Composite nano material and application thereof in disease treatment

Abstract

The invention relates to the field of nano biomedicine, and particularly discloses a composite nano material and application thereof in disease treatment. On the basis of the unique up-conversion optical characteristic of an up-conversion luminescent nano material, TPP is directly targeted to oxygen-enriched mitochondria, ROS generated by PDT induces APE1 to migrate from a cell nucleus to the mitochondria, an AP site of a nucleic acid double chain is recognized and cut, an anti-miR21 single chain is released to cytoplasm and is combined with a miR21 single chain in the cytoplasm, an apoptosis pathway based on Bcl-2 is started, and the apoptosis of the APE1 is inhibited. The anti-tumor effect is achieved by cooperating with PDT.

Description

Technical Field

[0001] The present invention belongs to the field of nano biomedicine, and particularly relates to an upconversion composite nanomaterial and its application in disease treatment. Background Art

[0002] At present, cancer remains one of the important reasons threatening human health. Traditional cancer treatment methods mainly include surgery, chemotherapy, radiotherapy, etc. However, traditional therapies have defects such as poor curative effect, large toxic and side effects, and immune tolerance. Therefore, there is an urgent need to develop effective new cancer treatment programs. Photodynamic therapy (PDT) is a non-invasive tumor treatment method and has been widely used in the field of clinical anti-tumor treatment. In addition to light and O 2 besides, photosensitizer (PS) is one of the key factors of PDT. The PDT method utilizes the ability of the photosensitizer to release heat energy under the stimulation of a specific wavelength, and then activates oxygen into singlet oxygen ( 1 O 2 ); or utilizes the photosensitizer to be converted from the ground state to the excited state under the action of an external light source, reacts with cell substrates through electron transfer, generates reactive oxygen species (ROS) products with endogenous oxygen, causes apoptosis of tumor cells and irreversibly damages the blood vessels in the tumor microenvironment, thereby achieving the purpose of inhibiting tumor growth. However, the tissue permeability of PS is poor, and self-aggregation makes it unable to penetrate the lipid bilayer, resulting in a reduction in the efficacy of PDT (GHEWALAT, SKWOR T, MUNIRATHINAM G. Photosensitizers in prostate cancer therapy [y]. Oncotarget, 2017, 8(18): 30524-30538.). Therefore, it is of great significance to develop near-infrared light-activated photosensitizers with high penetration depth.

[0003] Upconversion nanoparticles (UCNPs) are a class of optically active nanocrystals doped with lanthanide elements that can convert two or more low-energy photons into a single high-energy output photon upon near-infrared (NIR) light excitation. Compared with ultraviolet and visible light, near-infrared (NIR) light has the advantages of deeper penetration into biological tissues, reduced light damage during long-term irradiation, improved photostability, and low autofluorescence. When excited by NIR light, UCNPs can emit short-wave NIR light, visible light, or ultraviolet light, exhibiting unique upconversion optical properties, which can effectively solve the bottleneck of low penetration depth of the photosensitizer (PS) excitation light. Moreover, the ultraviolet or visible light emitted by UCNPs can activate the photosensitizer, thereby generating highly reactive reactive oxygen species (ROS), achieving the purpose of directly killing tumor cells or activating the immune system. Therefore, the combination of UCNPs and PS can not only solve the problem of poor tissue penetration ability of PS in photodynamic therapy (PDT), but also improve problems such as poor PS delivery and low targeting, showing good application prospects in the medical field. Research has shown that the combination of UCNPs and the photosensitizer M-540 can significantly increase the penetration depth of PDT and effectively induce cell death (ZHANG P, STEELANT W, KLAR M, et al. Versatile photosensitizers for photodynamic therapy at infrared excitation[J]. J Am Chem Soc, 2007, 129(15):4526-4527.); when the photosensitizer Ce6 is combined with UCNPs, the near-infrared light-triggered PDT tissue penetration depth of the UCNP-Ce6 nanocomposite is significantly increased (WANG C, TAO H Q, CHENG L, et al. Near-infrared light induced in vivo photodynamic therapy of cancer based on upconversion nanoparticles.[J] Biomaterials, 2011, 32(26):6145-6154.).

[0004] At present, the hypoxic characteristics of many solid tumors often limit the efficacy of PDT, and defects existing in the PDT process need to be improved to enhance the treatment effect. As the energy factory of cells, mitochondria have been recognized as the key subcellular targets of many photosensitizers (PSs) used in PDT. Mitochondria are rich in oxygen content. By using the compound triphenylphosphine (TPP) that specifically binds to mitochondria, PS can be targeted and delivered to mitochondria, significantly improving the generation efficiency of reactive oxygen species (ROS) in mitochondria and obviously enhancing the anti-tumor effect of PDT. Therefore, constructing a PDT nanoplatform with mitochondrial targeting has the potential to enhance the effect of PDT.

[0005] MicroRNA (miRNA) is a class of endogenous short-chain non-coding small RNAs, which generally exist in the genome in the form of single-copy, multi-copy or clusters. It regulates protein expression by fully or incompletely base-pairing with the 3'-untranslated region (3'UTR) of target mRNA, resulting in the degradation of target gene mRNA or the inhibition of mRNA translation, and is widely involved in processes such as cell growth, differentiation, proliferation, apoptosis, and metabolism. Therefore, miRNA is considered a promising therapeutic target in tumor treatment, such as miR21. However, ensuring the specific expression of therapeutic transgenes in the expected tissues while minimizing off-target effects elsewhere remains a challenge. Traditional gene delivery systems lack spatio-temporal specificity and cannot activate genes at specific lesion sites, severely limiting the efficacy of gene therapy. Although there is a near-infrared (NIR) light-activated gene therapy with deep penetration, its dependence on external activation still poses challenges in clinical implementation. Due to its significant programmability and excellent biocompatibility, DNA has become a general tool for engineering intelligent nanosystems.

[0006] Although significant progress has been made in current anti-tumor research, the effects of either PDT or gene therapy alone on tumors are insufficient. Therefore, it is necessary to combine PDT with other treatment methods. It has been found that based on UCNPs with the photosensitizer NH2-MIL-53(Fe) as the shell, loaded with DOX, under NIR irradiation, UCNPs activate NH2-MIL-53(Fe) to induce PDT and release DOX under the acidic conditions of the TME, which can synergistically inhibit the growth of gliomas (LVZ J, CAO Y, XUE D Z., et al. A multiphoton transition activated iron based metal organic framework for synergistic therapy of photodynamic therapy / chemodynamic therapy / chemotherapy for orthotopic gliomas[J]. J Mater Chem B, 2023, 11(5): 1100-1107.). Folic acid modification of UCNPs can actively target drugs to tumor sites by means of the interaction between folic acid and folic acid receptors, thereby guiding PDT to inhibit tumor growth. With UCNPs as the core and mSiO 2 as the shell, a novel delivery system (UCNPs@mSiO 2 -DOX / Ce6) is constructed. This system can activate Ce6 by absorbing near-infrared light, induce PDT to generate ROS, and release the chemotherapeutic drug DOX, achieving effective synergistic therapy and enhancing the tumor treatment effect (RAFIQUE R, GULAR, LEEIG, et al. Photo-induced reactions for disassembling of coloaded photosensitizer and drug molecules from upconversion-mesoporous silica nanoparticles. an effective synergistic cancer therapy[J] Mater Sci Eng C Mater Biol Appl, 2020, 110: 110545). Summary of the Invention

[0007] In the present invention, the AP site is modified on the single strand of miR21, and the anti-miR21 sequence anneals and hybridizes with it to form a double strand, which is then modified onto UCNPs. On the one hand, UCNPs deliver photosensitizers, which can be excited by NIR light, increasing the penetration depth of the excitation light and improving the PDT effect. Additionally, the material can be directly targeted to oxygen-rich mitochondria via TPP, enhancing the efficiency of PDT. The ROS generated by PDT can induce Apurinic / apyrimidinic endonuclease 1 (APE1) to migrate from the nucleus to the mitochondria, recognize the AP site on the nucleic acid double strand and cleave it, releasing the anti-miR21 single strand into the cytoplasm, where it binds to the miR21 single strand in the cytoplasm, initiating the apoptosis pathway based on Bcl-2 and synergistically exerting an anti-tumor effect with PDT( Figure 1 ). UCNPs, RB, anti-miR21 and TPP are controllably assembled together to combine the advantages of targeted therapy, PDT and gene therapy to achieve combined anti-tumor therapy, thus completing the present invention.

[0008] In a first aspect, the present invention provides a composite nanomaterial, and the structural formula of the composite nanomaterial is A@B@C, where A represents a nanomaterial carrier, B represents a photosensitizer, and C represents a targeting substance. The nanomaterial carrier is obtained by coating a layer of mesoporous silica on the upper surface of an upconversion nanomaterial. The targeting substance includes, but is not limited to, one or more of proteins, peptides (such as cyclic RGD), polysaccharides, and / or small biological molecules. The content ratio of the nanomaterial carrier, photosensitizer, and targeting substance is 5:1:2.

[0009] Further, the mesoporous silica is selected from C 8 H 20 O 4 Si, Cl 4 Si, HNaO 3 Si and / or SiO 2 or one or more of them.

[0010] Further, the upconversion luminescent nanomaterial is selected from NaErF doped with rare earth elements 4 , NaYF 4 , CaF 2 , Gd 2 (MoO4) 3 , Y 2 O 3 , Gd 2 O 2 S, BaY 2 F 8 , LiNbO 3 , Gd 2 O 2 , Y3 A l5 O 12 、TiO 2 , YF 3 、Lu 2 O 3 、LaCl 3 and / or Y 2 BZO 5 At least one or more of the up-conversion luminescent nanomaterials, wherein the rare earth element in the up-conversion luminescent nanomaterial can also be replaced by at least one or more of Yb, Tm, Er, Ln, Ho and / or Pr.

[0011] Furthermore, the structure of the upconversion luminescent nanomaterial includes a single-core structure or a core-shell nanostructure, wherein:

[0012] The upconversion luminescent nanomaterial with a single core structure can be NaErF 4 :Yb,Tm,Gd 2 O 2 :Er,Yb,Y 3 Al 5 O 12 :Er,Yb,TiO 2 :Er,Yb,YF 3 :Er,Yb,Lu 2 O 3 :Yb,Tm,NaYF 4 :Er,Yb,NaYF 4 :Yb,Tm or Y 2 BZO 5 :Yb, one of Ho;

[0013] The upconversion material with core-shell nanostructure can be NaGdF 4 :Yb,Tm@NaGdF 4 ,NaYF 4 :Yb,Er@NaYF 4 ,NaYb 4 :Er@NaLuF 4 ,NaYb 4 :Ho@NaYF 4 ,NaYF4:Yb,Tm@NaYF4:Yb,Er or NaYF 4 :Yb,Tm@NaGdF 4 :One of Yb.

[0014] Furthermore, the photosensitizer is selected from one or more of RB, 5-ALA, Ce6, Zn Pc, MB and / or MC540.

[0015] Furthermore, the composite nanomaterial has a core-shell structure with an average particle size of 100 nm, preferably 50 - 300 nm.

[0016] Furthermore, the mesoporous SiO 2 The shell thickness is 16.6 nm, preferably 10 - 20 nm.

[0017] Furthermore, the composite nanomaterial of the present invention is a drug.

[0018] In a second aspect, the present invention provides a pharmaceutical composition, which contains the composite nanomaterial described in the first aspect of the present invention and pharmaceutically acceptable excipients.

[0019] Furthermore, the pharmaceutical composition may also contain other anti-tumor active ingredients.

[0020] Furthermore, the pharmaceutical composition can be made into various forms such as injection, tablet, powder, granule, capsule or oral liquid, etc.; the drugs in the above various dosage forms can all be prepared according to the conventional methods in the pharmaceutical field.

[0021] In a third aspect, the present invention provides an application of the composite nanomaterial in the preparation of drugs for treating tumors.

[0022] Furthermore, the tumors include but are not limited to one or more of small cell lung cancer, non-small cell lung cancer, ovarian cancer, endometrial cancer, breast cancer, head and neck cancer, thymoma, colorectal cancer, pancreatic cancer, prostate cancer, bladder cancer and / or melanoma.

[0023] Furthermore, the structural formula of the composite nanomaterial is A@B@C, where A represents a nanomaterial carrier, B represents a photosensitizer, and C represents a targeting substance. The nanomaterial carrier is obtained by coating a layer of mesoporous silica on the upper surface of the upconversion nanomaterial. The targeting substance includes but is not limited to one or more of proteins, peptides (such as cyclic RGD), polysaccharides and / or small biological molecules. The content ratio of the nanomaterial carrier, photosensitizer and targeting substance is 5:1:2.

[0024] Furthermore, the mesoporous silica is selected from C 8 H 20 O 4 Si, Cl 4 Si, HNaO 3 Si and / or SiO 2 One or more of them.

[0025] Furthermore, the upconversion luminescent nanomaterial is selected from NaErF doped with rare earth elements 4 、NaYF 4 、CaF 2 、Gd 2(MoO4) 3 , Y 2 O 3 , Gd 2 O 2 S.BaY 2 F 8 、LiNbO 3 , Gd 2 O 2 , Y 3 A l5 O 12 、TiO 2 , YF 3 、Lu 2 O 3 、LaCl 3 and / or Y 2 BZO 5 At least one or more of the up-conversion luminescent nanomaterials, wherein the rare earth element in the up-conversion luminescent nanomaterial can also be replaced by at least one or more of Yb, Tm, Er, Ln, Ho and / or Pr.

[0026] Furthermore, the structure of the upconversion luminescent nanomaterial includes a single-core structure or a core-shell nanostructure, wherein:

[0027] The upconversion luminescent nanomaterial with a single core structure can be NaErF 4 :Yb,Tm,Gd 2 O 2 :Er,Yb,Y 3 Al 5 O 12 :Er,Yb,TiO 2 :Er,Yb,YF 3 :Er,Yb,Lu 2 O 3 :Yb,Tm,NaYF 4 :Er,Yb,NaYF 4 :Yb,Tm,or Y 2 BZO 5 :Yb, one of Ho;

[0028] The upconversion material with core-shell nanostructure can be NaGdF 4 :Yb,Tm@NaGdF 4 ,NaYF 4 :Yb,Er@NaYF 4 or NaYF 4 :Yb,Tm@NaGdF 4 :One of Yb.

[0029] Further, the photosensitizer is selected from one or more of RB, 5-ALA, Ce6, Zn Pc, MB, and / or MC540.

[0030] A specific embodiment of the composite nanomaterial of the present invention is UCNPs@RB@TPP-DNA, and the nanomaterial carrier is NaYF 4 :Yb,Er@NaYF 4 , the mesoporous silica is selected from SiO 2 , the photosensitizer is RB, and the targeting substance is mitochondrion-targeted TPP.

[0031] Beneficial effects

[0032] (1) The composite nanomaterial UCNPs@RB@TPP-DNA prepared by the method of the present invention has low cytotoxicity, good absorption in the near-infrared region, and good photodynamic effect.

[0033] (2) The present invention can target tumor cell mitochondria through TPP. After being excited by NIR light, ROS generated by the photosensitizer RB causes APE1 to migrate to the mitochondria, and then cleaves the anti-miR21 double-stranded DNA, activating gene therapy and synergistically exerting an anti-tumor effect with PDT.

[0034] (3) The present invention has the disciplinary intersection advantage of nanoscience and biomedicine, broadens the design and synthesis ideas of composite nanomaterials, and provides a new method for tumor treatment. Brief description of the drawings

[0035] Figure 1 Schematic diagram of a tumor gene therapy nanodrug platform activated by PDT.

[0036] Figure 2 is the transmission electron microscope (TEM) morphology (A) and particle size diagram (B) of UCNPs@SiO 2 -RB-DNA-TPP.

[0037] Figure 3 is the UCL spectrum of UCNPs excited by 980 nm near-infrared light and the ultraviolet-visible absorption spectrum of the photosensitizer RB.

[0038] Figure 4 is the ultraviolet / visible absorption spectra of UCNP@SiO 2 -NH 2 , UCNP@SiO 2 -NH 2 @TPP, UCNP@SiO 2 -NH 2 @DNA@TPP, the characteristic peaks of DNA and the characteristic peaks of TPP.

[0039] Figure 5 It is the fluorescence spectrum change of F-AM (100 nM) with the increase of APE1 concentration (A) and time (B). (C) is a schematic diagram of the release of Cy5-A from F-AM mediated by APE1.

[0040] Figure 6 It is the detection of the uptake (A) of nanomaterials by MCF-7 cells and its targeting property (B) by flow cytometry.

[0041] Figure 7 It is the detection of the change in the ROS level in MCF-7 cells after treatment with different materials using confocal imaging.

[0042] Figure 8 It is the immunofluorescence staining of APE1 in MCF-7 cells treated with different samples.

[0043] Figure 9 It is the detection of the miR21 mRNA expression in MCF-7 cells after different treatments by qRT-PCR.

[0044] Figure 10 It is the detection of the cytotoxicity of nanomaterials in different groups against tumor cells MCF-7 by the cck8 assay. Detailed implementation manners

[0045] To make the present invention more obvious and understandable, the following provides a detailed description in combination with examples and drawings. The following descriptions are only the preferred embodiments of the present invention, and do not limit the present invention in any formal or substantial way. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

[0046] Term explanations

[0047] Reactive oxygen species: (ROS) are a class of substances with high reactivity, including free radicals such as superoxide anion (O -2 ), hydroxyl radical (·OH), etc. and non-free radicals such as hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ). ROS in cells mainly come from mitochondria and NADPH oxidase.

[0048] Singlet oxygen: It is one type of reactive oxygen species. When the two unpaired electrons of the oxygen molecule in the ground state are excited by a certain amount of energy, the spin changes from parallel to antiparallel. When these two electrons occupy the same π * 2pWhen in the orbital, it is the relatively stable first excited state ( 1 O 2 , denoted as 1 Δ g (( 1 O 2 ). In the present invention, singlet oxygen refers to the active oxygen with high cytotoxicity formed by photosensitizer molecules absorbing light energy and transferring it to the surrounding oxygen under photoexcitation.

[0049] Photosensitizer: (photosensitizers, PS) is a substance that accumulates in tumor tissues and selectively makes them sensitive to light. A single pure compound with low cost and good stability, and a high absorption peak in the range of 600 - 800 nm (from red to dark red) is an ideal photosensitizer.

[0050] Upconversion luminescence: It is a kind of luminescence process that violates Stokes' law. After the luminescence center continuously absorbs two or more low - energy photons, it decays to the excited state through non - radiative relaxation, and then releases a high - energy photon when returning from the excited state to the ground state, generating upconversion luminescence.

[0051] Upconversion luminescence nanomaterials (UCNPs): mainly consist of three functional parts: matrix material, activator and sensitizer. It is a special optical functional material that follows Stokes' law, and its excitation light is generally low - energy near - infrared light in the wavelength range of the biological tissue optical window.

[0052] Biocompatibility: includes tissue compatibility and blood compatibility. Tissue compatibility reflects the degree of mutual regulation between a substance and living tissues, including physical properties such as the hardness and elastic modulus of the material; blood compatibility mainly refers to whether platelet aggregation, coagulation, thrombus formation or hemolysis will occur after the material comes into direct contact with blood. Once it occurs, the use efficiency and treatment effect of the material will be greatly reduced.

[0053] APE1: Apurinic / apyrimidinic endonuclease 1 is a protein that can perform DNA repair function and redox function, and can regulate a variety of transcription factors related to tumor - associated pathways.

[0054] TPP: Triphenylphosphine is a lipophilic cation that can cross the mitochondrial membrane and can enter the mitochondria under the drive of the mitochondrial transmembrane potential.

[0055] Example 1 Synthesis of composite nanomaterial UCNPs@SiO 2 -RB@TPP - DNA

[0056] 1. Synthesis of NaYF4:Yb,Er

[0057] A mixture containing 5.64 g of OA, 5.04 g of ODE, 0.78 mmol of YCl 3 , 0.2 mmol of YbCl 3 and 0.02 mmol of ErCl 3 was added to a 100 mL flask and heated to 120 °C. After cooling, 2.5 mmol of NaOH and 4 mmol of NH 4 F dissolved in methanol were added. The temperature was raised to 90 °C. After removing methanol, under vacuum, the temperature was raised to 120 °C, and then heated to 330 °C under nitrogen protection. After cooling, ethanol was added and the precipitate was collected by centrifugation and redissolved in 10 mL of cyclohexane.

[0058] 2. Synthesis of Trifluoroacetic Acid-Coated Core-Multishell Structure NaYF 4 :Yb,Er@NaYF 4 UCNP

[0059] The prepared NaYF 4 :Yb,Er (5 mL) colloidal solution was added to a solution containing a solvent (5.64 g of OA and 5.04 g of ODE) and a shell precursor mixture (0.5 mmol of CF 3 COONa and 0.5 mmol of Y(CF 3 COO) 3 ). It was heated to 120 °C under vacuum. Then, under nitrogen protection, it was heated to 315 °C. After cooling, ethanol was added, the precipitate was collected by centrifugation, and redispersed in 10 mL of cyclohexane.

[0060] 3. Synthesis of UCNPs@SiO 2 -RB

[0061] Take 0.1 g of CATB, 20 mL of water, 1 mL of UCNP and mix and stir overnight. Then add 40 mL of water, 6 mL of ethanol, 100 μL of sodium hydroxide (2 mol), raise the temperature to 60 °C, and dropwise add an ethanol solution containing TEOS (200 μL of TEOS, 1 mL of ethanol). After the reaction, APTES (100 μL of APTES, 1 mL of ethanol) was added and heated for 30 - 60 min. The precipitate was collected by centrifugation and redispersed in 2 mL of water. Take 1 mL of UCNPs@SiO 2 (aqueous solution) and 1 mL of RB (1 mg / mL, aqueous solution), stir for 24 h in the dark, collect the precipitate by centrifugation, and redisperse it in 1 mL of water.

[0062] 4. Synthesis of UCNPs@SiO 2 @RB@TPP-DNA (URMT)

[0063] Mix 2 mg of TPP-COOH, 2.4 mg of NHS, 7.2 mg of EDC, 480 μL of methanol and 120 μL of sodium carbonate buffer (0.05 M, pH 9.5). Then add 1 mL of UCNPs@SiO 2 @RB for reaction, collect the precipitate by centrifugation, and redisperse it in 1 mL of water. Prepare the stock solutions (100 mM) of the anti-miR21 strand and its complementary strand with water. Take 10 μL of anti-miR21 (100 mM), 10 μL of the complementary strand (100 mM) and 30 μL of HEPES buffer (20 mM), mix well, heat to 95 °C, react for 5 min, anneal, and then place it in the dark for 12 h. Add 20 μL of the above dsDNA solution to 50 μL of the UCNP@SiO 2 @RB@TPP solution, vortex for 1 min and then let it stand in the dark for 1 h.

[0064] Characterization of the materials in Example 2

[0065] Characterize the materials in each of the above synthesis steps. Characterize the morphology and particle size of the composite nanomaterials by electron microscopy; measure the infrared absorption spectrum to characterize the drug loading; measure the zeta potential and particle size distribution (DLS) to characterize the successful connection of each step of the materials.

[0066] The morphology and particle size of the composite nanomaterials under a transmission electron microscope (TEM) are as Figure 2 shown.

[0067] As Figure 3 shown, under 980 nm excitation, the emission spectrum of UCNPs matches the absorption spectrum of RB, indicating that RB can be effectively activated by UCNPs.

[0068] As Figure 4 shown, from the UV / visible absorption spectrum analysis of the characteristic peaks of UCNP@SiO 2 -NH 2 , UCNP@SiO 2 -NH 2 @TPP, UCNP@SiO 2 -NH 2 @TPP@DNA, the DNA characteristic peak and the TPP characteristic peak, it can be seen that DNA and TPP are successfully encapsulated on UCNPs.

[0069] Example 3 Evaluation of the effectiveness of the composite nanomaterials

[0070] 1. Evaluate the cleavage of the anti-miR21 complementary double strand by APE1

[0071] Modify the photosensitive group Cy5 on anti-miR21, and modify the quenching group BHQ3 and AP site (i.e., base deletion empty site) on its complementary strand. After annealing and hybridization into a double strand, add exogenous APE1 and detect the recovery of fluorescence.

[0072] As Figure 5 shown, after adding APE1, the phosphodiesterase bond on the 5'-side of the AP site is cleaved, resulting in the release of Cy5-A and an increase in fluorescence signal. In addition, the time-dependent fluorescence curve shows that the double-stranded FRET complementary strand responds rapidly to APE1-mediated cleavage.

[0073] 2. Mitochondrial targeting property

[0074] After incubating with different materials for 4 h, flow cytometry was used to detect the phagocytosis effect of the composite nanomaterials.

[0075] As Figure 6 shown, the single-stranded DNA loaded in the material was labeled with Cy5. Compared with the cells treated with URsM, URsMT showed stronger fluorescence intensity. This indicates that the uptake of URsMT by cells was enhanced through TPP-mediated endocytosis.

[0076] 3. Upconversion-mediated PDT property

[0077] After the material was irradiated with NIR light, DCFH-DA fluorescence was used to detect the generation of intracellular singlet oxygen to evaluate the effect of the nanomaterials in generating ROS.

[0078] As Figure 7 shown, in the absence of 980 nm laser irradiation, MCF-7 cells incubated with URT showed negligible green fluorescence. In contrast, MCF-7 cells showed obvious green fluorescence after 980 nm laser irradiation. In addition, after near-infrared light irradiation, compared with the cells treated with UR, the cells treated with URT produced more reactive oxygen species.

[0079] 4. Use immunofluorescence to evaluate whether singlet oxygen can cause the migration of APE1

[0080] MCF-7 cells were incubated in a glass culture dish for 24 h. After incubating with nURMT and URMT in Opti-MEM medium for 4 h, the light irradiation group was irradiated with a 980 nm laser for 10 minutes (1.2 W / cm 2After irradiation for 1 min with a 5-min interval and then incubation for 1 h, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and washed with PBS. Then they were blocked with 5% BSA for 30 min and incubated with anti-Bcl-2 (Immunoway, 1:500) and anti-APE1 (Abcam, 1:500) for 1 h. Subsequently, they were washed with PBS and incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody (Abcam, 1:500) for 1 h. Finally, they were stained with Hoechst 33342, and intracellular fluorescence was observed using a confocal laser scanning microscope (CLSM).

[0081] As Figure 8 shown, under near-infrared light irradiation, the level of APE1 in mitochondria in the nUMRT and UMRT groups was significantly increased compared with that in the non-irradiated group, and PDT stimulated the translocation of APE1 from the nucleus to mitochondria.

[0082] 5. Properties of miR21 inhibition

[0083] The AP site was labeled on the anti-miR21 complementary strand, hybridized into a double strand and assembled onto the composite nanomaterial. MCF-7 cells (4×10 5 cells) were seeded in a 6-well plate and incubated for 24 h. After treating the cells with different materials for 4 h, NIR light (980 nm, 1.2 W cm -2 ) was used to irradiate the cells for 10 min (irradiation for 1 min with a 5-min interval). After the irradiation, the cells were cultured for another 24 h. Total cellular RNA was extracted and reverse transcribed into cDNA, and then the change in miR21 expression in the cells was detected by fluorescence quantitative PCR.

[0084] As Figure 9 shown, in MCF-7 cells treated with URMT and URsMT plus 980-nm light irradiation, the expression of miR21 was significantly decreased. Cells treated with nURMT (miR21 double strand without AP site) showed no effect on miR21 expression. In addition, cells treated with URMT without NIR irradiation showed no change in miR21 expression.

[0085] 6. Cytotoxicity

[0086] After MCF-7 cells were incubated in a 96-well plate for 24 h, different materials were added to treat the cells for 4 h, and NIR light (980 nm, 1.2 W cm -2 ) was used to irradiate for 10 min (irradiation for 1 min with a 5-min interval). The cells were cultured for another 20 h. The cytotoxicity of the materials was detected using a CCK-8 kit, and the absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay reader.

[0087] AsFigure 10 As shown, in the absence of near-infrared light irradiation, the material exhibits good biocompatibility, while under 980 nm laser irradiation, the cell viability of cells treated with UR, URM, nURMT, URMT, and URsMT decreased to 70%, 47%, 43%, 20%, and 21% respectively.

Claims

1. A composite nanomaterial, wherein the structural formula of the composite nanomaterial is A@B@C, wherein A represents a nanomaterial carrier, B represents a photosensitizer, and C represents a targeting substance. The nanomaterial carrier is obtained by coating a layer of mesoporous silicon on the upper surface of an upconversion nanomaterial. The targeting substance includes but is not limited to one or more of proteins, peptides (such as cyclic RGD), polysaccharides and / or small biological molecules. The content ratio of the nanomaterial carrier, photosensitizer and targeting substance is 5:1:

2.

2. The composite nanomaterial according to claim 1, wherein the mesoporous silicon is selected from C8H 20 One or more of O4Si, Cl4Si, HNaO3Si and / or SiO2.

3. The composite nanomaterial according to claim 1, wherein the photosensitizer is selected from one or more of RB, 5-ALA, Ce6, ZnPc, MB and / or MC540.

4. The composite nanomaterial according to any one of claims 1 to 3, wherein the composite nanomaterial is a core-shell structure and has an average particle size of 100 nm.

5. A pharmaceutical composition comprising the composite nanomaterial according to claims 1-4 and a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, further comprising other anti-tumor active ingredients.

7. The pharmaceutical composition according to any one of claims 5 or 6, which can be prepared into various forms such as injection, tablets, powders, granules, capsules or oral liquids; the above-mentioned various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

8. Application of a composite nanomaterial in the preparation of drugs for treating tumors.

9. The use of claim 7 in preparing a drug for treating tumors, wherein the tumors include but are not limited to one or more of small cell lung cancer, non-small cell lung cancer, ovarian cancer, endometrial cancer, breast cancer, head and neck cancer, thymoma, colorectal cancer, pancreatic cancer, prostate cancer, bladder cancer and / or melanoma.

Images