Preparation method of light-driven nano plant flower transmission powder

Abstract

The invention relates to a preparation method of light-driven nano plant flower-transmission powder. The preparation method comprises the following steps: mixing a chitosan solution with a PLGA solution, adding a stabilizer, performing ultrasonic emulsification, adding a cross-linking agent, performing constant-temperature dispersion, performing centrifugal washing, and performing freeze drying to obtain porous chitosan-PLGA particles; dispersing the porous chitosan-PLGA particles in PBS (Phosphate Buffer Solution), adding sulfhydrylated SLG, oscillating and dialyzing to obtain functional particles of which the surfaces are grafted with targeting groups; mixing the porous chitosan-PLGA particles with the dried pollen, and oscillating in a vacuum adsorption device; and adding a photo-thermal response material dispersion liquid, continuing vacuum adsorption, and packaging a PLGA shell layer. Light energy is absorbed by the photo-thermal response material and then converted into heat energy, thermophoresis of a surrounding medium is triggered, the nano-particle material is pushed to move directionally, namely surface charges are generated under illumination, controllable movement is achieved through an external electric field or a self potential difference, and the photo-thermal response material can respond to the surface of the nano-particle material according to the local illumination intensity difference. And the nano-particle material is guided to move along the light intensity gradient, so that targeted pollination is realized.

Description

Technical Field

[0001] The present invention relates to a pollenizer, in particular to a method for preparing a light-driven nano-plant pollenizer, and belongs to the technical field of agricultural pollination. Background Art

[0002] Bee pollination is a core component of global ecological and agricultural security, but it currently faces severe challenges from a sharp decline in populations and degradation of its functions. Protecting bees requires a multifaceted approach, including reducing reliance on pesticides, restoring habitats, and promoting ecological agriculture. Technological innovations, such as precision pollination systems, should also be incorporated to address future risks. This issue not only concerns biodiversity but also directly impacts human food security and sustainable development. Traditional pollination methods remain the mainstream, but face multiple challenges.

[0003] 1. Insect pollination is dominant, with bees playing a central role in pollination

[0004] Insects (such as bees, butterflies, and moths) are the primary pollinators of angiosperms, with approximately 76% of food crops and 84% of plants relying on insect pollination. Bees are particularly crucial, influencing 35% of global crop yields and possessing significant economic value (for example, in China, bee pollination contributes over 300 billion yuan to 36 crops). Through cross-pollination, bees promote gene flow in plants, enhance genetic diversity, and improve plant adaptability to climate change, pests, and diseases. For example, apple trees rely on bees for pollination to produce fruit, and wild plants also rely on bees to maintain stable populations.

[0005] 2. Limitations of wind pollination and the crisis faced by bee pollination Wind-pollinated flowers (such as grasses and conifers) rely on wind to spread pollen, but this is inefficient and easily affected by climate. At the same time, wind-pollinated pollen is the main cause of hay fever (such as weeds and Artemisia pollen), which often causes health problems in areas with high allergy rates. Many countries (such as Beijing, Shanghai, and Tianjin in China) have established pollen monitoring systems to predict pollen concentrations through meteorological data and guide allergic people to protect themselves. For example, the pollen concentration in Chengdu reached 19,746 grains per thousand square millimeters in February 2025, triggering the highest level of warning.

[0006] 3. Sharp decline in pollinator populations: Pesticide overuse, habitat destruction, and climate change have led to a decline in pollinating insects like bees and wasps. A French study found that the proportion of self-pollination in modern plants (such as pansies) has increased from 50% to 80%, reflecting the pressure of insufficient pollinators.

[0007] A decline in pollinators could disrupt the food chain (for example, wasps preying on pests) and threaten agricultural security. 75% of global crops rely on animal pollination. A pollination crisis could lead to reduced fruit and vegetable production, exacerbating food security risks. Pesticides have a significant impact on bee pollination worldwide. Bees can carry viruses and bacteria through contact with diseased plants, transmitting pathogens to healthy plants during pollination. This forces farmers to use more toxic pesticides to control diseases, creating a vicious cycle. The overuse of pesticides has led to a sharp decline in bee populations and weakened natural pollination capacity.

[0008] 4. Limitations of traditional pollination methods: Passive pollination that relies on insects is inefficient and easily affected by environmental factors (such as climate and pesticides); the inability to precisely control pollination routes may exacerbate the spread of diseases.

[0009] 5. A vicious cycle in the ecological chain: Pesticide overuse, habitat fragmentation, and climate change are leading to a continuous decline in bee populations. Apis cerana cerana is now endangered in areas north of the Yellow River, with colony numbers declining by over 95%. European honey bee populations have also experienced regional collapses due to pesticide use.

[0010] The reduction in bees has forced plants to turn to self-pollination, resulting in a decrease in genetic diversity and a long-term weakening of plants' ability to adapt to environmental changes. The reduction in bees may also exacerbate pest outbreaks (such as the weakening of wasps' ability to prey on pests), further dependence on pesticides, and form a vicious cycle. Chinese honeybees perform key pollination tasks in high-altitude mountainous areas (such as the Qinghai-Tibet Plateau), and their reduction directly threatens the survival of endemic plants such as Rhodiola rosea and Meconopsis. In temperate regions (such as Europe), the intensification of agriculture has led to a decrease in wildflowers, limiting the food sources of bees. Hummingbirds, bats, etc. play an important role in the pollination of tropical plants, but habitat destruction also threatens their survival. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide a method for preparing a light-driven nano-nano plant pollenizer to solve the problem that the reduction of insects such as bees forces plants to turn to self-pollination, resulting in reduced genetic diversity, long-term weakening of plants' ability to adapt to environmental changes, and low natural pollination ability.

[0012] The technical solution of the present invention to solve the above technical problems is as follows: a method for preparing a light-driven nano-nano plant pollen agent, characterized in that it comprises the following steps: (1) mixing a chitosan solution with a PLGA solution, adding a stabilizer, ultrasonically emulsifying to form a W / O emulsion, adding a cross-linking agent and dispersing at a constant temperature, centrifugation washing, and freeze-drying to obtain porous chitosan-PLGA particles with a particle size of 200-500 nm;

[0013] Step (2): disperse the porous chitosan-PLGA particles in PBS, add thiolated SLG, shake, and dialyze to remove unreacted reagents to obtain functionalized particles with surface grafted targeting groups;

[0014] Step (3), spray drying the pollen and the pollen protectant solution to obtain dry pollen;

[0015] Step (4) is to mix the porous chitosan-PLGA particles and the dry pollen, place them in a vacuum adsorption device and shake them to allow the pollen to embed into the pores of the porous chitosan-PLGA particles; add a photothermal response material dispersion, continue vacuum adsorption, and then use a secondary emulsification method to encapsulate the PLGA shell layer to obtain a light-driven nano-plant pollen transfer agent.

[0016] The chitosan of the present invention can be dissolved in a 2% acetic acid solution to obtain a chitosan solution with a mass-to-volume solubility of 20-30%. PLGA (polylactic-co-glycolic acid, lactic acid (LA) and glycolic acid (GA) = 50:50-85:25, preferably 75:25, Mw, 10-30 kDa, preferably 20 kDa) is a linear polymer formed by alternating copolymerization of lactic acid (LA) and glycolic acid (GA) monomers via ester bonds. It is biodegradable, i.e., it is gradually degraded into lactic acid and glycolic acid in the body through ester hydrolysis, and ultimately metabolized into CO2 and H2O. It is biocompatible and FDA-approved for use in medical implants and drug delivery systems. It also has adjustable properties: by adjusting the LA / GA ratio and molecular weight (Mw), the degradation rate, mechanical strength, and drug release behavior can be controlled.

[0017] A higher LA:GA ratio results in slower degradation (LA is more hydrophobic and has a lower ester bond hydrolysis rate). At a 50:50 ratio, degradation occurs in approximately 1-2 months. At a 75:25 molar ratio, degradation occurs in approximately 4-6 months. A higher LA:GA ratio results in a longer sustained-release period (suitable for long-term controlled-release scenarios). Increasing the LA ratio increases material rigidity but reduces flexibility.

[0018] The weight-average molecular weight of the PLGA used in this invention is 20,000 Daltons. Higher molecular weights increase degradation time (longer chain breakage takes longer). High molecular weights (>30 kDa) may reduce drug encapsulation efficiency (lower porosity) and drug loading capacity. Low molecular weights (<10 kDa) are easier to process but have poor mechanical strength. 20 kDa is the equilibrium point, with a range of 10-30 kDa.

[0019] The PLGA solution is obtained by dissolving it in dichloromethane at a mass-to-volume concentration of 20-30%. Dichloromethane efficiently dissolves PLGA to form a uniform solution, facilitating emulsification processes (such as the W / O / W emulsion method). Its low boiling point (39.6°C) and high volatility allow for rapid solidification of nanoparticles. The rapid volatility of dichloromethane facilitates the efficient preparation of uniformly sized nanoparticles.

[0020] Table 1 below compares the performance parameters of different chitosan and PLGA parameters on particle degradation time, release half-life, particle hardness, and pollen encapsulation efficiency.

[0021] Table 1. Performance parameters of different chitosan and PLGA parameters affecting particle degradation time, release half-life, particle hardness, and pollen encapsulation efficiency

[0022] PLGA parameters LA=50:50, Mw=10kDa LA=75:25, Mw=20kDa Degradation time 1-2 months 4-6 months Drug release half-life 2 weeks 8 weeks Nanoparticle hardness (MPa) 8.5±1.2 12.3±1.8 Pollen encapsulation rate 70％±3.5 82％±2.7

[0023] The parameters of chitosan and PLGA of the present invention can be adjusted within a predetermined range according to the release and protection requirements to achieve precise control of the release scenario and adapt to different application needs.

[0024] Compared with traditional pollination methods, the light-driven nano-plant pollenizer of the present invention has high efficiency, reduces pest and disease carriers, has a pollen utilization rate of ≥85%, and is highly accurate. The comparative data are shown in Table 2.

[0025] Table 2. Parameter comparison with traditional pollination methods

[0026]

[0027]

[0028] On the basis of the above technical solution, the present invention can also be improved as follows.

[0029] The present invention is a light-driven nano-nano plant pollenizer as described above, further, in step (1), the volume ratio of the chitosan solution to the PLGA solution is 3:1, the mass ratio of the stabilizer to the volume ratio of the chitosan and PLGA solution is 0.5%; the mass ratio of the cross-linking agent to the volume ratio of the chitosan solution to the PLGA solution is 0.2%.

[0030] The present invention is a light-driven nano-plant pollen transfer agent as described above. Further, the ultrasonic emulsification conditions are 20kHz, 200W, and 10min; the constant temperature is 20-35°C, and the dispersion time is 3-5 hours.

[0031] The present invention is a light-driven nano-nano plant pollenizer as described above. Further, in step (2), the mass ratio of the porous chitosan-PLGA particles to the thiolated SLG particles is 100:1, and the thiolated SLG content of the thiolated SLG particles is 10 μg / mg.

[0032] The present invention is a light-driven nano-nano plant pollen transfer agent as described above. Further, in step (3), the pollen and the protective agent solution are mixed in a ratio of 1:4 to 1:6. The protective agent solution is obtained by dissolving the pollen protective agent in deionized water, adding 0.1% Tween-80 by weight of the protective agent solution, and magnetically stirring until completely dissolved.

[0033] The present invention is a light-driven nano-plant pollen agent as described above. Further, in step (4), the mass ratio of the porous chitosan-PLGA particles to the dry pollen is 4:1-6:1; the amount of the photothermal response material in the photothermal response material dispersion is 1% of the mass of the porous chitosan-PLGA particles.

[0034] The present invention is a light-driven nano-nano plant pollenizer as described above. Further, the stabilizer is one of PluronicF68, PluronicF127, Tween80, Poloxamer188, Span80, and lecithin. The comparative data of the use of different stabilizers are shown in Table 3.

[0035] Table 3. Experimental data of performance comparison of different stabilizers

[0036]

[0037]

[0038] The present invention provides a light-driven nano-plant pollenizer as described above, wherein the crosslinking agent is one of genipin, tannic acid, oxidized dextran (OxDex), glutaraldehyde, chondroitin sulfate, and a disulfide bond crosslinking agent (DTSSP). Comparative data using different crosslinking agents are shown in Table 4.

[0039] Table 4. Experimental data comparing the performance of different crosslinking agents

[0040]

[0041] The present invention provides a light-driven nano-nano plant pollenizer as described above, wherein the pollen protectant in the pollen protectant solution is one of a combination of trehalose and proline, sucrose and mannitol, ascorbic acid and glutathione, BSA and glycerol, PVP and sorbitol, or PEG and betaine; the pollen protectant solution is one of a combination of trehalose (10% w / v) and proline (5% w / v), an aqueous solution of sucrose 8% and mannitol 4%, an aqueous solution of ascorbic acid 1% and glutathione 0.5%, an aqueous solution of BSA 2% and glycerol 7%, an aqueous solution of PVP 3% and sorbitol 6%, or an aqueous solution of PEG 5% and betaine 3%. Comparative data on the use of different pollen protectants are shown in Table 5.

[0042] Table 5. Experimental data on performance comparison of different pollen protectants

[0043]

[0044] The present invention provides a light-driven nano-plant pollinator as described above. Furthermore, the photothermal responsive material is one of gold nanoparticles (AuNRs), Fe3O4@Au core-shell nanoparticles, carbon nanotubes (CNTs), copper sulfide (CuS) nanoparticles, black phosphorene (BP, full-spectrum absorption), and polydopamine nanoparticles (PDA). Comparative data using different photothermal responsive materials are shown in Table 6.

[0045] Table 6. Experimental data comparing the performance of different photothermal response materials

[0046]

[0047]

[0048] The beneficial effects of the present invention are as follows: the present invention converts light energy absorbed by the photothermal responsive material into heat energy, triggers thermophoresis of the surrounding medium, and promotes the directional movement of the nanoparticle material, that is, generates surface charge under light, and realizes controllable movement through an external electric field or its own potential difference, that is, the photothermal responsive material guides the nanoparticle material to move along the light intensity gradient according to the local light intensity difference, thereby realizing targeted pollination; the present invention prepares biocompatible functionalized particles that specifically adsorb pollen, and during use, the material structure changes are triggered by light (such as temperature increase, charge reversal), and the pollen is released to the target stigma.

[0049] The present invention utilizes photothermal responsive materials. First, they generate localized high-temperature zones under illumination, forming a controllable temperature gradient (typical value: 1-10K / μm) that induces thermal motion. Thermophoresis, also known as thermal diffusion or the Ludwig-Soret effect, refers to the directional migration of suspended particles or molecules due to thermodynamic imbalance in a temperature gradient field. Its essence is the result of the combined effects of local changes in medium properties caused by the temperature gradient and particle-medium interactions. That is, the photothermal responsive material absorbs light energy and converts it into heat, inducing thermophoresis in the surrounding medium and driving the directional movement of nanoparticles. Second, the material generates surface charge under illumination, enabling controlled motion through an external electric field or its own potential difference. Third, the material undergoes conformational changes under illumination, driving the movement direction of the nanocarrier or the release of pollen.

[0050] The light-driven pollination system of this invention allows for precise control, minimizing interference with non-target plants. It can be scalably manufactured to meet the needs of large-scale planting. Its efficiency is stable and unaffected by fluctuations in insect populations. Its biodegradable nature (e.g., a carrier based on plant polysaccharides) reduces ecological residues. Its clean energy (light energy) drive aligns with the philosophy of green agriculture.

[0051] The present invention can use gold nanoparticles (AuNPs), which absorb light energy of a specific wavelength (such as green light 532nm) through surface plasmon resonance (SPR), converting photon energy into heat energy, and the local temperature can reach 50-100°C (efficiency>90%).

[0052] The carbon-based materials used (such as graphene and carbon nanotubes) can absorb broad-spectrum light energy (ultraviolet-near-infrared) through the electron-phonon non-radiative relaxation process to generate a uniform thermal field; achieving energy conversion from light energy to electron excitation on the surface of the nanomaterial to energy release in the form of heat to the formation of a local temperature gradient (ΔT). That is, in a temperature gradient field, nanoparticles migrate to low-temperature areas due to the Soret effect (temperature gradient drives the diffusion of matter). For example: when a laser is focused near a nanoparticle, the local liquid (such as water or air medium) expands due to heat, resulting in a density difference; the particles are affected by the asymmetric force of collisions with the molecules of the surrounding medium (the kinetic energy of molecules in the high-temperature zone is large), which pushes the particles to move to the low-temperature zone at a speed of up to 10-100μm / s; the direction of movement is controlled by dynamically controlling the light source to change the direction of the temperature gradient, thereby achieving path planning for the nanoparticles.

[0053] The functionalized particles with surface grafted targeting groups formed by surface modification, namely hollow nanocarriers, can fix pollen by electrostatic adsorption or hydrogen bond combination.

[0054] The pollen release process includes: 1. Photothermal expansion release: local heating causes the carrier material to expand, breaking through the surface coating to release the pollen. 2. Photochemical response: using photosensitive molecules to modify the carrier, specific wavelengths of light (such as ultraviolet light) break the chemical bonds, achieving precise and timed release. DETAILED DESCRIPTION

[0055] The principles and features of the present invention are described below in conjunction with embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0056] The chitosan solution and PLGA solution used in the following examples were prepared as follows: chitosan was dissolved in 2% acetic acid solution to obtain a chitosan solution with a mass volume concentration of 30%, and PLGA (LA=75:25, Mw=20 kDa) was dissolved in dichloromethane to obtain a PLGA solution with a mass volume concentration of 30%.

[0057] Example 1

[0058] Chitosan solution and PLGA solution were mixed in a volume ratio of 3:1, and 0.5% w / v Pluronic F68 was added to the mixed volume; ultrasonic emulsification (20 kHz, 200 W, 10 min) was used to form a W / O emulsion; 0.2% w / v genipin was added to the mixed volume and the mixture was dispersed at a constant temperature for 4 h, washed by centrifugation, and freeze-dried to obtain porous particles with a particle size of 200 nm.

[0059] Porous chitosan-PLGA particles (100 mg) were dispersed in PBS (pH 7.4), and thiolated SLG (thiolated glycoprotein) (1 mg, 10 μg / mg particles) was added. The mixture was shaken at 37°C (200 rpm) for 12 h, and the unreacted reagents were removed by dialysis to obtain functionalized particles with surface grafted targeting groups.

[0060] Ascorbic acid and glutathione were dissolved in deionized water to obtain a pollen protectant solution, wherein the ascorbic acid solution had a mass volume concentration of 1% and the glutathione had a mass volume concentration of 0.5%. Tween-80 was added at a mass concentration of 0.1% by mass of the pollen protectant solution to enhance wettability, and the solution was magnetically stirred (30°C, 500 rpm) until completely dissolved. The pollen and the protectant solution were mixed in a ratio of 1:5 and spray dried (inlet temperature 35°C, atomization pressure 0.3 MPa) to obtain dry pollen.

[0061] 100 mg of porous chitosan-PLGA particles were mixed with 20 mg of dry pollen, placed in a vacuum adsorption device (-0.1 MPa) and shaken for 30 minutes to allow the pollen to embed into the pores of the particles to obtain pollen particles; carbon nanotube (CNTs) dispersion was added (carbon nanotubes were 1% w / w of the mass of the pollen particles), and vacuum adsorption was continued for 1 hour (adsorption rate ≥ 85%); the PLGA shell layer (thickness 50 nm) was encapsulated by the secondary emulsification method, and finally a light-driven nano-nano plant pollen agent with a particle size of about 800 nm was obtained.

[0062] Example 2

[0063] Chitosan solution and PLGA solution were mixed in a volume ratio of 3:1, and Poloxamer 188 was added at a mixing volume of 0.5% w / v; ultrasonic emulsification (20 kHz, 200 W, 10 min) was performed to form a W / O emulsion; tannic acid was added at a mixing volume of 0.2% w / v and dispersed at a constant temperature for 4 h, followed by centrifugal washing and freeze-drying to obtain porous particles with a particle size of 220 nm.

[0064] 100 mg of porous chitosan-PLGA particles were dispersed in PBS (pH 7.4), thiolated SLG (1 mg, 10 μg / mg of particles) was added, and the mixture was shaken at 37°C (200 rpm) for 12 h. The unreacted reagent was removed by dialysis to obtain functionalized particles with surface grafted targeting groups.

[0065] Sucrose and mannitol were dissolved in deionized water to a sucrose solution concentration of 8% by volume and a mannitol solution concentration of 4% by volume. A 0.1% (v / v) Tween-80 protectant solution was added to enhance wettability. The pollen was magnetically stirred until completely dissolved (30°C, 500 rpm) to prepare a pollen protectant solution. The pollen and the protectant solution were mixed in a ratio of 1:5 and spray-dried (inlet temperature 35°C, atomization pressure 0.3 MPa) to obtain dry pollen.

[0066] 100mg of porous chitosan-PLGA particles were mixed with 20mg of dry pollen and placed in a vacuum adsorption device (-0.1MPa) for 30 minutes to allow the pollen to embed into the pores of the particles. A nano-copper sulfide (CuS) dispersion (1% w / w of the pollen mass) was added, and vacuum adsorption was continued for 1 hour (adsorption efficiency ≥85%). A secondary emulsification method was used to encapsulate the PLGA shell (50nm thick), resulting in a light-driven nano-plant pollen agent with a particle size of approximately 800nm.

[0067] Example 3

[0068] Chitosan solution and PLGA solution were mixed in a volume ratio of 3:1, and 0.5% w / v Pluronic F127 was added to the mixture; ultrasonic emulsification (20 kHz, 200 W, 10 min) was performed to form a W / O emulsion; 0.2% w / v oxidized dextran was added to the mixture and dispersed at a constant temperature for 4 h, washed by centrifugation, and freeze-dried to obtain porous particles with a particle size of 250 nm.

[0069] 100 mg of porous chitosan-PLGA particles were dispersed in PBS (pH 7.4), thiolated SLG (1 mg, 10 μg / mg of particles) was added, and the mixture was shaken at 37°C (200 rpm) for 12 h. The unreacted reagent was removed by dialysis to obtain functionalized particles with surface grafted targeting groups.

[0070] Trehalose and proline were dissolved in deionized water with a trehalose solution concentration of 10% w / v and a proline solution concentration of 5% w / v. A protectant solution containing 0.1% (v / v) Tween-80 was added to enhance wettability. The pollen was magnetically stirred until completely dissolved (30°C, 500 rpm) to prepare a pollen protectant solution. The pollen and the protectant solution were mixed in a ratio of 1:5 and spray dried (inlet temperature 35°C, atomization pressure 0.3 MPa) to obtain dry pollen.

[0071] 100mg of porous chitosan-PLGA particles were mixed with 20mg of dry pollen and placed in a vacuum adsorption device (-0.1MPa) for 30 minutes to allow the pollen to embed into the pores of the particles, thereby obtaining pollen particles. A dispersion of Fe3O4@Au core-shell nanoparticles (Fe3O4@Au core-shell nanoparticles at 1% w / w of the pollen mass) was added, and vacuum adsorption was continued for 1 hour (adsorption efficiency ≥85%). A secondary emulsification method was used to encapsulate the PLGA shell (50nm thick), resulting in a final particle size of approximately 800nm ​​for a light-driven nano-plant pollen agent.

[0072] Example 4

[0073] Chitosan solution and PLGA solution were mixed in a volume ratio of 3:1, and 0.5% w / v Span80 was added; ultrasonic emulsification (20 kHz, 200 W, 10 min) was used to form a W / O emulsion; 0.2% w / v chondroitin sulfate was added and dispersed at a constant temperature for 4 h, centrifuged and washed, and freeze-dried to obtain porous particles with a particle size of 230 nm.

[0074] Porous chitosan-PLGA particles (100 mg) were dispersed in PBS (pH 7.4), and thiolated SLG (1 mg, 10 μg / mg particles) was added. The mixture was shaken at 37°C (200 rpm) for 12 h, and the unreacted reagents were removed by dialysis to obtain functionalized particles with surface grafted targeting groups.

[0075] PVP and sorbitol were dissolved in deionized water to a PVP solution concentration of 3% w / v and a sorbitol solution concentration of 6% w / v. 0.1% Tween-80 was added to enhance wettability and magnetic stirring was applied until completely dissolved (30°C, 500 rpm) to prepare a pollen protectant solution. The pollen and the protectant solution were mixed in a ratio of 1:5 and spray-dried (inlet temperature 35°C, atomization pressure 0.3 MPa) to obtain dry pollen.

[0076] 100mg of porous chitosan-PLGA particles were mixed with 20mg of dry pollen and placed in a vacuum adsorption device (-0.1MPa) for 30 minutes to allow the pollen to embed into the particle pores. A gold nanoparticle (AuNRs) dispersion (1% w / w of the pollen mass) was added, and vacuum adsorption was continued for 1 hour (adsorption efficiency ≥85%). A PLGA shell (50nm thick) was encapsulated using a secondary emulsification method, resulting in a light-driven nano-pollinator with a particle size of approximately 800nm.

[0077] Example 5

[0078] Chitosan solution and PLGA solution were mixed in a volume ratio of 3:1, and 0.5% w / v lecithin was added to the mixture. Ultrasonic emulsification (20 kHz, 200 W, 10 min) was performed to form a W / O emulsion. 0.2% w / v glutaraldehyde was added to the mixture and dispersed at a constant temperature for 4 h. The mixture was washed by centrifugation and freeze-dried to obtain porous particles with a particle size of 180 nm.

[0079] 100 mg of porous chitosan-PLGA particles were dispersed in PBS (pH 7.4), thiolated SLG (1 mg, 10 μg / mg of particles) was added, and the mixture was shaken at 37°C (200 rpm) for 12 h. The unreacted reagent was removed by dialysis to obtain functionalized particles with surface grafted targeting groups.

[0080] BSA and glycerol were dissolved in deionized water to a BSA solution concentration of 2% by volume and a glycerol solution concentration of 7% by volume. A 0.1% (v / v) Tween-80 protectant solution was added to enhance wettability. The solution was magnetically stirred (30°C, 500 rpm) until completely dissolved to prepare a pollen protectant solution. The pollen and protectant solution were mixed in a ratio of 1:5 and spray-dried (inlet temperature 35°C, atomization pressure 0.3 MPa) to obtain dry pollen.

[0081] 100mg of porous chitosan-PLGA particles were mixed with 20mg of dry pollen and placed in a vacuum adsorption apparatus (-0.1MPa) for 30 minutes to allow the pollen to embed into the pores of the particles. A phosphorene (BP) dispersion (1% w / w of the pollen mass) was added, and vacuum adsorption was continued for 1 hour (adsorption efficiency ≥85%). A PLGA shell (50nm thick) was encapsulated using a secondary emulsification method, resulting in a light-driven nano-plant pollen agent with a particle size of approximately 800nm.

[0082] Example 6

[0083] Chitosan solution and PLGA solution were mixed in a volume ratio of 3:1, 0.5% w / v Tween 80 was added, and ultrasonic emulsification (20 kHz, 200 W, 10 min) was performed to form a W / O emulsion; 0.2% w / v disulfide bond crosslinker (DTSSP) was added and dispersed at a constant temperature for 4 h, centrifuged and washed, and freeze-dried to obtain porous particles with a particle size of 180 nm.

[0084] 100 mg of porous chitosan-PLGA particles were dispersed in PBS (pH 7.4), thiolated SLG (1 mg, 10 μg / mg of particles) was added, and the mixture was shaken at 37°C (200 rpm) for 12 h. The unreacted reagent was removed by dialysis to obtain functionalized particles with surface grafted targeting groups.

[0085] PEG and betaine were dissolved in deionized water with a PEG solution concentration of 5% by volume and a betaine solution concentration of 3% by volume. A 0.1% (v / v) Tween-80 protectant solution was added to enhance wettability. The solution was stirred magnetically until completely dissolved (30°C, 500 rpm) to prepare a pollen protectant solution. The pollen and the protectant solution were mixed in a ratio of 1:5 and spray-dried (inlet temperature 35°C, atomization pressure 0.3 MPa) to obtain dry pollen.

[0086] 100mg of porous chitosan-PLGA particles were mixed with 20mg of dried pollen and placed in a vacuum adsorption device (-0.1MPa) and shaken for 30 minutes to allow the pollen to embed into the pores of the particles. A polydopamine nanoparticle (PDA) dispersion (1% w / w of the pollen mass) was added, and vacuum adsorption was continued for 1 hour (adsorption efficiency ≥85%). A secondary emulsification method was used to encapsulate the PLGA shell (50nm thick), resulting in a final particle size of approximately 800nm.

[0087] Comparative Example (CK)

[0088] Chitosan (2% acetic acid solution), PLGA (dichloromethane solution, no cross-linking agent optimization), pollen (not treated with a protective agent); chitosan solution and PLGA solution were mixed in a ratio of 3:1 and emulsified by mechanical stirring (no specific stabilizer was added); glutaraldehyde cross-linking (1% w / v, 24h); dry pollen was directly loaded without photothermal material encapsulation.

[0089] Table 7. Comprehensive performance comparison of Examples 1 to 6 and ck

[0090]

[0091] As can be seen from Table 7, the particle size of Examples 1 to 6 was significantly reduced, and the pollen survival rate, photothermal efficiency, adhesion strength and fruit setting rate were significantly improved, while the 72h release efficiency was significantly reduced.

[0092] Application method of the embodiment of the present invention

[0093] Spraying equipment: UAV equipped with an electrostatic spray system (such as DJI Agras T30), and a particle size control module to ensure uniform dispersion of nanoparticles.

[0094] Spraying time:

[0095] Early flowering period: spray the basic agent (loaded pollen + nutrients).

[0096] During the peak flowering period: spray a triggering agent (such as pH regulating solution to activate the release).

[0097] Dosage control: 200-300mL per mu, pollen concentration ≥10 6grains / mL; CK is natural pollination.

[0098] Table 8. Effects of Examples 1 to 6 and CK

[0099]

[0100] From the examples in Table 8, the single pollen load, pollen germination rate, pollen tube length and light energy efficiency are significantly improved.

[0101] The invention has biodegradability: the chitosan / PLGA carrier degrades into CO2 and H2O within 30 days.

[0102] Ecological compatibility: Nanomaterials are non-toxic to pollinating insects when the concentration is ≤0.1% (LD50>5000mg / kg).

[0103] No residue: degradation products can be absorbed by plants through metabolism.

[0104] 1. The pollen survival rate of the present invention is tested by the following test method:

[0105] In vitro germination experiment:

[0106] Culture medium preparation: Prepare a culture medium containing 15% sucrose, 0.01% boric acid and 1% agar (pH 6.5-7.0).

[0107] Pollen treatment: Spread the pollen treated with nano-pollinator evenly on the surface of the culture medium and culture it in the dark at 25℃ for 24 hours.

[0108] Microscopic observation: The proportion of pollen tube length ≥ 2 times of pollen diameter was counted and the survival rate was calculated (survival rate = number of germinated pollen / total number of pollen × 100%).

[0109] Key equipment: inverted microscope (40x objective lens), constant temperature incubator.

[0110] 2. The photothermal efficiency of the present invention is tested by the following test methods:

[0111] Photothermal heating curve determination:

[0112] Experimental setup: Using 808nm near-infrared laser (power density 1W / cm 2 ) irradiated the sample, and the temperature change was recorded in real time by an infrared thermal imager (resolution ±0.1°C)27.

[0113] Calculation formula: The photothermal conversion efficiency (η) is calculated by the formula:

[0114] η=∑(mCΔT) / (P×t)×100%

[0115] Where m is the mass of the solution, C is the specific heat capacity of water (4.2 J / g·°C), ΔT is the temperature rise, P is the laser power, and t is the irradiation time.

[0116] Free radical capture experiments: Detection of OH and O2 by ESR - The production amount verifies the photothermal driven catalytic activity.

[0117] 3. Leaf surface adhesion (adhesion strength (μN)) test method of the present invention:

[0118] Weighing method (quantification of residual rate)

[0119] Pretreatment: Wash and dry the leaves to be tested (such as tomato leaves), and weigh the initial weight (W0).

[0120] Spraying treatment: evenly spray the nanomaterial suspension (such as 0.01% photosynthesis agent solution), and weigh it after drying (W1).

[0121] Simulated scouring: Use a watering can to simulate rainfall (water volume 30 mm / h, duration 5 minutes), and weigh after drying (W2). Calculation: Adhesion (%) = [(W2-W0) / (W1-W0)] x 100.

[0122] 4. Testing method for fruit setting rate of the present invention

[0123] Field trial statistics:

[0124] Treatment group design: Randomly select plants in the flowering stage and spray them with nano-pollinator (the control group is sprayed with water).

[0125] Post-pollination tracking: Mark the flowers after artificial pollination and count the number of fruits set 7 days later.

[0126] Calculation formula: Fruit setting rate (%) = (number of fruits set / total number of treated flowers) × 100%.

[0127] Optimization conditions: Spraying should be done when the daily average temperature is ≥23℃ to avoid low temperature affecting pollen vitality.

[0128] 5. Testing method for 72-hour release efficiency of the present invention

[0129] In vitro sustained release simulation:

[0130] Release medium: simulates natural environment (such as pH 6.8 phosphate buffer) or targeted environment (such as pH 1.2, pH 6.8).

[0131] Dynamic sampling: Place the drug-loaded pollenizer in a dialysis bag (molecular weight cut-off 10kDa) and take samples at regular intervals to detect the release amount.

[0132] Detection technology: HPLC: quantitative analysis of the release concentration of the target ingredient.

[0133] UV spectrophotometry: Calculate the cumulative release rate based on the characteristic absorption peak.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a light-driven nano-plant pollen agent, characterized in that: The method comprises the following steps: (1) mixing a chitosan solution and a PLGA solution, adding a stabilizer, performing ultrasonic emulsification to form a W / O emulsion, adding a cross-linking agent, dispersing the mixture at a constant temperature, washing the mixture by centrifugation, and freeze-drying the mixture to obtain porous chitosan-PLGA particles with a particle size of 200-500 nm; Step (2): disperse the porous chitosan-PLGA particles in PBS, add thiolated SLG, shake, and dialyze to remove unreacted reagents to obtain functionalized particles with surface grafted targeting groups; Step (3), mixing the pollen with a pollen protectant solution, and spray drying to obtain dry pollen; Step (4) is to mix the porous chitosan-PLGA particles and the dry pollen, place them in a vacuum adsorption device and shake them to allow the pollen to embed into the pores of the porous chitosan-PLGA particles; add a photothermal response material dispersion, continue vacuum adsorption, and then use a secondary emulsification method to encapsulate the PLGA shell layer to obtain a light-driven nano-plant pollen transfer agent.

2. The light-driven nano-plant pollenizer according to claim 1, characterized in that: In step (1), the chitosan solution is a solution of chitosan dissolved in 2% acetic acid, with a mass volume concentration of 20-30%; the PLGA solution is a solution of PLGA dissolved in dichloromethane, with a mass volume concentration of 20-30%; the mixed volume ratio of the chitosan solution to the PLGA solution is 3:1, the mass ratio of the stabilizer to the mixed volume ratio of the chitosan solution to the PLGA solution is 0.5%; and the mass ratio of the cross-linking agent to the mixed volume ratio of the chitosan and PLGA is 0.2%.

3. The light-driven nano-plant pollenizer according to claim 1, characterized in that: The ultrasonic emulsification conditions are 20 kHz, 200 W, and 10 min; the constant temperature is 20-35° C., and the dispersion time is 3-5 hours.

4. The light-driven nano-plant pollenizer according to claim 1, characterized in that: In step (2), the mass ratio of the porous chitosan-PLGA particles to the thiolated SLG particles is 100:1, and the thiolated SLG content of the thiolated SLG particles is 10 μg / mg.

5. The light-driven nano-plant pollenizer according to claim 1, characterized in that: Step (3), the pollen and the protective agent solution are mixed in a mass ratio of 1:4 to 1:6, wherein the protective agent solution is prepared by dissolving the pollen protective agent in deionized water, adding 0.1% Tween-80 by mass of the pollen protective agent solution, and magnetically stirring until completely dissolved.

6. The light-driven nano-plant pollen transfer agent according to claim 1, characterized in that: In step (4), the mass ratio of the porous chitosan-PLGA particles to the dry pollen is 4:1-6:1; the amount of the photothermal response material in the photothermal response material dispersion is 1% of the mass of the porous chitosan-PLGA particles.

7. The light-driven nano-plant pollenizer according to any one of claims 1 to 6, characterized in that: The stabilizer is one of Pluronic F68, Pluronic F127, Tween 80, Poloxamer 188, Span 80 or lecithin.

8. The light-driven nano-plant pollenizer according to any one of claims 1 to 6, characterized in that: The cross-linking agent is one of genipin, tannic acid, oxidized dextran, glutaraldehyde, chondroitin sulfate or a disulfide bond cross-linking agent.

9. The light-driven nano-plant pollenizer according to any one of claims 1 to 6, characterized in that: The pollen protective agent is one of the combinations of trehalose and proline, sucrose and mannitol, ascorbic acid and glutathione, BSA and glycerol, PVP and sorbitol, and PEG and betaine.

10. The light-driven nano-plant pollenizer according to any one of claims 1 to 6, characterized in that: The photothermal response material is one of gold nanoparticles, Fe3O4@Au core-shell nanoparticles, carbon nanotubes, copper sulfide nanoparticles, black phosphorene and polydopamine nanoparticles.

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