Green alga microrobot and application thereof
By modifying the surface of microalgae with green algae microrobots containing macrophage membrane fragments, utilizing the recognition ability of macrophages and the phototaxis of Chlamydomonas, and combining white light and 637nm laser, the problem of the efficacy of microalgae microrobots in the hypoxic state of bacterial aggregation sites was solved, achieving efficient photodynamic antibacterial treatment effects.
Patent Information
- Application Number
- CN202510848075.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-24
- Publication Date
- 2025-09-23
AI Technical Summary
The efficacy of existing microalgae microrobots is significantly reduced under hypoxic conditions in areas where bacteria accumulate, making it difficult to achieve efficient photodynamic antibacterial treatment.
By modifying macrophage membrane fragments on the surface of microalgae, utilizing the recognition ability of macrophages and the phototaxis of Chlamydomonas, and combining white light and 637nm laser irradiation, the targeted delivery of green algae microrobots and the efficient production of reactive oxygen species are achieved, thereby enhancing the antibacterial effect.
It achieved highly efficient photodynamic antibacterial effects against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, reaching 96.5% and 97.4% respectively, and alleviated the bacterial hypoxia through photosynthesis.
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Figure CN120678917A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microalgae microrobots, and in particular relates to a green algae microrobot and a preparation method and application thereof. Background Art
[0002] Microalgae microrobots use microalgae as drug delivery vehicles, leveraging the microalgae's own motion to deliver drugs and release photosensitizers on demand for photodynamic antibacterial therapy. However, these microalgae microrobots often rely on exogenous oxygen supply to achieve efficient photodynamic antibacterial therapy. Areas where bacteria accumulate are often hypoxic, which significantly reduces the efficacy of the microalgae microrobots carrying photosensitizers in bacterial colonies. Summary of the Invention
[0003] The purpose of the present invention is to provide a green algae microrobot and its preparation method and application, the green algae micromachine of the present invention.
[0004] The present invention provides a green algae microrobot, comprising Chlamydomonas with macrophage membrane fragments modified on the surface.
[0005] The present invention also provides a method for preparing the green algae microrobot described in the above scheme, comprising the following steps:
[0006] The Chlamydomonas suspension and azide-tetraethylene glycol-active ester are mixed and incubated to obtain azide-modified Chlamydomonas; diphenylcyclooctyne-tetraethylene glycol-active ester and macrophage membrane fragments are mixed and incubated to obtain diphenylcyclooctyne-modified macrophage membrane fragments; the azide-modified Chlamydomonas and diphenylcyclooctyne-modified macrophage membrane fragments are mixed and subjected to click chemistry reaction to obtain a green algae microrobot.
[0007] Preferably, the concentration of Chlamydomonas in the Chlamydomonas suspension is 1×10 7 pieces / mL.
[0008] The present invention also provides the use of the green algae microrobot described in the above solution or the microalgae microrobot prepared by the preparation method in the preparation of antibacterial products.
[0009] Preferably, the antibacterial method includes photodynamic antibacterial method.
[0010] Preferably, the antimicrobial activity includes antibacterial activity.
[0011] Preferably, the bacteria include Escherichia coli and / or Staphylococcus aureus.
[0012] Preferably, the microalgae microrobot performs antibacterial activities by generating reactive oxygen species.
[0013] The present invention also provides an antibacterial product, comprising the green algae microrobot and the lighting device described in the above solution.
[0014] The present invention also provides a non-therapeutic antibacterial method for the green algae microrobots described in the above scheme, comprising: irradiating white light to aggregate the green algae microrobots to the antibacterial site; and then irradiating the aggregated green algae microrobots with 637nm laser for antibacterial effect.
[0015] The present invention provides a green algae microrobot (MCR), comprising Chlamydomonas, the surface of which is modified with macrophage membrane fragments. The present invention is based on the phototaxis of Chlamydomonas, and the green algae microrobot also has phototaxis. Under white light irradiation, the green algae microrobot will move toward the light source to achieve enrichment and continuously produce oxygen through photosynthesis. Under 637nm laser irradiation, the chlorophyll molecules of Chlamydomonas in the green algae microrobot will be stimulated to undergo photochemical conversion, generating dihydrochlorins with photodynamic activity. In addition, under laser irradiation, dihydrochlorins can produce reactive oxygen, thereby achieving efficient photodynamic antibacterial effect. In addition, the present invention utilizes the ability of macrophage fragments to recognize pathogens to promote the targeted delivery of the green algae microrobot, thereby further improving the antibacterial effect. The green algae microrobot of the present invention has a highly efficient antibacterial effect, with antibacterial effects against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus reaching 96.5% and 97.4%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0017] Figure 1 Characterization of Chlamydomonas reinhardtii, the raw material used to construct the green algae microrobot; a is a bright field image (I) and the corresponding fluorescence image (II) of Chlamydomonas reinhardtii; b is a pseudo-color scanning electron microscope image of Chlamydomonas reinhardtii, scale bar: 3 μm; c is a histogram of the short axis length of Chlamydomonas reinhardtii; d is a histogram of the long axis length of Chlamydomonas reinhardtii; e is a histogram of the flagella length of Chlamydomonas reinhardtii;
[0018] Figure 2 The morphological characterization of macrophages and their fragments; a is a bright field image of macrophages; b is a bright field image of macrophage membrane fragments; c is a fluorescence image of FITC-modified macrophage membrane fragments;
[0019] Figure 3 The morphology of the green algae microrobot is characterized; a is the fluorescence image of the green algae microrobot; b is the modification effect of macrophage membrane fragments on the green algae microrobot at different initial concentrations;
[0020] Figure 4To determine the dissolved oxygen of green algae microrobots under different treatments;
[0021] Figure 5 Fluorescence images of reactive oxygen species produced by green algae microrobots under different treatments; scale bar: 10 μm;
[0022] Figure 6 The results of the antibacterial ability identification of green algae microrobots; fluorescence images and scanning electron microscopy images of (a) Escherichia coli and (b) Staphylococcus aureus stained with DMAO (green, live bacteria) and EthD-III (red, dead bacteria) under different treatments, scale bar: 10 μm. DETAILED DESCRIPTION
[0023] The present invention provides a green algae microrobot, comprising Chlamydomonas with macrophage membrane fragments modified on the surface.
[0024] The green algae microrobot of the present invention is a phototactic membrane camouflaged green algae robot.
[0025] The present invention is based on the phototaxis of Chlamydomonas. Green algae microrobots also have phototaxis. Since the white light spectrum covers the absorption peak of chlorophyll, it can promote the efficient photosynthesis of green algae microrobots to produce oxygen. Chlamydomonas is rich in photosynthetic pigments such as chlorophyll, which is a natural photosensitizer. Under the irradiation of a laser with a wavelength of 637nm, the chlorophyll a in the MCRS can efficiently absorb light energy and excite electrons. These electrons leak when they are transferred through the photosynthetic electron transport chain and react with the surrounding oxygen molecules to produce superoxide (.O2 - Subsequently, under the catalysis of superoxide dismutase (SOD), chlorophyll a, a rich source of photosynthetic pigments in .O2 Chlamydomonas, can efficiently absorb light energy and excite electrons when irradiated by a laser with a wavelength of 637nm. These electrons leak when passing through the photosynthetic electron transport chain and react with surrounding oxygen molecules to generate superoxide (.O2 - ). Subsequently, under the catalysis of superoxide dismutase (SOD), O2 - It is converted into hydrogen peroxide (H2O2) through the Mehler reaction. Further, H2O2 generates hydroxyl radicals (.OH) through the Fenton reaction. At the same time, the excited chlorophyll molecules transfer energy to the ground state oxygen molecules during their own energy decay, prompting them to convert into singlet oxygen (1O2). These reactive oxygen species damage the bacterial cell membrane and DNA through oxidation, and cause protein leakage and a decrease in glutathione content, significantly weakening the antioxidant defense ability of bacteria, thereby achieving an antibacterial effect. In addition, the average size of Chlamydomonas is: the long axis length is 6.5μm, the short axis length is 4.9μm, and the flagella length is 10.6μm. As a single-cell green algae, it itself has low toxicity and degradability.
[0026] The present invention utilizes the ability of macrophage fragments to recognize pathogens, promoting targeted delivery of green algae microrobots, thereby further enhancing antibacterial efficacy. Furthermore, by modifying Chlamydomonas with macrophage membrane fragments, the present invention improves its biocompatibility. The green algae microrobots of the present invention possess excellent biocompatibility.
[0027] As an embodiment, the size of the macrophage membrane fragments is nanometer-scale; the macrophage fragments are extracted from macrophages; the present invention has no special limitation on the method for extracting the macrophage fragments, and conventional methods in the art can be used.
[0028] As an embodiment, macrophage membrane fragments are modified onto the surface of Chlamydomonas by click chemistry.
[0029] The present invention also provides a method for preparing the green algae microrobot described in the above scheme, comprising the following steps:
[0030] The Chlamydomonas suspension and azide-tetraethylene glycol-active ester are mixed and incubated to obtain azide-modified Chlamydomonas; diphenylcyclooctyne-tetraethylene glycol-active ester and macrophage membrane fragments are mixed and incubated to obtain diphenylcyclooctyne-modified macrophage membrane fragments; the azide-modified Chlamydomonas and diphenylcyclooctyne-modified macrophage membrane fragments are mixed and subjected to click chemistry reaction to obtain a green algae microrobot.
[0031] The invention mixes a Chlamydomonas suspension with an azide-tetraethylene glycol-active ester and incubates the mixture to obtain the azide-modified Chlamydomonas.
[0032] As an embodiment, the concentration of Chlamydomonas in the Chlamydomonas suspension is 1×10 7 / mL; the Chlamydomonas suspension is obtained by suspending Chlamydomonas in deionized water (DI); the volume ratio of the Chlamydomonas suspension and the azide-tetraethylene glycol-active ester is 1mL:4μL; the azide-tetraethylene glycol-active ester is used as a click chemistry tool to achieve azide modification on the surface of Chlamydomonas; the incubation time is 1h; the incubation temperature is 20-30°C, further 25-26°C; after obtaining the azide-modified Chlamydomonas, the azide-modified Chlamydomonas is also washed to remove unreacted click chemistry tools; the number of washings is 3 times.
[0033] The invention mixes diphenylcyclooctyne-tetraethylene glycol-active ester and macrophage membrane fragments, and incubates them to obtain diphenylcyclooctyne-modified macrophage membrane fragments.
[0034] As an embodiment, the volume ratio of the diphenylcyclooctyne-tetraethylene glycol-active ester and the macrophage membrane fragments is 4 μL:1 mL; the incubation time is 1 hour; the incubation temperature is 20-30°C, further 25-26°C; the diphenylcyclooctyne-tetraethylene glycol-active ester is used as a click chemistry tool to achieve diphenylcyclooctyne modification of macrophage membrane fragments; after obtaining the diphenylcyclooctyne-modified macrophage membrane fragments, the diphenylcyclooctyne-modified macrophage membrane fragments are also washed to remove unreacted click chemistry tools; the number of washings is 3 times.
[0035] After obtaining the azido-modified Chlamydomonas and the diphenylcyclooctyne-modified macrophage membrane fragments, the present invention mixes the azido-modified Chlamydomonas and the diphenylcyclooctyne-modified macrophage membrane fragments and performs a click chemistry reaction to obtain a green algae microrobot;
[0036] As an embodiment, the temperature of the click chemistry reaction is 25°C; the time of the click chemistry reaction is 1 hour; the rotation speed of the click chemistry reaction is 200 rpm; the click chemistry reaction conditions of the present invention can promote the occurrence of the click chemistry reaction; after the click chemistry reaction, the reaction product of the click chemistry reaction is also included in the cleaning; the number of cleaning times is 3 times.
[0037] The present invention also provides the use of the green algae microrobot described in the above solution or the microalgae microrobot prepared by the preparation method in the preparation of antibacterial products.
[0038] As an embodiment, the antibacterial method includes photodynamic antibacterial method.
[0039] As an embodiment, the antimicrobial activity includes antibacterial activity.
[0040] In one embodiment, the bacteria include Escherichia coli and / or Staphylococcus aureus.
[0041] As an embodiment, the microalgae microrobot performs antibacterial activities by generating reactive oxygen species.
[0042] As an embodiment, the antibacterial product includes an antibacterial drug.
[0043] The present invention also provides an antibacterial product, comprising the green algae microrobot and the lighting device described in the above solution.
[0044] As an embodiment, the illumination device can provide a white light source and a 637nm laser; the illumination device uses a power density of 0.5W / cm 2 The light emitting diode is used as the light source.
[0045] The antibacterial product device of the present invention is simple, and only requires a white light source and a 637nm laser to realize the photosynthesis oxygen production of the microalgae micromachine and generate active oxygen for photodynamic antibacterial.
[0046] The present invention also provides a non-therapeutic antibacterial method for the green algae microrobots described in the above scheme, comprising: irradiating white light to aggregate the green algae microrobots to the antibacterial site; and then irradiating the aggregated green algae microrobots with 637nm laser for antibacterial effect.
[0047] In the present invention, under white light irradiation, the green algae microrobots can, through the synergistic effect of their photoreceptors and flagella, move toward the light source to achieve enrichment and form a green algae microrobot group, and continuously produce oxygen through photosynthesis, achieving in situ oxygen production, effectively alleviating the hypoxic state of bacteria, and creating favorable conditions for subsequent antibacterial treatment. Subsequently, under 637nm laser irradiation, the Chlamydomonas chlorophyll molecules in the green algae microrobots will be stimulated to undergo photochemical conversion to generate dihydrochlorins with photodynamic activity. In addition, under laser irradiation, dihydrochlorins can produce reactive oxygen species, thereby achieving efficient photodynamic antibacterial. It has been verified that the method of the present invention can effectively inhibit the growth of Staphylococcus aureus and Escherichia coli.
[0048] In order to further illustrate the present invention, a green algae microrobot provided by the present invention, its preparation method and application are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.
[0049] Example 1 A green algae microrobot (MCR)
[0050] The preparation steps are as follows:
[0051] 1. Cultivation and Characterization of Chlamydomonas
[0052] The Chlamydomonas solution used in this example was purchased from the China Algae Seed Bank. After receiving the algae seeds, first shake the algae solution in the test tube thoroughly to ensure that the Chlamydomonas cells are evenly dispersed. Subsequently, the Chlamydomonas solution is placed in a light incubator with a day and night cycle for culture. During the culture process, strictly controlled culture parameters are used: the light intensity is stably maintained at 1000lx, the light and dark cycle is accurately set to a 12h light / 12h dark cycle mode, and the culture temperature is constant at 25°C. At the same time, the culture bottle is shaken regularly 3 times a day, each time for 2 minutes, to ensure that the algae cells obtain sufficient nutrition and light. Under normal growth conditions, such as Figure 1 As shown, its major axis length is 6.5 μm and its minor axis length is 4.9 μm. It is particularly noteworthy that two flagella structures of equal length were observed at the front end of the cell, and their average length was measured to be 10.6 μm.
[0053] 2. Extraction and Characterization of Macrophage Membranes
[0054] First, the growth status of macrophages was observed under an inverted microscope. Subsequently, macrophages cultured to the logarithmic growth phase were washed once with PBS buffer to remove residual culture medium. The cells were scraped with a cell scraper and centrifuged at 1500 rpm for 5 minutes to collect the macrophage pellet. Next, 1 mL of pre-chilled membrane protein extraction reagent A (containing phenylmethyl yellow fluoride) was added to the cell pellet, and after resuspending the cells, the mixture was incubated in an ice bath for 15 minutes to inhibit protease activity and protect the integrity of membrane proteins. Subsequently, the cells were disrupted using a liquid nitrogen-room temperature freeze-thaw cycle, repeated three times until the cell disruption rate was greater than 70%. The disrupted cell solution was then centrifuged (700g for 10 minutes), and the supernatant was collected to remove cell nuclei and unbroken cell debris. Finally, the supernatant was centrifuged again (14000g for 30 minutes) to obtain a highly pure macrophage membrane fragment pellet. The entire experimental process was performed at a low temperature of 4°C to ensure the structural integrity and biological activity of the membrane proteins. Since the size of the extracted macrophage membrane fragments is as low as nanometer level, in order to facilitate subsequent observation, e.g. Figure 2 As shown, macrophage membrane fragments were labeled with fluorescein isothiocyanate (FITC) to make them appear green fluorescent.
[0055] 3. Preparation and characterization of MCR
[0056] To prepare membrane-camouflaged green algae microrobots (MCRs), Chlamydomonas was first washed three times with deionized water (DI) to remove the culture medium residue. The washed Chlamydomonas was resuspended in DI to obtain a concentration of 1×10 7 / mL suspension. Next, 1mL of Chlamydomonas suspension was mixed with 4μL of azide-tetraethylene glycol-active ester (click chemistry tool) and incubated at room temperature for 1h to achieve azide modification on the CR surface. At the same time, 1mL of cell membrane fragments was modified with 4μL of diphenylcyclooctyne-tetraethylene glycol-active ester and incubated under the same conditions for 1h to introduce diphenylcyclooctyne groups. Subsequently, the modified Chlamydomonas and cell membrane fragments were washed three times respectively to remove unreacted click chemistry reagents. Finally, the diphenylcyclooctyne-modified cell membrane fragments were mixed with the azide-modified CR and incubated in a shaker at 25°C and 200rpm for 1h to promote the occurrence of click chemistry reactions. After the reaction was completed, three washes were performed again, and the MCR was finally collected and stored in PBS buffer for subsequent experiments. In order to verify the successful preparation of MCR, its morphology was characterized in detail. As Figure 3 As shown in the figure, the overlap of the two fluorescent signals observed under an inverted fluorescence microscope indicates that CR successfully binds to macrophage membrane fragments. In addition, by adjusting the initial concentration of macrophage membrane fragments, the binding rate of CR can be effectively controlled.
[0057] Test Example 1 Oxygen Generation in MCR
[0058] First, a power density of 0.5 W / cm 2 The light emitting diode was used as the light source to test the effect of different wavelengths of light on the oxygen production performance of MCR. The MCR was exposed to light of different wavelengths (red light: 620nm, blue light: 450nm, green light: 540nm, and broad spectrum white light), and the dissolved oxygen concentration was accurately measured using a dissolved oxygen meter. Figure 4 As shown in the figure, under green light irradiation, the oxygen production of the MCR was relatively low, only 9.67 mg / L. However, under white light irradiation, the oxygen production of the MCR reached 11.73 mg / L, significantly higher than the oxygen production under blue light (10.48 mg / L) and red light (10.21 mg / L). To ensure the accuracy and reliability of the experimental data, all measurements were conducted at a constant temperature (25°C) to eliminate the possible influence of temperature fluctuations on the experimental results.
[0059] Test Example 2: Active Oxygen Generation in MCR
[0060] In the experiment, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe was used to detect the level of reactive oxygen species (ROS) in MCR cells. DCFH-DA itself has no fluorescence. After entering the cells, it can be oxidized by ROS to produce DCF with green fluorescence. Its fluorescence intensity is positively correlated with the amount of ROS produced, thus being able to directly reflect the level of ROS in CR cells. Figure 5 As shown in the figure, under 637nm laser irradiation, a significant green fluorescence signal was observed in MCR cells, indicating the massive generation of ROS. In contrast, almost no green fluorescence signal was detected in the untreated group, and only a weak green fluorescence was observed in the white light irradiation group. This result confirms that 637nm laser can effectively stimulate the photodynamic reaction in MCR cells and promote the generation of ROS, providing important experimental basis for subsequent antibacterial experiments.
[0061] Experimental Example 3: In vitro antibacterial activity of membrane-camouflaged green algae microrobots
[0062] 1. Preparation of bacterial suspension
[0063] First, use a sterile syringe to pick up single Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) colonies and transfer them to sterile test tubes containing 5 mL of bacterial culture solution (Lysogeny-Broth nutrient solution). The test tubes were placed in a constant temperature shaker at 37 ° C and 200 rpm for 4 hours. This culture condition provides a stable and suitable growth environment for bacteria, which can promote the proliferation of bacteria. Next, the obtained bacterial solution is centrifuged to precipitate the bacteria and washed with PBS to remove metabolites. After washing, the colony is diluted with PBS to obtain a final concentration of 5×10 4 / μL of bacterial solution.
[0064] 2. To explore the antibacterial effect of MCR under different treatment conditions, a series of experiments were carried out using E. coli and S. aureus as examples. First, the effects of different treatment groups on bacterial survival were evaluated by live / dead staining experiments. Compared with the control group, 637nm laser irradiation group, MCR and bacteria co-culture group, and MCR combined with 637nm laser irradiation group (mainly green fluorescence), the proportion of dead bacteria (red fluorescence) was significantly higher when 637nm laser irradiation was used after sufficient oxygen was generated by white light irradiation, indicating that this treatment method has the strongest antibacterial effect. Further, scanning electron microscopy was used to observe the changes in the surface structure of E. coli and S. aureus in different treatment groups. The results showed that in the control group, 637nm laser irradiation group, and MCR and bacteria co-culture group, the bacterial surface was smooth and intact, indicating that these treatments did not cause significant damage to the physical structure of the bacteria. However, in the MCR combined with 637nm laser treatment group, some bacterial membranes showed obvious wrinkling and collapse; while in the MCRS combined with 637nm laser treatment group, wrinkling and collapse appeared on all bacterial surfaces, further confirming that this treatment method seriously damaged the bacterial structure. In general, MCR aggregated to form MCRS under white light irradiation and then combined with laser irradiation can effectively inhibit bacterial proliferation, and the survival rates of E. coli and S. aureus dropped to 3.5% and 2.6%, respectively. Figure 6 shown.
[0065] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.
Claims
1. A green algae microrobot, characterized in that: These include Chlamydomonas whose surfaces are decorated with fragments of macrophage membranes.
2. The method for preparing the green algae microrobot according to claim 1, comprising the following steps: The Chlamydomonas suspension and azide-tetraethylene glycol-active ester are mixed and incubated to obtain azide-modified Chlamydomonas; mixing diphenylcyclooctyne-tetraethylene glycol-active ester and macrophage membrane fragments, and incubating the mixture to obtain diphenylcyclooctyne-modified macrophage membrane fragments; Azide-modified Chlamydomonas and diphenylcyclooctyne-modified macrophage membrane fragments were mixed and subjected to click chemistry reaction to obtain green algae microrobots.
3. The preparation method according to claim 2, characterized in that The concentration of Chlamydomonas in the Chlamydomonas suspension is 1×10 7 pieces / mL.
4. Use of the green algae microrobot according to claim 1 or the microalgae microrobot prepared by the preparation method according to claim 2 or 3 in the preparation of antibacterial products.
5. The use according to claim 4, characterized in that The antibacterial method includes photodynamic antibacterial method.
6. The use according to claim 4, characterized in that The antimicrobial includes antibacterial.
7. The use according to claim 6, characterized in that The bacteria include Escherichia coli and / or Staphylococcus aureus.
8. The use according to claim 4, characterized in that The microalgae microrobot is antibacterial by generating active oxygen.
9. An antibacterial product, characterized in that: It comprises the green algae microrobot and the lighting device as described in claim 1.
10. A non-therapeutic antibacterial method based on the green algae microrobot according to claim 1, characterized in that: include: irradiating white light to cause the green algae microrobots to gather at the antibacterial site; The aggregated green algae microrobots are then irradiated with 637nm laser for antibacterial effects.