Ionizable triglyceride lipids, their preparation methods and applications
By designing triglyceride-like ionizable lipids and introducing triglyceride structures and fatty acid tail chains, the problems of low biodegradability and lysosomal escape efficiency of lipid nanoparticles were solved, achieving efficient delivery of nucleic acid drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENYANG PHARMA UNIV
- Filing Date
- 2024-10-29
- Publication Date
- 2026-05-26
Smart Images
Figure CN119390598B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of drug carrier technology, specifically relating to ionizable lipids based on triglyceride structures, their preparation methods, and applications. Background Technology
[0002] Lipid nanoparticles are considered one of the most promising platforms for nucleic acid drug delivery. However, most lipid nanoparticles still face problems such as poor biodegradability and low lysosomal escape efficiency in vivo. Introducing ester bonds into the lipid structure can improve the degradability of lipid materials. Triglycerides, formed by the esterification of the three hydroxyl groups of glycerol with three fatty acid molecules, exhibit excellent degradability due to the presence of multiple ester bonds. Based on the structure of triglycerides, two fatty acid tail chains and a tertiary amine head can be introduced to construct a universal structure conforming to ionizable lipids. The head and tail structures are important components of ionizable materials, affecting the lysosomal escape ability and transfection activity of lipid materials.
[0003] Based on the above, the inventors combined the degradable triglyceride structure with head chains of different lengths and tail chains of fatty acids of different lengths and saturation to design a triglyceride-like ionizable lipid, which not only improved the degradability of the lipid material, but also improved the lysosomal escape ability of the lipid nanoparticles. Summary of the Invention
[0004] This invention overcomes the defects and deficiencies of the prior art and provides a novel triglyceride-like ionizable lipid, its preparation method, and its application.
[0005] To achieve the objectives of this invention, the following technical solution is adopted:
[0006] The first objective of this invention is to provide a triglyceride-like ionizable lipid. The triglyceride-like ionizable lipid of this invention has the following structural formula:
[0007]
[0008] Where n is 2, 3, or 4, and R1 and R2 are each independently selected from -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CH(CH2)7CH3, and -(CH2) 12 CH3, -(CH2) 14 CH3 or -(CH2) 16 CH3.
[0009] Alternatively, in the above-mentioned triglyceride-like ionizable lipids, the tertiary amine head group is N,N-dimethyl tertiary amine, and is located at the β-, γ-, or δ- position of the ester bond, respectively; the linker is a triglyceride; and the fatty acid tail chain is myristic acid, palmitic acid, stearic acid, oleic acid, or linoleic acid.
[0010] Alternatively, in the above-mentioned triglyceride-like ionizable lipids, the triglyceride-like ionizable lipids are selected from the following compounds:
[0011]
[0012] A second objective of this invention is to provide a method for preparing the triglyceride-like ionizable lipids described in the first objective.
[0013] The present invention uses the following method to synthesize the above-mentioned triglyceride-like ionizable lipids, and the synthetic route is shown below:
[0014] The method for synthesizing triglyceride-like ionizable lipids is to link 1,3-dihydroxyacetone with fatty acids through esterification under the action of a catalyst and then reduce it to obtain a triglyceride-like tail structure. The tail structure is then linked to an acid containing a tertiary amino head through esterification to obtain triglyceride-like ionizable lipids.
[0015]
[0016] The catalyst is 4-dimethylaminopyridine (DMAP) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI).
[0017] A third objective of the present invention is to provide the application of the triglyceride-like ionizable lipids described in the first objective above in the preparation of nucleic acid drug delivery carriers.
[0018] Alternatively, in the above applications, the delivery vector is used for the delivery of one or more of pDNA, siRNA, ASO, or mRNA.
[0019] Alternatively, in the above applications, the delivery carrier is a lipid nanoparticle.
[0020] Alternatively, in the above applications, the nucleic acid drug delivery carrier may be administered locally via intramuscular, subcutaneous, intradermal, or intratumoral administration using microneedles, injection, or perfusion methods, or the nucleic acid drug delivery carrier may be administered via intravenous injection.
[0021] The degradability of ionizable lipids is a crucial factor influencing lipid applications; therefore, examining the release of ionizable lipids is beneficial for predicting their degradability in vivo. siRNA rapidly and efficiently escapes from lysosomes into the cytoplasm, forming an RNA-induced silencing complex, which is significant for gene silencing. Therefore, examining the lysosomal escape efficiency of lipid nanoparticles is helpful for predicting their gene silencing efficiency in vivo. This invention aims to provide lipids that significantly improve the degradability and lysosomal escape efficiency of ionizable lipids for nucleic acid drug delivery.
[0022] The list of partially ionizable triglyceride-like lipids synthesized in this invention is shown in Table 1.
[0023] Table 1. Structure and structural identification information of ionizable triglyceride lipids.
[0024]
[0025]
[0026] The beneficial effects of this invention are:
[0027] This invention introduces a triglyceride backbone to improve the degradability of ionizable lipids. By introducing head chains of different lengths and fatty acid tail chains of different lengths and saturation, it is of great significance to screen out ionizable lipids with high lysosomal escape ability for nucleic acid drug delivery. Attached Figure Description
[0028] Figure 1 Release of ionizable lipids containing triglycerides with different heads.
[0029] Figure 2 Pearson colocation coefficients of triglyceride-like ionizable lipids with different heads.
[0030] Figure 3 In vivo gene silencing effects of triglyceride-like ionizable lipids with different heads. Detailed Implementation
[0031] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.
[0032] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0033] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.
[0034] The following compounds were prepared according to the general synthetic route for ionizable triglyceride-like lipids.
[0035] Example 1
[0036] Linoleic acid (35.00 mmol), DMAP (17.80 mmol), and EDCI (35.70 mmol) were added to a round-bottom flask and dissolved in anhydrous dichloromethane. The mixture was activated in an ice bath for 2 h. Then, 1,3-dihydroxyacetone (15.90 mmol) was added to the reaction solution, and the reaction was carried out at room temperature for 12 h. All reactions were conducted under N2 protection. The product was purified by silica gel column chromatography to obtain a colorless oily product.
[0037] The above-mentioned colorless oily product (7.65 mmol) was dissolved in tetrahydrofuran:benzene (40 mL: 8 mL), and 7 mL of distilled water was added with stirring. Sodium borohydride (29.87 mmol) was added under ice bath conditions, and the mixture was stirred for 0.5 h. The reaction was then terminated by adding 3.39 mL of glacial acetic acid, followed by the addition of 40 mL of anhydrous chloroform. The reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was extracted and separated. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove water, and the anhydrous sodium sulfate was removed by filtration. The organic solvent was then removed by rotary evaporation to obtain the colorless oily product.
[0038] 3-Dimethylaminopropionate (3.20 mmol), DMAP (1.80 mmol), and EDCI (4.00 mmol) were added to a 100 mL round-bottom flask and dissolved in 60 mL of anhydrous chloroform. The mixture was activated in an ice bath for 2 h, and the resulting colorless oily product (1.60 mmol) was added to the reaction solution. The reaction was carried out at room temperature for 12 h. All reactions were conducted under N2 protection. The compound 2-TG-LA was obtained by silica gel column chromatography.
[0039] Example 2
[0040] Linoleic acid (35.00 mmol), DMAP (17.80 mmol), and EDCI (35.70 mmol) were added to a round-bottom flask and dissolved in anhydrous dichloromethane. The mixture was activated in an ice bath for 2 h. Then, 1,3-dihydroxyacetone (15.90 mmol) was added to the reaction solution, and the reaction was carried out at room temperature for 12 h. All reactions were conducted under N2 protection. The product was purified by silica gel column chromatography to obtain a colorless oily product.
[0041] The above colorless oily product (7.65 mmol) was dissolved in tetrahydrofuran:benzene (40 mL: 8 mL), and 7 mL of distilled water was added with stirring. Sodium borohydride (29.87 mmol) was added under ice bath conditions, and the mixture was stirred for 0.5 h. The reaction was then terminated by adding 3.39 mL of glacial acetic acid, followed by the addition of 40 mL of anhydrous chloroform. The reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was extracted and separated. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove water, and the anhydrous sodium sulfate was removed by filtration. The organic solvent was then removed by rotary evaporation to obtain the colorless oily product.
[0042] 12.92 mmol of 4-dimethylaminobutyrate, 7.30 mmol of DMAP, and 16.15 mmol of EDCI were added to a round-bottom flask and dissolved in anhydrous chloroform. The mixture was activated in an ice bath for 2 h, and the resulting colorless oily product (6.46 mmol) was added to the reaction solution. The reaction was carried out at room temperature for 12 h. All reactions were performed under N2 protection. The compound 3-TG-LA was obtained by silica gel column chromatography.
[0043] Example 3
[0044] Linoleic acid (35.00 mmol), DMAP (17.80 mmol), and EDCI (35.70 mmol) were added to a round-bottom flask and dissolved in anhydrous dichloromethane. The mixture was activated in an ice bath for 2 h. Then, 1,3-dihydroxyacetone (15.90 mmol) was added to the reaction solution, and the reaction was carried out at room temperature for 12 h. All reactions were conducted under N2 protection. The product was purified by silica gel column chromatography to obtain a colorless oily product.
[0045] The above-mentioned colorless oily product (7.65 mmol) was dissolved in tetrahydrofuran:benzene (40 mL: 8 mL), and 7 mL of distilled water was added with stirring. Sodium borohydride (29.87 mmol) was added under ice bath conditions, and the mixture was stirred for 0.5 h. The reaction was then terminated by adding 3.39 mL of glacial acetic acid, followed by the addition of 40 mL of anhydrous chloroform. The reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was extracted and separated. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove water, and the anhydrous sodium sulfate was removed by filtration. The organic solvent was then removed by rotary evaporation to obtain the colorless oily product.
[0046] 3.20 mmol of 5-dimethylaminovalerate, 1.80 mmol of DMAP, and 4.00 mmol of EDCI were added to a round-bottom flask and dissolved in anhydrous chloroform. The mixture was activated in an ice bath for 2 h, and then 1.60 mmol of the colorless oily product was added to the reaction solution. The reaction was carried out at room temperature for 12 h. All reactions were performed under N2 protection. The compound 4-TG-LA was obtained by silica gel column chromatography.
[0047] Example 4
[0048] Myristic acid (43.00 mmol), DMAP (17.20 mmol), and EDCI (54.00 mmol) were added to a round-bottom flask and dissolved in anhydrous chloroform. The mixture was activated in an ice bath for 2 h. Then, 1,3-dihydroxyacetone (14.00 mmol) was added to the reaction solution, and the mixture was reacted at room temperature for 12 h. All reactions were carried out under N2 protection. The reaction solution was evaporated to dryness, and 200 mL of anhydrous dichloromethane was added. The mixture was then frozen at -20°C for 4 h to allow crystals to precipitate. The crystals were filtered to obtain a white solid product.
[0049] The above-mentioned white solid product (10.78 mmol) was dissolved in tetrahydrofuran:benzene (70 mL: 14 mL), and 7 mL of distilled water was added with stirring. Sodium borohydride (33.00 mmol) was added under ice bath conditions, and the mixture was stirred for 0.5 h. The reaction was terminated by adding 3.75 mL of glacial acetic acid, and then 70 mL of anhydrous chloroform was added to the reaction solution. The reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was extracted and separated. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove water, and the anhydrous sodium sulfate was removed by filtration. The organic solvent was removed by rotary evaporation to obtain the white solid product.
[0050] 4-Dimethylaminobutyrate (19.54 mmol), DMAP (11.04 mmol), and EDCI (24.43 mmol) were added to a round-bottom flask and dissolved in anhydrous chloroform. The mixture was activated in an ice bath for 2 h, and the resulting white solid product (9.77 mmol) was added to the reaction solution. The reaction was allowed to proceed for 12 h at room temperature. The product was purified by silica gel column chromatography to give compound 3-TG-14.
[0051] Example 5
[0052] Palmitic acid (39.00 mmol), DMAP (16.20 mmol), and EDCI (49.00 mmol) were added to a round-bottom flask and dissolved in anhydrous chloroform. The mixture was activated in an ice bath for 2 h. Then, 1,3-dihydroxyacetone (13.00 mmol) was added to the reaction solution, and the mixture was reacted at room temperature for 12 h. All reactions were carried out under N2 protection. The reaction solution was evaporated to dryness, and 200 mL of anhydrous dichloroform was added. The mixture was then frozen at -20°C for 4 h to allow crystals to precipitate. The crystals were filtered to obtain a white solid product.
[0053] The above-mentioned white solid product (9.89 mmol) was dissolved in tetrahydrofuran:benzene (70 mL: 14 mL), and 7 mL of distilled water was added with stirring. Sodium borohydride (33.84 mmol) was added under ice bath conditions, and the mixture was stirred for 0.5 h. The reaction was terminated by adding 3.84 mL of glacial acetic acid, and then 70 mL of anhydrous chloroform was added to the reaction solution. The reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was extracted and separated. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove water, and the anhydrous sodium sulfate was removed by filtration. The organic solvent was removed by rotary evaporation to obtain the white solid product.
[0054] 14.08 mmol of 4-dimethylaminobutyrate, 7.96 mmol of DMAP, and 17.61 mmol of EDCI were added to a round-bottom flask and dissolved in anhydrous chloroform. The mixture was activated in an ice bath for 2 h, and the resulting white solid product (7.04 mmol) was added to the reaction solution. The reaction was allowed to proceed for 12 h at room temperature. The product was purified by silica gel column chromatography to give compound 3-TG-16.
[0055] Example 6
[0056] Stearic acid (35.00 mmol), DMAP (14.00 mmol), and EDCI (44.50 mmol) were added to a round-bottom flask and dissolved in anhydrous chloroform. The mixture was activated in an ice bath for 2 h. Then, 1,3-dihydroxyacetone (11.70 mmol) was added to the reaction solution, and the mixture was reacted at room temperature for 12 h. All reactions were carried out under N2 protection. The reaction solution was evaporated to dryness, and 200 mL of anhydrous dichloroform was added. The mixture was then frozen at -20°C for 4 h to allow crystals to precipitate. The crystals were filtered to obtain a white solid product.
[0057] The above-mentioned white solid product (9.65 mmol) was dissolved in tetrahydrofuran:benzene (70 mL: 14 mL), and 7 mL of distilled water was added with stirring. Sodium borohydride (36.21 mmol) was added under ice bath conditions, and the mixture was stirred for 0.5 h. The reaction was then terminated by adding 4.11 mL of glacial acetic acid, followed by the addition of 70 mL of anhydrous chloroform. The reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was extracted and separated. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove water, and the anhydrous sodium sulfate was removed by filtration. The organic solvent was then removed by rotary evaporation to obtain the white solid product.
[0058] 17.95 mmol of 4-dimethylaminobutyrate, 10.14 mmol of DMAP, and 22.43 mmol of EDCI were added to a round-bottom flask and dissolved in anhydrous chloroform. The mixture was activated in an ice bath for 2 h, and the resulting white solid product (8.97 mmol) was added to the reaction solution. The reaction was carried out at room temperature for 12 h. All reactions were performed under N2 protection. The compound 3-TG-18 was obtained by silica gel column chromatography.
[0059] Example 7
[0060] Oleic acid (35.40 mmol), DMAP (17.80 mmol), and EDCI (35.40 mmol) were added to a round-bottom flask and dissolved in anhydrous dichloromethane. The mixture was activated in an ice bath for 2 h. Then, 1,3-dihydroxyacetone (15.80 mmol) was added to the reaction solution, and the reaction was carried out at room temperature for 12 h. All reactions were conducted under N2 protection. The product was purified by silica gel column chromatography to obtain a colorless oily product.
[0061] The above-mentioned colorless oily product (9.44 mmol) was dissolved in tetrahydrofuran:benzene (40 mL: 8 mL), and 7 mL of distilled water was added with stirring. Sodium borohydride (35.16 mmol) was added under ice bath conditions, and the mixture was stirred for 0.5 h. The reaction was then terminated by adding 3.99 mL of glacial acetic acid, followed by the addition of 40 mL of anhydrous chloroform. The reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was extracted and separated. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove water, and the anhydrous sodium sulfate was removed by filtration. The organic solvent was then removed by rotary evaporation to obtain the colorless oily product.
[0062] 17.00 mmol of 4-dimethylaminobutyrate, 9.61 mmol of DMAP, and 21.25 mmol of EDCI were added to a round-bottom flask and dissolved in anhydrous chloroform. The mixture was activated in an ice bath for 2 h, and the resulting colorless oily product (8.50 mmol) was added to the reaction solution. The reaction was carried out at room temperature for 12 h. The compound 3-TG-OA was obtained by silica gel column chromatography.
[0063] Example 8: Release of lipid nanoparticles prepared from triglyceride-like ionizable lipids
[0064] The degradability of ionizable lipids is an important factor affecting lipid applications. Therefore, examining the release of ionizable lipids is helpful in predicting the degradability of lipids in vivo.
[0065] Lipid nanoparticles were dispersed in phosphate-buffered saline (PBS, pH 7.4) with or without 1000 IU lipase. After incubation at 37°C and 100 rpm for 24 h, the mixture was centrifuged at 13000 rpm. The concentration of free siRNA in the supernatant was detected and calculated using a microplate reader to obtain the release rate of the lipid nanoparticles.
[0066] The results are as follows Figure 1 As shown, under the condition of lipase addition, lipid nanoparticles prepared from triglyceride-like ionizable lipids released more siRNA compared to MC3-LNPs, with 2-TG-LA-LNPs releasing the most. This indicates that lipase promotes the cleavage of ester bonds in the triglyceride backbone, leading to rapid siRNA release. Therefore, triglyceride-like ionizable lipids have higher degradability.
[0067] Example 9: Lysosomal escape of lipid nanoparticles prepared from triglyceride-like ionizable lipids
[0068] The lysosomal escape efficiency of ionizable lipids is an important factor affecting lipid applications.
[0069] With 4×10 5RAW264.7 cells were seeded at a density of cells / well in 15 mm confocal dishes and cultured in a cell culture incubator for 12 h to allow for cell adhesion and growth. Then, the old culture medium was aspirated with a pipette, and lipid nanoparticles (siRNA, 200 nM) diluted in serum-free medium were added to each well, followed by incubation in a cell culture incubator for 6 h. After incubation, the drug-containing culture medium was discarded, and the cells were washed three times with cold pH 7.4 PBS. Lysosomes were stained green with a lysosomal fluorescent probe (LysoTrackerGreen DND-26) and washed three times with pH 7.4 PBS. Cell nuclei were stained blue with Hoechst 33342 and washed three times with pH 7.4 PBS. After adding 1 mL of PBS (pH 7.4) to the confocal dish, the co-localization of lipid nanoparticles and lysosomes was observed using a laser confocal microscope.
[0070] The results are as follows Figure 2 As shown in the figure. The results show that, compared with MC3-LNPs, 2-TG-LA-LNPs have a lower Pearson colocalization coefficient, and therefore have a higher lysosomal escape efficiency.
[0071] Example 10: Study on the in vivo gene silencing effect of lipid nanoparticles
[0072] Mice (C57BL / 6, purchased from the Animal Experiment Center of Shenyang Pharmaceutical University) were randomly divided into groups of four. The LPS / D-GalN (model group) and the Untreated (normal group) served as control groups. The treatment groups were administered magnesium isoglycyrrhizinate (MgIG) injection (4 mg / kg), free siRNA, 2-TG-LA-LNPs, and MC3-LNPs (siRNA, 0.75 mg / kg) via tail vein, respectively. Twenty-four hours after administration, except for the normal group, 200 μL of a mixed solution of lipopolysaccharide (10 μg / kg) and D-GalN (300 mg / kg) was injected intraperitoneally to establish a mouse ALF model. Five hours after induction, the mice were sacrificed. Serum IL-1β levels were detected using a mouse ELISA kit.
[0073] The results are as follows Figure 3 As shown in the figure. The results showed that both the 2-TG-LA-LNPs group and the MC3-LNPs group could effectively reduce the level of IL-1β in mouse serum, indicating that both lipid nanoparticles can effectively achieve gene silencing in vivo. Among them, the gene silencing effect of 2-TG-LA-LNPs was better and there was a significant difference compared with MC3-LNPs.
[0074] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A triglyceride-like ionizable lipid, characterized in that, Selected from the following compounds: 。 2. The method for preparing triglyceride-like ionizable lipids according to claim 1, characterized in that, The preparation method includes the following steps: Linoleic acid (35.00 mmol), DMAP (17.80 mmol), and EDCI (35.70 mmol) were added to a round-bottom flask and dissolved in anhydrous dichloromethane. The mixture was activated in an ice bath for 2 h. Then, 15.90 mmol of 1,3-dihydroxyacetone was added to the reaction solution, and the reaction was carried out at room temperature for 12 h. All reactions were performed under N2 protection. The product was purified by silica gel column chromatography to obtain a colorless oily product. 7.65 mmol of the above colorless oily product was dissolved in tetrahydrofuran:benzene = 40 mL: 8 mL. 7 mL of distilled water was added with stirring. 29.87 mmol of sodium borohydride was added under ice bath conditions. After stirring for 0.5 h, 3.39 mL of glacial acetic acid was added to terminate the reaction. 40 mL of anhydrous chloroform was then added to the reaction solution. The reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution. The organic layer was extracted and separated. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove water. The anhydrous sodium sulfate was removed by filtration. After removing the organic solvent using a rotary evaporator, a colorless oily product was obtained. 3.20 mmol of 3-dimethylaminopropionate, 1.80 mmol of DMAP, and 4.00 mmol of EDCI were added to a 100 mL round-bottom flask and dissolved in 60 mL of anhydrous chloroform. The mixture was activated in an ice bath for 2 h, and 1.60 mmol of the above colorless oily product was added to the above reaction solution. The reaction was carried out at room temperature for 12 h. All the above reactions were carried out under N2 protection. The product was purified by silica gel column chromatography to obtain compound 2-TG-LA.
3. The use of the triglyceride-like ionizable lipid of claim 1 in the preparation of nucleic acid drug delivery carriers.
4. The application according to claim 3, characterized in that, The delivery vector is used for delivery of one or more of pDNA, siRNA, ASO, or mRNA.
5. The application according to claim 3, characterized in that, The delivery carrier is lipid nanoparticles.
6. The application according to claim 3, characterized in that: The nucleic acid drug delivery carrier can be administered locally via intramuscular, subcutaneous, intradermal, or intratumoral administration using microneedles, injection, or perfusion methods, or administered via intravenous injection.