CN114634877B - Synthesis method of biological photosynthetic organelle, product and application thereof - Google Patents
Synthesis method of biological photosynthetic organelle, product and application thereof Download PDFInfo
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Abstract
The invention discloses a synthesis method of a biological photosynthetic cell device, which comprises the following steps: centrifugally collecting the microalgae suspension, adding a polyelectrolyte aqueous solution with surface modification, depositing polyelectrolyte on the surfaces of the microalgae by stirring, and centrifugally discarding the supernatant to obtain the microalgae with surface charge modification; adding the microalgae with the modified surface charge into an overnight hydrolyzed nano silicon dioxide particle precursor aqueous solution, stirring, centrifuging, and discarding the supernatant to obtain the microalgae with the surface modified nano silicon dioxide particles as a biological photosynthetic cell organelle. The invention also provides a biological photosynthetic cell device obtained by the synthesis method and application of the biological photosynthetic cell device in providing oxygen for mammalian cells in an anaerobic environment. The synthesized biological photosynthetic cell device generates oxygen through photosynthesis of the biological photosynthetic cell device in an anaerobic environment, so that the mammalian cells have good viability.
Description
Technical Field
The invention relates to the technical field of cell interface engineering, in particular to a synthesis method of a biological photosynthetic cell organ, a product and application thereof.
Background
Oxygen is the basis of human life activities, and human beings hardly survive under anaerobic conditions (e.g., deep sea, outer space, etc.). Since cells consume a lot of energy during growth and division or while maintaining various cell functions (e.g., macromolecular biosynthesis, cell movement, balance control of membrane potential and osmotic pressure, transmembrane and intracellular transport, and information processing). Typically, cells acquire a large amount of energy for their needs by aerobic respiration (including three stages of glycolysis, oxidative decarboxylation of pyruvate and citric acid cycle). However, under anaerobic conditions, cells can only perform lactic acid fermentation to obtain a small amount of ATP, and the survival requirement of the cells cannot be met. Microalgae are typical photoautotrophic organisms in nature, such as chlamydomonas reinhardtii, chlamydomonas noctiluca, diatoms, chlorella and the like, and can survive under anaerobic conditions by converting water and carbon dioxide into oxygen and biomass through photosynthesis. Thus, whether the autotrophic function of the photosynthetic organism can be transferred to the mammalian cell increases the survival rate of the mammalian cell in an anaerobic environment.
The organelle is used as an indispensable functional interval in living cells, so that the cells can accurately regulate a series of key biological processes. The artificial organelle is a natural organelle analogue synthesized by an artificial means, can be introduced into cells to replace or supplement specific biochemical functions of the cells, and provides a new way for correcting dysfunctional intracellular processes and adding new functions to a biological system. The construction of artificial organelles requires the establishment of a series of stringent requirements, such as stability in complex biological environments, biocompatibility, permeability for mass exchange, and maintenance of complete biological functions, to be provided between micro-segments. At present, the artificial organelle is mainly introduced into cells by means of endocytosis, electroporation, cell extrusion, nanoneedle, microinjection and the like. However, external force modes (such as electroporation, cell extrusion, nanoneedle and microinjection) often cause certain damage to cells, and the size of the opening has limitations on the introduction of artificial organelles. Therefore, the endocytosis can be utilized as a mode for widely introducing artificial organelles, and the good biocompatibility is advantageous. However, the endocytic efficiency depends on the heterogeneous endocytic activity of the cells themselves, and is not applicable to cells with limited endocytic capacity. There are a number of different endocytic pathways in eukaryotic cells, but the primary endocytic pathway in the life process of the cell (including nutrient absorption, signal transduction, synaptic vesicle cycling, and immune response) is clathrin mediated endocytosis. Research shows that the silica nanoparticles can enter cells through clathrin-mediated endocytosis and have good tolerance in severe acidic and alkaline environments.
Disclosure of Invention
The invention provides a synthesis method of a biological photosynthetic cell organ, a product and application thereof, and the synthesis method can generate oxygen through photosynthesis of the biological photosynthetic cell organ in an anaerobic environment, so that mammalian cells have good viability.
The invention provides the following technical scheme:
a method of synthesizing a biological photosynthetic organelle, the method comprising:
(1) Centrifugally collecting the microalgae suspension, adding a polyelectrolyte aqueous solution with surface modification, depositing polyelectrolyte on the surfaces of the microalgae by stirring, and centrifugally discarding the supernatant to obtain the microalgae with surface charge modification;
(2) Adding the microalgae with the modified surface charges prepared in the step (1) into an overnight hydrolyzed nano silicon dioxide particle precursor aqueous solution, stirring, centrifuging, and discarding the supernatant to obtain the microalgae with the surface modified nano silicon dioxide particles as a biological photosynthetic cell organelle.
In step (1), the polyelectrolyte is selected from polydimethyldiallylammonium chloride, polyethylenimine or polyallylamine hydrochloride. In the step (1), the microalgae are chlorella, chlamydomonas reinhardtii or chlamydomonas noctiluca.
Preferably, in the step (1), the cationic polyelectrolyte aqueous solution is polyethyleneimine, the concentration of which is 100-300mg/L, and the pH is adjusted to 6.5-7.5; the microalgae concentration is 1×10 6 -5×10 6 individual/mL; the speed of the centrifugation is 2000-5000rpm, the stirring speed is 200-500rpm, and the reaction time of the stirring is 10-20min.
In step (2), the aqueous nanosilica particle precursor solution is selected from sodium silicate, tetraethyl orthosilicate, or 3-mercaptopropyl trimethoxysilane.
Preferably, in the step (2), the nano silica particle precursor aqueous solution is a sodium silicate aqueous solution, the concentration of which is 5-20mmol/L, and the pH is adjusted to 6.5-7.5; the centrifugal speed is 2000-5000rpm, the stirring speed is 200-500rpm, and the stirring reaction time is 15-30min.
In order to ensure that the biological photosynthetic organelle keeps complete biological functions in the process of surface modification of nano silicon dioxide, the concentration and pH of polyelectrolyte aqueous solution and nano silicon dioxide particle precursor aqueous solution in the synthesis method are adjusted to proper ranges, and the concentration of microalgae in the modification process and the reaction time in different solutions are also adjusted. In order to ensure that the biological photosynthetic organelles can be safely and effectively introduced into mammalian cells, the concentration of the mammalian cells and microalgae and the incubation time of the mammalian cells and microalgae in the synthesis method are optimized.
The invention also provides a biological photosynthetic cell organ obtained by the synthesis method.
The photosynthetic organelle provides oxygen to mammalian cells in an anaerobic environment. The photosynthetic organelle of the organism is incubated with mammalian cells in a culture medium, and the photosynthetic organelle of the organism is introduced into the mammalian cells. The mammalian cells are human epidermal keratinocytes, human myocardial stem cells, human normal hepatocytes or human hepatoma cells. The culture medium is a high sugar Boke modified eagle (Dulbecco modified Eagle medium, DMEM) culture medium for culturing mammalian cells.
Preferably, the mammalian cell concentration is 1X 10 5 -2×10 5 The microalgae concentration is 1×10 per mL 5 -5×10 5 And (3) each mL, wherein the common incubation time is 2-12h.
Further preferably, in the step (1), the concentration of the aqueous solution of polyethyleneimine is 150mg/L and the pH is adjusted to 7.0, and the microalgae is Chlorella, the concentration of which is 4×10 6 The centrifugal speed is 5000rpm, the low-speed stirring speed is 400rpm, and the stirring reaction time is 15min; in the step (2), the concentration of the aqueous sodium silicate solution was 10mmol/L, and the pH was adjusted to 7.0, the centrifugation speed was 5000rpm, the low-speed stirring speed was 400rpm, and the stirring reaction time was 30min. When the biological photosynthetic cell organelle obtained by the synthesis method is applied in an anaerobic environment, oxygen is provided for mammalian cells, and the concentration of the mammalian cells is 1 multiplied by 10 5 The microalgae concentration is 2×10 per mL 5 And (3) per mL, wherein the common incubation time is 4h.
The optimal concentration of the cationic polyelectrolyte polyethyleneimine aqueous solution is selected to be 150mg/L, and if the concentration of the cationic polyelectrolyte is too low, the cationic polyelectrolyte aqueous solution can be deposited on the surface of microalgae too little, so that nano silicon dioxide particles cannot be modified on the surface. Too high a concentration of cationic polyelectrolyte can affect the biological activity of the microalgae, rendering it incapable of photosynthesis. The optimal concentration of the precursor aqueous solution of the nano silicon dioxide particles is 10mmol/L, and the precursor with too low concentrationThe solution causes little deposition of silica particles on the microalgae surface, but excessive growth of silica particles at too high a concentration can affect the biological function of the microalgae itself. The microalgae concentration is preferably 4×10 6 The nanometer silicon dioxide particles can be uniformly modified on the surface of a single microalgae per mL. In order to maintain the biological function of the biological photosynthetic organelle, the pH value of the precursor aqueous solution of the cationic polyelectrolyte and the nano silicon dioxide particles is regulated to about 7.0, the optimal reaction time is respectively 15min and 30min, and the nano silicon dioxide particles are uniformly modified on the surface of the precursor aqueous solution of the cationic polyelectrolyte and the nano silicon dioxide particles on the premise of not affecting the biological activity of the microalgae. In the process of introducing biological photosynthetic organelles into mammalian cells, the optimal concentration of mammalian cells is 1×10 5 Mammalian cells can be spread in culture dish to show monolayer growth, and the optimal concentration of photosynthetic organism organelle is 2×10 5 The average introduction of 1-2 photosynthetic organism organelles in mammalian cells growing in a single layer can be ensured per mL, and the effective uptake of the photosynthetic organism organelles by the mammalian cells can be ensured by incubating for 4h.
The technical concept of the invention is to select microalgae (which can be chlorella, chlamydomonas reinhardtii or chlamydomonas noctiluca) with photosynthesis as a biological photosynthetic cell device, modify cationic polyelectrolyte (which can be polydimethyl diallyl ammonium chloride, polyethylenimine or polyallylamine hydrochloride) on the surface of the microalgae through electrostatic adsorption to provide nucleation sites for the growth of nano silicon dioxide particles, and then select a precursor aqueous solution of the nano silicon dioxide particles (which can be sodium silicate, tetraethyl orthosilicate, 3-mercaptopropyl trimethoxysilane and the like) to modify the nano silicon dioxide particles on the surface of the microalgae. The endocytosis biological photosynthetic cell organ utilizing mammal cell can be introduced safely and effectively and maintain good biological function therein, and provides autotrophic function for mammal cell which is heterotrophic.
The method for introducing the biological photosynthetic cell organelle into the mammalian cells by modifying the nano silicon dioxide particles on the surfaces of the microalgae provided by the invention ensures that the biological photosynthetic cell organelle keeps complete biological functions in the mammalian cells, and can carry out photosynthesis in an anaerobic environment to provide oxygen for the mammalian cells, so that the mammalian cells have good viability, thereby solving the survival problem of the mammalian cells in the anaerobic environment.
Drawings
In fig. 1, a and E are scanning electron microscope images of natural microalgae and biological photosynthetic organelles, respectively; b and F are respectively the X-ray electron energy spectra of natural microalgae and biological photosynthetic organelles; C-D and G-H are transmission electron microscopy images of natural microalgae and biological photosynthetic organelles, respectively.
In FIG. 2, A is the maximum optical quantum yield B of the photosynthetic system II of the natural microalgae and the photosynthetic organelles of the organism, and B is the photosynthetic oxygen production rate of the natural microalgae and the photosynthetic organelles of the organism.
A and B in FIG. 3 are transmission electron microscope images of natural microalgae and biological photosynthetic cells introduced into mammalian cells, respectively; c is the endocytic efficiency of mammalian cells to natural microalgae and biological photosynthetic organelles.
A, B and C in FIG. 4 are the cellular activities of mammalian cells, mammalian cells mixed with natural microalgae, and mammalian cells introduced into a photosynthetic organelle of a living organism in an anaerobic environment, respectively.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the detailed description is presented by way of example only and is not intended to limit the scope of the invention.
Example 1 preparation of biological photosynthetic organelles Using Chlorella and introduction of human immortalized epidermal cells
Placing Chlorella cells at 25deg.C with illumination intensity of 40 μm -2 s -1 Is incubated with TAP (Tris/acetate/phosphate) medium in an illuminated incubator. After incubation for 24h, the OD was measured at 750nm using an ultraviolet spectrophotometer. Selection of cells in late logarithmic growth phase (OD 750 About 1.0) was centrifuged at 5000rpm for 5min, and the pellet was collected by discarding the supernatant and washed three times with PBS buffer. Resuspending Chlorella in polyethylenimine solution (150 mg/L, pH 7.0), diluting to ChlorellaCell concentration of 4X 10 6 After incubation at 400rpm for 15min with low-speed stirring, the chlorella cells were collected by centrifugation at 5000rpm for 5min, the supernatant was discarded, and washed twice with PBS buffer. Then dispersed in 10mmol/L sodium silicate aqueous solution (pH 7.0) of overnight hydrolysis, after low-speed stirring and incubation at 400rpm for 30min, centrifuged at 5000rpm for 5min, the supernatant was discarded, and the supernatant was collected and washed three times with PBS buffer to obtain the biological photosynthetic organelle. Human immortalized epidermal cells (HaCaT) were plated in 35mm glass bottom dishes at a cell concentration of 1X 10 in each dish 5 Each mL, in DMEM high-sugar medium (containing 10% fetal calf serum) in 5% CO 2 Is cultured at 37℃in a cell culture incubator. After the cells grow to about 60% of the bottom area of the dish, absorbing the cell culture medium, washing with PBS buffer solution for 2 times, respectively dispersing natural chlorella cells and biological photosynthetic cell organelles in the culture medium, adding the culture dish, and maintaining the concentration of chlorella cells at 2×10 5 Incubation for 4h at each mL.
TAP medium formulation (1L): NH (NH) 4 Cl 0.375g；MgSO 4 ·7H 2 O 0.1g；CaCl 2 ·7H 2 O 0.05g；K 2 HPO 4 1.08g；KH 2 PO 4 0.54g; tris hydrochloride 2.42g; hutner's trace element 1mL; glacial acetic acid 1mL, pH was adjusted to 7.0, and autoclaved at 121℃for 20min.
As shown in figure 1, the natural chlorella is compared with a scanning electron microscope image, an X-ray electron energy spectrum and a transmission electron microscope image of a biological photosynthetic cell device, the surface of the natural chlorella is smooth and has no silicon element signal, and the surface of the biological photosynthetic cell device is obviously modified by nano particles and has obvious silicon element signal; from FIG. 2, the maximum photochemical quantum yield (Fv/Fm) of the natural chlorella cells and the biological photosynthetic organelles are maintained in a stable state within 72 hours, and the photosynthetic oxygen production rate of the natural chlorella cells and the biological photosynthetic organelles within 72 hours are almost the same, which proves that the biological photosynthetic organelles have complete photosynthetic activity; as shown in FIG. 3, the natural chlorella is hardly phagocytized at the periphery of the human immortalized epidermal cells, the average endocytosis efficiency is 0.67%, the human immortalized epidermal cells have obvious endocytosis to the photosynthetic organelles of the organism, the photosynthetic organelles of the organism also keep a complete form, and the average endocytosis efficiency reaches 88.17% according to data statistics analysis.
Example 2 survival of human immortalized epidermal cells introduced into biological photosynthetic organelles in an anaerobic environment
Human immortalized epidermal cells (HaCaT) were plated in 35mm glass bottom dishes at a cell concentration of 1X 10 in each dish 5 Each mL, in DMEM high-sugar medium (containing 10% fetal calf serum) in 5% CO 2 Is cultured at 37℃in a cell culture incubator. After the cells grow to about 60% of the bottom area of the dish, absorbing the cell culture medium, washing with PBS buffer solution for 2 times, respectively dispersing natural chlorella cells and biological photosynthetic cell organelles in the culture medium, adding the culture dish, and maintaining the concentration of chlorella cells at 2×10 5 Incubation for 4h at each mL. The supernatant was aspirated and the human immortalized chlorella cells adhered to the surface of the epidermal cells were purged with PBS buffer until the wash became colorless and transparent. Three groups of samples of human immortalized epidermal cells, a mixture of human immortalized epidermal cells and natural chlorella and human immortalized epidermal cells introduced into biological photosynthetic cell organelles are respectively placed in an anaerobic sealing tank, and an anaerobic gas-producing bag (0%O) 2 ,5％CO 2 ) Sealing the culture dish in an anaerobic tank, and placing in an illumination incubator at 37deg.C under illumination intensity of 40 μm -2 s -1 Culturing for 24h.
As shown in FIG. 4, the cell activity of human immortalized epidermal cells, a mixture of human immortalized epidermal cells and natural chlorella, and human immortalized epidermal cells in an anaerobic environment after being introduced into a biological photosynthetic organelle, was detected by using an Annexin V-FITC apoptosis detection kit. After human immortalized epidermal cells were cultured under anaerobic conditions, only 38.78% of the cells were still active, 13.08% had early apoptosis and 48.04% had advanced apoptosis and necrosis as quantified by flow cytometry. After co-incubation of human immortalized epidermal cells with natural chlorella, the cells are cultured under anaerobic conditions, only 20.38% of the cells are active, 11.23% of the cells undergo early apoptosis, and 68.14% of the cells undergo late apoptosis and necrosis. The human immortalized epidermal cells are cultured under anaerobic condition after being introduced into biological cells for co-incubation, and have 65.93 percent of activity, 11.25 percent of early apoptosis and 22.42 percent of late apoptosis and necrosis. From this, it was demonstrated that the biological activity of mammalian cells in an anaerobic environment was significantly reduced, natural chlorella did not improve on the periphery of mammalian cells, and the introduction of biological photosynthetic organelles after the introduction of the biological photosynthetic organelles could increase the survival rate in an anaerobic environment.
The foregoing detailed description of the preferred embodiments and advantages of the invention will be appreciated that the foregoing description is merely illustrative of the presently preferred embodiments of the invention, and that no changes, additions, substitutions and equivalents of those embodiments are intended to be included within the scope of the invention.
Claims (6)
1. A method of synthesizing a photosynthetic organelle of a living organism, the method comprising:
(1) Centrifugally collecting chlorella suspension, adding a polyethyleneimine aqueous solution, depositing polyethyleneimine on the surface of the microalgae by stirring, and centrifugally discarding the supernatant to obtain the chlorella with modified surface charge;
(2) Adding the chlorella with modified surface charge prepared in the step (1) into an overnight hydrolyzed sodium silicate aqueous solution, stirring, centrifuging, and discarding the supernatant to obtain microalgae with modified surface of sodium silicate as a biological photosynthetic organelle;
the concentration of the polyethyleneimine aqueous solution is 150mg/L, and the concentration of the sodium silicate aqueous solution is 10 mmol/L.
2. A biological photosynthetic organelle obtained by the synthetic process of claim 1.
3. Use of the biological photosynthetic organelle according to claim 2 in an anaerobic environment, wherein the biological photosynthetic organelle provides oxygen to mammalian cells in the anaerobic environment.
4. Use of a biological photosynthetic organelle according to claim 3 in an anaerobic environment, wherein the biological photosynthetic organelle is co-incubated with mammalian cells in a culture medium to introduce the biological photosynthetic organelle into the mammalian cells.
5. The use of a biological photosynthetic organelle according to claim 4 in an anaerobic environment wherein the mammalian cell concentration is 1 x 10 5 -2×10 5 The chlorella concentration is 1X 10 per mL 5 -5×10 5 And (3) per mL, wherein the common incubation time is 2-12h.
6. The use of a photosynthetic organelle according to claim 3 in an anaerobic environment, wherein the mammalian cells are human epidermal keratinocytes, human cardiac stem cells, human normal hepatocytes or human hepatoma cells.
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