Method for promoting transdermal performance of nucleic acid-lipid nanoparticles through ultrasonic synergistic enhancement

Abstract

The invention provides a method for promoting the transdermal performance of nucleic acid-lipid nanoparticles through ultrasonic synergistic enhancement, and belongs to the technical field of transdermal drug delivery. The invention provides a method for promoting the transdermal performance of nucleic acid-lipid nanoparticles through ultrasonic synergistic enhancement, which comprises the following steps: (1) treating the surface of skin by using a transdermal agent containing ethanol and other components, and then coating the surface of skin with lipid nanoparticles loaded with nucleic acid; and (2) ultrasonic treatment is conducted on the coating area, the ultrasonic frequency is set to be 20 kHz-1 MHz, the ultrasonic intensity is set to be 0.3 W / cm to 2.0 W / cm, and treatment is conducted for 1 min to 3 min in a continuous or 10%-50% duty ratio. According to the method, the strongest and widest-range signal can be obtained in the aspect of delivery efficiency, meanwhile, the epidermal tissue is in a continuous strip shape and is obviously positive to the hair follicle outer root sheath, and the superficial dermis is visible and expanded; the apoptosis of the TUNEL is not increased; the method comprises the following steps: carrying out Masson and Hamp; e, the collagen is continuous, the structure is complete, the inflammation is slight, and no obvious difference exists from the contrast, so that the safety and the biocompatibility are high.

Description

Technical Field

[0001] This invention belongs to the field of transdermal drug delivery technology, specifically relating to a method for ultrasound-assisted enhancement of the transdermal performance of nucleic acid-lipid nanoparticles. Background Technology

[0002] Currently, mRNA therapy has many advantages over other treatment methods: (1) Safety; mRNA can be directly translated into proteins in the cytoplasm without entering the cell nucleus, thus avoiding the risk of chromosome insertion; (2) High efficiency; mRNA synthesis is simple, and it directly uses living cells to express target proteins. The target proteins can be modified post-translationally, thus more closely resembling the native conformation and properties of the target proteins. However, due to its large molecular weight and negative charge, mRNA is difficult to pass through the anionic lipid bilayer of the cell membrane. In addition, naked mRNA is easily degraded by RNases or phagocytosed by immune cells. Therefore, mRNA is usually delivered by loading it onto a vector.

[0003] Commonly used mRNA carriers include lipids and lipid-like materials, cationic polymers, and protamine. Ionizable lipid nanoparticles (LNPs) are recognized as a relatively advanced mRNA carrier. LNPs are mainly constructed using ionizable phospholipid materials and other helper phospholipids. They bind to negatively charged mRNA in an acidic buffer solution, thereby achieving efficient mRNA molecule encapsulation. LNPs are nearly electroneutrally neutral under physiological conditions, but become strongly positively charged under the acidic conditions of intracellular endolysosomes. Therefore, they can rapidly release mRNA into the cytoplasm through the proton sponge effect.

[0004] The efficiency of LNPs in delivering RNA needs to be improved, especially their poor transdermal performance. Therefore, how to provide a method that can improve the transdermal delivery of RNA by LNPs and has better biosafety has become an urgent technical problem to be solved. Summary of the Invention

[0005] This invention aims to solve the aforementioned technical problems by providing a method for ultrasound-assisted enhancement of the transdermal performance of nucleic acid-lipid nanoparticles. The technical objective of this invention is to provide a method that can be used to efficiently promote the transdermal delivery of mRNA via LNPs, while ensuring the safety and biocompatibility of the drug delivery system.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for ultrasound-assisted enhancement of the transdermal properties of nucleic acid-lipid nanoparticles, comprising the following steps: (1) The skin surface was treated with an ethanol-containing transdermal agent, and then lipid nanoparticles loaded with nucleic acids were coated on the skin surface for 0.5-2 min.

[0007] (2) Perform ultrasonic treatment on the coated area, set the ultrasonic frequency to 20 kHz-1 MHz, the ultrasonic intensity to 0.3-2.0 W / cm², and treat continuously or with a duty cycle of 10-50% for 1-3 min.

[0008] Furthermore, in step (1), the transdermal agent containing ethanol has an ethanol volume fraction of 50-85%; in addition to ethanol, the formulation also includes any one of the following: eucalyptus oil (volume fraction of 0.1-5%), oleic acid (volume fraction of 1-5%), polyethylene glycol (volume fraction of less than 1%), and glycerin (volume fraction of 5-10%), which are mixed with ethanol to form a pretreatment solution.

[0009] Furthermore, the conditions for ultrasonic treatment in step (2) are: ultrasonic frequency of 1 MHz and ultrasonic intensity of 1.5 W / cm. 2 Process for 2 minutes with a 50% duty cycle.

[0010] Furthermore, the nucleic acid-loaded lipid nanoparticles in step (1) are nucleic acid delivery carriers formulated based on ionizable lipids, cholesterol, cofactor phospholipids, and PEG-modified lipids. The ionizable lipid components include, but are not limited to, ALC-0315, SM-102, and DLin-MC3-DMA. The cofactor phospholipid components include, but are not limited to, distearylphosphatidylcholine and dioleoylphosphatidylethanolamine. The PEG-modified lipid components include, but are not limited to, ALC-0159 and DSPE-PEG2000.

[0011] Furthermore, the nucleic acids in step (1) include, but are not limited to: mRNA, plasmid, microRNA, siRNA, and shRNA.

[0012] Furthermore, in step (2), the ultrasound probe is moved to cover the entire drug delivery area.

[0013] The beneficial effects of this invention are as follows: (1) This invention provides a method for promoting the transdermal performance of nucleic acid-LNP transdermal drug delivery using "ethanol-ultrasound synergistic enhancement". The method involves first preparing lipid nanoparticles loaded with nucleic acid, then coating the skin with a certain concentration of an ethanol-containing transdermal agent, followed by appropriate ultrasound treatment, which greatly facilitates the transdermal delivery of nucleic acid-LNP drugs. The method of this invention achieves the strongest and widest signal in terms of delivery efficiency. Simultaneously, the epidermal tissue shows continuous band-like patterns and significant positivity in the outer root sheath of hair follicles, with expansion visible in the superficial dermis; TUNEL assays show no increase in apoptosis; Masson and H&E assays show continuous collagen, intact structure, mild inflammation, and no significant difference from the control, indicating high safety and biocompatibility.

[0014] (2) The transdermal agent formulation selected in this invention contains, in addition to the corresponding volume fraction of ethanol, any one of eucalyptus oil (0.1-5%, vol), oleic acid (1-5%, vol), PEG (less than 1%, vol) or glycerol (5-10%, vol) in combination with ethanol as an ethanol pretreatment solution. When combined with ultrasound, it can form a composite permeation-enhancing mechanism, providing a wider range of combination schemes for transdermal nucleic acid delivery. Attached Figure Description

[0015] Figure 1 To screen transdermal agents containing different concentrations of ethanol, it was found that pretreatment with transdermal agents containing 75% ethanol yielded the best results; the concentration range of transdermal agents containing ethanol used in this experiment was between 20% and 95% (vol).

[0016] Figure 2 Analysis of the effect of ethanol-containing transdermal pretreatment combined with different ultrasound treatments on transdermal LNP-EGFP delivery; (A) In vivo fluorescence imaging of mouse abdomen; (B) Immunofluorescence of paraffin / frozen sections of skin (EGFP, green; DAPI, blue), from left to right: low, medium, and high magnification fields, with the dashed box indicating the magnified area.

[0017] Figure 3 Effect of ethanol-containing transdermal pretreatment on skin apoptosis (TUNEL); (A) TUNEL staining of skin sections, rows in order: 1) Control group (Ctrl); 2) Transdermal pretreatment with 75% ethanol + ultrasound 1 (75% ethanol + Ultra1); 3) Transdermal pretreatment with 75% ethanol + ultrasound 2 (75% ethanol + Ultra2); 4) Transdermal pretreatment with 75% ethanol + ultrasound 3 (75% ethanol + Ultra3); 5) Transdermal pretreatment with 75% ethanol + ultrasound 4 (7) Transdermal pretreatment with 75% ethanol + Ultra4; columns from left to right represent low, medium, and high magnification fields; dashed boxes indicate magnified areas; nuclear staining with DAPI is blue; TUNEL-positive cells are red / magenta; no significant increase in positive cells was observed in any group. Representative images, scale bar shown in the figure.

[0018] Figure 4Masson's trichrome staining was used to evaluate the effects of ethanol and ultrasound on skin collagen. Mouse dorsal skin was treated with different methods: control (Ctrl); 75% ethanol transdermal agent + ultrasound 1 (75% ethanol transdermal agent + Ultra1); 75% ethanol transdermal agent + ultrasound 2 (75% ethanol transdermal agent + Ultra2); 75% ethanol transdermal agent + ultrasound 3 (75% ethanol transdermal agent + Ultra3); 75% ethanol transdermal agent + ultrasound 4 (75% ethanol transdermal agent + Ultra4). Masson's trichrome staining was performed on samples from each treatment group; collagen was blue, sarcoplasm / erythrocytes were red, and cell nuclei were dark. Each row corresponds to one treatment group, and columns from left to right represent low, medium, and high magnification fields. Dashed boxes indicate magnified areas. No significant collagen destruction or fibrosis was observed in any group.

[0019] Figure 5 To assess the effects of ethanol and ultrasound on skin histology using H&E, mouse dorsal skin was treated as follows: control (Ctrl); treatment with 75% ethanol transdermal agent + ultrasound 1 (75% ethanol transdermal agent + Ultra1); treatment with 75% ethanol transdermal agent + ultrasound 2 (75% ethanol transdermal agent + Ultra2); treatment with 75% ethanol transdermal agent + ultrasound 3 (75% ethanol transdermal agent + Ultra3); and treatment with 75% ethanol transdermal agent + ultrasound 4 (75% ethanol transdermal agent + Ultra4). After treatment, samples were collected for H&E staining. Each row corresponds to one treatment group; columns from left to right represent low, medium, and high magnification fields; dashed boxes indicate magnified areas. The epidermal and dermal structures of each group were well preserved, with no significant necrosis, hemorrhage, or inflammatory infiltration observed. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention. Example

[0021] I. Experimental Materials and Methods (a) Composition and material formulation LNP vector: Contains ionizable lipids, cholesterol, auxiliary lipids, and PEG-lipids; encapsulates nucleic acids (mRNA / siRNA / plasmid, EGFP optional as an example). Particle size is preferably 70–120 nm, PDI ≤ 0.2.

[0022] Ethanol-containing transdermal agents: Select 50–80 vol% ethanol in the aqueous phase, and then optionally add 0.1–5% eucalyptus oil, 1–5% oleic acid, up to 1% polyethylene glycol or 5–10% glycerol; pH 5.5–7.0.

[0023] LNP dosage form: The dosage form for administration is LNP aqueous dispersion or gel; it can be used in conjunction with medical coupling agents for ultrasound.

[0024] (II) Preparation and administration of materials Preparation of lipid nanoparticles (LNPs): The LNP formulation used in this experiment mainly consisted of Dlin-MC3-DMA (AVT, CAS: 1224606-06-7), PEG2000 (AVT, CAS: 160743-62-4), DSPC (AVT, CAS: 816-94-4), and cholesterol (aladdin, CAS: 57-88-5). These raw materials were mixed in a molar ratio of 50%: 1.5%: 10%: 38.5%, and negatively charged mRNA was electrostatically attracted to form mRNA-loaded LNPs. In the experiment, the four components were first dissolved in anhydrous ethanol according to the molar ratio, and then the appropriate mass of mRNA was added to a citrate buffer solution based on an N / P ratio of 6. Microfluidic mixing (UnigenBiotech, Nano S) was used in this experiment to allow the lipid solution and mRNA solution to fully and rapidly form uniformly sized LNPs in a micromixer. Since lipids are soluble in ethanol and nucleic acids are soluble in acidic buffer, residual ethanol was removed by ultrafiltration (30 kD) and the solution system was replaced with PBS.

[0025] Skin pretreatment: Wipe or spray the target area with an ethanol-containing transdermal agent for 0.5–2 minutes, and allow it to air dry until it stops dripping.

[0026] Administration: Apply LNP formulation (typical dose 0.5–2 mg nucleic acid / m²) to the pretreatment area. 2 skin).

[0027] Ultrasound: After applying the coupling agent, ultrasound is applied at a frequency of 20 kHz–1 MHz and a sound intensity of 0.3–2.0 W / cm². 2 Continuous or 10–50% duty cycle for 1–3 min; move the probe to cover the drug delivery area.

[0028] Coverage: Apply a semi-closed dressing for 30–60 minutes to reduce evaporation.

[0029] Indications and uses: Suitable for topical administration of nucleic acid drugs or vaccines that require epidermal / follicle-dermal localization, such as research and treatment exploration of hair follicle-related disease models.

[0030] Experimental results and safety (I) Experimental Conclusions: 1. Delivery efficiency: In vivo fluorescence imaging and EGFP immunofluorescence demonstration showed that the transdermal agent containing 75% ethanol plus ultrasound group obtained the strongest and widest signal, which was superior to ethanol alone or ultrasound alone. (See [link to relevant documentation]). Figure 1 and Figure 2 A.

[0031] 2. Tissue distribution: Continuous band-like patterns are observed in the epidermis and the outer root sheath of the hair follicle, with expansion visible in the superficial dermis. See [link / reference needed]. Figure 2 B.

[0032] 3. Biocompatibility: TUNEL showed no increase in apoptosis; Masson and H&E showed continuous collagen, intact structure, mild inflammation, and no significant difference from the control. See [link to relevant documentation]. Figure 3 – Figure 5 .

[0033] (II) The specific experimental results are as follows: 1. Effects of LNP transdermal formulation on mouse scalp To investigate whether the permeation-enhancing effect of ethanol exhibits a linear relationship, pretreatment solutions containing ethanol at volume fractions of 25%, 40%, 60%, 85%, and 95% were designed. The results showed that with increasing ethanol concentration, the transdermal delivery efficiency of LNP increased rapidly in the 60%–80% range. However, when the ethanol concentration in the transdermal agent exceeded 85%, mild dehydration and stratum corneum densification occurred in the skin surface, and the permeation efficiency decreased. These results suggest that the relationship between ethanol concentration and permeation-enhancing effect is non-linear, with an optimal range (preferably around 75%) that can achieve maximum transdermal flux while ensuring skin histological safety. These results indicate that the permeation-enhancing effect of ethanol is not simply concentration-dependent, but is influenced by the overall state of the skin barrier.

[0034] 2. Evaluation of the effect of combining LNP transdermal formulation with ultrasound In vivo imaging showed that ethanol pretreatment, in synergy with different ultrasound treatments, significantly enhanced percutaneous delivery efficiency. Figure 2 (A) Blank and control showed almost no fluorescence; Ultra1 with 75% ethanol transdermal agent showed a weak and focal signal; Ultra3 with 75% ethanol transdermal agent showed multifocal patchy enhancement; Ultra2 with 75% ethanol transdermal agent further enhanced the signal and showed a diffuse distribution; Ultra4 with 75% ethanol transdermal agent showed the weakest signal.

[0035] Immunofluorescence of tissue sections further validated the above trend. Figure 2 (B) The control section was negative; containing 75% ethanol transdermal agent + Ultra1 (frequency: 1 MHz, intensity: 0.5 W / cm²). 2(Duty cycle: 10%, processing time: 1 min) The formulation showed only scattered weak EGFP positivity at the epidermal / hair follicle opening; containing 75% ethanol transdermal agent + Ultra2 (frequency: 1 MHz, sound intensity: 1.0 W / cm²). 2 (Duty cycle: 30%, processing time: 2 min) Signal enhancement and extension to the outer root sheath of the hair follicle and superficial dermis; containing 75% ethanol transdermal agent + Ultra3 (frequency: 1 MHz, sound intensity: 1.5 W / cm²). 2 Duty cycle: 50%, processing time: 2 min) forms a continuous distribution along the epidermis and involves the hair follicle structure; contains 75% ethanol transdermal agent + Ultra4 (frequency: 1 MHz, sound intensity: 2.0 W / cm²). 2 The most significant signal was observed at a duty cycle of 50% (continuous mode can be substituted), with a processing time of 3 min. The signal covered the epidermis, the outer root sheath of the hair follicle, and extended into the dermis. These are representative images, suggesting that ethanol pretreatment improves skin permeability, and superimposed ultrasound can further promote delivery to deeper tissues and hair follicles.

[0036] 3. Effects of transdermal treatment on skin apoptosis (TUNEL) TUNEL staining was used to assess the skin on the back of mice. Figure 3 The results showed that in each treatment group (Ctrl, Ultra1 with 75% ethanol transdermal agent + Ultra2, Ultra3 with 75% ethanol transdermal agent + Ultra4), only sporadic TUNEL-positive cells were observed in the epidermis and hair follicle areas, with no increase in visibility compared to the control; the tissue structure was intact, with no signs of necrosis or shedding. Qualitative results indicate that under the experimental parameters, ethanol pretreatment and its combination with ultrasound did not induce detectable apoptosis of skin cells, demonstrating good biocompatibility.

[0037] 4. Masson trichrome evaluation of the effects of ethanol and ultrasound on skin collagen Masson trichrome staining shows ( Figure 4 In all treatment groups, dermal collagen remained continuous and stained uniformly, with no broken, hyalinated, or fibrotic bundles observed; the epidermal structure was intact, the basement membrane was continuous, and the morphology of hair follicles and appendages was well preserved. Overall, under these parameters, ethanol and its combined treatment with ultrasound did not cause detectable structural damage to collagen, demonstrating good histological safety.

[0038] 5. H&E assessment of the effects of ethanol and ultrasound on skin histology H&E displays ( Figure 5The skin structure of each treatment group remained intact. The epidermal layers were clear, the basement membrane was continuous, and there was no ulceration or necrosis; the dermal collagen and appendages retained their morphology, and no obvious edema or hemorrhage was observed; inflammatory cell infiltration was sparse and not significantly different from the control. Overall, this suggests that ethanol and ultrasound have good histological safety at these parameters.

Claims

1. A method for synergistically enhancing the transdermal properties of nucleic acid-lipid nanoparticles using ultrasound, characterized in that, Includes the following steps: (1) The skin surface was treated with an ethanol-containing transdermal agent, and then lipid nanoparticles loaded with nucleic acids were coated on the skin surface for 0.5-2 min.

2. (2) Perform ultrasonic treatment on the coated area, setting the ultrasonic frequency to 20 kHz-1 MHz and the ultrasonic intensity to 0.3-2.0 W / cm. 2 Process continuously or with a duty cycle of 10-50% for 1-3 minutes.

3. The method according to claim 1, characterized in that, The transdermal agent containing ethanol in step (1) has an ethanol volume fraction of 50-85%; the components of the transdermal agent containing ethanol, by volume fraction, include: 0.1-5% eucalyptus oil, 1-5% oleic acid, less than 1% polyethylene glycol or 5-10% glycerin, and a pretreatment solution composed of ethanol.

4. The method according to claim 1, characterized in that, The conditions for ultrasonic treatment in step (2) are: ultrasonic frequency of 1 MHz and ultrasonic intensity of 1.5 W / cm. 2 Process for 2 minutes with a 50% duty cycle.

5. The method according to claim 1, characterized in that, The nucleic acid-loaded lipid nanoparticles described in step (1) are nucleic acid delivery carriers based on ionizable lipids, cholesterol, cofactor phospholipids, and PEG-modified lipids; wherein, the ionizable lipid components include, but are not limited to: ALC-0315, SM-102, and DLin-MC3-DMA; the cofactor phospholipid components include, but are not limited to: distearylphosphatidylcholine and dioleoylphosphatidylethanolamine; and the PEG-modified lipid components include, but are not limited to: ALC-0159 and DSPE-PEG2000.

6. The method according to claim 1, characterized in that, The molar ratio of ionizable lipids, cholesterol, cofactor phospholipids and PEG-modified lipids in the lipid nanoparticles loaded with nucleic acids described in step (1) is 50-60%: 30-35%: 10-15%: 0.5-2%.

7. The method according to claim 1, characterized in that, The nucleic acids in the lipid nanoparticles loaded with nucleic acids described in step (1) include, but are not limited to: mRNA, plasmids, microRNA, siRNA, and shRNA.

8. The method according to claim 1, characterized in that, In step (2), a coupling agent is first applied to the coating area, followed by ultrasonic treatment.

9. The method according to claim 1, characterized in that, In step (2), the ultrasound probe is moved to cover the entire drug delivery area.

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