MRNA (messenger ribonucleic acid) lipid nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to the technical field of nano biology, and provides mRNA (messenger Ribonucleic Acid) lipid nanoparticles as well as a preparation method and application thereof. According to the mRNA lipid nanoparticle, hyaluronic acid in a tumor extracellular matrix is degraded through hyaluronidase (HAase) modification, the tumor microenvironment is improved, and the barrier effect of tumor tissue is reduced, so that the permeability of mRNA-LNP is enhanced. In addition, the system can be subjected to particle size overturning in a tumor slightly acidic environment, so that the delivery efficiency of tumor deep cells is improved. Experimental results show that the delivery system can effectively improve the infiltration capacity of mRNA in tumor tissues and the anti-tumor curative effect, and a new strategy is provided for tumor gene therapy.

Description

Technical Field

[0001] The present invention belongs to the field of nano-biotechnology, and particularly relates to mRNA lipid nanoparticles and a preparation method and application thereof. Background Art

[0002] Cancer has become the leading killer threatening people's health and life, and its effective clinical treatment faces significant challenges. Currently, chemotherapy, surgery, radiotherapy, and their combination are the mainstays of lung cancer treatment, but their effectiveness remains suboptimal, with issues such as poor tissue specificity and systemic side effects. Messenger RNA (mRNA) is a single-stranded RNA that carries genetic information and directs protein synthesis in the cytoplasm. Due to its short development cycle, flexible protein selection, rapid onset of action, and lack of genomic integration risk, mRNA therapeutics hold enormous potential for clinical cancer treatment and are a hot research topic for the global healthcare and drug development community. However, mRNA is a hydrophilic, anionic macromolecular compound that cannot penetrate the cell membrane barrier and enter cells. Furthermore, it is extremely unstable in the circulation and susceptible to nuclease degradation, making its drugability critically dependent on the delivery vehicle. mRNA-lipid nanoparticles (mRNA-LNPs) are core-shell nanoparticles composed of mRNA assembled from ionizable lipids, a co-lipid, cholesterol, and lipid-PEG. They have successfully enabled the commercialization of the first siRNA drug and two COVID-19 mRNA vaccines, and are considered the most ideal mRNA delivery vehicle. Although LNP can accumulate in tumors through its high permeability and retention effect (EPR effect), it is difficult for it to diffuse deep into the tumor due to the complex and dense interstitial matrix, unique vascular structure, hypoxia, high pressure and other physical barriers of tumor tissue. This makes it difficult to kill deep tumor cells, resulting in poor mRNA-LNP tumor treatment efficacy and causing tumor recurrence and metastasis. Therefore, mRNA-LNP has not yet achieved breakthroughs in its application in tumor treatment. How to enhance LNP's deep penetration into tumor tissue and improve its tumor infiltration rate are bottlenecks that need to be solved in mRNA therapy.

[0003] Furthermore, the tumor extracellular matrix, primarily composed of hyaluronic acid (HA), collagen, and fibronectin, significantly hinders the deep penetration of nanoparticles. Therefore, there is an urgent need to develop a new mRNA delivery system that can effectively overcome these issues and enhance anti-tumor efficacy. Summary of the Invention

[0004] The purpose of the present invention is to provide an mRNA lipid nanoparticle and its preparation method and application. The mRNA lipid nanoparticle can degrade hyaluronic acid in the extracellular matrix of tumor cells through HAase modification, improve the high-pressure microenvironment of the tumor interstitial tissue, and at the same time, the consumption of the outer layer HAase changes the particle size of the delivery system, thereby promoting the system to penetrate deep into the tumor. After entering the tumor cells, the system loosens in the slightly acidic environment of the tumor and releases mRNA.

[0005] The object of the present invention is achieved through the following technical solutions: The invention provides an mRNA lipid nanoparticle, which comprises an active lipid, modified polyethylene glycol and a hyaluronidase modification layer.

[0006] Furthermore, the structure of the modified polyethylene glycol is: .

[0007] The present invention also provides a method for preparing mRNA lipid nanoparticles, comprising the following steps: (1) PEG2000, p-carboxybenzaldehyde and 4-dimethylaminopyridine were dissolved in anhydrous dichloromethane. N,N'-dicyclohexylcarbodiimide was added dropwise under ice bath conditions. The mixture was stirred for reaction, filtered and evaporated to obtain CHO-PEG-CHO. (2) CHO-PEG-CHO and adipic acid dihydrazide were dissolved in anhydrous dimethylformamide, and triethylamine was added. After stirring and reacting, the unreacted substances were removed. After rotary evaporation and concentration, the mixture was precipitated into excess ether and washed several times. The obtained product was vacuum dried to obtain the modified polyethylene glycol. (3) Peptide lipids, auxiliary phospholipids, cholesterol, and post-modified polyethylene glycol are mixed with sodium citrate buffer containing mRNA in anhydrous ethanol solution, and nanoparticles are prepared using a microfluidic chip; (4) Vortexing the hyaluronidase and the nanoparticles to obtain mRNA lipid nanoparticles.

[0008] Furthermore, in step (1), the amount of PEG2000 is 3.0-5.0 g, the amount of p-carboxybenzaldehyde is 0.5-1.0 g, the amount of 4-dimethylaminopyridine is 0.1-0.5 g, the amount of anhydrous dichloromethane is 10-100 mL, and the amount of N,N'-dicyclohexylcarbodiimide is 1.0-2.0 g.

[0009] Furthermore, in step (1), the stirring reaction is carried out at 20-30° C. for 24-72 hours.

[0010] Furthermore, in step (2), the amount of CHO-PEG-CHO is 50-1000 mg, the amount of adipic acid dihydrazide is 10-1000 mg, the amount of anhydrous dimethylformamide is 1-5 mL, and the amount of triethylamine is 10-500 μL; and the stirring reaction is carried out at 20-60° C. for 24-72 h.

[0011] Furthermore, in step (3), the amount of the peptide lipid is 20-100 parts, the amount of the auxiliary phospholipid is 1-20 parts, the amount of cholesterol is 10-30 parts, and the amount of the modified polyethylene glycol is 1-2 parts.

[0012] Furthermore, in step (4), the concentration of the hyaluronidase is 1-5 mg / mL.

[0013] The present invention also provides an application of mRNA lipid nanoparticles in preparing medicine for treating tumors.

[0014] Furthermore, the mRNA lipid nanoparticles are modified with HAase to degrade hyaluronic acid in the extracellular matrix of tumor cells, thereby improving the high-pressure microenvironment of the tumor interstitial space. At the same time, the consumption of the outer layer of HAase changes the particle size of the delivery system, thereby promoting the system to penetrate deep into the tumor. After entering the tumor cells, the system loosens in the slightly acidic environment of the tumor and releases mRNA.

[0015] Beneficial effects

[0016] The hyaluronidase-modified layer of the mRNA lipid nanoparticles in this invention binds to the nanoparticle surface via electrostatic adsorption, specifically degrading hyaluronic acid in the interstitial spaces of tumor cells. This effectively improves the high-pressure microenvironment of the tumor interstitial space, creating favorable conditions for subsequent drug delivery. Upon depletion of the surface hyaluronidase, the nanoparticle size shrinks from a larger size (approximately 100 nm) to a smaller size (approximately 30 nm), significantly enhancing its ability to penetrate deep into tumor tissue. This overcomes the limitation of traditional mRNA-LNPs and improves their ability to kill deep-seated tumor cells. Furthermore, the benzyl imine-modified PEG breaks down in the weakly acidic conditions of the tumor microenvironment (pH 6.5), further loosening the delivery system and achieving efficient mRNA release, enabling the mRNA drug to exert its therapeutic effect at the tumor site.

[0017] The hyaluronidase-encapsulated mRNA lipid nanoparticles (HAase-LNPs) in this invention exhibit higher fluorescence intensity and significantly superior transfection efficiency compared to pure mRNA lipid nanoparticles (LNPs). This enhances the expression of mRNA drugs within tumor cells, improving therapeutic efficacy. Furthermore, their permeability into tumor spheres is significantly improved, allowing the drug to more fully penetrate tumor tissue and enhance anti-tumor efficacy. In vivo experiments in mice demonstrated that HAase-LNPs significantly reduced tumor volume, increasing tumor inhibition by 8.2% compared to pure LNPs, providing a more effective means for tumor treatment. Furthermore, they demonstrated no cytotoxicity towards normal lung epithelial cells (BEAS-2B), demonstrating the excellent biocompatibility of this delivery system, minimizing damage to normal cells and reducing side effects during treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Schematic diagram of the structure of mRNA lipid nanoparticles prepared by the present invention; Figure 2 Spectra of mRNA lipid nanoparticles prepared in the present invention, including (A) nuclear magnetic resonance hydrogen spectrum (NMR) of the final synthetic product coupled with lipids; (B, C) infrared spectra (IR) of the intermediate product and the final synthetic product coupled with lipids; Figure 3 Figure 1 shows the particle size of the mRNA lipid nanoparticles prepared in the present invention, including: (A) particle size comparison of three drug-carrying nanoparticles: LNPs, HAase-LNPs, and HA-HAase-LNPs; (B) potential comparison of LNPs and HAase-LNPs; (C) transmission electron micrographs of LNPs, HAase-LNPs, and HA-HAase-LNPs (1:100 nm); Figure 4 Schematic diagram of the cytotoxicity of the mRNA lipid nanoparticles prepared in the present invention on normal lung epithelial cells BEAS-2B (cultured for 48 h after transfection); Figure 5 This is a diagram showing the transfection efficiency of mRNA lipid nanoparticles prepared by the present invention for A549 cells; Figure 6 This is a flow cytometric analysis of apoptosis in A549 cells 48 hours after transfection with the mRNA lipid nanoparticles prepared by the present invention; Figure 7The figure shows the permeability of mRNA lipid nanoparticles prepared by the present invention to tumor spheres, where the scale bar on the figure is 100 nm; Figure 8 This is a diagram showing the tumor-suppressing effect of the mRNA lipid nanoparticles prepared in the present invention in mice. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] Unless otherwise specified, the chemical reagents, biochemical reagents and materials used in the present invention can be obtained from commercial channels.

[0026] The specific construction method of the mRNA lipid nanoparticles of the present invention includes: (1) Synthesis of polyethylene glycol aldehyde: Dissolve PEG2000 (3.0-5.0 g), p-carboxybenzaldehyde (0.5-1.0 g) and 4-dimethylaminopyridine (DMAP, 0.1-0.5 g) in 10-100 mL of anhydrous dichloromethane; add N,N'-dicyclohexylcarbodiimide (DCC) (1.0-2.0 g) dropwise under ice bath conditions; then, stir the mixture at 20-30 °C for 24-72 h; after the reaction, filter the solution and concentrate it by rotary evaporation to obtain CHO-PEG-CHO; (2) Synthesis of modified PEG: CHO-PEG-CHO (50-1000 mg) and adipic acid dihydrazide (ADH, 10-1000 mg) were dissolved in 1-5 mL of anhydrous dimethylformamide (DMF); 10-500 μL of triethylamine (TEA) was added, and the mixture was stirred at 20-60 °C for 24-72 hours; after the reaction was completed, the mixture was dialyzed with deionized water to remove unreacted substances; the resulting solution was concentrated by rotary evaporation and precipitated into excess ether, washed several times, and the product was vacuum dried to obtain polyethylene glycol modified with an ultra-pH-sensitive benzyl imine bond; (3) Take 20-100 parts of peptide lipid, 1-20 parts of auxiliary phospholipid, 10-30 parts of cholesterol, and 1-2 parts of modified polyethylene glycol, and mix them with sodium citrate buffer containing mRNA in anhydrous ethanol solution, wherein the molar ratio of peptide lipid to mRNA is 3:1-20:1, and the volume ratio of lipid alcohol phase to mRNA aqueous phase is 1:3. Prepare nanoparticles using a microfluidic chip; (4) Prepare 1-5 mg / mL hyaluronidase with PBS and vortex the nanoparticles. The volume ratio of hyaluronidase to nanoparticles is 1:1-3:1. mRNA lipid nanoparticles are obtained.

[0027] The invention comprises nanoparticles constructed by active lipids and modified polyethylene glycol and an outer-coated hyaluronidase modified layer.

[0028] The structure of polyethylene glycol modified with an ultra-pH-sensitive benzyl imine bond is shown below: .

[0029] The structural diagram of the mRNA lipid nanoparticles finally prepared by the present invention is as follows Figure 1 shown.

[0030] Example 1 The specific construction method of mRNA lipid nanoparticles in the present invention includes: (1) Synthesis of polyethylene glycol aldehyde: 4 g PEG2000, 0.5 g p-carboxybenzaldehyde, and 0.3 g 4-dimethylaminopyridine were dissolved in 50 mL of anhydrous dichloromethane; 1.5 g N,N'-dicyclohexylcarbodiimide was added dropwise under ice bath conditions; then, the mixture was stirred at 25 °C for 36 h; after the reaction, the solution was filtered and concentrated by rotary evaporation to obtain CHO-PEG-CHO; (2) Synthesis of modified PEG: 500 mg of CHO-PEG-CHO and 500 mg of adipic acid dihydrazide were dissolved in 3 mL of anhydrous dimethylformamide; 250 μL of triethylamine was added, and the mixture was stirred at 40°C for 36 hours; after the reaction was completed, the mixture was dialyzed with deionized water to remove unreacted substances; the resulting solution was concentrated by rotary evaporation and precipitated into excess ether, washed several times, and the product was vacuum dried to obtain polyethylene glycol modified with an ultra-pH-sensitive benzyl imine bond; (3) Take 80 parts of peptide lipid, 15 parts of auxiliary phospholipid, 20 parts of cholesterol, and 2 parts of modified polyethylene glycol, mix them with sodium citrate buffer containing mRNA in anhydrous ethanol solution, and prepare nanoparticles using a microfluidic chip; (4) 3 mg / mL hyaluronidase was prepared with PBS and vortexed with the above nanoparticles to obtain mRNA lipid nanoparticles. The nuclear magnetic resonance hydrogen spectrum and infrared spectrum of the synthesized product are shown in the figure. Figure 2 shown.

[0031] Example 2 The mRNA lipid nanoparticles: HAase-LNPs, mRNA lipid nanoparticles: LNPs without HAase, and mRNA lipid nanoparticles HA-HAase-LNPs reacted with HA were constructed in Example 1 to investigate the changes in their particle size. The results are shown in FIG. Figure 3 As shown by Figure 3 It can be seen that the particle size of LNPs changed significantly after binding with HAase, increasing by about 100 nm. At the same time, after reacting with HA, the particle size decreased significantly. A comparison of the potentials of LNPs and HAase-LNPs showed that the potential changed significantly after binding with HAase. The transmission electron microscopy images of LNPs, HAase-LNPs and HA-HAase-LNPs also intuitively showed the obvious change in particle size.

[0032] In addition, the results of exploring the cytotoxicity of the mRNA lipid nanoparticles in Example 1 to normal lung epithelial cells BEAS-2B are as follows Figure 4 As shown by Figure 4 It can be seen that the lipid carrier has no cytotoxicity to normal lung epithelial cells BEAS-2B.

[0033] The results of the mRNA lipid nanoparticle transfection efficiency of A549 cells and the apoptosis flow cytometry analysis of A549 cells 48 hours after transfection were as follows: Figure 5-6 As shown by Figure 5 It can be seen that the fluorescence intensity of LNPs is 27.1%, and the fluorescence intensity of HAase-LNPs is 88.8%. It can be seen that the fluorescence intensity of LNPs loaded with HAase is higher and the transfection effect is better. Figure 6 It can be seen that the transfection efficiency of HAase-LNPs is 14.8% higher than that of simple LNPs.

[0034] Example 3 Prepare 1% agarose gel and add 50 μL per well to a 96-well plate. The 96-well plate is then sterilized in a clean hood for 30 minutes under UV light. When A549 cells reach a density of approximately 80% in the culture flask, the cells are digested and centrifuged to prepare a cell suspension. Add 4000 cells / 50 μL per well to the agarose gel-lined wells. Shake the 96-well plate several times, both horizontally and vertically, using the cross-section method. Since the agarose solution forms a concave surface after solidification, cells cannot adhere to the agarose gel surface. After shaking, the cells aggregate on the concave surface of the agarose gel. The 96-well plate is cultured in an incubator. The medium is changed halfway every other day. After 2-3 days of culture, tumor spheres are essentially formed. After 3 days of culture, the tumor spheres fully express the extracellular matrix.

[0035] The tumor spheres were cultured with the culture medium containing the HAase-LNPs prepared in Example 1 for a predetermined incubation time (4 h, 8 h, 12 h, 24 h, and 36 h). The tumor spheres were then carefully aspirated and rinsed with ice-cold PBS. The tumor spheres were placed in a small dish containing ice-cold PBS for subsequent permeability and structural analysis using a confocal microscope. The final results are shown in Figure 2. Figure 7 As shown by Figure 7 It can be seen that the permeability can be effectively improved by culturing in a culture medium containing the HAase-LNPs prepared in Example 1.

[0036] Example 4 When the tumor volume of nude mice reached 80-100 mm 3 The nude mice were weighed at about 8, 11, 14, 17, and 20 days after cell inoculation (drugs were given every 2 days, for a total of 5 injections), and the mice were injected with saline (control group) (4 mice, half male and half female); LNPs (4 mice, half male and half female); and HAase-LNPs (4 mice, half male and half female). The weight and tumor volume of the mice were regularly monitored throughout the establishment of the permeation model. The final results are shown in the figure below. Figure 8 As shown by Figure 8It can be seen that LNPs can reduce tumor volume by 22.5%, while HAase-LNPs can reduce tumor volume by 30.7%. The overall tumor inhibition effect has increased by 8.2%. The tumor volume reduction in the HAase-LNPs group is the most obvious and the effect is the best.

[0037] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. An mRNA lipid nanoparticle, characterized in that The mRNA lipid nanoparticles include active lipids, modified polyethylene glycol and a hyaluronidase modification layer.

2. The mRNA lipid nanoparticle according to claim 1, wherein The structure of the modified polyethylene glycol is: 。 3. The preparation method of mRNA lipid nanoparticles as claimed in claim 1, wherein The following steps are involved: (1) PEG2000, p-carboxybenzaldehyde and 4-dimethylaminopyridine were dissolved in anhydrous dichloromethane. N,N'-dicyclohexylcarbodiimide was added dropwise under ice bath conditions. The mixture was stirred for reaction, filtered and evaporated to obtain CHO-PEG-CHO. (2) CHO-PEG-CHO and adipic acid dihydrazide were dissolved in anhydrous dimethylformamide, and triethylamine was added. After stirring and reacting, the unreacted substances were removed. After rotary evaporation and concentration, the mixture was precipitated into excess ether and washed several times. The obtained product was vacuum dried to obtain the modified polyethylene glycol. (3) Peptide lipids, auxiliary phospholipids, cholesterol, and post-modified polyethylene glycol are mixed with sodium citrate buffer containing mRNA in anhydrous ethanol solution, and nanoparticles are prepared using a microfluidic chip; (4) Vortexing the hyaluronidase and the nanoparticles to obtain mRNA lipid nanoparticles.

4. The preparation method according to claim 3, wherein In step (1), the amount of PEG2000 is 3.0-5.0 g, the amount of p-carboxybenzaldehyde is 0.5-1.0 g, the amount of 4-dimethylaminopyridine is 0.1-0.5 g, the amount of anhydrous dichloromethane is 10-100 mL, and the amount of N,N'-dicyclohexylcarbodiimide is 1.0-2.0 g.

5. The preparation method according to claim 3, wherein In step (1), the stirring reaction is carried out at 20-30° C. for 24-72 hours.

6. The preparation method according to claim 3, wherein In step (2), the amount of CHO-PEG-CHO is 50-1000 mg, the amount of adipic acid dihydrazide is 10-1000 mg, the amount of anhydrous dimethylformamide is 1-5 mL, and the amount of triethylamine is 10-500 μL; the stirring reaction is carried out at 20-60° C. for 24-72 hours.

7. The preparation method according to claim 3, wherein In step (3), the amount of the peptide lipid is 20-100 parts, the amount of the auxiliary phospholipid is 1-20 parts, the amount of cholesterol is 10-30 parts, and the amount of the modified polyethylene glycol is 1-2 parts.

8. The preparation method according to claim 3, wherein In step (4), the concentration of the hyaluronidase is 1-5 mg / mL.

9. Use of the mRNA lipid nanoparticles according to any one of claims 1 to 2 in the preparation of a medicament for treating tumors.

10. The use according to claim 9, characterized in that The mRNA lipid nanoparticles are modified with HAase to degrade hyaluronic acid in the extracellular matrix of tumor cells, thereby improving the high-pressure microenvironment of the tumor interstitial space. At the same time, the consumption of the outer layer of HAase changes the particle size of the delivery system, thereby promoting the system to penetrate deep into the tumor. After entering the tumor cells, the system loosens in the slightly acidic environment of the tumor and releases mRNA.

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