A targeted nano-photosensitizer carrier, and a preparation method and application thereof

Abstract

The application discloses a kind of targeted nano photosensitizer carrier and its preparation method and application, it is related to photosensitizer carrier technical field, the preparation method of the targeted nano photosensitizer carrier is as follows: after 4-carboxyl phenylboronic acid is activated, drop into the methanol solution of polyethyleneimine, phenylboronic acid modified polyethyleneimine is obtained by reaction;It is dissolved in phosphate buffer solution, drop into the methanol solution of 1,2,4-trihydroxy anthraquinone, and avoid light reaction to obtain anthraquinone-boronate modified polyethyleneimine;It is activated and reacted with folic acid solution, and is obtained;The targeted nano photosensitizer carrier prepared can stably load photosensitizer containing aromatic ring structure, realizes light-controlled release and pH response, can also specifically recognize tumor cells with high expression of folic acid receptor, and has wide application prospect in the field of photodynamic therapy.

Description

Technical Field

[0001] This invention relates to the field of photosensitizer carrier technology, specifically to a targeted nano photosensitizer carrier, its preparation method, and its application. Background Technology

[0002] Photodynamic therapy (PDT) is a novel cancer treatment method. Its basic principle is to achieve therapeutic effects through the synergistic effect of photosensitizers, oxygen, and light of specific wavelengths. Currently, clinically used photosensitizers mainly include hydrophobic photosensitizers containing large π-conjugated structures, such as porphyrins, phthalocyanines, and chlorophyll derivatives. These photosensitizers exert their effects primarily through two types of photodynamic reactions: Type I reactions involve the transfer of hydrogen atoms or electrons between the triplet excited-state photosensitizer and the substrate, generating free radicals or free radical ions. These products interact with activated oxygen in the tissue to produce cytotoxic oxides. Type II reactions involve the triplet excited-state photosensitizer directly transferring energy to oxygen molecules, forming highly oxidizing singlet oxygen, thereby leading to cancer cell death.

[0003] PDT, as an emerging treatment method, possesses significant advantages such as dual selectivity (due to differences in tissue distribution of photosensitizers and spatial limitations of light exposure), good tissue selectivity, controllable depth of action, strong effect on microvascular tissue damage, minimal systemic side effects, and flexible combination with other treatment modalities. With in-depth research, photosensitizers have undergone several generations of development. First-generation photosensitizers, represented by hematoporphyrin derivatives (HpD), suffer from limitations such as complex composition, unclear structure, unsatisfactory spectral performance, poor tissue penetration, and slow in vivo clearance. Second-generation photosensitizers, such as porphyrins and phthalocyanines, have shown significant improvements in terms of structural clarity, component homogeneity, and absorption wavelength.

[0004] However, these hydrophobic photosensitizers still face significant challenges in clinical applications. Their strong hydrophobicity and poor water solubility result in low bioavailability in vivo. Furthermore, the lack of effective targeted delivery systems makes selective enrichment at tumor sites difficult, and the photosensitizer molecules have a long distribution time in vivo, requiring 3–96 hours to reach maximum concentration. This not only affects treatment efficiency but also increases the risk of phototoxic reactions in normal tissues due to non-specific distribution. To address these issues, researchers have attempted to develop various nanocarrier systems, among which polymer nanocarriers have attracted widespread attention due to their good biocompatibility and modifiability.

[0005] CN113332262A discloses a tumor-targeting nanomedicine carrier that is easily taken up by cells, its preparation method, and its application. PFN nanoparticles are obtained by copolymerizing polymeric dopamine nanoparticles with folic acid molecules. These PFN nanoparticles have a particle size of approximately 150–170 nm, with a uniform and stable particle size distribution, which facilitates cellular uptake and improves drug bioavailability and efficacy. These PFN nanoparticles can be used to load the antiviral drug chloroquine phosphate and the antitumor drug zinc phthalocyanine photosensitizer, exhibiting good loading capacity for both, thus meeting the basic requirements of a nanomedicine carrier.

[0006] However, existing polymer nanocarriers are still not well-designed in terms of structure, making it difficult to achieve both efficient loading and controllable release at the same time; they also lack multiple responsiveness, making it impossible to achieve intelligent delivery based on the characteristics of the tumor microenvironment; at the same time, due to the single targeting modification strategy, the active targeting effect is not ideal, and the preparation process is complex and has poor reproducibility, which is not conducive to industrial application.

[0007] Therefore, there is an urgent need to develop a novel nano-photosensitizer carrier system. This carrier should effectively increase the water solubility of hydrophobic photosensitizers, achieve efficient loading of photosensitizers through rational structural design, possess multiple targeting functions, and ensure a simple and controllable preparation method. This has significant clinical application value for improving the therapeutic effect of phototherapy (PDT) and reducing side effects. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a targeted nanophotosensitizer carrier, its preparation method, and its application. The prepared targeted nanophotosensitizer carrier can stably load photosensitizers containing aromatic ring structures, achieving light-controlled release and pH response. It can also specifically recognize tumor cells with high folic acid receptor expression, and has broad application prospects in the field of photodynamic therapy.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A method for preparing a targeted photosensitizer carrier includes the following steps:

[0011] (1) Add 4-carboxyphenylboronic acid, EDC and NHS to anhydrous methanol, stir and mix to obtain 4-carboxyphenylboronic acid activation solution, add dropwise to the methanol solution of polyethyleneimine, stir and react, precipitate the product, wash, dialyze and freeze dry to obtain phenylboronic acid modified polyethyleneimine.

[0012] Carboxyl activation-amidation reaction of PEI modified with phenylboronic acid group: The carboxyl group in 4-carboxyphenylboronic acid first reacts with EDC to generate an unstable O-acyl isourea intermediate. This intermediate rapidly undergoes acyl transfer with NHS to obtain an active ester intermediate. The active ester intermediate then undergoes nucleophilic addition-elimination reaction with the primary amine (-NH2) and secondary amine (-NH-) groups on the PEI backbone to achieve covalent modification of the phenylboronic acid group on the PEI molecular chain by forming a stable amide bond. At the same time, the integrity of the boric acid group (-B(OH)2) is maintained during the reaction, providing active sites for subsequent reactions.

[0013] Preferably, in step (1), the stirring and mixing conditions are: stirring and mixing in the dark at room temperature for 30 to 60 minutes; and the stirring and reaction conditions are: stirring and reaction at room temperature for 12 to 24 hours.

[0014] Preferably, in step (1), the molecular weight of the polyethyleneimine is MW = 1500-2000.

[0015] PEI with a molecular weight of 1500-2000 was selected as the backbone. This molecular weight range ensures good water solubility and biocompatibility of the carrier, avoids cytotoxicity caused by excessively high molecular weight, and provides an appropriate amount of amino groups for modification.

[0016] Preferably, in step (1), the ratio of 4-carboxyphenylboronic acid, EDC, NHS, and anhydrous methanol is 150-200 mg: 230-280 mg: 140-170 mg: 8-15 mL; the ratio of polyethyleneimine to the methanol solution of polyethyleneimine is 180-220 mg: 5-10 mL; and the ratio of 4-carboxyphenylboronic acid to polyethyleneimine is 150-200 mg: 180-220 mg.

[0017] In step (1), the product was added dropwise to icy diethyl ether for precipitation, centrifuged, washed three times with icy diethyl ether, and the precipitate was dissolved in ultrapure water. Then, it was transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed for 48–72 h, with the dialysate being replaced every 12 h. After dialysis, the solution was freeze-dried for 48–72 h to obtain a white viscous phenylboronic acid-modified polyethyleneimine, which was sealed, protected from light, and stored at 4 °C.

[0018] (2) The phenylboronic acid-modified polyethyleneimine was dissolved in phosphate buffer, molecular sieve was added, and 1,2,4-trihydroxyanthraquinone methanol solution was added dropwise under stirring. The reaction was carried out under light-protected stirring. The product was filtered, dialyzed, and freeze-dried to obtain anthraquinone-boronic acid ester-modified polyethyleneimine.

[0019] Dynamic covalent condensation reaction of boric acid and anthraquinone: The boric acid group on the phenylboronic acid-modified polyethyleneimine molecular chain undergoes a reversible dehydration condensation reaction with the 1,2-1-diol structure of 1,2,4-trihydroxyanthraquinone to form a five-membered ring borate ester bond with a planar configuration. During this process, the 4-hydroxyl group remains in a free state due to steric hindrance and electronic effects. The presence of molecular sieves promotes the shift of the reaction equilibrium towards the product by absorbing water molecules generated in the reaction system, thereby improving the formation efficiency of the borate ester bond.

[0020] Preferably, in step (2), the pH of the phosphate buffer solution is 8.0–8.5, and the molecular sieve is…

[0021] Preferably, in step (2), the ratio of phenylboronic acid-modified polyethyleneimine, phosphate buffer, and molecular sieve is 100-150 mg: 15-20 mL: 1-2 g; the ratio of phenylboronic acid-modified polyethyleneimine to 1,2,4-trihydroxyanthraquinone is 100-150 mg: 80-100 mg; and the ratio of 1,2,4-trihydroxyanthraquinone to a methanol solution of 1,2,4-trihydroxyanthraquinone is 80-100 mg: 5 mL.

[0022] Preferably, in step (2), the reaction conditions for stirring in the dark are 20-30°C and 300-500 r / min for 12-20 h, and the pH is checked every 4 h during the reaction to maintain the pH of the solution at 8.0-8.5.

[0023] In step (2), the product was filtered to remove the molecular sieve, and the filtrate was transferred to a dialysis bag (MWCO = 2000 Da). The dialysis was performed with phosphate buffer at pH 8.0 for 24 h and ultrapure water for 48 h. The dialysis solution was changed every 12 h. The solution was freeze-dried for 48-72 h to obtain anthraquinone-boron ester modified polyethyleneimine. The solution was sealed, protected from light, and stored at -20 °C.

[0024] (3) Add anthraquinone-boron ester modified polyethyleneimine and N,N'-carbonyldiimidazole to anhydrous DMF, purge with nitrogen, stir in the dark to obtain an activation solution, add folic acid solution dropwise to the activation solution, stir to react, precipitate the product, wash, dialyze, freeze dry to obtain the targeted nano photosensitizer carrier.

[0025] Activation and coupling reaction of folic acid targeting group: The free 4-hydroxyl group on the anthraquinone structure first undergoes a nucleophilic substitution reaction with CDI to generate a highly reactive carbamate intermediate; this intermediate then undergoes an esterification reaction with the carboxyl group in the folic acid molecule, covalently linking the folic acid molecule to the photosensitizer carrier by forming a stable ester bond. In this process, CDI plays a dual role as an activator and a dehydrating agent, ultimately constructing a targeted nano-photosensitizer carrier system.

[0026] Preferably, in step (3), the ratio of anthraquinone-boron ester modified polyethyleneimine, N,N'-carbonyldiimidazole, and anhydrous DMF in the activation solution is 100 mg: 15-20 mg: 20-30 mL.

[0027] Preferably, in step (3), the ratio of folic acid, HEPES buffer solution with pH = 7.0 to 8.2, and DMF in the folic acid solution is 50 to 60 mg: 3 to 5 mL: 25 to 35 mL.

[0028] Preferably, in step (3), the ratio of anthraquinone-boron ester modified polyethyleneimine to folic acid is 100 mg: 50-60 mg.

[0029] Preferably, in step (3), the stirring conditions are: stirring at 20-25°C for 2-4 hours in the dark; and the stirring reaction conditions are: stirring at 20-35°C for 12-24 hours in the dark.

[0030] In step (3), the product is added dropwise to ice-cold ether for precipitation, centrifuged, washed three times with ether and DMF respectively, transferred to a dialysis bag (MWCO = 3500 Da), and dialyzed with PBS buffer containing a small amount of mannitol and pH = 7.4 for 48-72 h, changing the dialysate every 12 h. The solution is then freeze-dried for 24-48 h to obtain the targeted nano photosensitizer carrier, which is then sealed, protected from light, and stored at -20°C.

[0031] This invention also claims a targeted photosensitizer carrier prepared using the aforementioned preparation method.

[0032] This invention also claims the use of the aforementioned targeted nanophotosensitizer carrier in the preparation of photodynamic therapy drugs.

[0033] Preferably, the photosensitizer is a hydrophobic photosensitizer containing a large π-conjugated structure, including porphyrins, phthalocyanines, and chlorophyll derivatives.

[0034] Preferably, the photosensitizer is erythropoietin imide.

[0035] Preferably, the method includes the following steps: adding the targeted nano photosensitizer carrier and the hydrophobic photosensitizer to an organic solvent, ultrasonically treating to obtain a mixture, adding the mixture dropwise to an aqueous solution of a surfactant, stirring to evaporate the organic solvent, centrifuging, washing, and drying to obtain the drug for photodynamic therapy.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. This invention provides a method for preparing a targeted photosensitizer carrier. First, 4-carboxyphenylboronic acid is covalently grafted onto the PEI molecular chain via an EDC / NHS activation system. Then, a dynamic covalent borate ester bond is formed between the borate group and the vicinal diol structure of 1,2,4-trihydroxyanthraquinone. Finally, folic acid molecules are coupled to the carrier surface via CDI activation. The synthetic route used in this invention is rationally designed, the reaction conditions are controllable, and the post-processing method is simple and efficient.

[0038] 2. The targeted nanophotosensitizer carrier provided by this invention has the following technical effects: ① The introduction of the PEI backbone not only provides abundant modification sites but also effectively improves the water solubility of hydrophobic photosensitizers, reduces their aggregation, and enhances the photodynamic therapy effect; ② The boric acid group can form a stable cyclic borate ester complex with sialic acid overexpressed on the surface of tumor cells, enhancing cellular uptake; the borate ester bond will hydrolyze in the slightly acidic environment of the tumor, achieving pH-responsive release; ③ The anthraquinone structure has a large π-conjugated system, which can form a stable complex with photosensitizers containing aromatic ring structures through π-π stacking interactions. Simultaneously, it can generate singlet oxygen under light conditions, causing structural breakage and achieving light-controlled release, realizing efficient loading and controllable release of the photosensitizer; ④ The folic acid ligand can specifically recognize the highly expressed folic acid receptors on the surface of tumor cells, forming a synergistic effect with the targeting effect of the boric acid group, significantly improving the targeting efficiency of the carrier system. The entire carrier system has a reasonable structural design, good biocompatibility, and achieves precise delivery to tumor tissues, demonstrating significant application advantages in the field of photodynamic therapy. Attached Figure Description

[0039] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some schematic diagrams of certain embodiments of the present invention, and therefore should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 The right image shows a solution of the targeted nanophotosensitizer carrier from Example 1, and the left image shows a solution of the photodynamic therapy drug prepared using the same.

[0041] Figure 2 The absorption curve (200-600 nm) of the photodynamic therapy drug prepared by the targeted nano photosensitizer carrier in Example 1 is shown.

[0042] Figure 3 The absorption curve (350-730 nm) of the photodynamic therapy drug prepared by the targeted nano photosensitizer carrier in Example 1 is shown.

[0043] Figure 4The drug release curves of the photodynamic therapy drug prepared by the targeted nano photosensitizer carrier in Example 1 are shown in vitro at different pH values ​​over time.

[0044] Figure 5 The bar chart shows the enrichment effect of photodynamic therapy drugs prepared by photosensitizer carriers in different cells in Example 1 and Comparative Example 1. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Of course, the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0046] Unless otherwise specified, all chemical reagents and materials in this invention are purchased from the market or synthesized from raw materials purchased from the market.

[0047] A method for preparing a targeted photosensitizer carrier includes the following steps:

[0048] (1) Add 150-200 mg of 4-carboxyphenylboronic acid, 230-280 mg of EDC, and 140-170 mg of NHS to 8-15 mL of anhydrous methanol. Stir and mix in the dark at room temperature for 30-60 min to obtain 4-carboxyphenylboronic acid activation solution. Add 5-10 mL of methanol solution of polyethyleneimine (polyethyleneimine content 180-220 mg, molecular weight MW = 1500-2000) dropwise. Stir and react at room temperature for 12-24 h. Add the product dropwise to ice-cold ether for precipitation. Centrifuge and wash three times with ice-cold ether. Dissolve the precipitate in ultrapure water and then transfer it to a dialysis bag (MWCO = 1000 Da) for dialysis for 48-72 h. Change the dialysis solution every 12 h. After dialysis, freeze-dry the solution for 48-72 h to obtain white viscous phenylboronic acid modified polyethyleneimine. Seal, protect from light, and store at 4 °C.

[0049] (2) Dissolve 100–150 mg of phenylboronic acid-modified polyethyleneimine in 15–20 mL of phosphate buffer (pH 8.0–8.5), and add... Molecular sieves were used to add 5 mL of a methanol solution of 1,2,4-trihydroxyanthraquinone (1,2,4-trihydroxyanthraquinone content 80-100 mg) dropwise under stirring. The mixture was stirred at 20-30℃ and 300-500 r / min for 12-20 h in the dark. The pH was checked every 4 h during the reaction to maintain the pH of the solution at 8.0-8.5. The product was filtered to remove the molecular sieves, and the filtrate was transferred to a dialysis bag (MWCO = 2000 Da). The solution was dialyzed with phosphate buffer (pH 8.0) for 24 h and then with ultrapure water for 48 h, with the dialysate being changed every 12 h. The solution was freeze-dried for 48-72 h to obtain anthraquinone-boron ester modified polyethyleneimine, which was then sealed, protected from light, and stored at -20℃.

[0050] (3) 100 mg of anthraquinone-boron ester modified polyethyleneimine and 15-20 mg of N,N'-carbonyldiimidazole were added to 20-30 mL of anhydrous DMF. Nitrogen gas was introduced and the mixture was stirred in the dark at 20-25 °C for 2-4 h to obtain an activation solution. Folic acid solution (50-60 mg of folic acid, 3-5 mL of HEPES buffer with pH = 7.0-8.2, and 25-35 mL of DMF) was added dropwise to the activation solution. The mixture was stirred in the dark at 20-35 °C for 12-24 h. The product was added dropwise to ice-cold ether for precipitation. After centrifugation, the product was washed three times with ether and DMF, respectively. The product was then transferred to a dialysis bag (MWCO = 3500 Da) and dialyzed with PBS buffer containing a small amount of mannitol with pH = 7.4 for 48-72 h. The dialysis solution was changed every 12 h. The solution was freeze-dried for 24-48 h to obtain the targeted photosensitizer carrier. The carrier was sealed, protected from light, and stored at -20 °C.

[0051] The application of a photosensitizer carrier in the preparation of photodynamic therapy drugs includes the following steps:

[0052] The targeted nano-photosensitizer carrier and hydrophobic photosensitizer were added to an organic solvent and ultrasonically treated to obtain a mixture. The mixture was then added dropwise to an aqueous solution of a surfactant. After stirring to evaporate the organic solvent, the mixture was centrifuged, washed, and dried to obtain the drug for photodynamic therapy.

[0053] The present invention will be further described below through specific embodiments.

[0054] Example 1

[0055] A method for preparing a targeted photosensitizer carrier includes the following steps:

[0056] (1) 200 mg of 4-carboxyphenylboronic acid, 280 mg of EDC, and 170 mg of NHS were added to 15 mL of anhydrous methanol and stirred for 60 min in the dark at room temperature to obtain an activated solution of 4-carboxyphenylboronic acid. The solution was then added dropwise to a methanol solution of polyethyleneimine (polyethyleneimine content 220 mg, molecular weight MW = 1800) and stirred for 24 h at room temperature. The product was then added dropwise to ice-cold ether for precipitation. The product was separated by centrifugation and washed three times with ice-cold ether. The precipitate was dissolved in ultrapure water and then transferred to a dialysis bag (MWCO = 1000 Da) for dialysis for 72 h. The dialysis solution was changed every 12 h. After dialysis, the solution was freeze-dried for 72 h to obtain a white viscous phenylboronic acid-modified polyethyleneimine. The solution was sealed, protected from light, and stored at 4 °C.

[0057] (2) Dissolve 150 mg of phenylboronic acid-modified polyethyleneimine in 20 mL of phosphate buffer (pH 8.5), and add... Molecular sieves were used to add 5 mL of a methanol solution of 1,2,4-trihydroxyanthraquinone (100 mg of 1,2,4-trihydroxyanthraquinone) dropwise with stirring. The mixture was stirred at 30 °C and 500 rpm for 20 h in the dark, with the pH checked every 4 h to maintain the pH at 8.5. The product was filtered to remove the molecular sieves, and the filtrate was transferred to a dialysis bag (MWCO = 2000 Da). The mixture was dialyzed with phosphate buffer (pH 8.0) for 24 h and then with ultrapure water for 48 h, with the dialysate being changed every 12 h. The solution was then freeze-dried for 72 h to obtain anthraquinone-boron ester modified polyethyleneimine, which was sealed, protected from light, and stored at -20 °C.

[0058] (3) 100 mg of anthraquinone-boron ester modified polyethyleneimine and 20 mg of N,N'-carbonyldiimidazole were added to 30 mL of anhydrous DMF, nitrogen gas was introduced, and the mixture was stirred at 25 °C in the dark for 4 h to obtain an activation solution. Folic acid solution (60 mg of folic acid, 5 mL of HEPES buffer with pH = 8.2 and 35 mL of DMF were mixed) was added dropwise to the activation solution, and the mixture was stirred at 35 °C in the dark for 24 h. The product was added dropwise to ice-cold ether for precipitation, centrifuged, washed 3 times with ether and DMF respectively, and transferred to a dialysis bag (MWCO = 3500 Da). The mixture was dialyzed with PBS buffer containing a small amount of mannitol with pH = 7.4 for 72 h, and the dialysate was changed every 12 h. The solution was freeze-dried for 48 h to obtain the targeted nano photosensitizer carrier, which was sealed, protected from light, and stored at -20 °C.

[0059] Example 2

[0060] A method for preparing a targeted photosensitizer carrier includes the following steps:

[0061] (1) 180 mg of 4-carboxyphenylboronic acid, 260 mg of EDC, and 160 mg of NHS were added to 12 mL of anhydrous methanol and stirred for 50 min in the dark at room temperature to obtain an activated solution of 4-carboxyphenylboronic acid. The solution was then added dropwise to 8 mL of a methanol solution of polyethyleneimine (polyethyleneimine content 200 mg, molecular weight MW = 2000). The solution was stirred for 20 h at room temperature. The product was then added dropwise to ice-cold ether for precipitation. The product was separated by centrifugation and washed three times with ice-cold ether. The precipitate was dissolved in ultrapure water and then transferred to a dialysis bag (MWCO = 1000 Da) for dialysis for 60 h. The dialysis solution was changed every 12 h. After dialysis, the solution was freeze-dried for 60 h to obtain a white viscous phenylboronic acid-modified polyethyleneimine. The solution was sealed, protected from light, and stored at 4 °C.

[0062] (2) Dissolve 140 mg of phenylboronic acid-modified polyethyleneimine in 16 mL of phosphate buffer (pH 8.5), and add... Molecular sieves were used to add 5 mL of a methanol solution of 1,2,4-trihydroxyanthraquinone (containing 90 mg of 1,2,4-trihydroxyanthraquinone) dropwise under stirring. The mixture was stirred at 26 °C and 400 r / min for 16 h in the dark, with the pH checked every 4 h to maintain the pH at 8.5. The product was filtered to remove the molecular sieves, and the filtrate was transferred to a dialysis bag (MWCO = 2000 Da). The mixture was dialyzed with phosphate buffer (pH 8.0) for 24 h and then with ultrapure water for 48 h, with the dialysate being changed every 12 h. The solution was then freeze-dried for 60 h to obtain anthraquinone-boron ester modified polyethyleneimine, which was then sealed, protected from light, and stored at -20 °C.

[0063] (3) 100 mg of anthraquinone-boron ester modified polyethyleneimine and 16 mg of N,N'-carbonyldiimidazole were added to 24 mL of anhydrous DMF, nitrogen gas was introduced, and the mixture was stirred at 22 °C in the dark for 3 h to obtain an activation solution. Folic acid solution (55 mg of folic acid, 4 mL of HEPES buffer with pH = 8.0 and 30 mL of DMF were mixed) was added dropwise to the activation solution, and the mixture was stirred at 30 °C in the dark for 20 h. The product was added dropwise to ice-cold ether for precipitation, centrifuged, washed 3 times with ether and DMF respectively, and transferred to a dialysis bag (MWCO = 3500 Da). The mixture was dialyzed with PBS buffer containing a small amount of mannitol with pH = 7.4 for 60 h, and the dialysate was changed every 12 h. The solution was freeze-dried for 36 h to obtain the targeted nano photosensitizer carrier, which was sealed, protected from light, and stored at -20 °C.

[0064] Example 3

[0065] A method for preparing a targeted photosensitizer carrier includes the following steps:

[0066] (1) 160 mg of 4-carboxyphenylboronic acid, 240 mg of EDC, and 150 mg of NHS were added to 10 mL of anhydrous methanol and stirred for 40 min in the dark at room temperature to obtain an activated solution of 4-carboxyphenylboronic acid. The solution was then added dropwise to a methanol solution of polyethyleneimine (polyethyleneimine content 190 mg, molecular weight MW = 1700) and stirred for 16 h at room temperature. The product was then added dropwise to ice-cold ether for precipitation. The product was separated by centrifugation and washed three times with ice-cold ether. The precipitate was dissolved in ultrapure water and then transferred to a dialysis bag (MWCO = 1000 Da) for dialysis for 60 h. The dialysis solution was changed every 12 h. After dialysis, the solution was freeze-dried for 60 h to obtain a white viscous phenylboronic acid-modified polyethyleneimine. The solution was sealed, protected from light, and stored at 4 °C.

[0067] (2) Dissolve 120 mg of phenylboronic acid-modified polyethyleneimine in 17 mL of phosphate buffer (pH 8.0), and add... Molecular sieves were used to add 5 mL of a methanol solution of 1,2,4-trihydroxyanthraquinone (containing 90 mg of 1,2,4-trihydroxyanthraquinone) dropwise under stirring. The mixture was stirred at 24 °C and 400 r / min for 16 h in the dark, with the pH checked every 4 h to maintain the pH at 8.0. The product was filtered to remove the molecular sieves, and the filtrate was transferred to a dialysis bag (MWCO = 2000 Da). The mixture was dialyzed with phosphate buffer (pH 8.0) for 24 h and then with ultrapure water for 48 h, with the dialysate being changed every 12 h. The solution was then freeze-dried for 48 h to obtain anthraquinone-boron ester modified polyethyleneimine, which was sealed, protected from light, and stored at -20 °C.

[0068] (3) 100 mg of anthraquinone-boron ester modified polyethyleneimine and 16 mg of N,N'-carbonyldiimidazole were added to 24 mL of anhydrous DMF, nitrogen gas was introduced, and the mixture was stirred at 22 °C in the dark for 3 h to obtain an activation solution. Folic acid solution (54 mg of folic acid, 4 mL of pH 8.0 HEPES buffer, and 30 mL of DMF were mixed) was added dropwise to the activation solution, and the mixture was stirred at 25 °C in the dark for 16 h. The product was added dropwise to ice-cold ether for precipitation, centrifuged, washed 3 times with ether and DMF respectively, and transferred to a dialysis bag (MWCO = 3500 Da). The mixture was dialyzed with PBS buffer containing a small amount of mannitol and pH = 7.4 for 60 h, and the dialysate was changed every 12 h. The solution was freeze-dried for 36 h to obtain the targeted nano photosensitizer carrier, which was sealed, protected from light, and stored at -20 °C.

[0069] Example 4

[0070] A method for preparing a targeted photosensitizer carrier includes the following steps:

[0071] (1) 150 mg of 4-carboxyphenylboronic acid, 230 mg of EDC, and 140 mg of NHS were added to 8 mL of anhydrous methanol and stirred for 30 min in the dark at room temperature to obtain an activated solution of 4-carboxyphenylboronic acid. The solution was then added dropwise to a methanol solution of polyethyleneimine (polyethyleneimine content 180 mg, molecular weight MW = 1500) and stirred for 12 h at room temperature. The product was then added dropwise to ice-cold ether for precipitation. The product was separated by centrifugation and washed three times with ice-cold ether. The precipitate was dissolved in ultrapure water and then transferred to a dialysis bag (MWCO = 1000 Da) for dialysis for 48 h. The dialysis solution was changed every 12 h. After dialysis, the solution was freeze-dried for 48 h to obtain a white viscous intermediate. The intermediate was sealed, protected from light, and stored at 4 °C.

[0072] (2) Dissolve 100 mg of phenylboronic acid-modified polyethyleneimine in 15 mL of phosphate buffer (pH 8.0), and add... Molecular sieves were used to add 5 mL of a methanol solution of 1,2,4-trihydroxyanthraquinone (1,2,4-trihydroxyanthraquinone content 80 mg) dropwise under stirring. The mixture was stirred at 20 °C and 300 r / min for 12 h in the dark, with the pH checked every 4 h to maintain the pH at 8.0. The product was filtered to remove the molecular sieves, and the filtrate was transferred to a dialysis bag (MWCO = 2000 Da). The mixture was dialyzed with phosphate buffer (pH 8.0) for 24 h and then with ultrapure water for 48 h, with the dialysate changed every 12 h. The solution was then freeze-dried for 48 h to obtain the photosensitizer carrier, which was then sealed, protected from light, and stored at -20 °C.

[0073] (3) 100 mg of anthraquinone-boron ester modified polyethyleneimine and 15 mg of N,N'-carbonyldiimidazole were added to 20 mL of anhydrous DMF, nitrogen gas was introduced, and the mixture was stirred at 20 °C in the dark for 2 h to obtain an activation solution. Folic acid solution (50 mg of folic acid, 3 mL of pH 7.5 HEPES buffer, and 25 mL of DMF were mixed) was added dropwise to the activation solution, and the mixture was stirred at 20 °C in the dark for 12 h. The product was added dropwise to ice-cold ether for precipitation, centrifuged, washed 3 times with ether and DMF respectively, and transferred to a dialysis bag (MWCO = 3500 Da). The mixture was dialyzed with PBS buffer containing a small amount of mannitol and pH = 7.4 for 48 h, and the dialysate was changed every 12 h. The solution was freeze-dried for 24 h to obtain the targeted nano photosensitizer carrier, which was sealed, protected from light, and stored at -20 °C.

[0074] Comparative Example 1

[0075] A method for preparing a nano-photosensitizer carrier includes the following steps:

[0076] (1) 200 mg of 4-carboxyphenylboronic acid, 280 mg of EDC, and 170 mg of NHS were added to 15 mL of anhydrous methanol and stirred for 60 min in the dark at room temperature to obtain an activated solution of 4-carboxyphenylboronic acid. The solution was then added dropwise to a methanol solution of polyethyleneimine (polyethyleneimine content 220 mg, molecular weight MW = 1800) and stirred for 24 h at room temperature. The product was then added dropwise to ice-cold ether for precipitation. The product was separated by centrifugation and washed three times with ice-cold ether. The precipitate was dissolved in ultrapure water and then transferred to a dialysis bag (MWCO = 1000 Da) for dialysis for 72 h. The dialysis solution was changed every 12 h. After dialysis, the solution was freeze-dried for 72 h to obtain a white viscous phenylboronic acid-modified polyethyleneimine. The solution was sealed, protected from light, and stored at 4 °C.

[0077] (2) Dissolve 150 mg of phenylboronic acid-modified polyethyleneimine in 20 mL of phosphate buffer (pH 8.5), and add... Molecular sieves were used to add 5 mL of a methanol solution of 1,2,4-trihydroxyanthraquinone (100 mg of 1,2,4-trihydroxyanthraquinone) dropwise under stirring. The mixture was stirred at 30 °C and 500 rpm for 20 h in the dark. The pH was checked every 4 h during the reaction to maintain the pH at 8.5. The product was filtered to remove the molecular sieves, and the filtrate was transferred to a dialysis bag (MWCO = 2000 Da). The solution was dialyzed with phosphate buffer (pH 8.0) for 24 h and then with ultrapure water for 48 h, with the dialysate being changed every 12 h. The solution was then freeze-dried for 72 h to obtain the nano-photosensitizer carrier, which was then sealed, protected from light, and stored at -20 °C.

[0078] Comparative Example 2

[0079] A method for preparing a nano-photosensitizer carrier includes the following steps:

[0080] 200 mg of 4-carboxyphenylboronic acid, 280 mg of EDC, and 170 mg of NHS were added to 15 mL of anhydrous methanol and stirred for 60 min in the dark at room temperature to obtain an activated 4-carboxyphenylboronic acid solution. This solution was then added dropwise to 10 mL of a methanol solution of polyethyleneimine (polyethyleneimine content 220 mg, molecular weight MW = 1800), and stirred at room temperature for 24 h. The product was then added dropwise to refractory ether for precipitation, centrifuged, washed three times with refractory ether, and the precipitate was dissolved in ultrapure water. The precipitate was then transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed for 72 h, with the dialysate changed every 12 h. After dialysis, the solution was freeze-dried for 72 h to obtain a white, viscous nano-photosensitizer carrier, which was sealed, protected from light, and stored at 4 °C.

[0081] Comparative Example 3

[0082] Polyethyleneimine with a molecular weight of MW=1800 was used as a nano-photosensitizer carrier.

[0083] The targeted photosensitizer nanocarriers obtained in Examples 1-4 and Comparative Examples 1-3 were used in the preparation of photodynamic therapy drugs, and the steps are as follows:

[0084] 40 mg of targeted photosensitizer carrier and 6 mg of erythropoietin-18-imide were added to a 20 mL mixture of dichloromethane and methanol (volume ratio 1:1). The mixture was sonicated for 120 s to obtain a solution. The solution was then added dropwise to 75 mL of 2 wt% PVA aqueous solution. After stirring in a 25 °C water bath for 8 h to evaporate the organic solvent, the solution was centrifuged at 5000 r / min, washed with deionized water, and repeated 3 times. The solution was then vacuum dried to obtain the photodynamic therapy drug.

[0085] Weigh an appropriate amount of the prepared photodynamic therapy drug, dissolve it in a mixed solvent of dichloromethane and methanol, and measure its absorption curve and maximum absorption wavelength. Figure 2 , 3 To obtain the absorption curve of the photodynamic therapy drug prepared from the targeted photosensitizer carrier in Example 1, mixed solutions of rutin-18-imide in dichloromethane and methanol at different concentrations were prepared. The absorbance was measured at the maximum absorption wavelength to obtain a standard curve of absorbance and solution concentration. Then, 1 mg of the photodynamic therapy drug was weighed and dissolved in 5 mL of a mixed organic solvent of dichloromethane and methanol (volume ratio 1:1). After complete dissolution, the absorbance at the same absorption wavelength as the standard curve was measured using a UV spectrophotometer. The concentration of rutin-18-imide was then determined by comparing with the standard curve, and the drug loading and encapsulation efficiency in the photodynamic therapy drug were calculated using the formula (specific data are shown in Table 1).

[0086] Drug loading = [Weight of photosensitizer in the drug / Weight of the drug] × 100%;

[0087] Encapsulation efficiency = [weight of photosensitizer in the drug / amount of drug administered] × 100%.

[0088] 5 mL of the photodynamic therapy drug solution (solvent: dichloromethane, methanol, volume ratio 1:1; drug concentration: 20 mg / mL) prepared in Example 1 was poured into a dialysis bag. The dialysis bag was then placed in flasks containing 20 mL of PBS buffer solution (pH 7.4, simulating normal in vivo tissue) and acetate buffer solution (pH 5.0, simulating the weakly acidic environment of tumor tissue), respectively. The dialysis bag and flasks were kept at 37°C with stirring. Small amounts of solution were taken from the flasks periodically for UV detection. After detection, the solution in the cuvette was poured back into the flasks to ensure a constant total volume. Experimental data were recorded at 2 h, 4 h, 8 h, and 12 h. A drug release curve of the targeted nanophotosensitizer carrier over time was plotted. The results are as follows: Figure 4As shown, the photosensitizer loaded on the targeted nanophotosensitizer carrier prepared by this invention is released faster in the acidic microenvironment of tumors than in normal tissues.

[0089] The concentration of the drugs for photodynamic therapy prepared in Examples 1-4 and Comparative Examples 1-3 was determined after 12 hours of in vitro release in an acetic acid buffer solution at pH 5.0. The specific data are shown in Table 1.

[0090] Table 1 Performance testing of drugs prepared from photosensitizer carriers for photodynamic therapy

[0091]

[0092] The photodynamic therapy drug prepared from the targeted nanophotosensitizer carrier of Example 1 was used on human lung adenocarcinoma epithelial cells (A549) and human oral epithelial cancer cells (KB cells) with high folate receptor expression. The photodynamic therapy drug prepared from the photosensitizer carrier of Comparative Example 1 was used as a control. Laser fluorescence confocal imaging was performed, and the results are shown in [Figure 1]. Figure 5 After 1 hour and 3 hours of culture, the photodynamic therapy drug in Example 1 showed more significant enrichment in KB cells, indicating that the targeted photosensitizer carrier provided by this invention has a targeted selection effect on tumor cells with high folic acid receptor expression.

[0093] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a targeted photosensitizer nanocarrier, characterized in that, Includes the following steps: (1) Add 4-carboxyphenylboronic acid, EDC and NHS to anhydrous methanol, stir and mix to obtain 4-carboxyphenylboronic acid activation solution, add dropwise to the methanol solution of polyethyleneimine, stir and react, precipitate the product, wash, dialyze and freeze dry to obtain phenylboronic acid modified polyethyleneimine. (2) The phenylboronic acid-modified polyethyleneimine was dissolved in phosphate buffer, molecular sieve was added, and 1,2,4-trihydroxyanthraquinone methanol solution was added dropwise under stirring. The reaction was carried out under light-protected stirring. The product was filtered, dialyzed, and freeze-dried to obtain anthraquinone-boronic acid ester-modified polyethyleneimine. (3) Add anthraquinone-boron ester modified polyethyleneimine and N,N'-carbonyldiimidazole to anhydrous DMF, purge with nitrogen, stir in the dark to obtain an activation solution, add folic acid solution dropwise to the activation solution, stir to react, precipitate the product, wash, dialyze, freeze dry to obtain the targeted nano photosensitizer carrier.

2. The preparation method according to claim 1, characterized in that, In step (1), the stirring and mixing conditions are: stirring and mixing in the dark at room temperature for 30 to 60 minutes; the stirring and reaction conditions are: stirring and reaction at room temperature for 12 to 24 hours; the molecular weight of the polyethyleneimine is MW = 1500 to 2000.

3. The preparation method according to claim 1, characterized in that, In step (1), the ratio of 4-carboxyphenylboronic acid, EDC, NHS, and anhydrous methanol is 150-200 mg: 230-280 mg: 140-170 mg: 8-15 mL; the ratio of polyethyleneimine to the methanol solution of polyethyleneimine is 180-220 mg: 5-10 mL; and the ratio of 4-carboxyphenylboronic acid to polyethyleneimine is 150-200 mg: 180-220 mg.

4. The preparation method according to claim 1, characterized in that, In step (2), the pH of the phosphate buffer solution is 8.0–8.5, and the molecular sieve is… The ratio of phenylboronic acid-modified polyethyleneimine, phosphate buffer, and molecular sieve is 100–150 mg: 15–20 mL: 1–2 g; the ratio of phenylboronic acid-modified polyethyleneimine to 1,2,4-trihydroxyanthraquinone is 100–150 mg: 80–100 mg; and the ratio of 1,2,4-trihydroxyanthraquinone to a methanol solution of 1,2,4-trihydroxyanthraquinone is 80–100 mg: 5 mL.

5. The preparation method according to claim 1, characterized in that, In step (2), the reaction conditions for stirring in the dark are 20-30℃ and 300-500r / min for 12-20h. During the reaction, the pH is checked every 4h to maintain the pH of the solution at 8.0-8.

5.

6. The preparation method according to claim 1, characterized in that, In step (3), the ratio of anthraquinone-boron ester modified polyethyleneimine, N,N'-carbonyldiimidazole, and anhydrous DMF in the activation solution is 100 mg: 15-20 mg: 20-30 mL; the ratio of folic acid, HEPES buffer at pH 7.0-8.2, and DMF in the folic acid solution is 50-60 mg: 3-5 mL: 25-35 mL; and the ratio of anthraquinone-boron ester modified polyethyleneimine and folic acid is 100 mg: 50-60 mg.

7. The preparation method according to claim 1, characterized in that, In step (3), the stirring conditions are: stirring at 20-25°C for 2-4 hours in the dark; and the stirring reaction conditions are: stirring at 20-35°C for 12-24 hours in the dark.

8. A targeted nanophotosensitizer carrier prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the targeted nanophotosensitizer carrier as described in claim 8 in the preparation of photodynamic therapy drugs.

10. The application according to claim 9, characterized in that, The photosensitizer is a hydrophobic photosensitizer containing a large π-conjugated structure, including porphyrins, phthalocyanines, and chlorophyll derivatives.

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