Nano composite material based on UCNPs-photosensitizer as well as preparation method and application of nano composite material

Abstract

The invention belongs to the technical field of nano biological materials, and provides a nano composite material based on a UCNPs-photosensitizer and a preparation method and application of the nano composite material based on the UCNPs-photosensitizer, and the method comprises the following steps: 1, preparing TK-DOX through an amide reaction; 2, preparing an OA-UCNPs solution by a thermal coprecipitation method; 3, removing oleic acid by a hydrochloric acid method to prepare UCNPs; and 4, preparing the UCNPs-photosensitizer / TK-DOX by a coordination loading method. The nano composite material disclosed by the invention can realize high sensitization efficiency under low-power excitation, so that singlet oxygen is generated to realize cascade drug release, and synergistic treatment of photodynamic therapy and chemotherapy is realized by utilizing an EPR effect.

Description

Technical Field

[0001] The present invention relates to the technical field of nanobiomaterials, and particularly to a nanocomposite based on UCNPs-photosensitizer, its preparation method and application. Background Art

[0002] The latest data released by the International Agency for Research on Cancer (IARC) shows that there were approximately 19.3 million newly diagnosed cancer cases and approximately 10 million cancer deaths globally in 2020. The predicted data on the cancer burden shows that the number of newly diagnosed cancer cases globally will reach 28.4 million in 2040, a 47% increase compared to 2020. Therefore, if tumors can be accurately located and effectively treated at an early stage of cancer, the harm to patients will be significantly reduced, and early diagnosis and treatment can improve the cure rate and the quality of life of patients.

[0003] Currently, the main treatment methods include surgical treatment, chemotherapy, and photodynamic therapy (PDT), etc. Traditional surgical treatment has limitations in operation and is restricted in promotion. Chemotherapy lacks tumor targeting and can cause relatively serious toxic and side effects. Reducing toxic and side effects can be considered from two aspects. One is to improve the tumor-specific targeting of drugs. The loading of targeting molecules and the enhanced permeability and retention (EPR) effect of nanocomposites at the tumor site can enable drug-loaded substances to specifically target the tumor site and have a longer retention time. The other is to reduce the release of drugs at non-tumor locations, which can be achieved by taking certain means (such as near-infrared light irradiation, ultrasound irradiation) at the tumor site. Photodynamic therapy is a modern cancer treatment technology that combines photosensitizers and light of a specific wavelength to eliminate tumor cells, and has the characteristics of good controllability, small side effects, high selectivity, and strong specificity. However, commonly used photosensitizers generally have the disadvantage of weak tissue penetration ability, while rare-earth upconversion luminescent nanomaterials (Upconversion nanoparticles, abbreviated as UCNPs) have upconversion luminescence properties. Therefore, after combining photosensitizers with UCNPs, the tissue penetration ability can be improved and the treatment effect can be enhanced.

[0004] Chinese Patent CN 114836216 B discloses a rare-earth nanocomposite capable of enhancing singlet oxygen generation, its preparation method and application. It converts oil-soluble upconversion luminescent nanoparticles into water-soluble by coating with silica, and then continues to coat with a zinc oxide layer to form a multi-layer structure of UCNP@SiO 2 @ZnO. The designed heterogeneous upconversion nanomaterials in this invention can absorb near-infrared light energy and transfer the energy to the outer zinc oxide layer through the energy transfer mode of fluorescence resonance energy transfer (FRET), which can significantly enhance the generation of singlet oxygen. However, the energy transfer mode of FRET has the disadvantages of high excitation power and low sensitization efficiency.

[0005] Therefore, under low-power excitation, there is an urgent need for a nanocomposite material that can achieve high sensitization efficiency and generate singlet oxygen through sensitization to achieve cascade drug release, thereby improving the tumor treatment effect. Summary of the Invention

[0006] Aiming at the existing technical problems, the purpose of the present invention is to provide a UCNPs-photosensitizer-based nanocomposite material, its preparation method and application. The nanocomposite material of the present invention can achieve high sensitization efficiency under low-power excitation, thereby generating singlet oxygen to achieve cascade drug release, and realizing the synergistic treatment of photodynamic therapy and chemotherapy by using the EPR effect.

[0007] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a preparation method and application of a UCNPs-photosensitizer-based nanocomposite material, comprising the following steps:

[0009] 1. Preparation of TK-DOX by amide reaction, and its structural formula is as follows:

[0010]

[0011] 2. Preparation of OA-UCNPs solution by thermal co-precipitation method:

[0012] S1. Prepare aqueous solutions of GdCl 3 and NdCl 3 with concentrations of 1-1.2 mol / L respectively, and set aside; mix NaOH, NH 4 F and methanol to obtain a solution, and set aside;

[0013] S2. Mix the aqueous solutions of GdCl 3 and NdCl 3 , heat to 105-115 °C, stir until the water is evaporated, add oleic acid and octadecene, evacuate, raise the temperature gradient, cool to 45-50 °C, add the solution obtained in step S1, keep warm and stir for 1-1.5 h, heat to 85-95 °C, keep warm for 20-30 min and then raise the temperature to 105-115 °C, vacuum degas, then introduce argon for 2-3 min, evacuate for 4-5 min, and repeat 3 times; finally, under an argon atmosphere, raise the temperature to 300-310 °C, stir and react for 1-1.5 h, cool, wash, and dry to obtain OA-UCNPs; disperse OA-UCNPs in an organic solvent to obtain an OA-UCNPs solution;

[0014] 3. Preparation of UCNPs by removing oleic acid with hydrochloric acid method:

[0015] Mix the 0.1 - 0.15 mol / L hydrochloric acid aqueous solution with the OA - UCNPs solution obtained in Step 2, stir for 3 - 4 h, take the lower - layer liquid, centrifuge, wash, and dry to obtain UCNPs;

[0016] IV. Preparation of UCNPs - photosensitizer / TK - DOX by coordination loading method:

[0017] Q1. Mix the UCNPs obtained in Step 3 with deionized water to obtain a UCNPs solution with a concentration of 22 - 28 mg / mL for standby; mix the TK - DOX obtained in Step 1 with deionized water to obtain a TK - DOX solution with a concentration of 0.9 - 1.2 mg / mL for standby; mix the UCNPs solution and the TK - DOX solution, stir for 10 - 14 h, centrifuge, wash, and dry to obtain UCNPs - TK - DOX;

[0018] Q2. Mix the photosensitizer with deionized water to obtain a solution containing the photosensitizer for standby; mix the UCNPs - TK - DOX obtained in Q1 with deionized water to obtain a UCNPs - TK - DOX solution for standby; mix the solution containing the photosensitizer and the UCNPs - TK - DOX solution, stir for 10 - 14 h, centrifuge, and wash until the upper layer is basically colorless to obtain UCNPs - photosensitizer / TK - DOX, which is the nanocomposite material.

[0019] The reaction mechanism and function of the present invention are as follows:

[0020] 1. The traditional energy transfer method (fluorescence resonance energy transfer) of UCNPs binding with photosensitizer still has the disadvantages of high excitation power and low sensitization efficiency. The reason is that the energy of the excited - state energy level of lanthanide ions is higher than the S 1 energy level of the photosensitizer. In the process of realizing PDT, it is impossible to avoid the photon - energy loss caused by the inter - system crossing (ISC) between the S 1 and T 1 of the photosensitizer; while the present invention adopts the photosensitization method of lanthanide - triplet energy transfer (TET). The unique design makes the excited - state energy level of lanthanide ions located between the S 1 and T 1 energy levels of the photosensitizer, avoiding the inter - system crossing (ISC) process, improving the sensitization efficiency, and reducing the excitation power.

[0021] 2. The nanocomposite of the present invention is targeted to the tumor site by the EPR effect, absorbs energy under the irradiation of laser, transfers the energy to the photosensitizer by the TET energy transfer method, the photosensitizer absorbs the energy and converts triplet oxygen into singlet oxygen, and the released singlet oxygen can break the disulfide bond of propane-2,2-diylbis(sulfanyl)]diacetic acid, thereby releasing the chemotherapeutic drug doxorubicin hydrochloride, and finally realizing the synergistic treatment of chemotherapy and photodynamic therapy under low excitation power, thus improving the tumor treatment effect. In addition, the nanocomposite is particularly stable in the absence of light, has strong chemical inertness in blood, and has good biocompatibility, which can reduce the toxic and side effects on normal cells.

[0022] In some embodiments, the steps of preparing TK-DOX by the amide reaction in Step 1 are as follows:

[0023] Mix doxorubicin hydrochloride, propane-2,2-diylbis(sulfanyl)]diacetic acid, and dimethyl sulfoxide, add an activator, react at room temperature for 10-14 h, purify, and dry to obtain TK-DOX.

[0024] In some embodiments, the mass ratio of doxorubicin hydrochloride to propane-2,2-diylbis(sulfanyl)]diacetic acid is (1.9-2.3):1.

[0025] In some embodiments, the activator is a composition of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and the activator accounts for 2.5-2.8% of the total mass of doxorubicin hydrochloride and propane-2,2-diylbis(sulfanyl)]diacetic acid.

[0026] Preferably, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 3:1.

[0027] In some embodiments, in S1 of Step 2, the dosage ratio of NaOH, NH 4 F and methanol is 0.2 g:0.3 g:10 mL.

[0028] In some embodiments, the mode of gradient temperature increase in S2 of Step 2 is: until the solution has basically no bubbles at 105-115 °C, then heat up to 120-130 °C, until there are no bubbles completely, then heat up to 135-145 °C, and maintain for 1-1.5 h.

[0029] In some embodiments, in S2 of Step 2, the volume ratio of the GdCl 3 aqueous solution to the NdCl 3 aqueous solution is (1.3-1.7):1.

[0030] In some embodiments, the photosensitizer in step four is any one of chlorin, metallophthalocyanines containing different metals, and porphyrins containing different metals.

[0031] In some embodiments, the volume ratio of the UCNPs solution to the TK-DOX solution in Q1 of step four is 1:(4 - 6).

[0032] The second aspect of the present invention provides a nanocomposite based on UCNPs-photosensitizer prepared by the described preparation method.

[0033] The third aspect of the present invention provides the application of the nanocomposite obtained by the described preparation method in targeted treatment of tumor cells.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] 1. The present invention adopts the TET photosensitization method, which improves the sensitization efficiency and reduces the excitation power.

[0036] 2. The nanocomposite of the present invention targets to the tumor location by the EPR effect. The singlet oxygen generated under the irradiation of laser can break the disulfide bond of propane-2,2-diylbis(sulfanyl)]diacetic acid, and then release the chemical drug doxorubicin hydrochloride, finally realizing the synergistic treatment of chemotherapy and photodynamic therapy under low excitation power. In addition, the nanocomposite has good biocompatibility and can reduce the toxic and side effects on normal cells. Specific Embodiments

[0037] The following will describe the present invention in combination with specific implementation schemes. It should be noted that the following examples are examples of the present invention, only used to illustrate the present invention, rather than to limit the present invention. Without departing from the gist or scope of the present invention, other combinations and various improvements within the concept of the present invention can be made.

[0038] Prepare each nanocomposite based on UCNPs-photosensitizer according to the raw material ratios and preparation methods specified in the following examples and comparative examples.

[0039] For the convenience of those skilled in the art to implement the present invention, the sources of some raw materials in the examples and comparative examples are described as follows:

[0040] The raw materials used are not subject to special restrictions and can all be purchased from the market.

[0041] Example 1

[0042] A preparation method of a nanocomposite based on UCNPs-photosensitizer comprises the following steps:

[0043] I. Preparation of TK-DOX by amide reaction:

[0044] Mix 200 g of doxorubicin hydrochloride, 100 g of propane-2,2-diylbis(sulfanyl)diacetic acid, and 1800 mL of dimethyl sulfoxide, add 6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2 g of N-hydroxysuccinimide, react at room temperature for 12 h, purify using a permeable membrane with a molecular weight cut-off of 1000, and dry at 85 °C for 4 h to obtain TK-DOX;

[0045] II. Preparation of OA-UCNPs solution by thermal co-precipitation method:

[0046] S1. Prepare aqueous solutions of GdCl 3 and NdCl 3 with a concentration of 1 mol / L respectively for standby; Mix 2 g of NaOH, 3 g of NH 4 F, and 100 mL of methanol to obtain a solution for standby;

[0047] S2. Mix 12 mL of the GdCl 3 aqueous solution and 8 mL of the NdCl 3 aqueous solution, heat to 110 °C, stir until the water is evaporated, add 120 mL of oleic acid and 300 mL of octadecene, evacuate, heat to 125 °C until there are basically no bubbles in the solution, then heat to 140 °C, keep for 1 h, cool to 50 °C, add the solution prepared in step S1, keep warm and stir for 1 h, heat to 90 °C, keep warm and react for 25 min, then heat to 110 °C, degas under vacuum, then introduce argon for 3 min, evacuate for 5 min, repeat 3 times; Finally, under an argon atmosphere, heat to 305 °C, stir and react for 1 h, cool to room temperature, wash with cyclohexane and ethanol in turn, and dry at 85 °C for 4 h to obtain OA-UCNPs; Disperse OA-UCNPs in 200 mL of cyclohexane to obtain OA-UCNPs solution;

[0048] III. Removal of oleic acid by hydrochloric acid method to prepare UCNPs:

[0049] Mix 10 mL of 0.12 mol / L hydrochloric acid aqueous solution and 100 mL of the OA-UCNPs solution obtained in step II, stir for 3.5 h, take the lower layer liquid, centrifuge at 10000 r / min for 15 min, wash with deionized water 3 times, and dry at 85 °C for 4 h to obtain UCNPs;

[0050] IV. Preparation of UCNPs-photosensitizer / TK-DOX by coordination loading method:

[0051] Q1. Mix the UCNPs obtained in Step 3 with deionized water to obtain a UCNPs solution with a concentration of 25 mg / mL for standby; mix the TK-DOX obtained in Step 1 with deionized water to obtain a TK-DOX solution with a concentration of 1 mg / mL for standby; mix the UCNPs solution and the TK-DOX solution at a volume ratio of 1:5, stir for 12 h, centrifuge at 8000 r / min for 15 min, wash twice with deionized water, and dry at 85 °C for 4 h to obtain UCNPs-TK-DOX;

[0052] Q2. Mix chlorin e6 and deionized water to obtain a solution containing photosensitizer with a concentration of 2 mg / mL for standby; mix the UCNPs-TK-DOX obtained in Step Q1 with deionized water to obtain a UCNPs-TK-DOX solution with a concentration of 25 mg / mL for standby; mix the solution containing photosensitizer and the UCNPs-TK-DOX solution at a volume ratio of 1:3, stir for 12 h, centrifuge at 8000 r / min for 15 min, and wash with deionized water until the upper layer is basically colorless to obtain UCNPs-chlorin e6 / TK-DOX, which is the nanocomposite.

[0053] Example 2

[0054] A preparation method of a nanocomposite based on UCNPs-photosensitizer comprises the following steps:

[0055] I. Preparation of TK-DOX by amide reaction:

[0056] Mix 190 mg doxorubicin hydrochloride, 100 g propane-2,2-diylbis(sulfanyl)diacetic acid, and 1740 mL dimethyl sulfoxide, add 5.46 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.82 g N-hydroxysuccinimide, react at room temperature for 12 h, purify using a permeable membrane with a molecular weight cut-off of 1000, and dry at 85 °C for 4 h to obtain TK-DOX;

[0057] II. Preparation of OA-UCNPs solution by thermal co-precipitation method:

[0058] S1. Prepare aqueous solutions of GdCl 3 with a concentration of 1 mol / L and NdCl 3 for standby; mix 2 g NaOH, 3 g NH 4 F, and 100 mL methanol to obtain a solution for standby;

[0059] S2. Add 11.4 mL of the GdCl 3 aqueous solution and 8.6 mL of the NdCl 3Mix with an aqueous solution, heat to 105 °C, stir until the water is evaporated, add 120 mL of oleic acid and 300 mL of octadecene, evacuate the air, heat to 120 °C after the solution has basically no bubbles at 105 °C, and then heat to 135 °C after there are no bubbles at all, and keep it for 1.5 h. Cool to 45 °C, add the solution prepared in step S1, keep warm and stir for 1.5 h, heat to 85 °C, keep warm and react for 30 min, then heat to 105 °C, degas under vacuum, then introduce argon for 2 min, evacuate the air for 4 min, and repeat 3 times; Finally, under an argon atmosphere, heat to 300 °C, stir and react for 1.5 h, cool to room temperature, wash with cyclohexane and ethanol in turn, and dry at 85 °C for 4 h to obtain OA-UCNPs; Disperse OA-UCNPs in 200 mL of cyclohexane to obtain an OA-UCNPs solution;

[0060] III. Preparation of UCNPs by hydrochloric acid method to remove oleic acid:

[0061] Mix 10 mL of 0.1 mol / L hydrochloric acid aqueous solution with 100 mL of the OA-UCNPs solution obtained in step II, stir for 3 h, take the lower layer liquid, centrifuge at 10000 r / min for 15 min, wash with deionized water 3 times, and dry at 85 °C for 4 h to obtain UCNPs;

[0062] IV. Preparation of UCNPs-photosensitizer / TK-DOX by coordination loading method:

[0063] Q1. Mix the UCNPs obtained in step III with deionized water to obtain a UCNPs solution with a concentration of 22 mg / mL for standby; Mix the TK-DOX obtained in step I with deionized water to obtain a TK-DOX solution with a concentration of 0.9 mg / mL for standby; Mix the UCNPs solution and the TK-DOX solution in a volume ratio of 1:4, stir for 10 h, centrifuge at 8000 r / min for 15 min, wash with deionized water 2 times, and dry at 85 °C for 4 h to obtain UCNPs-TK-DOX;

[0064] Q2. Mix chlorin e6 with deionized water to obtain a solution containing photosensitizer with a concentration of 2 mg / mL for standby; Mix the UCNPs-TK-DOX obtained in step Q1 with deionized water to obtain a UCNPs-TK-DOX solution with a concentration of 25 mg / mL for standby; Mix the solution containing photosensitizer and the UCNPs-TK-DOX solution in a volume ratio of 1:3, stir for 10 h, centrifuge at 8000 r / min for 15 min, and wash with deionized water until the upper layer is basically colorless to obtain UCNPs-chlorin e6 / TK-DOX, which is the nanocomposite material.

[0065] Example 3

[0066] A preparation method of a nano-composite material based on UCNPs-photosensitizer, comprising the following steps:

[0067] I. Preparation of TK-DOX by amide reaction:

[0068] Mix 230 g of doxorubicin hydrochloride, 100 g of propane-2,2-diylbis(sulfanyl)diacetic acid, and 1980 mL of dimethyl sulfoxide, add 6.93 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2.31 g of N-hydroxysuccinimide, react at room temperature for 12 h, purify using a permeable membrane with a molecular weight cut-off of 1000, and dry at 85 °C for 4 h to obtain TK-DOX;

[0069] II. Preparation of OA-UCNPs solution by thermal co-precipitation method:

[0070] S1. Prepare aqueous solutions of GdCl 3 and NdCl 3 with a concentration of 1.2 mol / L respectively for standby; Mix 2 g of NaOH, 3 g of NH 4 F, and 100 mL of methanol to obtain a solution for standby;

[0071] S2. Mix 12.5 mL of GdCl 3 aqueous solution and 7.5 mL of NdCl 3 aqueous solution, heat to 115 °C, stir until the water is evaporated, add 120 mL of oleic acid and 300 mL of octadecene, evacuate, heat to 130 °C until there are basically no bubbles in the solution, then heat to 145 °C, and maintain for 1 h. Cool to 50 °C, add the solution prepared in step S1, keep stirring and reacting for 1 h, heat to 95 °C, keep reacting for 20 min and then heat to 115 °C, degas under vacuum, then introduce argon for 3 min and evacuate for 5 min, repeat 3 times; Finally, under an argon atmosphere, heat to 310 °C, stir and react for 1 h, cool to room temperature, wash with cyclohexane and ethanol in sequence, and dry at 85 °C for 4 h to obtain OA-UCNPs; Disperse OA-UCNPs in 200 mL of cyclohexane to obtain OA-UCNPs solution;

[0072] III. Preparation of UCNPs by removing oleic acid with hydrochloric acid method:

[0073] Mix 10 mL of 0.15 mol / L hydrochloric acid aqueous solution and 100 mL of OA-UCNPs solution obtained in step II, stir for 4 h, take the lower layer liquid, centrifuge at 10000 r / min for 15 min, wash with deionized water 3 times, and dry at 85 °C for 4 h to obtain UCNPs;

[0074] IV. Preparation of UCNPs-photosensitizer / TK-DOX by coordination loading method:

[0075] Q1. Mix the UCNPs obtained in Step 3 with deionized water to obtain a UCNPs solution with a concentration of 28 mg / mL for standby; mix the TK-DOX obtained in Step 1 with deionized water to obtain a TK-DOX solution with a concentration of 1.2 mg / mL for standby; mix the UCNPs solution and the TK-DOX solution at a volume ratio of 1:6, stir for 14 h, centrifuge at 8000 r / min for 15 min, wash twice with deionized water, and dry at 85 °C for 4 h to obtain UCNPs-TK-DOX;

[0076] Q2. Mix chlorin e6 and deionized water to obtain a solution containing photosensitizer with a concentration of 2 mg / mL for standby; mix the UCNPs-TK-DOX obtained in Step Q1 with deionized water to obtain a UCNPs-TK-DOX solution with a concentration of 25 mg / mL for standby; mix the solution containing photosensitizer and the UCNPs-TK-DOX solution at a volume ratio of 1:3, stir for 14 h, centrifuge at 8000 r / min for 15 min, and wash with deionized water until the upper layer is basically colorless to obtain UCNPs-chlorin e6 / TK-DOX, which is the nanocomposite material.

[0077] Comparative Example 1

[0078] A preparation method of a nanocomposite material based on UCNPs-photosensitizer, comprising the following steps:

[0079] I. Prepare OA-UCNPs solution by thermal co-precipitation method:

[0080] S1. Prepare aqueous solutions of GdCl 3 with a concentration of 1 mol / L and NdCl 3 for standby; mix 2 g of NaOH, 3 g of NH 4 F, and 100 mL of methanol to obtain a solution for standby;

[0081] S2. Add 12 mL of the GdCl 3 aqueous solution and 8 mL of the NdCl 3Mix with an aqueous solution, heat to 110 °C, stir until the water is evaporated, add 120 mL of oleic acid and 300 mL of octadecene, evacuate the air, heat to 125 °C until there are basically no bubbles in the solution at 110 °C, then heat to 140 °C after there are no bubbles at all, and maintain for 1 h. Cool to 50 °C, add the solution prepared in step S1, keep warm and stir for reaction for 1 h, heat to 90 °C, keep warm and react for 25 min, then heat to 110 °C, degas under vacuum, then introduce argon for 3 min, evacuate the air for 5 min, and repeat 3 times; Finally, under an argon atmosphere, heat to 305 °C, stir and react for 1 h, cool to room temperature, wash successively with cyclohexane and ethanol, and dry at 85 °C for 4 h to obtain OA-UCNPs; Disperse the OA-UCNPs in 200 mL of cyclohexane to obtain an OA-UCNPs solution;

[0082] II. Preparation of UCNPs by hydrochloric acid method to remove oleic acid:

[0083] Mix 10 mL of 0.12 mol / L hydrochloric acid aqueous solution with 100 mL of the OA-UCNPs solution obtained in step one, stir for 3.5 h, take the lower layer liquid, centrifuge at 10000 r / min for 15 min, wash 3 times with deionized water, and dry at 85 °C for 4 h to obtain UCNPs;

[0084] III. Preparation of UCNPs-photosensitizer / DOX by coordination loading method:

[0085] Q1. Mix the UCNPs obtained in step two with deionized water to obtain a UCNPs solution with a concentration of 25 mg / mL for standby; Mix doxorubicin hydrochloride and deionized water to obtain a doxorubicin hydrochloride solution with a concentration of 1 mg / mL for standby; Mix the UCNPs solution and the doxorubicin hydrochloride solution in a volume ratio of 1:5, stir for 12 h, centrifuge at 8000 r / min for 15 min, wash 2 times with deionized water, and dry at 85 °C for 4 h to obtain UCNPs-DOX;

[0086] Q2. Mix chlorin e6 and deionized water to obtain a solution containing photosensitizer with a concentration of 2 mg / mL for standby; Mix the UCNPs-DOX obtained in step Q1 and deionized water to obtain a UCNPs-DOX solution with a concentration of 25 mg / mL for standby; Mix the solution containing photosensitizer and the UCNPs-DOX solution in a volume ratio of 1:3, stir for 12 h, centrifuge at 8000 r / min for 15 min, and wash with deionized water until the upper layer is basically colorless to obtain UCNPs-chlorin e6 / DOX, which is the nanocomposite material.

[0087] Comparative Example 2

[0088] A preparation method of a nano-composite material based on UCNPs-photosensitizer, the specific implementation is the same as that of Example 1, except for the step S2:

[0089] Mix 12 mL of YCl 3 aqueous solution and 8 mL of YbCl 3 aqueous solution, heat to 110 °C, stir until the water is evaporated, add 120 mL of oleic acid and 300 mL of octadecene, evacuate, at 110 °C until there are basically no bubbles in the solution, then raise the temperature to 125 °C, after completely no bubbles, raise the temperature to 140 °C, and keep for 1 h, cool to 50 °C, add the solution prepared in step S1, keep warm and stir for 1 h, heat to 90 °C, keep warm and react for 25 min, then raise the temperature to 110 °C, vacuum degas, then introduce argon for 3 min, evacuate for 5 min, repeat 3 times; finally, under the argon atmosphere, raise the temperature to 305 °C, stir and react for 1 h, cool to room temperature, wash with cyclohexane and ethanol in turn, dry at 85 °C for 4 h to obtain OA-UCNPs; disperse OA-UCNPs in 200 mL of cyclohexane to obtain an OA-UCNPs solution.

[0090] Effect evaluation:

[0091] Test the nano-composite materials prepared in the above Examples 1-3 and Comparative Examples 1-2, and the specific results are shown in Table 1.

[0092] Therapeutic test:

[0093] (1) Cytotoxicity test:

[0094] Inoculate HeLa cells in a sterile 96-well microplate (8000 cells per well), and incubate in an incubator at 37 °C for 24 h. Dilute the nano-composite materials in Examples 1-3 and Comparative Examples 1-2 with deionized water into UCNPs-chlorin e6 / TX-DOX solution and UCNPs-chlorin e6 / DOX solution with a concentration of 200 μg / mL. Incubate the cells with fresh medium containing 100 μL of the above solution at 37 °C for 12 h. Subsequently, add 100 μL of 10 wt% CCK-8 solution diluted with fresh medium to each well, and incubate in an incubator for 1 h to calculate the cell survival rate. It can be seen from the results that the HeLa cells are basically not dead and still maintain a very high cell survival rate.

[0095] (2) Cell lethality test:

[0096] HeLa cells were seeded in a sterile 96-well microplate (8000 cells per well) and incubated in an incubator at 37 °C for 24 h. The solutions of UCNPs-chlorin / TX-DOX and UCNPs-chlorin / DOX in Examples 1-3 and Comparative Examples 1-2 were diluted with deionized water to a concentration of 200 μg / mL. The cells were co-incubated with fresh medium containing 100 μL of the above solutions at 37 °C for 12 h, then irradiated with 808 nm near-infrared light at a power density of 1.5 W / cm2 for 5 min, and further cultured for 24 h. Subsequently, 100 μL of 10 wt% CCK-8 solution diluted with fresh medium was added to each well and incubated in an incubator for 1 h, and the cell viability was calculated.

[0097] Combining tests (1) and (2), when not irradiated, the solutions of UCNPs-chlorin / TX-DOX and UCNPs-chlorin / DOX had no obvious toxicity to HeLa cells, but after irradiation, the viability of HeLa cells decreased, indicating that they had a strong lethality to cancer cells; in addition, it could be clearly found that the lethality of Examples 1-3 (the cell viability was less than 10%) to cancer cells was much higher than that of Comparative Example 1 and Comparative Example 2.

[0098] The above are only the preferred embodiments of the present invention and do not limit the present application in any form. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any person skilled in the art, without departing from the scope of the technical solution of the present application, makes some changes or modifications using the disclosed technical content, which are equivalent to equivalent implementation cases. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution.

Claims

1. A method for preparing a nanocomposite material based on UCNPs-photosensitizer, characterized in that: The following steps are included:

1. Preparation of TK-DOX by amide reaction, the structural formula of which is as follows:

2. Preparation of OA-UCNPs solution by thermal co-precipitation method: S1. Prepared respectively a 1-1.2 mol / L concentration of GdCl3 aqueous solution, NdCl3 aqueous solution, set aside; NaOH, NH4F, methanol mixed to obtain a solution, set aside; S2. Mix the GdCl3 aqueous solution and the NdCl3 aqueous solution, heat to 105-115°C, stir until the water evaporates, add oleic acid and octadecene, evacuate, increase the temperature gradually, cool to 45-50°C, add the solution obtained in step S1, keep warm and stir for 1-1.5h, heat to 85-95°C, keep warm for 20-30min, then heat to 105-115°C, vacuum degassing, then pass argon for 2-3min, evacuate for 4-5min, repeat 3 times; finally, in an argon atmosphere, heat to 300-310°C, stir for 1-1.5h, cool, wash, and dry to obtain OA-UCNPs; disperse the OA-UCNPs in an organic solvent to obtain an OA-UCNPs solution; 3. Preparation of UCNPs by removing oleic acid using hydrochloric acid method: Mix 0.1-0.15 mol / L hydrochloric acid aqueous solution and the OA-UCNPs solution obtained in step 2, stir for 3-4 hours, take the lower layer of liquid, centrifuge, wash, and dry to obtain UCNPs; 4. Preparation of UCNPs-photosensitizer / TK-DOX by coordination loading method: Q1. The UCNPs obtained in step 3 were mixed with deionized water to obtain a UCNPs solution with a concentration of 22-28 mg / mL for standby use; the TK-DOX obtained in step 1 was mixed with deionized water to obtain a TK-DOX solution with a concentration of 0.9-1.2 mg / mL for standby use; the UCNPs solution and the TK-DOX solution were mixed, stirred for 10-14 h, centrifuged, washed, and dried to obtain UCNPs-TK-DOX; Q2. Mix the photosensitizer and deionized water to obtain a photosensitizer-containing solution for use; mix the UCNPs-TK-DOX obtained in step Q1 and deionized water to obtain a UCNPs-TK-DOX solution for use; mix the photosensitizer-containing solution and the UCNPs-TK-DOX solution, stir for 10-14 hours, centrifuge, and wash until the upper layer is basically colorless to obtain UCNPs-photosensitizer / TK-DOX, which is a nanocomposite material.

2. The method for preparing a nanocomposite material based on UCNPs-photosensitizer according to claim 1, characterized in that: The steps for preparing TK-DOX by the amide reaction described in step 1 are as follows: Doxorubicin hydrochloride, propane-2,2-diylbis(sulfide)]diacetic acid and dimethyl sulfoxide are mixed, an activator is added, the mixture is reacted at room temperature for 10-14 hours, purified and dried to obtain TK-DOX.

3. The method for preparing a nanocomposite material based on UCNPs-photosensitizer according to claim 2, characterized in that: The mass ratio of doxorubicin hydrochloride to propane-2,2-diylbis(sulfide)]diacetic acid is (1.9-2.3):

1.

4. The method for preparing a nanocomposite material based on UCNPs-photosensitizer according to claim 2, characterized in that: The activator is a composition of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and the activator accounts for 2.5-2.8% of the total mass of doxorubicin hydrochloride and propane-2,2-diylbis(sulfide)]diacetic acid.

5. The method for preparing a nanocomposite material based on UCNPs-photosensitizer according to claim 1, characterized in that: The gradient temperature increase mode in step 2 is: at 105-115° C. until the solution is substantially free of bubbles, then increase the temperature to 120-130° C., and then increase the temperature to 135-145° C. after there are no bubbles at all, and maintain for 1-1.5 hours.

6. The method for preparing a nanocomposite material based on UCNPs-photosensitizer according to claim 1, characterized in that: The volume ratio of the GdCl3 aqueous solution to the NdCl3 aqueous solution in S2 of step 2 is (1.3-1.7):

1.

7. The method for preparing a nanocomposite material based on UCNPs-photosensitizer according to claim 1, characterized in that: The photosensitizer in step 4 is any one of dihydrochlorin, phthalocyanine containing different metals and porphyrin containing different metals.

8. The method for preparing a nanocomposite material based on UCNPs-photosensitizer according to claim 1, characterized in that: The volume ratio of the UCNPs solution and the TK-DOX solution in Q1 of step 4 is 1:(4-6).

9. A nanocomposite material based on UCNPs-photosensitizer obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the nanocomposite material obtained by the preparation method according to any one of claims 1 to 8 in targeted treatment of tumor cells.

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