Rare earth up-conversion nanoparticle carrier as well as preparation method and application thereof

Abstract

The invention relates to a rare earth up-conversion nano-particle carrier and a preparation method and application thereof, belongs to the technical field of nano-drug carriers, and aims to solve the problems that the dynamic distribution law and biological distribution characteristics of the conventional nano-carrier cannot be fully investigated, and the stability of the nano-carrier is poor. The existing carrier has at least one of the problems of poor stability, poor water solubility, low drug loading capacity and the like. The rare earth up-conversion nano-particle carrier is of a multi-layer core-shell structure, macromolecular chains on the surface of the rare earth up-conversion nano-particle carrier obtained through the method can fully stretch, on one hand, a better steric hindrance effect is provided, and the water dispersibility and stability of the rare earth up-conversion nano-particle carrier are improved; on the other hand, mutual overlapping and sinking between surface adsorption macromolecular chains are reduced, so that more anchoring points are provided, and efficient loading of vaccines / drugs is achieved.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine carrier technology, and in particular to a rare earth upconversion nanoparticle carrier, its preparation method, and its application. Background Technology

[0002] Malignant tumors have become a major factor affecting human health, with their incidence and mortality rates rising year by year. Traditional treatments for malignant tumors include surgical resection, chemotherapy, and radiotherapy. Among these, chemotherapy, which can be used alone or in combination with other therapies, has been used to treat various types of tumors. However, due to their poor water solubility, systemic distribution, and lack of tumor-targeting ability, chemotherapeutic drugs can damage normal tissues while killing tumor cells, leading to poor treatment efficacy and severe toxic side effects. Encapsulating chemotherapeutic drugs in drug delivery carriers can effectively improve their water solubility, increase bioavailability, and achieve targeted delivery to cells and specific tissues. Therefore, the use of nanomedicine carriers can significantly improve the anti-tumor efficiency of chemotherapeutic drugs and has great clinical application prospects in tumor treatment.

[0003] To evaluate the protective efficacy of various vaccines and the antitumor activity of chemical drugs, it is essential to develop an in vivo tracking and measurement technique. This technique, combined with nanocarriers, would allow the biodistribution characteristics of vaccines or chemical drugs to be observed using instruments such as cameras, mobile phones, and microscopes after they enter the body. Existing nanomaterials suitable for vaccine / chemical drug delivery include phospholipid nanomaterials, polymer nanomaterials, and inorganic nanocarriers. While fluorescent labeling technology can be used to track these nanocarriers, it is limited by the depth of tissue penetration. In other words, nanocarriers deeply embedded in body tissues and organs cannot be precisely located using traditional fluorescent labeling techniques. Summary of the Invention

[0004] Based on the above analysis, the present invention aims to provide a rare earth upconversion nanoparticle carrier, its preparation method and application, in order to solve at least one of the following problems of existing nanocarriers: they cannot fully investigate the dynamic distribution law and biodistribution characteristics of nanocarriers, and existing carriers have poor stability, poor water solubility and low drug loading capacity.

[0005] In a first aspect, the present invention provides a method for preparing rare earth upconversion nanoparticle carriers, the method comprising the following steps: (1) The hydrophobic rare earth upconversion nanoparticles were dispersed in a nonpolar solvent to obtain a first mixture; (2) The amphiphilic polymer is dispersed in a polar solvent to obtain a second mixture; (3) The first mixture and the second mixture are mixed and the nonpolar solvent is removed to obtain the rare earth upconversion nanoparticle carrier.

[0006] Furthermore, in step (1), the hydrophobic rare earth upconversion nanoparticles are hydrophobic upconversion nanoparticles capable of emitting light.

[0007] Furthermore, in step (1), the nonpolar solvent includes cyclohexane and / or chloroform.

[0008] Furthermore, in step (1), the concentration of the hydrophobic rare earth upconversion nanoparticles in the nonpolar solvent is 0.1 mg / mL to 20 mg / mL.

[0009] Furthermore, in step (2), the amphiphilic polymer includes DSPE-PEG. 100~20000 One or more of Tween-80 and TPGS.

[0010] Furthermore, in step (2), the polar solvent includes secondary water and physiological solutions.

[0011] Furthermore, in step (2), the concentration of the amphiphilic polymer in the polar solvent is 0.1 mg / mL to 100 mg / mL.

[0012] Furthermore, in step (3), the volume ratio of the first mixture and the second mixture is 2:10 to 10:2; And / or, in step (3), rotary evaporation or vacuum pumping is used to remove nonpolar solvents.

[0013] Secondly, the present invention provides a rare earth upconversion nanoparticle carrier prepared by the above method.

[0014] Thirdly, the present invention provides an application of the aforementioned rare earth upconversion nanoparticle carrier in fluorescence imaging and / or drug loading.

[0015] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects: (1) The rare earth upconversion nanoparticle carrier described in this invention has a multi-layer core-shell structure. The polymer chains on the surface of the rare earth upconversion nanoparticle carrier obtained by the method of this invention can be fully extended. On the one hand, it provides better steric hindrance to improve the water dispersibility and stability of the rare earth upconversion nanoparticle carrier. On the other hand, it reduces the mutual overlap and trapping between the surface adsorbed polymer chains to provide more anchoring points and achieve efficient loading of vaccines / drugs. (2) The rare earth upconversion nanoparticle carrier prepared by the method of the present invention has excellent upconversion fluorescence performance, good in vivo penetration depth, small light scattering, and less tissue absorption of excitation light, which makes it have a higher signal-to-noise ratio and better imaging quality when used for imaging. Therefore, it can effectively trace vaccines / drugs located in deep tissues. The rare earth upconversion nanoparticle carrier prepared by the method of the present invention has good biosafety and has important research significance and broad application prospects in the field of nanobiomedicine. The rare earth upconversion nanoparticle carrier prepared by the method of the present invention has universal applicability for the tracing and measurement of vaccines / drugs and can be applied to the labeling and measurement analysis of other similar vaccines and drugs.

[0016] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0017] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0018] Figure 1 Scanning electron microscope image of OA-NaYF4:Yb,Er upconversion nanoparticles prepared in Example 1 of this invention; Figure 2 Scanning electron microscope image of OA-NaYF4:Mn,Yb,Er upconversion nanoparticles prepared in Example 2 of this invention; Figure 3 The DSPE-PEG prepared in Example 1 of this invention 5000 Scanning electron microscope image of NaYF4:Yb,Er nanoparticle carrier; Figure 4 The DSPE-PEG prepared in Example 5 of this invention 5000 Scanning electron microscope image of NaYF4:Yb,Er nanoparticle carrier; Figure 5 DSPE-PEG in Application Example 1 of this invention 5000 -NaYF4:Yb,Er nanoparticle support and DSPE-PEG 5000 Hydration diameter data of NaYF4:Yb,Er@mRNA nanovaccine; Figure 6 The DSPE-PEG in Embodiment 1 of the present invention 5000-NaYF4:Yb,Er nanoparticle carriers and DSPE-PEG in Application Example 1 5000 Upconversion fluorescence spectrum of NaYF4:Yb,Er@mRNA nanovaccine; Figure 7 The DSPE-PEG in Embodiment 1 of the present invention 5000 -NaYF4:Yb,Er nanoparticle carriers and DSPE-PEG in Application Example 1 5000 Toxicity data for the -NaYF4:Yb,Er@mRNA nanovaccine; Figure 8 This is a map showing the in vivo location of the nano-vaccine in experimental mice under 980nm near-infrared light irradiation, as illustrated in Example 1 of the present invention, using a mobile phone. Figure 9 This is a diagram of the tissues and organs of a mouse used in the anatomical experiment of Example 1 of the present invention; Figure 10 In Example 1 of this invention, after the experimental mice were dissected, the distribution of the nano-fluorescent carrier in the organs was monitored using a mobile phone under 980nm near-infrared light irradiation. Figure 11 The results of the cytotoxicity test in Example 3 of the present invention are shown. Detailed Implementation

[0019] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which constitute a part of the present invention and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0020] A specific embodiment of the present invention discloses a method for preparing rare earth upconversion nanoparticle carriers, the method comprising the following steps: (1) The hydrophobic rare earth upconversion nanoparticles were dispersed in a nonpolar solvent to obtain a first mixture; (2) The amphiphilic polymer is dispersed in a polar solvent to obtain a second mixture; (3) The first mixture and the second mixture are mixed and the nonpolar solvent is removed to obtain the rare earth upconversion nanoparticle carrier.

[0021] Specifically, in step (1), the hydrophobic rare earth upconversion nanoparticles are hydrophobic upconversion nanoparticles capable of emitting light. Preferably, they are upconversion nanoparticles that emit red, green, or blue light.

[0022] It should be noted that the hydrophobic rare earth (core-shell structure) upconversion nanoparticles described in this invention are commercially available products or prepared using existing methods. For example, there are green-emitting hydrophobic OA-NaYF4:Yb,Er upconversion nanoparticles, green-emitting hydrophobic OA-NaYF4:Yb,Ho upconversion nanoparticles, red-emitting hydrophobic OA-NaYF4:Mn,Yb,Er upconversion nanoparticles, blue-emitting hydrophobic OA-NaYF4:Yb,Tm upconversion nanoparticles, yellow-green-emitting hydrophobic OA-NaYF4:Yb,Tm,Er upconversion nanoparticles, and 800nm ​​near-infrared light-excited OA-NaYF4:Yb,Ln@NaYF4:Nd multilayer core-shell structure upconversion nanoparticles (Ln=Er, Ho, Tm), etc., which are coated with oleic acid and / or oleylamine on the surface and have good hydrophobicity. They can be dispersed in nonpolar solvents such as cyclohexane and chloroform. The diameter of the hydrophobic rare earth upconversion nanoparticles ranges from 5nm to 200nm, and the morphologies include spheres and hexagonal prisms.

[0023] The rare earth upconversion nanoparticle carrier of the present invention has a multi-layer core-shell structure, specifically a matrix material: rare earth luminescent ions@hydrophobic layer@amphiphilic polymer layer structure. Preferably, the matrix material is NaYF4, the rare earth luminescent ions include Yb, Tm / Er / Ho, etc., the hydrophobic layer includes oleic acid and / or oleylamine molecules, and the amphiphilic polymer layer mainly includes amphiphilic polymers.

[0024] The polymer chains on the surface of the rare earth upconversion nanoparticle carrier obtained by the method of this invention can be fully extended. On the one hand, this provides better steric hindrance to improve the water dispersibility and stability of the rare earth upconversion nanoparticle carrier; on the other hand, it reduces the overlap and trapping between the surface adsorbed polymer chains to provide more anchoring points and achieve efficient loading of vaccines / drugs.

[0025] The amphiphilic polymer of this invention comprises hydrophilic and hydrophobic chains. The hydrophobic chains bind to the surface of rare earth upconversion nanoparticles through hydrophobic interactions, while the hydrophilic chains effectively improve the dispersibility and stability of rare earth upconversion nanoparticles in polar solutions such as water and physiological fluids. The polymer adsorption layer formed by the amphiphilic polymer on the surface of rare earth upconversion nanoparticles adsorbs DNA, mRNA, or chemical drugs through electrostatic interactions, van der Waals forces, and other forces, thereby obtaining novel coronavirus nanovaccines / nanodrugs.

[0026] The rare-earth upconversion nanoparticle carriers prepared by the method of this invention exhibit excellent upconversion fluorescence performance, good in vivo penetration depth, low light scattering, and less tissue absorption of excitation light, resulting in a higher signal-to-noise ratio and better imaging quality when used for imaging. Therefore, they can effectively trace vaccines / drugs located in deep tissues. The rare-earth upconversion nanoparticle carriers prepared by the method of this invention also have good biosafety, which is of significant research value and has broad application prospects in the field of nanobiomedicine. The rare-earth upconversion nanoparticle carriers prepared by the method of this invention are universally applicable to the tracking and measurement of vaccines / drugs and can be applied to the labeling and measurement analysis of other similar vaccines and drugs.

[0027] Specifically, in step (1), the nonpolar solvent includes cyclohexane and / or chloroform.

[0028] Specifically, in step (1), the concentration of the hydrophobic rare earth upconversion nanoparticles in the nonpolar solvent is 0.1 mg / mL to 20 mg / mL, for example, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, and 20 mg / mL.

[0029] It should be noted that the concentration of the hydrophobic rare earth upconversion nanoparticles in the nonpolar solvent of the present invention is selected within the above range because if the concentration is too low, the yield will be affected; if the concentration is too high, they will easily agglomerate, affecting the subsequent modification and the dispersibility and water solubility of the carrier.

[0030] Preferably, in step (1), the hydrophobic rare earth upconversion nanoparticles are prepared by the following method: Sodium fluoride, yttrium oleate precursor, ytterbium oleate precursor, erbium oleate precursor, oleic acid, and 1-octadecene were stirred evenly, heated under vacuum, cooled, centrifuged, and washed to obtain the hydrophobic rare earth on-conversion nanoparticles.

[0031] More preferably, in step (1), the hydrophobic rare earth upconversion nanoparticles are prepared by the following method: Oleic acid and ethanol were mixed, and NaOH solution was added. Then, MnCl2 solution, Y(NO3)3 solution, Yb(NO3)3 solution and Er(NO3)3 solution were added. The mixture was stirred, NaF solution was added, the mixture was heated to react, centrifuged, the sediment was collected, and washed to obtain the hydrophobic rare earth upconversion nanoparticles.

[0032] Specifically, in step (2), the amphiphilic polymer includes DSPE-PEG. 100~20000 One or more of Tween-80 and TPGS (vitamin E polyethylene glycol succinate).

[0033] Specifically, in step (2), the polar solvent includes deionized water and physiological solution, preferably, the physiological solution includes phosphate buffer.

[0034] Specifically, in step (2), the concentration of the amphiphilic polymer in the polar solvent is 0.1 mg / mL to 100 mg / mL, for example, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, and 95 mg / mL.

[0035] It should be noted that the concentration of the amphiphilic polymer in the polar solvent in this invention is within the range described above. If the concentration is too low, the surface of the upconversion nanoparticles cannot be sufficiently modified, affecting their dispersibility, water solubility, and loading capacity; if the concentration is too high, it wastes polymer materials and increases costs.

[0036] Specifically, in step (3), the volume ratio of the first mixture and the second mixture is 2:10 to 10:2, for example, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1.

[0037] It should be noted that if the volume ratio is too small, on the one hand, the content of polar solvent in the system will be low, which will affect the phase transfer efficiency; on the other hand, it will prolong the time for removing non-polar solvent, which is not conducive to reducing costs. If the volume ratio is too large, the content of amphiphilic polymer will be low, which will also affect the phase transfer efficiency.

[0038] Specifically, in step (3), rotary evaporation or vacuum pumping is used to remove nonpolar solvents.

[0039] It should be noted that during the conventional process of removing nonpolar solvents by high-temperature heating, the adsorbed polymer chains on the surface are prone to overlap and become trapped, thereby reducing the efficient loading of vaccines / drugs. Therefore, in this invention, rotary evaporation or depressurized gas extraction is used to remove nonpolar solvents.

[0040] Preferably, the pressure of rotary evaporation or vacuum pumping is 0.01~200Pa, for example, 10Pa, 20Pa, 30Pa, 40Pa, 50Pa, 60Pa, 70Pa, 80Pa, 90Pa, 100Pa, 110Pa, 120Pa, 130Pa, 140Pa, 150Pa, 160Pa, 170Pa, 180Pa, 190Pa, and the temperature is 4℃~37℃, for example, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃.

[0041] Too low a pressure places high demands on the pump, increasing costs; too high a pressure affects the removal efficiency of non-polar solvents.

[0042] Another specific embodiment of the present invention discloses a rare earth upconversion nanoparticle carrier prepared by the above method.

[0043] The polymer chains on the surface of the rare earth upconversion nanoparticle carrier obtained by the method of the present invention can be fully extended. On the one hand, this provides better steric hindrance to improve the water dispersibility and stability of the rare earth upconversion nanoparticle carrier; on the other hand, it reduces the mutual overlap and trapping between the surface adsorbed polymer chains to provide more anchoring points and achieve efficient loading of vaccines / drugs. The rare earth upconversion nanoparticle carrier described in this invention is a hydrophilic carrier with excellent upconversion fluorescence performance, good in vivo penetration depth, low light scattering, and less tissue absorption of excitation light. When used for imaging, it has a higher signal-to-noise ratio and better imaging quality, thus it can effectively trace vaccines / drugs located in deep tissues.

[0044] Another specific embodiment of the present invention is the application of the aforementioned rare-earth upconversion nanoparticle carrier in fluorescence imaging and / or drug loading.

[0045] Specifically, the drugs include nucleic acid vaccines or chemical drugs. The nucleic acid vaccines are DNA, mRNA, etc., that can induce cellular and humoral immunity in the body by expressing antigen proteins within cells. The chemical drugs are small molecule drugs that can kill malignant tumor cells.

[0046] Preferably, the nucleic acid vaccine is a COVID-19 vaccine, and more preferably, the COVID-19 vaccine is an mRNA vaccine. The chemical drug also includes an anti-tumor drug.

[0047] Specifically, the application includes dispersing the rare earth upconversion nanoparticle carrier in a solvent, adding the drug, mixing, stirring, washing, and performing in vivo drug tracing.

[0048] Specifically, the rare earth upconversion nanoparticle carrier has a size of 5nm to 200nm in the solvent, for example, 10nm, 20nm, 30nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150nm, 155nm, 160nm, 165nm, 170nm, 175nm, 180nm, 185nm, 190nm, and 195nm.

[0049] Specifically, the concentration of the rare earth upconversion nanoparticle carrier in the solvent is 0.1 mg / mL to 20 mg / mL, for example, 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, and 18 mg / mL, and the concentration of the drug in the solvent is 0.1 mg / mL to 100 mg / mL, for example, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, and 95 mg / mL.

[0050] It should be noted that if the concentration of rare earth upconversion nanoparticle carriers in the solvent is too low, they cannot effectively load vaccine / drug molecules; if the concentration is too high, on the one hand, vaccine / drug molecules are wasted and costs are increased, and on the other hand, the carriers are prone to aggregation, which affects subsequent loading.

[0051] If the concentration of the drug in the solvent is too low, it cannot effectively load the vaccine / drug molecules; if the concentration is too high, it will waste vaccine / drug molecules and increase costs.

[0052] Specifically, the solvent includes deionized water and physiological solutions, preferably phosphate buffer.

[0053] Specifically, at temperatures of 4℃-37℃, for example, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, and pH of 5.00-9.00 (for example, 6.00, 7.00, 8.00), stirring is carried out for 0.5h-48h, for example, 5h, 10h, 15h, 20h, 25h, 30h, 35h, 40h, 45h.

[0054] It should be noted that if the temperature is too low, the solvent is prone to solidification; if the temperature is too high, it will affect the loading efficiency and the activity or efficacy of the vaccine / drug. A pH value that is too high or too low will cause vaccine inactivation or protonation of drug molecules, affecting loading efficiency. If the stirring time is too short, the vaccine / drug will not be fully loaded, affecting loading efficiency; stirring for a certain time (not exceeding 48 hours) will reach loading equilibrium.

[0055] The rare earth upconversion nanoparticle carrier described in this invention utilizes the surface properties of rare earth upconversion nanoparticles, amphiphilic polymers, and drugs. By combining rare earth upconversion nanoparticles and drugs together using amphiphilic polymers, fluorescent tracers or drug loading can be achieved, and the drug loading amount can reach 36%.

[0056] It should be noted that all raw materials used in this invention are existing raw materials or prepared using existing methods. The technical solution of this invention will be further explained below with reference to specific embodiments.

[0057] Preparation Example 1 This preparation example describes a method for preparing hydrophobic rare earth upconversion nanoparticles. Utilizing an existing high-temperature pyrolysis method, the method includes the following steps: using rare earth oleate as a raw material and a mixed solution of oleic acid and 1-octadecene (volume ratio 2:3) as a solvent, upconversion nanoparticles capable of emitting red, green, and blue light are prepared using a high-temperature pyrolysis method; the excitation and emission spectra of the upconversion nanoparticles are controlled by doping with different ions and designing a core-shell structure.

[0058] The specific preparation method used in this example is as follows: 4 mmol of sodium fluoride was weighed, and 0.8 mmol of yttrium oleate precursor, 0.18 mmol of ytterbium oleate precursor and 0.02 mmol of erbium oleate precursor were transferred into a 100 mL double-necked flask; oleic acid and 1-octadecene were added to make the total volume reach 6 mL and 9 mL, respectively; the mixture was heated to 115 °C under continuous stirring and vacuum and held for 1.5 h; it was heated to 310 °C under Ar atmosphere and held for 2 h; when the reaction cooled to room temperature, it was centrifuged at 12000 rpm for 3 min, the precipitate was collected, and the product was washed three times with a mixture of cyclohexane and ethanol (volume ratio of 1:1) to obtain hydrophobic OA-NaYF4:Yb,Er upconversion nanoparticles emitting green light.

[0059] Scanning electron microscopy results show that the OA-NaYF4:Yb,Er upconversion nanoparticles have uniform size and morphology. Figure 1 (i.e., the average size is 75nm, and they are all hexagonal crystal phases).

[0060] Preparation Example 2 This preparation example describes a method for preparing hydrophobic rare earth upconversion nanoparticles using an existing solvothermal method, comprising the following steps: using rare earth nitrates as raw materials and a mixed solution of oleic acid and ethanol (volume ratio 1:1) as solvent, upconversion nanoparticles capable of emitting red, green, and blue light are prepared using a solvothermal method; the emission spectrum of the upconversion nanoparticles is controlled by adjusting the concentration and type of doped rare earth ions.

[0061] The specific preparation method used in this example is as follows: 10 mL of oleic acid and 10 mL of ethanol were mixed, followed by the addition of 1.5 mL of NaOH solution (5 M). After stirring evenly, 0.6 mL of MnCl2 solution (0.5 M), 1.0 mL of Y(NO3)3 solution (0.5 M), 0.9 mL of Yb(NO3)3 solution (0.2 M), and 0.1 mL of Er(NO3)3 solution (0.2 M) were added successively. After stirring for 10 min, 2 mL of NaF solution (2 M) was added. After stirring for another 15 min, the resulting suspension was transferred to a polytetrafluoroethylene reactor and reacted at 200 °C for 8 h. After the reaction was completed, the precipitate was collected by centrifugation at 12000 rpm for 5 min, and the product was washed three times with ethanol and deionized water to obtain hydrophobic OA-NaYF4:Mn,Yb,Er upconversion nanoparticles emitting red light.

[0062] Scanning electron microscopy results show that the OA-NaYF4:Mn,Yb,Er upconversion nanoparticles have uniform size and morphology. Figure 2 The average size is about 30 nm, and they are all cubic crystal phases.

[0063] Example 1 This embodiment discloses a method for preparing rare earth upconversion nanoparticle carriers, the method comprising the following steps: (1) The green-emitting hydrophobic OA-NaYF4:Yb,Er upconversion nanoparticles prepared in Preparation Example 1 were dispersed in cyclohexane at a concentration of 2 mg / mL and ultrasonically dispersed to obtain the first mixture; (2) DSPE-PEG 2000 Dispersed in secondary water at a concentration of 10 mg / mL, a second mixture is obtained; (3) The first mixture and the second mixture were mixed at a volume ratio of 1:1 and stirred at 25°C for 6 hours. Cyclohexane was evaporated by rotary evaporation at a pressure of 0.05 Pa and a temperature of 15°C. After complete evaporation, the remaining solution was centrifuged, and the precipitate was washed three times with deionized water to obtain DSPE-PEG. 2000 -NaYF4:Yb,Er nanoparticle carrier.

[0064] The DSPE-PEG prepared in this embodiment 2000-NaYF4:Yb,Er nanoparticle carriers were tested using electron microscopy, such as... Figure 3 As shown, DSPE-PEG 2000 The average size of the NaYF4:Yb,Er nanoparticle carrier is approximately 75 nm, and its average hydrated particle size is approximately 90 nm.

[0065] Example 2 This embodiment discloses a method for preparing rare earth upconversion nanoparticle carriers, the method comprising the following steps: (1) The hydrophobic OA-NaYF4:Yb,Er upconversion nanoparticles emitting green light prepared in Preparation Example 1 were dispersed in chloroform at a concentration of 10 mg / mL and ultrasonically dispersed to obtain the first mixture; (2) DSPE-PEG 2000 Dispersed in secondary water at a concentration of 50 mg / mL, a second mixture is obtained; (3) The first mixture and the second mixture were mixed at a volume ratio of 5:1 and stirred at 25°C for 24 hours. The chloroform was evaporated by rotary evaporation (pressure 10 Pa, temperature 25°C). After complete evaporation, the remaining solution was centrifuged, and the precipitate was washed three times with deionized water to obtain DSPE-PEG. 2000 -NaYF4:Yb,Er nanoparticle carrier.

[0066] Example 3 This embodiment discloses a method for preparing rare earth upconversion nanoparticle carriers, the method comprising the following steps: (1) The green-emitting hydrophobic OA-NaYF4:Yb,Er upconversion nanoparticles prepared in Preparation Example 1 were dispersed in chloroform at a concentration of 10 mg / mL and ultrasonically dispersed to obtain the first mixture. (2) DSPE-PEG 2000 Dispersed in secondary water at a concentration of 50 mg / mL, a second mixture is obtained; (3) The first mixture and the second mixture were mixed at a volume ratio of 1:5 and stirred at 4°C for 12 hours. The chloroform was evaporated by rotary evaporation (pressure 0.05 Pa, temperature 4°C). After complete evaporation, the remaining solution was centrifuged, and the precipitate was washed three times with deionized water to obtain DSPE-PEG. 2000 -NaYF4:Yb,Er nanoparticle carrier.

[0067] Example 4 The method for preparing a rare earth upconversion nanoparticle carrier in this embodiment is the same as that in Example 1, except that in step (3), cyclohexane is removed by depressurization and pumping, specifically, the pressure is 2 Pa and the temperature is 15 °C.

[0068] Example 5 This embodiment discloses a method for preparing rare earth upconversion nanoparticle carriers, the method comprising the following steps: (1) The hydrophobic OA-NaYF4:Mn,Yb,Er upconversion nanoparticles emitting red light prepared in Preparation Example 2 were dispersed in cyclohexane at a concentration of 2 mg / mL and ultrasonically dispersed to obtain the first mixture; (2) DSPE-PEG 5000 Dispersed in secondary water at a concentration of 10 mg / mL, a second mixture is obtained; (3) The first mixture and the second mixture were mixed at a volume ratio of 1:1 and stirred at 4°C for 6 hours. Cyclohexane was evaporated by rotary evaporation (pressure 2 Pa, temperature 4°C). After complete evaporation, the remaining solution was centrifuged, and the precipitate was washed three times with deionized water to obtain DSPE-PEG. 5000 -NaYF4:Mn,Yb,Er nanoparticle carrier.

[0069] The DSPE-PEG prepared in this embodiment 5000 -NaYF4:Mn,Yb,Er nanoparticle carriers were tested using electron microscopy, such as... Figure 4 As shown, DSPE-PEG 5000 - The size of the NaYF4:Mn,Yb,Er nanoparticle carrier is approximately 30 nm.

[0070] Comparative Example 1 The preparation method of the rare earth upconversion nanoparticle carrier in this comparative example is as follows: The green-emitting hydrophobic OA-NaYF4:Yb,Er upconversion nanoparticles prepared in Example 1 and DSPE-PEG were used. 2000 The concentration of hydrophobic OA-NaYF4:Yb,Er upconversion nanoparticles, dispersed in secondary water, is 1 mg / mL, along with DSPE-PEG. 2000 The concentration was 5 mg / mL. After centrifugation, the precipitate was washed three times with water to obtain rare earth upconversion nanoparticle carriers.

[0071] Comparative Example 2 The preparation method of the rare earth upconversion nanoparticle carrier in this comparative example is the same as that in Example 1, except that in step (3), cyclohexane is removed by heating at a temperature of 70°C.

[0072] Comparative Example 3 The preparation method of the rare earth upconversion nanoparticle carrier in this comparative example is the same as that in Example 1, except that in step (3), the volume ratio of the first mixture and the second mixture is 6:1.

[0073] Comparative Example 4 The preparation method of the rare earth upconversion nanoparticle carrier in this comparative example is the same as that in Example 1, except that in step (1), the concentration of the hydrophobic OA-NaYF4:Yb,Er upconversion nanoparticles dispersed in cyclohexane is 25 mg / mL.

[0074] Comparative Example 5 The preparation method of the rare earth upconversion nanoparticle carrier in this comparative example is the same as that in Example 1, except that in step (2), DSPE-PEG... 2000 The concentration dispersed in secondary water is 0.5 mg / mL.

[0075] Experimental Example 1 The stability, dispersibility, and drug loading of the carriers prepared in Examples 1-5 and Comparative Examples 1-5 were tested respectively, and the results are shown in Table 1.

[0076] The drug loading test method is as follows: First, a standard solution of doxorubicin hydrochloride was prepared, and the absorbance at 490 nm was measured using a UV-Vis spectrophotometer to create a concentration-absorbance standard curve A. Second, the mixtures of the carriers (total mass m1) and doxorubicin hydrochloride (total mass m2) prepared in Examples 1-5 and Comparative Examples 1-2 were centrifuged, and the supernatant was collected and its absorbance was measured. The mass m3 of residual doxorubicin hydrochloride in the supernatant was calculated using standard curve A. Then, the drug loading was calculated using the following formula: (m2-m3) / m1*100% Table 1

[0077] The rare earth upconversion nanoparticle carriers prepared by this invention have an average hydrated particle size of 43~102 nm, a Zeta potential of -31.51~-19.96 mV, and a drug loading of 19~36%.

[0078] Compared with Example 1, Comparative Example 1 showed that, due to the absence of cyclohexane, the hydrophobic OA-NaYF4:Yb,Er upconversion nanoparticles aggregated in secondary water, even with the addition of DSPE-PEG. 2000 The surface of hydrophobic OA-NaYF4:Yb,Er upconversion nanoparticles cannot be sufficiently modified, resulting in poorly dispersed DSPE-PEG. 2000 -NaYF4:Yb,Er nanoparticle carrier. Therefore, cyclohexane acts as a dispersant for the hydrophobic OA-NaYF4:Yb,Er upconversion nanoparticles.

[0079] Compared with Example 1, Comparative Example 2 showed that heating to remove cyclohexane easily leads to insufficient extension of polymer chains on the surface of rare earth upconversion nanoparticle carriers, with partial overlap and trapping, which in turn affects the loading of vaccines / drugs. Compared with Example 1, Comparative Example 3 showed that increasing the volume of cyclohexane had an effect on DSPE-PEG. 2000 The stability and drug loading of the -NaYF4:Yb,Er nanoparticle carrier were not significantly affected; the main effect was an increase in the time required to remove cyclohexane.

[0080] Compared with Example 1, Comparative Example 4 showed that the high concentration of hydrophobic OA-NaYF4:Yb,Er upconversion nanoparticles prevented sufficient dispersion, affecting DSPE-PEG. 2000 Stability and drug loading capacity of NaYF4:Yb,Er nanoparticle carriers.

[0081] Compared with Example 1, Comparative Example 5 showed that low concentration DSPE-PEG 2000 This prevents the surface of the hydrophobic OA-NaYF4:Yb,Er upconversion nanoparticles from being sufficiently modified, affecting DSPE-PEG. 2000 Stability and drug loading capacity of NaYF4:Yb,Er nanoparticle carriers.

[0082] Application Example 1 The DSPE-PEG prepared in Example 1 2000-NaYF4:Yb,Er nanoparticles were further dispersed in phosphate buffer (pH=7.40) at a concentration of 2 mg / mL. The model mRNA (manufactured by Shanghai Weihuan Biotechnology Co., Ltd., sequence [sequence name missing]) was dispersed in phosphate buffer (pH=7.40) at a concentration of 0.2 mg / mL. The above solutions were mixed at a volume ratio of 1:1, stirred at 25℃ for 6 h, centrifuged, and washed. The precipitate was washed three times with deionized water to obtain DSPE-PEG. 2000 -NaYF4:Yb,Er@mRNA nanovaccine.

[0083] (1) The results of dynamic light scattering show that, Figure 5 As shown, after the vector binds to mRNA, DSPE-PEG2000 The hydration diameter of the NaYF4:Yb,Er nanoparticle carrier increased by approximately 30 nm, such as Figure 6 As shown, it retains its good upconversion fluorescence performance.

[0084] (2) The DSPE-PEG prepared in Example 1 2000 -NaYF4:Yb,Er nanoparticle support and DSPE-PEG 2000 The -NaYF4:Yb,Er@mRNA nanovaccine was dispersed in the culture medium. After the cells adhered, different concentrations of the nanovaccine were added, and the cells were cultured for another 24 hours. The cell viability was then tested using the CCK-8 assay.

[0085] Toxicity data such as Figure 7 As shown, the results indicate that DSPE-PEG 2000 -NaYF4:Yb,Er nanoparticle support and DSPE-PEG 2000 -NaYF4:Yb,Er@mRNA nanovaccines all exhibit good biocompatibility.

[0086] (3) DSPE-PEG 2000 The -NaYF4:Yb,Er@mRNA nanovaccine was dispersed in physiological saline and injected subcutaneously into the right hind leg of mice. The mice were then irradiated with 980nm near-infrared light, and real-time images were taken using a mobile phone. Figure 8 As shown in the figure. The results indicate that DSPE-PEG can be clearly observed. 2000 The distribution of the NaYF4:Yb,Er nanofluorocarrier in vivo was observed; and upconversion fluorescence signals were detected in the liver 30 min after subcutaneous injection, indicating that this nanofluorocarrier can be successfully used to study DSPE-PEG. 2000 Biodistribution and in vivo metabolism of the -NaYF4:Yb,Er@mRNA nanovaccine.

[0087] The tissues and organs obtained by dissecting the mice in this experiment are as follows: Figure 9 As shown, under 980nm near-infrared light irradiation, the distribution of this nano-fluorescent carrier in organs was monitored using a mobile phone. Figure 10 As shown, 30 minutes after subcutaneous injection of the fluorescent nanocarrier, an upconversion signal was detected in the liver, indicating that the fluorescent nanocarrier can be successfully used to study the biodistribution and in vivo metabolism of nanovaccines.

[0088] Application Example 2 The DSPE-PEG prepared in Example 5 5000- The NaYF4:Mn,Yb,Er nanoparticle carrier was further dispersed in phosphate buffer (pH=7.40) at a concentration of 2 mg / mL. The model mRNA (manufactured by Shanghai Weihuan Biotechnology Co., Ltd., sequence not specified) was dispersed in phosphate buffer (pH=7.40) at a concentration of 0.2 mg / mL. The above solutions were mixed at a volume ratio of 1:1, stirred at 25℃ for 6 h, centrifuged and washed, and the precipitate was washed three times with deionized water to obtain DSPE-PEG. 5000 -NaYF4:Mn,Yb,Er@mRNA nanovaccine.

[0089] The results of dynamic light scattering indicate that after the vector binds to mRNA, DSPE-PEG 5000- The hydration diameter of the NaYF4:Mn,Yb,Er nanoparticle support increased by approximately 28 nm while retaining its excellent upconversion fluorescence properties.

[0090] Application Example 3 The DSPE-PEG prepared in Example 1 2000 The NaYF4:Yb,Er nanoparticle carrier was further dispersed in phosphate buffer (pH=7.40) at a concentration of 1 mg / mL. Doxorubicin hydrochloride was also dispersed in phosphate buffer (pH=7.40) at a concentration of 1 mg / mL. The above solutions were mixed at a volume ratio of 1:1, stirred at 25°C for 6 h, centrifuged and washed, and the precipitate was washed three times with deionized water to obtain DSPE-PEG. 2000 -NaYF4:Yb,Er@DOX Nanomedicine.

[0091] (1) The results of dynamic light scattering indicate that after the carrier binds to doxorubicin hydrochloride, DSPE-PEG 2000 The hydration diameter of the NaYF4:Yb,Er nanoparticle support is increased by approximately 8 nm, while retaining its excellent upconversion fluorescence properties.

[0092] (2) DSPE-PEG 2000 The NaYF4:Yb,Er@DOX nanomedicine was dispersed in the culture medium. After cell adhesion, different concentrations of nanomedicine were added, and the cells were cultured for another 24 hours. Cell viability was then tested using the CCK-8 assay. Toxicity data are as follows: Figure 11 As shown, the results indicate that DSPE-PEG 2000 -NaYF4:Yb,Er@mRNA nanomedicine exhibits good cell-killing effects.

[0093] Application Example 4 The DSPE-PEG prepared in Example 5 5000 The NaYF4:Mn,Yb,Er nanoparticle support was further dispersed in phosphate buffer (pH=7.40) at a concentration of 1 mg / mL. Doxorubicin hydrochloride was also dispersed in phosphate buffer (pH=7.40) at a concentration of 1 mg / mL. The solutions were mixed at a volume ratio of 1:1, stirred at 25°C for 6 h, centrifuged, and washed. The precipitate was washed three times with deionized water to obtain DSPE-PEG. 5000 -NaYF4:Mn,Yb,Er@DOX Nanomedicine.

[0094] Dynamic light scattering results indicate that after the carrier binds to doxorubicin, DSPE-PEG... 5000 The hydration diameter of the NaYF4:Mn,Yb,Er nanoparticle support is increased by approximately 7 nm, while retaining its excellent upconversion fluorescence properties.

[0095] The present invention also conducted the above-mentioned experiments on nanoparticle carriers prepared in other embodiments, and the results were basically the same. Due to space limitations, they will not be listed one by one.

[0096] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing rare earth upconversion nanoparticle carriers, characterized in that, The method includes the following steps: (1) The hydrophobic rare earth upconversion nanoparticles were dispersed in a nonpolar solvent to obtain a first mixture; (2) The amphiphilic polymer is dispersed in a polar solvent to obtain a second mixture; (3) The first mixture and the second mixture are mixed and the nonpolar solvent is removed to obtain the rare earth upconversion nanoparticle carrier.

2. The method for preparing a rare earth upconversion nanoparticle carrier according to claim 1, characterized in that, In step (1), the hydrophobic rare earth upconversion nanoparticles are hydrophobic upconversion nanoparticles that can emit light.

3. The method for preparing a rare earth upconversion nanoparticle carrier according to claim 1, characterized in that, In step (1), the nonpolar solvent includes cyclohexane and / or chloroform.

4. A method for preparing a rare earth upconversion nanoparticle carrier according to any one of claims 1-3, characterized in that, In step (1), the concentration of the hydrophobic rare earth upconversion nanoparticles in the nonpolar solvent is 0.1 mg / mL to 20 mg / mL.

5. A method for preparing a rare earth upconversion nanoparticle carrier according to any one of claims 1-3, characterized in that, In step (2), the amphiphilic polymer includes DSPE-PEG. 100~20000 One or more of Tween-80 and TPGS.

6. The method for preparing a rare earth upconversion nanoparticle carrier according to claim 1, characterized in that, In step (2), the polar solvent includes deionized water and physiological solutions.

7. The method for preparing a rare earth upconversion nanoparticle carrier according to claim 1, characterized in that, In step (2), the concentration of the amphiphilic polymer in the polar solvent is 0.1 mg / mL to 100 mg / mL.

8. The method for preparing a rare earth upconversion nanoparticle carrier according to claim 1, characterized in that, In step (3), the volume ratio of the first mixture and the second mixture is 2:10 to 10:2; And / or, in step (3), rotary evaporation or vacuum pumping is used to remove nonpolar solvents.

9. A rare earth upconversion nanoparticle carrier prepared by the method according to any one of claims 1-8.

10. The application of the rare earth upconversion nanoparticle carrier of claim 9 in fluorescence imaging and / or drug loading.

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