Three-layer rare earth fluoride, up-conversion nanoparticles, tumor in-situ vaccine and application

Abstract

The invention discloses a three-layer rare earth fluoride, an up-conversion nanoparticle, a tumor in-situ vaccine and application. The three-layer rare earth fluoride has a core-shell structure and comprises a core nanocrystal, a first shell layer and a second shell layer from inside to outside, wherein the crystal phase of the core nanocrystal is alpha-NaYbF4, and the core nanocrystal comprises a doping element; the doping element comprises Tm; the composition of the first shell layer comprises NaYbF4; and the composition of the second shell layer comprises NaYF4. The three-layer rare earth fluoride can realize conversion between near-infrared light and ultraviolet light. Furthermore, the up-conversion nanoparticles containing the three layers of rare earth fluorides and the photo-crosslinking agent can be used for efficiently capturing and enriching tumor antigens in situ under the near-infrared light activation condition, and excellent tumor antigen presentation efficiency is ensured; in addition, the tumor in-situ vaccine containing the three-layer rare earth fluoride, the photo-crosslinking agent and the active medicine can obviously improve the in-situ treatment effect.

Description

Technical Field

[0001] This invention relates to a three-layer rare earth fluoride, upconversion nanoparticles, an in situ tumor vaccine, and their applications, specifically to a three-layer rare earth fluoride, upconversion nanoparticles, an in situ tumor vaccine based on a light-controlled antigen capture system, its preparation method, and its applications. Background Technology

[0002] Cancer vaccines enhance anti-tumor specific immunotherapy through antigen-driven T-cell activation, showing promise as a powerful weapon in cancer treatment. Based on their physical composition, cancer vaccines can be categorized into whole tumor tissue, tumor cells, proteins, peptides, RNA or DNA, and immune adjuvants that introduce antigens. The origins of cancer vaccines can be traced back to the 1910s when scientists injected inactivated streptococci and Serratia into tumor tissue. In recent years, with breakthroughs in immunotherapy and continuous innovation in omics technologies, research on cancer therapeutic vaccines has become a hot topic. Therapeutic vaccines targeting solid tumors such as breast cancer, liver cancer, and lung cancer have entered clinical trials, potentially offering hope for cancer patients.

[0003] Cancer vaccines can be divided into traditional cancer vaccines and in situ cancer vaccines.

[0004] Traditional cancer vaccines consist of pre-selected tumor antigens and immunostimulatory adjuvants. After in vitro synthesis, the vaccine formulation is injected into the body, delivering the antigens and adjuvants to antigen-presenting cells in lymph nodes for antigen presentation and activation of antigen-specific CD8+ T cells. The activated cytotoxic T lymphocytes can directly recognize and kill tumor cells expressing the tumor antigen. For example, Toni K. Choueiri performed whole-exome sequencing, RNA sequencing, and neoantigen prediction on renal cell carcinoma cells, identifying up to 20 neoantigens. These neoantigens were then produced into a neoantigen-mixed vaccine, and poly-ICLCs were used as adjuvants to stimulate the immune system, injected subcutaneously into patients. In nine subjects, the vaccine demonstrated an immune response against the neoantigens, and this immune response lasted for months to years. Besides antigens or peptides, research on mRNA vaccines encoding tumor antigens is also progressing rapidly. Among them, small circular RNA (circRNA) vaccines, as a novel vaccine platform, have unique structures and advantages, and are expected to overcome the challenges of poor stability and uncontrollable immunogenicity of mRNA vaccines, bringing new hope to cancer immunotherapy. Guizhi Zhu et al. used lipid nanodelivery to encode antigen circRNA. This vaccine induced up to 10 times more antigen-specific T cells in mice, resulting in a strong anti-tumor cellular immune response.

[0005] Nevertheless, the efficacy of traditional cancer vaccines may vary due to the differential expression of specific tumor antigens in different patients. In order to meet the needs of different patients, there is an urgent need to develop in situ loaded cancer vaccines that target personalized antigens.

[0006] Cancer patients' own tumors contain abundant tumor-associated antigens. Therefore, novel personalized in situ tumor vaccines with stronger immune response induction capabilities than traditional tumor vaccines can be constructed using the patient's own tumor tissue, realizing a vaccine application strategy of "taken from the patient, used for the patient." Recent research progress shows that traditional cancer treatments (including radiotherapy, chemotherapy, and photodynamic therapy) can induce immunogenic cell death (ICD) in tumor cells, thereby triggering the release of tumor-specific antigens. Short peptides and proteins released during this process are effectively taken up, processed, and presented by antigen-presenting cells (APCs).

[0007] Nevertheless, achieving safe and effective in situ cancer vaccines still faces some challenges, such as insufficient tumor antigen release limiting antigen uptake by antigen-presenting cells (APCs), and low-immunogenicity non-specific proteins that may even inhibit the presentation function of APCs.

[0008] Nanoparticle-based delivery systems show particular promise in the release, capture, and transport of tumor antigens. First, nanoparticles enhance the accumulation of chemotherapeutic drugs in tumor tissues by improving penetration and retention effects. Long-chain PEG or electrically neutral liposomes are used to prolong the blood circulation time of chemotherapeutic drugs, further improving drug accumulation in tumors. After tumor antigen release, nanoparticles can utilize physicochemical interactions (including electrostatic adsorption, hydrophobic forces, and covalent bonds) to achieve molecular binding, thereby capturing and enriching tumor antigens. Andrew Z. Wang et al. compared the antigen capture effects of cationic modified nanoparticles, PLGA-modified nanoparticles, and maleimide-modified nanoparticles. The results showed that PLGA-modified nanoparticles, which capture proteins through hydrophobic interactions, and maleimide-modified nanoparticles, which capture proteins through chemical bonding, were the most effective, maximizing the improvement of αPD-1 efficacy. Finally, nanoparticles with motility or APC-targeting properties can load these tumor antigens and deliver them to APCs, achieving efficient APC presentation. Jinhui Wu et al. designed an antigen capture system based on attenuated Salmonella, utilizing the motility of bacterial flagella. After the release of tumor antigens during radiotherapy, these bacteria were able to efficiently transport the antigens to CD103 at the tumor margin. + CD11C + Significantly increased CD8 in DC cells + The degree of T cell activation.

[0009] However, current platforms exhibit key limitations, namely the inability to achieve spatiotemporal control over the initiation of antigen capture, and these deficiencies ultimately affect the efficiency of antigen enrichment and subsequent antigen presentation.

[0010] Therefore, to address these challenges, there is an urgent need in this field to develop precise engineering systems for tumor-specific targeting that can control antigen isolation capabilities in both time and space. Summary of the Invention

[0011] To address the aforementioned technical deficiencies in existing technologies, this invention proposes a three-layer rare-earth fluoride, upconversion nanoparticles, an in situ tumor vaccine, its preparation method, and its applications. The three-layer rare-earth fluoride can achieve the conversion between 980nm near-infrared light and ultraviolet light, exhibiting excellent luminescent properties and stability. Furthermore, the upconversion nanoparticles can efficiently capture and enrich tumor antigens in situ under near-infrared light activation conditions, ensuring excellent tumor antigen presentation efficiency. Simultaneously, this in situ tumor vaccine can significantly improve the efficacy of in situ therapy.

[0012] To achieve the above objectives, the present invention adopts the following technical solution.

[0013] This invention provides a three-layer rare-earth fluoride, which has a core-shell structure and comprises, from the inside out, a core nanocrystal, a first shell layer, and a second shell layer; wherein,

[0014] The crystal phase of the nuclear nanocrystal is α-NaYbF4, and it includes a doping element, Tm (thulium).

[0015] The first shell layer comprises NaYbF4;

[0016] The second shell is composed of NaYF4.

[0017] In this invention, the three-layer rare earth fluoride is in the form of nanocrystals, and the NaYbF4 in the first shell has an amorphous phase structure, which can be used as a sensitizing layer; those skilled in the art will know that it is significantly different from the α-NaYbF4 in the core nanocrystals. The core nanocrystals can convert 980nm near-infrared light into ultraviolet light; while the Yb (ytterbium) in the first shell and the Y (yttrium) in the second shell can be used to enhance luminescence and improve the stability of the nanocrystals.

[0018] In some embodiments, the molar doping of Tm in the nuclear nanocrystals is 0.1%-1%, for example, 0.5%, based on the molar amount of Na.

[0019] In some preferred embodiments, the three-layer rare earth fluoride has a core-shell structure, and from the inside out are a core nanocrystal, a first shell layer, and a second shell layer; wherein, the crystal phase of the core nanocrystal is α-NaYbF4, and includes a doping element; the doping element is Tm; the composition of the first shell layer is NaYbF4; the composition of the second shell layer is NaYF4; in the core nanocrystal, based on the molar amount of Na, the molar doping amount of Tm is 0.5%.

[0020] The present invention also provides a method for preparing the three-layer rare earth fluoride as described above, which includes the following steps:

[0021] I. Nuclear nanocrystals are prepared by reaction 1 using a raw material composition 1 comprising a sodium fluoride source, a ytterbium fluoride source, a thulium fluoride source, and a solvent;

[0022] II. The raw material composition 2, comprising the said core nanocrystals, a sodium fluoride source, a ytterbium fluoride source, and a solvent, is subjected to reaction 2 to coat the first shell of the core nanocrystals, thereby obtaining a precursor;

[0023] III. The raw material composition 3, comprising the core nanocrystals coated with the first shell, a sodium fluoride source, a yttrium fluoride source, and a solvent, undergoes reaction 3 to coat the core nanocrystals with the second shell.

[0024] The three-layer rare earth fluoride was obtained.

[0025] In this invention, in steps I, II, and III, the raw material compositions 1, 2, and 3 are used only to distinguish different raw material compositions, and the reactions 1, 2, and 3 are used only to distinguish different reactions.

[0026] In this invention, in steps I, II, and III, the sodium fluoride source, ytterbium fluoride source, yttrium fluoride source, thulium fluoride source, and solvent can each be independently selected from conventional reagents in the art.

[0027] In some implementations, in steps I, II, and III, the fluorinated sodium source is independently sodium trifluoroacetate; the fluorinated ytterbium source is independently ytterbium trifluoroacetate; the fluorinated yttrium source is independently yttrium trifluoroacetate; and the fluorinated thulium source is independently thulium trifluoroacetate.

[0028] In some implementations, in step I, the molar ratio of the fluorinated sodium source, the fluorinated ytterbium source, and the fluorinated thulium source is 0.5:(0.4-0.6):(0.002-0.003), for example, 0.5:0.4975:0.0025, based on the molar amounts of sodium, ytterbium, and thulium.

[0029] In some implementations, in step II, the molar ratio of the fluorinated sodium source to the fluorinated ytterbium source is 1:(0.8-1.2), for example, 1:1, based on the molar amounts of sodium and ytterbium. The amounts of the fluorinated sodium source and fluorinated ytterbium source mentioned here do not include the amounts used in step I; that is, the amounts mentioned here refer only to the relative amounts of the newly added raw materials in this step, as follows.

[0030] In some implementations, in step III, the molar ratio of the fluorinated sodium source to the fluorinated yttrium source, based on the molar amounts of sodium and yttrium, is 1:(0.8-1.2), for example, 1:1. The amounts of fluorinated sodium sources mentioned here do not include the amounts used in steps I and II; that is, the amounts mentioned here refer only to the relative amounts of newly added raw materials in this step, as follows.

[0031] In some embodiments, in steps I, II, and III, the solvent is each independently selected from one or more of 1-octadecene, oleic acid, and oleylamine.

[0032] In some embodiments, in steps I, II and III, the solvent is each independently 1-octadecene, oleic acid and oleylamine; the volume ratio of 1-octadecene, oleic acid and oleylamine is, for example, 10:5:5.

[0033] In some implementations, in step I, the ratio of the amount of "the fluorinated sodium source, the fluorinated ytterbium source and the fluorinated thulium source" to the solvent is 1 mmol: 20 mL.

[0034] In some implementations, in step II, the ratio of the amount of "the sodium fluoride source and the ytterbium fluoride source" to the amount of solvent is 2 mmol: 20 mL.

[0035] In some implementations, in step III, the ratio of the amount of "the sodium fluoride source and the yttrium fluoride source" to the amount of solvent is 2 mmol: 20 mL.

[0036] In some specific embodiments, in step I, the raw material composition 1 includes the following components: sodium trifluoroacetate, ytterbium trifluoroacetate, yttrium trifluoroacetate, thulium trifluoroacetate, 1-octadecene, oleic acid, and oleylamine; wherein, the molar ratio of sodium trifluoroacetate, ytterbium trifluoroacetate, and thulium trifluoroacetate is, for example, 0.5:0.4975:0.0025; the volume ratio of 1-octadecene, oleic acid, and oleylamine is, for example, 10:5:5; and the ratio of the amount of the fluorinated sodium source, fluorinated ytterbium source, and fluorinated thulium source to the solvent is, for example, 1 mmol:20 mL.

[0037] In some specific embodiments, in step II, the raw material composition 2 includes the following components: sodium trifluoroacetate, ytterbium trifluoroacetate, 1-octadecene, oleic acid, and oleylamine; wherein, the molar ratio of sodium trifluoroacetate and ytterbium trifluoroacetate is, for example, 1:1; the volume ratio of 1-octadecene, oleic acid, and oleylamine is, for example, 10:5:5; and the ratio of the amount of "the fluorinated sodium source and the fluorinated ytterbium source" to the amount of solvent is, for example, 2 mmol:20 mL.

[0038] In some specific embodiments, in step III, the raw material composition 3 includes the following components: sodium trifluoroacetate, yttrium trifluoroacetate, 1-octadecene, oleic acid, and oleylamine; wherein, the molar ratio of sodium trifluoroacetate and yttrium trifluoroacetate is, for example, 1:1; the volume ratio of 1-octadecene, oleic acid, and oleylamine is, for example, 10:5:5; and the ratio of the amount of "the fluorinated sodium source and the fluorinated yttrium source" to the amount of solvent is, for example, 2 mmol:20 mL.

[0039] In this invention, in steps I, II, and III, reactions 1, 2, and 3 can each be carried out independently according to conventional reactions for preparing rare earth fluorides in the art.

[0040] In some implementations, step I, reaction 1, comprises a two-stage heating reaction:

[0041] In the first stage, the reaction temperature is preferably increased from 30°C to 100-120°C, for example, 110°C.

[0042] The optimal reaction time for the first stage is 5-20 minutes, for example, 10 minutes.

[0043] The first stage is preferably carried out in a vacuum environment.

[0044] The heating rate of the first stage reaction is preferably 8-11℃ / min, for example 10℃ / min.

[0045] In particular, the reaction temperature of the second stage is preferably increased from the reaction temperature of the first stage to 280-300℃, for example 290℃.

[0046] The optimal reaction time for the second stage is 30-60 minutes.

[0047] The second stage is preferably carried out in an inert atmosphere, such as an argon atmosphere.

[0048] The heating rate of the second stage reaction is preferably 8-11 °C / min, for example 10 °C / min.

[0049] In some implementations, step II, reaction 2, comprises a two-stage heating reaction.

[0050] In the first stage, the reaction temperature is preferably increased from 30°C to 100-120°C, for example, 110°C.

[0051] The optimal reaction time for the first stage is 5-20 minutes, for example, 10 minutes.

[0052] The first stage is preferably carried out in a vacuum environment.

[0053] The heating rate of the first stage reaction is preferably 8-11℃ / min, for example 10℃ / min.

[0054] In particular, the reaction temperature of the second stage is preferably increased from the reaction temperature of the first stage to 280-300℃, for example 290℃.

[0055] The optimal reaction time for the second stage is 30-60 minutes.

[0056] The second stage is preferably carried out in an inert atmosphere, such as an argon atmosphere.

[0057] The heating rate of the second stage reaction is preferably 8-11 °C / min, for example 10 °C / min.

[0058] In some implementations, step III, reaction 3, comprises a two-stage heating reaction.

[0059] In the first stage, the reaction temperature is preferably increased from 30°C to 100-120°C, for example, 110°C.

[0060] The optimal reaction time for the first stage is 5-20 minutes, for example, 10 minutes.

[0061] The first stage is preferably carried out in a vacuum environment.

[0062] The heating rate of the first stage reaction is preferably 8-11℃ / min, for example 10℃ / min.

[0063] The reaction temperature in the second stage is preferably 280-300℃, for example 290℃.

[0064] The optimal reaction time for the second stage is 30-60 minutes.

[0065] The second stage is preferably carried out in an inert atmosphere, such as an argon atmosphere.

[0066] The heating rate of the second stage reaction is preferably 8-11 °C / min, for example 10 °C / min.

[0067] In some specific implementations, step I, after reaction 1, further includes cooling to room temperature, precipitating with ethanol, centrifuging to collect the precipitate, washing with cyclohexane and ethanol, and then dispersing the resulting precipitate in cyclohexane.

[0068] The step of precipitation with ethanol preferably includes adding ethanol to the coolant.

[0069] The amount of cyclohexane used for dispersion is, for example, 1 mL.

[0070] In some specific implementations, step II, after reaction 2, further includes cooling to room temperature, precipitating with ethanol, centrifuging to collect the precipitate, washing with ethanol, and then dispersing the obtained precipitate in cyclohexane.

[0071] The amount of cyclohexane used for dispersion is, for example, 1 mL.

[0072] In some specific embodiments, step III, after reaction 3, further includes cooling to room temperature, precipitation with ethanol, washing with cyclohexane and / or ethanol, and then dispersing the resulting precipitate in cyclohexane.

[0073] The amount of cyclohexane used for dispersion is, for example, 5 mL.

[0074] The present invention also provides a three-layer rare earth fluoride, which is prepared by the preparation method of the three-layer rare earth fluoride as described above.

[0075] In some implementations, the three-layer rare earth fluoride is as defined above.

[0076] The present invention also provides an upconversion nanoparticle, which has a core of three rare earth fluorides as described above and a surface coated with a silica shell; the silica shell includes mesoporous channels; and the upconversion nanoparticle has the following structure one or structure two:

[0077] Structure 1:

[0078] The silica shell surface is covalently bonded with a photocrosslinking agent; the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups;

[0079] Structure 2:

[0080] The silica shell has a coating film on its surface, and a photocrosslinking agent is covalently bonded to the surface of the film; the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups.

[0081] Preferably, in the second structure, the surface of the silicon dioxide shell is also modified with amino groups, and the presence of amino groups makes the SiO2 surface positively charged, which is more conducive to the coating of negatively charged films.

[0082] In practical applications of this invention, the photocrosslinking agent can activate the cis-trans isomerization of the three rare earth fluorides based on the light conversion energy, thereby converting the light energy into mechanical energy and forming a "molecular motor".

[0083] Furthermore, during the research, the present invention unexpectedly discovered that, in addition to the above-mentioned characteristics, the upconversion nanoparticles also have the effect of in-situ capture and enrichment of tumor antigens, and ensure excellent tumor antigen presentation efficiency; that is, based on the characteristics of the upconversion nanoparticles, the present invention can achieve the effects of light-controlled antigen capture and enrichment with simple technical means.

[0084] In some embodiments, the photocrosslinker is a primary amine-nitrobenzene azide crosslinker, such as sulfo-SANPAH, with the following structural formula:

[0085] .

[0086] In some embodiments, the upconversion nanoparticles have a particle size of 100-150 nm, for example 132 nm.

[0087] In some embodiments, the zeta potential of the upconversion nanoparticles is -5mV to +10mV.

[0088] In some embodiments, the membrane is a bacterial membrane or a lipid membrane, preferably a bacterial membrane.

[0089] The bacterial membrane is preferably selected from pathogenic bacterial membranes, such as Escherichia coli membranes. The bacterial membrane can act as an immune-activating adjuvant, promoting the targeted delivery of tumor antigens to tumor-draining lymph nodes. Specifically, in a bilateral MC38 xenograft mouse model, tumor orthotopic vaccines coated with bacterial membranes can induce a robust CD8+ T cell response and significantly inhibit distant metastasis through a telescoping effect. Furthermore, the bacterial membrane retains the native lipopolysaccharide (LPS)-toll-like receptor 4 (TLR4) interaction, promoting rapid maturation of dendritic cells, co-expression of CD80 / CD86, and enabling cross-presentation of captured tumor antigens under major histocompatibility complex class I (MHC-I) restriction to initiate a cytotoxic T lymphocyte response.

[0090] The lipid membrane preferably comprises phospholipids and cholesterol, such as DOPC, DOPE, cholesterol, and DSPE-mPEG2000-NH2.

[0091] In some specific embodiments, the composition of the lipid membrane, by mass ratio, is: DOPC:DOPE:cholesterol:DSPE-mPEG2000-NH2=25:50:2:1.

[0092] In some preferred embodiments, the upconversion nanoparticles have the three-layer rare earth fluoride core as the core and are coated with a silica shell. The silica shell includes mesoporous channels, and the surface of the silica shell has an E. coli membrane. The membrane surface is covalently linked with a photocrosslinking agent, and the silica shell surface is amino-modified. The photocrosslinking agent is sulfo-SANPAH.

[0093] The present invention also provides a method for preparing upconversion nanoparticles as described above, which includes the following steps:

[0094] S1. The surface of the three-layer rare earth fluoride is coated with silica, and the template agent is removed to obtain the first precursor.

[0095] S2. Prepared using either method one or method two as follows:

[0096] When the upconversion nanoparticles possess the first structure, the following method is used:

[0097] The first precursor is modified with amino groups and then reacted with a photocrosslinking agent to obtain the upconversion nanoparticles; wherein the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups; the photocrosslinking agent is covalently linked through the amino groups on the surface of the silica shell and the sulfonic acid groups in the phenyl azide compound;

[0098] When the upconversion nanoparticles possess the second structure, the following method two is adopted:

[0099] The first precursor is coated with a film and then reacted with a photocrosslinking agent to obtain the upconversion nanoparticles; wherein the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups; the photocrosslinking agent is covalently linked through the sulfonic acid groups in the phenyl azide compound;

[0100] Preferably, in Method 2, the step of modifying the first precursor with amino groups before membrane coating is further included; the photocrosslinking agent is covalently linked to the amino groups on the membrane and the sulfonic acid groups in the phenyl azide compound.

[0101] In this invention, in step S1, the coating can be performed using conventional methods in the art, such as the Stöber method; wherein, after removing the template agent used in the coating step, corresponding mesoporous channels can be generated in the silica shell simultaneously.

[0102] In some implementations, step S1, the coating includes the following steps:

[0103] The mixture of the three rare earth fluorides, template agent, silicon source, catalyst, aqueous solvent and oil solvent is reacted to obtain the product.

[0104] Preferably, the three-layer rare earth fluoride is first dissolved in a portion of the oily solvent before being mixed with other materials.

[0105] The template agent is, for example, hexadecyltrimethylammonium bromide.

[0106] The silicon source is, for example, tetraethyl orthosilicate.

[0107] The catalyst is, for example, urea.

[0108] The aqueous solvent is, for example, water and / or pentanol.

[0109] The oily solvent is, for example, cyclohexane.

[0110] The preferred amounts of the three rare earth fluorides, template agent, silicon source and catalyst are (0.01-0.05)mol:(0.5-2)g:(0.5-2)mL:(0.1-0.5)g, for example 0.02mol:1g:1mL:0.3g.

[0111] The volume ratio of the aqueous solvent to the oily solvent is preferably (10-20): (10-20), for example, 15.01:15.

[0112] The reaction temperature is preferably 60-80°C, for example 70°C; the reaction time is preferably 6-10 hours, for example 8 hours.

[0113] In some implementations, step S1 further includes centrifugation to collect the precipitate, washing to collect the precipitate, and dispersion.

[0114] The centrifugation speed is, for example, 12,000 rpm, and the time is, for example, 10 min.

[0115] The cleaning is performed, for example, with ethanol.

[0116] The dispersion is carried out, for example, with a 1% NaCl methanol solution.

[0117] In this invention, the removal of the template agent in step S1 can be carried out using conventional procedures in the art; for example, reacting at 50°C for 4 hours is sufficient.

[0118] In some implementations, step S1, after removing the template agent, further includes centrifugation to collect the precipitate, washing, and drying.

[0119] The centrifugation speed is, for example, 12,000 rpm, and the time is, for example, 10 min.

[0120] The cleaning is performed, for example, with ethanol.

[0121] The drying temperature is, for example, 60°C.

[0122] In this invention, in both methods of step S2, the amino modification can be carried out independently using conventional steps in the art; for example, the first precursor can be reacted with a silane coupling agent in a solvent; the solvent is, for example, anhydrous ethanol, and the silane coupling agent is, for example, (3-aminopropyl)triethoxysilane.

[0123] In some embodiments, in both methods of step S2, the amino modification further includes a step of centrifugation to collect the precipitate and washing. The washing is performed, for example, with ethanol.

[0124] In some embodiments, in one of the steps of S2, the step of reacting with the photocrosslinker includes: incubating the amino-modified product and the photocrosslinker at room temperature in a dark environment, for example, for 24 hours.

[0125] In some implementations, in step S2 of the first method, the reaction with the photocrosslinking agent further includes centrifugation to collect the precipitate and washing. The washing is performed, for example, with deionized water.

[0126] In some preferred embodiments, in step S2, the preparation of the bacterial membrane includes the following steps: lysing the bacteria and centrifuging to obtain the membrane.

[0127] The pyrolysis is performed, for example, under ice bath conditions using an ultrasonic method.

[0128] Preferably, the centrifugation includes a first centrifugation to collect the supernatant and a second centrifugation to collect the supernatant; the first centrifugation is performed at a speed of, for example, 6000g, for a time of, for example, 10min, and at a temperature of, for example, 4℃; the second centrifugation is performed at a speed of, for example, 20000g, for a time of, for example, 10min, and at a temperature of, for example, 4℃.

[0129] In some embodiments, in the second method of step S2, the membrane coating includes: mixing the first precursor or the amino-modified product with the membrane in a solvent; the solvent is, for example, an aqueous PBS solution.

[0130] In some implementations, in step S2, the reaction with the photocrosslinking agent includes: incubating the product obtained by film coating and the photocrosslinking agent at room temperature in a dark environment, for example, for 24 hours.

[0131] In some embodiments, in the second method of step S2, the reaction with the photocrosslinking agent further includes centrifugation to collect the precipitate and washing it. The washing is performed, for example, with deionized water.

[0132] The present invention also provides an upconversion nanoparticle, which is prepared by the upconversion nanoparticle preparation method described above.

[0133] In some implementations, the upconversion nanoparticles are as previously defined.

[0134] This invention also provides an in situ tumor vaccine, which has a core of three rare earth fluorides as described above, and a surface coated with a silica shell, wherein the silica shell includes mesoporous channels; the mesoporous channels are loaded with an active drug; and the in situ tumor vaccine has the following structure three or structure four:

[0135] Structure 3:

[0136] The silica shell surface is covalently bonded with a photocrosslinking agent; the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups;

[0137] Structure Four:

[0138] The silica shell has a coating film on its surface, and a photocrosslinking agent is covalently bonded to the surface of the film; the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups;

[0139] In the fourth structure, the surface of the silica shell is preferably modified with amino groups.

[0140] In this invention, the active drug is an active drug that can induce immunogenic cell death in tumor cells.

[0141] In some embodiments, the active pharmaceutical ingredient is doxorubicin hydrochloride (DOX).

[0142] In some embodiments, the active drug loading rate in the tumor in situ vaccine is not less than 85%, preferably not less than 90%.

[0143] In some embodiments, the photocrosslinker is a primary amine-nitrobenzene azide crosslinker, such as sulfo-SANPAH.

[0144] In some embodiments, the membrane is a bacterial membrane or a lipid membrane, preferably a bacterial membrane.

[0145] The bacterial membrane is preferably selected from pathogenic bacterial membranes, such as Escherichia coli membranes.

[0146] The lipid membrane preferably comprises phospholipids and cholesterol, such as DOPC, DOPE, cholesterol, and DSPE-mPEG2000-NH2.

[0147] In some specific embodiments, the composition of the lipid membrane, by mass ratio, is: DOPC:DOPE:cholesterol:DSPE-mPEG2000-NH2=25:50:2:1.

[0148] In some preferred embodiments, the tumor in situ vaccine has a core of the aforementioned three-layer rare earth fluoride, with a silica shell covering the surface. The silica shell includes mesoporous channels, which are loaded with an active drug, namely doxorubicin hydrochloride. The surface of the silica shell has an E. coli membrane, and the membrane surface is covalently linked with a photocrosslinking agent. The surface of the silica shell is amino-modified, and the photocrosslinking agent is sulfo-SANPAH.

[0149] In some preferred embodiments, the tumor in situ vaccine uses the aforementioned three-layer rare earth fluoride as a core, coated with a silica shell. The silica shell includes mesoporous channels loaded with an active drug, namely doxorubicin hydrochloride. The surface of the silica shell has an E. coli membrane, covalently linked to a photocrosslinking agent. The silica shell surface is amino-modified, and the photocrosslinking agent is sulfo-SANPAH. The three-layer rare earth fluoride has a core-shell structure, consisting of a core nanocrystal, a first shell, and a second shell from the inside out. The core nanocrystal has the crystal phase α-NaYbF4 and includes a dopant element, Tm. The first shell is composed of NaYbF4, and the second shell is composed of NaYF4. In the core nanocrystal, based on the molar amount of Na, the molar doping amount of Tm is 0.5%.

[0150] The present invention also provides a method for preparing the above-described tumor in situ vaccine, which includes the following steps:

[0151] A. The surface of the three-layer rare earth fluoride is coated with silica, and the template agent is removed to obtain the first precursor.

[0152] B. Prepared using method three or method four as follows:

[0153] When the tumor in situ vaccine possesses structure three, the following method three is adopted:

[0154] The first precursor is modified with amino groups, then reacted with a photocrosslinking agent, and then loaded with a drug to prepare the tumor in situ vaccine; wherein the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups; the photocrosslinking agent is covalently linked to the amino groups on the surface of the silica shell and the sulfonic acid groups in the phenyl azide compound;

[0155] When the tumor in situ vaccine has the fourth structure, the following method four is adopted:

[0156] The first precursor is loaded with a drug, then coated with a membrane, and then reacted with a photocrosslinking agent to prepare the tumor in situ vaccine; wherein the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups; the photocrosslinking agent is covalently linked through the sulfonic acid groups in the phenyl azide compound;

[0157] Preferably, in Method 4, the step of modifying the first precursor with amino groups before drug loading is further included; the photocrosslinking agent is covalently linked to the amino groups on the membrane and the sulfonic acid groups in the phenyl azide compound.

[0158] In some implementations, step A, the coating can be performed using conventional methods in the art, such as the Stöber method; preferably, it includes the following steps:

[0159] The mixture of the three rare earth fluorides, template agent, silicon source, catalyst, aqueous solvent and oil solvent is reacted to obtain the product.

[0160] Preferably, the three-layer rare earth fluoride is first dissolved in a portion of the oily solvent before being mixed with other materials.

[0161] The template agent is, for example, hexadecyltrimethylammonium bromide.

[0162] The silicon source is, for example, tetraethyl orthosilicate.

[0163] The catalyst is, for example, urea.

[0164] The aqueous solvent is, for example, water and / or pentanol.

[0165] The oily solvent is, for example, cyclohexane.

[0166] The preferred amounts of the three rare earth fluorides, template agent, silicon source and catalyst are (0.01-0.05)mol:(0.5-2)g:(0.5-2)mL:(0.1-0.5)g, for example 0.02mol:1g:1mL:0.3g.

[0167] The volume ratio of the aqueous solvent to the oily solvent is preferably (10-20): (10-20), for example, 15.01:15.

[0168] The reaction temperature is preferably 60-80°C, for example 70°C; the reaction time is preferably 6-10 hours, for example 8 hours.

[0169] In some implementations, step A further includes centrifugation to collect the precipitate, washing to collect the precipitate, and dispersion.

[0170] The centrifugation speed is, for example, 12,000 rpm, and the time is, for example, 10 min.

[0171] The cleaning is performed, for example, with ethanol.

[0172] The dispersion is carried out, for example, with a 1% NaCl methanol solution.

[0173] In this invention, in step A, the removal of the template agent can be carried out using conventional procedures in the art; for example, reacting at 50°C for 4 hours is sufficient.

[0174] In some implementations, step A, after removing the template agent, further includes centrifugation to collect the precipitate, washing, and drying.

[0175] The centrifugation speed is, for example, 12,000 rpm, and the time is, for example, 10 min.

[0176] The cleaning is performed, for example, with ethanol.

[0177] The drying temperature is, for example, 60°C.

[0178] In some embodiments, in steps B, methods three and four, the amino modification step each independently includes: reacting the first precursor with a silane coupling agent in a solvent; the solvent being, for example, anhydrous ethanol, and the silane coupling agent being, for example, (3-aminopropyl)triethoxysilane.

[0179] In some embodiments, in steps B, specifically in methods three and four, the amino modification independently further includes a step of centrifugation to collect the precipitate and washing. The washing is performed, for example, with ethanol.

[0180] In some embodiments, in step B, method three, the step of reacting with the photocrosslinker includes: incubating the amino-modified product and the photocrosslinker at room temperature in a dark environment, for example, for 24 hours.

[0181] In some implementations, step B, in method three, further includes centrifugation to collect the precipitate and washing it after the reaction with the photocrosslinking agent. The washing is performed, for example, with deionized water.

[0182] In some preferred embodiments, in step B, method four, the preparation of the bacterial membrane includes the following steps: lysing the bacteria and centrifuging to obtain the membrane.

[0183] The pyrolysis is performed, for example, under ice bath conditions using an ultrasonic method.

[0184] Preferably, the centrifugation includes a first centrifugation to collect the supernatant and a second centrifugation to collect the supernatant; the first centrifugation is performed at a speed of, for example, 6000g, for a time of, for example, 10min, and at a temperature of, for example, 4℃; the second centrifugation is performed at a speed of, for example, 20000g, for a time of, for example, 10min, and at a temperature of, for example, 4℃.

[0185] In some implementations, in step B, method four, the membrane coating step includes:

[0186] The drug-loaded product is reacted with the membrane in a solvent, for example, an aqueous solution of PBS.

[0187] In some implementations, in step B, method four, the step of reacting with the photocrosslinker includes: incubating the product obtained by film coating and the photocrosslinker at room temperature in a dark environment, for example, 24 hours.

[0188] In some implementations, in step B, method four, the reaction with the photocrosslinking agent further includes centrifugation to collect the precipitate and washing. The washing is performed, for example, with deionized water.

[0189] The present invention also provides an in situ tumor vaccine, which is prepared by the method for preparing an in situ tumor vaccine as described above.

[0190] In some implementations, the tumor in situ vaccine is as defined above.

[0191] The present invention also provides the use of the aforementioned three-layer rare earth fluoride or the aforementioned upconversion nanoparticles in the preparation of drugs for the prevention and / or treatment of tumors.

[0192] In some implementations, the tumor is one of colon cancer, melanoma, lung cancer, and rectal cancer.

[0193] In some embodiments, the tumor drug is activated by irradiation with a 980nm laser.

[0194] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0195] The reagents and raw materials used in this invention are all commercially available.

[0196] The positive and progressive effects of this invention are as follows:

[0197] The three-layer rare earth fluoride of the present invention can achieve the conversion of 980nm near-infrared light to ultraviolet light, and has excellent luminescence performance and stability. Furthermore, the upconversion nanoparticles containing the three-layer rare earth fluoride and photocrosslinking agent can activate the cis-trans isomerization of the photocrosslinking agent through the light conversion energy from near-infrared to ultraviolet light, and can also perform in-situ efficient capture and enrichment of tumor antigens, and ensure excellent tumor antigen presentation efficiency.

[0198] Furthermore, the tumor in situ vaccine of the present invention, which contains the three layers of rare earth fluoride, photocrosslinking agent and active drug, has good tumor cell compatibility. While triggering the release of a large number of tumor antigens, it achieves efficient in situ capture, enrichment and presentation of tumor antigens, thereby effectively stimulating T cell activation and improving the in situ treatment effect. Attached Figure Description

[0199] Figure 1 The crystal phase and morphology of UCNP, UCNP-SiO2, US, ULS, and UMS in Example 1 are characterized. a) XRD patterns of UCNP, UCNP-SiO2, and standard cards; b) Particle size of US, ULS, and UMS; c) TEM image of UMS, scale bar 100 nm.

[0200] Figure 2 XPS spectrum of UCNP-SiO2 and elemental mapping of UMS in Example 1. a) XPS spectrum of UCNP-SiO2; b) Elemental mapping image of UMS, scale bar is 100nm.

[0201] Figure 3 The upconversion emission spectra and images of UCNPs in Example 1 are shown. a) Upconversion emission spectra of UCNPs; b) Emission of UCNPs under 980nm laser irradiation.

[0202] Figure 4 The results of SDS-PAGE images and zeta potential analysis in Example 1 are shown. a) SDS-PAGE bands of E. coli (EcN), BM and UMS; b) zeta potentials of US, ULS and UMS.

[0203] Figure 5 The results of nitrogen adsorption-desorption tests on US in Example 1 are shown. a) Pore size distribution diagram; b) Adsorption-desorption isotherm.

[0204] Figure 6 The results of drug release characterization of UMS-DOX in Example 1 are shown.

[0205] Figure 7The results of the evaluation of the uptake performance of US, ULS, and UMS by tumor cells in Example 2 are shown. a) Confocal image of tumor cells uptake of the material, scale bar 50 μm; b) Average fluorescence intensity (MFI) of tumor cells after uptake of the material by flow cytometry.

[0206] Figure 8 To assess the effects of free DOX, US-DOX, ULS-DOX, and UMS-DOX on tumor cell killing and antigen release in Example 2, the following methods were used: a) Cck-8 assay was used to detect the killing efficiency of free DOX, US-DOX, ULS-DOX, and UMS-DOX on tumor cells; b) Taking HMGB1 as an example, the degree to which free DOX, US-DOX, ULS-DOX, and UMS-DOX promoted the ICD effect in tumor cells was detected.

[0207] Figure 9 The concentration of adsorbed proteins in UM, US, ULS, UMS, UM+L, US+L, ULS+L, and UMS+L in Example 2 is used to measure the effect.

[0208] Figure 10 The image shows the CLSM image (scale bar 10μm) captured by BMDC for US, ULS, and UMS in Example 2.

[0209] Figure 11 The results of flow cytometry fluorescence quantification of BMDC uptake of US, ULS, and UMS in Example 2 are shown in (a); and the polarization of BMDCs in PBS, UMS, CL, UM+CL, US+CL+L, ULS+CL+L, UMS+CL-L, and UMS+CL+L are shown in (b).

[0210] Figure 12 For the effect, Example 2 included the safety evaluation of BMDCs and the in vitro polarization of T cells. a) BMDC cell viability was detected by Cck-8 assay after co-incubation with US, ULS, and UMS, respectively; b) CD3 concentrations in PBS, UMS, CL, UM+CL, US+CL+L, ULS+CL+L, UMS+CL-L, and UMS+CL+L. + CD4 + T cell polarization ratio; c) CD3 in PBS, UMS, CL, UM+CL, US+CL+L, ULS+CL+L, UMS+CL-L, and UMS+CL+L + CD8 + T cell polarization ratio. Detailed Implementation

[0211] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0212] The specific information regarding the reagents used in the following examples and comparative examples is as follows:

[0213] Ytterbium trifluoroacetate (Yb-TFA, 99.99%), Yttrium trifluoroacetate (Y-TFA, 99.99%), Sodium trifluoroacetate (Na-TFA, 99.99%), Thulium trifluoroacetate (Tm-TFA, 99.99%), 1-Octadecene (ODE, 90%, Merck), Oleic acid (OA, 90%, Merck), Oleylamine (OM, 90%, Merck), Anhydrous ethanol (CH3CH2OH, 99.5%, Shanghai Guoyao), Cyclohexane (99.6%, Shanghai Guoyao), Urea (99%, Aladdin), Tetraethyl orthosilicate (TEOS, 99.5%, Merck), n-Pentanol (99%, Merck), Hexadecyltrimethylammonium bromide (CTAB, 99%, Merck), (3-aminopropyl)triethoxysilane (APTES, 98.5%, Merck), Sulfo-SANPAH (Thermo Fisher), deionized water, Escherichia coli (Beina Biotechnology), LB medium (Sangon Biotech), dipalmitoylphosphatidylcholine (DPPC, Meilun), L-cysteine, egg yolk lecithin (DEPC, Solarbio), doxorubicin hydrochloride (doxorubicin, DOX, MCE), PAGE gel rapid preparation kit (10%), SDS-PAGE protein loading buffer, BCA kit (Yamei), Coomassie brilliant blue rapid staining solution (Beyotime), tricolor pre-stained protein marker (10~250kDa, ABclonal).

[0214] The specific information of the instruments used in the following embodiments and comparative examples is shown in Table 1:

[0215] Table 1

[0216]

[0217] Example 1

[0218] 1. Preparation of three-layer rare earth fluorides:

[0219] 1.1 Preparation of nuclear nanocrystals (α-NaYbF4:0.5%Tm):

[0220] Add 10 mL ODE, 5 mL OA, 5 mL OM, 0.4975 mmol Yb-TFA, 0.0025 mmol Tm-TFA, and 0.5 mmol Na-TFA to a 100 mL three-necked flask. In a vacuum environment, increase the temperature from 30 °C to 110 °C at 10 °C / min and hold for 10 min. Then, in an argon atmosphere, increase the temperature to 290 °C at 10 °C / min and heat for 30 min. After the reaction is complete, cool to room temperature, add ethanol to the coolant to precipitate, centrifuge to remove the supernatant, and wash several times with cyclohexane-ethanol. Finally, redisperse the precipitate in 1 mL of cyclohexane.

[0221] 1.2. First shell layer (NaYbF4):

[0222] 1 mmol Yb-TFA, 1 mmol Na-TFA, 5 mL OA, 5 mL OM, 10 mL ODE, and 1 mL of the product obtained in step (1) were mixed and added to a 100 mL three-necked flask. The temperature was increased from 30 °C to 110 °C at 10 °C / min in a vacuum environment and held for 10 min. Then, the temperature was increased to 290 °C at 10 °C / min in an argon atmosphere and reacted for 1 h. After the reaction was completed, the mixture was cooled to room temperature, washed several times with ethanol, and the precipitate was redispersed in 1 mL cyclohexane.

[0223] 1.3. Covering with a second shell (NaYF4):

[0224] 1 mmol Y-TFA, 1 mmol Na-TFA, 5 mL OA, 5 mL OM, 10 mL ODE, and 1 mL of the product obtained in step (2) were mixed and added to a 100 mL three-necked flask. The temperature was increased from 30 °C to 110 °C at 10 °C / min in a vacuum environment and held for 10 min. Then, the temperature was increased to 290 °C at 10 °C / min in an argon atmosphere and reacted for 1 h. After the reaction was completed, the mixture was cooled to room temperature, washed several times with ethanol, and the precipitate was redispersed in 5 mL of cyclohexane.

[0225] Finally, 1 mmol of three-layer rare earth fluoride (α-NaYbF4:0.5%Tm@2NaYbF4@2NaYF4, UCNPs) was obtained.

[0226] 2. Preparation of upconversion nanoparticles:

[0227] 2.1 Silica Coating (Preparation of UCNPs-SiO2)

[0228] Silica was coated onto the surface of oil-phase UCNPs using the Stöber method, and then converted into water-soluble UCNPs-SiO2. Specifically:

[0229] First, 1 mmol of the synthesized UCNPs from step 1 was dissolved in 5 mL of cyclohexane and sonicated to ensure uniform dispersion. Then, 0.02 mmol of the UCNPs cyclohexane solution, 1 g of CTAB, 0.3 g of urea, 15 mL of water, 10 μL of pentanol, 1 mL of TEOS, and 15 mL of cyclohexane were added to a 100 mL round-bottom flask. The mixture was stirred at room temperature for 1 h, then heated to 70 °C and stirred for 8 h. After the reaction was complete, the mixture was centrifuged at 12000 rpm for 10 min. The precipitate was dispersed with anhydrous ethanol and washed twice. The precipitate was then dispersed in a methanol solution containing 1% NaCl and reacted at 50 °C for 4 h to remove the template (TEOS). After the reaction was complete, the mixture was centrifuged at 12000 rpm for 10 min, washed with anhydrous ethanol, and then dried in a 60 °C oven.

[0230] 2.2 Connection of photocrosslinking agents

[0231] Method 1

[0232] (1) Disperse 1 mg of the obtained UCNPs-SiO2 in 10 mL of anhydrous ethanol, add 1 mL of APTES and stir at room temperature for 24 h to carry out amino modification, then centrifuge to collect, and wash the precipitate several times with ethanol to obtain UM.

[0233] (2) 1 mg of the obtained UM was dissolved in water with 2 mg of sulfo-SANPAH in the dark, incubated at room temperature in the dark for 24 h, and then centrifuged and washed to obtain US.

[0234] Method 2

[0235] (1) Escherichia coli was collected in PBS and lysed by sonication in an ice bath, followed by centrifugation (6000 g, 10 min, 4 °C) to remove undisturbed bacteria. The supernatant was centrifuged (20000 g, 10 min, 4 °C) to obtain bacterial membrane fragments (BM). The total protein content of the membrane was determined by the BCA method.

[0236] (2) Prepare UM according to the previous steps, mix the aqueous solution containing 1 mg UM with BM, sonicate in an ice water bath for 5 min, centrifuge and wash the precipitate, incubate with 2 mg sulfo-SANPAH at room temperature in the dark for 24 h, centrifuge and wash to obtain UMS.

[0237] Method 3

[0238] UM was prepared following the steps above. An aqueous solution containing 1 mg of UM was mixed with a lipid membrane (LIP) prepared by rotary evaporation with a mass ratio of DOPC:DOPE:cholesterol:DSPE-mPEG2000-NH2 = 25:50:2:1. The mixture was sonicated in an ice-water bath for 5 min, centrifuged and washed, and the precipitate was incubated with 2 mg of sulfo-SANPAH at room temperature in the dark for 24 h. After centrifugation and washing, ULS was obtained.

[0239] 3. Preparation of in situ tumor vaccines

[0240] Method 1

[0241] Add 2 mg of DOX dissolved in PBS to the obtained 1 mg US and stir for 12 h. Then centrifuge and wash with deionized water to obtain DOX-loaded US (US-DOX).

[0242] Method 2

[0243] (1) Prepare UM according to the previous steps, add DOX (2 mg) dissolved in PBS to 1 mg UM and stir for 12 h, then centrifuge and wash with deionized water to obtain DOX-loaded UM, i.e. UM-DOX.

[0244] (2) Mix 500 μg UM-DOX with 1 mg / mL BM, sonicate in an ice bath for 5 min, centrifuge and wash the precipitate, incubate with 2 mg sulfo-SANPAH in the dark at room temperature for 24 h, centrifuge and wash to obtain UMS-DOX.

[0245] Method 3

[0246] (1) Prepare UM-DOX by following the steps above.

[0247] (2) Mix 1 mg UM-DOX with LIP, sonicate in an ice bath for 5 min, centrifuge and wash, then incubate the precipitate with 2 mg sulfo-SANPAH at room temperature in the dark for 24 h, centrifuge and wash to obtain ULS-DOX.

[0248] Example 1

[0249] The following characterizations were performed on UCNPs, UCNPs-SiO2, US, UMS, ULS, BM, US-DOX, UMS-DOX, and ULS-DOX, respectively:

[0250] 1. Characterization methods

[0251] (1) Characterization of structure and phase purity: X-ray powder diffraction (XRD) was used to determine the structure and phase purity of the sample.

[0252] (2) Particle size characterization: The particle size of the sample was determined using a Malvern laser particle size analyzer at a 90° angle.

[0253] (3) Zeta potential characterization: The sample to be tested was injected into the U-shaped gold-plated sample cell with a syringe, and the zeta potential of the sample to be tested was measured with a Malvern laser particle size analyzer.

[0254] (4) Characterization of bacterial membrane surface proteins: Characterization was performed using SDS-PAGE. Specifically, the bacterial membrane extract, US, and UMS were boiled in SDS-PAGE loading buffer. Then, samples containing equal amounts of protein (40 μg / well) were loaded onto 10% SDS-PAGE gels and electrophoresed at 100V. The resulting gels were stained with Coomassie Brilliant Blue, washed overnight, and then imaged using a GBOX system.

[0255] (5) Characterization of mesoporous structure: For US, nitrogen adsorption-desorption experiment was used. Specifically, the temperature was raised to 120℃ in a nitrogen environment, degassed for 4-6 hours, and the desorption peak was recorded.

[0256] (6) Drug loading rate characterization: For UMS-DOX, tests were conducted. Specifically, using DOX dilution as a reference solution, a UV spectrophotometer was used to scan the wavelength range of 200~800 nm. The results showed that the DOX dilution had a large absorption at a wavelength of 488 nm. Therefore, 488 nm was used as the detection wavelength for the drug loading rate of UMS-DOX. Using a blank dilution as a blank solution, the absorbance of the DOX stock solution and the supernatant after centrifugation of UMS drug-loaded solution was measured at a wavelength of 488 nm. Drug loading rate = (absorbance of stock solution - absorbance of supernatant) / absorbance of stock solution * 100%.

[0257] (7) Drug release characterization: For UMS-DOX, the drug release process was simulated in vitro under the slightly acidic environment of the tumor. Specifically, buffer systems with pH=7.0 and pH=6.4 were prepared to detect the DOX release process. 0.5 mL of UMS-DOX was added to a dialysis bag, and a 50 mL buffer system was placed in it. 100 μL of the buffer system was taken at different time points for absorbance detection at 488 nm. The release amount was calculated and plotted.

[0258] 2. Characterization Results

[0259] (1) Characterization of structure, light conversion performance, etc.

[0260] Figure 1 Figure 'a' shows the XRD patterns of UCNPs and UCNPs-SiO2. As can be seen from the figure, the XRD patterns of both correspond to the diffraction peaks of the standard cubic phase NaYF4 (α phase) standard card PDF#06-0342, with almost no impurity peaks appearing, proving that the crystalline phase in both UCNPs and UCNPs-SiO2 is α-NaYF4.

[0261] Figure 1 b represents the particle size analysis results for US, ULS, and UMS, respectively. Figure 1 c is a TEM image of UMS. As can be seen from the image, UMS consists of uniformly dispersed spheres with a particle size of approximately 132 nm.

[0262] Figure 2 Figure a shows the XPS energy spectrum of UCNPs-SiO2. As can be seen from the figure, UCNPs-SiO2 is composed of elements such as carbon, silicon, oxygen, fluorine, sodium, yttrium, and ytterbium, indicating that rare earth elements such as ytterbium have been successfully doped into UCNPs-SiO2.

[0263] Figure 2 b represents the elemental mapping results of UMS. This further confirms the presence of elements such as carbon, silicon, oxygen, fluorine, sodium, yttrium, and ytterbium in UMS, and detects Tm, indicating that Tm was also successfully doped.

[0264] Figure 3 Figures a and b show the beam characteristics of UCNPs under 980nm laser (a) and infrared light irradiation (b), respectively. The results indicate that under 980nm laser excitation, UCNPs exhibit distinct emission peaks in both the ultraviolet and blue light regions; while under infrared light irradiation, UCNPs show a distinct violet beam. This demonstrates that UCNPs possess significant ultraviolet and blue light upconversion luminescence characteristics, which is consistent with high Yb... 3+ Tm at ion concentration 3+ The ion population is closely related to the energy levels of 3H5, 1I6, and 1D2.

[0265] (2) Characterization of membrane proteins and electrical potential of bacterial films

[0266] This invention analyzes the membrane proteins of the extracted bacterial membrane and detects the corresponding surface potential. Electrophoresis results show that, compared with *E. coli* lysate (EcN), the extracted bacterial membrane (BM) and the protein components of UMS exhibit different enrichment patterns at the same total mass. Figure 4 (a). This indicates that during the extraction of the bacterial membrane, certain components of the membrane are retained, while the protein contents of *E. coli* are removed. Based on this, the present invention hypothesizes that UMS can exert the immunostimulatory function of the *E. coli* bacterial membrane. Furthermore, the potentials of US, ULS, and UMS are all between -5 and +10 mV, exhibiting an overall electrically neutral state. This is beneficial for stable transport in systemic circulation and reduces the formation of protein corona (a). Figure 4 (b).

[0267] (3) Characterization of mesoporous structure

[0268] Depend on Figure 5 The results show that the obtained US has a mesoporous dominant structure with a uniform core pore size (10 nm); the sharpness of the main peak indicates that the synthesis process has good control over the pore size. Figure 5The adsorption isotherm of b generally exhibits typical Type IV characteristics. In the low relative pressure region (P / P0 < 0.1), the initial adsorption capacity is high, indicating the possible presence of micropores or strong adsorption sites in the material. As the relative pressure increases to the medium range (P / P0 ≈ 0.1–0.8), the adsorption capacity shows a gradual upward trend, reflecting a multilayer adsorption process. In the high relative pressure region (P / P0 > 0.8), the adsorption capacity increases sharply. Simultaneously, the desorption and adsorption branches form a significant hysteresis loop in the range of P / P0 ≈ 0.4–0.95. This hysteresis loop closes in the low-pressure region, further confirming the mesoporous structure. Therefore, this curve type indicates that the material is predominantly mesoporous.

[0269] (4) Characterization of drug release characteristics

[0270] In an in vitro simulation of the drug release process under a slightly acidic environment within a tumor, it was found that UMS-DOX was almost completely released within 10 hours at a pH of 6.4, while no significant release was observed in a neutral environment. Figure 6 ).

[0271] Example 2

[0272] 1. Characterization methods

[0273] (1) Cck-8 assay for cell killing

[0274] MC38 mouse colon cancer cells were obtained from the stem cell bank of the Chinese Academy of Sciences Cell Bank and cultured in a cell culture incubator at 37°C and 5% CO2. The culture medium used was 1640 medium supplemented with 10% fetal bovine serum.

[0275] MC38 cells were added to 96-well plates at a density of 5000 cells / well, 100 μL per well. After culturing for 24 h, free DOX, US, ULS, and UMS (diluted with 5% fetal bovine serum in 1640 medium) at DOX concentrations of 0, 2.5, 5, 10, 20, and 40 μg / mL were added to the 96-well plates, respectively. After incubation for 24 h, the medium was discarded and replaced with 100 μL of 1640 basal medium containing 10% CCK-8 solution. The plates were incubated at 37°C for 1 h, and the absorbance at 450 nm was measured using a microplate reader, and the cell viability of each group was calculated.

[0276] (2) Detection of tumor cell uptake of materials

[0277] First, Cy5 dye was applied to each group of materials.

[0278] UM was prepared according to the steps in Example 1. 1 mg / mL of UM was incubated with Cy5 dye in the dark for 4 h. Then, the UM precipitate labeled with Cy5 dye was obtained by centrifugation at 12000 rpm. After washing three times, it was resuspended in PBS.

[0279] Similarly, Cy5-labeled US, ULS, and UMS were prepared respectively.

[0280] The corresponding characteristics are as follows:

[0281] ① The uptake of US, ULS, and UMS by MC38 cells was observed using laser confocal microscopy. After digestion, MC38 cells were seeded at a density of 20,000 cells / well in 24-well plates coated with cell spreaders. Cy5-labeled materials of the same fluorescence intensity (diluted in 1640 medium with 5% fetal bovine serum) were added to each well, and the plates were cultured for 4 hours. The plates were then thoroughly washed with PBS, fixed with 4% paraformaldehyde at room temperature for 20 minutes, and discarded. After DAPI staining, the spreaders were removed, placed on glass slides, mounted, and observed.

[0282] ② Quantitative analysis of cell uptake results was performed using flow cytometry. MC38 cells were digested and seeded at a density of 50,000 cells / well in 12-well plates. Cy5-labeled materials (diluted in 1640 medium with 5% fetal bovine serum) of the same fluorescence intensity were added to each well, and the plates were cultured for 4 hours. Cells were then digested and collected from the wells, washed with PBS, and the average fluorescence intensity of Cy5 in each well was detected by flow cytometry.

[0283] (3) Detection of antigen release capacity of tumor cells after material intervention

[0284] HMGB1 is the most common tumor-associated antigen (TAA), and was selected as the representative antigen to detect the antigen release level of MC38 cells after intervention. MC38 cells were seeded at a density of 100,000 cells / well in 6-well plates and cultured for 24 h. Afterward, free DOX, US-DOX, ULS-DOX, and UMS-DOX were added to each well (DOX concentration was 10 μg / mL, diluted in 1640 medium with 5% fetal bovine serum). After culturing in a cell culture incubator at 37°C for 18 h, the cell supernatant was collected, centrifuged at 12,000 rpm for 20 min, and the HMGB1 content of the supernatant was detected using an ELISA kit.

[0285] (4) Detection of the material's ability to capture antigens released by tumor cells

[0286] MC38 cells were seeded at a density of 100,000 cells / well in 6-well plates and cultured for 24 h. UM-DOX, US-DOX, ULS-DOX, and UMS-DOX were then added to each well (DOX concentration 10 μg / mL, diluted with 5% fetal bovine serum in 1640 medium). After culturing at 37°C for 18 h, the cell supernatant was collected and centrifuged at 12,000 rpm for 20 min. The cell supernatant was mixed with 100 μg of fresh material corresponding to each well, irradiated with a 980 nm laser for 5 min, and co-incubated for 4 h. The pellet was then collected by centrifugation at 12,000 rpm, washed with PBS, and the protein content of each pellet was determined using the BCA method.

[0287] (5) Detection of material uptake levels by mouse bone marrow-derived dendritic cells (BMDCs)

[0288] First, the model antigen, chicken ovalbumin (OVA), was labeled with Cy5. Cy5 and OVA were co-incubated at 4°C in the dark for 24 hours. Then, the mixture was dialyzed for 5 days at 4°C in the dark using a 50 kDa dialysis bag to remove free Cy5. The liquid from the dialysis bag was then collected and concentrated using a 100 kDa ultrafiltration tube. US, ULS, and UMS were mixed with the concentrated Cy5-OVA, irradiated with a 980 nm laser for 5 minutes, and then co-incubated for 4 hours. Finally, the mixture was centrifuged at 12000 rpm for 10 minutes to collect the precipitate.

[0289] BMDCs were extracted from mouse bone marrow: Intact femurs and tibias were isolated from 6-8 week old male C57bl / 6 mice. The contents of the bone marrow cavity were flushed with a syringe and filtered through a 70 μm cell filter. The contents of the bone marrow cavity were centrifuged at 1600 rpm for 5 min, and the pellet was resuspended in erythrocyte lysis buffer. After standing for 2 min, the pellet was collected by centrifugation again. The cell pellet was seeded at a density of 10⁶ cells per well in 6-well plates, designated as day 0 of culture. On days 1, 3, and 5, the medium was replaced halfway with 1640 complete medium containing 100 ng / mL GM-CSF. Mature BMDCs were obtained after 5 days.

[0290] The corresponding characteristics are as follows:

[0291] ① The uptake of US, ULS, and UMS by MC38 cells was observed using laser confocal microscopy. BMDCs were seeded at a density of 20,000 cells / well in 24-well plates. Each group of materials labeled with the same fluorescence intensity of Cy5-OVA (diluted in 1640 medium with 5% fetal bovine serum) were added, and the cells were cultured in a cell culture incubator for 4 h. Cells were then collected and thoroughly washed with PBS. Each well was fixed with 4% paraformaldehyde at room temperature for 20 min and then discarded. Cells were stained successively with DAPI and FITC-labeled anti-mouse CD11c, and the cell solution was dropped onto cell slides. After drying, the slides were mounted and observed.

[0292] ② Flow cytometry was used to quantitatively analyze the cell uptake results. BMDCs were seeded at a density of 50,000 cells / well in 12-well plates. Cy5-OVA-labeled materials (diluted in 1640 medium with 5% fetal bovine serum) of the same fluorescence intensity were added to each well, and the plates were cultured for 4 hours. Cells were then digested and collected from the wells, washed with PBS, fixed with 4% paraformaldehyde at room temperature for 20 minutes, and discarded. After incubation with FITC-labeled anti-mouse CD11C, CD11C levels in each well were detected by flow cytometry. + The average fluorescence intensity of Cy5 in cells.

[0293] (6) Functional evaluation of materials in vitro to stimulate immune cell differentiation

[0294] Primary T cells were isolated and cultured from mouse spleens: Spleens were collected from 6-8 week old male C57bl / 6 mice, ground into a fine powder using a blunt instrument, and filtered through a 70 μm cell strainer. After centrifugation at 1600 rpm for 5 min, the cell pellet was collected, resuspended, and dispersed in 6-well plates at a concentration of 10⁶ cells per well. The culture medium was 1640 complete medium containing the Rapid-Act T Cell Activation Kit mixture. Subsequent material interventions were performed after culturing in a cell incubator for 2 days.

[0295] Equal masses of UM, US, ULS, and UMS were mixed with tumor cell lysis buffer (CL) and incubated under 980 nm laser irradiation for 5 min (+L) or in darkness (-L). The precipitates were collected and resuspended in 1640 complete medium containing 100 ng / ml GM-CSF to culture mature BMDCs. After 48 h of culture, cells were collected, fixed with 4% paraformaldehyde at room temperature for 20 min, and stained with flow cytometry antibodies. After washing with PBS, the expression of CD80 and CD86 was detected by flow cytometry. T cells were added to the BMDCs system cultured for 48 h at a cell ratio of 10:1, and cultured for another 24 h. All cells in the wells were collected, fixed with 4% paraformaldehyde at room temperature for 20 min, and stained with flow cytometry antibodies. After washing with PBS, the expression of CD4 and CD8 was detected by flow cytometry.

[0296] 2. Characterization Results

[0297] To investigate the delivery effects of US, ULS, and UMS on the chemotherapeutic drug DOX and their tumor cell killing function, this invention first prepared various Cy5-labeled materials, co-incubated them with tumor cells, and then analyzed them by flow cytometry. Figure 7 a) and confocal microscopy were used to study the uptake efficiency of tumor cells ( Figure 7(b) Compared with the US group, the uptake efficiency of both ULS and UMS was significantly improved, with the phagocytic efficiency of UMS being 4 times that of the US group. This suggests that BM has good tumor cell compatibility. This good fusion property allows UMS to more effectively kill tumor cells, release large amounts of tumor antigens, and induce ICD effects.

[0298] To detect the ability of each group to induce tumor cells to release tumor antigens, HMGB1 was selected as the model antigen. The release level of tumor antigens was inferred by detecting the HMGB1 content in the supernatant after incubation of tumor cells in each group. 10 μg / mL DOX was selected as the dose for subsequent in vitro experiments. After MC38 cells were incubated for 18 h, the tumor cell supernatant was collected, and the HMGB1 release level was detected. The results showed that, compared with US, ULS and UMS were better phagocytosed by tumor cells, mediating a stronger cell-killing effect. At a DOX concentration of 5 μg / ml, the killing rate of ULS-DOX was twice that of the US-DOX group. Figure 8 (a). Furthermore, UMS-DOX releases approximately three times more HMGB1 than free DOX, indicating that UMS-DOX can enhance tumor cell phagocytosis of DOX, more effectively trigger ICD, and release a large amount of tumor antigen, which is beneficial for initiating the subsequent antigen capture process. Figure 8 (b).

[0299] Subsequently, new materials were added to the supernatant to obtain UM+L, US+L, UMS+L, and ULS+L, to simulate antigen capture after antigen release. Figure 9 As shown, protein enrichment significantly increased in the US, ULS, and UMS groups after 980nm laser irradiation. UM, serving as the positive control group, captures negatively charged proteins through the attraction between positive and negative charges. The results showed that the protein capture levels of each group were no lower than those of the UM group. Furthermore, only a small amount of protein was attracted under dark conditions, and protein enrichment only occurred under 980nm laser irradiation, indicating that all other groups exhibited a certain degree of photoselectivity compared to the UM group.

[0300] Furthermore, this invention explores whether UMS can be taken up by DC cells and induce DC polarization. Specifically:

[0301] This invention uses Cy5-labeled OVA as a model antigen to simulate the uptake of various materials by DC cells after capture. Figure 10 and Figure 11 (a). Under the same mass, UMS loaded with OVA were more significantly engulfed by BMDCs and induced BMDC polarization, and induced BMDCs to express CD80 and CD86 (a). Figure 11 (b).

[0302] After APC cells process and present tumor antigens, they further induce T cell differentiation. The direction and degree of T cell differentiation are detected by observing the expression levels of CD4 and CD8 in T cells. T cells extracted from the spleen are incubated with polarized BMDCs for 24 hours, and the polarization direction of T cells after 24 hours is observed. It was observed that T cells tend to polarize towards CD8+. + The tendency for T cell polarization is less towards CD4. + The trend of T cell polarization may be related to tumor antigen-induced antigen cross-presentation and the role of MHC-1 molecules. Figure 12 (b and c). It is noteworthy that none of the materials showed significant killing effects on BMDCs, indicating that the materials themselves are safe for APC cells, and the phagocytosis of the materials by BMDCs does not affect their cell viability. Figure 12 (a).

Claims

1. A three-layer rare earth fluoride, characterized in that, It possesses a core-shell structure, comprising, from the inside out, core nanocrystals, a first shell, and a second shell; wherein, The crystal phase of the core nanocrystal is α-NaYbF4, and it includes doping elements; the doping elements include Tm. The first shell layer comprises NaYbF4; The second shell is composed of NaYF4.

2. The three-layer rare earth fluoride as described in claim 1, characterized in that, In the nuclear nanocrystals, based on the molar amount of Na, the molar doping amount of Tm is 0.1%-1%, for example, 0.5%.

3. The three-layer rare earth fluoride as described in claim 1 or 2, characterized in that, The three-layer rare earth fluoride has a core-shell structure, consisting of a core nanocrystal, a first shell, and a second shell from the inside out. The core nanocrystal has a crystal phase of α-NaYbF4 and includes a dopant element, Tm. The first shell is composed of NaYbF4, and the second shell is composed of NaYF4. In the core nanocrystal, the molar doping of Tm is 0.5% based on the molar amount of Na.

4. A method for preparing a three-layer rare earth fluoride as described in any one of claims 1-3, characterized in that, It includes the following steps: I. Nuclear nanocrystals are prepared by reaction 1 using a raw material composition 1 comprising a sodium fluoride source, a ytterbium fluoride source, a thulium fluoride source, and a solvent; II. The raw material composition 2, comprising the said core nanocrystals, a sodium fluoride source, a ytterbium fluoride source, and a solvent, is subjected to reaction 2 to coat the first shell of the core nanocrystals, thereby obtaining a precursor; III. The raw material composition 3, including the precursor, sodium fluoride source, yttrium fluoride source and solvent, is subjected to reaction 3 to coat the core nanocrystals with a second shell layer, thereby obtaining the three-layer rare earth fluoride.

5. The method for preparing the three-layer rare earth fluoride as described in claim 4, characterized in that, It meets one or more of the following conditions: (1) In step I, the molar ratio of the sodium-containing source, the ytterbium-containing source and the thulium-containing source is 0.5:(0.4-0.6):(0.002-0.003), for example 0.5:0.4975:0.0025, based on the molar amounts of sodium, ytterbium and thulium. (2) In step II, the molar ratio of the sodium-containing source and the ytterbium-containing source is 1:(0.8-1.2), for example, 1:1, based on the molar amounts of sodium and ytterbium. (3) In step III, the molar ratio of the sodium fluoride source and the yttrium fluoride source is 1:(0.8-1.2), for example 1:1, based on the molar amounts of sodium and yttrium. (4) In steps I, II and III, each solvent independently includes one or more of 1-octadecene, oleic acid and oleylamine; preferably, the volume ratio of 1-octadecene, oleic acid and oleylamine is 10:5:5; (5) In step I, the ratio of the amount of "the sodium fluoride source, the ytterbium fluoride source and the thulium fluoride source" to the amount of solvent is 1 mmol: 20 mL; (6) In step II, the ratio of the amount of "the sodium fluoride source and the ytterbium fluoride source" to the amount of solvent is 2 mmol: 20 mL; (7) In step III, the ratio of the amount of "the sodium fluoride source and the yttrium fluoride source" to the amount of solvent is 2 mmol: 20 mL; (8) In step I, reaction 1 includes a two-stage heating reaction: In particular, the reaction temperature in the first stage is preferably increased from 30°C to 100-120°C, for example, 110°C; The optimal reaction time for the first stage is 5-20 minutes, for example, 10 minutes; The first stage is preferably carried out in a vacuum environment; The heating rate of the first stage reaction is preferably 8-11℃ / min, for example 10℃ / min; In particular, the reaction temperature of the second stage is preferably increased from the reaction temperature of the first stage to 280-300℃, for example 290℃; The optimal reaction time for the second stage is 30-60 minutes. The second stage is preferably carried out in an inert atmosphere, such as an argon atmosphere; The heating rate of the second stage reaction is preferably 8-11℃ / min, for example 10℃ / min; (9) In step II, reaction 2 includes a two-stage heating reaction: In particular, the reaction temperature in the first stage is preferably increased from 30°C to 100-120°C, for example, 110°C; The optimal reaction time for the first stage is 5-20 minutes, for example, 10 minutes; The first stage is preferably carried out in a vacuum environment; The heating rate of the first stage reaction is preferably 8-11℃ / min, for example 10℃ / min; In particular, the reaction temperature of the second stage is preferably increased from the reaction temperature of the first stage to 280-300℃, for example 290℃; The optimal reaction time for the second stage is 30-60 minutes. The second stage is preferably carried out in an inert atmosphere, such as an argon atmosphere; The heating rate of the second stage reaction is preferably 8-11℃ / min, for example 10℃ / min; (10) In step III, reaction 3 includes a two-stage heating reaction: In particular, the reaction temperature in the first stage is preferably increased from 30°C to 100-120°C, for example, 110°C; The optimal reaction time for the first stage is 5-20 minutes, for example, 10 minutes; The first stage is preferably carried out in a vacuum environment; The heating rate of the first stage reaction is preferably 8-11℃ / min, for example 10℃ / min; The preferred reaction temperature for the second stage is 280-300℃, for example, 290℃; The optimal reaction time for the second stage is 30-60 minutes. The second stage is preferably carried out in an inert atmosphere, such as an argon atmosphere; The heating rate of the second stage reaction is preferably 8-11 °C / min, for example 10 °C / min.

6. An upconversion nanoparticle, characterized in that, The upconversion nanoparticles have a core of three rare-earth fluoride as described in any one of claims 1-3, and are coated with a silica shell, wherein the silica shell includes mesoporous channels; and the upconversion nanoparticles have either the following structure one or structure two: Structure 1: The silica shell surface is covalently bonded with a photocrosslinking agent; the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups; Structure 2: The silica shell has a coating film on its surface, and a photocrosslinking agent is covalently bonded to the surface of the film; the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups; In the second structure, the surface of the silica shell is preferably modified with amino groups.

7. The upconversion nanoparticles as described in claim 6, characterized in that, It meets one or more of the following conditions: (1) The photocrosslinking agent is a primary amine-nitrobenzene azide crosslinking agent, such as sulfo-SANPAH; (2) The particle size of the upconversion nanoparticles is 100-150 nm, for example 132 nm; (3) The Zeta potential of the upconversion nanoparticles is -5mV to +10mV; (4) The membrane is a bacterial membrane or a lipid membrane, preferably a bacterial membrane; Preferably, the bacterial membrane is a pathogenic bacterial membrane, such as an Escherichia coli membrane; The lipid membrane preferably comprises phospholipids and cholesterol, such as DOPC, DOPE, cholesterol, and DSPE-mPEG2000-NH2. The lipid membrane is preferably composed of DOPC:DOPE:cholesterol:DSPE-mPEG2000-NH2 in a mass ratio of 25:50:2:

1.

8. The upconversion nanoparticles as described in claim 6 or 7, characterized in that, It has a core of three rare earth fluorides, a surface covered with a silica shell, the silica shell includes mesoporous channels, the surface of the silica shell has an E. coli membrane, the surface of the membrane is covalently linked with a photocrosslinking agent, the surface of the silica shell is amino-modified, and the photocrosslinking agent is sulfo-SANPAH.

9. A method for preparing upconversion nanoparticles as described in any one of claims 6-8, characterized in that, Includes the following steps: S1. The surface of the three-layer rare earth fluoride is coated with silica, and the template agent is removed to obtain the first precursor. S2. Prepared using either method one or method two as follows: When the upconversion nanoparticles possess the first structure, the following method is used: The first precursor is modified with amino groups and then reacted with a photocrosslinking agent to obtain the upconversion nanoparticles; wherein the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups; the photocrosslinking agent is covalently linked through the amino groups on the surface of the silica shell and the sulfonic acid groups in the phenyl azide compound; When the upconversion nanoparticles possess the second structure, the following method two is adopted: The first precursor is coated with a film and then reacted with a photocrosslinking agent to obtain the upconversion nanoparticles; wherein the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups; the photocrosslinking agent is covalently linked through the sulfonic acid groups in the phenyl azide compound; Preferably, in Method 2, the step of modifying the first precursor with amino groups before membrane coating is further included; the photocrosslinking agent is covalently linked to the amino groups on the membrane and the sulfonic acid groups in the phenyl azide compound.

10. The method for preparing upconversion nanoparticles as described in claim 9, characterized in that, One or more of the following conditions must be met: (1) In step S1, the coating is performed using the Stöber method; preferably, it includes the following steps: The mixture of the three rare earth fluorides, template agent, silicon source, catalyst, aqueous solvent and oil solvent is reacted to obtain the product. Preferably, the three-layer rare earth fluoride is first dissolved in a portion of the oily solvent before being mixed with other materials; The template agent is, for example, hexadecyltrimethylammonium bromide; The silicon source is, for example, tetraethyl orthosilicate; The catalyst is, for example, urea; The aqueous solvent is, for example, water and / or pentanol; The oily solvent is, for example, cyclohexane; The preferred amounts of the three-layer rare earth fluoride, template agent, silicon source and catalyst are (0.01-0.05)mol:(0.5-2)g:(0.5-2)mL:(0.1-0.5)g, for example 0.02mol:1g:1mL:0.3g; The volume ratio of the aqueous solvent to the oily solvent is preferably (10-20): (10-20), for example, 15.01:15; The reaction temperature is preferably 60-80°C, for example 70°C; the reaction time is preferably 6-10 hours, for example 8 hours. (2) In both S2 method one and method two, the amino modification step independently includes: reacting the first precursor with a silane coupling agent in a solvent; the solvent is, for example, anhydrous ethanol, and the silane coupling agent is, for example, (3-aminopropyl)triethoxysilane. (3) In step S2, the second method, the membrane coating step includes: The first precursor or the amino-modified product is reacted with the membrane in a solvent, for example, an aqueous solution of PBS.

11. A tumor in situ vaccine, characterized in that, It has a three-layer rare-earth fluoride core as described in any one of claims 1-3, and a silica shell layer on the surface, wherein the silica shell layer includes mesoporous channels; the mesoporous channels are loaded with active drugs; and the tumor in situ vaccine has the following structure three or structure four: Structure 3: The silica shell surface is covalently bonded with a photocrosslinking agent; the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups; Structure Four: The silica shell has a coating film on its surface, and a photocrosslinking agent is covalently bonded to the surface of the film; the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups; In the fourth structure, the surface of the silica shell is preferably modified with amino groups.

12. The tumor in situ vaccine as described in claim 11, characterized in that, It meets one or more of the following conditions: (1) The photocrosslinking agent is a primary amine-nitrobenzene azide crosslinking agent, such as sulfo-SANPAH; (2) The membrane is a bacterial membrane or a lipid membrane, preferably a bacterial membrane; Preferably, the bacterial membrane is a pathogenic bacterial membrane, such as an Escherichia coli membrane; The lipid membrane preferably comprises phospholipids and cholesterol, such as DOPC, DOPE, cholesterol, and DSPE-mPEG2000-NH2. The composition of the lipid membrane, by mass ratio, is preferably DOPC:DOPE:cholesterol:DSPE-mPEG2000-NH2=25:50:2:1; (3) The active drug is an active drug that induces immunogenic cell death in tumor cells; Preferably, the active pharmaceutical ingredient is doxorubicin hydrochloride; (4) The drug loading rate of the active drug is not less than 85%, preferably not less than 90%.

13. The tumor in situ vaccine as described in claim 11 or 12, characterized in that, It has a core of three rare earth fluorides and a surface covered with a silica shell. The silica shell includes mesoporous channels, and the mesoporous channels are loaded with an active drug, namely doxorubicin hydrochloride. The surface of the silica shell has an Escherichia coli membrane, and the membrane surface is covalently linked with a photocrosslinking agent. The surface of the silica shell is amino-modified, and the photocrosslinking agent is sulfo-SANPAH.

14. A method for preparing a tumor in situ vaccine as described in any one of claims 11-13, characterized in that, It includes the following steps: A. The surface of the three-layer rare earth fluoride is coated with silica, and the template agent is removed to obtain the first precursor. B. Prepared using method three or method four as follows: When the tumor in situ vaccine possesses structure three, the following method three is adopted: The first precursor is modified with amino groups, then reacted with a photocrosslinking agent, and then loaded with a drug to prepare the tumor in situ vaccine; wherein the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups; the photocrosslinking agent is covalently linked to the amino groups on the surface of the silica shell and the sulfonic acid groups in the phenyl azide compound; When the tumor in situ vaccine has the fourth structure, the following method four is adopted: The first precursor is loaded with a drug, then coated with a membrane, and then reacted with a photocrosslinking agent to prepare the tumor in situ vaccine; wherein the photocrosslinking agent is a phenyl azide compound containing sulfonic acid groups; the photocrosslinking agent is covalently linked through the sulfonic acid groups in the phenyl azide compound; Preferably, in Method 4, the step of modifying the first precursor with amino groups before drug loading is further included; the photocrosslinking agent is covalently linked to the amino groups on the membrane and the sulfonic acid groups in the phenyl azide compound.

15. The method for preparing the tumor in situ vaccine as described in claim 14, characterized in that, One or more of the following conditions must be met: (1) In step A, the coating is performed using the Stöber method; preferably, it includes the following steps: The mixture of the three rare earth fluorides, template agent, silicon source, catalyst, aqueous solvent and oil solvent is reacted to obtain the product. Preferably, the three-layer rare earth fluoride is first dissolved in the oily solvent and then mixed with other materials; The template agent is, for example, hexadecyltrimethylammonium bromide; The silicon source is, for example, tetraethyl orthosilicate; The catalyst is, for example, urea; The aqueous solvent is, for example, water and / or pentanol; The oily solvent is, for example, cyclohexane; The preferred amounts of the three-layer rare earth fluoride, template agent, silicon source and catalyst are (0.01-0.05)mol:(0.5-2)g:(0.5-2)mL:(0.1-0.5)g, for example 0.02mol:1g:1mL:0.3g; The volume ratio of the aqueous solvent to the oily solvent is preferably (10-20): (10-20), for example, 15.01:15; The reaction temperature is preferably 60-80°C, for example 70°C; the reaction time is preferably 6-10 hours, for example 8 hours. (2) In steps B, methods three and four, the amino modification steps each independently include: reacting the first precursor with a silane coupling agent in a solvent; the solvent is, for example, anhydrous ethanol, and the silane coupling agent is, for example, (3-aminopropyl)triethoxysilane. (3) In step B, method four, the membrane coating step includes: The drug-loaded product is reacted with the membrane in a solvent, for example, an aqueous solution of PBS.

16. The use of a trilayer rare earth fluoride as described in any one of claims 1-3, or an upconversion nanoparticle as described in any one of claims 6-8, in the preparation of drugs for the prevention and / or treatment of tumors; in, The tumor is preferably one of colon cancer, melanoma, lung cancer, and rectal tumor; The tumor drug is preferably activated by 980nm laser irradiation.

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