Gelled cell as well as preparation method and application thereof

Abstract

The invention discloses a gelled cell as well as a preparation method and application thereof. Relates to the field of synthetic biology and biomedicine. The gelated cell is a cell structure without cell viability, and comprises: a carrier cell framework comprising a cell membrane which retains the membrane protein content, the type and the lipid order of an original carrier cell; the intracellular hydrogel is formed in situ in the carrier cell framework through supramolecular host-guest interaction or cross-linking reaction, and is fused with the inner surface of a cell membrane; the antibacterial substance is loaded in the intracellular hydrogel; wherein pores are distributed on the cell membrane, the intracellular hydrogel is communicated with the external environment through the pores, and the loaded antibacterial substances are exposed. The gelated cell provided by the invention can simultaneously realize targeted delivery, contact sterilization and endotoxin adsorption, and avoids the problem of secondary inflammation caused by release of endotoxin in traditional antibiotic therapy and the problem of membrane protein loss or disordered arrangement in a cell membrane coating technology.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and biomedicine, and in particular to a gelled cell, its preparation method, and its application. Background Technology

[0002] Bacterial infections pose a significant threat to human health, with widespread and profound harm. From causing acute, highly contagious diseases to leading to persistent chronic infections, from local tissue damage to inducing systemic sepsis and even multiple organ failure, the pathogenic mechanisms of bacteria are complex and diverse. Bacteria can not only directly damage the structure and function of host cells by secreting exotoxins, but also release large quantities of their own structural components, such as lipopolysaccharides (endotoxins) in the cell wall, after bacterial death and lysis, continuously and strongly activating the host's innate immune system. This activation leads to the overactivation of immune cells such as macrophages, releasing massive amounts of inflammatory mediators such as tumor necrosis factor-α, interleukin-1β, and interleukin-6, forming a "cytokine storm," thereby causing systemic inflammatory response syndrome and secondary damage to tissues and organs.

[0003] Traditional antibiotic therapy plays a crucial role in killing bacteria; however, it cannot eliminate the endotoxins released after bacterial death. In fact, the killing of a large number of bacteria in a short period can lead to a massive release of endotoxins, exacerbating the condition. Clinically, despite various treatment strategies aimed at neutralizing or eliminating endotoxins, the complex molecular structure, diverse mechanisms of action, and extensive interactions with the host immune system of endotoxins limit the effectiveness of existing methods, making it difficult to fundamentally block the inflammatory cascade.

[0004] On the other hand, in response to the excessive inflammatory response itself, the modern biomedical field has developed targeted agents such as monoclonal antibodies against specific inflammatory cytokines. These agents can precisely block the signaling pathways of key inflammatory factors such as interleukin-6 or tumor necrosis factor-α, showing good efficacy in the treatment of some inflammatory diseases. However, in the complex systemic inflammatory environment caused by severe infections (such as severe pneumonia), tissue damage is usually the result of the networked and synergistic effects of multiple inflammatory mediators. After a single pathway is inhibited, other inflammatory factors may continue to drive the inflammatory process through bypass or feedback mechanisms, leading to a significant reduction in treatment efficacy. Therefore, developing a therapeutic agent that can broadly and efficiently adsorb or neutralize multiple different inflammatory mediators, thereby more comprehensively regulating the inflammatory network from upstream, has become a highly challenging and urgent need in the field of anti-inflammatory therapy.

[0005] Against this backdrop, cell-derived formulations offer new avenues for anti-inflammatory therapy: utilizing cell membrane coating technology, natural cell membranes are coated onto the surface of nanomaterials, allowing them to inherit the biological functions of cell membranes (such as selective permeation and cell recognition) to achieve targeted drug delivery. For example, researchers have used the cell membranes of macrophages (MAs), which are rich in cytokine-binding receptors, to coat reactive oxygen species-responsive nanoparticles, enabling them to mimic the adsorption and neutralization of inflammatory cytokines by MAs.

[0006] However, this technological approach faces fundamental technical bottlenecks in practical preparation and application. The preparation process typically involves multiple complex steps, including cell membrane separation, purification, vesicle formation, and fusion or coating with nanoparticles. During this series of in vitro operations, the cell membrane structure is inevitably subjected to physical and chemical disturbances, leading to the loss, denaturation, or loss of activity of membrane proteins. Even more challenging is the difficulty in precisely controlling the reassembly of the cell membrane on the nanoparticle surface; the original transmembrane topology, spatial orientation, and lateral distribution of membrane proteins within the membrane plane are easily disrupted. Protein function is highly dependent on its precise three-dimensional conformation and correct anchoring and arrangement on the membrane. Such spatial structural misalignment directly leads to a significant decrease in its binding affinity and specificity to ligands, or even complete loss of function. Summary of the Invention

[0007] A gelled cell, a cell-free cellular structure, comprising:

[0008] The carrier cell framework includes a cell membrane, and the cell membrane retains the content, types, and lipid sequence of the original carrier cell membrane proteins;

[0009] Intracellular hydrogels are formed in situ within the carrier cell framework through supramolecular host-guest interactions or cross-linking reactions, and fuse with the inner surface of the cell membrane.

[0010] Antibacterial substances are loaded into the intracellular hydrogel;

[0011] The cell membrane has pores, and the intracellular hydrogel communicates with the external environment through these pores, exposing the loaded antibacterial substance.

[0012] According to embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects:

[0013] The gelled cells of this invention can simultaneously achieve targeted delivery, contact sterilization, and endotoxin adsorption, avoiding the secondary inflammation caused by endotoxin release in traditional antibiotic treatment, as well as the loss or disordered arrangement of membrane proteins in cell membrane coating technology.

[0014] Specifically, firstly, by using a cell-free cell framework, the immune risks that live cells may cause are eliminated, but the content, types and lipid sequences of the original cell membrane proteins are fully preserved, which allows the gelled cells to inherit the biological functions of the original cell.

[0015] Secondly, intracellular hydrogels are formed in situ through supramolecular host-guest interactions or cross-linking reactions and fuse with the inner surface of the cell membrane. This provides a stable mechanical support structure and serves as a reservoir for antibacterial substances. The fusion ensures a tight connection between the gel and the membrane, which is beneficial for the transfer of antibacterial substances to the membrane surface.

[0016] Third, the pores formed on the cell membrane allow the intracellular hydrogel to communicate with the external environment without disrupting the membrane protein structure, exposing the loaded antimicrobial substances. These pores enable the antimicrobial substances in the gel to be locally exposed. When bacteria approach, the natural adsorption properties of the cell membrane capture the bacteria near the pores, and the exposed antimicrobial substances directly contact the bacterial membrane, disrupting the bacterial membrane potential and integrity through electrostatic interactions, leading to bacterial death. Simultaneously, the endotoxins released by the dead bacteria are bound to and neutralized by receptors on the intact membrane, thereby blocking inflammation.

[0017] According to one embodiment of the present invention, the carrier cell framework is derived from original carrier cells; preferably, the original carrier cells are immune cells; more preferably, the immune cells include at least one of monocytes, macrophages, dendritic cells, neutrophils, and mast cells. Immune cells express a variety of chemokine receptors and pathogen recognition receptors on their surface. Using the immune cell framework allows gelled cells to inherit these homing abilities, achieving targeted delivery.

[0018] In this invention, the cell membrane has the same membrane protein content as the original carrier cells. This ensures that the gelled cells have a similar membrane protein abundance to the original cells, thereby maintaining the integrity of membrane function, particularly the adsorption and targeted recognition capabilities for endotoxins. More specifically, this invention avoids significant loss of membrane proteins through a mild preparation process (such as freeze-thaw and photopolymerization).

[0019] According to one embodiment of the present invention, the pores distributed on the cell membrane are formed by a freeze-thaw operation.

[0020] According to one embodiment of the present invention, the immune cells are macrophages. Macrophage membranes are rich in TLR4, scavenger receptors, etc., enabling them to efficiently adsorb endotoxins and bacteria. Simultaneously, macrophages exhibit strong chemotaxis towards sites of inflammation. After gelation, these membrane proteins are retained, allowing the carrier to target the infection focus and neutralize endotoxins.

[0021] According to one embodiment of the present invention, the intracellular hydrogel is formed by a photo-initiated free radical polymerization reaction.

[0022] According to one embodiment of the present invention, the antibacterial substance is a cationic monomer, which is one of the cross-linking monomers of an intracellular hydrogel. Integrating antibacterial function and structural cross-linking function into the same molecule allows the antibacterial substance to also participate in the formation of the intracellular hydrogel, thereby ensuring that the antibacterial substance is stably fixed in the gel network.

[0023] According to one embodiment of the present invention, the cationic monomer is selected from at least one of 2-aminoethyl methacrylate hydrochloride (AEMA) and (3-acrylamidopropyl)trimethylammonium chloride. AEMA's amino group is protonated and positively charged at physiological pH, enabling electrostatic disruption of bacterial membranes; its methacrylate group facilitates free radical polymerization. AEMA was specifically chosen because its charge density and polymerization activity are suitable for this system.

[0024] According to one embodiment of the present invention, the cationic monomer is 2-aminoethyl methacrylate hydrochloride, and the concentration of 2-aminoethyl methacrylate hydrochloride in the gel precursor solution is 10-30 wt%; preferably, the concentration is one of 10 wt%, 15 wt%, 20 wt%, 25 wt%, and 30 wt%.

[0025] According to one embodiment of the present invention, the raw material component of the intracellular hydrogel includes polyethylene glycol diacrylate (PEG-DA); preferably, the average molecular weight of the polyethylene glycol diacrylate is 700-800. PEG-DA with an average molecular weight of 700-800 has shorter chains, higher cross-linking density, and forms a more robust gel.

[0026] According to one embodiment of the present invention, the concentration of polyethylene glycol diacrylate in the gel precursor solution is 2-20 wt%; preferably, the concentration is 4-12 wt%. A concentration of 4-12 wt% is suitable; too low a concentration will not gel, while too high a concentration will result in excessive hardness and may damage cell structure.

[0027] According to one embodiment of the present invention, the average size of the pores is in the nanometer to submicrometer range. The nanometer to submicrometer pore size allows for partial exposure of the intracellular hydrogel while maintaining the overall integrity and continuity of the cell membrane.

[0028] According to one embodiment of the present invention, the antibacterial substance further includes at least one of antibiotics, antimicrobial peptides, and metal nanoparticles.

[0029] According to one embodiment of the present invention, the cell-inactive cell structure is determined by the fact that the cells have completely lost their cell viability as detected by the CCK-8 assay. The gelled cells of the present invention are cell-inactive, which ensures the biosafety of the therapeutic carrier and completely eliminates the risk of live cells potentially proliferating, mutating, or triggering uncontrolled immune responses.

[0030] A method for preparing the gelled cells includes the following steps:

[0031] S1. Mix the gel precursor solution, which includes hydrogel monomers, antibacterial substances and photoinitiators, with the original carrier cells to obtain a mixed system;

[0032] S2. Perform at least one freeze-thaw operation on the mixed system to allow the gel precursor to penetrate into the original carrier cells and form pores on the cell membrane;

[0033] S3. Trigger a polymerization reaction in an ice bath to crosslink the gel precursor that has infiltrated the original carrier cells in situ to form an intracellular hydrogel that fuses with the inner surface of the cell membrane, thereby obtaining gelled cells without cell activity.

[0034] According to embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects:

[0035] This invention provides a simple and controllable preparation method that enables large-scale production of stable antibacterial gelled cells.

[0036] The method of this invention consists of three key steps:

[0037] First, a gel precursor solution including hydrogel monomers, antibacterial substances and initiators is mixed with carrier cells to ensure that the precursor and cells are in full contact, laying the foundation for subsequent infiltration.

[0038] Secondly, the mixed system is subjected to at least one freeze-thaw operation. During the freeze-thaw process, when the cells are frozen at low temperature, the water inside the cells forms ice crystals, which leads to volume expansion and increased membrane mechanical stress, forming nanopores. After thawing, the ice crystals melt, leaving permanent pores. These pores allow precursor small molecules and polymers to diffuse into the cell interior while maintaining the structural integrity of macromolecular membrane proteins. Repeated freeze-thaw operations can adjust the pore density and size.

[0039] Third, the polymerization reaction is triggered, causing the infiltrated gel precursor to crosslink in situ, forming an intracellular hydrogel that fuses with the inner surface of the cell membrane. In this step, the photoinitiator absorbs ultraviolet light to generate active free radicals, initiating the chain polymerization of monomers and forming a three-dimensional crosslinked network within the cell. The polymerization reaction is carried out in a low-temperature ice bath to dissipate heat and prevent protein denaturation. The formed gel network interacts with cytoskeleton residues and phospholipids in the inner membrane layer through physical entanglement, and even chemically binds to membrane components through unreacted functional groups, achieving tight fusion. The final product is cell-free but retains the complete membrane structure, and the gel exposes antibacterial substances through its pores. The entire method synergistically achieves pore formation, precursor infiltration, and in-situ gelation, ensuring the structural stability and functional integration of the gelled cells.

[0040] According to one embodiment of the present invention, the freezing temperature of the freeze-thaw operation is not higher than -20 °C.

[0041] According to one embodiment of the present invention, the freezing temperature of the freeze-thaw operation is -20 ℃ to -180 ℃.

[0042] According to one embodiment of the present invention, the freezing time of the freeze-thaw operation is ≥15 min, preferably, the freezing time of the freeze-thaw operation is 15 to 60 min.

[0043] According to one embodiment of the present invention, the freeze-thaw operation is followed by thawing at room temperature.

[0044] According to one embodiment of the present invention, in step S3, the polymerization reaction is a photo-initiated free radical polymerization reaction.

[0045] According to one embodiment of the present invention, the concentration of the hydrogel monomer in the gel precursor solution is 2-20 wt%; preferably, the concentration of the hydrogel monomer in the gel precursor solution is 4-12 wt%.

[0046] According to one embodiment of the present invention, the photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone (I2959). I2959 has good water solubility, is easy to mix with the precursor solution, and its decomposition products have low toxicity.

[0047] According to one embodiment of the present invention, the concentration of the photoinitiator in the gel precursor solution is 0.5-2 wt%. An appropriate concentration provides sufficient initiation efficiency while avoiding cytotoxicity or side effects caused by excessive initiator.

[0048] According to one embodiment of the present invention, the concentration of the photoinitiator in the gel precursor solution is one of 0.5wt%, 1.0wt%, 1.5wt%, and 2.0wt%.

[0049] In this invention, the cell membrane retains the content, types, and lipid sequence of the original carrier cells' membrane proteins. This description aims to define the structural integrity of the cell membrane in the "gelled cells" described in this invention. In the context of this invention:

[0050] "Preservation of membrane protein content and types" means that when compared and analyzed by protein quantification methods (such as the BCA method) and protein separation and staining methods (such as SDS-PAGE and Coomassie Brilliant Blue staining), the membrane protein extracts of the gelled cells and the membrane protein extracts of the original carrier cells show no statistically significant difference in total protein content, and exhibit high similarity in the main protein band patterns.

[0051] "Preservation of lipid order" is a relative and functional description. Its core meaning is that the cell membrane of the gelled cells obtained by the specific preparation method described in this invention (especially the process involving controlled freeze-thaw operation and in-situ photopolymerization) has its lipid bilayer arrangement ordered in a substantial manner, thereby avoiding the severely disordered arrangement of membrane lipids caused by the cell membrane extraction and reassembly process as described in the background art.

[0052] This "substantially maintained" lipid order manifests functionally as follows: the cell membrane of the gelled cells effectively supports membrane proteins (such as the TLR4 receptor) in maintaining their native conformation and spatial orientation, thereby enabling the gelled cells to inherit and exhibit the bioactivity based on membrane protein function possessed by the original carrier cells, such as the highly efficient adsorption capacity for specific pathogen-related molecular patterns (such as bacterial endotoxin LPS). This functional result can be verified through the adsorption and neutralization experiments described in the examples.

[0053] In this invention, the intracellular hydrogel is fused with the inner surface of the cell membrane; this description refers to the interaction between the intracellular hydrogel and the cell membrane. In this invention, observation using confocal fluorescence microscopy (e.g., labeling the cell membrane and intracellular hydrogel with different fluorescent dyes) reveals that the network structure of the intracellular hydrogel is in close contact with the inner surface of the cell membrane, and co-localization regions of the dyes exist at the interface, indicating the formation of a continuous and stable interfacial layer, rather than a simple physical filling or encapsulation relationship. This "fusion" helps ensure that the antimicrobial substances loaded in the hydrogel can be effectively delivered to the membrane region.

[0054] Another aspect of the invention relates to the use of the gelled cells in the preparation of medicaments for treating bacterial infections and clearing bacterial endotoxins.

[0055] According to one embodiment of the present invention, the endotoxin comprises lipopolysaccharide (LPS).

[0056] According to one embodiment of the present invention, the bacterial infection includes infections caused by Escherichia coli or Staphylococcus aureus.

[0057] In another aspect, the present invention provides a pharmaceutical composition comprising the gelled cells and a pharmaceutically acceptable carrier or excipient.

[0058] According to one embodiment of the present invention, the pharmaceutically acceptable carrier or excipient includes at least one of physiological saline, glucose solution, and phosphate buffer used in injectable formulations; lactose, microcrystalline cellulose, and magnesium stearate used in oral formulations; carbomer and hydroxyethyl cellulose used in topical gel formulations; and sucrose and mannitol used in lyophilized formulations.

[0059] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0060] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0061] Figure 1 This is a flowchart of the preparation method of gelled cells in Example 1.

[0062] Figure 2 SEM images of gelled cells prepared in Example 1, non-gelled cells prepared in Comparative Example 1, and macrophage leukemia cells.

[0063] Figure 3 This is a confocal fluorescence image of gelled cells from Example 1.

[0064] Figure 4 The graphs show the storage modulus test results of gelled cells in Examples 1-5.

[0065] Figure 5 The rheological frequency scan is shown for the hydrogel prepared using polyethylene glycol diacrylate and I2959 in Example 1.

[0066] Figure 6 The image shows a SEM image of the hydrogel prepared using polyethylene glycol diacrylate and I2959 in Example 1.

[0067] Figure 7 A bar chart comparing the membrane protein content of macrophages and gelled cells from Example 1, as determined by the BCA protein quantification method.

[0068] Figure 8 This is a comparison image of Coomassie brilliant blue staining of macrophages and gelled cell membrane proteins from Example 1.

[0069] Figure 9 The bar chart shows the cell viability of macrophages measured using the CCK-8 assay, macrophages mixed with gel solution, and gelled cells from Example 1.

[0070] Figure 10 This is a confocal microscope image superimposed of the gelled cells from Example 1 after staining with membrane dyes and gel dyes.

[0071] Figure 11 This is a comparison diagram of membrane zeta potential measurements between macrophages and gelled cells from Example 1.

[0072] Figure 12 This is a confocal microscope image superimposed of the gelled cells from Example 6 after staining with membrane dyes and gel dyes.

[0073] Figure 13 This is a comparison diagram of the membrane zeta potential of gelled cells in Example 6 and gelled cells in Example 1.

[0074] Figure 14 The results of plate culture of gelled cells from Comparative Example 2 and gelled cells from Example 1 after co-incubation with Escherichia coli are shown.

[0075] Figure 15 The graph shows the quantitative statistics of the gelled cells of Comparative Example 2 and the gelled cells of Example 1 after co-incubation with Escherichia coli.

[0076] Figure 16 The results of plate culture of gelled cells from Comparative Example 2 and gelled cells from Example 1 after co-incubation with Staphylococcus aureus are shown.

[0077] Figure 17 The graph shows the quantitative statistics of the gelled cells of Comparative Example 2 and the gelled cells of Example 1 after co-incubation with Escherichia coli. Detailed Implementation

[0078] The terms "preferred," "more preferred," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0079] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0080] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of the present invention.

[0081] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0082] In the examples, the polyethylene glycol diacrylate (PEG-DA) had an average molecular weight of 700 and was purchased from Maclean's.

[0083] Example 1

[0084] A gelled cell, a cell-free cellular structure, comprising:

[0085] The carrier cell framework includes a cell membrane, and the cell membrane retains the content, types, and lipid sequence of the original carrier cell membrane proteins;

[0086] Intracellular hydrogels are formed in situ within the aforementioned carrier cell framework through supramolecular host-guest interactions or cross-linking reactions, and fuse with the inner surface of the cell membrane.

[0087] Antibacterial substances are loaded into the above-mentioned intracellular hydrogel;

[0088] The cell membrane has pores, through which the intracellular hydrogel communicates with the external environment and exposes the loaded antibacterial substance.

[0089] A method for preparing the above-mentioned gelled cells, the flowchart of which is shown below. Figure 1 As shown, specifically, it includes the following steps:

[0090] S1. Mix 10 wt% polyethylene glycol diacrylate and 0.5 wt% photoinitiator I2959 at 60℃, and sonicate at 100 kHz for 20 min. Then add 10 wt% cationic monomer AEMA (2-aminoethyl methacrylate hydrochloride), dilute with PBS, and filter through a 0.22 μm filter membrane to obtain the gel precursor solution. Add 1 mL of the gel precursor solution to a 1×10⁻⁶ centrifuge tube. 7 RAW 264.7 mouse mononuclear macrophage leukemia cells (maintained under 5% CO2, 37 ℃, and 10% FBS culture conditions) were mixed to obtain a mixed system;

[0091] S2. Place the mixture in a -80°C freezer for pre-freezing to allow the gel precursor to infiltrate the cells. Thaw at room temperature after 15 minutes to form pores on the cell membrane. After centrifugation, add 2 mL of PBS.

[0092] S3. Place 2cm of crushed ice at the bottom of the container, place the sample on the ice, and irradiate it under 20W ultraviolet light for 15 minutes to solidify it. Through freeze-thaw-ultraviolet irradiation, the gel precursor that has penetrated into the cells is cross-linked in situ to form an intracellular hydrogel that is fused with the inner surface of the cell membrane, thereby obtaining gelled cells without cell activity.

[0093] Example 2

[0094] The only difference between Example 2 and Example 1 is that in step S1 of Example 2, the amount of polyethylene glycol diacrylate used is 4 wt%, while in step S1 of Example 1, the amount of polyethylene glycol diacrylate used is 10 wt%.

[0095] Example 3

[0096] The only difference between Example 3 and Example 1 is that in step S1 of Example 3, the amount of polyethylene glycol diacrylate used is 6 wt%, while in step S1 of Example 1, the amount of polyethylene glycol diacrylate used is 10 wt%.

[0097] Example 4

[0098] The only difference between Example 4 and Example 1 is that the amount of polyethylene glycol diacrylate used in step S1 of Example 4 is 8 wt%, while the amount of polyethylene glycol diacrylate used in step S1 of Example 1 is 10 wt%.

[0099] Example 5

[0100] The only difference between Example 5 and Example 1 is that in step S1 of Example 5, the amount of polyethylene glycol diacrylate used is 12 wt%, while in step S1 of Example 1, the amount of polyethylene glycol diacrylate used is 10 wt%.

[0101] Example 6

[0102] The only difference between Example 6 and Example 1 is that the amount of cationic monomer AEMA used in step S1 of Example 6 is 30 wt%, while the amount of cationic monomer AEMA used in step S1 of Example 1 is 10 wt%.

[0103] Comparative Example 1

[0104] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 uses non-gelled macrophages.

[0105] Specifically:

[0106] A method for preparing non-gelled cells includes the following steps:

[0107] S1. Add 1 mL of PBS to a 15 mL centrifuge tube containing 1×10⁻⁶ ppm of PBS. 7 RAW 264.7 mouse mononuclear macrophage leukemia cells (maintained under 5% CO2, 37 ℃, and 10% FBS culture conditions) were mixed to obtain a mixed system;

[0108] S2. Place the mixture in a -80℃ freezer for pre-freezing, thaw at room temperature after 15 min, centrifuge, and add 2 mL of PBS;

[0109] S3. Place a 2cm layer of crushed ice at the bottom of the container, place the sample on the ice, and irradiate it under 20W ultraviolet light for 15 minutes to solidify it and obtain non-gelled cells.

[0110] Comparative Example 2

[0111] The difference between Comparative Example 2 and Example 1 is that the cationic monomer AEMA is not added in step S1 of Comparative Example 2, while the cationic monomer AEMA is added in Example 1.

[0112] Performance testing:

[0113] SEM images of gelled cells prepared in Example 1, non-gelled cells prepared in Comparative Example 1, and RAW 264.7 mouse mononuclear macrophage leukemia cells are shown below. Figure 2 As shown. Among them, Figure 2 Image a is a SEM image of RAW 264.7 mouse mononuclear macrophage leukemia cells. Figure 2 b is a SEM image of the gelled cells prepared in Example 1. Figure 2 c is a SEM image of the non-gelled cells prepared in Comparative Example 1. Figure 2The scale bars for b and c are consistent with those for a. The instrument used was a HITACHI Regulus 8100 (high-resolution cold field emission scanning electron microscope). As can be seen from the figures, the gelled cells prepared in Example 1 have the same morphology and size as W 264.7 mouse mononuclear macrophage leukemia cells, indicating that the gelled cells prepared in Example 1 of this invention retain a stable cell structure. In contrast, the non-gelled cells prepared in Comparative Example 1, due to the lack of gelation, cannot retain their cell structure and morphology.

[0114] The gelled cells prepared in Example 1 were stained with membrane dye (DiO) and nuclear dye (DAPI) and obtained using a confocal microscope (ZEISS-LSM880 ultra-high resolution laser confocal microscope) as shown. Figure 3 The confocal fluorescence image shown. Figure 3 In the figure, 'a' represents the graph with a scale bar of 10 μm. Figure 3 Figure b is a graph with a scale bar of 1 μm. The morphology of the cells and the positional relationship between the cell nucleus and the cell membrane can be clearly observed from the graph, indicating that the gelled cells of Example 1 of the present invention inherit the complete cell membrane of macrophages.

[0115] The gelled cells prepared in Examples 1-5 were used to test their storage modulus using a rheometer. The test results are as follows: Figure 4 As shown. Figure 4 The storage modulus is shown in the figure. As can be seen from the figure, the storage modulus of the hydrogel increases with the increase of the amount of polyethylene glycol diacrylate.

[0116] Figure 5 The rheological frequency scan is shown for the hydrogel prepared with 10 wt% polyethylene glycol diacrylate and 0.5 wt% I2959 in Example 1, where angular frequency is the angular frequency. Figure 5 This demonstrates that the hydrogel it forms possesses typical and stable solid gel properties.

[0117] Figure 6 The image shows a SEM image of the hydrogel prepared with 10 wt% polyethylene glycol diacrylate and 0.5 wt% I2959 in Example 1. As can be seen from the image, the hydrogel of the present invention has a uniform porous structure, which is the microscopic basis for its ability to load and slowly release antibacterial substances.

[0118] Figure 7 This is a bar chart comparing the membrane protein content of macrophages (MA) and gelled cells (AEMA-GM) from Example 1, as determined by the BCA protein quantification method. In the figure, memberance protein represents the number of membrane proteins. Figure 7Data shows that there is essentially no difference in the content of membrane proteins between the two, therefore the gelled cells of this invention highly retain the membrane proteins of the cells.

[0119] Figure 8 This is a comparison image of Coomassie Brilliant Blue staining (SDS-PAGE gel electrophoresis) of membrane proteins from macrophages (MA) and gelled cells (AEMA-GM) from Example 1. The protein band patterns shown in the image are highly similar, demonstrating that the gelled cells from Example 1 not only retain the content of membrane proteins from macrophages (MA), but also the types of membrane proteins.

[0120] Figure 9 Bar graphs showing cell viability measurements of macrophages (MA), macrophages mixed with gel solution (the culture medium for macrophages is gel solution, named MA+Material), and gelled cells (AEMA-GM) from Example 1, measured using the CCK-8 assay. Cell proliferation is defined as cell proliferation. The CCK-8 assay was performed by adding 20 μL of CCK-8 solution to each well of a 96-well plate containing 3000 cells in a 200 μL culture volume, incubating for 40 min in a cell culture incubator, and then detecting the cells using a microplate reader at a wavelength of 450 nm. The results showed that both macrophages and macrophages mixed with gel solution exhibited cell viability; therefore, the gel solution material used in this invention is non-cytotoxic. Furthermore, the gelled cells (AEMA-GM) prepared in Example 1 completely lost their cell viability, confirming that they were inactive "cell" carriers.

[0121] Figure 10 This is a confocal microscopy overlay image of gelled cells (AEMA-GM) from Example 1 stained with membrane dye (DiO, green) and gel dye (NHS-Cy3, red). The yellow areas (red and green merged) in the image indicate that the intracellular hydrogel is exposed through pores in the cell membrane and has fused with the cell membrane.

[0122] Figure 11 This is a comparison of membrane zeta potential measurements between macrophages (MA) and gelled cells (AEMA-GM) from Example 1. Figure 11 The results showed that the membrane surface potential of AEMA-GM was significantly higher than that of normal MA. This is because the cationic AEMA loaded in the hydrogel is exposed through the membrane pores, leading to an increase in the positive charge on the membrane surface, which electrochemically confirms the presence of pores and the exposure of antibacterial substances.

[0123] Figure 12 This is a confocal microscopy overlay image of gelled cells (AEMA-GM) from Example 6 stained with membrane dye (DiO, green) and gel dye (NHS-Cy3, red), compared with... Figure 10The same pattern shows the fusion and exposure of the gel and membrane.

[0124] Figure 13 This is a comparison of the membrane zeta potential of the gelled cells of Example 6 and the gelled cells of Example 1. The results show that the gelled cells of Example 6, which are loaded with a higher concentration (30 wt%) of cationic monomer (AEMA), have a higher membrane potential. This further confirms that the increase in membrane potential is directly related to the amount of exposed cations, and proves that surface properties can be controlled by adjusting the AEMA concentration.

[0125] Figure 14 The results of co-incubating Escherichia coli with gelled cells (PEG-GM) from Comparative Example 2 and AEMA-GM from Example 1, followed by plating, are shown below. The culture conditions were as follows: temperature 37°C, LB liquid medium, and incubation times of 0, 3, 6, 12, and 24 hours. After incubation, the bacterial suspension was collected and plated using the following method: the bacterial suspension was diluted 10... 7 After doubling, add 100 µL to an LB agar plate, spread evenly with 4-6 glass beads (4 mm in diameter), and then incubate at 37°C for 13 hours. Perform colony counting analysis. Figure 14 It is evident that the number of colonies co-incubated with AEMA-GM decreased significantly over time, while the colonies in the PEG-GM group continued to grow, directly demonstrating the contact antibacterial effect of AEMA-GM.

[0126] Figure 15 The quantitative statistical graphs of the gelled cells (PEG-GM) of Comparative Example 2 and the gelled cells (AEMA-GM) of Example 1 after co-incubation with Escherichia coli were plotted by measuring the turbidity of the bacterial solution at OD600nm. Figure 15 In this context, absorbance refers to the absorbance. Figure 15 The curves showed that bacterial growth was significantly inhibited in the AEMA-GM group, and the turbidity was much lower than that in the PEG-GM group, quantifying its antibacterial efficiency in the form of data.

[0127] Figure 16 The results of co-incubation of gelled cells (PEG-GM) from Comparative Example 2 and gelled cells (AEMA-GM) from Example 1 with Staphylococcus aureus followed by plating were shown. The culture conditions were as follows: temperature 37°C, LB liquid medium, and incubation times of 0, 3, 6, 12, and 24 hours. After incubation, the bacterial suspension was collected and plated using the following method: the bacterial suspension was diluted 10... 7After double-distillation, 100 µL was added to LB agar plates, and 4-6 glass beads (4 mm in diameter) were used to spread the mixture evenly. The plates were then incubated at 37°C for 13 hours, followed by colony counting analysis. The figure shows that co-incubation with AEMA-GM significantly reduced the number of Staphylococcus aureus colonies, demonstrating that the gelled cells also have significant antibacterial activity against Gram-positive bacteria.

[0128] Figure 17 Quantitative statistical graphs of gelled cells (PEG-GM) from Comparative Example 2 and gelled cells (AEMA-GM) from Example 1 after co-incubation with Escherichia coli. The quantitative data further confirm that AEMA-GM can effectively inhibit the growth of Staphylococcus aureus.

[0129] The above are merely embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A gelled cell, characterized in that: Cellular structures that are not cell-active include: The carrier cell framework includes a cell membrane, and the cell membrane retains the content, types, and lipid sequence of the original carrier cell membrane proteins; Intracellular hydrogels are formed in situ within the carrier cell framework through supramolecular host-guest interactions or cross-linking reactions, and fuse with the inner surface of the cell membrane. Antibacterial substances are loaded into the intracellular hydrogel; The cell membrane has pores, and the intracellular hydrogel communicates with the external environment through these pores, exposing the loaded antibacterial substance.

2. The gelled cell according to claim 1, characterized in that: The carrier cell framework is derived from the original carrier cells; preferably, the original carrier cells are immune cells; more preferably, the immune cells include at least one of monocytes, macrophages, dendritic cells, neutrophils and mast cells.

3. The gelled cell according to claim 2, characterized in that: The immune cells mentioned are macrophages.

4. The gelled cell according to claim 1, characterized in that: The antibacterial substance is a cationic monomer, which is one of the cross-linking monomers of intracellular hydrogel.

5. A gelled cell according to claim 4, characterized in that: The cationic monomer is selected from at least one of 2-aminoethyl methacrylate hydrochloride and (3-acrylamidopropyl)trimethylammonium chloride.

6. A method for preparing gelled cells as described in any one of claims 1 to 5, characterized in that: Includes the following steps: S1. Mix the gel precursor solution, which includes hydrogel monomers, antibacterial substances and photoinitiators, with the original carrier cells to obtain a mixed system; S2. Perform at least one freeze-thaw operation on the mixed system to allow the gel precursor to penetrate into the original carrier cells and form pores on the cell membrane; S3. Trigger a polymerization reaction in an ice bath to crosslink the gel precursor that has infiltrated the original carrier cells in situ to form an intracellular hydrogel that fuses with the inner surface of the cell membrane, thereby obtaining gelled cells without cell activity.

7. The method according to claim 6, characterized in that: The freezing temperature of the freeze-thaw operation shall not exceed -20 ℃.

8. The method according to claim 6, characterized in that: The concentration of the hydrogel monomer in the gel precursor solution is 10-30 wt%.

9. The method according to claim 6, characterized in that: The freezing time of the freeze-thaw operation is ≥15 min, preferably 15 to 60 min.

10. A pharmaceutical composition comprising the gelled cells of any one of claims 1 to 5, and a pharmaceutically acceptable carrier or excipient.

Images