Targeted nano photosensitizer carrier as well as preparation method and application thereof

Abstract

The invention discloses a targeted nano photosensitizer carrier and a preparation method and application thereof, and relates to the technical field of photosensitizer carriers, the preparation method of the targeted nano photosensitizer carrier comprises the following steps: activating 4-carboxyphenylboronic acid, dropwise adding the activated 4-carboxyphenylboronic acid into a methanol solution of polyethyleneimine, and reacting to obtain phenylboronic acid modified polyethyleneimine; dissolving the polyethyleneimine into a phosphate buffer solution, dropwise adding a methanol solution of 1, 2, 4-trihydroxy anthraquinone, and reacting in a dark place to obtain anthraquinone-boric acid ester modified polyethyleneimine; and activating and reacting with a folic acid solution to obtain the folic acid. The prepared targeted nano photosensitizer carrier can stably load a photosensitizer containing an aromatic ring structure, realizes light-controlled release and pH response, can specifically recognize tumor cells with high folate receptor expression, and has a wide application prospect in the field of photodynamic therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of photosensitizer carriers, and in particular to a targeted nano photosensitizer carrier and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) is a new type of tumor treatment method. Its basic principle is to use the synergistic effect of photosensitizers, oxygen and light of specific wavelengths to achieve therapeutic effects. The photosensitizers currently used in clinical practice mainly include hydrophobic photosensitizers containing large π conjugated structures, such as porphyrins, phthalocyanines, and chlorophyll derivatives. This type of photosensitizer mainly works through two types of photodynamic reactions: Type I reactions involve the transfer of hydrogen atoms or electrons between triplet excited photosensitizers and substrates to generate free radicals or free radical ions, which interact with activated oxygen in tissues to produce cytotoxic oxides; Type II reactions are triplet excited photosensitizers that transfer energy directly to oxygen molecules to form singlet oxygen with strong oxidizing properties, thereby causing cancer cell death.

[0003] As an emerging treatment method, PDT has significant advantages such as dual selectivity (differences in tissue distribution of photosensitizers and spatial limitations of light), good tissue selectivity, controllable depth of action, strong damage to microvascular tissue, small systemic side effects, and flexible combination with other treatment methods. With the deepening of research, photosensitizers have gone through multiple generations of development. The first generation of photosensitizers is represented by hematoporphyrin derivatives (HpD), but they have limitations such as complex components, unclear structures, unsatisfactory spectral performance, poor tissue penetration, and slow clearance in the body. The second generation of photosensitizers, such as porphyrins and phthalocyanines, have made significant improvements in structural clarity, component uniformity, and absorption wavelength.

[0004] However, these hydrophobic photosensitizers still face serious challenges in clinical applications. Due to their strong hydrophobicity and poor water solubility, their bioavailability in the body is low; at the same time, due to the lack of an effective targeted delivery system, it is difficult to achieve selective enrichment at the tumor site. The photosensitizer molecules have a long distribution time in the body, and it takes 3 to 96 hours to reach the maximum enrichment concentration, which not only affects the treatment efficiency, but also increases the risk of phototoxic reactions in normal tissues due to nonspecific distribution. To solve these problems, researchers have tried to develop various nanocarrier systems, among which polymer nanocarriers have received widespread attention due to their good biocompatibility and modifiability.

[0005] CN113332262A provides a tumor-targeted nano drug carrier that is easily taken up by cells, and its preparation method and application. PFNs nano particles are obtained by copolymerizing polymerized dopamine nano particles with folic acid molecules. The particle size of the PFNs nano particles is about 150-170nm, and the particle size distribution is uniform and stable, which is conducive to cell uptake, thereby improving the bioavailability and effectiveness of the drug. The PFNs nano particles can be used to load the antiviral drug chloroquine phosphate and the antitumor drug phthalocyanine zinc photosensitizer, and have good loading capacity for both, which can meet the basic requirements of nano drug carriers.

[0006] However, the existing polymer nanocarriers are still not reasonable in terms of structural design, making it difficult to achieve efficient loading and controlled release at the same time; they also lack multiple responsiveness and cannot achieve intelligent delivery based on the characteristics of the tumor microenvironment; at the same time, due to the single targeted modification strategy, the active targeting effect is not ideal, and the preparation process is complex and has poor repeatability, which is not conducive to industrial application.

[0007] Therefore, it is urgent to develop a new type of nanophotosensitizer carrier system, which should be able to effectively increase the water solubility of hydrophobic photosensitizers, achieve efficient loading of photosensitizers through reasonable structural design, and have multiple targeting functions, while ensuring that the preparation method is simple and controllable. This has important clinical application value for improving the PDT treatment effect and reducing side effects. Summary of the invention

[0008] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a targeted nano photosensitizer carrier and a preparation method and application thereof. The prepared targeted nano photosensitizer carrier can stably load photosensitizers containing aromatic ring structures, achieve light-controlled release and pH response, and can also specifically identify tumor cells with high expression of folate receptors, and has broad application prospects in the field of photodynamic therapy.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

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

[0011] (1) adding 4-carboxyphenylboronic acid, EDC, and NHS to anhydrous methanol, stirring and mixing to obtain a 4-carboxyphenylboronic acid activated solution, adding dropwise to a methanol solution of polyethyleneimine, stirring for reaction, precipitating the product, washing, dialyzing, and freeze-drying to obtain polyethyleneimine modified with phenylboronic acid;

[0012] Carboxyl activation-amidation reaction of PEI modified by phenylboronic acid group: the carboxyl group in 4-carboxyphenylboronic acid first reacts with EDC to form an unstable O-acylisourea intermediate, which 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, while maintaining the integrity of the boronic acid group (-B(OH)2) during the reaction to provide an active site 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 reaction conditions are stirring and reacting 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 to 2000 is selected as the backbone. This molecular weight range not only ensures good water solubility and biocompatibility of the carrier, but also avoids cytotoxicity caused by too high molecular weight, and at the same time provides an appropriate amount of amino groups for modification.

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

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

[0018] (2) dissolving the phenylboronic acid-modified polyethyleneimine in a phosphate buffer, adding molecular sieves, and dropping a methanol solution of 1,2,4-trihydroxyanthraquinone under stirring, stirring to react in the dark, and filtering, dialyzing, and freeze-drying the product to obtain anthraquinone-boronate-modified polyethyleneimine;

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

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

[0022] Preferably, in step (2), the dosage ratio of phenylboronic acid-modified polyethyleneimine, phosphate buffer and molecular sieve is 100-150 mg: 15-20 mL: 1-2 g; the dosage ratio of phenylboronic acid-modified polyethyleneimine and 1,2,4-trihydroxyanthraquinone is 100-150 mg: 80-100 mg; the ratio of 1,2,4-trihydroxyanthraquinone and methanol solution of 1,2,4-trihydroxyanthraquinone is 80-100 mg: 5 mL.

[0023] Preferably, in step (2), the reaction conditions are: stirring the reaction at 20-30° C. and 300-500 r / min in a dark environment for 12-20 hours, and checking the pH every 4 hours during the reaction to maintain the solution pH at 8.0-8.5.

[0024] In step (2), the product is filtered to remove the molecular sieve, and the filtrate is transferred to a dialysis bag (MWCO = 2000Da), and dialyzed with a phosphate buffer solution with a pH of 8.0 for 24 hours and ultrapure water for 48 hours, and the dialysate is replaced every 12 hours. The solution is freeze-dried for 48 to 72 hours to obtain anthraquinone-boronic acid ester-modified polyethyleneimine, which is sealed, protected from light, and stored at -20°C.

[0025] (3) Adding anthraquinone-boronic acid ester-modified polyethyleneimine and N,N'-carbonyldiimidazole to anhydrous DMF, introducing nitrogen, stirring in the dark to obtain an activation solution, adding folic acid solution dropwise to the activation solution, stirring to react, precipitating the product, washing, dialyzing, and freeze-drying to obtain the targeted nano photosensitizer carrier.

[0026] Activation 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; the intermediate then undergoes an esterification reaction with the carboxyl group in the folic acid molecule to covalently link the folic acid molecule to the photosensitizer carrier by forming a stable ester bond. CDI plays a dual role as an activator and a dehydrating agent in this process, ultimately constructing a targeted nano-photosensitizer carrier system.

[0027] Preferably, in step (3), the dosage ratio of anthraquinone-boric acid ester-modified polyethyleneimine, N,N'-carbonyldiimidazole, and anhydrous DMF in the activation solution is 100 mg: 15-20 mg: 20-30 mL.

[0028] Preferably, in step (3), the usage ratio of folic acid, HEPES buffer with pH=7.0-8.2, and DMF in the folic acid solution is 50-60 mg: 3-5 mL: 25-35 mL.

[0029] Preferably, in step (3), the dosage ratio of anthraquinone-boronate modified polyethyleneimine to folic acid is 100 mg:50-60 mg.

[0030] Preferably, in step (3), the stirring condition in the dark is 20-25° C. and the stirring reaction condition is 20-35° C. and the stirring reaction is 12-24 h.

[0031] In step (3), the product is added dropwise to ice ether for precipitation, centrifuged, washed three times with ether and DMF respectively, transferred to a dialysis bag (MWCO = 3500Da), dialyzed with PBS buffer containing a small amount of mannitol and pH = 7.4 for 48 to 72 hours, the dialysate is replaced every 12 hours, and the solution is freeze-dried for 24 to 48 hours to obtain the targeted nano photosensitizer carrier, which is sealed, protected from light, and stored at -20°C.

[0032] The present invention also claims protection for a targeted nano photosensitizer carrier prepared by the preparation method.

[0033] The present invention also claims to protect the use of the targeted nano photosensitizer carrier in the preparation of photodynamic therapy drugs.

[0034] Preferably, the photosensitizer is a hydrophobic photosensitizer containing a large π-conjugated structure, including porphyrins, phthalocyanines, and chlorophyll derivatives.

[0035] Preferably, the photosensitizer is purpurin imide.

[0036] Preferably, the method comprises the following steps: adding a targeted nano photosensitizer carrier and a hydrophobic photosensitizer to an organic solvent, ultrasonically treating the mixture to obtain a mixed solution, dropping the mixed solution into an aqueous solution of a surfactant, stirring to volatilize the organic solvent, centrifuging, washing, and drying to obtain the drug for photodynamic therapy.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. The present invention provides a method for preparing a targeted nano photosensitizer carrier, firstly, 4-carboxyphenylboronic acid is covalently grafted onto the PEI molecular chain through an EDC / NHS activation system; then, a dynamic covalent borate ester bond is formed by using the boronic acid group and the vicinal diol structure of 1,2,4-trihydroxyanthraquinone; finally, folic acid molecules are coupled to the carrier surface through CDI activation. The synthetic route adopted by the present invention is reasonably designed, the reaction conditions are controllable, and the post-treatment method is simple and efficient.

[0039] 2. The targeted nano photosensitizer carrier provided by the present invention has the following technical effects: ① The introduction of the PEI skeleton not only provides abundant modification sites, but also effectively improves the water solubility of the hydrophobic photosensitizer, reduces its aggregation, and enhances the photodynamic therapy effect; ② The boric acid group can form a stable cyclic boronate complex with the sialic acid overexpressed on the surface of tumor cells to enhance cell uptake; the boronate bond will hydrolyze in the slightly acidic environment of the tumor to achieve pH responsive release; ③ The anthraquinone structure has a large π conjugated system, which can form a stable complex with the photosensitizer containing an aromatic ring structure through π-π stacking, and can generate singlet oxygen under light conditions, causing structural fracture, achieving light-controlled release, and achieving efficient loading and controllable release of the photosensitizer; ④ The folic acid ligand can specifically recognize the folic acid receptor highly expressed on the surface of tumor cells, and form 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, which has significant application advantages in the field of photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show schematic diagrams of certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 The solution of the targeted nano photosensitizer carrier of Example 1 (right) and the solution of the photodynamic therapy drug prepared using the same (left);

[0042] Figure 2 The absorption curve (200-600 nm) of the photodynamic therapy drug prepared by the targeted nano photosensitizer carrier in Example 1;

[0043] Figure 3 is the absorption curve (350-730 nm) of the photodynamic therapy drug prepared by the targeted nano photosensitizer carrier in Example 1;

[0044] Figure 4The drug release curve of the photodynamic therapy drug prepared by the targeted nano photosensitizer carrier in Example 1 at different pH values ​​in vitro over time;

[0045] Figure 5 This is a bar graph showing the enrichment effect of the photodynamic therapy drug prepared by the photosensitizer carrier of Example 1 and Comparative Example 1 in different cells. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. Of course, the specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.

[0047] Unless otherwise specified, the chemical reagents and materials in the present invention are purchased from commercial sources or synthesized from raw materials purchased from commercial sources.

[0048] A method for preparing a targeted nano photosensitizer carrier comprises the following steps:

[0049] (1) 150-200 mg 4-carboxyphenylboronic acid, 230-280 mg EDC, and 140-170 mg NHS were added to 8-15 mL anhydrous methanol, and the mixture was stirred and mixed for 30-60 min in the dark at room temperature to obtain a 4-carboxyphenylboronic acid activated solution, which was added dropwise to 5-10 mL of a methanol solution of polyethyleneimine (polyethyleneimine content: 180-220 mg, molecular weight: MW=1500-2000), and the mixture was stirred and reacted at room temperature for 12-24 h. The product was added dropwise to icy ether for precipitation, centrifuged, and washed three times with icy ether. The precipitate was dissolved in ultrapure water, and then transferred to a dialysis bag (MWCO=1000 Da) for 48-72 h of dialysis, and the dialysis solution was replaced every 12 h. After the dialysis was completed, the solution was freeze-dried for 48-72 h to obtain a white viscous phenylboronic acid-modified polyethyleneimine, which was sealed and stored in a dark place at 4° C.

[0050] (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 sieve, add dropwise 5 mL of 1,2,4-trihydroxyanthraquinone methanol solution (1,2,4-trihydroxyanthraquinone content 80-100 mg) under stirring, react for 12-20 hours at 20-30°C and 300-500 r / min in a dark environment, check the pH every 4 hours during the reaction to maintain the solution pH at 8.0-8.5, filter the product to remove the molecular sieve, transfer the filtrate to a dialysis bag (MWCO=2000Da), dialyze with pH 8.0 phosphate buffer for 24 hours and ultrapure water for 48 hours, replace the dialysate every 12 hours, freeze-dry the solution for 48-72 hours to obtain anthraquinone-boronic acid ester modified polyethyleneimine, seal it, protect it from light, and store it at -20°C;

[0051] (3) 100 mg of anthraquinone-boronic acid ester-modified polyethyleneimine and 15-20 mg of N,N'-carbonyldiimidazole were added to 20-30 mL of anhydrous DMF, nitrogen was introduced, and the mixture was stirred at 20-25° C. in the dark for 2-4 h to obtain an activation solution. A folic acid solution (mixed with 50-60 mg of folic acid, 3-5 mL of HEPES buffer having a pH value of 7.0-8.2, and 25-35 mL of DMF) was added dropwise to the activation solution, and the mixture was stirred at 20-35° C. in the dark for 12-24 h. The product was added dropwise to ice ether for precipitation, centrifuged, washed with ether and DMF for three times, respectively, and transferred to a dialysis bag (MWCO=3500Da). The product was dialyzed with a PBS buffer containing a small amount of mannitol and a pH value of 7.4 for 48-72 h, and the dialyzate was replaced every 12 h. The solution was freeze-dried for 24-48 h to obtain the targeted nano photosensitizer carrier, which was sealed and stored in the dark at -20° C.

[0052] A use of a photosensitizer carrier in the preparation of a photodynamic therapy drug comprises the following steps:

[0053] The targeted nano photosensitizer carrier and the hydrophobic photosensitizer are added to an organic solvent, subjected to ultrasonic treatment to obtain a mixed solution, and the mixed solution is added dropwise to an aqueous solution of a surfactant, stirred to volatilize the organic solvent, centrifuged, washed, and dried to obtain the drug for photodynamic therapy.

[0054] The present invention will be further described below through specific embodiments.

[0055] Example 1

[0056] A method for preparing a targeted nano photosensitizer carrier comprises the following steps:

[0057] (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 the mixture was stirred and mixed for 60 min in the dark at room temperature to obtain a 4-carboxyphenylboronic acid activated solution, which was added dropwise to a methanol solution of 10 mL of polyethyleneimine (polyethyleneimine content: 220 mg, molecular weight: MW=1800), and the mixture was stirred and reacted at room temperature for 24 h. The product was added dropwise to icy ether for precipitation, centrifuged, and washed with icy ether for 3 times. The precipitate was dissolved in ultrapure water, and then transferred to a dialysis bag (MWCO=1000Da) for 72 h of dialysis, and the dialysis solution was replaced every 12 h. After the dialysis was completed, the solution was freeze-dried for 72 h to obtain a white viscous phenylboronic acid-modified polyethyleneimine, which was sealed and stored in a dark place at 4°C.

[0058] (2) Dissolve 150 mg of phenylboronic acid-modified polyethyleneimine in 20 mL of phosphate buffer (pH 8.5) and add molecular sieve, add dropwise 5mL of 1,2,4-trihydroxyanthraquinone methanol solution (1,2,4-trihydroxyanthraquinone content 100mg) under stirring, react for 20h at 30℃ and 500r / min in a dark place, check the pH every 4h during the reaction to maintain the solution pH at 8.5, filter the product to remove the molecular sieve, transfer the filtrate to a dialysis bag (MWCO=2000Da), dialyze with pH 8.0 phosphate buffer for 24h and ultrapure water for 48h, replace the dialysate every 12h, freeze-dry the solution for 72h to obtain anthraquinone-boronic acid ester modified polyethyleneimine, seal, protect from light, and store at -20℃;

[0059] (3) 100 mg of anthraquinone-boronic acid ester-modified polyethyleneimine and 20 mg of N,N'-carbonyldiimidazole were added to 30 mL of anhydrous DMF, nitrogen was introduced, and the mixture was stirred at 25°C in the dark for 4 h to obtain an activation solution. A folic acid solution (mixed with 60 mg of folic acid, 5 mL of HEPES buffer with a pH of 8.2, and 35 mL of DMF) 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 ether for precipitation, centrifuged, washed with ether and DMF for 3 times, respectively, and transferred to a dialysis bag (MWCO = 3500 Da). It was dialyzed with a PBS buffer containing a small amount of mannitol and a pH of 7.4 for 72 h, and the dialyzate was replaced every 12 h. The solution was freeze-dried for 48 h to obtain the targeted nano photosensitizer carrier, which was sealed and stored in the dark at -20°C.

[0060] Example 2

[0061] A method for preparing a targeted nano photosensitizer carrier comprises the following steps:

[0062] (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 the mixture was stirred and mixed for 50 min in the dark at room temperature to obtain a 4-carboxyphenylboronic acid activated solution, which was added dropwise to 8 mL of a methanol solution of polyethyleneimine (polyethyleneimine content: 200 mg, molecular weight: MW=2000), and the mixture was stirred and reacted at room temperature for 20 h. The product was added dropwise to icy ether for precipitation, and the mixture was centrifuged and washed three times with icy ether. The precipitate was dissolved in ultrapure water, and then transferred to a dialysis bag (MWCO=1000Da) for 60 h of dialysis, and the dialysis solution was replaced every 12 h. After the dialysis was completed, the solution was freeze-dried for 60 h to obtain a white viscous phenylboronic acid-modified polyethyleneimine, which was sealed and stored in a dark place at 4°C.

[0063] (2) Dissolve 140 mg of phenylboronic acid-modified polyethyleneimine in 16 mL of phosphate buffer (pH 8.5) and add molecular sieve, add dropwise 5mL of 1,2,4-trihydroxyanthraquinone methanol solution (1,2,4-trihydroxyanthraquinone content 90mg) under stirring, react for 16h at 26℃ and 400r / min in a dark place, check the pH every 4h during the reaction to maintain the solution pH at 8.5, filter the product to remove the molecular sieve, transfer the filtrate to a dialysis bag (MWCO=2000Da), dialyze with pH 8.0 phosphate buffer for 24h and ultrapure water for 48h, replace the dialysate every 12h, freeze-dry the solution for 60h to obtain anthraquinone-boronic acid ester modified polyethyleneimine, seal, protect from light, and store at -20℃;

[0064] (3) 100 mg of anthraquinone-boronic acid ester-modified polyethyleneimine and 16 mg of N,N'-carbonyldiimidazole were added to 24 mL of anhydrous DMF, nitrogen was introduced, and the mixture was stirred at 22°C in the dark for 3 h to obtain an activation solution. A folic acid solution (mixed with 55 mg of folic acid, 4 mL of HEPES buffer with a pH value of 8.0, and 30 mL of DMF) 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 ether for precipitation, centrifuged, washed with ether and DMF for three times, respectively, and transferred to a dialysis bag (MWCO = 3500 Da). The product was dialyzed with a PBS buffer containing a small amount of mannitol and a pH value of 7.4 for 60 h, and the dialyzate was replaced every 12 h. The solution was freeze-dried for 36 h to obtain the targeted nano photosensitizer carrier, which was sealed and stored in the dark at -20°C.

[0065] Example 3

[0066] A method for preparing a targeted nano photosensitizer carrier comprises the following steps:

[0067] (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 the mixture was stirred and mixed for 40 min in the dark at room temperature to obtain a 4-carboxyphenylboronic acid activated solution, which was added dropwise to a methanol solution of 6 mL of polyethyleneimine (polyethyleneimine content: 190 mg, molecular weight: MW=1700), and the mixture was stirred and reacted at room temperature for 16 h. The product was added dropwise to icy ether for precipitation, centrifuged, and washed three times with icy ether. The precipitate was dissolved in ultrapure water, and then transferred to a dialysis bag (MWCO=1000Da) for 60 h of dialysis, and the dialysis solution was replaced every 12 h. After the dialysis was completed, the solution was freeze-dried for 60 h to obtain a white viscous phenylboronic acid-modified polyethyleneimine, which was sealed and stored in a dark place at 4°C.

[0068] (2) Dissolve 120 mg of phenylboronic acid-modified polyethyleneimine in 17 mL of phosphate buffer (pH 8.0) and add molecular sieve, add dropwise 5mL of 1,2,4-trihydroxyanthraquinone methanol solution (1,2,4-trihydroxyanthraquinone content 90mg) under stirring, react for 16h at 24℃ and 400r / min in a dark place, check the pH every 4h during the reaction to maintain the solution pH at 8.0, filter the product to remove the molecular sieve, transfer the filtrate to a dialysis bag (MWCO=2000Da), dialyze with pH 8.0 phosphate buffer for 24h and ultrapure water for 48h, replace the dialysate every 12h, freeze-dry the solution for 48h to obtain anthraquinone-boronic acid ester modified polyethyleneimine, seal, protect from light, and store at -20℃;

[0069] (3) 100 mg of anthraquinone-boronic acid ester-modified polyethyleneimine and 16 mg of N,N'-carbonyldiimidazole were added to 24 mL of anhydrous DMF, nitrogen was introduced, and the mixture was stirred at 22°C in the dark for 3 h to obtain an activation solution. A folic acid solution (mixed with 54 mg of folic acid, 4 mL of HEPES buffer with a pH value of 8.0, and 30 mL of DMF) 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 ether for precipitation, centrifuged, washed with ether and DMF for three times, respectively, and transferred to a dialysis bag (MWCO = 3500 Da). The product was dialyzed with a PBS buffer containing a small amount of mannitol and a pH value of 7.4 for 60 h, and the dialyzate was replaced every 12 h. The solution was freeze-dried for 36 h to obtain the targeted nano photosensitizer carrier, which was sealed and stored in the dark at -20°C.

[0070] Example 4

[0071] A method for preparing a targeted nano photosensitizer carrier comprises the following steps:

[0072] (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 the mixture was stirred and mixed for 30 min in the dark at room temperature to obtain a 4-carboxyphenylboronic acid activation solution, which was added dropwise to a methanol solution of 5 mL of polyethyleneimine (polyethyleneimine content: 180 mg, molecular weight: MW=1500), and the mixture was stirred and reacted at room temperature for 12 h. The product was added dropwise to icy ether for precipitation, centrifuged, and washed three times with icy ether. The precipitate was dissolved in ultrapure water, and then transferred to a dialysis bag (MWCO=1000Da) for 48 h of dialysis. The dialysis solution was replaced every 12 h. After the dialysis was completed, the solution was freeze-dried for 48 h to obtain a white viscous intermediate, which was sealed, protected from light, and stored at 4°C;

[0073] (2) Dissolve 100 mg of phenylboronic acid-modified polyethyleneimine in 15 mL of phosphate buffer (pH 8.0) and add molecular sieve, dropwise add 5mL of 1,2,4-trihydroxyanthraquinone methanol solution (1,2,4-trihydroxyanthraquinone content 80mg) under stirring, react for 12h at 20℃ and 300r / min in a dark environment, check the pH every 4h during the reaction to maintain the solution pH at 8.0, filter the product to remove the molecular sieve, transfer the filtrate to a dialysis bag (MWCO=2000Da), dialyze with pH 8.0 phosphate buffer for 24h and ultrapure water for 48h, replace the dialysate every 12h, freeze-dry the solution for 48h to obtain the photosensitizer carrier, seal, protect from light, and store at -20℃;

[0074] (3) 100 mg of anthraquinone-boronic acid ester-modified polyethyleneimine and 15 mg of N,N'-carbonyldiimidazole were added to 20 mL of anhydrous DMF, nitrogen was introduced, and the mixture was stirred at 20°C in the dark for 2 h to obtain an activation solution. A folic acid solution (mixed with 50 mg of folic acid, 3 mL of HEPES buffer with a pH of 7.5, and 25 mL of DMF) 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 ether for precipitation, centrifuged, washed with ether and DMF for three times, respectively, and transferred to a dialysis bag (MWCO = 3500 Da). The product was dialyzed with a PBS buffer containing a small amount of mannitol and a pH of 7.4 for 48 h, and the dialyzate was replaced every 12 h. The solution was freeze-dried for 24 h to obtain the targeted nano photosensitizer carrier, which was sealed and stored in the dark at -20°C.

[0075] Comparative Example 1

[0076] A method for preparing a nano photosensitizer carrier comprises the following steps:

[0077] (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 the mixture was stirred and mixed for 60 min in the dark at room temperature to obtain a 4-carboxyphenylboronic acid activated solution, which was added dropwise to a methanol solution of 10 mL of polyethyleneimine (polyethyleneimine content: 220 mg, molecular weight: MW=1800), and the mixture was stirred and reacted at room temperature for 24 h. The product was added dropwise to icy ether for precipitation, centrifuged, and washed with icy ether for 3 times. The precipitate was dissolved in ultrapure water, and then transferred to a dialysis bag (MWCO=1000Da) for 72 h of dialysis, and the dialysis solution was replaced every 12 h. After the dialysis was completed, the solution was freeze-dried for 72 h to obtain a white viscous phenylboronic acid-modified polyethyleneimine, which was sealed and stored in a dark place at 4°C.

[0078] (2) Dissolve 150 mg of phenylboronic acid-modified polyethyleneimine in 20 mL of phosphate buffer (pH 8.5) and add Molecular sieves, 5 mL of 1,2,4-trihydroxyanthraquinone methanol solution (1,2,4-trihydroxyanthraquinone content: 100 mg) was added dropwise with stirring, and the reaction was stirred at 30 ° C and 500 r / min in a dark environment for 20 hours. The pH was checked every 4 hours during the reaction to maintain the solution pH at 8.5. The product was filtered to remove the molecular sieves, and the filtrate was transferred to a dialysis bag (MWCO = 2000Da), dialyzed with phosphate buffer with a pH of 8.0 for 24 hours and ultrapure water for 48 hours, and the dialysate was replaced every 12 hours. The solution was freeze-dried for 72 hours to obtain a nano photosensitizer carrier, which was sealed and stored in a dark environment at -20 ° C.

[0079] Comparative Example 2

[0080] A method for preparing a nano photosensitizer carrier comprises the following steps:

[0081] 200 mg 4-carboxyphenylboronic acid, 280 mg EDC, and 170 mg NHS were added to 15 mL anhydrous methanol, stirred and mixed for 60 min in the dark at room temperature to obtain a 4-carboxyphenylboronic acid activated solution, which was added dropwise to a methanol solution of 10 mL polyethyleneimine (polyethyleneimine content: 220 mg, molecular weight: MW=1800), stirred and reacted at room temperature for 24 h, and the product was added dropwise to ice ether for precipitation, centrifuged, washed with ice ether for 3 times, dissolved in ultrapure water, and then transferred to a dialysis bag (MWCO=1000Da) for 72 h of dialyzing, and the dialysate was replaced every 12 h. After the dialysis, the solution was freeze-dried for 72 h to obtain a white viscous nano photosensitizer carrier, which was sealed and stored in a dark place at 4°C.

[0082] Comparative Example 3

[0083] Polyethyleneimine with a molecular weight of MW=1800 was used as the nano-photosensitizer carrier.

[0084] The targeted nano photosensitizer carriers obtained in Examples 1 to 4 and Comparative Examples 1 to 3 are used in the preparation of photodynamic therapy drugs, and the steps are as follows:

[0085] 40 mg of targeted nano photosensitizer carrier and 6 mg of purpurin-18-imide were added to a mixed solution of 20 mL of dichloromethane and methanol (volume ratio of 1:1), and ultrasonically treated for 120 s to obtain a mixed solution. The mixed solution was dropwise added into 75 mL of a 2 wt% PVA aqueous solution, stirred in a water bath at 25° C. for 8 h to volatilize the organic solvent, centrifuged at 5000 r / min in a centrifuge, washed with deionized water, repeated 3 times, and vacuum dried to obtain the photodynamic therapy drug.

[0086] 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 The absorption curve of the photodynamic therapy drug prepared by the targeted nano photosensitizer carrier in Example 1) was prepared, and a mixed solution of dichloromethane and methanol of purpurin-18-imide of different concentrations was prepared, and its absorbance was measured at the maximum absorption wavelength to obtain a standard curve of its absorbance and solution concentration. Then 1 mg of the drug for photodynamic therapy was weighed and dissolved in 5 mL of a mixed organic solvent of dichloromethane and methanol (volume ratio 1:1). After complete dissolution, its absorbance at the same absorption wavelength as the standard curve was measured with an ultraviolet spectrophotometer, and then its purpurin-18-imide concentration was found by comparing it with the standard curve, and the drug loading and encapsulation rate in the drug for photodynamic therapy were calculated according to the formula (see Table 1 for specific data):

[0087] Drug loading = [weight of photosensitizer in drug / weight of drug] × 100%;

[0088] Encapsulation efficiency = [weight of photosensitizer in drug / dose] × 100%.

[0089] 5 mL of the drug solution for photodynamic therapy prepared in Example 1 (the solvent is dichloromethane and methanol in a volume ratio of 1:1, and the drug concentration is 20 mg / mL) was poured into a dialysis bag, and the dialysis bags were placed in flasks containing 20 mL of PBS buffer solution with pH = 7.4 (simulating normal tissue in the body) and acetate buffer solution with pH = 5.0 (simulating the weak acid environment of tumor tissue), respectively. The dialysis bag and the flask were kept at 37° C. and stirred. A small amount of solution was taken out from the flask for ultraviolet detection every once in a while. After the detection, the solution in the cuvette was poured back into the flask to ensure that the total volume of the flask was constant. The experimental data of 2h, 4h, 8h, and 12h were recorded, and the drug release curve of the targeted nano-photosensitizer carrier in vitro over time was plotted. The results are shown as follows: Figure 4As shown, it can be seen that the photosensitizer loaded by the targeted nano photosensitizer carrier prepared by the present invention is released faster in the acidic microenvironment of the tumor than in normal tissues.

[0090] The concentrations of the drugs for photodynamic therapy prepared in Examples 1 to 4 and Comparative Examples 1 to 3 released in vitro for 12 hours in an acetate buffer solution at pH = 5.0 were measured. The specific data are shown in Table 1.

[0091] Table 1 Performance test of drugs for photodynamic therapy prepared by photosensitizer carrier

[0092]

[0093] The photodynamic therapy drug prepared by the targeted nano photosensitizer carrier of Example 1 was used for human lung adenocarcinoma epithelial cells (A549) and human oral epithelial cancer cells (KB cells) with high expression of folate receptors, and the photodynamic therapy drug prepared by the photosensitizer carrier of Comparative Example 1 was used as a comparison to measure laser fluorescence confocal imaging. The results are shown in FIG. Figure 5 After culturing for 1 hour and 3 hours, the photodynamic therapy drug of Example 1 was more obviously enriched in KB cells, indicating that the targeted nano photosensitizer carrier provided by the present invention has a targeted selective effect on tumor cells with high expression of folate receptors.

[0094] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a targeted nano photosensitizer carrier, characterized in that: The steps include: (1) adding 4-carboxyphenylboronic acid, EDC, and NHS to anhydrous methanol, stirring and mixing to obtain a 4-carboxyphenylboronic acid activated solution, adding dropwise to a methanol solution of polyethyleneimine, stirring for reaction, precipitating the product, washing, dialyzing, and freeze-drying to obtain polyethyleneimine modified with phenylboronic acid; (2) dissolving the phenylboronic acid-modified polyethyleneimine in a phosphate buffer, adding molecular sieves, and dropping a methanol solution of 1,2,4-trihydroxyanthraquinone under stirring, stirring to react in the dark, and filtering, dialyzing, and freeze-drying the product to obtain anthraquinone-boronate-modified polyethyleneimine; (3) Adding anthraquinone-boronic acid ester-modified polyethyleneimine and N,N'-carbonyldiimidazole to anhydrous DMF, introducing nitrogen, stirring in the dark to obtain an activation solution, adding folic acid solution dropwise to the activation solution, stirring to react, precipitating the product, washing, dialyzing, and freeze-drying 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 for 30 to 60 minutes in the dark and at room temperature; the stirring reaction conditions are stirring and reacting for 12 to 24 hours at room temperature; and 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 usage ratio of 4-carboxyphenylboric 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; the usage ratio of 4-carboxyphenylboric acid and 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 is 8.0 to 8.5, and the molecular sieve is The dosage ratio of phenylboronic acid-modified polyethyleneimine, phosphate buffer, and molecular sieve is 100-150 mg: 15-20 mL: 1-2 g; the dosage ratio of phenylboronic acid-modified polyethyleneimine and 1,2,4-trihydroxyanthraquinone is 100-150 mg: 80-100 mg; the ratio of 1,2,4-trihydroxyanthraquinone and the 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 are: stirring the reaction at 20-30° C. and 300-500 r / min in a dark environment for 12-20 hours. During the reaction, the pH value is checked every 4 hours to maintain the pH value of the solution at 8.0-8.

5.

6. The preparation method according to claim 1, characterized in that: In step (3), the dosage ratio of anthraquinone-boronate modified polyethyleneimine, N,N'-carbonyldiimidazole, and anhydrous DMF in the activation solution is 100 mg: 15-20 mg: 20-30 mL; the dosage ratio of folic acid, HEPES buffer with pH = 7.0-8.2, and DMF in the folic acid solution is 50-60 mg: 3-5 mL: 25-35 mL; the dosage ratio of anthraquinone-boronate 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 condition in the dark is 20-25° C. and the stirring reaction condition is 20-35° C. and the stirring reaction is 12-24 h.

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

9. Use of the targeted nano photosensitizer carrier according to claim 8 in the preparation of photodynamic therapy drugs.

10. The use 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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