Engineered nanoparticles for regulating obesity-related ion channel and preparation method and application method thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to engineered nanoparticles for regulating and controlling obesity-related ion channels and a preparation method and application method thereof, and the engineered nanoparticles are composed of brown adipose cell membrane vesicles, water-soluble embedded membrane conjugated oligomerization electrolyte, amphiphilic molecules and Aifm2 plasmids. According to the engineered nanoparticles for regulating and controlling the obesity-related ion channel, by catalyzing conversion from NADH to NAD < + >, accelerating electron transfer, promoting glycolysis and regulating and controlling the cell membrane ion channel function, intracellular Ca < 2 + > signal transduction is affected, UCP1 heat production is promoted, energy consumption is achieved, and BAT non-trembling heat production is enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an engineered nanoparticle for regulating obesity-related ion channels, and a preparation method and an application method thereof. Background Art

[0002] Obesity, as the basic pathological feature of many metabolic diseases, is a major hidden danger to global public health. In response to this challenge, promoting the thermogenic effect of uncoupling protein 1 (UCP1) and activating the thermogenic potential of brown adipose tissue (BAT) are key strategies to regulate obesity by accelerating energy consumption. In this process, targeting ion channels can help promote the glycolysis process and finely regulate the signaling pathways closely related to obesity, which is of far-reaching significance for in-depth revelation of the thermogenic mechanism of UCP1.

[0003] Intracellular calcium ions (Ca 2 There is a close relationship between the steady state of Ca + concentration and the body's heat production capacity. 2 + signal transduction plays a key regulatory role in targeting UCP1, regulating obesity and related metabolic processes. 2 The fine regulation of Ca levels depends on the Ca2+ on the cell membrane and various organelles 2 + ion channels, among which transient receptor potential (TRP) ion channels, especially TRPV1 ion channels, act as Ca 2 + signal transduction mediator, capable of responding to external stimuli and regulating Ca 2 + signal, and therefore has attracted much attention in the field of obesity and its related metabolism. By activating TRPV1 ion channels highly expressed on the BAT cell membrane, it promotes Ca 2 + influx is an effective strategy to combat diet-induced obesity by enhancing metabolism and increasing energy expenditure. 2 + Targeting the TRPV1 ion channel related to signal transduction and synergistically regulating its related signaling pathways is of great significance in the treatment of obesity and related metabolic diseases caused by promoting UCP1 heat production.

[0004] In the exploration of targeting disease-related proteins such as ion channels to regulate cell physiological functions, organic conjugated molecules have shown significant advantages due to their excellent photophysical properties endowed by their unique π-electron conjugated skeleton. However, organic conjugated molecules still face challenges in interacting well with cells. In order to enhance their biomimetic functions and lesion targeting capabilities, nanomedicines based on cell membrane engineering have shown broad application prospects. The core of building a cell membrane engineering molecular platform is to design membrane-embedded conjugated oligoelectrolytes. These molecules are composed of a hydrophobic conjugated main chain and polar ionic side groups. The distribution of their hydrophobic and hydrophilic groups is similar to that of phospholipid bilayers. They can spontaneously embed into the cell membrane through electrostatic and hydrophobic interactions. By adjusting their chemical structure, their interactions with the cell membrane can be controlled, thereby achieving applications with different functions. Furthermore, by combining advanced technologies such as lipid insertion, chemical conjugation, metabolic engineering and genetic engineering to modify the cell membrane, it is possible to endow cell membrane-engineered nanoparticles with specific biological application functions based on membrane-embedded conjugated oligoelectrolytes. These cell membrane engineered nanoparticles, with their excellent design flexibility and biocompatibility, provide an attractive option for addressing major challenges in the biomedical field, and also provide an innovative solution strategy for in-depth analysis of the complex mechanism of UCP1 heat production during obesity metabolism. Summary of the invention

[0005] The purpose of the present invention is to provide an engineered nanoparticle for regulating obesity-related ion channels and a preparation method thereof in order to solve at least one of the above technical problems.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] An engineered nanoparticle for regulating obesity-related ion channels, the nanoparticle is composed of brown adipocyte membrane vesicles, water-soluble membrane-embedded conjugated oligoelectrolytes, amphiphilic molecules and Aifm2 plasmids.

[0008] Furthermore, the water-soluble membrane-inserted conjugated oligoelectrolyte is an organic conjugated molecule composed of a hydrophobic conjugated main chain and polar ionic side groups, and the distribution of its hydrophobic groups and hydrophilic groups is similar to that of a phospholipid bilayer.

[0009] A method for preparing engineered nanoparticles for regulating obesity-related ion channels comprises the following steps:

[0010] Produce target cell membrane vesicles;

[0011] The target cell membrane vesicles and the water-soluble membrane-embedded conjugated oligoelectrolyte are mixed, and stirred at room temperature to embed the water-soluble membrane-embedded conjugated oligoelectrolyte into the target cell membrane vesicle carrier to obtain solution A;

[0012] Solution A is mixed with the amphiphilic molecule solution and stirred, and then ultrafiltered and concentrated to obtain solution B;

[0013] Solution B was mixed with an equal volume of the target plasmid and vortexed for 15 seconds, and allowed to stand for 30 minutes to prepare the final target cell membrane engineered nanoparticle solution; the target plasmid was selected according to the site of application of the engineered nanoparticle.

[0014] Further, preparing target cell membrane vesicles includes:

[0015] Collecting target cells and extracting target cell membranes by differential centrifugation; the target cells are selected according to the site where the engineered nanoparticles are applied;

[0016] The target cell membrane is dispersed in water, and the target cell membrane is squeezed into vesicles using a membrane squeezer to prepare the target cell membrane vesicles.

[0017] Furthermore, the target cells are brown fat cells.

[0018] Furthermore, the water-soluble membrane-inserted conjugated oligoelectrolyte has a linear conjugated skeleton, a flexible chain modified with polar groups at both ends, and contains a quaternary ammonium salt structure at both ends.

[0019] Furthermore, the amphiphilic molecule solution is obtained by the following method:

[0020] The amphiphilic molecule DSPE-PEG2000-PEI25000 is dissolved in an organic solvent to obtain solution C, and solution C is added into water and ultrasonicated for 5-20 minutes. The solution is stirred in the dark while passing argon gas until the organic solvent is completely removed, and impurities are removed by dialyzing using a dialysis bag.

[0021] Furthermore, the organic solvent is one of DMSO, methanol or ethanol.

[0022] Further, when solution C is added to water, the ratio of solution C to water is 1:2-1:10.

[0023] Furthermore, the target plasmid is Aifm2 plasmid.

[0024] A method for applying engineered nanoparticles for regulating obesity-related ion channels, wherein the engineered nanoparticles for regulating obesity-related ion channels prepared by any of the preparation methods described above act on brown fat cells, and the method comprises the following steps:

[0025] Use near-infrared light to activate TRPV1 ion channels;

[0026] Regulates intracellular Ca 2+ concentration, promoting UCP1 thermogenesis.

[0027] The beneficial effects of the present invention are:

[0028] The engineered nanoparticles for regulating obesity-related ion channels and the preparation method thereof provided by the present invention modify chemical molecules to design a series of water-soluble membrane-embedded conjugated oligoelectrolytes with good photophysical properties to achieve stable interaction with cell membranes. Aiming at the characteristics of obesity and related metabolic processes, starting from two major directions, BAT metabolism and non-shivering thermogenesis, cell membrane engineered nanoparticles with uniform size, stable performance, high photothermal conversion efficiency, strong homologous targeting ability, accurate fluorescence imaging positioning and high gene delivery efficiency are constructed.

[0029] The present invention regulates TRPV1-mediated Ca 2+ Signal transduction, promoting UCP1-mediated uncoupled respiratory heat production and catalyzing the conversion of intracellular NADH to NAD + The transformation process promotes mitochondrial electron transfer, accelerates glycolysis, and thus realizes energy consumption.

[0030] The cell membrane engineered nanoparticles with good photophysical properties designed and prepared by the present invention provide a new strategy for precisely regulating TRP channels, regulating intracellular electron transfer, promoting UCP1 heat production in BAT, and regulating obesity and its related metabolic processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a flow chart of a method for preparing engineered nanoparticles for regulating obesity-related ion channels according to one embodiment of the present invention;

[0032] Figure 2 A structural diagram of a water-soluble membrane-intercalated conjugated oligoelectrolyte according to an embodiment of the present invention;

[0033] Figure 3 A COE-BBT synthesis route diagram for one embodiment of the present invention;

[0034] Figure 4 This is a schematic diagram of the preparation of cell membrane engineered nanoparticles in Embodiment 1 of the present invention;

[0035] Figure 5 The oil red staining images of fat cells and the qRT-PCR data analysis of brown fat cells in Example 1 of the present invention;

[0036] Figure 6 The ultraviolet absorption spectrum, membrane embedding image and photothermal temperature rise curve of the cell membrane engineered nanoparticles in Example 1 of the present invention;

[0037] Figure 7This is an immunofluorescence image of the TRPV1 ion channel in Example 2 of the present invention;

[0038] Figure 8 This is a patch clamp data diagram of cell membrane engineered nanoparticles regulating TRP channels in Example 2 of the present invention;

[0039] Fig. 9 The cell membrane engineered nanoparticles in Example 2 of the present invention affect intracellular Ca 2+ Concentration data graph;

[0040] Fig.10 This is a data diagram showing the effect of cell membrane engineered nanoparticles on intracellular electron transfer in Example 3 of the present invention. DETAILED DESCRIPTION

[0041] The content of the present invention will now be discussed with reference to exemplary embodiments. It should be understood that the embodiments discussed are only to enable those skilled in the art to better understand and thus implement the content of the present invention, rather than implying any limitation on the scope of the present invention.

[0042] As used herein, the term “including” and variations thereof are to be interpreted as open-ended terms meaning “including, but not limited to.” The term “based on” is to be interpreted as “based, at least in part, on.” The terms “one embodiment” and “an embodiment” are to be interpreted as “at least one embodiment.”

[0043] Embodiment 1

[0044] According to one embodiment of the present invention, an engineered nanoparticle for regulating obesity-related ion channels is composed of brown adipocyte membrane vesicles, water-soluble membrane-embedded conjugated oligoelectrolytes, amphiphilic molecules and Aifm2 plasmids.

[0045] Preferably, the water-soluble membrane-inserting conjugated oligoelectrolyte is an organic conjugated molecule composed of a hydrophobic conjugated main chain and polar ionic side groups, and the distribution of the hydrophobic group and the hydrophilic group is similar to that of a phospholipid bilayer.

[0046] In this embodiment, an engineered nanoparticle for regulating obesity-related ion channels is mainly composed of four parts: a water-soluble membrane-embedded conjugated oligoelectrolyte, a brown adipocyte membrane vesicle, a positively charged amphiphilic molecule DSPE-PEG2000-PEI25000, and an Aifm2 plasmid. First, the brown adipocyte membrane is converted into a nanoscale cell membrane vesicle carrier by mechanical extrusion technology. The carrier can achieve efficient targeting and long circulation in vivo by virtue of its homologous targeting ability to brown adipocytes. The brown adipocyte membrane vesicle is a vesicle carrier prepared from a cell membrane, which has good homologous targeting properties, can target the target site, and reduce the side effects on normal tissues. Subsequently, a water-soluble membrane-embedded conjugated oligoelectrolyte with a linear conjugated skeleton and a polar ion side group is embedded in the cell membrane vesicle. Its unique structure enables the electrolyte to spontaneously embed into the cell membrane through electrostatic and hydrophobic interactions, thereby regulating its function. Then, the amphiphilic molecule DSPE-PEG2000-PEI25000 was modified onto the surface of cell membrane vesicles embedded with conjugated oligoelectrolytes using lipid membrane fusion technology. This molecule not only enhanced the water solubility and stability of the nanoparticles, but also adsorbed the negatively charged Aifm2 plasmid through its positive charge characteristics. Finally, the prepared cell membrane engineered nanoparticles precisely targeted brown adipocytes under homologous targeting, catalyzing NADH to NAD + The nanoparticles can also photothermally activate TRPV1 ion channels on the cell membrane, thereby affecting the intracellular Ca 2+ Signal transduction promotes UCP1 thermogenesis and significantly enhances the non-shivering thermogenesis of brown adipose tissue (BAT), thereby effectively regulating obesity and its related metabolic processes, showing great potential in the treatment of obesity and the management of related metabolic diseases.

[0047] The present invention proposes an engineered nanoparticle for regulating obesity-related ion channels, which is composed of a water-soluble membrane-embedded conjugated oligoelectrolyte, brown adipocyte membrane vesicles, an amphiphilic molecule DSPE-PEG2000-PEI25000 and an Aifm2 plasmid. It is prepared by mechanical extrusion, electrostatic and hydrophobic embedding, lipid membrane fusion and other technical means, and achieves efficient homologous targeting to brown adipocytes and long circulation in vivo. By promoting the conversion of NADH to NAD + It accelerates electron transfer and glycolysis, and photothermally activates TRPV1 ion channels to promote UCP1 thermogenesis, significantly enhances BAT non-shivering thermogenesis, effectively regulates obesity and related metabolic processes, and shows great potential for obesity treatment and metabolic disease management.

[0048] Figure 1 The flowchart of the method for preparing engineered nanoparticles for regulating obesity-related ion channels according to one embodiment of the present invention is shown in FIG. Figure 1As shown, according to one embodiment of the present invention, a method for preparing engineered nanoparticles for regulating obesity-related ion channels comprises the following steps:

[0049] Step S102, preparing target cell membrane vesicles;

[0050] Step S104, mixing the target cell membrane vesicles with the water-soluble membrane-embedded conjugated oligoelectrolyte, stirring at room temperature, embedding the water-soluble membrane-embedded conjugated oligoelectrolyte into the target cell membrane vesicle carrier, and obtaining a solution A;

[0051] Step S106, mixing solution A with the amphiphilic molecule solution, and concentrating the mixture after ultrafiltration to obtain solution B;

[0052] Step S108, solution B is mixed with an equal volume of the target plasmid and vortexed for 15 seconds, and then allowed to stand for 30 minutes to prepare the final target cell membrane engineered nanoparticle solution; the target plasmid is selected according to the site of application of the engineered nanoparticles.

[0053] Preferably, the target plasmid is Aifm2 plasmid.

[0054] In this embodiment, a method for preparing engineered nanoparticles for regulating obesity-related ion channels utilizes the targeting of cell membrane vesicles, the embedding of water-soluble membrane-embedded conjugated oligoelectrolytes, the stability of amphiphilic molecules, and the coding of target plasmids, and successfully prepares engineered nanoparticles that can accurately deliver genes and promote energy consumption through a series of delicate steps and chemical reactions. These nanoparticles have broad application prospects in the fields of biomedicine, gene therapy, etc.

[0055] In step S102, first, it is necessary to extract the cell membrane from the target cell (such as brown fat cells) and prepare it into cell membrane vesicles by certain technical means (such as physical fragmentation, chemical treatment or biological methods). These vesicles retain the structure and characteristics of the original cell membrane and are the basis for subsequent steps. As a carrier, the cell membrane vesicles can simulate the surface characteristics of the target cell and provide precise targeting for subsequent gene delivery.

[0056] In step S104, the prepared target cell membrane vesicles are mixed with water-soluble membrane-embedded conjugated oligoelectrolytes and stirred at room temperature to obtain solution A. The water-soluble membrane-embedded conjugated oligoelectrolytes can interact with the cell membrane vesicles and embed therein. The water-soluble membrane-embedded conjugated oligoelectrolytes have unique chemical structures and properties, and can interact with the lipid bilayer of the target cell membrane vesicles to achieve embedding. This embedding process not only enhances the stability of the vesicles, but also provides the possibility for subsequent gene loading.

[0057] In step S106, the solution A obtained in the previous step (target cell membrane vesicle carrier solution embedded with water-soluble membrane-embedded conjugated oligoelectrolytes) is mixed and stirred with an amphiphilic molecule solution. The amphiphilic molecule has hydrophilic and hydrophobic ends, which can further stabilize the vesicle structure. After mixing, solution B is obtained by ultrafiltration and concentration. The addition of amphiphilic molecules can further enhance the stability and dispersibility of the vesicles, and ultrafiltration and concentration are used to remove excess solvents and impurities and improve the purity of the solution.

[0058] In step S108, solution B is mixed with an equal volume of target plasmid (such as Aifm2 plasmid), vortexed for 15 seconds, and then left to stand for 30 minutes. In this way, the target plasmid can be loaded into the target cell membrane vesicles by lipid membrane fusion to form the final target cell membrane engineered nanoparticle solution. Vortexing helps the plasmid to mix and contact with the vesicle, while standing allows the plasmid to have enough time to embed into the vesicle through mechanisms such as electrostatic action and hydrophobic action. As a specific gene carrier, the Aifm2 plasmid can encode a protein related to energy metabolism, which can be accurately delivered to the target cells to achieve the effects of accelerating electron transfer, promoting glycolysis, and enhancing the thermogenesis of UCP1.

[0059] Through sophisticated steps and chemical reactions, the present invention utilizes the targeting of cell membrane vesicles, the embedding of water-soluble membrane-embedded conjugated oligoelectrolytes, the stability of amphiphilic molecules, and the coding of Aifm2 plasmids to successfully prepare engineered nanoparticles that can accurately deliver genes and promote energy consumption, providing innovative solutions for the fields of biomedicine and gene therapy, and achieving significant effects such as accelerating electron transfer, promoting glycolysis, and enhancing UCP1 thermogenesis.

[0060] According to one embodiment of the present invention, step S102 includes:

[0061] collecting target cells and extracting target cell membranes by differential centrifugation; the target cells are selected according to the site where the engineered nanoparticles are applied;

[0062] The target cell membrane is dispersed in water, and the target cell membrane is squeezed into vesicles using a membrane squeezer to prepare the target cell membrane vesicles.

[0063] Preferably, the target cells are brown adipocytes.

[0064] In this embodiment, first, cells of different tissues, i.e., target cells, are selected according to the site of application of the nanoparticles, and specific target cells (preferably brown fat cells, because they are closely related to energy metabolism) are collected, and the target cell membranes of these target cells are accurately extracted using differential centrifugation. Differential centrifugation can effectively separate pure cell membranes based on the differences in size and density of cells and their components. Subsequently, these extracted target cell membranes are dispersed in water, and appropriate pressure is applied using a film extruder to squeeze the target cell membranes into a tiny and uniform vesicle structure, thereby preparing target cell membrane vesicles. This process not only ensures that the vesicles retain the characteristics and targeting of the original cell membrane, but also provides an accurate and stable carrier for subsequent gene delivery. The cell membrane vesicles prepared by this method have broad application prospects in the fields of biomedicine and gene therapy, especially in situations where precise gene delivery and regulation of energy metabolism are required.

[0065] The present invention collects target cells from specific tissues (such as brown fat cells), uses differential centrifugation to accurately extract cell membranes, and prepares tiny and uniform cell membrane vesicles through a membrane extruder. These vesicles not only retain the characteristics and targeting of the original cell membrane, but also provide stable and precise carriers for subsequent gene delivery, thereby showing broad application prospects in the fields of biomedicine and gene therapy, especially in the precise delivery of genes and regulation of energy metabolism.

[0066] Figure 2 FIG. 1 is a structural diagram of a water-soluble membrane-inserted conjugated oligoelectrolyte according to an embodiment of the present invention. Figure 2 As shown, according to one embodiment of the present invention, the water-soluble membrane-embedded conjugated oligoelectrolyte in step S104 has a linear conjugated skeleton, a flexible chain modified with polar groups at both ends, and contains a quaternary ammonium salt structure at both ends.

[0067] In this embodiment, a special water-soluble membrane-inserted conjugated oligoelectrolyte is designed and synthesized according to the needs. Its structural characteristics are that it has a linear conjugated skeleton, both ends are modified by flexible chains and carry polar groups, and a quaternary ammonium salt structure is specially introduced at the end to enhance water solubility. This electrolyte is similar to the topological structure of the phospholipid bilayer, and is intended to achieve good interaction with the cell membrane. By finely regulating the donor-acceptor structure and frontier orbital energy levels of the conjugated skeleton, the electrolyte can absorb a spectral range from ultraviolet visible light to near-infrared light. The quaternary ammonium salt structure at both ends not only improves the water solubility of the electrolyte, but also ensures good affinity with water molecules. During the design process, the effects of molecular topology, molecular length, hydrophobic-hydrophilic balance and functional group modification on function are fully considered. By adjusting these parameters, the interaction between the electrolyte and the cell membrane can be precisely controlled. When the length of the electrolyte molecule matches the thickness of the lipid bilayer, it can be stably bound to the cell membrane, providing a solid foundation for realizing different functional applications. The conjugated oligoelectrolyte structure described in this scheme is only an example and is not limited to Figure 4 The specific form shown.

[0068] The water-soluble membrane-embedded conjugated oligoelectrolyte designed by the present invention achieves good interaction with cell membranes and wide spectrum absorption by finely regulating its structure, thus providing a solid foundation for functional applications.

[0069] According to one embodiment of the present invention, the amphiphilic molecule solution in step S106 is obtained by:

[0070] The amphiphilic molecule DSPE-PEG2000-PEI25000 is dissolved in an organic solvent to obtain solution C, and solution C is added into water and ultrasonicated for 5-20 minutes. The solution is stirred in the dark while passing argon gas until the organic solvent is completely removed, and impurities are removed by dialyzing using a dialysis bag.

[0071] Preferably, the organic solvent is DMSO, methanol or ethanol.

[0072] Preferably, when solution C is added to water, the ratio of solution C to water is 1:2-1:10.

[0073] In the present embodiment, the amphiphilic molecule DSPE-PEG2000-PEI25000 is dissolved in a preferred organic solvent (such as DMSO, methanol or ethanol, wherein DMSO is the abbreviation form of dimethyl sulfoxide) to form a solution C. Subsequently, solution C is slowly added to water in a ratio of 1:2 to 1:10, and is ultrasonically treated for 5-20min to promote the uniform dispersion of amphiphilic molecules. Then, the solution is stirred under luminescence conditions, and argon gas is introduced simultaneously to thoroughly remove the residual organic solvent. Finally, the solution is dialyzed using a dialysis bag to effectively remove impurities, thereby obtaining a pure amphiphilic molecule solution. This process ensures the stability and dispersibility of the amphiphilic molecules, and provides a key material basis for the subsequent preparation of engineered nanoparticles.

[0074] The present invention efficiently prepares a stable and pure amphiphilic molecule solution by precisely controlling the dissolution, ultrasound, light-shielding stirring and dialysis steps, laying a solid foundation for the synthesis of engineered nanoparticles.

[0075] According to one embodiment of the present invention, a method for applying engineered nanoparticles for regulating obesity-related ion channels uses any engineered nanoparticle for regulating obesity-related ion channels of the present invention to act on brown fat cells, and the application method comprises the following steps:

[0076] Use near-infrared light to activate TRPV1 ion channels;

[0077] Regulates intracellular Ca 2+ concentration, promoting UCP1 thermogenesis.

[0078] In this embodiment, an application scheme of engineered nanoparticles for regulating obesity-related ion channels is proposed. The scheme uses carefully designed engineered nanoparticles for regulating obesity-related ion channels, and activates photothermal response ion channels such as TRPV1 under near-infrared light irradiation by precisely targeting brown adipose tissue (BAT), thereby regulating obesity-related signaling pathways and promoting thermogenesis of UCP1. The nanoparticles use the photothermal properties of conjugated oligoelectrolytes to heat up and activate TRPV1 ion channels on the BAT cell membrane under the action of near-infrared light, triggering extracellular Ca 2+A large amount of influx activates CaMKII, which then activates AMPK phosphorylation and SIRT-1 phosphorylation, deacetylates PPARγ and PRDM-16 and stabilizes the interaction. At the same time, SIRT-1 activates PGC-1α, transcriptionally activates PPARα, and finally activates UCP1 to significantly enhance the non-shivering thermogenesis of BAT. This scheme reduces the impact on other parts of the body by gradually targeting the cell membrane to the organelle ion channel, not only achieving the coordinated regulation of obesity-related signaling pathways, but also providing a new strategy for the design and preparation of cell membrane engineered nanoparticles with good photophysical properties, opening up new ideas for the effective regulation of obesity and its related metabolic processes.

[0079] The application method of engineered nanoparticles for regulating obesity-related ion channels proposed in the present invention activates TRPV1 ion channels under near-infrared light irradiation by precisely targeting brown adipose tissue and utilizing the photothermal properties of conjugated oligoelectrolytes, thereby achieving coordinated regulation of obesity-related signaling pathways, promoting UCP1 heat production, and effectively reducing the impact on other parts of the body, providing a new strategy for designing and preparing cell membrane engineered nanoparticles with good photophysical properties and precisely regulating obesity and its related metabolic processes.

[0080] Embodiment 2

[0081] The implementation example in this embodiment takes the brown adipocyte vesicle carrier embedded with conjugated oligoelectrolyte COE-BBT as an example to illustrate the preparation method of cell membrane engineered nanoparticles and its regulation of cell metabolism and physiological functions by regulating TRP channels, so as to introduce the patent of the present invention.

[0082] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, biological materials, etc. used in the following examples are all commercially available unless otherwise specified.

[0083] The conjugated oligoelectrolyte COE-BBT used in the following examples is a "wing" of a stilbene, ie, a trialkoxy-substituted stilbene (Compound 1), prepared by the Horner-Wadsworth-Emmons reaction between an aldehyde and a phosphate. Figure 3 A COE-BBT synthesis route diagram of an embodiment of the present invention is shown in FIG. Figure 3 As shown, a multi-step Stille coupling reaction is used to introduce different conjugated components and extend the conjugated skeleton to obtain an intermediate BBT-Br with a DAD structure. Finally, COE-BBT is obtained by replacing the bromine atom using a simplified reaction.

[0084] Example 1: Preparation of cell membrane engineered nanoparticles

[0085] Figure 4Schematic diagram of the preparation of cell membrane engineered nanoparticles in Example 1 of the present invention. Figure 4 As shown, according to one embodiment of the present invention, an engineered nanoparticle for regulating obesity-related ion channels is mainly composed of four parts: a membrane-embedded conjugated oligoelectrolyte, a brown fat cell membrane vesicle, an amphiphilic molecule and an Aifm2 plasmid. The transformed brown fat cell membrane is prepared into a nanoscale cell membrane vesicle carrier by mechanical extrusion technology, and the membrane-embedded conjugated oligoelectrolyte is spontaneously embedded in the cell membrane through electrostatic and hydrophobic interactions, and through close interaction with the cell membrane, it exerts its photothermal and fluorescence imaging functions. The cell membrane is ligand-modified by lipid membrane fusion, and the Aifm2 plasmid is loaded by electrostatic interaction to enable it to have a gene delivery function, thereby obtaining a cell membrane engineered nanoparticle (see Figure 4 ).

[0086] A method for preparing engineered nanoparticles for regulating obesity-related ion channels comprises the following steps:

[0087] (a) Figure 5 The oil red staining images of fat cells and the qRT-PCR data analysis of brown fat cells in Example 1 of the present invention; wherein Figure 5 a is the oil red staining image before adipocyte transformation. Figure 5 b is the oil red staining image after adipocyte transformation. Figure 5 c is the qRT-PCR data analysis of brown adipocytes. Figure 5 As shown, mesenchymal stem cells in the logarithmic growth phase were inoculated into a culture dish, and the cells were differentiated when contact inhibition occurred. On day 0, differentiation medium was added to the culture dish full of cells, and the maintenance medium was replaced on the second day. The medium was changed every two days, and mature brown fat cells were obtained after the seventh day. The lipid droplets in the cells were stained with a fat-soluble oil red dye, and it was found that the number of red lipid droplets in the transformed cells increased, proving that the cells were transformed into adipocytes ( Figure 5 a- Figure 5 b). Further verification using qRT-PCR technology revealed that the levels of brown fat-specific highly expressed mRNAs such as Aifm2 and UCP1 were significantly increased, proving the transformation of brown fat cells ( Figure 5 c).

[0088] (b) Lyse the cells using a cell lysis buffer and collect the cell membrane using differential centrifugation. The cell membrane concentration is determined using the BCA protein quantification method, and the cell membrane concentration is calculated using the BCA working curve equation and the absorbance value of the cell membrane. The cell membrane fragments dispersed in ultrapure water are prepared into nanoscale cell membrane vesicles using a liposome extruder, and the particle size and potential are detected using a light scattering particle size analyzer, and the morphology of the vesicles is observed using a transmission electron microscope.

[0089] (c) The vesicles were mixed with 20 μM water-soluble membrane-embedded conjugated oligoelectrolytes and stirred at room temperature for 2 h to embed the conjugated oligoelectrolytes into the cell membrane vesicle carriers.

[0090] (d) The amphiphilic molecule DSPE-PEG2000-PEI25000 was dissolved in DMSO, and the above solution was added to 9 mL of ultrapure water, and ultrasonicated for 10 min. The solution was then stirred for 5 h in the dark while passing argon gas until the organic solvent was completely removed, and impurities were removed by dialysis using a dialysis bag.

[0091] (e) The cell membrane vesicle carrier solution embedded with conjugated oligoelectrolytes was mixed with the DSPE-PEG2000-PEI25000 solution and stirred for 4 h, and then concentrated after ultrafiltration.

[0092] (f) 1 mL of the above solution was mixed with an equal volume of Aifm2 plasmid and vortexed for 15 seconds. After standing for 30 minutes, the final cell membrane engineered nanoparticle solution was prepared.

[0093] Figure 6 The ultraviolet absorption spectrum, membrane embedding image and photothermal heating curve of the cell membrane engineered nanoparticles in Example 1 of the present invention are shown; wherein, Figure 6 a is the UV absorption spectrum of cell membrane engineered nanoparticles, Figure 6 b is the membrane embedding image of cell membrane engineered nanoparticles. Figure 6 c is the photothermal heating curve of cell membrane engineered nanoparticles. Figure 6 As shown, cell membrane engineered nanoparticles (CENs in the figure) have similar characteristic absorption peaks as COE-BBT ( Figure 6 a), and the green fluorescence of COE-BBT was observed using a laser confocal microscope ( Figure 6 b), proving that COE-BBT can be well embedded in cell membrane vesicles ( Figure 6 b). Figure 6 c shows that cell membrane engineered nanoparticles have good photothermal properties.

[0094] Example 2: Regulation of obesity-related ion channels by cell membrane engineered nanoparticles

[0095] This example is based on the good photothermal properties of conjugated oligoelectrolytes. Cell membrane engineered nanoparticles can heat up and activate TRPV1 ion channels on the BAT cell membrane under the irradiation of near-infrared light, promoting extracellular Ca 2+A large amount of influx occurs, thereby activating CaMKII. Subsequently, AMPK phosphorylation and SIRT-1 phosphorylation are activated, deacetylation of PPARγ and PRDM-16 is achieved, and their stability and interaction are promoted. At the same time, SIRT-1 activates PGC-1α, transcriptionally activates PPARα, and promotes thermogenesis of BAT by activating UCP1.

[0096] Figure 7 This is an immunofluorescence image of the TRPV1 ion channel in Example 2 of the present invention; Figure 8 This is a patch clamp data diagram of cell membrane engineered nanoparticles regulating TRP channels in Example 2 of the present invention;

[0097] Fig. 9 The cell membrane engineered nanoparticles in Example 2 of the present invention affect intracellular Ca 2+ Concentration data plot. Figure 7-9 As shown, an application method of engineered nanoparticles for regulating obesity-related ion channels, and its application effects are as follows:

[0098] (a) Whole-cell patch clamp technique was used to detect the current changes through TRPV1 ion channels on the brown adipocyte membrane to verify the regulation of ion channel function by nanoparticles. Figure 8 As shown, cell membrane engineered nanoparticles can significantly enhance the current of TRPV1 ion channels under the irradiation of near-infrared light, proving that cell membrane engineered nanoparticles have a good activation effect on TRPV1 ion channels.

[0099] (b) Utilization of Ca 2+ Indicator Fluo-4 to monitor intracellular Ca 2+ The concentration changes verified the regulatory effect of cell membrane engineered nanoparticles on TRPV1 ion channels. Figure 8 As shown, under the irradiation of near-infrared light, the Ca in brown adipocytes treated with cell membrane engineered nanoparticles 2+ The brightest fluorescence indicates that the cell membrane engineered nanoparticles mediate the extracellular Ca2+ transport by activating TRPV1 ion channels. 2+ A large amount of influx of proteins is achieved, thus regulating the TRPV1 ion channel-related signaling pathways.

[0100] Example 3: Effects of cell membrane engineered nanoparticles on intracellular electron transport

[0101] This embodiment is that cell membrane engineered nanoparticles achieve energy consumption by promoting glycolysis. Under the guidance of the homologous targeting ability of brown fat cell membranes, cell membrane engineered nanoparticles target BAT sites, where DSPE-PEG2000-PEI25000 can form a stable complex with DNA to protect DNA and improve its stability. After entering the cell, the protonation of amines will lead to a large influx of counterions and a decrease in osmotic potential, achieving the release of Aifm2 plasmids in brown fat cells in a low pH environment. Because Aifm2 has NADH oxidase activity, it can promote the oxidation of NADH to NAD + , and NAD in cells + The level is positively correlated with the rate of cellular glycolysis. By detecting NAD + / NADH level verifies the effect of cell membrane engineered nanoparticles on intracellular electron transfer.

[0102] Fig.10 This is a data diagram of the effect of cell membrane engineered nanoparticles on intracellular electron transfer in Example 3 of the present invention. A method for detecting the effect of engineered nanoparticles that regulate obesity-related ion channels on intracellular electron transfer comprises the following steps:

[0103] Using NAD + / NADH Detection Kit detects NAD in cells by colorimetry + / NADH changes. Collect cells, remove culture medium by centrifugation, and add NAD + / NADH extract for cell lysis. Pipette the sample to be tested into the well plate, add the colorimetric solution, and measure the absorbance of the sample at 450nm to measure NAD + To detect the ratio of the two, add a certain volume of the sample to be tested into the centrifuge tube and heat it to decompose NAD + , aspirate the supernatant into the well plate, add the color developing solution, measure the absorbance of the sample at 450nm, and perform data analysis.

[0104] like Fig.10 As shown, brown adipocytes treated with cell membrane engineered nanoparticles have NAD + The ratio of NADH / NADH increased significantly, proving that cell membrane engineered nanoparticles promoted the electron transfer process in cells.

[0105] The above description is only a preferred embodiment of the present application and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the present application is not limited to the technical solution formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the above features are replaced with (but not limited to) technical features with similar functions disclosed in the present application.

[0106] It should be understood that the size of the serial numbers of each step in the content of the invention and the embodiments of the present invention does not absolutely mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

Claims

1. An engineered nanoparticle for regulating obesity-related ion channels, characterized in that: The nanoparticles consist of brown adipocyte membrane vesicles, water-soluble membrane-embedded conjugated oligoelectrolytes, amphiphilic molecules and Aifm2 plasmids.

2. A method for preparing engineered nanoparticles for regulating obesity-related ion channels, characterized in that: The following steps are involved: Produce target cell membrane vesicles; The target cell membrane vesicles and the water-soluble membrane-embedded conjugated oligoelectrolyte are mixed, and stirred at room temperature to embed the water-soluble membrane-embedded conjugated oligoelectrolyte into the target cell membrane vesicle carrier to obtain solution A; Solution A is mixed with the amphiphilic molecule solution and stirred, and then ultrafiltered and concentrated to obtain solution B; Solution B was mixed with an equal volume of the target plasmid and vortexed for 15 seconds, and allowed to stand for 30 minutes to prepare the final target cell membrane engineered nanoparticle solution; the target plasmid was selected according to the site of application of the engineered nanoparticle.

3. The method for preparing engineered nanoparticles for regulating obesity-related ion channels according to claim 2, characterized in that: Produce target cell membrane vesicles, including: Collecting target cells and extracting target cell membranes by differential centrifugation; the target cells are selected according to the site where the engineered nanoparticles are applied; The target cell membrane is dispersed in water, and the target cell membrane is squeezed into vesicles using a membrane squeezer to prepare the target cell membrane vesicles.

4. The method for preparing engineered nanoparticles for regulating obesity-related ion channels according to claim 3, characterized in that: The target cells are brown fat cells.

5. The method for preparing engineered nanoparticles for regulating obesity-related ion channels according to claim 2, characterized in that: The water-soluble membrane-embedded conjugated oligoelectrolyte has a linear conjugated skeleton, flexible chains modified with polar groups at both ends, and quaternary ammonium salt structures at both ends.

6. The method for preparing engineered nanoparticles for regulating obesity-related ion channels according to claim 2, characterized in that: The amphiphilic molecule solution is obtained by the following method: The amphiphilic molecule DSPE-PEG2000-PEI25000 is dissolved in an organic solvent to obtain solution C, and solution C is added into water and ultrasonicated for 5-20 minutes. The solution is stirred in the dark while passing argon gas until the organic solvent is completely removed, and impurities are removed by dialyzing using a dialysis bag.

7. The method for preparing engineered nanoparticles for regulating obesity-related ion channels according to claim 6, characterized in that: The organic solvent is DMSO, methanol or ethanol.

8. The method for preparing engineered nanoparticles for regulating obesity-related ion channels according to claim 6, characterized in that: When solution C is added to water, the ratio of solution C to water is 1:2-1:

10.

9. The method for preparing engineered nanoparticles for regulating obesity-related ion channels according to claim 2, characterized in that: The target plasmid is Aifm2 plasmid.

10. A method for applying engineered nanoparticles for regulating obesity-related ion channels, wherein the engineered nanoparticles for regulating obesity-related ion channels prepared by the preparation method according to claims 2-9 act on brown fat cells, characterized in that: The application method comprises the following steps: Use near-infrared light to activate TRPV1 ion channels; Regulates intracellular Ca 2+ concentration, promoting UCP1 thermogenesis.

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