Nanoparticles for photodynamic therapy and their preparation method

Abstract

This invention relates to the field of drug delivery systems, and discloses a nanoparticle for photodynamic therapy and its preparation method. The nanoparticle has a core-shell-corona composite structure, comprising, from the inside out: a core layer composed of NaYF4 upconversion nanoparticles; a middle layer: a mesoporous Zr-MOF layer; a shell layer: a pH-responsive polymer layer formed by linking folic acid to polyethylene glycol-polycaprolactone block copolymer via hydrazone bonds; and a targeting corona layer comprising dual targeting ligands, namely a bicyclic [6.1.0]nonyne-functionalized iRGD peptide and an azide-functionalized anti-EGFR nanobody 7D12. The nanoparticles of this invention possess advantages such as high stability, strong targeting, and high photosensitizer loading, and can efficiently generate reactive oxygen species.

Description

Technical Field

[0001] This invention relates to the field of drug delivery systems, and more specifically, to a nanoparticle for photodynamic therapy and its preparation method. Background Technology

[0002] Photodynamic therapy (PDT) is a novel therapeutic technique that uses photosensitizers to generate reactive oxygen species (ROS) under irradiation with a specific wavelength of light to kill diseased cells. It boasts advantages such as high selectivity, minimal invasiveness, and low side effects, and has been widely used in the treatment of diseases such as tumors and condyloma acuminata. Its core principle is as follows: after the photosensitizer accumulates at the lesion site, it is excited by light of a specific wavelength, transitioning from the ground state to the excited state. This excited state reacts with surrounding oxygen to generate singlet oxygen and other reactive oxygen species, inducing apoptosis or necrosis of cells.

[0003] However, the photosensitizers used in traditional photodynamic therapy have many limitations:

[0004] Poor water solubility: Most photosensitizers (such as porphyrins and phthalocyanines) are lipid-soluble compounds, which tend to aggregate in the physiological environment, resulting in low bioavailability;

[0005] Insufficient targeting: Free photosensitizers are easily taken up by normal tissues, resulting in low accumulation at the lesion site. This not only reduces the therapeutic effect but may also cause phototoxic damage to normal tissues.

[0006] Short circulation time: Unmodified photosensitizers are easily cleared by the reticuloendothelial system (such as the liver and spleen) and are difficult to remain at the lesion site for a long time;

[0007] Limited penetration depth: Traditional photosensitizers are mostly excited in the ultraviolet-visible region (400-600nm), and the tissue penetration depth is only a few millimeters, which is not effective for treating deep tumors.

[0008] To address these issues, nanoparticle-based drug delivery systems have become a research hotspot. Nanoparticles can encapsulate photosensitizers through hydrophobic interactions, improving their water solubility; surface modification with targeting molecules can enhance lesion site selectivity; furthermore, the "high permeability and retention effect (EPR)" of nanoparticles can enhance tumor tissue uptake. However, existing nanoparticle-based drug delivery systems still suffer from problems such as insufficient stability (e.g., easy disintegration in the blood), uncontrollable photosensitizer release, and low targeting efficiency.

[0009] Based on this, we provide nanoparticles that are highly stable, highly targeted, have a high photosensitizer loading rate, and can efficiently generate reactive oxygen species, which is of great significance for improving the effect of photodynamic therapy. Summary of the Invention

[0010] In view of this, the present invention proposes a nanoparticle for photodynamic therapy, aiming to solve at least one of the aforementioned background problems.

[0011] This invention proposes a nanoparticle for photodynamic therapy, wherein the nanoparticle has a core-shell-corona composite structure, comprising, from the inside out:

[0012] Core layer: Composed of NaYF4 upconversion nanoparticles, doped with Yb 3+ and Er 3+ Yb 3+ The doping amount is 15-20 mol%, Er 3+ The doping concentration is 1-2 mol%, with the remainder being Y. 3+ ;

[0013] Intermediate layer: Mesoporous Zr-MOF layer, wherein the Zr-MOF is UiO-66, with a pore size of 2-5 nm and a specific surface area of ​​1000-1200 m². 2 / g, via Zr 4+ Coordination with photosensitizers to load photosensitizers;

[0014] Outer shell: pH-responsive polymer layer, formed by linking folic acid through hydrazone bonds of polyethylene glycol-polycaprolactone block copolymer, wherein the molecular weight of polyethylene glycol is 2000-3000 Da, the molecular weight of polycaprolactone is 4000-6000 Da, and the thickness of polymer layer is 10-15 nm.

[0015] Targeting the canopy: This includes dual targeting ligands, namely a bicyclic [6.1.0]nonyne-functionalized iRGD peptide and an azide-functionalized anti-EGFR nanobody 7D12, with a molar ratio of 2:1.

[0016] Preferably, the NaYF4 upconversion nanoparticles in the core layer have a particle size of 20-30 nm. When excited by near-infrared light at 980 nm ± 5 nm, they can emit 540 nm ± 2 nm green light and 660 nm ± 2 nm red light, with the emission intensity ratio of green light to red light being 3:1-4:1.

[0017] Preferably, the photosensitizer loaded in the intermediate layer is selected from bamboo red fungus A (HMME), dihydroporphyrin e6 (Ce6) or zinc phthalocyanine (ZnPc).

[0018] Preferably, the loading of dihydroporphyrin e6 (Ce6) or zinc phthalocyanine (ZnPc) is 30-40 wt%, and the loading of bamboo red fungus A is 35-38 wt%.

[0019] Preferably, the dual-targeting ligand is obtained by bioorthogonal reaction modification, wherein the bioorthogonal reaction is a copper-free azide-alkyne cycloaddition reaction of BCN and N3, and the density of targeting ligands on the surface of the modified nanoparticles is 1.2-1.5 per nm².

[0020] The present invention also provides a method for preparing the nanoparticles described in the above technical solution, comprising the following steps:

[0021] S1. Synthesis of NaYF4 upconversion nanoparticles: Rare earth nitrates were mixed with oleic acid and octadecene and reacted at 160°C for 1 hour under nitrogen protection to form a complex. After adding NaF methanol solution, the temperature was increased to 300°C at a rate of 5°C / min and reacted for 2 hours. After washing with ethanol, the mixture was dispersed in cyclohexane to obtain NaYF4 upconversion nanoparticles.

[0022] S2. Layer-by-layer self-assembly coating of Zr-MOF and loading of photosensitizer: NaYF4 upconversion nanoparticles were dispersed in DMF, ZrCl4 and terephthalic acid were added, the pH was adjusted to 3.0, and after sonication for 30 minutes, the reaction was carried out at 60℃ for 24 hours. After centrifugation and washing, the photosensitizer DMF solution was sonicated for 2 hours.

[0023] S3. Modification of pH-responsive polymer shell: Polyethylene glycol-polycaprolactone and folic acid were reacted in DMF for 24 hours under DCC / DMAP catalysis to synthesize polyethylene glycol-polycaprolactone-folic acid, which was then mixed with the chloroform dispersion of the product from step S2, ultrasonicated to form micelles, and purified by dialysis.

[0024] S4. Coupling with a targeting ligand: The bicyclic [6.1.0]nonyne-functionalized iRGD peptide and the azide-functionalized anti-EGFR nanobody 7D12 were dissolved in PBS at a molar ratio of 2:1, and the product of step S3 was added. The mixture was stirred at 37°C for 4 hours, and after centrifugation and washing, the nanoparticles for photodynamic therapy were obtained.

[0025] Preferably, in step S1, the total concentration of rare earth nitrates is 0.04 mol / L, composed of Yb(NO3)3·6H2O, Er(NO3)3·6H2O and Y(NO3)3·6H2O in a molar ratio of 18:1:81, and the molar ratio of NaF to total rare earth ions is 3:1.

[0026] Preferably, in step S2, the molar ratio of ZrCl4 to terephthalic acid is 1:1, the mass ratio of NaYF4 upconversion nanoparticles to ZrCl4 is 1:4, the ultrasonic power is 400W, the ultrasonic power is 200W when the photosensitizer is loaded, and the mass ratio of photosensitizer DMF to MOF is 1:3.

[0027] Preferably, in step S3, the mass ratio of polyethylene glycol-polycaprolactone to folic acid is 4:1, the molar ratio of DCC to folic acid is 1.2:1, the amount of DMAP used is 20% of the mass of folic acid, the molecular weight cutoff of the dialysis bag is 10,000 Da, the dialysis medium is chloroform, and it is changed every 6 hours.

[0028] The present invention also provides the application of the nanoparticles described above in the preparation of photodynamic therapy drugs, which are used to treat liver cancer, breast cancer or lung cancer.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] (1) High stability: The porous structure of MOF and the PEG-PCL shell effectively protect the photosensitizer from external environmental interference. It still maintains more than 90% of its integrity after being placed in PBS buffer for 7 days.

[0031] (2) Strong targeting: The dual targeting mechanism of iRGD and 7D12 increases the binding efficiency of nanoparticles on the surface of tumor cells by more than 3 times and significantly reduces the uptake by normal tissues.

[0032] (3) High photosensitizer loading rate: The high specific surface area of ​​mesoporous MOF (>1000 m²) 2 The HMME loading rate reached 38.7% due to the combination of HMME loading rate and coordination, which far exceeded that of traditional liposomes.

[0033] (4) Highly efficient ROS generation: The upconversion luminescence of UCNPs is highly matched with the absorption spectrum of HMME, and singlet oxygen (ROS) is generated through fluorescence resonance energy transfer (FRET). 1 The O2 quantum yield was increased to 0.66, which is higher than that of free HMME.

[0034] (5) Adaptation to hypoxic microenvironment: The mesoporous structure of MOF can load perfluorocarbons (PFCs), which release oxygen under light, alleviate tumor hypoxia, and further enhance ROS generation. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0036] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] This invention proposes a nanoparticle for photodynamic therapy, wherein the nanoparticle has a core-shell-corona composite structure, comprising, from the inside out:

[0041] Core layer: Composed of NaYF4 upconversion nanoparticles, doped with Yb 3+ and Er 3+ Yb 3+ The doping amount is 15-20 mol%, Er 3+ The doping concentration is 1-2 mol%, with the remainder being Y. 3+ ;

[0042] Intermediate layer: Mesoporous Zr-MOF layer, wherein the Zr-MOF is UiO-66, with a pore size of 2-5 nm and a specific surface area of ​​1000-1200 m² / g, through which Zr... 4 ⁺ Coordination with photosensitizer to load photosensitizer;

[0043] Outer shell: pH-responsive polymer layer, formed by linking folic acid through hydrazone bonds of polyethylene glycol-polycaprolactone block copolymer, wherein the molecular weight of polyethylene glycol is 2000-3000 Da, the molecular weight of polycaprolactone is 4000-6000 Da, and the thickness of polymer layer is 10-15 nm.

[0044] Targeting the canopy: This includes dual targeting ligands, namely a bicyclic [6.1.0]nonyne-functionalized iRGD peptide and an azide-functionalized anti-EGFR nanobody 7D12, with a molar ratio of 2:1.

[0045] Specifically:

[0046] Core layer: Composed of NaYF4 upconversion nanoparticles, doped with Yb 3+ and Er 3+ Yb 3+ The doping amount is 15-20 mol%, Er 3+ The doping concentration is 1-2 mol%, with the remainder being Y. 3+ ;

[0047] In this invention, the NaYF4 upconversion nanoparticles in the core layer have a particle size of 20-30 nm. Under near-infrared light excitation of 980 nm ± 5 nm, they can emit 540 nm ± 2 nm green light and 660 nm ± 2 nm red light, with the emission intensity ratio of green light to red light being 3:1-4:1.

[0048] This invention employs NaYF4 upconversion nanoparticles as the core layer, where NaYF4 is the matrix material with the highest known upconversion efficiency, and Yb 3+ As a sensitizer, it can efficiently absorb 980 nm near-infrared light (tissue penetration depth up to 5-10 mm), Er 3+ As an activator, it can emit 540 nm and 660 nm light that matches the absorption of most photosensitizers. The 18:1 doping ratio of the two can maximize the energy transfer efficiency and improve the upconversion luminescence intensity by 40% compared with the traditional doping ratio (such as 20:2).

[0049] Intermediate layer: Mesoporous Zr-MOF layer, wherein the Zr-MOF is UiO-66, with a pore size of 2-5 nm and a specific surface area of ​​1000-1200 m². 2 / g, via Zr 4+ Coordination with photosensitizers to load photosensitizers;

[0050] In this invention, the photosensitizer loaded in the intermediate layer is selected from bamboo red fungus A (HMME), dihydroporphyrin e6 (Ce6) or zinc phthalocyanine (ZnPc).

[0051] In this invention, the loading of dihydroporphyrin e6 (Ce6) or zinc phthalocyanine (ZnPc) is 30-40 wt%, and the loading of bamboo red fungus A is 35-38 wt%.

[0052] The Zr-MOF (UiO-66) in this invention possesses ultra-high chemical stability (tolerant to buffer solutions with pH 2-11) and biocompatibility. Its 2-5 nm mesoporous structure matches the molecular size of photosensitizers, enabling Zr... 4+ The coordination with the hydroxyl group of the photosensitizer achieves high-efficiency loading, increasing the loading rate by 2-3 times compared to physical adsorption; at the same time, its 1000-1200 m 2 The high specific surface area of ​​ / g can simultaneously load perfluorocarbons (PFCs) as oxygen carriers to alleviate tumor hypoxia.

[0053] Outer shell: pH-responsive polymer layer, formed by linking folic acid with polyethylene glycol-polycaprolactone block copolymer via hydrazone bonds, wherein the molecular weight of PEG is 2000-3000 Da, the molecular weight of PCL is 4000-6000 Da, and the polymer layer thickness is 10-15 nm;

[0054] The polyethylene glycol-polycaprolactone (PEG-PCL) of this invention is an FDA-approved biodegradable material. The PEG chain (molecular weight 2000-3000 Da) can reduce the phagocytosis of nanoparticles by the reticuloendothelial system (RES) and prolong blood circulation time to more than 12 hours (compared to only 4-6 hours for traditional nanoparticles). The PCL chain (molecular weight 4000-6000 Da) can enhance the hydrophobic interaction with MOF and improve the coating stability. The hydrazone bond is rapidly hydrolyzed in the acidic microenvironment of tumors (pH 6.0-6.8) and stable in normal tissues (pH 7.4), enabling targeted and controllable release of photosensitizers.

[0055] Targeting the canopy: This includes dual targeting ligands, namely a bicyclic [6.1.0]nonyne-functionalized iRGD peptide and an azide-functionalized anti-EGFR nanobody 7D12, with a molar ratio of 2:1.

[0056] In this invention, the dual-targeting ligand is obtained through a bioorthogonal reaction modification, wherein the bioorthogonal reaction is a copper-free azide-alkyne cycloaddition reaction of BCN and N3. After modification, the density of targeting ligands on the surface of the nanoparticles is 1.2-1.5 ligands / nm. 2 .

[0057] iRGD peptide can target integrin αvβ3 in tumor angiogenesis, and 7D12 nanobody can specifically bind to EGFR highly expressed in tumor cells. The 2:1 modification ratio of the two can synergistically improve the uptake rate of tumor cells (2.5 times higher than single targeting). The copper-free Click reaction efficiency of BCN and N3 reaches over 90%, avoiding the cytotoxicity of traditional copper catalytic systems, and the reaction conditions are mild (physiological pH and temperature), which does not affect the structural integrity of nanoparticles.

[0058] The present invention also provides a method for preparing the nanoparticles described in the above technical solution, comprising the following steps:

[0059] S1. Synthesis of NaYF4 upconversion nanoparticles, i.e., UCNPs: Rare earth nitrates were mixed with oleic acid and octadecene and reacted at 160°C for 1 hour under nitrogen protection to form a complex. After adding NaF methanol solution, the temperature was increased to 300°C at a rate of 5°C / min and reacted for 2 hours. After washing with ethanol, the mixture was dispersed in cyclohexane to obtain NaYF4 upconversion nanoparticles.

[0060] In this invention, the total concentration of rare earth nitrates in step S1 is 0.04 mol / L, which is composed of Yb(NO3)3·6H2O, Er(NO3)3·6H2O and Y(NO3)3·6H2O in a molar ratio of 18:1:81, and the molar ratio of NaF to total rare earth ions is 3:1.

[0061] Oleic acid, as a surfactant, can coordinate with rare earth ions through carboxyl groups, controlling the growth of nanoparticles into uniform spheres of 20-30 nm. Octadecylene, as a high-boiling-point solvent (315℃), provides a stable high-temperature reaction environment, avoiding particle size unevenness caused by solvent evaporation. Heating at 160℃ removes moisture and oxygen from the system, preventing rare earth ion hydrolysis. Nitrogen protection prevents oleic acid oxidation, ensuring stable complex formation. A slow heating rate of 5℃ / min avoids particle aggregation caused by local overheating. A reaction temperature of 300℃ promotes the formation of hexagonal phase crystals of NaYF4 (upconversion efficiency is 3 times higher than that of the cubic phase). Ethanol, as an antisolvent, can destroy the surface coating of oleic acid, causing UCNPs to aggregate and precipitate. Centrifugation at 8000 rpm can effectively separate nanoparticles from excess surfactant. Cyclohexane dispersion can prevent UCNPs aggregation, and storage at 4℃ ensures stability for more than 6 months.

[0062] S2. Layer-by-layer self-assembly coating of Zr-MOF and loading of photosensitizer: NaYF4 upconversion nanoparticles were dispersed in DMF, ZrCl4 and terephthalic acid were added, the pH was adjusted to 3.0, and after sonication for 30 minutes, the reaction was carried out at 60℃ for 24 hours. After centrifugation and washing, the photosensitizer DMF solution was sonicated for 2 hours.

[0063] In this invention, in step S2, the molar ratio of ZrCl4 to terephthalic acid is 1:1, the mass ratio of NaYF4 upconversion nanoparticles to ZrCl4 is 1:4, the ultrasonic power is 400W, the ultrasonic power is 200W when the photosensitizer is loaded, and the mass ratio of photosensitizer DMF to MOF is 1:3.

[0064] The present invention utilizes 200 W ultrasound to promote the diffusion of HMME into the mesopores of MOF, increasing the loading rate by 15% compared to static adsorption; room temperature loading avoids the thermal degradation of HMME (HMME is easily decomposed above 60℃).

[0065] S3. Modification of pH-responsive polymer shell: Polyethylene glycol-polycaprolactone and folic acid were reacted in DMF for 24 hours under DCC / DMAP catalysis to synthesize polyethylene glycol-polycaprolactone-folic acid, which was then mixed with the chloroform dispersion of the product from step S2, ultrasonicated to form micelles, and purified by dialysis.

[0066] In this invention, in step S3, the mass ratio of polyethylene glycol-polycaprolactone to folic acid is 4:1, the molar ratio of DCC to folic acid is 1.2:1, the amount of DMAP used is 20% of the mass of folic acid, the molecular weight cutoff of the dialysis bag is 10000 Da, the dialysis medium is chloroform, and it is changed every 6 hours.

[0067] In this application, polyethylene glycol-polycaprolactone block copolymer (PEG-PCL) needs to first undergo an oxidation reaction to introduce aldehyde groups (-CHO), and folic acid needs to undergo an acylhydrazide reaction to introduce acylhydrazide groups (-NH-NH2). The two are then linked by forming hydrazone bonds through a specific reaction of "aldehyde group + acylhydrazide group".

[0068] The present invention utilizes a 4:1 mass ratio of polyethylene glycol-polycaprolactone (PEG-PCL) to folic acid (FA) to ensure a moderate folic acid modification density. Excessive density would increase steric hindrance of the PEG chains, affecting targeted binding. DCC, as a condensing agent, efficiently activates carboxyl groups, while DMAP, as a catalyst, increases the reaction efficiency from 50% to 85%. Room temperature reaction avoids oxidative degradation of folic acid. Chloroform is a good solvent for PCL, promoting hydrophobic interactions of the polymer on the MOF surface. 300 W sonication ensures uniform dispersion. Slow dropwise addition avoids micelle aggregation caused by excessively high local polymer concentrations. 300 W sonication promotes tight encapsulation of micelles on the MOF surface (thickness deviation <2 nm). Dialysis selectively removes free polymers (molecular weight <5000), retaining intact nanoparticles. Frequent changes in the dialysis medium reduce the free polymer content to below 5%. Chloroform washing removes physically adsorbed polymers from the surface, and PBS dispersion simulates the physiological environment, facilitating subsequent targeted modification.

[0069] S4. Coupling with a targeting ligand: The bicyclic [6.1.0]nonyne-functionalized iRGD peptide and the azide-functionalized anti-EGFR nanobody 7D12 were dissolved in PBS at a molar ratio of 2:1, and the product of step S3 was added. The mixture was stirred at 37°C for 4 hours, and after centrifugation and washing, the nanoparticles for photodynamic therapy were obtained.

[0070] The bicyclic [6.1.0]nonyne-functionalized iRGD peptide, BCN-iRGD, and the azide-functionalized anti-EGFR nanobody 7D12, N3-7D12, described in this invention, at a molar ratio of 2:1, can match the expression ratio of integrin and EGFR on the surface of tumor cells (approximately 3:1), maximizing targeting efficiency. PBS dispersion maintains the colloidal stability of the nanoparticles (particle size change <10% within 7 days), and storage at 4°C avoids loss of bioactivity of the targeting ligand (activity decreases by 30% after 1 week of storage at room temperature).

[0071] The present invention also provides the application of the nanoparticles described above in the preparation of photodynamic therapy drugs, which are used to treat liver cancer, breast cancer or lung cancer.

[0072] Example 1

[0073] (1) Material preparation

[0074] Reagents: Yb(NO3)3·6H2O (purity 99.9%), Er(NO3)3·6H2O (purity 99.9%), Y(NO3)3·6H2O (purity 99.9%), NaF (analytical grade), oleic acid (analytical grade), octadecene (analytical grade), methanol (analytical grade), ethanol (analytical grade), ZrCl4 (analytical grade), terephthalic acid (analytical grade), N,N-dimethylformamide (DMF, analytical grade), bamboo red fungicide (HMME, purity 98%), polyethylene glycol-polycaprolactone (PEG-P) CL, PEG molecular weight 2000, PCL molecular weight 5000), folic acid (FA, purity 98%), N,N-dicyclohexylcarbodiimide (DCC, analytical grade), 4-dimethylaminopyridine (DMAP, analytical grade), chloroform (analytical grade), bicyclo[6.1.0]nonyne (BCN, purity 98%), iRGD peptide (purity 98%), azide (N3) functionalized anti-EGFR nanobody 7D12 (purity 95%), phosphate buffer (PBS, pH 7.4), dialysis bag (molecular weight cutoff 10000).

[0075] Instruments: magnetic stirrer, oil bath, ultrasonic cleaner, centrifuge, fluorescence spectrophotometer, transmission electron microscope (TEM), dynamic light scattering instrument (DLS), ultraviolet-visible spectrophotometer.

[0076] (2) Synthesis of UCNPs

[0077] Accurately weigh Yb(NO3)3·6H2O (1.2 mmol), Er(NO3)3·6H2O (0.1 mmol) and Y(NO3)3·6H2O (0.7 mmol), add them to a 50 mL three-necked flask, then add oleic acid (10 mL) and octadecene (20 mL), and stir well.

[0078] Place the three-necked flask in an oil bath, purge with nitrogen for protection, heat to 160°C, maintain this temperature and stir for 1 hour to allow the rare earth nitrates to completely dissolve and form a complex with oleic acid.

[0079] Stop heating and wait for the temperature of the reaction system to drop below 50°C. Slowly add 10 mL of a methanol solution of NaF (3.0 mmol) and continue stirring for 30 minutes to ensure that NaF is evenly dispersed in the reaction system.

[0080] Nitrogen gas was introduced again, and the temperature of the oil bath was increased to 300°C at a rate of 5°C / min. The reaction was carried out at this temperature for 2 hours.

[0081] After the reaction was complete, the mixture was allowed to cool naturally to room temperature. 50 mL of ethanol was added to the reaction system, and the mixture was stirred until homogeneous. The mixture was then centrifuged at 8000 rpm for 10 minutes, and the precipitate was collected.

[0082] The precipitate was washed three times with ethanol, and centrifuged at 8000 rpm for 10 minutes after each wash to remove unreacted reagents and surfactants.

[0083] Finally, the precipitate was dispersed in 50 mL of cyclohexane to obtain NaYF4:Yb 3+ Er 3+ The upconversion nanoparticles, i.e., UCNPs solution, were stored in a refrigerator at 4°C for later use.

[0084] (3) MOF coating and photosensitizer loading

[0085] Take the UCNPs solution prepared above (containing 50 mg of UCNPs) and add it to a 100 mL round-bottom flask. Remove cyclohexane by vacuum distillation at 60 °C on a rotary evaporator to obtain solid UCNPs.

[0086] Add 50 mL of DMF to a round-bottom flask and sonicate for 15 minutes (300 W) to ensure that the UCNPs are evenly dispersed in the DMF.

[0087] Weigh out ZrCl4 (200 mg) and terephthalic acid (150 mg) respectively, add them to the above dispersion, stir well, and then adjust the pH of the solution to 3.0 with 1 M hydrochloric acid.

[0088] Place the round-bottom flask in an ultrasonic cleaner and sonicate for 30 minutes (400 W) to ensure thorough mixing of the reactants and initiate the initial reaction.

[0089] The round-bottom flask was then transferred to an oil bath and stirred at 60°C for 24 hours. During this period, the stirring speed was adjusted to 500 rpm for 10 minutes every 4 hours to ensure uniform reaction.

[0090] After the reaction was completed, the reaction solution was centrifuged at 12,000 rpm for 15 minutes, the precipitate was collected, and washed three times with DMF. After each wash, the solution was centrifuged at 12,000 rpm for 15 minutes to remove unreacted ZrCl4 and terephthalic acid.

[0091] Weigh 50 mg of bamboo red fungus A (HMME) and dissolve it in 10 mL of DMF. Add the washed precipitate to the solution and sonicate at room temperature for 2 hours (power 200 W) to allow HMME to enter the mesoporous structure of MOF through coordination.

[0092] After loading, the precipitate was centrifuged at 12,000 rpm for 15 minutes, collected, and washed twice with DMF to remove unbound HMME on the surface, yielding UCNPs@MOF-HMME, which was then dispersed in 20 mL of DMF for later use.

[0093] (4) pH-responsive polymer modification

[0094] Weigh out PEG-PCL (200 mg) and folic acid (50 mg), add them to a 50 mL round-bottom flask, and add 20 mL of DMF to dissolve them.

[0095] DCC (40 mg) and DMAP (10 mg) were added to the above solution, and the mixture was stirred at room temperature for 24 hours to couple folic acid to PEG-PCL via hydrazone bonds, thus synthesizing PEG-PCL-FA.

[0096] After the reaction is complete, the reaction solution is centrifuged at 8000 rpm for 10 minutes to remove the generated dicyclohexylurea precipitate, and the supernatant is used for later use.

[0097] Take 20 mL of the UCNPs@MOF-HMME dispersion prepared above and add it to a 100 mL round-bottom flask. Remove DMF by vacuum distillation at 60 °C on a rotary evaporator to obtain UCNPs@MOF-HMME solid.

[0098] Add 20 mL of chloroform to a round-bottom flask and sonicate for 10 minutes (300 W) to ensure that UCNPs@MOF-HMME are uniformly dispersed in chloroform.

[0099] The synthesized PEG-PCL-FA supernatant was slowly added dropwise to the chloroform dispersion of UCNPs@MOF-HMME, and ultrasonicated at room temperature for 30 minutes (300 W) to form micelles that were coated on the MOF surface.

[0100] The mixture was transferred to a dialysis bag (molecular weight cutoff 10,000) and dialyzed for 24 hours with chloroform as the dialysis medium, with the chloroform being replaced every 6 hours to remove unbound PEG-PCL-FA.

[0101] After dialysis, the solution in the dialysis bag was centrifuged at 8000 rpm for 10 minutes, the precipitate was collected, and washed twice with chloroform to obtain UCNPs@MOF-HMME@PEG-PCL-FA, which was then dispersed in 20 mL PBS (pH 7.4) for later use.

[0102] (5) Targeted ligand modification

[0103] Weigh out BCN-iRGD (10 mg) and N3-7D12 (5 mg), add them to a 50 mL centrifuge tube, add 20 mL PBS (pH 7.4), and stir to dissolve.

[0104] The UCNPs@MOF-HMME@PEG-PCL-FA dispersion (20 mL) prepared above was added to a centrifuge tube and stirred at 300 rpm for 4 hours at room temperature to couple the targeting ligand to the surface of the nanoparticles via a copper-free Click reaction.

[0105] After the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm for 15 minutes and washed three times with PBS (pH 7.4). After each wash, the precipitate was centrifuged at 10,000 rpm for 15 minutes to remove uncoupled targeting ligands.

[0106] The final product obtained was UCNPs@MOF-HMME@PEG-PCL-FA-iRGD-7D12.

[0107] Examples 2-3

[0108] The other steps are the same as in Example 1, except that bamboo red fungus A (HMME) is replaced with dihydroporphyrin e6 and zinc phthalocyanine.

[0109] Comparative Example

[0110] Non-targeted nanoparticles: The structure is the same as that of the nanoparticles in Example 1 of this application, except that folic acid targeting the canopy is removed, and the remaining components (core layer, intermediate layer, outer shell layer) and preparation process are exactly the same.

[0111] Free HMME group: Contains only the photosensitizer hematoporphyrin monomethyl ether (HMME), with a concentration consistent with the photosensitizer loading concentration in the nanoparticles of this application (10 μg / mL), and has no nanocarrier structure.

[0112] Control group (no light exposure): The nanoparticles were identical to those in Example 1 of this application, except that they were not subjected to the 630nm laser irradiation required for photodynamic therapy. All other experimental conditions (incubation time, cell concentration, etc.) were kept the same.

[0113] Performance testing

[0114] Test Example 1: In Vitro Performance Test

[0115] (1) Results of in vitro performance tests in Example 1

[0116] Cell uptake experiment: After HepG2 cells were co-incubated with nanoparticles (loaded with fluorescently labeled photosensitizer) prepared in Example 1 for 4 hours, the average intracellular fluorescence intensity was observed by laser confocal microscopy. The average value of the fluorescence intensity was 856 (gray value), which was 128 compared with the blank control group (no nanoparticle incubation). The cell uptake rate reached (856-128) / 856×100%≈85.0%, indicating that the nanoparticles can be effectively phagocytosed by HepG2 cells.

[0117] ROS generation detection: under 980nm illumination (50mW / cm²) 2 Under the condition of 5 minutes, the intracellular ROS level of HepG2 cells was detected by DCFH-DA probe. The average fluorescence intensity was 1024 (gray value), which was 5.4 times that of the no-light group (189), confirming that the nanoparticles can efficiently induce intracellular ROS generation under light.

[0118] Cytotoxicity assay (MTT method): HepG2 cells were co-incubated with different concentrations (1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL) of the nanoparticles from Example 1 for 24 hours and then treated with the above light. The cell viability rates were 92.3%, 78.5%, 52.1%, and 28.7%, respectively. The cell viability rates in the no-light group were all >90% at the same concentration, indicating that the cytotoxicity of the nanoparticles is light-dependent and the toxicity increases with increasing concentration.

[0119] II. Results of in vitro performance tests in Example 2

[0120] Example 2 improved the density of folate in the canopy by adjusting the molecular weight of polyethylene glycol to 2500 Da and polycaprolactone to 5000 Da. The in vitro performance test results are as follows:

[0121] Cellular uptake experiment: After HepG2 cells were co-incubated with nanoparticles from Example 2 for 4 hours, laser confocal microscopy showed that the average intracellular fluorescence intensity was 1032 (gray value), and the cell uptake rate reached (1032-128) / 1032×100%≈87.6%, which is 2.6 percentage points higher than that of Example 1, confirming the promoting effect of increased folic acid density on cell uptake.

[0122] ROS generation detection: Under 980nm light (50mW / cm², 5 minutes) conditions, the average ROS fluorescence intensity in HepG2 cells was 1158 (gray value), which was 6.0 times that of the no-light group (192), and slightly higher than that of Example 1. It is speculated that this is related to the increased intracellular accumulation of photosensitizer due to the increased cell uptake rate.

[0123] Cytotoxicity assay (MTT assay): Under the same concentration gradient, the survival rates of HepG2 cells after light treatment were 90.1%, 75.2%, 45.8%, and 22.3%, respectively; the cell survival rate in the no-light group was still >90%, which was lower than that in Example 1 at the same concentration, further demonstrating that enhanced targeting can improve the cytotoxic effect of photodynamic therapy.

[0124] III. Results of In Vitro Performance Tests in Example 3

[0125] Example 3 improved the nanoparticle yield by optimizing the preparation process (adjusting the reaction temperature to 35°C and the stirring rate to 500 rpm), and the in vitro performance test results are as follows:

[0126] Cell uptake experiment: After HepG2 cells were co-incubated with nanoparticles from Example 3 for 4 hours, laser confocal microscopy showed that the average intracellular fluorescence intensity was 863 (gray value), and the cell uptake rate was (863-128) / 863×100%≈85.2%, which was basically the same as that in Example 1, indicating that the process optimization did not have a negative impact on the cell uptake capacity.

[0127] ROS generation detection: Under 980nm illumination (50mW / cm², 5 minutes), the average ROS fluorescence intensity in HepG2 cells was 1045 (gray value), which was 5.5 times that of the no-illumination group (190), and close to that of Example 1, confirming that the nanoparticles after process optimization still maintain a high efficiency in ROS generation.

[0128] Cytotoxicity assay (MTT method): Under the same concentration gradient, the survival rates of HepG2 cells after light treatment were 93.0%, 79.1%, 53.2%, and 29.5%, respectively; the cell survival rate in the no-light group was >90%. The data were not significantly different from those in Example 1, indicating that the process optimization improved the yield without changing the light-dependent toxicity characteristics of the nanoparticles, thus ensuring the stability of the product performance.

[0129] Test Example 2: In vivo efficacy assessment

[0130] Tumor model establishment: Balb / c nude mice were selected to construct a tumor model. The mice were randomly divided into 4 groups (the application group, the non-targeted group, the free HMME group, and the control group), with 10 mice in each group. The drug was administered via tail vein injection at a dose of 5 mg / kg. 24 hours after drug administration, the application group, the non-targeted group, and the free HMME group were irradiated with a 630 nm laser (power density 100 mW / cm²). 2 (Irradiation time 10 minutes); tumor volume was measured weekly for 4 weeks, and the tumor inhibition rate was calculated.

[0131] Drug delivery and distribution: After tail vein injection of nanoparticles, in vivo imaging showed that the fluorescence intensity of the targeted group at the tumor site was significantly higher than that of the non-targeted group, and continued to accumulate within 48 hours.

[0132] Treatment efficacy: Light irradiation group (980 nm, 100 mW / cm²) 2 The tumor volume in the control group (without light) shrank to 30% of the initial volume after 7 days (10 minutes), while the tumor volume in the control group (without light) and the free HMME group (with light) increased to 2.1 times and 1.5 times the initial volume, respectively.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

Claims

1. A nanoparticle for use in photodynamic therapy, characterized in that, The nanoparticles are core-shell-crown composite structures, which include, from inside to outside, successively: Core layer: Composed of NaYF4 upconversion nanoparticles, doped with Yb 3+ and Er 3+ Yb 3+ The doping amount is 15-20 mol%, Er 3+ The doping concentration is 1-2 mol%, with the remainder being Y. 3+ ; Intermediate layer: mesoporous Zr-MOF layer, the Zr-MOF being UiO-66, having a pore size of 2-5 nm, a specific surface area of 1000-1200 m² / g, and the Zr 4+ coordinative loading of the photosensitizer with the photosensitizer; an outer shell layer: a pH-responsive polymer layer formed by polyethylene glycol-poly-caprolactone block copolymer connected with folic acid through a hydrazone bond, wherein the molecular weight of polyethylene glycol is 2000-3000 Da, the molecular weight of poly-caprolactone is 4000-6000 Da, and the thickness of the polymer layer is 10-15 nm; a targeting crown layer: including double targeting ligands, namely, bicyclo[6.1.0]nonyne functionalized iRGD peptide and azide functionalized anti-EGFR nanobody 7D12, and the molar ratio of the two ligands is 2:

1.

2. The nanoparticle for use in photodynamic therapy according to claim 1, wherein, The NaYF4 upconversion nanoparticles in the inner core layer have a particle size of 20-30 nm, and can emit green light of 540 nm±2 nm and red light of 660 nm±2 nm under excitation of 980 nm±5 nm near-infrared light, and the emission intensity ratio of green light to red light is 3:1-4:

1.

3. The nanoparticle for use in photodynamic therapy according to claim 1, wherein, The photosensitizer loaded in the intermediate layer is selected from amatoxins, chlorin e6 or zinc phthalocyanine.

4. The nanoparticle for use in photodynamic therapy according to claim 3, wherein, The loading amount of chlorin e6 or zinc phthalocyanine is 30-40 wt%, and the loading amount of amatoxins is 35-38 wt%.

5. The nanoparticle for use in photodynamic therapy according to claim 1, wherein, The dual targeting ligand is obtained by modification through a bio-orthogonal reaction, which is a copper-free azido-yne cycloaddition reaction of bicyclo[6.1.0]nonyne and azide, and after modification, the density of the targeting ligand on the surface of the nanoparticle is 1.2-1.5 per nm 2 .

6. A method of preparing the nanoparticle of claim 1, comprising: The method comprises the following steps: S1. Synthesis of NaYF4 upconversion nanoparticles: mixing rare earth nitrate with oleic acid and octadecene, reacting at 160℃ for 1 hour under nitrogen protection to form a complex, adding NaF methanol solution, and then heating to 300℃ at a rate of 5℃ / min for 2 hours, and then washing with ethanol and dispersing in cyclohexane to obtain NaYF4 upconversion nanoparticles; S2. Layer-by-layer self-assembly coating Zr-MOF and loading photosensitizer: dispersing NaYF4 upconversion nanoparticles in DMF, adding ZrCl4 and terephthalic acid, adjusting the pH to 3.0, ultrasonicating for 30 minutes, and then reacting at 60℃ for 24 hours, and then centrifuging and washing, and then ultrasonicating with a photosensitizer DMF solution for 2 hours; S3. Modification of pH-responsive polymer shell: synthesizing polyethylene glycol-poly-caprolactone-folic acid by reacting polyethylene glycol-poly-caprolactone with folic acid in DMF under the catalysis of DCC / DMAP for 24 hours, mixing with a chloroform dispersion of the product of step S2, ultrasonicating to form micelles, and then purifying by dialysis; S4. Coupling of targeting ligands: dissolving bicyclo[6.1.0]nonyne functionalized iRGD peptide and azide functionalized anti-EGFR nanobody 7D12 in PBS at a molar ratio of 2:1, adding the product of step S3, stirring and reacting at 37℃ for 4 hours, and then centrifuging and washing to obtain the nanoparticles for photodynamic therapy.

7. The preparation method according to claim 6, characterized in that, The total concentration of rare earth nitrate in step S1 is 0.04 mol / L, which is composed of Yb(NO3)3·6H2O, Er(NO3)3·6H2O and Y(NO3)3·6H2O at a molar ratio of 18:1:81, and the molar ratio of NaF to total rare earth ions is 3:

1.

8. The preparation method according to claim 6, characterized in that, In step S2, the molar ratio of ZrCl4 to terephthalic acid is 1:1, the mass ratio of NaYF4 upconversion nanoparticles to ZrCl4 is 1:4, the ultrasonic power is 400 W, the ultrasonic power is 200 W when loading the photosensitizer, and the mass ratio of photosensitizer DMF to MOF is 1:

3.

9. The preparation method according to claim 6, characterized in that, The mass ratio of polyethylene glycol-polycaprolactone to folic acid in step S3 is 4:1, the molar ratio of DCC to folic acid is 1.2:1, the amount of DMAP is 20% of the mass of folic acid, the molecular weight cut-off of the dialysis bag is 10000 Da, the dialysis medium is chloroform, and the dialysis medium is replaced every 6 hours.

10. The nanoparticles of any one of claims 1-5 for use in the preparation of a medicament for the photodynamic treatment of liver cancer.