A chlorin e6-loaded nanocluster activatable by x-rays and a preparation method thereof

Abstract

The application belongs to the technical field of biological medicine preparation, and particularly relates to a chlorin e6-loaded nanocluster activated by X-rays and a preparation method thereof. Hydroxyethyl starch and succinic anhydride are reacted in the presence of N,N-dimethylaminopyridine to obtain carboxylated hydroxyethyl starch, zinc oxide nanoparticles are assembled on the carboxylated hydroxyethyl starch to form zinc oxide nanoclusters, and then chlorin e6 is assembled on the zinc oxide nanoclusters. The obtained zinc oxide nanocluster loaded with chlorin e6 has a cluster structure, can generate singlet oxygen under X-ray irradiation, and can play an X-ray-induced photodynamic therapy role, and has potential application value in the photodynamic therapy of deep tumors.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical formulation technology, and specifically relates to an X-ray activatable dihydroporphyrin E6 nanocluster and its preparation method. Background Technology

[0002] Effective treatment of deep tumors remains a major challenge for traditional photodynamic therapy (PDT) due to the limited penetrating power of the light sources used. X-rays, however, possess strong tissue penetrating power, making them a promising excitation source for activating photosensitizers within deep tumors. To this end, researchers have developed various X-ray-excited luminescent nanoparticles loaded with multiple photosensitizers, thereby transferring X-ray energy to the photosensitizers. Among these luminescent nanoparticles, the ultraviolet luminescence of zinc oxide nanoparticles largely matches the ultraviolet absorption of porphyrin compounds. Therefore, the combination of zinc oxide nanoparticles and photosensitizers is considered one of the optimal combinations for X-ray-induced photodynamic therapy. However, zinc oxide nanoparticles are difficult to disperse in water, present challenges in drug loading, and exhibit high toxicity, making it difficult to combine them with photosensitizers for effective X-ray-induced photodynamic therapy. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a method for preparing X-ray-activated dihydroporphyrin E6 nanoclusters:

[0004] (1) Preparation of carboxylated hydroxyethyl starch

[0005] Hydroxyethyl starch and succinic anhydride were dissolved together in dimethyl sulfoxide, and N,N-dimethylaminopyridine was added to the mixture for reaction. After the reaction, the resulting reaction solution was dialyzed in ultrapure water and then freeze-dried to obtain carboxylated hydroxyethyl starch.

[0006] (2) Preparation of zinc oxide nanoclusters

[0007] The carboxylated hydroxyethyl starch obtained in step (1) was added to ultrapure water, and zinc oxide nanoparticles were added to it. The mixture was then ultrasonically dispersed to obtain a zinc oxide nanocluster dispersion.

[0008] (3) Preparation of dihydroporphyrin E6 nanoclusters

[0009] After dissolving dihydroporphyrin E6 in dimethyl sulfoxide, it was added to the zinc oxide nanocluster dispersion obtained in step (2). After being ultrasonically dispersed, it was dialyzed and then centrifuged to obtain zinc oxide nanoclusters loaded with dihydroporphyrin E6.

[0010] As a preferred embodiment: in step (1), the molecular weight of hydroxyethyl starch is 40,000 to 450,000, the degree of hydroxyethyl substitution is 0.4 to 0.5, the mass ratio of hydroxyethyl starch to succinic anhydride is 6 to 20:1, and the molar ratio of succinic anhydride to N,N-dimethylaminopyridine is 1:0.5.

[0011] As a preferred option: in step (1), the mixing reaction is carried out by stirring at room temperature for 24 hours.

[0012] As a preferred embodiment: in step (2), the diameter of the zinc oxide nanoparticles is 30±10nm, the mass ratio of carboxylated hydroxyethyl starch to zinc oxide nanoparticles is 2~6:1, the ultrasonic power of ultrasonic dispersion is 37.5W, and the ultrasonic time is 5 minutes.

[0013] As a preferred embodiment: in step (3), the mass ratio of dihydroporphyrin E6 to zinc oxide nanoclusters is 0.05 to 0.1:1, the ultrasonic power for ultrasonic dispersion is 37.5W and the ultrasonic time is 5 minutes, and the centrifugal separation speed is 12000 rpm and the centrifugal separation time is 10 minutes.

[0014] The present invention also provides an X-ray-activated dihydroporphyrin E6 nanocluster obtained by the above preparation method.

[0015] The beneficial effects of this invention are as follows:

[0016] In this invention, zinc oxide nanoparticles are first aggregated together using the abundant functional groups and high molecular weight of carboxylated hydroxyethyl starch to form stable zinc oxide nanoclusters. Then, the photosensitizer dihydroporphyrin E6 is introduced, allowing it to be loaded into the pores between the clusters of zinc oxide nanoclusters on the carboxylated hydroxyethyl starch. This creates a stable physical bond between dihydroporphyrin E6 and the zinc oxide nanoclusters, and improves the loading capacity of dihydroporphyrin E6 when combined with zinc oxide. Under X-ray irradiation, the zinc oxide nanoclusters absorb X-rays, transferring energy to dihydroporphyrin E6, thereby activating the photosensitizer to produce singlet oxygen, achieving X-ray-induced photodynamic therapy. Using X-rays to induce photodynamic therapy overcomes the bottleneck of insufficient tissue penetration when using conventional light sources in photodynamic therapy. Attached Figure Description

[0017] Figure 1 The above is the 1H NMR spectrum of the carboxylated hydroxyethyl starch prepared in step (1) of Example 1 of this invention in deuterated water;

[0018] Figure 2 This is a transmission electron microscope image of the dihydroporphyrin E6 nanoclusters prepared in Example 1 of the present invention;

[0019] Figure 3This is a transmission electron microscope image of the dihydroporphyrin E6 nanoclusters prepared in Example 2 of the present invention;

[0020] Figure 4 This is a transmission electron microscope image of the dihydroporphyrin E6 nanoclusters prepared in Example 3 of the present invention;

[0021] Figure 5 The photoluminescence spectra of the zinc oxide nanoclusters prepared in step (2) of Example 1 and the dihydroporphyrin E6 nanoclusters prepared in step (3) of Example 1 are shown in the figure (the spectrum pointed to by the arrow of "Example 4" in the figure represents the light emission spectrum of the zinc oxide nanoclusters prepared in step (2) of Example 1; the spectrum pointed to by the arrow of "Example 7" in the figure represents the light emission spectrum of the dihydroporphyrin E6 nanoclusters prepared in step (3) of Example 1). Detailed Implementation

[0022] A method for preparing X-ray activated dihydroporphyrin E6 nanoclusters:

[0023] (1) Preparation of carboxylated hydroxyethyl starch

[0024] Hydroxyethyl starch with a molecular weight of 40,000 to 450,000 and a degree of hydroxyethyl substitution of 0.4 to 0.5 and succinic anhydride were dissolved together in dimethyl sulfoxide at a mass ratio of 6 to 20:1. N,N-dimethylaminopyridine with a molar ratio of 0.5:1 to succinic anhydride was added to the solution and the mixture was stirred at room temperature for 24 hours. After the reaction, the resulting reaction solution was dialyzed in ultrapure water for 3 days and then freeze-dried to obtain carboxylated hydroxyethyl starch.

[0025] (2) Preparation of zinc oxide nanoclusters

[0026] The carboxylated hydroxyethyl starch obtained in step (1) was added to ultrapure water, and zinc oxide nanoparticles with a mass ratio of 1:2 to 6 with a diameter of 30±10 nm were added to it. The mixture was then ultrasonically dispersed at an ultrasonic power of 37.5 W for 5 minutes to obtain a zinc oxide nanocluster dispersion.

[0027] (3) Preparation of dihydroporphyrin E6 nanoclusters

[0028] Dihydroporphyrin E6, with a mass ratio of 0.05 to 0.1:1 to the zinc oxide nanoclusters obtained in step (2), was dissolved in dimethyl sulfoxide and added to the zinc oxide nanocluster dispersion obtained in step (2). After ultrasonic dispersion at 37.5W for 5 minutes, the mixture was dialyzed and then centrifuged at 12000 rpm for 10 minutes to obtain zinc oxide nanoclusters loaded with dihydroporphyrin E6.

[0029] Example 1

[0030] (1) Preparation of carboxylated hydroxyethyl starch

[0031] 1.8 g of hydroxyethyl starch (molecular weight 130 kDa, degree of hydroxyethyl substitution 0.4) and 0.3 g of succinic anhydride were dissolved together in 10 mL of dimethyl sulfoxide. 0.183 g of N,N-dimethylaminopyridine was added, and the mixture was stirred at room temperature (25°C) for 24 hours. After the reaction, the resulting solution was transferred to a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed in ultrapure water for 3 days. Finally, it was freeze-dried to obtain carboxylated hydroxyethyl starch.

[0032] The carboxylated hydroxyethyl starch synthesized in this embodiment was characterized by proton nuclear magnetic resonance spectroscopy (¹H NMR), see [link to original text]. Figure 1 The results showed that, in addition to the characteristic hydrogen signal of hydroxyethyl starch, carboxylated hydroxyethyl starch also exhibited a characteristic peak of succinic anhydride methylene hydrogen at around 2.5 ppm, indicating that succinic anhydride successfully reacted with hydroxyethyl starch. The degree of carboxyl substitution was calculated to be 0.28 by the ratio of the integral of the succinic anhydride methylene hydrogen signal to the integral of the characteristic hydrogen signal of hydroxyethyl starch.

[0033] (2) Preparation of zinc oxide nanoclusters

[0034] 0.2g of the carboxylated hydroxyethyl starch prepared in step (1) was added to 100mL of ultrapure water and fully dissolved and dispersed. Then, 100mg of zinc oxide nanoparticles were added to it and ultrasonically treated for 5 minutes using a probe-type ultrasonic crusher with an ultrasonic power of 37.5W to obtain a carboxylated hydroxyethyl starch-stabilized zinc oxide nanocluster dispersion.

[0035] (3) Preparation of dihydroporphyrin E6 nanoclusters

[0036] Take 5 mL of a 2 mM dihydroporphyrin E6 dimethyl sulfoxide solution and add it to the zinc oxide nanocluster dispersion prepared in step (2). Use a probe-type ultrasonic disruptor to sonicate for 5 minutes with an ultrasonic power of 37.5 W. Then transfer the resulting dispersion to a dialysis bag (molecular weight cutoff 3500 Da), dialyze in ultrapure water for 3 days, and then centrifuge (12000 rpm) for 10 minutes. Take the precipitate as the dihydroporphyrin E6 nanocluster and disperse it in 10 mL of ultrapure water for later use.

[0037] The concentration of dihydroporphyrin E6 dispersed in ultrapure water obtained in this embodiment was 0.92 mM, as determined by ultraviolet-visible spectrophotometry.

[0038] Example 2

[0039] (1) Preparation of carboxylated hydroxyethyl starch

[0040] 1.84 g of hydroxyethyl starch (molecular weight 40 kDa, degree of hydroxyethyl substitution 0.5) and 0.2 g of succinic anhydride were dissolved together in 10 mL of dimethyl sulfoxide. 0.122 g of N,N-dimethylaminopyridine was added and the mixture was stirred at room temperature for 24 hours. After the reaction, the resulting reaction solution was transferred to a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed in ultrapure water for 3 days. The solution was then freeze-dried to obtain carboxylated hydroxyethyl starch.

[0041] The carboxylated hydroxyethyl starch synthesized in this embodiment was characterized by proton nuclear magnetic resonance spectroscopy. The degree of carboxyl substitution was calculated to be 0.19 by the ratio of the integral of the methylene hydrogen signal of succinic acid monoester to the integral of the characteristic hydrogen signal of hydroxyethyl starch.

[0042] (2) Preparation of zinc oxide nanoclusters

[0043] 0.3g of the carboxylated hydroxyethyl starch prepared in step (1) was added to 100mL of ultrapure water and fully dissolved and dispersed. Then, 100mg of zinc oxide nanoparticles were added to it and ultrasonically treated for 5 minutes using a probe-type ultrasonic crusher with an ultrasonic power of 37.5W to obtain a carboxylated hydroxyethyl starch-stabilized zinc oxide nanocluster dispersion.

[0044] (3) Preparation of dihydroporphyrin E6 nanoclusters

[0045] Take 5 mL of a 2 mM dihydroporphyrin E6 dimethyl sulfoxide solution and add it to the zinc oxide nanocluster dispersion prepared in step (2). Use a probe-type ultrasonic disruptor to sonicate for 5 minutes with an ultrasonic power of 37.5 W. Then transfer the resulting dispersion to a dialysis bag (molecular weight cutoff 3500 Da), dialyze in ultrapure water for 3 days, and then centrifuge (12000 rpm) for 10 minutes. Take the precipitate as the dihydroporphyrin E6 nanocluster and disperse it in 10 mL of ultrapure water for later use.

[0046] The concentration of dihydroporphyrin E6 dispersed in ultrapure water obtained in this embodiment was 0.75 mM, as determined by ultraviolet-visible spectrophotometry.

[0047] Example 3

[0048] (1) Preparation of carboxylated hydroxyethyl starch

[0049] 1.84 g of hydroxyethyl starch (molecular weight 450 kDa, degree of hydroxyethyl substitution 0.5) and 0.1 g of succinic anhydride were dissolved together in 10 mL of dimethyl sulfoxide. 0.061 g of N,N-dimethylaminopyridine was added and the mixture was stirred at room temperature for 24 hours. After the reaction, the resulting reaction solution was transferred to a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed in ultrapure water for 3 days. The solution was then freeze-dried to obtain carboxylated hydroxyethyl starch.

[0050] The carboxylated hydroxyethyl starch synthesized in this embodiment was characterized by proton nuclear magnetic resonance spectroscopy. The degree of carboxyl substitution was calculated to be 0.08 by the ratio of the integral of the methylene hydrogen signal of succinic acid monoester to the integral of the characteristic hydrogen signal of hydroxyethyl starch.

[0051] (2) Preparation of zinc oxide nanoclusters

[0052] 0.6g of the carboxylated hydroxyethyl starch prepared in step (1) was added to 100mL of ultrapure water and fully dissolved and dispersed. Then, 100mg of zinc oxide nanoparticles were added to it and ultrasonically treated for 5 minutes using a probe-type ultrasonic crusher with an ultrasonic power of 37.5W to obtain a carboxylated hydroxyethyl starch-stabilized zinc oxide nanocluster dispersion.

[0053] (3) Preparation of dihydroporphyrin E6 nanoclusters

[0054] Take 5 mL of a 2 mM dihydroporphyrin E6 dimethyl sulfoxide solution and add it to the zinc oxide nanocluster dispersion prepared in step (2). Use a probe-type ultrasonic disruptor to sonicate for 5 minutes with an ultrasonic power of 37.5 W. Then transfer the resulting dispersion to a dialysis bag (molecular weight cutoff 3500 Da), dialyze in ultrapure water for 3 days, and then centrifuge (12000 rpm) for 10 minutes. Take the precipitate as the dihydroporphyrin E6 nanocluster and disperse it in 10 mL of ultrapure water for later use.

[0055] The concentration of dihydroporphyrin E6 dispersed in ultrapure water obtained in this embodiment was 0.93 mM, as determined by ultraviolet-visible spectrophotometry.

[0056] Comparative Example 1

[0057] Without using carboxylated hydroxyethyl starch to stabilize the zinc oxide nanoparticles (the missing zinc oxide was replenished by an equal mass of carboxylated hydroxyethyl starch), dihydroporphyrin E6 was loaded directly onto the carboxylated hydroxyethyl starch, and all other operations were the same as in Example 1:

[0058] (1) Preparation of carboxylated hydroxyethyl starch

[0059] Same as step (1) in Example 1;

[0060] (2) Preparation of carboxylated hydroxyethyl starch dispersion

[0061] Add 0.3g of the carboxylated hydroxyethyl starch prepared in step (1) to 100mL of ultrapure water to fully dissolve and disperse it, and then use a probe-type ultrasonic crusher to ultrasonically treat it for 5 minutes with an ultrasonic power of 37.5W to obtain a carboxylated hydroxyethyl starch dispersion.

[0062] (3) Preparation of dihydroporphyrin E6 starch

[0063] Take 5 mL of a 2 mM dihydroporphyrin e6 dimethyl sulfoxide solution and add it to the carboxylated hydroxyethyl starch dispersion prepared in step (2). Use a probe-type ultrasonic homogenizer to sonicate for 5 minutes at an ultrasonic power of 37.5 W. Then transfer the resulting dispersion to a dialysis bag (molecular weight cutoff 3500 Da), dialyze in ultrapure water for 3 days, and then centrifuge (12000 rpm) for 10 minutes. Take the precipitate as the dihydroporphyrin e6-loaded carboxylated hydroxyethyl starch and disperse it in 10 mL of ultrapure water for later use.

[0064] The concentration of dihydroporphyrin E6 dispersed in ultrapure water obtained in this comparative example was 0.08 mM, as determined by ultraviolet-visible spectrophotometry.

[0065] Comparative Example 2

[0066] Carboxylated hydroxyethyl starch was not used (the missing carboxylated hydroxyethyl starch was supplemented by an equal mass of nano-zinc oxide), and dihydroporphyrin E6 was loaded directly onto nano-zinc oxide. All other operations were the same as in Example 1.

[0067] (1) Preparation of nano zinc oxide dispersion

[0068] 300 mg of zinc oxide nanoparticles were added to 100 mL of Tris buffer (pH 8.5) and ultrasonically treated for 5 minutes using a probe-type ultrasonic homogenizer with an ultrasonic power of 37.5 W to obtain a zinc oxide nanoparticle dispersion.

[0069] (2) Preparation of zinc oxide nanoparticles loaded with dihydroporphyrin E6

[0070] Take 5 mL of a 2 mM dihydroporphyrin E6 dimethyl sulfoxide solution and add it to the zinc oxide nanocluster dispersion prepared in step (1). Use a probe-type ultrasonic disruptor to sonicate for 5 minutes at an ultrasonic power of 37.5 W. Then, transfer the resulting dispersion to a dialysis bag (molecular weight cutoff 3500 Da) and dialyze in ultrapure water for 3 days. After that, centrifuge (12000 rpm) for 10 minutes and take the precipitate as dihydroporphyrin E6-loaded zinc oxide nanoparticles. Sonicately disperse it in 10 mL of ultrapure water and measure the concentration of dihydroporphyrin E6 dispersed in ultrapure water by UV-Vis spectrophotometry. The concentration of dihydroporphyrin E6 dispersed in ultrapure water obtained in this comparative example is 0.21 mM.

[0071] Comparative Examples 1 and 2 were conducted by the applicant to further verify the loading behavior of dihydroporphyrin E6 on zinc oxide nanoclusters obtained by carboxylated hydroxyethyl starch in this application. Comparative Example 1 shows that the loading effect of carboxylated hydroxyethyl starch on dihydroporphyrin E6 is very limited, while the loading capacity of the nano-zinc oxide in Comparative Example 2 is also low. In contrast, the zinc oxide nanoclusters formed by carboxylated hydroxyethyl starch in this application can load a larger amount of dihydroporphyrin E6. This is because after the nano-zinc oxide is first combined into clusters by carboxylated hydroxyethyl starch, the zinc oxide nanoparticles in the nanoclusters form interstitial structures on the carboxylated hydroxyethyl starch, allowing dihydroporphyrin E6 to enter and form a stable loading of dihydroporphyrin E6.

[0072] Comparing Examples 1-3, it can be seen that the zinc oxide nanoclusters prepared in Examples 1 and 3 have stronger drug loading capacity. This may be because the carboxylated hydroxyethyl starch used in Examples 1 and 3 has a larger molecular weight, which makes the zinc oxide nanoclusters more firmly bound and more conducive to drug encapsulation.

[0073] Particle size and morphology detection

[0074] Preparation of test solutions: The spare solutions of dihydroporphyrin E6 nanoclusters prepared in Examples 1-3 and dispersed in 10 mL of ultrapure water were further diluted 50 times with ultrapure water to obtain the test solutions of each dihydroporphyrin E6 nanocluster.

[0075] Dynamic light scattering and transmission electron microscopy tests: The particle size and potential (n=3) of each prepared nanocluster test solution were determined by dynamic light scattering, as shown in Table 1:

[0076] Table 1: Particle size and potential of dihydroporphyrin E6 nanoclusters

[0077] Hydrated diameter (nm) polydispersity index Potential (mV) Example 1 168.5±13.6 0.265±0.032 0.23±0.06 Example 2 254.8±15.5 0.281±0.038 0.52±0.08 Example 3 175.4±17.4 0.252±0.041 0.28±0.05

[0078] As shown in Table 1, the hydrated diameters of the dihydroporphyrin E6 nanoclusters prepared in Examples 1-3 ranged from 100 to 300 nm, and the polydispersity index was less than 0.3, indicating a narrow particle size distribution. The potentials were all near neutral. The dihydroporphyrin E6 nanoclusters prepared in Example 2 had a larger particle size than those prepared in Examples 1 and 3, possibly due to the smaller molecular weight of the carboxylated hydroxyethyl starch used to stabilize the zinc oxide nanoclusters in Example 2. Combined with the drug loading performance results, it can be seen that a larger molecular weight of the carboxylated hydroxyethyl starch used to stabilize the zinc oxide nanoclusters is beneficial for preparing nanoclusters with smaller particle sizes and also for drug loading.

[0079] The prepared nanocluster test solutions were dropped onto copper grids covered with carbon films, allowed to air dry, and the morphology of the nanoclusters was observed using a transmission electron microscope, as shown in the attached figure. Figure 2 To be continued Figure 4 .

[0080] Photoluminescence detection

[0081] The zinc oxide nanocluster dispersion prepared in step (2) of Example 1 and the dihydroporphyrin E6 nanocluster preparation dispersed in 10 mL of ultrapure water prepared in step (3) were diluted with ultrapure water to a zinc oxide concentration of 0.2 mg / mL. The emission spectra of the two were then measured by a fluorescence spectrophotometer with an excitation wavelength of 250 nm.

[0082] like Figure 5 As shown, the zinc oxide nanoclusters prepared in step (2) exhibit a strong emission peak at approximately 400 nm under 250 nm ultraviolet light excitation, while the emission peak at approximately 400 nm of the dihydroporphyrin E6 nanoclusters prepared in step (3) is significantly reduced under 250 nm ultraviolet light excitation. This indicates that the light emitted by zinc oxide at approximately 400 nm in the dihydroporphyrin E6 nanoclusters prepared in step (3) is absorbed by dihydroporphyrin E6, confirming that when zinc oxide in the dihydroporphyrin E6 nanoclusters prepared in step (3) is excited, it can transfer energy to dihydroporphyrin E6.

[0083] X-ray induced singlet oxygen generation detection

[0084] Test solution preparation

[0085] Prepare a 100 μg / mL solution of 1,3-diphenylisobenzofuran in dimethyl sulfoxide by dissolving 1,3-diphenylisobenzofuran in dimethyl sulfoxide. Prepare a 1 mM solution of dihydroporphyrin E6 in dimethyl sulfoxide by dissolving dihydroporphyrin E6 in dimethyl sulfoxide.

[0086] Using the above-mentioned 100 μg / m of 1,3-diphenylisobenzofuran dimethyl sulfoxide solution and 1 mM dihydroporphyrin e6 dimethyl sulfoxide solution as stock solutions, and ultrapure water as diluent, the following solutions were prepared:

[0087] 20 μg / mL 1,3-Diphenylisobenzofuran test solution 1

[0088] Test solution 2, containing both 2 μM dihydroporphyrin E6 and 20 μg / mL 1,3-diphenylisobenzofuran.

[0089] Test solution 3 containing 3 μM dihydroporphyrin e6 and 20 μg / mL 1,3-diphenylisobenzofuran; test solution 4 containing 4 μM dihydroporphyrin e6 and 20 μg / mL 1,3-diphenylisobenzofuran.

[0090] Using the above-mentioned 100 μg / m 1,3-diphenylisobenzofuran dimethyl sulfoxide solution and the dihydroporphyrin E6 nanoclusters dispersed in 10 mL of ultrapure water prepared in step (3) of Example 1 as stock solutions, and ultrapure water as a diluent, the following solutions were prepared separately:

[0091] 5. Test solution of 20 μg / mL 1,3-diphenylisobenzofuran

[0092] Test solution 6, containing both 2 μM dihydroporphyrin E6 and 20 μg / mL 1,3-diphenylisobenzofuran.

[0093] Test solution 7 containing 3 μM dihydroporphyrin e6 and 20 μg / mL 1,3-diphenylisobenzofuran; test solution 8 containing 4 μM dihydroporphyrin e6 and 20 μg / mL 1,3-diphenylisobenzofuran.

[0094] Using the above-mentioned 100 μg / m 1,3-diphenylisobenzofuran dimethyl sulfoxide solution and the zinc oxide nanocluster dispersion prepared in step (2) of Example 1 as mother liquors, and ultrapure water as a diluent, the following preparations were made:

[0095] 9. Test solution of 20 μg / mL 1,3-diphenylisobenzofuran

[0096] Test solution 10 containing both 20 μg / mL zinc oxide and 20 μg / mL 1,3-diphenylisobenzofuran

[0097] Test solution 11 containing 30 μg / mL zinc oxide and 20 μg / mL 1,3-diphenylisobenzofuran, and test solution 12 containing 40 μg / mL zinc oxide and 20 μg / mL 1,3-diphenylisobenzofuran.

[0098] Singlet oxygen detection

[0099] Take 2 mL of each of the above test solutions and irradiate them with 100 kV X-rays at a dose of 5 Gy. The amount of singlet oxygen produced is determined by measuring the decrease in UV absorption of 1,3-diphenylisobenzofuran at 405 nm (singlet oxygen reacts with 1,3-diphenylisobenzofuran, causing a decrease in its UV absorption at 405 nm; the greater the decrease in UV absorption, the more singlet oxygen is generated). See Table 2:

[0100] Table 2: Relative changes in UV absorption of 1,3-diphenylisobenzofuran at 405 nm

[0101]

[0102]

[0103] (In Table 2, in the "UV absorption retention" item, "1" means that the UV absorption of 1,3-diphenylisobenzofuran at 405 nm has not decreased, and the rest represent the remaining proportion after reduction.)

[0104] As shown in Table 2, the free dihydroporphyrin E6 and the zinc oxide nanoclusters prepared in step (2) of Example 1 could not effectively generate singlet oxygen under X-ray irradiation, while the dihydroporphyrin E6-loaded nanoclusters prepared in step (3) of Example 1 showed significant singlet oxygen generation under X-ray irradiation, which was concentration-dependent. This indicates that under X-ray irradiation, zinc oxide absorbs X-ray energy and transfers it to dihydroporphyrin E6, activating dihydroporphyrin E6 and generating X-ray-induced photodynamic effects.

[0105] Evaluation of the effect of X-ray-induced photodynamic killing of tumor cells

[0106] Preparation of test solution

[0107] Using a 1 mM dihydroporphyrin E6 solution in dimethyl sulfoxide as the stock solution and RPMI 1640 medium as the diluent, test solutions with a concentration of 0.5 μM dihydroporphyrin E6 were prepared.

[0108] 2. Test solution with a dihydroporphyrin E6 concentration of 1 μM.

[0109] 3. Test solution with a dihydroporphyrin E6 concentration of 2 μM.

[0110] 4. Test solution with a dihydroporphyrin E6 concentration of 3 μM.

[0111] Test solution 5 with a dihydroporphyrin E6 concentration of 4 μM;

[0112] Using the dihydroporphyrin E6 nanoclusters dispersed in 10 mL of ultrapure water prepared in step (3) of Example 1 as the stock solution, and RPMI 1640 medium as the diluent, separate preparations were made.

[0113] 6. Test solution with a dihydroporphyrin E6 concentration of 0.5 μM

[0114] 7. Test solution with a dihydroporphyrin E6 concentration of 1 μM.

[0115] 8. Test solution with a dihydroporphyrin E6 concentration of 2 μM.

[0116] 9. Test solution with a dihydroporphyrin E6 concentration of 3 μM.

[0117] Test solution 10 with a dihydroporphyrin E6 concentration of 4 μM;

[0118] Using the zinc oxide nanocluster dispersion prepared in step (2) of Example 1 as the stock solution and RPMI 1640 medium as the diluent, further preparations were made separately.

[0119] Test solution 11 with a zinc oxide concentration of 2.5 μg / mL

[0120] Test solution 12 with a zinc oxide concentration of 5 μg / mL

[0121] Test solution 13 with a zinc oxide concentration of 10 μg / mL

[0122] Test solution 14 with a zinc oxide concentration of 15 μg / mL

[0123] Test solution 15 with a zinc oxide concentration of 20 μg / mL.

[0124] Using phosphate buffer as a solvent, prepare a 5 mg / mL solution of 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT), filter it through a 0.22 μm filter membrane, and obtain the MTT solution.

[0125] X-ray-induced photodynamic killing of tumor cells

[0126] Mouse breast cancer 4T1 cells were seeded at a density of 5000 cells per well in 96-well plates and cultured overnight for cell adhesion. The cells were then co-incubated with the prepared experimental solutions described above for 12 hours (n=4), followed by X-ray irradiation at a dose of 5 Gy; alternatively, without X-ray irradiation, the cells were cultured for another 24 hours, then 20 μL of the above-mentioned MTT solution was added, and the cells were cultured for another 4 hours. The culture medium was then aspirated, and 150 μL of dimethyl sulfoxide was added to each well. The cells were shaken to dissolve the precipitate, and the absorbance at 490 nm was measured using a microplate reader. The cell viability in the blank culture medium was considered 100%, and the average cell viability in each experimental solution was calculated. See Tables 3 and 4 for details.

[0127] Table 3: Cell viability in various experimental solutions without X-ray irradiation

[0128]

[0129]

[0130] Table 4: Cell viability in various experimental solutions under X-ray irradiation

[0131]

[0132] Under no radiation exposure, within the measured concentration range, free dihydroporphyrin E6, the dihydroporphyrin E6-loaded nanoclusters prepared in step (3) of Example 1, and the zinc oxide nanoclusters prepared in step (2) of Example 1 all showed no significant cytotoxicity. After 5 Gy of X-ray irradiation at 100 KV, free dihydroporphyrin E6 and the zinc oxide nanoclusters prepared in step (2) of Example 1 still showed no significant cytotoxicity, while the dihydroporphyrin E6-loaded nanoclusters prepared in step (3) of Example 1 exhibited significant tumor cell killing activity in a concentration-dependent manner. This indicates that the singlet oxygen generated by the dihydroporphyrin E6-loaded nanoclusters prepared in step (3) of Example 1 under X-ray irradiation can effectively kill tumor cells and exert X-ray-induced photodynamic therapy.

Claims

1. A method for preparing X-ray-activated dihydroporphyrin E6 nanoclusters, characterized in that: The preparation method is as follows: (1) Preparation of carboxylated hydroxyethyl starch Hydroxyethyl starch and succinic anhydride were dissolved together in dimethyl sulfoxide, and N,N-dimethylaminopyridine was added to the mixture for reaction. After the reaction, the resulting reaction solution was dialyzed in ultrapure water and then freeze-dried to obtain carboxylated hydroxyethyl starch. (2) Preparation of zinc oxide nanoclusters The carboxylated hydroxyethyl starch obtained in step (1) was added to ultrapure water, and zinc oxide nanoparticles were added in a mass ratio of 2 to 6:

1. After ultrasonic dispersion, zinc oxide nanocluster dispersion was obtained. (3) Preparation of dihydroporphyrin e6 nanoclusters According to the mass ratio of dihydroporphyrin E6 to zinc oxide nanoclusters of 0.05–0.1:1, dihydroporphyrin E6 was dissolved in dimethyl sulfoxide and then added to the zinc oxide nanoclusters dispersion obtained in step (2). After thorough ultrasonic dispersion, the mixture was dialyzed and then centrifuged to obtain zinc oxide nanoclusters loaded with dihydroporphyrin E6. In step (1), the molecular weight of hydroxyethyl starch is 130,000 to 450,000 and the degree of hydroxyethyl substitution is 0.4 to 0.

5. The mass ratio of hydroxyethyl starch to succinic anhydride is 6 to 20:1, the molar ratio of succinic anhydride to N,N-dimethylaminopyridine is 1:0.5, and the mixing reaction is carried out by stirring at room temperature for 24 hours. In step (2), the diameter of the zinc oxide nanoparticles is 30±10nm.

2. The method for preparing X-ray-activated dihydroporphyrin E6 nanoclusters as described in claim 1, characterized in that: In step (2), the ultrasonic power of the ultrasonic dispersion is 37.5W and the ultrasonic time is 5 minutes.

3. The method for preparing X-ray-activated dihydroporphyrin E6 nanoclusters as described in claim 1, characterized in that: In step (3), the ultrasonic power of the ultrasonic dispersion is 37.5W and the ultrasonic time is 5 minutes.

4. The method for preparing X-ray-activated dihydroporphyrin E6 nanoclusters as described in claim 1, characterized in that: In step (3), the centrifugation speed is 12,000 rpm and the centrifugation time is 10 minutes.

5. An X-ray-activated dihydroporphyrin E6 nanocluster obtained by the preparation method according to any one of claims 1 to 4.

Images