Preparation method of erythrocyte membrane coated framework nucleic acid nanogel

Abstract

The invention belongs to but not limited to the technical field of gene synthesis, and discloses a preparation method of framework nucleic acid nanogel coated with an erythrocyte membrane, NG is formed through complementary pairing of bases, and compounding of RBCm and NG is realized by using an Avanti small extruder. SiRNA and TDN are complementarily combined through bases and cross-linked to form NG, TDN provides a first layer of protection for siRNA, and the stability and efficacy of siRNA are enhanced; and the erythrocyte membrane is a second layer of protection to prevent enzymatic degradation and protein adsorption. The M (at) NG combines the bionic property and biocompatibility of RBCm and the efficient drug loading and stability of NG, and has the characteristics of targeted drug delivery, immune escape and the like. The system is suitable for treating various diseases, and the application field of the RNA interference technology is expanded.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the field of gene synthesis technology, and in particular relates to a method for preparing a framework nucleic acid nanogel coated with an erythrocyte membrane. Background Art

[0002] Nanogels, as a porous, regulatable material, are widely used in drug delivery and biomedicine. Traditional nanogels are usually composed of polymers or inorganic materials, with good biocompatibility and drug carrier functions. Nucleic acid drugs, such as small interfering RNA (siRNA) and micromolecular RNA (miRNA), have been widely studied and applied in the field of disease treatment because of their ability to interfere with gene expression. However, due to problems such as physiological stability, cell targeting and intracellular release, the delivery of nucleic acid drugs still faces huge challenges. At present, viral or non-viral vectors for gene delivery have made great progress, but there are still major challenges. For example, although viral vectors can effectively transfect target gene cells, their immunogenicity and mutagenicity seriously hinder their application. Although non-viral vectors such as micelles, liposomes and inorganic nanoparticles can deliver nucleic acid drugs in vivo and in vitro, there are still problems with efficiency and safety. At the same time, the gene silencing effect is closely related to the cationic properties of the carrier. The current dilemma is that the higher the cationic charge of the carrier, the more effectively it can compress the nucleic acid drug, thereby effectively silencing the expression of the target gene, but at this time the toxicity of the cationic material is enhanced, resulting in serious side effects. In addition, cationic nanoparticles interact strongly with negatively charged proteins in plasma, which makes the nanocarrier unstable and rapidly cleared during circulation, reducing the therapeutic effect of nucleic acid drugs. Therefore, it is urgent to break the cationic carrier delivery mode and find new strategies for nucleic acid drug delivery to achieve safe and effective delivery.

[0003] In view of the above analysis, the technical problems that urgently need to be solved in the existing technology are: cationic nanoparticles interact strongly with negatively charged proteins in plasma, resulting in instability of nanocarriers, which are quickly cleared during circulation and reduce the therapeutic effect of nucleic acid drugs. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for preparing siRNA-mediated framework nucleic acid nanogel coated with red blood cell membrane, breaking the traditional viral vector and cationic carrier delivery mode, and at the same time using red blood cell membrane as the coating material of nucleic acid nanogel to construct a new type of bio-inspired nano drug carrier, expanding the application field of RNA interference technology.

[0005] The present invention is achieved by a method for preparing a red blood cell membrane-coated framework nucleic acid nanogel, comprising:

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

[0007] S2, heating the mixture solution at 95°C for 10-20 minutes, then quickly transferring it to 4°C to cool it to obtain a DNA tetrahedral framework nucleic acid nanostructure, and then storing it at 4°C;

[0008] S3, preparing siRNA with sticky ends as a cross-linking agent (the present invention takes siTOX as an example);

[0009] S4, siRNA and TDN were mixed at a molar ratio of 1:2, shaken vigorously at 37°C for 3 h, and NG was obtained and stored at 4°C;

[0010] S5, whole blood of mice was centrifuged at 3000 rpm for 5 min at below 4°C to remove plasma and buffy coat;

[0011] S6, collect the red blood cell pellet, wash it three times in ice-cold 1x phosphate buffer, and resuspend it in 10 volumes of 0.25x PBS, and then place the solution at 4°C for 3-4 hours to hemolyze and permeabilize;

[0012] S7, centrifugation at 12000 rpm for 5 min to remove released hemoglobin, the lower precipitate was resuspended in a hypotonic solution, and washed several times until the supernatant was colorless to obtain red blood cell membrane (RBCm);

[0013] S8, re-dispersing the RBCm in 1x PBS solution, and performing water bath sonication for 5 minutes, and using an Avanti mini extruder to extrude the RBCm through 400 nm and 200 nm polycarbonate porous membranes, respectively, to prepare uniform RBCm vesicles;

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

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

[0016] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0017] First, the delivery vector of the present invention combines the biological characteristics of red blood cell membranes, the structural advantages of framework nucleic acids and nanogels, and successfully uses siRNA as a cross-linking chain to connect tetrahedral framework nucleic acids through base complementary pairing to achieve the construction of nanogels based on framework nucleic acids, and construct a bionic framework nucleic acid nanogel carrier integrating 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, and has broad application prospects in the fields of RNA interference technology and precision medicine.

[0018] Second, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

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

[0020] This invention is helpful for the industrialization and promotion of drug controllable delivery carriers, and is expected to become a universal strategy for achieving nucleic acid drug delivery. It is of great significance to improve the long-term circulation of carriers in vivo, the bioavailability of drugs, and the delivery of nucleic acid drugs, and will greatly broaden the application areas of drug controllable delivery and RNA interference technology. At the same time, this invention will actively promote industry-university-research cooperation, promote the transformation and application of scientific research results, promote the active exploration of new drug research and development and clinical trials, and bring huge economic benefits.

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

[0022] The physiological stability of siRNA, the pharmacodynamic properties of nanocomplexes, and the long-term biosafety of carrier materials have greatly hindered the clinical application of RNA interference therapy. At present, viral or non-viral vectors for gene delivery have made great progress, but there are still major challenges such as mutagenicity, transfection efficiency, and safety issues. Micelles and liposomes composed of cationic polymers and lipids are the most commonly used non-viral vectors, which can effectively compress siRNA and deliver it to target cells through electrostatic effects. However, the gene silencing effect is closely related to the cationic nature of the carrier. The usual dilemma is that the higher the cationic charge of the carrier, the more effectively it can compress siRNA, thereby effectively silencing the expression of the target gene, but at this time the toxicity of the cationic material is enhanced, which will produce serious side effects. At the same time, cationic nanoparticles interact strongly with negatively charged proteins in plasma, resulting in the instability of nanocarriers, which are quickly cleared during the circulation process, reducing the therapeutic effect of RNA interference technology. Therefore, it is urgent to break the cationic carrier delivery mode and find new strategies for siRNA delivery.

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

[0024] The present invention breaks through the technical prejudice of the traditional cationic carrier delivery mode of nucleic acid drugs by introducing a nanogel formed by combining a DNA tetrahedral framework nucleic acid structure with siRNA. Traditional technologies mostly rely on electrostatic interactions to deliver nucleic acid drugs through cationic liposomes or polymer carriers, but there are many problems such as low delivery efficiency, high cytotoxicity and immune response that limit 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 the present invention provides a novel self-assembly platform with excellent biocompatibility and efficient nucleic acid delivery capabilities, thereby breaking the traditional mode of using cationic-related carriers to deliver nucleic acid drugs, realizing a new strategy for delivering nucleic acid drugs based on nucleic acid structures, and achieving more stable and efficient nucleic acid drug delivery.

[0025] Third, the technical solution of the present invention 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 through the preparation method of red blood cell membrane-coated framework nucleic acid nanogel (M@NG), and has achieved significant technical progress, which is 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 the body, resulting in a significant reduction in their stability and efficacy. The present invention uses a double protective barrier design, using framework nucleic acid (TDN) to provide the first layer of protection for siRNA, and red blood cell membrane (RBCm) to provide the second layer of protection for the entire nanogel, avoiding the problems of enzyme degradation and protein adsorption in the external environment, thereby significantly improving the physiological stability and biocompatibility of nucleic acid drugs.

[0028] 2. Prolongs the circulation time in the body and enhances immune escape

[0029] Nucleic acid drugs have short circulation time in the body and poor physiological stability, which are key problems that need to be overcome in the prior art. The present invention uses red blood cell membranes to coat nanogels, and utilizes the biomimetic and immune escape properties of red blood cell membranes to prolong the circulation time of nucleic acid drugs in the blood, reduce the risk of drugs being recognized and cleared by the immune system, and thus significantly enhance the efficacy and duration of action of drugs in the body.

[0030] 3. Achieved an efficient targeted drug delivery system

[0031] Traditional nucleic acid drug delivery systems lack targeting and are often difficult to accurately target specific lesions. The present invention uses the dual structure of M@NG to not only protect nucleic acid drugs, but also achieve targeted delivery, which can more effectively deliver drugs to target tissues or cells. This technology is particularly suitable for the treatment of diseases such as tumors and inflammation, and provides an efficient and accurate nucleic acid drug delivery route.

[0032] 4. It has multifunctional adaptability and expands the application field of RNA interference technology

[0033] The present invention uses a flexible cross-linking agent design to allow siRNA to be replaced according to the needs of different diseases, and is suitable for the treatment of a variety of diseases, such as tumors and inflammatory diseases. This flexible drug delivery system greatly expands the application field of RNA interference (RNAi) technology, improves the wide adaptability of nucleic acid drugs, and provides strong support for the development of RNAi technology in clinical applications.

[0034] In summary, the present invention not only solves the problems of poor stability, short circulation time, and poor targeting of nucleic acid drugs in the prior art, but also significantly improves the stability, targeting, and in vivo biocompatibility of drugs 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a flow chart of a method for preparing a framework nucleic acid nanogel coated with an erythrocyte membrane provided by an embodiment of the present invention;

[0036] Figure 2 It is a transmission electron microscope image of the framework nucleic acid nanogel coated with red blood cell membrane and its particle size diagram provided by an embodiment of the present invention;

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

[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 being treated with different materials provided in the embodiments of the present invention, so as to reflect the successful delivery of siRNA and its successful release from the nucleic acid gel and exert its effect. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] The present invention provides a method for preparing a framework nucleic acid nanogel coated with an erythrocyte membrane, forming NG through base complementary pairing and realizing the compounding of RBCm and NG by using an Avanti small extruder. siRNA and TDN are combined and cross-linked to form NG through base complementary pairing, TDN provides a first layer of protective barrier for siRNA, which can effectively protect the physiological stability and drug efficacy of siRNA; the erythrocyte membrane provides a second layer of protective barrier for TDN and siRNA, so that the nucleic acid structure is protected from the influence of the external environment, such as enzyme degradation and protein adsorption. The construction of this drug delivery system not only improves the stability of the nucleic acid structure and nucleic acid drugs, but also helps to prolong its circulation time and drug efficacy in the body. M@NG utilizes a dual combination of erythrocyte membrane (RBCm) and nucleic acid nanogel (NG), which not only retains the biomimetic and biocompatibility of RBCm, but also utilizes the advantages of NG in drug delivery and therapeutic applications, such as the characteristics of efficient drug loading and protection of nucleic acids from degradation. Due to the combination of the characteristics of RBCm and NG, M@NG can not only be used as a targeted drug delivery system, but also has potential biocompatibility and immune escape. At the same time, the cross-linking agent used in the synthetic gel can be replaced by other siRNA, which can be used to treat a variety of diseases, such as tumors, inflammatory diseases, etc., and is of great significance for the effective delivery of nucleic acid drugs and the expansion of the application field of RNA interference technology.

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

[0042] The preparation of nanogel (NG) was achieved by base complementary 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 and stored at 4°C.

[0043] Take whole blood from mice and centrifuge at 3000rpm for 5 minutes below 4°C to remove plasma and buffy coat. Collect the red blood cell precipitate, wash it 3 times in ice-cold 1x phosphate buffer (PBS), and resuspend it in 10 times the volume of 0.25x PBS, then place the solution at 4°C for hemolysis and membrane permeation for 3-4 hours. After that, centrifuge at 12000rpm for 5min to remove the released hemoglobin, resuspend the lower precipitate in a hypotonic solution, wash it several times until the supernatant is colorless, and obtain the red blood cell membrane (RBCm). Finally, the RBCm was re-dispersed in a 1x PBS solution, and ultrasonicated in a water bath for 5 minutes. The above RBCm was extruded through 400nm and 200nm polycarbonate porous membranes using an Avanti small extruder (Avanti Polar Lipids) to prepare uniform RBCm vesicles. The obtained RBCm vesicles were mixed with NG and coextruded 14 times through a 100 nm polycarbonate porous membrane using an Avanti mini-extruder to obtain red blood cell membrane-coated drug-loaded nucleic acid nanogels (M@NG).

[0044] The present invention provides a safer and more accurate nucleic acid drug delivery solution due to its advantages such as efficient delivery capability, good biocompatibility, controllable drug release characteristics, and significantly reduced side effects. It can be used for targeted delivery of siRNA to silence pathogenic genes and treat related diseases. In cancer treatment, in addition to the siTOX shown in the present invention, the relevant siRNA in the nanogel (such as siRNA that inhibits hypoxia) can also be replaced and effectively delivered to tumor cells to improve the cancer treatment effect. In addition, the delivery of anti-inflammatory factor siRNA can also be achieved for the treatment of osteoarthritis or other inflammation-related diseases. The invention aims to achieve a general strategy and mode for siRNA delivery, achieve efficient nucleic acid drug delivery, and expand its application in the biomedical field, such as cancer treatment, inflammation treatment, antibacterial treatment, etc.

[0045] Transmission electron microscopy images showed that M@NG was spherical without obvious aggregation. Malvern particle size analyzer (DLS) measured its size to be about 106nm, which laid the foundation for the nanoparticles to be enriched in the tumor site through the EPR effect ( Figure 2). RBCm coating not only provides compatibility with organisms, but also enhances the stability and long-term storage of NG through its natural cell membrane properties, thereby extending the shelf life of the drug. The stability of TDN, NG and M@NG in 10% FBS was tested by gel electrophoresis. The results showed that M@NG was stable in 10% FBS for at least 24 hours, showing good serum stability ( Figure 3 ).

[0046] In order to verify whether siRNA can be effectively released to achieve gene silencing function, we selected RAW 264.7 cells that can express green fluorescent protein (RAW 264.7-GFP cells) to verify that nucleic acid nanogels successfully deliver siRNA and that siRNA is released from nanogels to exert gene silencing effects. In this experiment, siGFP, which inhibits GFP expression, was selected to construct nucleic acid nanogels. If the green fluorescence intensity or protein expression in the cells decreases after siGFP is delivered to cells via nanogels, it indicates that siGFP has exerted a gene silencing effect. Liposome 2000-transfected siGFP (Lipo 2000siGFP) and NG synthesized with disordered siRNA (siGFP-NC) were used as positive and negative controls, respectively. The results of protein immunoblotting experiments showed that all preparations loaded with siGFP could reduce the 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 the present invention.

[0048]

[0049] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a framework nucleic acid nanogel coated with an erythrocyte membrane, characterized in that: The method comprises the following steps: S1, equimolar amounts of oligonucleotides were mixed in TM buffer; S2, heating the mixture solution at 95°C for 10-20 minutes, then quickly transferring to 4°C for cooling to obtain a DNA tetrahedral framework nucleic acid nanostructure, and storing it at 4°C; S3, siRNA with sticky ends was prepared, mixed with TDN at a molar ratio of 1:2, and vigorously shaken at 37 °C for 3 h to obtain nanogel NG; S4, collecting red blood cells, preparing red blood cell membranes (RBCm), and obtaining uniform RBCm vesicles by ultrasound and extrusion; S5, RBCm vesicles were mixed with NG and coextruded through a 100 nm polycarbonate porous membrane for multiple times to obtain red blood cell membrane-coated drug-loaded nucleic acid nanogels.

2. The method for preparing the erythrocyte membrane-coated nucleic acid nanogel according to claim 1, characterized in that: The tetrahedral framework nucleic acid nanostructure in step S2 is formed by mixing, heating, rapidly cooling and storing at 4° C. the oligonucleotides in TM buffer.

3. The method for preparing the erythrocyte membrane-coated nucleic acid nanogel according to claim 1, characterized in that: The siRNA used to form NG in the S3 step is siTOX with sticky ends, which is formed by gradient annealing of the sense strand and the antisense strand to room temperature and stored at -20°C.

4. The method for preparing the erythrocyte membrane-coated nucleic acid nanogel according to claim 1, characterized in that: The RBCm vesicles in step S5 are extruded through an Avanti small extruder, through 400nm and 200nm polycarbonate porous membranes respectively, and finally co-extruded through a 100nm polycarbonate porous membrane 14 times to form a uniform M@NG structure.

5. A red blood cell membrane-coated drug-loaded nucleic acid nanogel, characterized in that: The nanogel is formed by coating the framework nucleic acid nanogel NG with the red blood cell membrane RBCm, wherein the NG is formed by base complementary pairing and cross-linking of TDN and siRNA, and the RBCm provides biocompatibility and immune escape for the NG.

6. The erythrocyte membrane-coated drug-loaded nucleic acid nanogel according to claim 5, characterized in that: The cross-linking agent siRNA of the nucleic acid nanogel can be replaced by other siRNAs with sticky ends according to treatment needs, and is suitable for the treatment of various diseases, including tumors or inflammatory diseases.

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