A method for preparing a red blood cell membrane-coated framework nucleic acid nanogel

Abstract

The application belongs to but is not limited to the technical field of gene synthesis, and discloses a preparation method of a red blood cell membrane coated framework nucleic acid nanogel, which forms NG through base complementary pairing, and realizes the compounding of RBCm and NG by using an Avanti small extruder.SiRNA is combined and cross-linked with TDN through base complementary to form NG, and the TDN provides the first layer of protection for the siRNA, enhances the stability and drug efficacy of the siRNA, and the red blood cell membrane provides the second layer of protection to prevent enzyme degradation and protein adsorption.M@NG combines the bionics and biocompatibility of RBCm and the high drug loading and stability of NG, and has the characteristics of targeted drug delivery, immune escape and the like.The system is suitable for the treatment of various diseases, and expands the application field of RNA interference technology.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of gene synthesis technology, and particularly relates to a method for preparing a framework nucleic acid nanogel coated with a red blood cell membrane. Background Technology

[0002] Nanogels, as porous and tunable materials, have wide applications in drug delivery and biomedicine. Traditional nanogels are typically composed of polymers or inorganic materials, exhibiting good biocompatibility and drug carrier functions. Nucleic acid drugs, such as small interfering RNA (siRNA) and small RNA (miRNA), have been extensively studied and applied in disease treatment due to their ability to intervene in gene expression. However, the delivery of nucleic acid drugs still faces significant challenges due to issues such as physiological stability, cell targeting, and intracellular release. Currently, significant progress has been made in viral and non-viral vectors for gene delivery, but major challenges remain. For example, while viral vectors can effectively transfect target gene cells, their immunogenicity and mutagenicity severely hinder their application. Non-viral vectors such as micelles, liposomes, and inorganic nanoparticles can all deliver nucleic acid drugs in vitro and in vivo, but efficiency and safety issues still exist. Furthermore, the gene silencing effect is closely related to the cationic properties of the vector. The current dilemma is that the higher the cationic charge of the vector, the more effectively it can compress nucleic acid drugs, thereby effectively silencing the expression of target genes. However, this increases the toxicity of the cationic material, leading to serious side effects. Furthermore, cationic nanoparticles interact strongly with negatively charged proteins in plasma, leading to instability of the nanocarriers and rapid clearance during circulation, thus reducing the therapeutic efficacy of nucleic acid drugs. Therefore, there is an urgent need to break away from the cationic carrier delivery model, and exploring new strategies for nucleic acid drug delivery represents a promising future for achieving safe and effective delivery.

[0003] Based on the above analysis, the urgent technical problem to be solved by the existing technology is that the cationic nanoparticles interact strongly with the negatively charged proteins in the plasma, which leads to the instability of the nanocarrier, rapid clearance during circulation, and reduced therapeutic effect of nucleic acid drugs. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for preparing siRNA-mediated framework nucleic acid nanogels coated with red blood cell membranes, breaking away from the traditional delivery modes of viral and cationic carriers. At the same time, by utilizing red blood cell membranes as the coating material for nucleic acid nanogels, a novel bio-inspired nanodrug carrier is constructed, expanding the application field of RNA interference technology.

[0005] This invention is achieved as follows: a method for preparing a framework nucleic acid nanogel coated with a red blood cell membrane, comprising:

[0006] S1, Mix equimolar amounts of oligonucleotides in TM buffer;

[0007] S2, the mixture solution is heated at 95℃ for 10-20 min, and then quickly transferred to 4℃ to cool to obtain DNA tetrahedral framework nucleic acid nanostructures, which are then stored at 4℃.

[0008] S3, Prepare siRNA with sticky ends as a cross-linking agent (siTOX is used as an example in this invention);

[0009] S4, mix siRNA and TDN at a molar ratio of 1:2, shake vigorously at 37°C for 3 hours to obtain NG and store at 4°C;

[0010] S5, whole blood from mice was centrifuged at 3000 rpm for 5 minutes at 4°C or below to remove plasma and the brownish-yellow layer of erythrocyte sedimentation rate;

[0011] S6. Collect the red blood cell precipitate, wash it three times in ice-cold 1x phosphate buffer, and resuspend it in 10 volumes of 0.25x PBS. Then place the solution at 4°C for 3-4 hours to hemolyze and break down the membrane.

[0012] S7, centrifuge at 12000 rpm for 5 min to remove the released hemoglobin, resuspend the lower precipitate in hypotonic solution, wash repeatedly until the supernatant is colorless, and obtain red blood cell membrane (RBCm);

[0013] S8, RBCm was redispersed in 1x PBS solution and sonicated in a water bath for 5 minutes. The RBCm was then extruded through 400nm and 200nm polycarbonate porous membranes using an Avanti mini extruder to prepare uniform RBCm vesicles.

[0014] S9, the obtained RBCm vesicles were mixed with NG and extruded 14 times through a 100nm polycarbonate porous membrane using an Avanti mini extruder to obtain drug-loaded nucleic acid nanogels coated with erythrocyte membranes.

[0015] The specific steps for preparing siRNA with sticky ends as a cross-linking agent are as follows: dissolve sense siTOX and antisense siTOX separately in DEPC water, mix them in an equimolar ratio, perform gradient annealing of the mixed liquid to room temperature in a PCR instrument, and store the assembled siRNA at -20℃.

[0016] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0017] First, the delivery carrier of this invention combines the biological characteristics of red blood cell membranes, the structural advantages of framework nucleic acids and nanogels. It successfully uses siRNA as a cross-linking chain to connect tetrahedral framework nucleic acids through complementary base pairing, thereby realizing the construction of framework nucleic acid-based nanogels. This constructs a biomimetic framework nucleic acid nanogel carrier that integrates gene-immunotherapy, aiming to overcome the limitations of traditional nucleic acid drug delivery systems and improve the stability of nucleic acid materials and nucleic acid drugs. It has broad application prospects in the fields of RNA interference technology and precision medicine.

[0018] Secondly, as supplementary evidence of the inventive step of the claims of this invention, it is also reflected in the following important aspects:

[0019] (1) The expected benefits and commercial value of the technical solution of this invention after transformation are as follows:

[0020] This invention facilitates the industrialization and promotion of controllable drug delivery vectors, and is expected to become a universal strategy for nucleic acid drug delivery. It is of great significance for improving the long-term circulation of vectors in vivo, drug bioavailability, and nucleic acid drug delivery, and will greatly broaden the application areas and development of controllable drug delivery and RNA interference technology. Simultaneously, this invention will actively promote industry-academia-research collaboration, facilitate the transformation and application of scientific research results, drive active exploration in new drug development and clinical trials, and bring significant economic benefits.

[0021] (2) The technical solution of the present invention solves a technical problem that people have long wanted to solve but have never been able to solve successfully:

[0022] The physiological stability of siRNA, the pharmacodynamic properties of nanocomposites, and the long-term biosafety of carrier materials have significantly hindered the clinical application of RNA interference therapy. While significant progress has been made in viral and non-viral vectors for gene delivery, major challenges remain, including mutagenicity, transfection efficiency, and safety issues. Micelles and liposomes, composed of cationic polymers and lipids, are the most commonly used non-viral vectors, effectively compressing siRNA and delivering it to target cells via electrostatic interactions. However, the gene silencing effect is closely related to the cationic properties of the vector. A common dilemma is that while a higher cationic charge on the vector leads to more effective compression of siRNA and thus more effective silencing of target gene expression, this also increases the toxicity of the cationic material, potentially causing severe side effects. Furthermore, cationic nanoparticles interact strongly with negatively charged proteins in plasma, leading to nanocarrier instability and rapid clearance during circulation, thus reducing the therapeutic efficacy of RNA interference technology. Therefore, breaking away from the cationic vector delivery model and exploring new strategies for siRNA delivery is urgently needed.

[0023] (3) The technical solution of the present invention overcomes technical bias:

[0024] This invention overcomes the technical limitations of traditional cationic carrier-based nucleic acid drug delivery by introducing a nanogel formed by the binding of a DNA tetrahedral framework nucleic acid structure with siRNA. Traditional techniques often rely on cationic liposomes or polymer carriers to deliver nucleic acid drugs through electrostatic interactions, but these methods often suffer from low delivery efficiency, high cytotoxicity, and immune responses, limiting their application. Unlike traditional cationic carriers that rely on charge interactions, negatively charged DNA nanostructures rely on covalent binding or molecular recognition to compress functional nucleic acids and form delivery complexes. The DNA tetrahedral framework nucleic acid structure of this invention provides a novel self-assembly platform with excellent biocompatibility and high-efficiency nucleic acid delivery capabilities, thus breaking the traditional model of nucleic acid drug delivery using cationic carriers and realizing a new strategy for nucleic acid drug delivery based on nucleic acid structure, while achieving more stable and efficient nucleic acid drug delivery.

[0025] Third, the technical solution of this invention, through the preparation method of red blood cell membrane-coated framework nucleic acid nanogels (M@NG), solves the problems of poor stability, short in vivo circulation time, easy enzymatic degradation, and insufficient targeting of nucleic acid drugs in the prior art, and achieves significant technical progress, mainly reflected in the following aspects:

[0026] 1. Improved the stability and biocompatibility of nucleic acid drugs.

[0027] Traditional nucleic acid drugs are easily degraded by enzymes in vivo, leading to a significant reduction in their stability and efficacy. This invention employs a dual protective barrier design, utilizing framework nucleic acids (TDN) to provide the first layer of protection for siRNA, and erythrocyte membranes (RBCm) to provide the second layer of protection for the entire nanogel. This avoids problems of enzymatic degradation and protein adsorption in the external environment, thereby significantly improving the physiological stability and biocompatibility of nucleic acid drugs.

[0028] 2. It prolongs the circulation time in the body and enhances immune evasion.

[0029] The short circulation time and poor physiological stability of nucleic acid drugs in vivo have always been key problems that need to be overcome in existing technologies. This invention utilizes the biomimetic and immune escape properties of erythrocyte membrane-coated nanogels to prolong the circulation time of nucleic acid drugs in the blood, reducing the risk of the drug being recognized and cleared by the immune system, thereby significantly enhancing the efficacy and duration of action of the drug in vivo.

[0030] 3. A highly efficient targeted drug delivery system has been achieved.

[0031] Traditional nucleic acid drug delivery systems often lack targeting capabilities, making it difficult to precisely target specific lesions. This invention, through the dual structure of M@NG, not only protects the nucleic acid drug but also achieves targeted delivery, enabling more efficient delivery of drugs to target tissues or cells. This technology is particularly suitable for the treatment of diseases such as tumors and inflammation, providing an efficient and precise nucleic acid drug delivery route.

[0032] 4. It has multifunctional adaptability, expanding the application areas of RNA interference technology.

[0033] This invention, through flexible cross-linking agent design, allows siRNA to be replaced according to different disease requirements, making it suitable for the treatment of various diseases, such as tumors and inflammatory diseases. This flexible drug delivery system greatly expands the application field of RNA interference (RNAi) technology, improves the broad applicability of nucleic acid drugs, and provides strong support for the development of RNAi technology in clinical applications.

[0034] In summary, this invention not only solves the problems of poor stability, short cycle time, and poor targeting of nucleic acid drugs in the prior art, but also significantly improves the stability, targeting, and in vivo biocompatibility of the drug through innovative nanogel structure and red blood cell membrane coating technology, bringing significant technological progress to the effective delivery of nucleic acid drugs and the application of RNA interference technology. Attached Figure Description

[0035] Figure 1 This is a flowchart of the method for preparing erythrocyte membrane-coated framework nucleic acid nanogels provided in this embodiment of the invention;

[0036] Figure 2 These are transmission electron microscopy images and particle size distributions of the framework nucleic acid nanogels coated with red blood cell membranes provided in this embodiment of the invention.

[0037] Figure 3 The stability of the framework nucleic acid nanostructure, nanogel, and red blood cell membrane-coated framework nucleic acid nanogel provided in the embodiments of the present invention when incubated with 10% fetal bovine serum at 37°C for different times;

[0038] Figure 4 The expression level of green fluorescent protein (GFP) in RAW 264.7-GFP cells (RAW264.7 cells expressing green fluorescent protein) was detected by Western blot after different material treatments provided in this embodiment of the invention, so as to reflect the successful delivery of siRNA and its successful release from the nucleic acid gel and exert its effect. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] This invention provides a method for preparing a framework nucleic acid nanogel coated with a erythrocyte membrane. The method involves forming an NG (nucleic acid nanogel) through complementary base pairing and using an Avanti miniature extruder to composite RBCm with the NG. siRNA and TDN bind and cross-link through complementary base pairing to form the NG. TDN provides the first protective barrier for siRNA, effectively protecting its physiological stability and efficacy. The erythrocyte membrane provides a second protective barrier for both TDN and siRNA, protecting the nucleic acid structure from external environmental influences such as enzymatic degradation and protein adsorption. This drug delivery system not only improves the stability of the nucleic acid structure and the nucleic acid drug but also helps prolong its circulation time and efficacy in vivo. M@NG utilizes the dual combination of erythrocyte membrane (RBCm) and nucleic acid nanogel (NG), retaining the biomimetic and biocompatibility of RBCm while leveraging the advantages of NG in drug delivery and therapeutic applications, such as efficient drug loading and protection against nucleic acid degradation. Due to the combined properties of RBCm and NG, M@NG can serve not only as a targeted drug delivery system but also possesses potential biocompatibility and immune evasion capabilities. Meanwhile, the cross-linking agent used to synthesize the gel can be replaced with other siRNAs, which can be used to treat a variety of diseases, such as tumors and inflammatory diseases. This is of great significance for the effective delivery of nucleic acid drugs and the expansion of the application fields of RNA interference technology.

[0041] The DNA or RNA oligonucleotides used in this invention are shown in Table 1 (in this invention, siTOX, a small interfering RNA that inhibits TOX protein expression, is used as an example). Figure 1 As shown, equimolar amounts of oligonucleotides (S1, S2, S3, S4 or S1-L, S2-L, S3-L, S4-L) were mixed in TM buffer (20 mM Tris base, 5 mM MgCl2, pH = 8.0). The mixture was heated at 95°C for 10-20 min, then rapidly transferred to 4°C to cool and obtain DNA tetrahedra (TDN, final concentration 2 μM), which were then stored at 4°C. Subsequently, siRNA with sticky ends (siTOX in this invention) was prepared as a cross-linking agent. The specific steps are as follows: positive and negative siTOX were dissolved separately in DEPC water, then mixed in an equimolar ratio. The mixed liquid was subjected to gradient annealing to room temperature in a PCR instrument, and the assembled siRNA was stored at -20°C.

[0042] The nanogel (NG) was prepared by base complementarity pairing. siRNA (4 μM) and TDN (2 μM) were mixed at a molar ratio of 1:2 and vigorously shaken at 37 °C for 3 h to obtain NG, which was then stored at 4 °C.

[0043] Whole blood from mice was centrifuged at 3000 rpm for 5 minutes at below 4°C to remove plasma and the brownish-yellow layer of erythrocyte sedimentation rate. The erythrocyte precipitate was collected, washed three times in ice-cold 1x phosphate-buffered saline (PBS), and resuspended in 10 volumes of 0.25x PBS. The solution was then incubated at 4°C for 3-4 hours to lyse and disrupt the membrane. Afterward, centrifugation at 12000 rpm for 5 minutes was used to remove released hemoglobin. The lower precipitate was resuspended in hypotonic solution and washed repeatedly until the supernatant was colorless, yielding erythrocyte membranes (RBCm). Finally, the RBCm was redispersed in 1x PBS and sonicated in a water bath for 5 minutes. The RBCm was then extruded through 400 nm and 200 nm polycarbonate porous membranes using an Avanti Polar Lipids mini extruder to prepare homogeneous RBCm vesicles. The obtained RBCm vesicles were mixed with NG and extruded 14 times through a 100 nm polycarbonate porous membrane using an Avanti mini extruder to obtain drug-loaded nucleic acid nanogels (M@NG) coated with erythrocyte membranes.

[0044] This invention offers a safer and more precise nucleic acid drug delivery solution due to its high delivery efficiency, good biocompatibility, controllable drug release characteristics, and significantly reduced side effects. It can be used to target and deliver siRNA to silence pathogenic genes, treating related diseases. In cancer treatment, in addition to the siTOX shown in this invention, the relevant siRNA in the nanogel (such as hypoxia-inhibiting siRNA) can be replaced and effectively delivered to tumor cells, improving cancer treatment efficacy. Furthermore, it can also deliver anti-inflammatory siRNA for the treatment of osteoarthritis or other inflammatory diseases. This invention aims to realize a universal strategy and mode for siRNA delivery, achieving efficient nucleic acid drug delivery and expanding its applications in the biomedical field, such as cancer treatment, inflammation treatment, and antibacterial therapy.

[0045] Transmission electron microscopy images showed that M@NG was spherical with no obvious aggregation, and its size was measured to be approximately 106 nm using a Malvern particle size analyzer (DLS). This lays the foundation for the enrichment of nanoparticles at the tumor site through the EPR effect. Figure 2The coating of RBCm not only provides biocompatibility but also enhances the stability and long-term storage of NG through its natural cell membrane properties, thereby extending the drug's effective period. Gel electrophoresis was used to test the stability of TDN, NG, and M@NG in 10% FBS, and the results showed that M@NG was stable in 10% FBS for at least 24 hours, demonstrating good serum stability. Figure 3 ).

[0046] To verify whether siRNA can effectively release and achieve gene silencing, we selected RAW 264.7 cells (RAW 264.7-GFP cells) that express green fluorescent protein to verify the successful delivery of siRNA by nucleic acid nanogels and the release of siRNA from the nanogels to exert a gene silencing effect. In this experiment, siGFP, which inhibits GFP expression, was selected to construct the nucleic acid nanogel. If the green fluorescence intensity or protein expression in the cells decreased after siGFP was delivered to the cells via the nanogel, it indicated that siGFP exerted a gene silencing effect. Lipo 2000 transfected siGFP (Lipo 2000siGFP) and NG synthesized with disordered siRNA (siGFP-NC) were used as positive and negative controls, respectively. Western blotting results showed that all siGFP-loaded formulations reduced green fluorescence intensity, while the PP-FNAG (siScram) group loaded with disordered siRNA had no significant effect on the reduction of green fluorescence. Figure 4 ).

[0047] Table 1. DNA or RNA oligonucleotides used in this invention.

[0048]

[0049] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a framework nucleic acid nanogel coated with a red blood cell membrane, characterized in that, The method includes the following steps: S1, Mix equimolar amounts of oligonucleotides in TM buffer; S2, the mixture solution is heated at 95℃ for 10-20 minutes, and then rapidly transferred to 4℃ for cooling to obtain DNA tetrahedral framework nucleic acid nanostructures, namely TDN, which are stored at 4℃; S3, prepare siRNA with sticky ends, mix it with TDN at a molar ratio of 1:2, and shake vigorously at 37°C for 3 hours to obtain framework nucleic acid nanogel, i.e. NG; S4. Collect red blood cells, prepare red blood cell membrane RBCm, and obtain uniform RBCm vesicles by ultrasound and extrusion. S5, RBCm vesicles are mixed with NG and co-extruded multiple times through a 100nm polycarbonate porous membrane to obtain drug-loaded nucleic acid nanogels coated with erythrocyte membranes, namely, framework nucleic acid nanogels coated with erythrocyte membranes. In step S3, the siRNA used to form NG is siTOX with sticky ends, which is formed by gradient annealing of the sense and antisense strands to room temperature and stored at -20°C.

2. The method for preparing erythrocyte membrane-coated framework nucleic acid nanogels according to claim 1, characterized in that, In step S5, the mixture of RBCm vesicles and NG is extruded through a small Avanti extruder through 400nm and 200nm polycarbonate porous membranes, and finally extruded through a 100nm polycarbonate porous membrane a total of 14 times to form a uniform red blood cell membrane-coated framework nucleic acid nanogel, namely the M@NG structure.

3. A drug-loaded nucleic acid nanogel coated with a red blood cell membrane, characterized in that, The nanogel is composed of a framework nucleic acid nanogel NG and a red blood cell membrane RBCm. NG is formed through base complementarity and cross-linking of TDN and siRNA, while RBCm provides biocompatibility and immune escape for NG.

4. The drug-loaded nucleic acid nanogel coated with erythrocyte membrane according to claim 3, characterized in that, The siRNA in the nucleic acid nanogel can be replaced with other siRNAs with sticky ends according to treatment needs, making it suitable for the treatment of a variety of diseases, including tumors or inflammatory diseases.

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