related genes in the cortex and hippocampus of TBI mice at the site of brain injury. The results are as follows: Figure 23 As shown, the expression of pro-apoptotic genes, including Caspase3 and Bax, in the cortical and hippocampal brain injury areas of TBI mice (G3 and G4) treated with mBDNF&mIL-10@INBT LNP was significantly lower than that in the LNP-only group, indicating that mBDNF&mIL-10@INBT LNP treatment can significantly inhibit the expression of pro-apoptotic genes in the brain injury areas of TBI mice.

[0128] We used qPCR to detect the expression of anti-apoptosis-related genes in the cortex and hippocampus of TBI mice at the site of brain injury. The results are as follows: Figure 24 As shown, the expression of pro-apoptotic genes, including Bcl2, in the cortical and hippocampal brain injury areas of TBI mice (G3, G4) treated with mBDNF&mIL-10@INBT LNP was significantly higher than that in the LNP-only group, indicating that mBDNF&mIL-10@INBT LNP treatment can significantly enhance the expression of anti-apoptotic genes in the brain injury areas of TBI mice.

[0129] We calculated the Bax / Bcl-2 ratio after qPCR, and the results are as follows: Figure 25 As shown, the Bax / Bcl-2 ratio in the cortex and hippocampus of TBI mice (G3, G4) treated with mBDNF & mIL-10@INBT LNP was significantly reduced, even comparable to that in the sham-operated G1 group. This indicates that after TBI, brain cells are more inclined to choose survival after receiving death signals, thus inhibiting neuronal apoptosis.

[0130] Next, on day nine, we collected brain tissue from TBI mice and further evaluated the therapeutic effect of mBDNF & mIL-10@INBT LNP treatment on subacute TBI mice by performing immunofluorescence staining on the brain tissue sections. The results are as follows: Figure 26 Immunofluorescence colocalization analysis showed that the number of CD68+Iba1+ microglia (representing the pro-inflammatory phenotype) in the cortex and hippocampus of the damaged area mice was significantly increased in the G2 (TBI model) group, while the number of this activated cell population was significantly reduced in the G3 and G4 treatment groups, proving that the therapy can effectively regulate the neuroinflammatory response after TBI and effectively reverse the excessive activation of microglia.

[0131] We further evaluated the therapeutic effect of mBDNF & mIL-10@INBT LNP treatment on subacute TBI mice by performing immunofluorescence staining on brain tissue sections from the experiment. The results are as follows: Figure 27 As shown, compared with the G2 (TBI model) group, the activation level of astrocytes in the cortex and hippocampus of the damaged area mice in the G3 and G4 treatment groups was significantly inhibited (manifested as GFAP fluorescence intensity), indicating that this therapy may further inhibit the formation of glial scars by inhibiting the activation of astrocytes.

[0132] We further evaluated the therapeutic effect of mBDNF & mIL-10@INBT LNP treatment on subacute TBI mice by performing immunofluorescence staining on brain tissue sections from the experiment. The results are as follows: Figure 28As shown, compared with the G2 (TBI model) group, the number of surviving neurons in the cortex and hippocampus of the brain injury area was significantly increased in the G3 and G4 treatment groups. This indicates that the therapy can effectively prevent neuronal loss after TBI and has potential neuroprotective effects.

[0133] The above experiments showed that mBDNF & mIL-10@INBT LNP treatment effectively inhibited neuronal loss after TBI. Next, we investigated whether this therapy could promote neurogenesis. We labeled the newly generated cells by intraperitoneal injection of BrdU, such as... Figure 29 As shown, immunofluorescence staining experiments on brain tissue sections revealed that, compared with the G2 (TBI model) group, the combined treatment of mBDNF & mIL-10@INBT LNP in the G3 and G4 groups significantly increased the number of DCX-positive cells in the brains of TBI mice. More importantly, the number of double-positive cells, simultaneously marking the cell proliferation marker BrdU and the immature neuron marker DCX, also significantly increased. This result indicates that we successfully activated and enhanced post-traumatic endogenous neurogenesis through the strategy of endogenous BDNF expression. Exogenous BDNF effectively promoted the proliferation of neural progenitor cells, guided their differentiation into neuronal lineages, and supported the survival of newly generated neurons. This therapy not only protects the existing neuronal regulatory injury microenvironment through previously validated anti-inflammatory and anti-apoptotic mechanisms, but also further supplements and repairs damaged neural networks by promoting neurogenesis, providing a novel and far-reaching therapeutic approach for achieving long-term functional recovery after TBI.

[0134] Given the anti-inflammatory and neuroprotective effects of mBDNF & mIL-10@INBT LNP in acute and subacute TBI mice, we further investigated its long-term therapeutic potential in TBI mice. The experiment was divided into three groups (n=10): sham mice were given PBS intranasally (G1), TBI mice were given LNP only intranasally (G2), and TBI mice were given mBDNF & mIL-10@INBT LNP intranasally at a dose of 1 mg / kg (G3). Intranasal administration began 6 hours after the first TBI impact and was repeated every three days. Figure 30 ).

[0135] We first conducted an open field test to assess the motor function of the mice in each group. The experimental procedure was as follows: A mouse was gently placed in the center of a box and allowed to explore freely for 10 minutes. After the test, another mouse was placed in the box. The results are as follows: Figure 31 As shown, there were no significant differences in average speed and total distance traveled among the three groups of mice, indicating that the motor function of the mice remained intact after TBI.

[0136] In the open field experiment, we also statistically analyzed the time the mice spent in the central region of the open field and the number of times they entered the center. The results are as follows: Figure 32 As shown, there were no significant differences among the three groups of mice, indicating that TBI modeling and subsequent multiple nasal drops did not induce anxiety-like behavior in the mice.

[0137] To test the effects of mBDNF & mIL-10@INBT LNP treatment on cognition, learning, and memory in TBI mice, we conducted a 6-day water maze test. First, we conducted 5 days of learning and memory training using a circular water tank with a diameter of 120 cm. An appropriate amount of titanium dioxide was dissolved in the tank, and the water level was 0.5-1 cm above the platform, with a water temperature of 23-25℃. The circular tank was divided into four quadrants, defined as east, west, south, and north, with a simple graphic marker affixed to the inner wall of each quadrant. The four quadrants were distinct. Mice received four rounds of training daily. Mice were removed from the platform if the cumulative time spent on the platform within the 1-minute training period was ≥15 seconds. Mice with insufficient time were guided through the training. The training continued for 5 days, and the results are as follows: Figure 33 As shown, both G1 and G3 mice showed increasingly shorter time required to find the platform, with no significant difference between the two groups. However, G2 mice required significantly longer time to find the platform than the other two groups.

[0138] On the sixth day, the platform was removed for testing of the mice. During the test, the platform was removed, and the mice were allowed to explore freely for one minute. The time spent in the original platform area was recorded. The results are as follows: Figure 34 As shown in the left figure, after treatment with mBDNF & mIL-10@INBT LNP, the time required for the G3 group TBI mice to find the platform for the first time was significantly shorter than that of the G2 LNP-only group. The number of times the G3 group mice found the platform was significantly higher than that of the G2 group throughout the test. It is worth noting that there was no significant difference between the G3 group and the G1 group. There was no significant difference in the total distance traveled and the average speed among the three groups of mice during the test. Figure 34 (Right figure) This illustrates that mBDNF & mIL-10@INBT LNP treatment can significantly improve the learning and memory abilities of TBI mice, and this improvement in cognitive behavior is not due to motor ability.

[0139] To test the effects of mBDNF & mIL-10@INBT LNP on conditioned fear memory in mice, we conducted a three-day conditioned fear test. In short, Day 1 (training phase): Mice were placed in the test environment, a sound signal was played for 20 seconds, followed by a 0.7 mA electric shock to the paw for 2 seconds, with both the sound and shock signals ending simultaneously. This was repeated three times. Day 2 (Contextual Fear Conditioning): 24 hours later, mice were placed in the same test environment, and a 5-minute video recording was performed. The proportion of rigid responses was used to assess the mice's memory of the contextual condition. Day 3 (Cued Fear Conditioning): 48 hours later, mice were placed in a test environment with modified inner walls, and a 5-minute video recording was performed, followed by a 3-minute sound signal. The proportion of rigid responses was used to assess the mice's memory of the contextual condition. Figure 35 ).

[0140] Conditioned fear experiments were conducted on TBI mice, and the results were as follows: Figure 37 As shown, the G3 group mice performed a situational fear test on the second day. Figure 36 (Left) and the third day's cue fear test ( Figure 36 The freezing rate in the right group was significantly higher than that in the untreated G2 group. This indicates that mBDNF & mIL-10@INBT LNP treatment significantly improved fear memory in TBI mice.

[0141] To investigate the persistence of neuroinflammation in a chronic TBI model, we performed CD45 immunofluorescence staining analysis on the cortical injury areas in mice. The results are as follows: Figure 37 As shown, mBDNF & mIL-10@INBT LNP treatment significantly reduced CD45+ immune cell infiltration in the cortical region. This finding indicates that this combination therapy not only improves cognitive function but also effectively alleviates chronic neuroinflammation after TBI. In conclusion, our results strongly validate the therapeutic potential of mBDNF & mIL-10@INBT LNP in promoting long-term functional recovery after TBI.

[0142] Besides efficacy, safety and immunogenicity are also key factors influencing the clinical performance of lipid nanoparticles. Uncontrolled immune responses can lead to serious adverse events, including tissue inflammation, allergic reactions, and organ damage. Therefore, the in vivo safety of INBT LNP was tested. TBI mice were administered 1 mg / kg mBDNF & mIL-10@INBT LNP via intranasal instillation. T24 hours after administration, serum levels of liver function markers (aspartate aminotransferase, AST; alanine aminotransferase, ALT) and kidney function markers (urea, UREA; blood urea nitrogen, BUN) were measured. Results are as follows: Figure 38As shown, 24 hours after intranasal administration of TBI mice, there was no significant difference in LNP administration compared with the G1 PBS group, indicating that it was well tolerated in vivo.

[0143] Next, we measured the levels of liver and kidney function markers in the serum of TBI mice 48 hours after intranasal administration. The results are as follows. Figure 39 As shown, there was no significant difference in LNP levels 48 hours after administration compared to the G1 group, indicating that it was well tolerated in vivo.

[0144] Damage to major organs after nasal administration was also assessed by H&E staining of tissue sections. Results are as follows: Figure 40 As shown, the mBDNF & mIL-10@INBT LNP treatment group did not produce significant toxicity in vivo, indicating that INBT LNP has good safety.

[0145] Considering that repeated intranasal administration may damage the nasal cavity in mice, H&E staining of tissue sections was used to assess the damage to the nasal cavity after intranasal administration. Results are as follows: Figure 41 As shown, no significant toxicity was observed in the nasal cavity in the mBDNF&mIL-10@INBT LNP treatment group, indicating that INBT LNP has good safety.

[0146] Currently, achieving efficient brain delivery of therapeutic mRNA remains a significant challenge due to the blood-brain barrier. To address this issue, this invention develops a lipid nanoparticle delivery system that enables efficient brain expression of mRNA via nasal drops. Research has revealed that the surface charge of the lipid nanoparticle (LNP) is a key parameter determining its naso-brain delivery efficiency. Positively charged LNPs exhibit optimal brain-targeting ability, with delivery efficiency significantly superior to FDA-approved lipids SM-102 and ALC-0315. Based on this, we named the optimized LNP with the highest naso-brain delivery efficiency INBT LNP. Mechanistic studies show that INBT LNP primarily enters the brain via the olfactory nerve and trigeminal nerve pathways within the nasal cavity. In therapeutic applications, we utilized INBT LNP to co-deliver BDNF mRNA and IL-10 mRNA, significantly improving cognitive and learning abilities in a mouse model of traumatic brain injury. This study demonstrates that INBT LNP is a promising brain-targeting mRNA delivery platform, providing a new strategy and feasible pathway for the treatment of TBI and other central nervous system diseases.

[0147] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A lipid nanoparticle that targets the brain via the nasal route, the raw materials of which include peptide-based ionizable lipids, cofactor phospholipids, steroidal lipids, and polyethylene glycol lipids; wherein The structural formula of the peptide-based ionizable lipid is shown in Formula I: in, n is an integer between 1 and 30. m and o are integers between 0 and 30. p is an integer between 0 and 10. X is either O or S independently each time it appears. A1, A2, and A3 are each independently represented by a hydrogen atom or an alkyl group when they appear. R2 and R3, each time they appear, independently represent the side groups contained in the amino acid building blocks of ionizable lipids, where R2 is any amino acid side group with a pKa greater than or equal to about 6.0, and R3 is any amino acid side group with a pKa less than about 6.

0. A4 and A5 are hydrophobic tails and are independently substituted alkyl chains of 4 to 25 carbon atoms, either saturated or unsaturated, linear or branched, wherein the alkyl chains optionally contain one or more linking groups L, the linking groups L being selected from amide bonds, ester bonds, disulfide bonds, ketithiolide bonds, ether bonds, or combinations thereof. R1 represents the N-terminus of the peptide group that can ionize the lipid, and R1 is a hydrogen atom or an acetyl group. R4 represents the C-terminus of the peptide-ionizable lipid, and R4 is a hydroxyl group or has an amino group, amino acid group, and / or other functional group modification.

2. The lipid nanoparticle of claim 1, wherein: The peptide-based ionizable lipid is any of the following lipids: a12Dab4, Am-Ka12K4, Ac-Ka12K4, Am-R3a12K4, Am-K3a12K4, a12K4, Ha12K4, Ca12K2, a12K4D-Ca, a12K4E2-Ca, a12K2E-Ca, a12K4E-Ca; Its structural formula is as follows: 。 3. The lipid nanoparticles according to claim 1 or 2, characterized in that: The auxiliary phospholipids include, but are not limited to: DOPC (1,2) Dioleoyl sn glycerol 3 Phosphatidylcholine), DSPC (1,2) Distearate acyl sn glycerol 3 Phosphatidylcholine), POPE (1-palmitoyl-2-oleoyl-phosphatidylethanolamine), DPPC (1,2) Dipalmitoyl sn glycerol 3 Phosphatidylcholine), POPC (2) oleoyl 1 Palmitoyl sn glycerol 3 Phosphatidylcholine), DOPE (1,2) Dioleoyl sn glycerol 3 Phosphatidylethanolamine), DSPE (1,2) Distearate acyl sn glycerol 3 Phosphatidylethanolamine and DPPE (1,2) Dipalmitoyl sn glycerol 3 Phosphatidylethanolamine); And / or, the steroidal lipids include, but are not limited to: cholesterol, cholesterolanol, 7α-hydroxylamine, and hydroxylamine. Hydroxycholesterol, β sitosterol, 7β Hydroxycholesterol, cholesterol, and cholesterolone; And / or, the polyethylene glycol lipids include, but are not limited to: DMG-PEG2000 (1,2... Dimyristic acid rac glycerin 3 Methoxylated polyethylene glycol 2000), DSG PEG2000 (1,2) Distearate rac glycerin 3 Methoxylated polyethylene glycol 2000), DSPE mPEG2000 (distearylphosphatidylacetamide-methoxy polyethylene glycol 2000) and ALC 0159 (Methoxylated polyethylene glycol bis(tetradecyl)acetamide); And / or, the peptide-based ionizable lipids account for 35-60% of the raw materials in molar percentage; the auxiliary phospholipids account for 5-25% of the raw materials in molar percentage; the steroidal lipids account for 30-50% of the raw materials in molar percentage; and the polyethylene glycol lipids account for 1-5% of the raw materials in molar percentage.

4. A lipid nanoparticle carrying nucleic acid for brain targeting via the nasal route, comprising the lipid nanoparticle of any one of claims 1-3 and the nucleic acid molecule encapsulated in the lipid nanoparticle.

5. The lipid nanoparticles loaded with nucleic acid according to claim 4, characterized in that: The nucleic acid is mRNA; And / or, the mRNA expresses a protein with therapeutic function or a protein with fluorescent reporter function; And / or, the therapeutic function is to treat diseases of the central nervous system; And / or, the molar ratio of ionizable nitrogen atoms in the lipid molecules of the lipid nanoparticles to phosphate molecules in the nucleic acid molecules is fixed at (5-15):1; And / or, the lipid nanoparticles carrying nucleic acids are administered via intranasal administration.

6. The use of the lipid nanoparticles according to any one of claims 1-3 as a carrier in drug delivery; wherein the drug is a nucleic acid; Alternatively, the use of the lipid nanoparticles according to any one of claims 1-3 or the nucleic acid-loaded lipid nanoparticles according to claim 4 or 5 in the preparation of products having the functions described in A1) and / or A2): A1) This allows the drug to be delivered intranasally and targeted to the brain; A2) Treatment of central nervous system diseases.

7. A product having the functions described in A1) and / or A2) below, comprising the nucleic acid-loaded lipid nanoparticles as described in claim 4 or 5; A1) This allows the drug to be delivered intranasally and targeted to the brain; A2) Treatment of central nervous system diseases.

8. The method for preparing the nucleic acid-loaded lipid nanoparticles according to claim 4 or 5, comprising the following steps: Step (a1): Dissolve peptide-based ionizable lipids, cofactor phospholipids, steroidal lipids, and polyethylene glycol lipids in an organic solvent according to the molar ratio to obtain a lipid organic phase; Step (a2): Dissolve the nucleic acid molecules in a buffer solution to obtain a nucleic acid molecule solution; Step (a3): The lipid organic phase from step (a1) and the nucleic acid molecule solution from step (a2) are mixed and incubated to obtain a solution of lipid nanoparticles loaded with nucleic acid.

9. The preparation method according to claim 8, characterized in that: The organic solvent includes any one or a combination of at least two of methanol, ethanol, propanol, tetrahydrofuran, and diethyl ether; And / or, the buffer solution is a citrate-sodium citrate buffer solution; And / or, the molar ratio of ionizable nitrogen atoms in the total lipids to phosphate molecules in the nucleic acids in the lipid organic phase is fixed at (5-15):1; And / or, the volume ratio of the lipid organic phase to the nucleic acid molecule solution is 1:(1-6). And / or, the incubation conditions are static incubation at room temperature for 10-30 minutes.

10. A method for delivering brain-targeted, nucleic acid-loaded lipid nanoparticles, comprising the following steps: 1) Prepare the nucleic acid-loaded lipid nanoparticles as described in claim 4 or 5; 2) The nucleic acid-loaded lipid nanoparticles are administered intranasally to achieve brain-targeted delivery of nucleic acid-loaded nucleic acid lipid nanoparticles.

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