A delivery carrier with nucleic acid time sequence release function, and a preparation method and application thereof

Abstract

The application discloses a delivery carrier with nucleic acid time sequence release function and a preparation method and application thereof, and belongs to the technical field of nucleic acid drug delivery. The nucleic acid time sequence release delivery carrier comprises, from inside to outside, an inner water phase layer, a middle oil phase layer and an outer water phase layer; the inner water phase layer contains an aqueous solution of nucleic acid molecules; the middle oil phase layer contains a hydrophobic segment of an active targeting nucleic acid delivery polymer and an amphiphilic block copolymer; and the outer water phase layer contains a hydrophilic segment of the amphiphilic block copolymer. After the delivery nucleic acid enters a cell, the nucleic acid molecules on the surface of the delivery carrier are released first, and then the nucleic acid molecules in the middle oil phase layer and the inner water phase are released in turn, so that the time sequence regulation or protein expression of the nucleic acid is realized. By using the technical scheme of the application, the nucleic acid is released in time sequence, the protein is regulated or expressed in a certain order in the cell, the problem that a traditional nucleic acid carrier cannot time sequence control the protein expression in the cell is overcome, and the delivery carrier can be used for preparing a drug for treating a neural stem cell gene disease.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid drug delivery technology, specifically relating to a delivery carrier with nucleic acid time-sequential release function, its preparation method, and its application. Background Technology

[0002] Neural stem cells (NSCs) are central nervous system stem cells with self-renewal and multi-lineage differentiation potential, capable of differentiating into neurons, astrocytes, and oligodendrocytes. Studies have shown that most transplanted NSCs ultimately differentiate into astrocytes rather than neurons, significantly limiting their ability to replace neuronal function. Therefore, current technologies often use exogenous plasmids to genetically modify neural stem cells to improve their survival, migration, differentiation, and neural repair capabilities. On the other hand, the proliferation, migration, and differentiation of neural stem cells is a highly temporal physiological process, requiring precise expression of different functional genes at specific time points for targeted regulation. This places higher demands on the release modes of gene delivery vectors.

[0003] Currently, methods for introducing exogenous genes into neural stem cells mainly rely on gene delivery vectors. Based on vector type, they can be divided into two main categories: viral vectors and non-viral vectors. Among them, viral vectors (such as lentiviruses, adenoviruses, and adeno-associated viruses) are widely used in neural stem cell gene modification due to their inherent gene transduction capabilities. Viral vectors can mediate the efficient integration and long-term expression of exogenous genes; however, the application of viral vectors presents significant safety risks, including: First, immunogenicity is a prominent issue; viral vectors may trigger a host immune response, leading to the elimination of transfected cells or causing an inflammatory response; second, the potential risk of insertional mutations is worrying; random integration by integrative vectors may activate proto-oncogenes or destroy tumor suppressor genes, posing a carcinogenic risk; third, the gene loading capacity of viral vectors is limited, making them unsuitable for larger therapeutic genes; fourth, the large-scale production of viral vectors is complex and costly, and biosafety requirements limit their clinical translation and widespread adoption. For example, Chinese invention patent CN102174578A discloses a lentiviral vector and a method for preparing a bone marrow mesenchymal stem cell line that secretes brain-derived neurotrophic factor mediated by it, but its safety issues remain a major obstacle to clinical translation.

[0004] Non-viral vectors, particularly cationic polymers (such as polyethyleneimine and poly-β-amino esters) and lipid nanoparticles, have become a research hotspot as alternatives to viral vectors due to their advantages such as good biosafety, ease of large-scale production, and functionalization. However, these traditional non-viral vectors still face significant challenges when applied to neural stem cells: First, there is an irreconcilable contradiction between transfection efficiency and cytotoxicity; the high charge density required for efficient transfection often leads to significant cell damage. Second, the vector / nucleic acid complex is not stable enough in the physiological environment and is easily degraded by nucleases. Third, and most challengingly, existing non-viral vectors mostly employ a simple model of nucleic acid adsorption or encapsulation, and their release kinetics are mostly characterized by rapid burst release or sustained release at a single rate. This means that all delivered nucleic acids are released and begin expressing proteins almost simultaneously after entering the cell, making it impossible to achieve sequential expression and regulation of different genes over time. Therefore, it is difficult to simulate or guide complex cellular behavior sequences and to directly apply them to gene therapy for neurological diseases.

[0005] Chinese invention patent CN111632039A discloses a multifunctional nanodelivery system composed of an amphiphilic polymer and a plasmid gene delivery polymer, possessing a hydrophilic shell and a hydrophobic core, which can be used for gene regulation and tracing of therapeutic cells such as stem cells. However, this carrier is prepared using a single-emulsion (O / W) method, where nucleic acid molecules mainly bind to the carrier surface or shell layer, making it easily degraded by nucleases, resulting in insufficient in vivo stability and the inability to achieve sequential release of nucleic acids.

[0006] Cationic polymers such as polyethyleneimine (PEI), polylysine, and polyamide-amine dendritic molecules have become the most widely studied non-viral gene carriers due to their ability to bind negatively charged nucleic acid molecules to form nanocomposites through electrostatic interactions. However, the application of PEI in neural stem cells still faces several technical bottlenecks. Firstly, transfection efficiency is low. Neural stem cells have limited endocytosis capacity for nanoparticles, and traditional cationic polymer-nucleic acid complexes are easily captured and degraded by endosomes / lysosomes after entering the cell, lacking an efficient "lysosomal escape" mechanism, resulting in the final gene expression level failing to meet therapeutic needs. Secondly, cytotoxicity is a prominent issue. Neural stem cells are highly sensitive to changes in the microenvironment. Under the nitrogen-to-phosphorus ratio required to achieve high transfection efficiency, high-branched PEI (25 kDa) significantly induces cell membrane damage, mitochondrial dysfunction, and apoptosis, severely impairing the viability and differentiation potential of neural stem cells. The proportion of neural stem cells that differentiate into neurons after PEI transfection decreases significantly. Thirdly, there is a lack of active targeting capability. Most non-viral vectors in the existing technology lack the function of specifically recognizing neural stem cells. After systemic administration, they are easily captured non-specifically by the reticuloendothelial system such as the liver and spleen, making it difficult to enrich neural stem cells in the lesion area of ​​the central nervous system. Fourthly, the release mode is singular and uncontrollable, mostly a one-time burst release, which cannot achieve the sequential release of nucleic acids that matches the survival, migration, and differentiation sequence of neural stem cells.

[0007] To address the aforementioned issues, researchers have proposed various improvement strategies, such as enhancing cellular uptake by modifying cell-penetrating peptides; promoting lysosomal escape by introducing components with proton sponge effects, such as imidazole groups, histidine, or chloroquine; and extending in vivo circulation time by using polyethylene glycol modification. However, these strategies often only solve a single problem and are difficult to achieve multi-objective synergistic optimization of "efficient transfection, low toxicity, stable delivery, targeted enrichment, and time-sequential release".

[0008] Nanocarriers prepared by double emulsion method (especially water-in-oil-in-water, W / O / W) have a unique multilayered structure. The W / O / W double emulsion structure can encapsulate nucleic acid molecules in the innermost aqueous phase, while the middle oil phase acts as a hydrophobic barrier, effectively isolating them from external nuclease erosion. This provides a new approach for the efficient encapsulation and controlled release of hydrophilic nucleic acid drugs. However, the application research of existing double emulsion carriers in neural stem cells is still very limited, and there are problems such as unreasonable component selection, incomplete solidification of the oil phase, and poor carrier stability. For example, Chinese invention patent CN120860231A discloses a multi-level liver-targeted drug delivery system based on ionizable amino lipids. It uses a W / O / W dual-phase water-in-oil-in-water nanoemulsion as the molding framework, compounded with ionizable amino lipids and targeted modified lipids, which self-assemble into lipid nanoparticles. In the emulsion's layered structure, liver-targeting modified groups such as galactose and bile acids are covalently linked to the outer aqueous phase, achieving precise targeted recognition of hepatocytes. The middle oil phase is enriched with ionizable amino lipids, enabling intracellular subcellular level targeted release. Therefore, this patent, through its W / O / W three-layer structure, sequentially endows the delivery system with long-circulation capability, liver particle size adaptability, hepatocyte targeted recognition capability, and intracellular pH-responsive drug release capability. However, this drug delivery system exhibits strong overall drug release synchronization, making it unable to achieve phased or time-sequential release. Consequently, in practical applications, it cannot fully meet the phased requirements of hepatocyte metabolic regulation, instead relying on overall liver-targeted release. Furthermore, the non-specific release of nucleic acid drugs into various hepatic cells can easily interfere with normal hepatocyte physiological functions.

[0009] Based on existing technical problems, this invention makes targeted technical improvements to the W / O / W double-lactam structure, developing a novel delivery system capable of efficiently loading nucleic acids, specifically targeting neural stem cells, and, in particular, utilizing the carrier's own structural design to achieve nucleic acid release "from the surface inward" or "stepwise sequentially." This system achieves efficient plasmid loading, stable protection, and efficient, low-toxicity transfection of neural stem cells, overcoming the technical challenges of existing delivery carriers such as easy degradation of nucleic acids, low transfection efficiency, high cytotoxicity, and poor targeting. It meets the key technical needs for promoting the development of time-series gene therapy, especially in the field of precise regulation of neural stem cells. Summary of the Invention

[0010] The main objective of this invention is to provide a delivery vector with nucleic acid time-sequential release function, its preparation method and application, so as to overcome the shortcomings of the prior art.

[0011] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0012] As the first objective of this invention, it provides a delivery carrier with a timed release function for nucleic acids, which has a water-in-oil-in-water multiple emulsion structure. The multiple emulsion structure comprises, from the inside out: an inner aqueous phase layer containing an aqueous solution of nucleic acid molecules; an intermediate oil phase layer containing a hydrophobic segment of an actively targeted nucleic acid delivery polymer and an amphiphilic block copolymer; and an outer aqueous phase layer containing a hydrophilic segment of the amphiphilic block copolymer. The actively targeted nucleic acid delivery polymer comprises a cationic polymer and a hydrophobic backbone polymer. The cationic polymer is protonated under physiological pH conditions to carry a positive charge. The hydrophobic backbone polymer and the hydrophobic segments of the amphiphilic block copolymer self-assemble to form a hydrophobic shell, which encapsulates the polyelectrolyte complex inside the carrier. The surface of the delivery carrier adsorbs nucleic acid molecules through electrostatic interaction, so that the nucleic acid molecules form a gradient distribution in the multiple emulsion structure. After the nucleic acid is delivered into the cell, the nucleic acid molecules on the surface of the delivery carrier are released first, and then the nucleic acid molecules in the intermediate oil phase and the inner aqueous phase are released in sequence, so as to realize the temporal regulation of nucleic acid or protein expression.

[0013] In a preferred embodiment, the cationic polymer is poly(β-amino ester) PBAE, whose tertiary amine groups on the molecular chain are protonated under physiological pH conditions to carry a positive charge, forming a polyelectrolyte complex with negatively charged nucleic acid molecules.

[0014] In a preferred embodiment, the hydrophobic backbone polymer is polylactic acid-glycolic acid copolymer (PLGA).

[0015] In a preferred embodiment, the amphiphilic block copolymer is polyethylene glycol-polycaprolactone block copolymer (PEG-PCL).

[0016] Preferably, the molecular weight of PBAE is 5000~15000 Da.

[0017] Preferably, the molar ratio of lactic acid to glycolic acid monomers in PLGA is 50:50, and the molecular weight is 10,000 to 30,000 Da.

[0018] Preferably, in PEG-PCL, the molecular weight of the PEG segment is 2000~5000 Da, and the molecular weight of the PCL segment is 4000~10000 Da.

[0019] In a preferred embodiment, the nucleic acid molecule includes, but is not limited to, any one or a combination of two or more of plasmid DNA, siRNA, mRNA, and antisense oligonucleotides.

[0020] In a preferred embodiment, the delivery vector with nucleic acid sequential release function has a positively charged surface, an average particle size of 150±20 nm, a polydispersity index (PDI) of 0.15±0.05, and a zeta potential of +25±5 mV.

[0021] As a second aspect of the invention, the present invention also provides a method for preparing a delivery vector with nucleic acid timing release function as described above, the preparation steps including:

[0022] S1. Dissolve nucleic acid molecules in sterile water to prepare an internal aqueous phase solution, denoted as solution A;

[0023] S2. Dissolve the actively targeted delivery polymer in an organic solvent to obtain an oil phase solution, denoted as solution B;

[0024] S3. Dissolve the amphiphilic block copolymer in sterile water to obtain an external aqueous solution, denoted as solution C;

[0025] S4. Under ultrasonic conditions, solution A is slowly added to solution B, and a single emulsion is formed to obtain a W / O primary emulsion, denoted as solution D.

[0026] S5. Under ultrasonic conditions, solution D is slowly added to solution C, and secondary emulsification is performed to obtain a crude W / O / W double emulsion, denoted as solution E.

[0027] S6. Purify solution E to obtain the delivery vector with nucleic acid time-sequential release function.

[0028] In a preferred embodiment, in S2, the organic solvent is any one or a combination of two or more of tetrahydrofuran, dichloromethane, trichloromethane, and dimethyl sulfoxide.

[0029] In a preferred embodiment, in S4, the volume ratio of solution A to solution B is 1:(20~100).

[0030] In a preferred embodiment, the rate at which solution A is added to solution B is 10~50 μL / min.

[0031] As a preferred embodiment, the primary emulsification method includes any one of ultrasonic dispersion, microfluidic method, ultrasonic emulsification method, or mechanical stirring method.

[0032] In a preferred embodiment, in S5, the volume ratio of solution D to solution C is 1:(5~20).

[0033] In a preferred embodiment, the solution D is added to the solution C at a rate of 100~500 μL / min.

[0034] As a preferred embodiment, the secondary emulsification method includes any one of ultrasonic dispersion, microfluidic method, ultrasonic emulsification method, or mechanical stirring method.

[0035] In a preferred embodiment, in S6, the purification process is ultrafiltration purification, using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30~100 kDa and a centrifugation speed of 8,000~12,000 rpm.

[0036] As a third aspect of the invention, the present invention also provides a dual-gene time-sequential delivery vector, comprising: a delivery vector having a nucleic acid time-sequential release function as described above and a second nucleic acid molecule adsorbed onto the surface of the delivery vector by electrostatic interaction.

[0037] Preferably, the delivery vector with nucleic acid timing release function is loaded with a first nucleic acid molecule.

[0038] Preferably, the delivery vector with nucleic acid timing release function is mixed with the second nucleic acid molecule and then incubated to obtain the dual-gene timing delivery vector.

[0039] Preferably, the first nucleic acid molecule and the second nucleic acid molecule are the same or different.

[0040] Preferably, the first nucleic acid molecule and the second nucleic acid molecule include, but are not limited to, any one or a combination of two or more of plasmid DNA, siRNA, mRNA, and antisense oligonucleotides.

[0041] As a fourth aspect of the invention, the present invention also provides a delivery vector with nucleic acid timing release function as described above, or the application of a dual-gene timing delivery vector as described above in the preparation of a drug for treating neural stem cell gene diseases.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] (1) This invention uses biodegradable materials (PBAE, PLGA, and PEG-PCL) as raw materials for neural stem cell targeted therapy delivery carriers. The degradation products are non-toxic and can be metabolized and cleared by the body. At the effective transfection concentration, the cell survival rate of neural stem cells exceeds 85%, which is far superior to that of traditional cationic polymer PEI (cell survival rate <60%), and has excellent biocompatibility. At the same time, PBAE has an acid-responsive proton sponge effect, which is conducive to the lysosomal escape of the carrier after endocytosis by the cell, and promotes the sequential release of nucleic acids into the cytoplasm, thereby achieving efficient cell transfection.

[0044] (2) The preparation method provided by the present invention also has the advantages of simple and readily available raw materials, convenient and controllable operation process, mild conditions (room temperature ultrasound), avoiding harsh conditions such as residual organic solvents and high temperature and high pressure, and is suitable for industrial production with small batch-to-batch differences, and has good industrialization prospects.

[0045] (3) This invention is the first to combine three polymers, PBAE, PLGA and PEG-PCL, through the W / O / W double emulsion method to construct a multi-layered neural stem cell targeted therapy delivery vector. The five advantages of targeted delivery, time-sequential release, long-term expression, low toxicity and safety and industrialization work together to break through the technical bottleneck of existing non-viral vectors in neural stem cell transfection. It provides a safe, efficient and controllable new delivery platform for the treatment of nervous system diseases based on neural stem cell gene modification, and has significant clinical translational value and industrial application prospects. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 This is a schematic diagram of the simulated structure of the nucleic acid nanocarrier prepared by the present invention, showing the W / O / W three-layer structure design, which can realize the time-sequential release of nucleic acids.

[0048] Figure 2 This is a transmission electron microscope image of the nucleic acid delivery vector prepared in Example 1 of the present invention.

[0049] Figure 3 This is a potential detection diagram of the nucleic acid delivery vector prepared in Example 1 of the present invention.

[0050] Figure 4 This is a bar chart showing the cell viability detection results of neural stem cells after incubating the nucleic acid delivery vector prepared in Example 3 of this invention for 24 hours.

[0051] Figure 5 This is a comparison of the fluorescence results of nucleic acid expression in cells transfected with the nucleic acid nanocarrier prepared in Example 2 of this invention.

[0052] Figure 6 These are agarose gel electrophoresis images of the nucleic acid nanocarrier prepared in Example 1 of this invention, showing the sequential release of nucleic acid at 0h, 2h, 4h, 6h, 12h, 24h, and 48h.

[0053] Figure 7This is a comparison image of immunofluorescence staining for the regulation of neural stem cell differentiation in Example 4 of the present invention.

[0054] Figure 8 Images showing the healing morphology of mouse wounds after treatment with WOW nanocarriers at different times in Example 5 of this invention. Detailed Implementation

[0055] Together with Figure 1 Reading the following specific embodiments will provide a more complete understanding of the invention. It should be understood that the detailed embodiments disclosed herein are merely illustrative of the invention, which can be embodied in various forms. Therefore, the specific functional details disclosed herein should not be construed as limiting, but rather as the basis for the claims and as a representative basis for teaching those skilled in the art to employ the invention in different ways in fact.

[0056] Addressing the core shortcomings of existing liposome delivery systems—reliant on microfluidics and click chemistry, difficult to scale, and unable to adapt to the time-sequential delivery of nucleic acid macromolecules—as well as the technical bottlenecks of traditional non-viral vectors—low transfection efficiency, high toxicity, and uncontrollable release, this invention provides a neural stem cell targeted therapy delivery vector. This vector possesses excellent neural stem cell targeting capability, high transfection efficiency, and low cytotoxicity, effectively delivering therapeutic nucleic acids to neural stem cells. Without external stimulation, nucleic acids are autonomously and sequentially released through gradient degradation of the vector. Upon entering the cell, it first rapidly releases surface-adsorbed nucleic acids to initiate early gene expression, followed by a slow release of the inner encapsulated nucleic acids to maintain long-term gene expression, thereby regulating the time-sequential expression of genes. This perfectly matches the physiological cycle of neural stem cell proliferation, migration, and differentiation, achieving gene modification and functional regulation. This method can be used for applications such as stem cell differentiation regulation and tissue repair.

[0057] Poly(β-amino esters) (PBAEs) are a class of biodegradable cationic polymers with low cytotoxicity and high nucleic acid binding capacity. However, when used alone, they suffer from poor colloidal stability and short in vivo circulation time. Polylactic acid-glycolic acid copolymer (PLGA) and polyethylene glycol-polycaprolactone block copolymer (PEG-PCL) are FDA-approved biodegradable materials for drug delivery. However, traditional PLGA nanoparticles have low nucleic acid encapsulation efficiency and are difficult to achieve efficient intracellular delivery and time-controlled release. This invention assembles PBAE, PLGA, and PEG-PCL polymers using a W / O / W double emulsion method to construct a neural stem cell targeted therapy delivery carrier with a multi-layered septate structure. At the effective transfection concentration, the cell survival rate of neural stem cells exceeds 85%, which is far superior to that of traditional cationic polymer PEI (cell survival rate <60%), demonstrating excellent biocompatibility. Meanwhile, PBAE has an acid-responsive proton sponge effect, which facilitates the escape of lysosomes after the vector is endocytosed by the cell, promotes the sequential release of nucleic acids into the cytoplasm, and thus achieves efficient cell transfection.

[0058] Specifically, the neural stem cell targeted therapy delivery carrier provided by this invention can be used to target and deliver nucleic acid drugs into neural stem cells. It mainly consists of an active targeted delivery polymer (including the cationic polymer PBAE and the hydrophobic backbone polymer PLGA) and a shell stabilizing component (the amphiphilic block copolymer PEG-PCL). These components interact through a water-in-oil-in-water (W / O / W) double emulsion to form a three-layered structure: simultaneously, the carrier surface can adsorb some nucleic acid molecules through electrostatic interactions; the intermediate oil phase consists of hydrophobic segments of PBAE, PLGA, and PEG-PCL, while the outer aqueous phase consists of hydrophilic segments of PEG-PCL. PBAE binds tightly to nucleic acids through electrostatic interactions to form a polyelectrolyte complex; PLGA and the hydrophobic segments of PEG-PCL self-assemble to form a hydrophobic shell; and PEG segments are enriched on the carrier surface. See also... Figure 1 The figure shows a schematic diagram of the three-layer structure of the W / O / W double emulsion nanocarrier provided by the present invention. As can be seen from the figure, the carrier consists of the following layers from the inside to the outside: an inner aqueous phase (loaded with nucleic acid molecules), an intermediate oil phase (PBAE, PLGA and PEG-PCL hydrophobic segments), and an outer aqueous phase (PEG-PCL hydrophilic segments). At the same time, some nucleic acid molecules are adsorbed on the surface of the carrier. The nucleic acid molecules are efficiently encapsulated in the inner aqueous phase and partially adsorbed on the surface of the carrier. The gradient degradation characteristics of the three-layer structure endow the carrier with the ability to release nucleic acids in a sequential manner, which breaks through the limitation of existing nested liposomes that require external stimulation to achieve multi-stage release.

[0059] In some more specific embodiments, the cationic polymer PBAE is a polyβ-amino ester with optimized side chains and a molecular weight of 5000~15000 Da. The tertiary amine groups on its molecular chain are protonated at physiological pH and carry a positive charge.

[0060] In some more specific embodiments, the hydrophobic backbone polymer PLGA has a lactic acid to glycolic acid monomer molar ratio of 50:50 and a molecular weight of 10,000~30,000 Da.

[0061] In some more specific embodiments, in the amphiphilic block copolymer PEG-PCL, the molecular weight of the PEG segment is 2000~5000 Da, and the molecular weight of the PCL segment is 4000~10000 Da.

[0062] In some more specific implementations, the nucleic acid molecules include, but are not limited to, any one or a combination of two or more of plasmid DNA, small interfering RNA (siRNA), messenger RNA (mRNA), and antisense oligonucleotides.

[0063] In a preferred embodiment, the plasmid DNA is a GFP reporter gene plasmid, used to verify the transfection efficiency of the vector (see Appendix). Figure 4 As another preferred embodiment, the plasmid DNA is a functional plasmid carrying therapeutic genes (such as brain-derived neurotrophic factor BDNF, glial cell-derived neurotrophic factor GDNF, etc.) for the time-sequential functional modification of neural stem cells and disease treatment.

[0064] The delivery carrier of this invention is a W / O / W double emulsion structure, in which nucleic acid molecules are encapsulated in an inner aqueous phase. The preparation process does not require microfluidic chips or click chemistry reactions; it can be completed simply through conventional ultrasonic emulsification. The preparation method includes the following steps:

[0065] Step 1: Dissolve nucleic acid molecules in sterile water to prepare internal aqueous phase solution A;

[0066] Step 2: Dissolve the actively targeted delivery polymers (PBAE and PLGA) in an organic solvent to obtain oil phase solution B;

[0067] Step 3: Dissolve the shell stabilizing component PEG-PCL in sterile water to obtain the outer aqueous phase solution C;

[0068] Step 4: Under ultrasonic conditions, slowly add solution A to solution B to emulsify and obtain a W / O primary emulsion, denoted as solution D;

[0069] Step 5: Under ultrasonic conditions, slowly add solution D to solution C to emulsify and obtain a crude W / O / W double emulsion, denoted as solution E;

[0070] Step 6: Purify solution E to obtain the neural stem cell targeted therapy delivery vector.

[0071] In some more specific embodiments, the organic solvent is a volatile organic solvent, including any one or a combination of two or more of tetrahydrofuran, dichloromethane, trichloromethane, and dimethyl sulfoxide, but not limited thereto.

[0072] In some more specific embodiments, the preparation method includes: slowly adding solution A to solution B, controlling the addition rate at 10~50 μL / min, and emulsifying under ultrasonic conditions.

[0073] In some more specific embodiments, the preparation method includes: slowly adding the solution D to the solution C, controlling the addition rate at 100~500 μL / min, and performing re-emulsification under ultrasonic conditions.

[0074] In some more specific implementations, the mass ratio of the nucleic acid molecule to the active targeted delivery polymer is 1:(5~50).

[0075] In some more specific implementations, the volume ratio of solution A to solution B is 1:(20~100).

[0076] In some more specific implementations, the mixing method of solution A and solution B includes any one of ultrasonic dispersion, microfluidic method, ultrasonic emulsification or mechanical stirring; preferably ultrasonic emulsification.

[0077] In some more specific implementations, the volume ratio of solution D to solution C is 1:(5~20).

[0078] In some more specific implementations, the mixing method of solution D and solution C includes any one of ultrasonic dispersion, microfluidic method, ultrasonic emulsification or mechanical stirring; preferably ultrasonic emulsification.

[0079] Furthermore, the ultrasonic treatment time is 0.5~2 min; the ultrasonic power is 100~300 W; and the ultrasonic temperature is room temperature (25℃).

[0080] Furthermore, the purification process is an ultrafiltration purification process, using ultrafiltration centrifuge tubes with a molecular weight cutoff of 30~100 kDa, centrifuging at a speed of 8,000~12,000 rpm, and repeatedly washing with sterile water 2~4 times.

[0081] The water-in-oil-in-water (W / O / W) double emulsion structure neural stem cell targeted therapy delivery carrier provided by this invention can efficiently deliver nucleic acid drugs to neural stem cells, overcoming the bottleneck of traditional liposomes and polymer carriers that cannot achieve sequential nucleic acid release. It also overcomes the shortcomings of advanced microfluidic nested liposomes, such as difficulty in large-scale production and incompatibility with nucleic acid delivery, achieving autonomous sequential and gradual release of nucleic acids from 0 to 72 hours. This overcomes the technical difficulties of low delivery efficiency and easy degradation of nucleic acids in existing non-viral carriers during neural stem cell transfection. Agarose gel electrophoresis results show that the carrier of this invention exhibits sequential nucleic acid release bands at 0h, 2h, 4h, 6h, 12h, 24h, and 48h, with no significant burst release, demonstrating the carrier's excellent sequential controlled release and protection capabilities for nucleic acids.

[0082] The neural stem cell targeted therapy delivery vector provided by this invention has a uniform particle size distribution, with the particle size within an appropriate range, a small polydispersity index (PDI), good colloidal stability, and a suitable positive charge on its surface, which is conducive to cell uptake. After 72 hours of transfection into neural stem cells, the vector showed significant expression of GFP (green fluorescent protein), a high proportion of positive cells, and strong fluorescence intensity, indicating that the vector can efficiently mediate the entry of nucleic acid molecules into neural stem cells through time-sequential release and achieve high-level, time-sequential expression.

[0083] The vector provided by this invention can effectively regulate the differentiation direction of neural stem cells by precisely releasing neurotrophic factor plasmids. The proportion of GFAP-positive cells, a marker of astrocytes, is significantly reduced, while the number of Tuj1-positive cells, a neuron-specific marker, is greatly increased. Furthermore, the neurons have slender processes and abundant branches, forming a complex neural network-like structure. The results indicate that the vector provided by this invention can effectively inhibit the non-specific differentiation of neural stem cells into astrocytes and significantly promote their directional differentiation into functional neurons, providing key technical support for the repair of central nervous system injuries.

[0084] The W / O / W nanocarrier constructed in this invention is highly versatile and has a wide range of applications. It is not only suitable for gene modification and regulation of neural stem cells, but also for efficient local delivery and long-term expression of functional genes in skin wounds, overcoming the clinical pain points of traditional wound medications, such as short half-life, short-term efficacy, and the need for repeated administration. Animal wound experiments show that the carrier of this invention can stably load functional therapeutic plasmids and continuously release and express them locally in the wound, significantly accelerating the wound healing process. On day 7 after administration, the residual wound area in the carrier treatment group was only 3.2% of the initial area, far lower than the 15.6% in the blank control group, and the time to complete wound epithelialization was shortened by about 2 days. Simultaneously, this carrier can effectively promote the proliferation of granulation tissue, the orderly arrangement of collagen fibers, and increase the density of local neovascularization, significantly optimizing the quality of wound repair. The invention confirms that the nanodelivery platform possesses excellent in vivo gene delivery efficiency and tissue repair regulation capabilities. It can be used for the directed differentiation regulation of neural stem cells, as well as for gene therapy of soft tissue injuries such as skin trauma and refractory ulcers. This greatly expands the clinical applicability of the composite nanocarrier and has strong technical extensibility and broad clinical translational value.

[0085] The technical solution of the present invention will be described in detail below through specific embodiments. Unless otherwise specified, the experimental materials used in the embodiments can be purchased from conventional biochemical reagent companies.

[0086] Example 1

[0087] This embodiment uses a GFP reporter gene plasmid to prepare a neural stem cell targeted delivery vector to evaluate the vector's physicochemical properties, transfection efficiency, and nucleic acid release behavior.

[0088] Specifically, the preparation method of the neural stem cell targeted delivery vector includes the following steps:

[0089] (1) Extraction of GFP plasmid: Take the E. coli culture carrying the GFP plasmid, first activate the bacterial culture with 5 mL of Amp-resistant LB medium by shaking, then transfer it to 100 mL of LB medium containing ampicillin, and culture at 37 ℃ and 200 rpm for 14-16 h, and collect 50 mL of bacterial culture; centrifuge the bacterial culture at 4 ℃ and 5000×g for 15 min to enrich the bacterial cell pellet, fully resuspend the bacterial cells with 8 mL of RES buffer on ice, then add 8 mL of LYS lysis buffer and gently invert to mix, and let stand at room temperature for 5 min to lyse the bacteria. During this interval, use 12 mL of EQU buffer to equilibrate the chromatography column along the side wall of the filter; then add 8 mL of NEU neutralization solution and mix well. A white flocculent precipitate will precipitate in the system. Filter the entire mixture onto the column by gravity, and the plasmid binds to the column matrix and impurities are retained; wash away the impurities and small molecules stepwise with 5 mL of EQU and 8 mL of WASH buffer, and then wash with 5 mL of ELU. The plasmid solution was obtained by elution with buffer solution; 3.5 mL of isopropanol was added to the eluent and mixed well. The mixture was centrifuged at 4 °C and 6000 × g for 30 min to obtain plasmid precipitate. The precipitate was gently washed with 2 mL of 70% ethanol and then air-dried in a fume hood for 10–15 min. Finally, the plasmid was reconstituted with 500 μL of sterile water, and the concentration was determined by spectrophotometer before aliquoting and storage at -20 °C.

[0090] (2) Preparation of the inner aqueous phase: Dissolve 1000 ng GFP plasmid DNA in sterile ultrapure water and make up to a total volume of 10 μL to obtain solution A (plasmid concentration 100 ng / μL).

[0091] (3) Preparation of oil phase: In EP tube No. 1, add 250 μL of tetrahydrofuran (THF), 4 μL of PBAE polymer solution (10 mg / mL), 5 μL of PLGA solution (10 mg / mL), and 0.7 μL of PEG-PCL solution (10 mg / mL) in sequence, and mix thoroughly by gentle blowing to obtain solution B.

[0092] (4) Preparation of external aqueous phase: Add 3 μL of PEG-PCL solution (10 mg / mL) to EP tube No. 2, and make up to 1 mL of total volume with sterile water. Gently pipette to mix and obtain solution C (PEG-PCL concentration 0.03 mg / mL).

[0093] (5) Preparation of W / O colostrum: Under normal temperature (25℃) ultrasonic conditions, the solution A from step (1) is slowly added to the solution B from step (2), and ultrasonic treatment is continued for 1 min to obtain W / O colostrum, which is denoted as solution D.

[0094] (6) Preparation of W / O / W double emulsion: Under normal temperature (25℃) ultrasonic conditions, the solution D in step (4) is slowly transferred to the solution C in step (3), and ultrasonic treatment is continued for 1 min to obtain crude W / O / W double emulsion, which is denoted as solution E.

[0095] (7) Ultrafiltration concentration and purification: Transfer solution E in 400 μL fractions to ultrafiltration centrifuge tubes (molecular weight cutoff 50 kDa), and concentrate by centrifugation at 10,000 rpm for 10 min at room temperature. Repeat until the sample is concentrated to approximately 100 μL. Add an appropriate amount of sterile water to the concentrated sample to the original volume, gently resuspend, and centrifuge at 10,000 rpm for 5 min at room temperature. Discard the filtrate. Repeat this washing step three times to remove uncoated free nucleic acids, organic solvents, and impurities.

[0096] (8) Ultrasonic resuspension and dispensing: Add sterile water to the outer tube of the cleaned ultrafiltration tube until it reaches 2 / 3 of the tube's volume. Ultrasonicate for 2 min to resuspend the carrier, shaking the tube during the process to ensure full dispersion. Aspirate and mix the resuspended carrier solution, dispense it into PCR tubes, and sonicate again for 10 s to obtain the final product, W / O / W nanocarrier.

[0097] See Figure 2 and Figure 3 The figures show the particle size distribution and potential detection of the W / O / W nanocarriers prepared in this embodiment. Dynamic light scattering (MALEN Zetasizer Nano ZS) was used for detection. The results showed that the average particle size of the nanocarriers was 150±20 nm, the polydispersity index (PDI) was 0.15±0.05, and the zeta potential was +25±5 mV, indicating that the carrier particles were uniform in size and had a suitable positive charge on the surface, which is beneficial for cell uptake.

[0098] See Figure 6 The following are the agarose gel electrophoresis results of the plasmid encapsulation efficiency and sequential release of nucleic acids by the W / O / W nanocarrier prepared in this embodiment. The W / O / W nanocarrier was placed in the release medium, and samples were taken at 0h, 2h, 4h, 6h, 12h, 24h, and 48h for electrophoresis. The results showed that no free nucleic acid bands were observed at 0h, proving that the W / O / W nanocarrier encapsulated plasmid DNA at a rate exceeding 90%, demonstrating excellent nucleic acid encapsulation and protection capabilities. Clear plasmid bands appeared at each time point from 2h to 48h, and the band brightness gradually increased with time, exhibiting typical sequential release characteristics without obvious burst release, directly confirming that the W / O / W nanocarrier can achieve stable, continuous, and autonomous sequential release of nucleic acids.

[0099] Example 2

[0100] In this embodiment, the expression of GFP in neural stem cells transfected with the W / O / W nanocarrier prepared in Example 1 was observed using a fluorescence microscope to evaluate the transfection efficiency.

[0101] (1) Isolation and culture of neural stem cells: Hippocampal tissue from newborn SD rats within 24 hours was digested with 0.25% trypsin for 15 min, dispersed by mechanical pipetting, and passed through a 200-mesh cell sieve. The cell suspension was collected and cultured at 1×10⁻⁶ cells / mL. 5 The cells were seeded at a density of 1 / mL in culture flasks containing DMEM / F-12 (purchased from Hyclone) (1% SP) neural stem cell culture medium and incubated at 37°C in a 5% CO2 incubator. The medium was changed every 3 days and the cells were passaged every 5-7 days.

[0102] (2) Preparation scheme of surface adsorption plasmid W / O / W nanocarrier: no plasmid in the inner aqueous phase, the middle oil is the same as in Example 1, 1000 ng GFP plasmid is added to the outer aqueous phase, and other operations are the same as in Example 1.

[0103] (3) Transfection experiment: 3rd-5th generation neural stem cells were used to transfect the cells at a concentration of 5×10⁻⁶ cells / mL. 4 The cells were seeded at a density of cells / well in 24-well plates. After culturing for 24 h, the old culture medium was discarded, and the cells were divided into two groups for transfection. One group was added with 500 μL of serum-free culture medium containing the nanocarrier (containing GFP plasmid) prepared in Example 1, and was denoted as the inner plasmid group. The other group was added with the nanocarrier (containing GFP plasmid) prepared in step (2) of this example, and was denoted as the outer plasmid group.

[0104] Four hours after transfection, both groups were replaced with complete culture medium, and samples were taken at 0h, 12h, 24h, 36h, 48h, and 72h post-transfection for observation.

[0105] (4) Fluorescence microscopy observation: 72 h after transfection, the culture medium was discarded and the cells were washed twice with PBS. The expression of GFP green fluorescence was observed under a fluorescence microscope (Olympus IX73), and cell morphology and density were observed in bright field. Superimposed images were also taken.

[0106] See Figure 5 Figure A shows the time-series fluorescence images of GFP expression after transfection of neural stem cells with W / O / W nanocarriers in this embodiment from 0 to 72 hours. It includes a comparison of transfection with two delivery vectors obtained after core-encapsulated plasmids and surface-adsorbed plasmids. Figure A shows the green fluorescence expression of the inner-encapsulated plasmid group after transfection with GFP plasmids in the inner aqueous phase. As can be seen from the figure, gene expression initiation is delayed, peak is delayed, and decay is slow. There is no obvious fluorescence from 0 to 12 hours, and only a very weak signal is seen at 24 hours, which is significantly weaker than the surface group. The fluorescence slowly increases from 36 to 48 hours, and the fluorescence continues to increase steadily after 72 hours.

[0107] Figure B shows the green fluorescence expression of the outer plasmid group with added GFP plasmid in the external aqueous phase. Due to the adsorption of plasmid on the surface, gene expression started quickly, peaked early, and declined rapidly. There was no fluorescence signal from 0 to 12 h, scattered green fluorescence appeared at 24 h, the signal was significantly enhanced at 36 h, and covered most of the field of view from 48 to 72 h (early expression peak). GFP-positive cells were close to 100%, and the expression level reached its highest point. Moreover, the gene expression level of the outer plasmid group was significantly higher than that of the inner plasmid group at the same time, and the expression period of the inner plasmid group was also significantly longer than that of the outer plasmid group.

[0108] The results showed that the plasmid loading method directly determines the gene expression sequence: surface-adsorbed plasmids release rapidly and achieve early and efficient expression; core-encapsulated plasmids rely on polymer slow degradation to achieve delayed, long-lasting, and high-abundance expression. Moreover, the cells in both groups showed normal morphology and no obvious damage, indicating that the delivery vectors obtained by both loading methods have good biocompatibility. Through the dual-compartment layered structure, this water-in-oil-in-water vector can achieve timed gene expression by regulating plasmid distribution, providing a flexible solution for precise intervention in neural regeneration.

[0109] Example 3

[0110] In this embodiment, the MTT assay was used to detect the cytotoxicity of the nanocarrier prepared in Example 1 to neural stem cells, and the NP group without plasmid (prepared in the same way as the WOW nanocarrier prepared in Example 1, except that no GFP plasmid was added in step 1) and the naked GFP plasmid group at the same concentration were used as controls.

[0111] (1) Neural stem cell seeding: 3rd-5th generation neural stem cells were seeded at a concentration of 1×10⁻⁶. 4 The cells were seeded at a density of 100 μL / well in 96-well plates, and 100 μL of neural stem cell culture medium (formulation: 50 mL Neurobasal medium (Gibco), 1 mL B27 (2%), 0.5 mL Gln (1%), 20 ng / mL bFGF, 20 ng / mL EGF and 0.5% SP) was added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 h.

[0112] (2) Vector treatment: Discard the old culture medium and add 100 μL of serum-free culture medium to the W / O / W nanovector (NP+GFP) group, the NP group without GFP plasmid, and the naked GFP plasmid group provided in Example 1, respectively. A negative control group (cells and culture medium only, no vector) and a blank control group (no cells, culture medium only, no vector) were also set up. Five replicates were set up for each group. After 4 h of transfection, the medium was replaced with complete culture medium, and the culture was continued for another 24 h.

[0113] (3) MTT assay: Add 20 μL of thiazolyl blue MTT solution (5 mg / mL) to each well and continue culturing for 4 h. After terminating the culture, carefully aspirate the supernatant, add 150 μL of DMSO to each well, and shake for 10 min to fully dissolve the crystals.

[0114] The absorbance (OD value) of each well was measured at a wavelength of 490 nm using a microplate reader (BioTek Synergy H1).

[0115] The cell survival rate is calculated as follows: Cell survival rate (%) = (OD value of experimental group - OD value of blank control group) / (OD value of negative control group - OD value of blank control group) × 100%.

[0116] See Figure 4 The figure shows a comparison of the results of the MTT assay for detecting the cytotoxicity of the W / O / W nanocarrier in this embodiment. The results show that at a plasmid content of 0.5 μg (effective transfection concentration), the cell viability of the nanocarrier group (NP+GFP) provided by this invention is 102.3% ± 0.2%, indicating that the delivery vector provided in this embodiment has extremely low cytotoxicity to neural stem cells at the effective transfection concentration and has good biosafety.

[0117] Example 4

[0118] This embodiment utilizes the double-layer nucleic acid delivery vector function of the W / O / W complex structure of the present invention to construct a time-sequential release delivery vector with the SOX9 silencing plasmid siSOX9-mCherry plasmid adsorbed on the surface and the neural differentiation transcription factor NeuroD1 encapsulated in the inner layer. This enables precise time-sequential regulation of early inhibition of astrocyte differentiation and late initiation of directed neuronal differentiation during the differentiation process of neural stem cells.

[0119] Specifically, the preparation method of the neural stem cell directed differentiation dual-gene time-sequential delivery vector is as follows:

[0120] (1) Dissolve 1200 ng NeuroD1 plasmid DNA (purchased from Addgene, plasmid number: 173480) in sterile ultrapure water and make up to a total volume of 10 μL to obtain an inner aqueous phase solution A (plasmid concentration 120 ng / μL).

[0121] (2) Preparation of oil phase: Same as step (3) in Example 1.

[0122] (3) Preparation of external water phase: Same as step (4) in Example 1.

[0123] (4) Preparation of W / O colostrum and W / O / W double emulsion: Same as steps (5) and (6) in Example 1, to prepare the core nanocarrier encapsulating NeuroD1 plasmid.

[0124] (5) Ultrafiltration purification: Same as step (7) in Example 1, to obtain purified nanocarriers encapsulating NeuroD1 plasmids.

[0125] (6) The purified nanocarrier was mixed with 600 ng siSOX9-mCherry plasmid DNA (purchased from Addgene, plasmid number: #128744) and incubated at room temperature for 30 min, so that NeuroD1 plasmid was quantitatively adsorbed onto the surface of the carrier by electrostatic interaction, thus obtaining a dual-gene time-release vector carrying NeuroD1 (surface) and SiSOX9-mCherry (inner layer).

[0126] (7) Resuspension and repackaging: Same as step (8) in Example 1.

[0127] (8) Isolation and culture of neural stem cells: Hippocampal tissue from neonatal SD rats within 24 hours was digested with 0.25% trypsin for 15 min, dispersed by mechanical pipetting, and passed through a 200-mesh cell sieve. The cell suspension was collected and cultured at 1×10⁻⁶ cells / mL. 5 NSCs were seeded at a density of 12 cells / mL in culture flasks containing neural stem cell culture medium and cultured in a 37°C, 5% CO2 incubator. The medium was changed once a day, and the cells were passaged every 2-5 days. NSCs in good condition from the 2nd to the 5th generation were selected for subsequent experiments.

[0128] (9) Transfection and differentiation induction experiment: Take the 2nd-5th generation neural stem cells from step (8) and use 1×10 5 Seeds were inoculated at a density of 100 cells / well in laser confocal microscopy culture dishes, and the old culture medium was discarded after 24 h of culture. In the experimental group, 500 μL of serum-free culture medium containing the prepared dual-gene time-series vector (containing neurotrophic-related plasmid) was added to each well. After 4 h of transfection, the medium was replaced with differentiation induction medium containing 1% FBS. In the control group, an equal amount of plasmid was transfected with a conventional single-emulsion nanocarrier and differentiation induction culture was performed simultaneously. The culture was placed in a 37℃, 5% CO2 incubator for 7 days to induce differentiation.

[0129] (10) Immunofluorescence staining detection: After 7 days of differentiation induction, the culture medium was discarded, the cells were washed 3 times with PBS, and fixed with 4% paraformaldehyde at room temperature for 15 min; 0.3% Triton X-100 was used for permeabilization for 10 min, and 5% goat serum was used for blocking at room temperature for 1 h; rabbit anti-GFAP primary antibody (1:500, astrocyte marker) and mouse anti-Tuj 1 primary antibody (1:300, neuron-specific marker) were added respectively, and incubated overnight in a humidified chamber at 4℃; the next day, after washing with PBS, Cy3-labeled goat anti-rabbit secondary antibody (1:500, red fluorescence) and FITC-labeled goat anti-mouse secondary antibody (1:500, green fluorescence) were added, and incubated at room temperature in the dark for 1 h; Hoechst 33342 (1 μg / mL) was added to stain the nuclei for 5 min in the dark, and after mounting with anti-fluorescence quenching mounting medium, the slides were placed in a laser confocal microscope (Zeiss LSM). Images were observed and captured under 880°C to detect the expression of neuronal marker Tuj1 and astrocyte marker GFAP, and to evaluate the directed differentiation effect of neural stem cells.

[0130] A control group and an experimental group were set up. The experimental group was transfected with an equal amount of plasmid using the W / O / W nanocarrier prepared in this embodiment, and the differentiation was induced and cultured simultaneously. The control group was transfected with an equal amount of plasmid using a conventional single emulsion nanocarrier, and the differentiation was induced and cultured simultaneously.

[0131] (11) Preparation method of monoemulsion nanocarrier in the control group: 1200 ng NeuroD1 plasmid DNA and 600 ng siSOX9-mCherry plasmid DNA were dissolved in an inner aqueous phase solution (plasmid concentration 120 ng / μL), and the operation method was the same as step (1) of this embodiment. Oil phase preparation: same as step (2) of Example 1. W / O water emulsion preparation: same as steps (5) and (6) of Example 1, to prepare a core nanocarrier encapsulating NeuroD1 plasmid. Ultrafiltration purification: same as step (7) of Example 1, to obtain purified nanocarriers encapsulating NeuroD1 and siSOX9-mCherry plasmid. Resuspension and dispensing: same as step (8) in Example 1.

[0132] See Figure 7 These are immunofluorescence staining comparison images of neural stem cell differentiation regulation using monoemulsion nanocarriers and W / O / W nanocarriers, respectively; among them, Figure 7 Figure A in the diagram represents the control group, which was transfected with an equal amount of plasmid using the single emulsion nanocarrier in step (11) of this embodiment. Figure 7Figure B in the diagram represents the experimental group, which was transfected with an equal amount of plasmid using the nanocarrier prepared in this embodiment. Both groups underwent differentiation induction culture simultaneously. After 7 days of induction differentiation, there were significant differences in the differentiation phenotypes of neural stem cells in each group. Comparison between Figures A and B shows that the control group had a large number of GFAP-positive astrocytes, which were widely distributed and exhibited a typical astral morphology with enlarged cell bodies. Tuj1-positive neurons were few in number, with short and sparse processes, and their differentiation tendency was mainly astrocytes. Furthermore, quantitative analysis showed that the proportion of Tuj1-positive neurons in the control group was only 18.3%, while the proportion of GFAP-positive astrocytes was relatively high. Hoechst nuclear staining results showed that the cells in both groups were evenly distributed, morphologically intact, and showed no obvious signs of apoptosis or necrosis, indicating that the time-release nucleic acid delivery vector provided in this embodiment has good biocompatibility.

[0133] The above results confirm that the time-release nucleic acid delivery vector provided in this embodiment can effectively inhibit the non-specific differentiation of neural stem cells into astrocytes by precisely regulating the time-series expression of the target gene, while significantly promoting their directed differentiation into functional neurons, providing an efficient and safe delivery strategy for the directed induction of neural stem cells in the repair of central nervous system injuries.

[0134] Example 5

[0135] This embodiment uses the W / O / W dual nucleic acid nanocarrier constructed in Example 4 to verify the local delivery efficiency, retention capacity, and in vivo expression of the carrier in a mouse skin wound model, providing a visual tracing basis for subsequent research on functional gene delivery and wound repair.

[0136] Specifically, the preparation method of the mouse skin wound healing dual-gene time-sequential delivery vector transfection is as follows:

[0137] (1) Dissolve 1200 ng of angiogenesis factor (VEGF) plasmid DNA (purchased from Addgene, plasmid number: 200947) in sterile ultrapure water and make up to a total volume of 10 μL to obtain an inner aqueous phase solution A (plasmid concentration 120 ng / μL).

[0138] (2) Oil phase preparation: Same as step (3) in Example 1.

[0139] (3) Preparation of external water phase: Same as step (4) in Example 1.

[0140] (4) Preparation of W / O colostrum and W / O / W double emulsion: Same as steps (5)-(6) in Example 1, to prepare the core nanocarrier encapsulating NeuroD1 plasmid.

[0141] (5) Ultrafiltration purification: Same as step (7) in Example 1, to obtain purified nanocarriers encapsulating NeuroD1 plasmids.

[0142] (6) The purified nanocarrier was mixed with the inflammatory factor gene (IL-1, purchased from Addgene, plasmid number: 214341) and incubated at room temperature for 30 min, so that the IL-1 plasmid was quantitatively adsorbed onto the surface of the carrier through electrostatic interaction, thus obtaining a dual-gene time-release vector carrying IL-1 (surface) and VEGF (inner layer).

[0143] (7) Resuspension and repackaging: Same as step (8) in Example 1.

[0144] Specifically, 8-week-old male C57BL / 6 mice were selected as experimental subjects. After isoflurane inhalation anesthesia, the backs were shaved, dehaired, and disinfected with iodine. Two 6 mm diameter shear wounds were created on the back of each mouse, using the midline of the back as the axis. Mice were randomly divided into two groups (n=10 per group): a control group (CK group, simultaneously expressing inflammatory cytokine plasmid (IL-1) and angiogenesis factor (VEGF)) and a W / O / W nanocarrier group (Medicine group, with W / O / W nanoparticles adsorbing the inflammatory cytokine gene (IL-1) and W / O / W nanoparticles encapsulating VEGF; IL-1 expression was prioritized to promote inflammation, followed by VEGF to promote angiogenesis). 20 μL of the corresponding solution was injected intradermally at four sites in the skin surrounding the wounds. Wound photographs were taken every two days. The wound area was calculated using ImageJ software and compared with the initial area on day 0. Statistical analysis was performed using GraphPad 10.0 software.

[0145] The results show:

[0146] See Figure 8 The images show a comparison of the healing morphology of mouse wounds after treatment with Wow nanocarriers at different times. In Figure A, the images show the macroscopic morphology of mouse wounds after treatment with Wow nanocarriers at different times. In Figure B, the images show a comparison of the quantitative statistical results of the change in residual wound area over time between the control group and the W / O / W nanocarrier group.

[0147] On day 0 after modeling, there was no statistically significant difference in wound area between the two groups, indicating good homogeneity of the model construction. As the observation period lengthened, the wound area of ​​both groups showed a decreasing trend, but the wound healing rate of the W / O / W nanocarrier group (Medicine group) of this invention was significantly faster than that of the control group (CK group).

[0148] Morphological observation showed that the wound healing process in the control group was slow, with obvious unclosed wound tissue still visible on day 7, and delayed drying and scab formation. In contrast, the wound shrinkage in the carrier group of this invention was significantly accelerated, showing significant wound reduction as early as day 3, and by day 7 the wound was almost completely epithelialized, with only light red new tissue remaining, and no obvious signs of inflammatory exudation or infection. Quantitative analysis showed that on day 7, the residual wound area in the carrier group of this invention was close to 0 mm², while the control group still had approximately 5.0 mm². 2 The wound was not closed.

[0149] The above results confirm that the W / O / W nanocarrier provided by this invention can precisely match the physiological process of wound healing through a time-sequential release pattern of adsorbing IL-1 inflammatory factor genes on the surface and encapsulating VEGF angiogenesis factor genes in the inner layer: early moderate expression of IL-1 mediates the inflammatory response, providing a basis for the initiation of the subsequent repair process; late slow release of VEGF promotes angiogenesis, avoiding the antagonism between inflammatory signals and pro-angiogenesis signals, thereby significantly accelerating the wound contraction and epithelialization process, achieving efficient synergistic regulation of wound healing, and providing a more efficient technical solution for stem cell gene therapy of central nervous system diseases.

[0150] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims.

Claims

1. A delivery vector with nucleic acid timing release function, characterized in that, It has a multi-layered emulsion structure consisting of water in oil and water in water; The multiple emulsion structure, from the inside out, includes: The inner aqueous phase layer contains an aqueous solution of nucleic acid molecules; The intermediate oil phase layer contains an active targeted nucleic acid delivery polymer and an amphiphilic block copolymer; The outer aqueous phase layer contains amphiphilic block copolymers; The active targeted nucleic acid delivery polymer includes cationic polymers and hydrophobic backbone polymers; The hydrophobic segments of the amphiphilic block copolymer are distributed in the intermediate oil phase layer, and the hydrophilic segments of the amphiphilic block copolymer are distributed in the outer aqueous phase layer. The cationic polymer is protonated under physiological pH conditions to carry a positive charge, and forms a dense polyelectrolyte complex with negatively charged nucleic acid molecules through electrostatic interaction. The hydrophobic backbone polymer and the hydrophobic segments of the amphiphilic block copolymer self-assemble to form a hydrophobic shell, which encapsulates the polyelectrolyte complex inside the carrier. The surface of the delivery carrier adsorbs nucleic acid molecules through electrostatic interaction, causing the nucleic acid molecules to form a gradient distribution in the multiple emulsion structure. After the nucleic acid is delivered into the cell, the nucleic acid molecules on the surface of the delivery carrier are released first, and then the nucleic acid molecules in the middle oil phase layer and the inner aqueous phase are released in sequence, thereby realizing the time-sequential regulation of nucleic acid or protein expression. The cationic polymer is poly-β-amino ester PBAE, whose tertiary amine groups on the molecular chain are protonated under physiological pH conditions to carry a positive charge, forming a polyelectrolyte complex with negatively charged nucleic acid molecules. The hydrophobic backbone polymer is polylactic acid-glycolic acid copolymer (PLGA). The amphiphilic block copolymer is polyethylene glycol-polycaprolactone block copolymer (PEG-PCL).

2. The delivery vector with nucleic acid timing release function according to claim 1, characterized in that, The molecular weight of PBAE is 5000~15000 Da; And / or, the molar ratio of lactic acid to glycolic acid monomers in PLGA is 50:50, and the molecular weight is 10,000~30,000 Da; And / or, in PEG-PCL, the molecular weight of the PEG segment is 2000~5000 Da, and the molecular weight of the PCL segment is 4000~10000 Da.

3. The delivery vector with nucleic acid timing release function according to any one of claims 1-2, characterized in that, The nucleic acid molecules include any one or a combination of two or more of plasmid DNA, siRNA, mRNA, and antisense oligonucleotides.

4. The delivery vector with nucleic acid timing release function according to any one of claims 1-2, characterized in that, Its surface carries a positive charge, with an average particle size of 150±20 nm; a polydispersity index (PDI) of 0.15±0.05, and a zeta potential of +25±5 mV.

5. A method for preparing a delivery vector with nucleic acid timing release function as described in any one of claims 1-4, characterized in that, The preparation steps include: S1. Dissolve nucleic acid molecules in sterile water to prepare an internal aqueous phase solution, denoted as solution A; S2. Dissolve the active targeted delivery polymer and the amphiphilic block copolymer in an organic solvent to obtain an oil phase solution, denoted as solution B; S3. Dissolve the amphiphilic block copolymer in sterile water to obtain an external aqueous solution, denoted as solution C; S4. Under ultrasonic conditions, solution A is slowly added to solution B, and a single emulsion is formed to obtain a W / O primary emulsion, denoted as solution D. S5. Under ultrasonic conditions, solution D is slowly added to solution C, and secondary emulsification is performed to obtain a crude W / O / W double emulsion, denoted as solution E. S6. Purify solution E to obtain the delivery vector with nucleic acid time-sequential release function.

6. The preparation method according to claim 5, characterized in that, In S2, the organic solvent is any one or a combination of two or more of tetrahydrofuran, dichloromethane, trichloromethane, and dimethyl sulfoxide; In S4, the volume ratio of solution A to solution B is 1:(20~100). The rate at which solution A is added to solution B is 10~50 μL / min; The method of primary emulsification includes any one of ultrasonic dispersion, microfluidic method, ultrasonic emulsification method or mechanical stirring method.

7. The preparation method according to claim 5, characterized in that, In S5, the volume ratio of solution D to solution C is 1:(5~20). The rate at which solution D is added to solution C is 100~500 μL / min; The secondary emulsification method includes any one of ultrasonic dispersion, microfluidic method, ultrasonic emulsification or mechanical stirring method; In S6, the purification process is ultrafiltration purification, using ultrafiltration centrifuge tubes with a molecular weight cutoff of 30~100 kDa and a centrifugation speed of 8,000~12,000 rpm.

8. A dual-gene time-series delivery vector, characterized in that, include: The delivery carrier having a nucleic acid time-sequential release function as described in any one of claims 1-4, and a second nucleic acid molecule adsorbed on the surface of the delivery carrier by electrostatic interaction; The delivery vector with nucleic acid sequential release function is loaded with a first nucleic acid molecule; The delivery vector with nucleic acid timing release function is mixed with the second nucleic acid molecule and then incubated to obtain the dual-gene timing delivery vector; The first nucleic acid molecule and the second nucleic acid molecule may be the same or different; The first nucleic acid molecule and the second nucleic acid molecule are each selected from any one or a combination of two or more of plasmid DNA, siRNA, mRNA, and antisense oligonucleotides.

9. The use of a delivery vector having nucleic acid timing release function as described in any one of claims 1-4, or the dual-gene timing delivery vector as described in claim 8, in the preparation of a medicament for treating neural stem cell gene diseases.

Images