Ionizable lipid compounds containing a borneol structure, methods of making and use thereof

Abstract

The present application relates to ionizable lipid compounds containing borneol structure and its preparation method and application, and belongs to the technical field of biological medicine. The present application introduces polyamine structure and hydrophobic tail chain by modifying the hydroxyl group at C2 position of borneol molecule, and prepares a new type of ionizable lipid compounds containing borneol structure. The compound has the following advantages: (i) the borneol structure can effectively break through the skin keratin layer barrier and significantly improve the transdermal absorption efficiency; (ii) the polyamine structure can be ionized under acidic conditions, efficiently carry nucleic acid drugs through electrostatic adsorption, help to enhance the stability of nucleic acid and promote lysosome escape; (iii) the tail chain modification enhances the liposolubility of the compound, endows it with lipid characteristics, and can be used as a raw material for the preparation of solid lipid nanoparticles. The present application also provides a preparation method of the compound, and the ionizable lipid compounds containing borneol structure show good application prospect in the field of transdermal delivery of nucleic acid drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, and relates to ionizable lipid compounds containing borneol structure, their preparation methods, and applications. Background Technology

[0002] Transdermal drug delivery systems (TDDS) have garnered significant attention in clinical applications due to their non-invasive nature, avoidance of injection pain and needle phobia, and suitability, particularly for children, the elderly, and long-term users. Compared to oral administration, the transdermal route completely bypasses the first-pass metabolism in the liver, significantly improving drug bioavailability; for some drugs, transdermal bioavailability can be 2-5 times higher than oral administration. However, the skin, as the largest organ in the human body, is composed of multiple barriers to transdermal drug absorption, formed by the epidermis, dermis, and subcutaneous tissue. The stratum corneum, located on the outermost layer of the epidermis, is composed of 10-15 layers of dead keratinocytes and a highly ordered lipid bilayer primarily composed of ceramides, cholesterol, and free fatty acids, forming the core barrier for transdermal drug absorption. Therefore, effectively overcoming the stratum corneum barrier has become the primary challenge in the development of TDDS formulations.

[0003] To overcome the aforementioned barriers, various transdermal penetration enhancers have been developed in existing technologies, mainly divided into two categories: chemically synthesized and naturally derived. While chemically synthesized penetration enhancers (such as azone) have certain penetration-enhancing effects, they generally suffer from high toxicity, damage to the skin barrier, easy induction of irritation or allergic reactions, and slow onset of action. In contrast, naturally derived penetration enhancers such as borneol, peppermint oil, and eucalyptus oil not only reduce the skin barrier's resistance to drugs by disrupting the lipid bilayer structure of the stratum corneum, but also exhibit good safety, with low irritation and toxicity. Among them, borneol, as a traditional Chinese medicine used for "guide drugs" and "opening orifices," has demonstrated unique application value in TDDS research. Its penetration-enhancing mechanism mainly stems from the high compatibility between borneol's unique lipid-soluble bridged ring structure and the stratum corneum lipid matrix: borneol can reversibly disrupt the orderly arrangement of the lipid bilayer in the stratum corneum, forming microchannels within the barrier structure that allow drug molecules to diffuse. In addition, borneol has the dual functions of promoting permeation and enhancing efficacy. It can significantly increase the transdermal throughput of various drugs such as ligustrazine, fluorouracil, and tripterygium wilfordii. Its own anti-inflammatory, analgesic, and mind-clearing pharmacological activities can also exert a synergistic therapeutic effect with the main drugs.

[0004] In recent years, nucleic acid drugs such as siRNA, mRNA, and DNA vaccines have made significant progress in the treatment of TDDS (Treatment-Dependent Disease). Transdermal delivery of nucleic acid drugs offers advantages such as being non-invasive, having high compliance, and bypassing the first-pass effect of the liver, making it particularly suitable for the treatment of skin-related diseases such as psoriasis, melanoma, scarring, and hereditary skin diseases. Current research mainly relies on nanocarriers such as lipid nanoparticles, polymer nanoparticles, and exosomes, or physical permeation techniques such as microneedles and electroporation to achieve intradermal delivery of nucleic acids. Among these, ionizable lipid nanoparticles have attracted considerable attention due to their combined properties of efficient encapsulation, protection of nucleic acids, promotion of cell internalization, and lysosomal escape. However, existing ionizable lipids are mostly designed for intravenous or intramuscular injection scenarios, and their structures have not yet been optimized for the penetration ability of the skin's stratum corneum barrier, lacking deep adaptation to the specific needs of transdermal drug delivery. Therefore, developing novel ionizable lipids that retain efficient nucleic acid loading and delivery performance while possessing the ability to actively cross the stratum corneum barrier, enhance skin penetration depth, and improve local transfection efficiency has become a key scientific issue and technological breakthrough for promoting the clinical translation of nucleic acid drugs through transdermal delivery. Based on this, designing novel ionizable lipids containing natural permeation-enhancing structural units such as borneol holds promise for achieving an integrated "barrier penetration-nucleic acid delivery" delivery strategy, providing a new technical pathway and valuable exploration for transdermal nucleic acid drug delivery. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide an ionizable lipid compound containing a camphor structure; a second objective of the present invention is to provide a method for preparing the said ionizable lipid compound containing a camphor structure; and a third objective of the present invention is to provide the application of the said ionizable lipid compound containing a camphor structure in the preparation of a transdermal drug delivery system.

[0006] To achieve the above objectives, the present invention provides the following technical solution: 1. An ionizable lipid compound containing a camphor structure, the structural formula of which is shown below:

[0007] I Where R is or or .

[0008] Preferably, the structural formula of the compound is as follows: , Ⅰ-1 , Ⅰ-2 .

[0009] Ⅰ-3 2. The method for preparing the above-mentioned ionizable lipid compound containing a camphor structure, wherein the reaction formula of the method is as follows:

[0010] Where R is or or .

[0011] Preferably, the method includes the following steps: (1) Borneol, succinic anhydride, 4-dimethylaminopyridine (DMAP) and organic base were dissolved in dichloromethane (CH2Cl2) and stirred at room temperature for 24-72 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was acidified with dilute hydrochloric acid (HCl). The organic phase was washed successively with saturated brine (NaCl solution) and saturated sodium bicarbonate (NaHCO3 solution). The mixture was separated and dried with anhydrous sodium sulfate (Na2SO4). After filtration, the solvent was evaporated under vacuum and purified by column chromatography to obtain carboxylic acid derivatives of borneol. (2) The carboxylic acid derivative of borneol, N-hydroxysuccinimide (NHS) and condensing agent obtained in step (1) were dissolved in dichloromethane (CH2Cl2) and stirred at room temperature for 2-4 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the solvent was removed by evaporation under vacuum. The product was dissolved in a mixed solution of n-hexane / ethyl acetate (n-hexane / EtOAc), washed with saturated brine (NaCl solution), dried with anhydrous sodium sulfate (Na2SO4), filtered, and the organic phase was refrigerated to crystallize. The product was then dried under vacuum to obtain the borneol active ester intermediate. (3) N-(2-aminoethyl)-1,4-piperazine diethylamine, the borneol active ester intermediate obtained in step (2) and the organic base are dissolved in an organic solvent and stirred at 30~50℃ for 2 h. Then, excess alkyl epoxide is added and the temperature is raised to 60~80℃ and stirred for 48~72 h. The solvent is removed by evaporation under vacuum and purified by column chromatography to obtain an ionizable lipid compound containing a borneol structure.

[0012] More preferably, the molar ratio of borneol, succinic anhydride, 4-dimethylaminopyridine (DMAP) to organic base in step (1) is 1:2~4:1~3:1~5; The organic base is triethylamine (Et3N) or N,N-diisopropylethylamine (DIPEA). The mass-to-volume ratio of borneol to dichloromethane (CH2Cl2) is 1:0.2~10 g / mL; The volume ratio of dichloromethane (CH2Cl2) to dilute hydrochloric acid (HCl), saturated saline solution (NaCl solution), and saturated sodium bicarbonate (NaHCO3 solution) is independently 1:0.5~2. The mass-to-volume ratio of anhydrous sodium sulfate (Na2SO4) to dichloromethane (CH2Cl2) is 1:5~20 g / mL; The eluent used in column chromatography purification is a mixed solution of n-hexane and ethyl acetate (EtOAc) in a volume ratio of 2 to 6:1.

[0013] More preferably, in step (2), the molar ratio of the carboxylic acid derivative of borneol obtained in step (1), N-hydroxysuccinimide (NHS), and the condensing agent is 1:0.5~2.5:1.1~2.5; The condensing agent is N,N-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl). The n-hexane / ethyl acetate mixed solution is a mixed solution composed of n-hexane (n-hexane) and ethyl acetate (EtOAc) in a volume ratio of 2~6:1; The volume ratio of the mixed solution to saturated saline solution (NaCl solution) is 1:1~4; The mass-to-volume ratio of anhydrous sodium sulfate (Na2SO4) to the mixed solution is 1:5~20 g / mL.

[0014] More preferably, the molar ratio of N-(2-aminoethyl)-1,4-piperazine diethyldiethylamine, borneol active ester intermediate, and organic base in step (3) is 1~5:1:1.2~2.5; The organic base is triethylamine (Et3N) or N,N-diisopropylethylamine (DIPEA). The molar ratio of the borneol active ester intermediate to the alkyl epoxide compound is 1:2.5~8; The alkyl epoxide is 1,2-epoxydodecane, 1,2-epoxytetradecane, or 1,2-epoxyhexadecane; The eluent used for purification by column chromatography was a mixed solution of dichloromethane (CH2Cl2), methanol (MeOH), and ammonia (NH3·H2O) in a volume ratio of 72:25:3.

[0015] 3. The ionizable lipid compound containing a camphor structure can be used to prepare transdermal drug delivery carriers.

[0016] Preferably, the transdermal drug delivery carrier is a solid lipid nanoparticle.

[0017] 4. The ionizable lipid compound containing the camphor structure can also be used to prepare nucleic acid drug delivery materials.

[0018] Preferably, the nucleic acid drug includes siRNA and / or mRNA.

[0019] The beneficial effects of this invention are as follows: (1) This invention utilizes the unique molecular structure of borneol and modifies its C2 position hydroxyl group to obtain a compound that can actively break through the skin stratum corneum barrier, significantly improve the transdermal absorption efficiency of drugs (especially nucleic acid drugs), and overcome the problem of poor transdermal permeability of nucleic acid drugs.

[0020] (2) The polyamine structure in the complex can be protonated and become positively charged in an acidic environment. It can efficiently encapsulate negatively charged nucleic acid drugs (such as siRNA and mRNA) through electrostatic adsorption to form a stable complex. At the same time, this ionization property helps to promote the escape of the complex from the lysosome, avoid the degradation of nucleic acid, and thus improve the intracellular delivery efficiency.

[0021] (3) By introducing hydrophobic tail chain modification, the compounds of the present invention have good lipid solubility and can be used alone or in combination with other lipid materials to construct lipid-based nanodelivery systems such as solid lipid nanoparticles, providing a novel carrier material for the development of transdermal drug delivery formulations.

[0022] (4) The present invention uses borneol from natural sources as raw material and designs and synthesizes a class of novel ionizable lipid compounds by introducing polyamine structure and hydrophobic tail chain; the preparation method is mild, simple and controllable, and suitable for large-scale production.

[0023] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0024] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 The present invention discloses the general structural formula of an ionizable lipid compound containing a camphor structure; Figure 2 Ionizable lipid compounds containing borneol structure prepared in Examples 1-3 1. I 2. I Mass spectrum of 3; Figure 3 I 1. TEM image of lipid nanoparticles prepared from ionizable lipids; Figure 4 I as determined by TNS fluorescence spectroscopy 1. I 2 and I 3. pKa value of the prepared lipid nanoparticles; Figure 5 I was determined using a gel retardation assay. 1. The N / P ratio of the prepared lipid nanoparticles encapsulating siRNA; Figure 6 MTT assay for I 1. I 2 and I 3. Cytotoxicity of the prepared lipid nanoparticles; Figure 7 In vitro transfection efficiency of lipid nanoparticles prepared by I-1, I-2 and I-3 was investigated. Figure 8 Lysosomal escape characterization of lipid nanoparticles prepared by I-1; Figure 9 I 1. I 2. I 3. In vivo study of the transdermal permeability of the prepared lipid nanoparticles; Figure 10 HE staining pathological sections of skin tissue from mouse models of psoriasis after treatment with different formulations. Detailed Implementation

[0025] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0026] The raw materials and reagents used in the embodiments of this invention are all commercially available analytical grade / pharmaceutical grade products, and the experimental methods used are all conventional experimental methods in the biomedical field unless otherwise specified.

[0027] Example 1 Preparation of structural formula I The ionizable lipid compound containing a camphor structure, I-1, has the following structural formula: , The specific preparation method is as follows: (1) Weigh 500 mg (3.25 mmol, 1 eq) of borneol, 975 mg (9.75 mmol, 3 eq) of succinic anhydride, 795 mg (6.5 mmol, 2 eq) of 4-dimethylaminopyridine (DMAP) and 657.5 mg (6.5 mmol, 2 eq) of triethylamine (Et3N) and dissolve them in 20 mL of dichloromethane (CH2Cl2). Stir the reaction at room temperature for 24 h and monitor the reaction by thin-layer chromatography (TLC). After the reaction is complete, add dilute hydrochloric acid (HCl, 20 mL) to acidify three times, separate the liquid and organic phase, wash three times with saturated saline (NaCl solution, 20 mL) and saturated sodium bicarbonate (NaHCO3 solution, 20 mL) respectively, separate the liquid and organic phase, and wash with anhydrous sodium sulfate (Na2SO4, 150 mL) to dissolve the organic phase. g) Dry, filter, evaporate under vacuum to remove solvent, and purify by column chromatography (using a mixed solution of n-hexane and ethyl acetate (EtOAc) in a 3:1 volume ratio) to obtain a carboxylic acid derivative of borneol, whose ¹H NMR (600 MHz, Chloroform-d) δ 4.75 (ddt, J = 6.8, 5.2, 1.5 Hz, 1H), 2.73– 2.60 (m, 2H), 2.62– 2.54 (m, 2H), 1.93– 1.81 (m, 2H), 1.72– 1.55 (m, 3H), 1.43 (ddt, J = 12.5, 7.2, 5.1 Hz, 1H), 1.34 (ddd, J = 12.6, 7.1, 5.3 Hz). Hz,1H), 0.93 (d, J = 1.3 Hz, 3H), 0.88 (d, J = 1.5 Hz, 3H), 0.83 (d, J = 1.5 Hz,3H).

[0028] (2) Weigh 100 mg (0.39 mmol, 1 eq) of the carboxylic acid derivative of borneol obtained in step (1), 49.5 mg (0.43 mmol, 1.1 eq) of N-hydroxysuccinimide (NHS) and 88.5 mg (0.43 mmol, 1.1 eq) of N,N-dicyclohexylcarbodiimide (DCC) and dissolve them in 10 mL of dichloromethane (CH2Cl2). Stir the reaction at room temperature for 4 h and monitor the reaction by thin-layer chromatography (TLC). After the reaction is complete, evaporate the solvent under vacuum, add 5 mL of a mixed solution of n-hexane and ethyl acetate (EtOAc) in a volume ratio of 4:1 to dissolve the product, wash three times with saturated saline (NaCl solution, 10 mL), add anhydrous sodium sulfate (Na2SO4, 120 g) to dry, filter, refrigerate the organic phase at 4 °C to precipitate crystals, dry under vacuum to obtain the borneol active ester intermediate, whose ¹H NMR (600 MHz, Chloroform-d) δ 4.75 (ddt, J = 6.8, 5.2, 1.5Hz, 1H), 2.80 (s, 4H), 2.84 – 2.65 (m, 4H), 1.93 – 1.81 (m, 2H), 1.72 – 1.55(m, 3H), 1.43 (ddt, J = 12.5, 7.2, 5.1 Hz, 1H), 1.34 (ddd, J = 12.6, 7.1, 5.2Hz, 1H), 0.93 (d, J = 1.3 Hz, 3H), 0.88 (d, J = 1.5 Hz, 3H), 0.83 (d, J = 1.5Hz, 3H).

[0029] (3) Weigh 50 mg (0.23 mmol, 1 eq) of N-(2-aminoethyl)-1,4-piperazine diethyldiethylamine. Disperse 81.5 mg (0.23 mmol, 1 eq) of the borneol active ester intermediate obtained in step (2) in 5 mL of ethanol (EtOH). Then add 28 mg (0.28 mmol, 1.2 eq) of triethylamine (Et3N) and heat and stir at 40 °C for 2 h. Then add 154 mg (0.84 mmol, 3.6 eq) of 1,2-epoxydodecane and heat to 80 °C and stir for 48 h. Evaporate the solvent from the reactants under vacuum and purify by column chromatography (the eluent used is a mixed solution of dichloromethane (CH2Cl2), methanol (MeOH) and ammonia (NH3·H2O) in a volume ratio of 72:25:3) to obtain the product with the structural formula I. 1. An ionizable lipid compound containing a camphor structure, its ¹H NMR (600 MHz, Chloroform-d) δ 4.04 – 3.95 (m, 1H), 3.47 (qd, J = 4.8, 0.8 Hz, 1H), 3.32 (dd, J = 6.0, 1.9 Hz, 1H), 3.01 (ddd, J = 31.0, 11.9, 5.4 Hz, 1H), 2.95 – 2.84 (m, 2H), 2.85 (s, 3H), 2.86 – 2.71 (m, 4H), 2.13 – 2.01 (m, 1H), 1.93 – 1.79 (m, 1H), 1.82 – 1.75 (m, 1H), 1.67 – 1.57 (m, 1H), 1.60 – 1.51(m, 2H), 1.53 – 1.48 (m, 2H), 1.51 – 1.41 (m, 12H), 1.13 (d, J = 1.3 Hz, 1H), 1.14 – 1.06 (m, 4H), 1.03 (d, J = 1.5 Hz, 1H).

[0030] Example 2 Following the reaction steps in Example 1 (i.e., replacing some reactants as in Example 1, while maintaining the same addition ratios and other reaction conditions as in Example 1), a structure with the following formula I was prepared. The ionizable lipid compound containing a camphor structure, I-2, has the following structural formula: , Specific steps: Replace 1,2-epoxytetradecane in Example 1 (3) with 1,2-epoxytetradecane to prepare a structure with the following formula: The ionizable lipid compound containing a camphor structure, ¹H NMR (600 MHz, Chloroform-d) δ 3.83 – 3.75 (m, 1H), 3.30 – 3.24 (m, 1H), 3.12 (dd, J = 6.0, 1.9 Hz, 1H), 2.81 (ddd, J = 31.0, 11.9, 5.4 Hz, 1H), 2.74 – 2.66 (m, 1H), 2.69 – 2.64 (m, 1H), 2.65 (s, 3H), 2.64 – 2.50 (m, 4H), 1.93 – 1.81 (m, 1H), 1.72 – 1.59 (m, 1H), 1.62 – 1.55 (m, 1H), 2. 1.47 – 1.38 (m, 1H), 1.40 – 1.34 (m, 1H), 1.36 –1.29 (m, 2H), 1.31 – 1.21 (m, 18H), 0.93 (d, J = 1.3 Hz, 1H), 0.93 – 0.86 (m,4H), 0.83 (d, J = 1.5 Hz, 1H).

[0031] Example 3 Following the reaction steps in Example 1 (i.e., replacing some reactants as in Example 1, while maintaining the same addition ratios and other reaction conditions as in Example 1), a structure with the following formula I was prepared. 3. An ionizable lipid compound containing a camphor structure, the structural formula of I-3 is: , The specific preparation method is as follows: 1,2-epoxyhexadecane is replaced with 1,2-epoxydodecane in Example 1 (3) to prepare the structure with the following formula: I The ionizable lipid compound containing camphor structure 3 was ionized by ¹H NMR (600 MHz, Chloroform-d) δ 3.79 (tqd, J = 7.3, 5.4, 2.0 Hz, 1H), 3.12 (dd, J = 6.0, 1.9Hz, 1H), 2.81 (ddd, J = 31.0, 11.9, 5.4 Hz, 1H), 2.74 – 2.63 (m, 2H), 2.65 (s, 3H), 2.65 – 2.50 (m, 4H), 1.93 – 1.81 (m, 1H), 1.72 – 1.59 (m, 1H), 1.62– 1.55 (m, 1H), 1.47 – 1.37 (m, 1H). 1.39 – 1.33 (m, 1H), 1.36 – 1.28 (m,3H), 1.31 – 1.21 (m, 21H), 0.93 (d, J = 1.3 Hz, 1H), 0.94 – 0.86 (m, 4H), 0.83 (d, J = 1.5 Hz, 1H).

[0032] The ionizable lipid compound containing a camphor structure disclosed in this invention has the following general structural formula: Figure 1 As shown. The mass spectra of the ionizable lipid compounds I-1, I-2, and I-3 containing a camphor structure prepared in Examples 1-3 are shown below. Figure 2 As shown.

[0033] Example 4 Ionizable lipid compound containing borneol structure 1. I 2. I 3. Lipid nanoparticles (LNPs) were prepared, and their related physicochemical properties and in vitro and in vivo activities were tested, as detailed below: (1) Screening of pharmaceutical formulations First, lipid nanoparticles were prepared according to the different formulations in Table 1, and I... 1. Prepared as an ionizable lipid, along with cofactors DSPC, cholesterol, and DMG-PEG. 2k Combination formation based on I 1. Different lipid nanoparticles. The specific preparation method is as follows: I 1. Assists in the production of lipids DSPC, cholesterol, and DMG-PEG. 2kDissolve and mix the siRNA in an organic solvent according to the appropriate molar ratio. Using a microfluidic device, transfer the mixture to a 6.25 mM sodium acetate buffer (pH = 5) containing siRNA (Antisense: 5′-UUCAUUAUCAGUGCAAUUGdTdT-3′) at a water:organic solvent volume ratio. v / v The mixture was prepared in a 3:1 ratio, allowed to stand, and then transferred to a dialysis bag for dialyzing with PBS buffer (pH = 7.4) overnight. Finally, it was concentrated using an ultracentrifuge filter. The particle size and PDI (polymer dispersibility index) of the prepared formulations are shown in Table 2.

[0034] Table 1. Formulations of lipid nanoparticles with different molar ratios

[0035] Table 2. Particle size and PDI of lipid nanoparticles with different molar ratios.

[0036] As can be seen from Table 2, the lipid nanoparticles prepared under formulation No. 5 have a particle size of <100nm and good dispersibility, and have greater potential for transdermal absorption in terms of particle size, which can be used for subsequent experiments.

[0037] Compound I prepared in Examples 1-3 1. I 2. I 3. Lipid nanoparticles formed from different ionizable lipids according to formulation number 5 in Table 1 were prepared. The particle size and PDI are shown in Table 3. As can be seen from Table 3, I... 1. I 2. I The LNP particles obtained were all <100nm in size and had good dispersion, which can be used for subsequent experiments.

[0038] Table 3. Particle size and PDI of lipid nanoparticles formed from different ionizable lipids of the same formulation

[0039] (2) Morphological characterization of lipid nanoparticles Compound I prepared in Example 2 1. Lipid nanoparticles, prepared according to formulation 5 in Table 1, were used as ionizable lipids. The size and morphology of the particles were then observed using transmission electron microscopy (TEM). The specific sample preparation steps were as follows: the prepared lipid nanoparticle sample was dropped onto a copper grid covered with a support film and incubated at room temperature for 15 min. Then, 1% phosphotungstic acid solution was added, and the sample was incubated at room temperature for 10 min. After drying, the sample was observed under a TEM and the images were recorded. The morphological characterization results are as follows: Figure 3 As shown, by I 1. The lipid nanoparticles prepared from ionizable lipids are spherical, with a round morphology, uniform size, and a particle size of about 90 nm.

[0040] (3) Determination of pka value Compound I prepared in Examples 1-3 1. I 2. I 3. Lipid nanoparticles prepared according to the formulation of formulation 5 in Table 1 were used as ionizable lipids, and the pKa value of the lipid nanoparticles was determined by TNS fluorescence irradiation.

[0041] The specific experimental method was as follows: First, the prepared lipid nanoparticles were diluted to 2 mM with PBS buffer solution. Then, a buffer solution containing 130 mM NaCl, 10 mM ammonium acetate, 10 mM Hepes, and 10 mM MES was prepared. The lipid nanoparticle solution was further diluted to 100 μM, and the solution was divided into 8 groups. The pH value of each group of solutions was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 using NaOH or HCl solution. Anionic fluorescent dye 2... (p toluidino) 6 Naphthalenesulfonic acid (TNS) was dissolved in distilled water to form a 100 μM solution, and the final concentration of lipid nanoparticles was adjusted to 1 μM. At room temperature, with an excitation wavelength of 321 nm and an emission wavelength of 445 nm, the fluorescence intensity of each group was measured using a multi-functional microplate reader. pKa was defined as the fluorescence intensity at which the pH value rises to half of the maximum fluorescence intensity. The final results are as follows: Figure 4 As shown. From Figure 4 It can be seen that the I synthesized by this invention 1. I 2. I 3. As an ionizable lipid, the pka value of LNP prepared is 6~6.5, which is beneficial for the efficient loading of nucleic acids and lysosomal escape.

[0042] (4) Determination of siRNA encapsulation efficiency Using quantitative methods The iT RiboGreen RNA Assay kit was used to detect the RNA samples listed in Table 3 above. 1. I 2. I 3. Encapsulation of siRNA by lipid nanoparticles prepared from ionizable lipids. The specific experimental method was as follows: lipid nanoparticles before dialysis were diluted in Tris-125 microparticles in 96-well plates. The dialyzed lipid nanoparticles were diluted in EDTA(TE) buffer to a concentration of 2% Triton X. The sample was immersed in 100 μL TE buffer for 30 min, and then RiboGreen working solution was added to the corresponding wells. The fluorescence intensity was measured using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. A standard curve for siRNA was prepared, and the encapsulation efficiency was calculated by measuring the amount of free siRNA and siRNA after dialysis. The results are shown in Table 4. The I synthesized by this invention... 1. I 2. I 3. As an ionizable lipid, LNP has a good encapsulation rate of nucleic acid, which is higher than 95%, and there is no leakage problem.

[0043] Table 4, I 1. I 2. I 3. Encapsulation efficiency of siRNA by lipid nanoparticles prepared from ionizable lipids

[0044] (5) Determination of N / P ratio by gel retardation experiment The gel retardation assay was used to determine the I-100% concentration of the formulation prepared from the formulation number 5 in Table 1. 1. N / P ratio of siRNA-loaded lipid nanoparticles. The specific experimental method was as follows: siRNA was dissolved in DEPC water. Using microfluidic technology, the concentrations of the organic and aqueous phases were mixed according to the formulation specified in Table 1 (number 5) at N / P ratios of 1:4, 1:2, 1:1, 2:1, 4:1, 8:1, 16:1, and 32:1 to prepare siRNA-loaded lipid nanoparticle solutions. The binding ability of LNPs to siRNA was investigated by agarose gel electrophoresis. The nanoparticle solution sample was mixed with 6× loading buffer and then loaded for electrophoresis. The electrophoresis conditions were set to 100 V and 60 mA. Electrophoresis was stopped when the bromophenol blue indicator reached the lower edge of the gel, and the sample was observed and images were acquired using a gel imaging system. The results are as follows: Figure 5 As shown, the I synthesized in this invention 1. As an ionizable lipid, LNP can be prepared, and complete encapsulation of siRNA can be achieved when the N / P ratio is greater than 4:1.

[0045] (6) MTT assay for the cytotoxicity of lipid nanoparticles HaCaT cells in logarithmic growth phase were trypsinized and seeded at a density of 5 × 10³ cells / well in 96-well plates, with a final volume of 200 μL per well. The plates were incubated overnight at 37°C with 5% CO2 until cell attachment. When cell confluence reached 60%-70%, the plates were removed, the original culture medium was discarded, and an appropriate amount of blood-free and antibiotic-free culture medium was added. Then, different concentrations of LNPs prepared from ionizable lipids I-1, I-2, and I-3 were added, and the plates were returned to the 37°C, 5% CO2 incubator for further incubation. After 24 hours, the drug-containing medium was discarded, and 180 μL of fresh culture medium and 20 μL of MTT solution (5 mg / mL) were added to each well. The plates were then incubated again for 4 hours. After incubation, the 96-well plates were removed, the supernatant was discarded, and 150 μL of LDMSO was added to each well. The plates were then shaken at 37°C for 15 minutes. Finally, the absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The absorbance values ​​were recorded, and a cell growth inhibition rate curve was plotted with drug concentration on the x-axis and cell viability on the y-axis. The results are as follows: Figure 6 As shown, I synthesized by the present invention 1. I 2. I 3. LNPs prepared as ionizable lipids exhibit low cytotoxicity and high biocompatibility.

[0046] (7) Evaluation of in vitro transfection efficiency of Luc siRNA loaded on lipid nanoparticles HaCaT-Luc cells were seeded at a density of 5 × 10³ cells per well in 96-well plates and cultured for 24 h. The culture plates were removed, the original culture medium was discarded, and an appropriate amount of serum- and antibiotic-free DMEM medium was added. Then, LNPs (containing Luc siRNA, with a final siRNA concentration of 50 nM and an antisense sequence of 5′-UUCAUUAUCAGUGCAAUUGdTdT-3′) prepared from ionizable lipids I-1, I-2, and I-3 were added, and the cells were cultured for another 24 h. Lipo2000 transfection reagent was used as a positive control. After culture, cells were collected and analyzed using the Firefly luciferase reporter gene assay kit, and protein concentration was simultaneously determined using the BCA protein assay kit. The relative light units (RLU) percentage of luciferase was calculated using the following formula: RLU (%) = luciferase luminescence intensity / relative protein concentration × 100%. Results are as follows: Figure 7As shown, compared with Lipo2000, the gold standard reagent for cell transfection, LNP prepared using I-1, I-2, and I-3 as ionizable lipids can efficiently carry siRNA into cells and exhibits better target gene silencing efficiency.

[0047] (8) Characterization of lysosomal escape of lipid nanoparticles HaCaT cells were stored at a density of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 12-well plates and cultured at 37°C in a 5% CO2 incubator. When cells adhered and reached approximately 50% confluence, the original culture medium was discarded, and 1 mL of serum- and antibiotic-free DMEM medium was added. Then, LNP (containing FAM-labeled siRNA at a final siRNA concentration of 200 nM) prepared using I-1 as an ionizable lipid was added, and the plates were returned to the 37°C, 5% CO2 incubator for another 6 hours. After culture, the drug-containing medium was discarded, and each well was washed three times with PBS. Then, a suitable amount of pre-warmed lysosomal fluorescent probe Lyso-Tracker was added, and the plates were incubated at 37°C for 30 minutes. Cells were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 30 minutes, rinsed three times with PBS, and incubated with DAPI nuclear staining solution in the dark for 10 minutes. After washing three times with PBS again, fluorescence co-localization imaging was performed using a high-content imaging system in confocal mode. The results are as follows: Figure 8 As shown, LNPs prepared using I-2 as an ionizable lipid can efficiently escape lysosomes after entering the cell and carrying siRNA.

[0048] (9) In vivo study of the transdermal permeability of lipid nanoparticles Nine BALB / c mice were anesthetized and their back hair was removed using a depilatory cream. After the skin's stratum corneum recovered, the mice were randomly divided into three groups of three. Lipid nanoparticles containing FAM-labeled siRNA, prepared according to formulas I-1, I-2, and I-3, were dispersed in hyaluronic acid hydrogels and applied evenly to the back skin of each mouse at a rate of approximately 0.2 mL (approximately 2 cm × 2 cm area). Six hours after administration, the mice were sacrificed, and the skin from the treatment site was harvested. The skin was repeatedly rinsed with PBS to remove surface residue and cut into 0.5 cm × 0.5 cm pieces. After OCT embedding, longitudinal sections with a thickness of approximately 10 μm were prepared using a cryostat. The sections were fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed three times with PBS, incubated with DAPI staining solution in the dark for 10 minutes, washed three more times with PBS, and then observed and photographed using a confocal laser scanning microscope (CLSM). The results are as follows: Figure 9 As shown, LNPs prepared using I-1, I-2, and I-3 as ionizable lipids can effectively penetrate the stratum corneum barrier of the skin and deliver nucleic acid drugs to the epidermis and dermis, demonstrating their great potential as transdermal drug delivery carriers for nucleic acid drugs.

[0049] (10) Pharmacological evaluation of lipid nanoparticles in the treatment of psoriasis First, an imiquimod (IMQ)-induced psoriasis mouse model was established: BALB / c mice were anesthetized, and their back hair was removed with depilatory cream. 5% imiquimod cream (IMQ dosage 3.125 mg / day) was then applied daily to a 2cm × 2cm area. After 3 days of treatment, typical psoriatic-like skin inflammation, including erythema and desquamation, appeared on the backs of the mice, indicating successful model induction. The successfully modeled mice were randomly divided into 6 groups of 5 mice each: ① Model group; ② Clobetasol propionate cream (CP) treatment group; ③ siRNA-loaded liposomes prepared with commercially available DLin-MC3-DMA ionizable lipids (MC3); ④ siRNA-loaded liposomes prepared with commercially available SM-102 ionizable lipids (SM102); ⑤ siRNA-loaded liposomes prepared with I-2 ionizable lipids (I-2); ⑥ siRNA-loaded liposomes prepared with I-3 ionizable lipids (I-3). The therapeutic target for psoriasis was a CD93-targeting siRNA sequence (antisense strand: 5′-CCACACCTGAAGGAACCATCTGdTdT-3′), administered at a dose of 1 mg / kg every two days in all groups. A healthy control group (n=5) was also included; mice were shaved but not treated with IMQ. At the end of day 9, skin samples from the backs of mice in each group were collected for HE staining to assess epidermal thickness and pathological improvement. Results are as follows: Figure 10 As shown, compared with the CP, MC3 and SM102 treatment groups, lipid nanoparticles (LNPs) formulated with I-1 or I-3 ionizable lipids can more effectively improve the skin pathological symptoms of psoriasis, and significantly reduce epidermal thickness, epidermal hyperplasia and skin edema; among them, the I-3 treatment group has the most significant effect.

[0050] In summary, this invention discloses a class of ionizable lipid compounds containing a borneol structure, their preparation methods, and applications. Through systematic studies from the molecular level to in vivo animal studies, the following advantages of these compounds have been confirmed: (i) the borneol structure can effectively penetrate the stratum corneum barrier of the skin, significantly improving the transdermal absorption efficiency of drugs; (ii) the polyamine structure ionizes under acidic conditions, enabling efficient loading of nucleic acid drugs through electrostatic adsorption, enhancing nucleic acid stability, and promoting lysosomal escape; (iii) tail chain modification improves the lipid solubility of the compounds, endowing them with excellent lipid properties, and making them suitable as raw materials for the preparation of solid lipid nanoparticles. Therefore, the ionizable lipid compounds containing a borneol structure disclosed in this invention show promising application prospects in the field of transdermal delivery of nucleic acid drugs.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An ionizable lipid compound containing a camphor structure, characterized in that, The structural formula of the compound is shown below: I Where R is or or .

2. The ionizable lipid compound containing a camphor structure according to claim 1, characterized in that, The structural formula of the compound is shown below: ， Ⅰ-1 ， Ⅰ-2 Ⅰ-3。 3. A method for preparing the ionizable lipid compound containing a camphor structure as described in any one of claims 1 to 2, characterized in that, The reaction formula of the method is: Where R is or or .

4. The preparation method according to claim 3, characterized in that, The method includes the following steps: (1) Borneol, succinic anhydride, 4-dimethylaminopyridine (DMAP) and organic base were dissolved in dichloromethane (CH2Cl2) and stirred at room temperature for 24-72 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was acidified with dilute hydrochloric acid (HCl). The organic phase was washed successively with saturated brine (NaCl solution) and saturated sodium bicarbonate (NaHCO3 solution). The mixture was separated and dried with anhydrous sodium sulfate (Na2SO4). After filtration, the solvent was evaporated under vacuum and purified by column chromatography to obtain carboxylic acid derivatives of borneol. (2) The carboxylic acid derivative of borneol, N-hydroxysuccinimide (NHS) and condensing agent obtained in step (1) were dissolved in dichloromethane (CH2Cl2) and stirred at room temperature for 2-4 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the solvent was removed by evaporation under vacuum. The product was dissolved in a mixed solution of n-hexane / ethyl acetate (n-hexane / EtOAc), washed with saturated brine (NaCl solution), dried with anhydrous sodium sulfate (Na2SO4), filtered, and the organic phase was refrigerated to crystallize. The product was then dried under vacuum to obtain the borneol active ester intermediate. (3) N-(2-aminoethyl)-1,4-piperazine diethylamine, the borneol active ester intermediate obtained in step (2) and the organic base are dissolved in an organic solvent and stirred at 30~50℃ for 2 h. Then, excess alkyl epoxide is added and the temperature is raised to 60~80℃ and stirred for 48~72 h. The solvent is removed by evaporation under vacuum and purified by column chromatography to obtain an ionizable lipid compound containing a borneol structure.

5. The preparation method according to claim 4, characterized in that, In step (1), the molar ratio of borneol, succinic anhydride, 4-dimethylaminopyridine (DMAP) to the organic base is 1:2~4:1~3:1~5; the organic base is triethylamine (Et3N) or N,N-diisopropylethylamine (DIPEA); the mass-to-volume ratio of borneol to dichloromethane (CH2Cl2) is 1:0.2~10 g / mL; the volume ratio of dichloromethane (CH2Cl2) to dilute hydrochloric acid (HCl), saturated saline solution (NaCl solution), and saturated sodium bicarbonate (NaHCO3 solution) is independently 1:0.5~2; the mass-to-volume ratio of anhydrous sodium sulfate (Na2SO4) to dichloromethane (CH2Cl2) is 1:5~20 g / mL; the eluent used for column chromatography purification is a mixed solution of n-hexane and ethyl acetate (EtOAc) in a volume ratio of 2~6:1; In step (2), the molar ratio of the carboxylic acid derivative of borneol obtained in step (1), N-hydroxysuccinimide (NHS), and the condensing agent is 1:0.5~2.5:1.1~2.5; the condensing agent is N,N-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl); the n-hexane / ethyl acetate mixed solution is a mixed solution composed of n-hexane (n-hexane) and ethyl acetate (EtOAc) in a volume ratio of 2~6:1; the volume ratio of the mixed solution to saturated saline solution (NaCl solution) is 1:1~4; the mass-volume ratio of anhydrous sodium sulfate (Na2SO4) to the mixed solution is 1:5~20 g / mL; In step (3), the molar ratio of N-(2-aminoethyl)-1,4-piperazine diethyldiethylamine, borneol active ester intermediate, and organic base is 1~5:1:1.2~2.5; the organic base is triethylamine (Et3N) or N,N-diisopropylethylamine (DIPEA); the molar ratio of borneol active ester intermediate to alkyl epoxide is 1:2.5~8; the alkyl epoxide is 1,2-epoxydodecane, 1,2-epoxytetradecane, or 1,2-epoxyhexadecane; the eluent used for column chromatography purification is a mixed solution of dichloromethane (CH2Cl2), methanol (MeOH), and ammonia (NH3·H2O) in a volume ratio of 72:25:

3.

6. The use of the ionizable lipid compound containing a camphor structure as described in any one of claims 1 to 2 in the preparation of transdermal drug delivery materials.

7. The use of the ionizable lipid compound containing a camphor structure as described in any one of claims 1 to 2 in the preparation of nucleic acid drug delivery materials.

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