Preparation method of cell membrane-based nucleic acid drug-loaded biological vesicle inhalation preparation as well as product and application of cell membrane-based nucleic acid drug-loaded biological vesicle inhalation preparation

Abstract

The invention discloses a preparation method of a cell membrane-based nucleic acid drug-loaded biological vesicle inhalation preparation. The preparation method comprises the following steps: preparing target cells into a cell suspension; repeatedly extruding the cell suspension through an extrusion device to obtain extrusion liquid; carrying out differential centrifugation on the extruded liquid, and collecting the biological vesicles; loading a nucleic acid drug into the biological vesicles; wherein the differential centrifugation comprises two centrifugation steps, and the centrifugation conditions are as follows: 1) the centrifugal force is 2,000-10,000 g, and the centrifugation time is 10-60 minutes; and (2) the centrifugal force is 16,000 to 100,000 g, and the centrifugal time is 10 to 120 minutes. The invention also discloses application of the cell membrane-based nucleic acid drug-loaded biological vesicle inhalation preparation obtained by the preparation method in preparation of drugs for treating lung diseases. The nucleic acid drug-loaded biological vesicle inhalation preparation prepared by the invention can be used for efficiently loading a nucleic acid drug and efficiently delivering the nucleic acid drug to the lung through atomization administration, and the basic character characterization and the drug-loading capability of the atomized preparation are basically consistent with those of the preparation before atomization.

Description

Technical Field

[0001] The present invention relates to the field of drug delivery preparations, and in particular to a preparation method of a cell membrane-based nucleic acid drug-carrying biological vesicle inhalation preparation, and a product and application thereof. Background Art

[0002] Inhalation administration is widely used as a clinical administration route for lung diseases due to its directness and locality. For example, asthma is mainly treated by inhaling steroidal anti-inflammatory drugs and airway dilators. The advantage of inhalation administration is that the drug can accurately reach the lung lesions and take effect quickly, while significantly reducing systemic side effects. However, inhalation administration still faces some challenges, including how to more effectively protect the drug from the influence of atomization shear force and environmental factors, how to prolong the residence time of the drug in the lungs to enhance the efficacy, and how to improve the biocompatibility of the drug with the human body to ensure the safety and effectiveness of the treatment.

[0003] In recent years, more and more studies have confirmed that nucleic acid therapy offers the potential to treat diseases that are difficult to reach with traditional drugs, expanding the scope of treatment, including genetic diseases, rare diseases, and chronic diseases. Nucleic acid therapy includes a variety of RNA therapeutics, such as antisense oligonucleotides (ASO), mRNA vaccines, small interfering RNA (siRNA), microRNA (miRNA), aptamers, and small activating RNA (saRNA). Among them, siRNA therapy is suitable for a variety of lung diseases due to its high precision, wide target range, and reduced drug resistance. However, to date, no inhaled siRNA therapy has been approved for clinical use. This is because siRNA is difficult to withstand the severe shear force generated during the atomization process, is easily degraded by nucleases in the environment, and lacks good biocompatibility. Therefore, how to construct an efficient delivery system to improve the stability of nucleic acid drugs, promote the targeted cell uptake of nucleic acid molecules, and help them escape from cells has become a crucial part of the application of nucleic acid therapy in lung diseases.

[0004] Extruded vesicles based on cell membranes have good biocompatibility, low immunogenicity and low toxicity. Compared with extracellular vesicles secreted by cells, extruded vesicles have higher yields, a relatively shorter production process and a simpler process. Therefore, they have the potential to become good nucleic acid drug carriers. However, after the solution of nucleic acid drug-carrying biological vesicles is atomized into an aerosol, whether the particle size and potential of the biological vesicles change; whether the labeled proteins on the surface of the biological vesicles are retained; whether the nucleic acid loading capacity of the biological vesicles changes; and whether the ability of the biological vesicles to promote the cellular uptake of nucleic acid molecules has changed have not yet been explored. This is crucial for the preparation of inhalation preparations of nucleic acid drug-carrying biological vesicles, and is an important part of nucleic acid therapy based on the biological vesicle delivery platform in the inhalation administration of lung diseases. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing a cell membrane-based biological vesicle inhalation preparation containing nucleic acid drugs. The prepared biological vesicle inhalation preparation containing nucleic acid drugs can efficiently load nucleic acid drugs and be efficiently delivered to the lungs through aerosol administration, and the basic properties and drug loading capacity of the preparation after aerosolization are basically consistent with those before aerosolization.

[0006] The present invention provides the following technical solutions to achieve the above-mentioned purpose: A method for preparing a cell membrane-based nucleic acid drug-carrying biological vesicle inhalation preparation, the preparation method comprising the following steps: (1) Prepare target cells into cell suspension; (2) repeatedly extruding the cell suspension through an extrusion device to obtain an extrudate; (3) performing differential centrifugation on the extrudate to collect the biological vesicles; (4) loading the nucleic acid drug into the biological vesicle obtained in step (3); Wherein, in step (3), the differential centrifugation includes two centrifugation steps, and the centrifugation conditions are: centrifugal force of 2,000-10,000 g, centrifugation time of 10-60 min; centrifugal force of 16,000-100,000 g, centrifugation time of 10-120 min.

[0007] In step (1), the target cells are derived from human cells or non-human mammalian cells.

[0008] In step (1), the target cells are derived from stem cells or immune cells that have an immunomodulatory response or an inflammatory tendency.

[0009] In step (1), the source of the target cells is stem cells.

[0010] In step (1), the target cells are resuspended in a buffer system. The buffer system needs to be non-toxic to the cells, not affect the basic functions of the cells, and not damage the biological activity of the vesicles.

[0011] In step (2), the pore size of the filter hole of the extrusion device is 0.4-10 μm, and the number of extrusions is 1-20 times. The pore size of the filter hole is preferably 5 μm, and the number of repeated extrusions is preferably 20 times.

[0012] In step (3), the purpose of the first centrifugation step is to separate cell debris and apoptotic bodies, and the purpose of the second centrifugation step is to purify and collect biological vesicles. The centrifugation conditions are preferably: centrifugation at a centrifugal force of 2,000 g for 30 min to separate cell debris and apoptotic bodies, and centrifugation at a centrifugal force of 100,000 g for 60 min to purify and collect biological vesicles.

[0013] In step (4), the nucleic acid drug is selected from antisense oligonucleotides (ASO), small interfering RNA (siRNA), nucleic acid aptamers, messenger RNA (mRNA), micro RNA (miRNA), self-amplifying RNA (saRNA), CRISPR / Cas9, etc.

[0014] Furthermore, the differential centrifugation needs to be performed under low temperature conditions to protect the morphology and activity of the biological vesicles, preferably at 4°C.

[0015] The present invention also provides a cell membrane-based nucleic acid drug-carrying biological vesicle inhalation preparation obtained by the above preparation method. The average particle size of the biological vesicle is 30-500 nm. Preferably, the average particle size is 100-250 nm. The biological vesicle is resuspended in a buffer solution and then atomized for administration, which can be achieved for any atomization form for aqueous solution, so it is not limited.

[0016] The present invention also provides an application of the above-mentioned cell membrane-based nucleic acid drug-carrying biological vesicle inhalation preparation in the preparation of drugs for treating lung diseases.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention achieves process optimization of extruded vesicles by exploring several key steps of the extrusion process. While ensuring basic characterizations such as morphology and potential, it greatly reduces the complexity of production and improves the yield, and gives full play to the yield advantage of extruded vesicles compared to exosomes, thereby achieving control of vesicle particle size and maximization of yield. In the cell membrane-based nucleic acid drug-carrying biovesicle inhalation preparation prepared by the present invention: the biovesicles can not only inherit the characteristics of the source cells and exert corresponding physiological effects, but also can load drugs to achieve efficient drug delivery; the preparation after atomization is basically consistent with that before atomization in morphology, size, potential and particle size distribution dispersion (PDI), and the marker protein on the membrane surface is also well retained; there is no significant difference in the loading amount of nucleic acid in the biovesicles after atomization and the ability of the biovesicles to promote the cellular uptake of nucleic acid molecules; The exogenous nucleic acid drug carried by the cell membrane-based nucleic acid drug-loaded biological vesicle inhalation preparation prepared by the present invention can synergistically treat lung diseases with the immunomodulatory effect of the vesicle itself, can be administered by inhalation (atomization), and can be further applied to scientific research and clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A schematic diagram of the preparation method of the cell membrane-based nucleic acid drug-carrying biological vesicle inhalation preparation provided by the present invention; Figure 2The yield, particle size and PDI of the biovesicles prepared in Example 1 using filter membranes with different pore sizes, different extrusion times and different centrifugation conditions; Figure 3 Transmission electron microscope images of the biological vesicles prepared in Example 2 before and after atomization; Figure 4 The potential, particle size and particle size distribution of the biological vesicles prepared in Example 2 before and after atomization; Figure 5 The changes of surface marker proteins of the biological vesicles prepared in Example 2 before and after atomization; Figure 6 This is a statistical diagram of the loading of Cy5-siRNA into the biological vesicles prepared in Example 2 before and after atomization; Figure 7 This is a diagram showing the nucleic acid delivery efficiency of the biological vesicles loaded with Cy5-siRNA to in vitro airway epithelial cells before and after atomization prepared in Example 2; Figure 8 Fluorescence images of the biological vesicles loaded with Cy5-siRNA prepared in Example 2 and naked Cy5-siRNA being taken up by epithelial cells; Fig. 9 The distribution and retention of the biological vesicles loaded with Cy5-siRNA and naked Cy5-siRNA in the lungs after aerosol administration prepared in Example 2; Fig.10 The therapeutic effect of siTSLP loaded by biological vesicles prepared in Example 2 in the lungs after aerosol inhalation. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with specific embodiments, application examples and accompanying drawings.

[0020] Example 1: Preparation of biological vesicle inhalation preparation Taking human umbilical cord blood-derived mesenchymal stem cells (hMSCs) as an example, Figure 1 The biological vesicles are prepared by the preparation method shown, and the specific steps are as follows: 1) hMSCs (provided by Ningbo Sinocell Biotechnology Co., Ltd., ethics approval number 2021-R108) were cultured to a cell density of 70-80%, the cells were digested with trypsin, and the cells were resuspended in phosphate buffered saline (PBS) without calcium and magnesium ions; 2) Using an extrusion device with a specific pore structure to extrude the cell suspension, and controlling the cell density to about 1.5 million / mL; 3) The extrudate was subjected to differential centrifugation to collect the biological vesicles, which were recorded as MSC-NVs.

[0021] In this example, the pore size, extrusion times and differential centrifugation conditions are as follows: Figure 2 As shown, the number of particles in each group was obtained by nanoflow analysis, and the particle size and PDI of each group were obtained by dynamic light scattering (DLS) analysis; the average particle size obtained by DLS detection shows that the particle size is within the range of aerosol administration. Combining the yield and PDI, the most preferred conditions are: passing through a 5 μm pore size filter membrane, extruding 20 times, centrifuging at 2,000 g to remove cell debris, and collecting biological vesicles at 100,000 g.

[0022] Example 2: Preparation of a biological vesicle inhalation preparation containing nucleic acid drugs Taking hMSCs as an example, Figure 1 The preparation method shown is used to prepare a cell membrane-based drug-loaded biological vesicle inhalation preparation, and the specific steps are as follows: 1) hMSCs were cultured to a cell density of 70-80%, the cells were digested with trypsin, and the cells were resuspended in PBS without calcium and magnesium ions; 2) Repeatedly extrude the cell suspension 20 times using an extrusion device with a specific pore structure (pore size: 5 μm); 3) Collect the extrudate and centrifuge at 2000 g for 30 min at 4 °C, discard the precipitate to remove most of the cell debris and apoptotic bodies; 4) Take the supernatant and centrifuge at 100,000 g for 60 min to precipitate the biovesicles; 5) Load siRNA into vesicles using transfection reagent (Exo-FectTM, Exosome Transfection Kit, SBI).

[0023] The biocapsule prepared in Example 2 was subjected to pressure atomization and then tested and characterized, including: The morphology of the biological vesicles before and after atomization was photographed by transmission electron microscopy. Figure 3 It can be seen that the “cup-shaped” morphology of the vesicles is retained after atomization; The potential, average particle size and PDI of the biovesicles before and after atomization were measured by a dynamic light scattering instrument. Figure 4 It can be seen that the potential, particle size, and PDI of the vesicles after atomization have no significant differences from those before atomization; The changes of the surface marker proteins of biological vesicles before and after atomization were detected by Western blotting. Figure 5 It can be seen that the marker proteins (CD9, TSG101, CD63, and CD105) on the surface of the vesicles are well preserved after nebulization; The biological vesicles loaded with Cy5-siRNA before and after atomization were purified by centrifugation again, and the obtained biological vesicles were resuspended and the fluorescence intensity was detected under a fluorescence microplate reader. Figure 6 It can be seen that there is no significant difference in the Cy5-siRNA loading capacity of biological vesicles after atomization; The biovesicles loaded with Cy5-siRNA before and after atomization were inoculated into a 6-well plate pre-plated with BEAS-2B cells (2×10 5 The cells were digested and collected, and the proportion of Cy5-positive cells in the total cell population was analyzed and quantified by flow cytometry. Figure 7 It can be seen that the biological vesicles loaded with Cy5-siRNA provided by the present invention can achieve nucleic acid delivery to cells, and the delivery efficiency is not affected by the atomization process.

[0024] Application Example 1: Cell membrane-based biovesicle inhalation preparation for in vitro cellular delivery of nucleic acid drug siRNA Taking human BEAS-2B cells as an example, the biological vesicles loaded with siRNA prepared in Example 2 were used to investigate the delivery of siRNA to in vitro airway epithelial cells under the protection and delivery of biological vesicles. The siRNA was fluorescently labeled with Cy5. The naked Cy5-siRNA and the Cy5-siRNA encapsulated by the biological vesicles (referred to as NVs-Cy5-siRNA) were inoculated in confocal dishes pre-plated with epithelial cells (1×105). The cells were co-incubated and cultured for 6 h at 5% CO2 and 37°C. The culture solution was discarded, stained with lysosomal dye (Lyso-Tracker Green) for 30 min, the dye was discarded, washed three times with PBS, and then stained with nuclear dye (Hoechst). After 10 min, it was also washed three times with PBS, and the delivery of nucleic acid from biological vesicles loaded with Cy5-siRNA to epithelial cells was observed and photographed under a confocal microscope.

[0025] Depend on Figure 8 It can be seen that naked siRNA cannot be taken up by cells or cannot pass through the cell membrane into the cell, while biological vesicles loaded with siRNA can efficiently deliver siRNA to epithelial cells.

[0026] Application Example 2: Cell membrane-based biovesicle inhalation formulations can be used for pulmonary delivery Taking the ovalbumin-induced mouse asthma model as an example, the residence of the biological vesicles loaded with siRNA prepared in Example 2 in the lungs of asthmatic mice was investigated. Female BALB / C mice weighing 18 to 22 g were sensitized by intraperitoneal injection of 80 μg of ovalbumin mixed with immune adjuvant on days 0 and 14, and an asthma mouse model was constructed by aerosol inhalation of 5% ovalbumin on days 21-23. The siRNA was labeled with Cy5, and the naked Cy5-siRNA and the biological vesicles loaded with Cy5-siRNA were diluted to the same fluorescence intensity after fluorescence quantification and administered to asthmatic mice by pressure atomization. The mice were euthanized by cervical dislocation at 4 h, 24 h, and 48 h after administration. Subsequently, the main organs of the mice (heart, liver, spleen, lung, and kidney) were removed, and the Cy5 fluorescence signal intensity in each organ was analyzed using a small animal in vivo imaging system to determine the distribution of Cy5-siRNA in each organ.

[0027] Depend on Fig. 9 It can be seen that 48 hours after nebulization, the Cy5 fluorescence signal in the lungs of mice in the naked siRNA group was significantly reduced. In contrast, there was still an obvious Cy5 fluorescence signal in the lungs of mice in the biological vesicle group loaded with Cy5-siRNA, indicating that the biological vesicles loaded with siRNA described in the present invention can effectively prolong the residence time of siRNA in the lungs of asthmatic mice.

[0028] Application Example 3: Cell membrane-based drug-loaded biovesicles can achieve precise treatment of asthma Taking the ovalbumin-induced mouse asthma model as an example, the biological vesicle inhalation preparation (referred to as MSC-NVs-siTSLP) loaded with siRNA (siTSLP for the asthma inflammatory factor thymic stromal lymphopoietin, i.e., TSLP) prepared in Example 2 was used to downregulate the pulmonary inflammatory factors of asthmatic mice. Female BALB / C mice weighing 18 to 22 g were sensitized by intraperitoneal injection of 80 μg of ovalbumin mixed with immune adjuvant on days 0 and 14, and an asthma mouse model was constructed by aerosol inhalation of 5% ovalbumin on days 21-23. On the 21st day, PBS and 20 μg of MSC-NVs / MSC-NVs-siNC / MSC-NVs-siTSLP were inhaled by aerosol respectively. On the 23rd day, the mice were euthanized within 24 h after nebulization challenge, and the bronchoalveolar lavage fluid was collected and centrifuged at 2,800 rpm. The supernatant was collected and the levels of thymic stromal lymphopoietin (TSLP), interleukin-4 (IL-4), and interleukin-13 (IL-13) were detected by enzyme-linked immunosorbent assay.

[0029] Depend on Fig.10It was found that the level of TSLP was significantly downregulated, and the downstream inflammatory factors IL-4 and IL-13 of TSLP were also downregulated synchronously, indicating that the delivery of siTSLP to airway epithelial cells and the silencing effect were successfully achieved by encapsulating siTSLP in biological vesicles.

Claims

1. A method for preparing a cell membrane-based nucleic acid drug-carrying biological vesicle inhalation preparation, characterized in that: The preparation method comprises the following steps: (1) Prepare target cells into cell suspension; (2) repeatedly extruding the cell suspension through an extrusion device to obtain an extrudate; (3) performing differential centrifugation on the extrudate to collect the biological vesicles; (4) loading the nucleic acid drug into the biological vesicle obtained in step (3); Wherein, in step (3), the differential centrifugation includes two centrifugation steps, and the centrifugation conditions are: centrifugal force of 2,000-10,000 g, centrifugation time of 10-60 min; centrifugal force of 16,000-100,000 g, centrifugation time of 10-120 min.

2. The method for preparing the cell membrane-based nucleic acid drug-carrying biological vesicle inhalation preparation according to claim 1, characterized in that: The target cells are derived from human cells or non-human mammalian cells.

3. The method for preparing the cell membrane-based nucleic acid drug-carrying biological vesicle inhalation preparation according to claim 1, characterized in that: The target cells are derived from stem cells or immune cells with immunomodulatory ability or inflammatory tropism.

4. The method for preparing the cell membrane-based nucleic acid drug-carrying biological vesicle inhalation preparation according to claim 1, characterized in that: In step (2), the pore size of the filter pores of the extrusion device is 0.4-10 μm, and the number of extrusions is 1-20 times.

5. The method for preparing the cell membrane-based nucleic acid drug-carrying biological vesicle inhalation preparation according to claim 1, characterized in that: In step (3), the differential centrifugation includes two centrifugation steps, and the centrifugation conditions are: centrifugal force of 2,000 g, centrifugal time of 30 min and centrifugal force of 100,000 g, centrifugal time of 60 min.

6. The method for preparing the cell membrane-based nucleic acid drug-carrying biological vesicle inhalation preparation according to claim 5, characterized in that: In step (4), the nucleic acid drug is selected from antisense oligonucleotides, small interfering RNA, nucleic acid aptamers, messenger RNA, microRNA, self-amplifying RNA or CRISPR / Cas9.

7. A cell membrane-based nucleic acid drug-loaded biological vesicle inhalation preparation obtained according to the preparation method of any one of claims 1 to 6.

8. The cell membrane-based nucleic acid drug-carrying biological vesicle inhalation preparation according to claim 7, characterized in that: The particle size of the biological vesicle is 30-500 nm.

9. Use of the cell membrane-based nucleic acid drug-loaded biological vesicle inhalation preparation according to claim 7 in the preparation of drugs for treating lung diseases.

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