Composite nanoparticles loaded with doxorubicin

Abstract

A composite nanoparticle loaded with doxorubicin belongs to the technical field of nano-biomedical materials. It uses mesoporous dopamine as a carrier, modifies reduced gadolinium phosphomolybdate on its surface, further wraps chitosan on the surface, and finally loads the drug doxorubicin to make composite nanoparticles, wherein the reduced gadolinium phosphomolybdate is dissolved in water, glutathione is added, stirred in the dark at room temperature, then anhydrous ethanol is added to precipitate a blue precipitate, centrifuged, washed with water and ethanol, and vacuum dried to obtain the obtained composite nanoparticles. The amount ratio of gadolinium phosphomolybdate, water, and GSH is 20 mg: 10~12 mL: 90~110 mg. The nanoparticles of the present invention have excellent photothermal conversion efficiency, reaching 48.6%. In the composite nanoparticles, the drug loading of DOX reaches 42.5%, and the release efficiency in the tumor environment is nearly 81%. The composite nanoparticles effectively play the synergistic treatment of photothermal-chemodynamic-chemotherapy and improve the therapeutic effect on tumors.

Description

[0001] This invention is a divisional application of patent application number "202410637176.1", and the invention name is "A kind of mesoporous polydopamine-based composite drug-loaded nanoparticles and their preparation and application". Technical Field

[0002] The invention relates to the technical field of nano biomedical materials, and in particular to composite nanoparticles loaded with doxorubicin. Background Art

[0003] Commonly used tumor treatments include chemotherapy, surgery, and radiotherapy. These traditional treatments cannot completely cure tumors and lack specificity, and there are still some problems. Therefore, it is necessary to develop some new treatments to overcome the above shortcomings. Compared with traditional cancer drug molecules, a major advantage of nanomaterials is that they can integrate multiple treatment modes into one particle. By utilizing the high permeability and high retention effect (Enhanced permeation and retention, EPR) of nanomaterials or grafting targeting molecules, nanomaterials can be transported to the lesion site. At the same time, multiple combined treatment modes can work synergistically to achieve better anti-cancer effects and reduce unnecessary side effects.

[0004] Chemodynamic therapy (CDT) can catalyze hydrogen peroxide (H2O2) in the tumor area to produce highly toxic hydroxyl radicals (·OH). This method shows high tumor specificity due to its intrinsic characteristics of pH and H2O2 dependence. Photothermal therapy (PTT) is a minimally invasive treatment method that uses near-infrared light to irradiate materials with high photothermal conversion efficiency to increase the local temperature of the tumor to above 42°C, thereby killing tumor cells. Therefore, the invention of a multifunctional nanocomposite material that can synergistically treat tumors with multiple treatment modes is crucial to achieve effective tumor treatment. It also provides an efficient and economical strategy for the application of nanocomposites in the field of synergistic tumor treatment. Summary of the invention

[0005] The present invention aims to provide a composite drug-loaded nanoparticle based on mesoporous polydopamine, specifically a mesoporous polydopamine / gadolinium phosphomolybdate@chitosan composite nanoparticle loaded with doxorubicin.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned mesoporous polydopamine / gadolinium phosphomolybdate@chitosan composite nanoparticles loaded with doxorubicin.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A composite nanoparticle loaded with doxorubicin, characterized in that: the composite nanoparticle uses mesoporous dopamine as a carrier (MPDA), and the surface of the composite nanoparticle is modified with reduced gadolinium phosphomolybdate (r-GdPMo) to form MPDA-GdPMo nanoparticles, and chitosan (Cs) is further coated on the surface to load the drug doxorubicin (DOX) to prepare MPDA-GdPMo@CS-DOX composite nanoparticles, the reduced gadolinium phosphomolybdate is prepared by dissolving gadolinium phosphomolybdate in water, adding glutathione (GSH), stirring at room temperature in the dark for 10-12 hours, then adding anhydrous ethanol to precipitate a blue precipitate, centrifuging, washing with water and ethanol, and vacuum drying, and the amount ratio of the gadolinium phosphomolybdate, water, and GSH is 20 mg: 10-12 mL: 90-110 mg.

[0009] Furthermore, the mesoporous polydopamine is prepared by dissolving poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (F127) and 1,3,5-trimethylbenzene (TMB) in a mixed solvent of water and ethanol to form a mixed solution, adding trimethylamine hydrochloride (TRIS) aqueous solution and dopamine hydrochloride to the mixed solution, stirring at room temperature for 20 to 24 hours, centrifuging and washing to obtain nanospheres.

[0010] Furthermore, the amount ratio of F127, TMB and mixed solvent is 1g:1g:330~340mL, the volume ratio of water and ethanol in the mixed solvent is 1:1, the amount ratio of the mixed solution, TRIS aqueous solution and dopamine hydrochloride is 12~14mL:0.8~1mL:5.5~6.5mg, and the mass concentration of TRIS aqueous solution is 90%.

[0011] Furthermore, the MPDA-GdPMo nanoparticles are prepared by dispersing MPDA nanoparticles in water, then adding r-GdPMo, stirring for 20-24 hours, centrifuging, and washing to obtain MPDA-GdPMo nanoparticles, wherein the amount ratio of MPDA nanoparticles, water, and r-GdPMo is 8-10 mg:18-24 mL:8-10 mg.

[0012] A method for preparing mesoporous polydopamine / gadolinium phosphomolybdate / chitosan / adriamycin composite nanoparticles, characterized in that it comprises the following steps:

[0013] (1) Synthesis of mesoporous polydopamine nanocarriers (MPDA)

[0014] F127 and TMB were dissolved in a mixed solvent of water and ethanol to form a mixed solution, TRIS aqueous solution and dopamine hydrochloride were added to the mixed solution, stirred at room temperature for 20-24 hours, centrifuged and washed to obtain mesoporous polydopamine (MPDA) nanospheres;

[0015] (2) Synthesis of reduced gadolinium phosphomolybdate (r-GdPMo)

[0016] Dissolve gadolinium phosphomolybdate (GdPMo) in water, add glutathione (GSH), stir at room temperature in the dark for 10-12 h, then add anhydrous ethanol to precipitate, centrifuge, wash, and dry to obtain reduced gadolinium phosphomolybdate (GdPMo);

[0017] (3) Synthesis of MPDA-GdPMo nanoparticles

[0018] MPDA nanoparticles were dispersed in water, and then r-GdPMo was added, stirred for 20-24 h, centrifuged, and washed to obtain MPDA-GdPMo nanoparticles;

[0019] (4) Synthesis of MPDA-GdPMo@CS-DOX nanoparticles

[0020] Take chitosan (CS) solution, adjust its pH to 6.0, add MPDA-GdPMo nanoparticles and doxorubicin (DOX) aqueous solution in sequence, stir for 20-24 h, centrifuge, and wash with water to obtain MPDA-GdPMo@CS-DOX NPs nanoparticles.

[0021] Furthermore, in the mixed solution of step (1), the ratio of F127, TMB and mixed solvent is 1 g:1 g:330-340 mL, and the volume ratio of water to ethanol in the mixed solvent is 1:1.

[0022] Furthermore, in the step (1), the dosage ratio of the mixed solution, the TRIS aqueous solution, and dopamine hydrochloride is 12-14 mL: 0.8-1 mL: 5.5-6.5 mg, and the mass concentration of the TRIS aqueous solution is 90%.

[0023] Furthermore, in step (2), gadolinium phosphomolybdate (GdPMo) is prepared by dissolving Na2MoO4·2H2O in water, adding H3PO4 solution with a mass concentration of 85% and concentrated HCl, mixing, boiling and refluxing, adding NH4Cl after cooling, collecting the precipitate by suction filtration, dissolving the precipitate in water, adding concentrated HCl and ether for extraction, and obtaining H6[P2Mo 18 O 62 ] etherate, add the etherate into water, remove ether with cold air, add Gd(NO3)3·6H2O solution, adjust pH to 3.8-4.2, leave for 2 h under stirring, then add KCl, generate precipitate under constant stirring, cool in ice bath for 15-25 min, filter, wash, recrystallize and dry to obtain the product.

[0024] Furthermore, the usage ratio of the Na2MoO4·2H2O, water, H3PO4 solution, concentrated HCl and NH4Cl is 100g: 400-500mL: 12-18mL: 70-90mL: 100-110g, and the mass concentration of the H3PO4 solution is 85%.

[0025] Furthermore, the usage ratio of the etherate, water, Gd(NO3)3·6H2O and KCl is 50 mL: 50-70 mL: 7-9 g: 25 g.

[0026] Furthermore, in the step (2), the usage ratio of GdpMo, water and GSH is 20 mg: 10-12 mL: 90-110 mg.

[0027] Furthermore, in the step (3), the ratio of the amount of MPDA nanoparticles, water and r-GdPMo is 8-10 mg:18-24 mL:8-10 mg.

[0028] Furthermore, in step (4), the mass concentration of the CS solution is 2%, the dosage ratio of the CS solution, the MPDA-GdPMo nanoparticles and the doxorubicin aqueous solution is 0.8-1.2 mL: 1-1.2 mg: 0.8-1.2 mL, and the concentration of the DOX aqueous solution is 0.8-1.2 mg·mL.

[0029] Polydopamine (PDA) has good biocompatibility and good photothermal conversion ability, and can be used as a drug carrier for both photothermal therapy and chemotherapy. At the same time, its structure contains abundant benzene rings, amino groups and hydroxyl groups, making it easy to be modified. Mo-containing polyacid salts have a small size and good water solubility, and are highly sensitive to pH and reducing environments. Glutathione (GSH) can reduce gadolinium molybdate to convert Mo 6+ Reduction to Mo 5+ , reduced gadolinium phosphomolybdate can be used as Fenton reagent, Mo 5+ It can react with H2O2 to produce ·OH. The mesoporous polydopamine / gadolinium phosphomolybdate nanoparticles are further loaded with doxorubicin hydrochloride (DOX) anti-tumor drugs, which simultaneously play an anti-tumor role in the synergistic treatment of PTT, CDT and chemotherapy, achieving enhanced tumor treatment effects.

[0030] In the process of using polydopamine as a carrier to load DOX, it was found that CS was wrapped on the surface of polydopamine to give the drug release a pH response, thereby improving its drug release efficiency. However, this resulted in an unsatisfactory DOX loading amount and the synergistic anti-tumor effect did not meet expectations.

[0031] In order to solve the problem of DOX loading efficiency, the present invention prepares polydopamine with a mesoporous structure, and then modifies the surface of polydopamine with reduced gadolinium phosphomolybdate, adjusts the surface structure of polydopamine, and subsequently encapsulates chitosan, which also affects the surface structure of chitosan accordingly, thereby improving the loading of DOX and increasing the loading amount of DOX on the CS surface. In addition to the newly added chemical kinetic properties, the reduced structure of gadolinium phosphomolybdate also has photothermal conversion properties. When it modifies mesoporous polydopamine, it produces a synergistic effect with mesoporous polydopamine, enhancing the release rate of DOX in the tumor environment, thereby enhancing the chemotherapy performance of nanoparticles. The photothermal conversion ability of mesoporous polydopamine and the continuous consumption of GSH in tumor cells further promote the CDT effect. This structure effectively synergizes PTT, CDT and chemotherapy on tumor cells, achieving excellent anti-tumor effects.

[0032] Most specifically, a method for preparing mesoporous polydopamine / gadolinium phosphomolybdate / chitosan / adriamycin composite nanoparticles is characterized by comprising the following steps:

[0033] (1) Synthesis of MPDA

[0034] Take 0.3-0.4 g F127 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) and 0.3-0.4 g TMB (1,3,5-trimethylbenzene) and dissolve them in a mixed solution of 100-120 mL water and ethanol in a volume ratio of 1:1, stir for 30 minutes, add 8-10 mL of 90% mass concentration of TRIS (trimethylamine hydrochloride) aqueous solution and 55-65 mg dopamine hydrochloride to the above solution, stir at room temperature for 20-24 h, centrifuge, and wash with ethanol and acetone to obtain mesoporous polydopamine (MPDA) nanospheres;

[0035] (2) Synthesis of GdPMo

[0036] Dissolve 100 g Na2MoO4·2H2O in 400-500 mL water, add 12-18 mL 85% H3PO4 solution and 70-90 mL 12 mol / L concentrated HCl, boil the mixed solution under reflux for 6-9 h, cool, add 100-110 g NH4Cl, filter to obtain a precipitate, dissolve the precipitate in water, add concentrated HCl and ether to extract, and obtain H6[P2Mo 18 O 62] etherate. Take 50 mL of the etherate and add water to 100~120 mL, remove the ether with cold air, add 7~9gGd(NO3)3·6H2O, adjust the pH to 3.8~4.2, place under stirring for 2 h, then add 25g KCl solid or an equivalent amount of KCl saturated solution to the solution, and a precipitate will form under constant stirring. After cooling in an ice bath for 20 min, filter, wash, and recrystallize three times with sulfuric acid aqueous solution with a pH of 3.7, and obtain the product GdPMo after vacuum drying;

[0037] (3) Synthesis of r-GdPMo

[0038] Take 20 mg of GdPMo and dissolve it in 10-12 mL of water, add 90-110 mg of glutathione (GSH), stir at room temperature in the dark for 10-12 h, then add anhydrous ethanol to precipitate a blue precipitate, centrifuge, wash with water and ethanol, and dry in vacuum;

[0039] (4) Synthesis of MPDA-GdPMo nanoparticles

[0040] Weigh 8-10 mg of MPDA nanoparticles and disperse them in 18-24 mL of water, then add 8-10 mg of r-GdPMo, stir for 20-24 h, centrifuge, and wash three times to obtain MPDA-GdPMo nanoparticles;

[0041] (5) Synthesis of MPDA-GdPMo@CS-DOX nanoparticles

[0042] Take 8-12 mL of 2% chitosan (CS) solution, adjust its pH to 5.8-6.2, and add 10-12 mg of MPDA-GdPMo NPs and 8-12 mL, 0.8-1.2 mg mL -1 The doxorubicin (DOX) aqueous solution was stirred for 20-24 h, centrifuged, and washed three times with water to obtain MPDA-GdPMo@CS-DOX nanoparticles.

[0043] The present invention has the following technical effects:

[0044] The present invention uses mesoporous polydopamine modified with gadolinium phosphomolybdate as a carrier, and then coats the surface with chitosan and loads DOX. It has an excellent photothermal conversion efficiency of 48.6%, effectively adjusts the drug loading efficiency of DOX, and improves the release rate of DOX in the tumor environment. The drug loading of DOX reaches 42.5%, and the release efficiency in the tumor environment is nearly 81%. The drug-loaded nanoparticles have uniform particle size and good stability and dispersibility in biological water. The nanoparticles effectively play a synergistic role in photothermal-chemodynamic-chemotherapy and improve the therapeutic effect on tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 :TEM images of different nanoparticles (ac); SEM images of different nanoparticles (df).

[0046] Figure 2 : Hydration particle size distribution of different nanoparticles.

[0047] Figure 3 : Zeta potential diagram of different nanoparticles.

[0048] Figure 4 : Temperature rise curves of MPDA-GdPMo@CS nanoparticle aqueous solutions with different concentrations under laser irradiation.

[0049] Figure 5 : Photostability of MPDA-GdPMo@CS nanoparticles with different concentrations under laser irradiation.

[0050] Figure 6 : The cooling curve of MPDA-GdPMo@CS nanoparticles after laser irradiation for 600 s and the thermal conversion time constant of the cooling process were calculated.

[0051] Figure 7 :The effects of different nanoparticles on ROS production.

[0052] Figure 8 : UV absorption spectra of MPDA-GdPMo@CS nanoparticles before and after the action of DOX solution.

[0053] Fig. 9 : DOX release curves of MPDA-GdPMo@CS-DOX nanoparticles at different pH values ​​(a), and DOX release curves under laser irradiation (b).

[0054] Fig.10 : Cell viability of three tumor cells after co-incubation with MPDA-GdPMo@CS nanoparticles for 24 and 48 h.

[0055] Fig.11 :The hemolysis percentage after the interaction between MPDA-GdPMo@CS nanoparticles and red blood cells at different concentrations

[0056] Fig.12 : CLSM images of ROS produced in HepG2 cells under different treatment conditions.

[0057] Fig.13 : Synergistic therapeutic effect of MPDA-GdPMo@CS nanoparticles loaded with DOX drug.

[0058] Fig.14: Fluorescence microscopy images of HepG2 cells treated with different treatments (scale bar: 100 μm).

[0059] Fig.15 : Example 2 Synergistic therapeutic effect of MPDA-GdPMo@CS nanoparticles loaded with DOX drug.

[0060] Fig.16 : Example 3 Synergistic therapeutic effect of MPDA-GdPMo@CS nanoparticles loaded with DOX drug. DETAILED DESCRIPTION

[0061] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0062] Example 1

[0063] A composite nanoparticle loaded with doxorubicin is prepared according to the following steps:

[0064] (1) Synthesis of MPDA

[0065] 0.36 g F127 and 0.36 g TMB were dissolved in a mixed solution of water (60 mL) and ethanol (60 mL), stirred for 30 minutes, 10 mL of 90% mass concentration TRIS aqueous solution and 60 mg dopamine hydrochloride were added to the above solution, stirred at room temperature for 24 h, centrifuged, and washed with ethanol and acetone to obtain mesoporous polydopamine (MPDA) nanospheres;

[0066] (2) Synthesis of GdPMo

[0067] Dissolve 100 g Na2MoO4·2H2O in 450 mL water, add 15 mL 85% H3PO4 solution and 80 mL concentrated HCl, boil the mixed solution under reflux for 8 h, cool, add 100 g NH4Cl, filter to obtain a precipitate, dissolve the precipitate in water, add concentrated HCl and ether to extract, and obtain H6[P2Mo 18 O 62] etherate. Take 50 mL of the etherate and add water to 100 mL, remove the ether with cold air, add 8g Gd(NO3)3·6H2O, add 50% NaAc solution while stirring at room temperature until the pH of the solution is close to 4.0, place it under stirring for 2 h, and then add 25 g KCl to the solution. A precipitate is generated under constant stirring. After cooling in an ice bath for 20 min, filter, wash, and recrystallize three times with a sulfuric acid aqueous solution with a pH of 3.7. After vacuum drying, the product GdPMo is obtained;

[0068] (3) Synthesis of r-GdPMo

[0069] Take 20 mg of GdPMo and dissolve it in 10 mL of water, add 100 mg of glutathione (GSH), stir at room temperature in the dark for 12 h, then add anhydrous ethanol to precipitate a blue precipitate, centrifuge, wash with water and ethanol, and dry in vacuum;

[0070] (4) Synthesis of MPDA-GdPMo nanoparticles

[0071] 10 mg of MPDA nanoparticles were dispersed in 20 mL of water, and then 10 mg of r-GdPMo was added. After stirring for 24 h, the mixture was centrifuged and washed three times with water to obtain MPDA-GdPMo nanoparticles.

[0072] (5) Synthesis of MPDA-GdPMo@CS-DOX nanoparticles

[0073] Take 10 mL of 2% chitosan (CS) solution, adjust its pH to 6.0 with 0.5 M NaOH, and add 10 mg of MPDA-GdPMo NPs and 10 mL, 1 mg mL -1 The doxorubicin (DOX) aqueous solution was stirred for 24 h, centrifuged, and washed three times with water to obtain MPDA-GdPMo@CS-DOX nanoparticles.

[0074] Synthesis and characterization of MPDA-GdPMo@CS-R particles:

[0075] The morphology of nanoparticles was characterized by Figure 1As shown in the figure, ac represents the TEM images of MPDA, MPDA-GdPMo and MPDA-GdPMo@CS nanoparticles, respectively, and df represents the SEM images of MPDA, MPDA-GdPMo and MPDA-GdPMo@CS nanoparticles, respectively. It can be seen from the TEM and SEM images that the MPDA surface has a mesoporous structure and the particle size is about 110 nm. When the surface is loaded with GdPMo polyacid, the amino groups in MPDA can be combined with the oxygen atoms in GdPMo polyacid through hydrogen bonds, and the mesopores on the MPDA surface are covered to a certain extent. The mesopores on the surface of MPDA-GdPMo particles are not as obvious as those on the surface of MPDA, and the particle size is about 120 nm. After the MPDA-GdPMo nanoparticles are coated with chitosan (CS), it can be seen that the mesoporous structure on the surface of the MPDA-GdPMo nanoparticles completely disappears, and a layer of chitosan is coated on the outside, and the size of the MPDA-GdPMo@CS nanoparticles increases to 165 nm.

[0076] The hydrated particle sizes of MPDA nanoparticles, MPDA-GdPMo nanoparticles, and MPDA-GdPMo@CS nanoparticles were further measured. Figure 2 As shown in the figure, they are 125 nm, 164 nm and 223 nm respectively. The particle size results are slightly larger than the nanoparticle size results characterized by TEM. This is because the nanoparticles associate with water molecules in the solution to form a hydration film. The above results also show that the particle sizes of MPDA nanoparticles, MPDA-GdPMo nanoparticles, and MPDA-GdPMo@CS nanoparticles are uniform and have good stability and dispersibility in water.

[0077] Figure 3 The figure is the Zeta potential diagram of each particle. It can be seen that the Zeta potential of MPDA nanoparticles is -48 mV, and the Zeta potential of MPDA-GdPMo is -30 mV. After MPDA is loaded with GdPMo polyacid, the potential increases, indicating that GdPMo polyacid is loaded on MPDA; in addition, after MPDA-GdPMo is coated with chitosan (CS) on the surface, the Zeta potential increases to 14.6 mV, which also shows that chitosan successfully wraps MPDA-GdPMo. After the anti-tumor drug doxorubicin (DOX) is loaded on the surface of the nanocarrier MPDA-GdPMo@CS, the Zeta potential increases to 23.2 mV. The results show that DOX is loaded on the MPDA-GdPMo@CS nanoparticles in large quantities.

[0078] Performance Testing:

[0079] 1. Photothermal conversion effect of MPDA-GdPMo@CS nanospheres

[0080] To investigate the photothermal conversion effect of MPDA-GdPMo@CS nanoparticles, near-infrared (NIR) laser (808 nm, 1.0 W cm -2 Five groups of 200 µL of different concentrations (0, 50, 100, 200 and 500 µg mL -1 ) of MPDA-GdPMo@CS nanoparticle solution for 600 s, and record the temperature change every 30 s. After completion, stop irradiation and record the cooling process of the solution. Under the same conditions, deionized water was irradiated with laser as a control group. The photothermal conversion efficiency ( η ):

[0081]

[0082] h is the heat transfer coefficient, S is the surface area of ​​the container, T max is the equilibrium temperature, T sur is the ambient temperature, Q dis is the heat absorbed by the solvent, I is the power of the irradiating light (1.0 W cm -2 ), A 808 is the UV-vis absorbance value of MPDA-GdPMo@CS nanoparticle solution at 808 nm. The value of hS can be calculated according to formula (2):

[0083]

[0084] τ s is the sample system time constant, m D and C D is the mass and specific heat capacity of water as solvent.

[0085] The photothermal conversion ability of MPDA-GdPMo@CS nanoparticles was analyzed. Figure 4 Figure 2 shows the temperature change of MPDA-GdPMo@CS nanoparticles with different concentrations under 808 nm laser irradiation. After 10 min of irradiation, the concentration was 500 g·mL -1 The MPDA-GdPMo@CS nanoparticle solution can be heated to 71.5°C, while the temperature of deionized water has hardly changed. And after four repeated laser irradiation cooling cycles, the temperature rise of MPDA-GdPMo@CS nanoparticles has not decreased significantly. Figure 5 The results show that MPDA-GdPMo@CS nanoparticles have good photothermal stability and exhibit concentration / irradiation time-dependent photothermal behavior. Figure 6(a) The cooling curve of MPDA-GdPMo@CS nanoparticles after 808 nm laser irradiation for 600 s and then turning off the laser. (b) The thermal conversion time constant of the system is calculated from the system cooling process. τ s The photothermal conversion efficiency of MPDA-GdPMo@CS nanoparticles is 48.6% according to the calculation formula.

[0086] 2. Chemical kinetics performance test

[0087] In order to test the ROS generation effect of MPDA-GdPMo@CS nanoparticles, we selected o-phenylenediamine (OPD) as a ROS scavenger. This is because ROS can catalyze the oxidation of OPD into yellow 2,3-diaminophenazine (DAP), causing the solution to change from colorless to yellow. DAP has a characteristic absorption peak at 420 nm, and the generated ROS can be detected by UV-vis.

[0088] MPDA-GdPMo@CS nanoparticles were prepared at different concentrations (400, 200, 100, 50 and 25 µg mL -1 ) were dispersed into 1 mL of a mixed solution in which the concentration of H2O2 was 100 mM and the concentration of OPD was 1.0 mM. After reacting in the dark for 2 h, the mixture was centrifuged at 9000 rpm for 10 min. The absorbance of the supernatant was measured to demonstrate the effect of particle concentration on the production of ROS.

[0089] Figure 7 Effects of different nanoparticles on ROS production (a: blank; b: 100 μg / mL MPDA; c: 100 μg / mL CS; dh: 25, 50, 100, 200 and 400 µg·mL -1 It can be seen that the reaction of MPDA, CS and OPD does not produce an obvious absorption peak at 425 nm, indicating that neither MPDA nor CS can react with OPD to produce ROS. However, the reaction of MPDA-GdPMo@CS nanoparticles with OPD produces an absorption peak at 425 nm, and the intensity of the absorption peak at 425 nm increases with the increase of particle concentration, indicating that the reaction of MPDA-GdPMo@CS nanoparticles with H2O2 produces ROS, which has a chemical kinetic effect.

[0090] 3. Drug loading efficiency of MPDA-GdPMo@CS-DOX nanoparticles

[0091] The synthesized MPDA-GdPMo@CS-DOX nanoparticles were centrifuged and washed three times to remove the unloaded DOX, and the supernatant was collected. The supernatant and the original drug solution were tested by UV spectroscopy. The UV absorption peak of DOX was located at 480 nm. By comparing with the standard curve, the content of DOX loaded into the nanoparticles was calculated. The drug loading efficiency can be calculated according to formula (3):

[0092]

[0093] Figure 8 The UV absorption spectra of DOX before (red) and after (black) mixing with MPDA-GdPMo@CS nanoparticles. As can be seen from the figure, after DOX is mixed with the particles, the absorption intensity at 480 nm is significantly reduced, indicating that MPDA-GdPMo@CS nanoparticles are successfully loaded with DOX, and the drug loading is calculated to be about 42.5%.

[0094] 4. In vitro drug release

[0095] The drug release rate of MPDA-GdPMo@CS-DOX nanoparticles was evaluated by dialysis. First, 3 ml of PBS solution containing 3 mg of drug-loaded MPDA-GdPMo@CS-DOX nanoparticles was loaded into two dialysis bags (MWCO 4000 Da) at room temperature, where the pH values ​​of the PBS solutions were 7.4 and 5.3, respectively. Then, the dialysis bags were placed in PBS solutions with corresponding pH values. Within the specified time period, the release efficiency of MPDA-GdPMo@CS-DOX nanoparticles was measured by UV at 480 nm.

[0096] To verify the efficacy of the drug under 808 nm laser irradiation (intensity 1.0 W cm -2 , time 5 min) can improve the release efficiency. We used the same experimental method as above to prepare the MPDA-GdPMo@CS-DOX nanoparticle solution with a pH value of 5.3 and irradiated it within the specified time. The release efficiency is calculated by formula (4):

[0097] (4)

[0098] Fig. 9a shows the release of DOX from MPDA-GdPMo@CS nanoparticles loaded with DOX in the environment of normal tissue pH = 7.4 and tumor environment pH = 5.3 simulated by PBS. When there is no 808 nm laser irradiation and the release time reaches 108 h, the DOX release amount is about 10.9% at pH = 7.4, while at pH = 5.3, DOX is continuously released with a cumulative release amount of about 65.2%. It can be seen that the MPDA-GdPMo@CS nanoparticles loaded with DOX have a DOX release behavior that responds to pH changes, reducing the damage of DOX to normal tissues while enhancing the killing effect on tumors. In addition, we also used a power of 1 W·cm -2 The results are shown in Figure 2. Fig. 9 As shown in b, nearly 81% of DOX was released. This confirms that the photothermal effect increases the temperature to promote the release of DOX. The accelerated drug release rate can be attributed to the photothermal effect of MPDA-GdPMo@CS nanoparticles, which can convert light into heat to stimulate drug release. The above results confirm that MPDA-GdPMo@CS nanoparticles have the properties of pH / NIR dual response to release drugs, which improves traditional chemotherapy.

[0099] 5. Cytotoxicity and hemolysis analysis:

[0100] (1) Cytotoxicity:

[0101] The cytotoxicity of MPDA-GdPMo@CS nanoparticles was detected by MTT method. HepG2, MCF-7 and Hela cells were selected for detection. The three cultured cells were inoculated at a cell density of 1.0×10 4 The cells were plated on a 96-well plate and incubated overnight. Then different concentrations of nanoparticles (3.125, 6.25, 12.5, 25, 50, and 100 μg mL -1 ) were added to the cells and cultured for 24 h and 48 h. After the culture was completed, the cell activity detected by MTT method was calculated using formula (5):

[0102] (5)

[0103] The biosafety of MPDA-GdPMo@CS nanoparticles was tested by incubating them with tumor cells for 24 h and 48 h. Fig.10 As shown in Figure 2, when the concentration of MPDA-GdPMo@CS nanoparticles is as high as 100 g mL -1The activity of the three cell lines (HeLa, MCF-7, and HepG2) was as high as 90%, confirming that MPDA-GdMo@CS nanoparticles have high biosafety.

[0104] (2) Hemolysis test:

[0105] The blood compatibility of the nanoparticles was evaluated by a human blood hemolysis test. First, red blood cells were washed five times by centrifugation with normal saline. Then, 0.3 ml of red blood cell suspension diluted 10 times with PBS was taken, and 1.2 mL of PBS (-) and 1.2 mL of distilled water (+) were added as positive and negative controls. In addition, the nanoparticles were prepared into 1.2 mL of PBS suspensions of different concentrations (12.5, 25, 50, 100, 200, 400 and 800 g mL -1 ), add 0.3 mL of diluted red blood cells respectively. After 2 h, collect the supernatant by centrifugation, place the supernatant in a UV-vis spectrophotometer, and measure its absorbance. Use formula (6) to calculate the hemolysis percentage of red blood cells:

[0106] (6)

[0107] Fig.11 The hemolysis percentages of MPDA-GdPMo@CS nanoparticles and erythrocytes after different concentrations of the nanoparticles are reacted. The inset is the corresponding hemolysis test picture. It can be observed that in the erythrocyte hemolysis test, even if the concentration of MPDA-GdPMo@CS nanoparticles is 800 g·mL -1 , and no large amount of hemolysis occurred. This phenomenon further proves that MPDA-GdPMo@CS nanoparticles have good blood compatibility.

[0108] 6. Analysis of the ROS production effect of MPDA-GdPMo@CS nanoparticles at the cellular level

[0109] Since the H2O2 content in tumor cells is higher than that in normal cells, in order to confirm the effect of MPDA-GdPMo@CS nanoparticles in producing highly toxic ROS at the cellular level, we used 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) reagent to detect the ROS content in HepG2 cells. DCFH-DA can react with ROS and show green fluorescence under CLSM.

[0110] First, select a 6-well plate and culture 1 mL of HepG2 cells in the plate to make the cell concentration 1×10 4 1 mL of nanoparticles (25 and 50 μg mL -1) and 10 mM H2O2 were dissolved in the cell culture medium after washing with PBS, and incubated for another 6 h. After the incubation, the cells were washed three times with cold PBS, and then 10 mM DCFH-DA indicator was added and incubated for 30 minutes at room temperature. Finally, the cells were washed three times with PBS, and the cells were observed under a fluorescence microscope.

[0111] like Fig.12 As shown in the figure, compared with the control group, the MPDA group did not produce green fluorescence signals, and the intracellular H2O2 6+ / Mo 5+ The MPDA-GdPMo@CS nanoparticles catalyze redox reactions to generate highly toxic ROS. The green fluorescence signal is strong, and the higher the concentration, the stronger the green fluorescence signal, which means that more ROS are produced.

[0112] 7. Photothermal-chemokinetic-chemotherapy synergistic effect of MPDA-GdPMo@CS-DOX nanoparticles

[0113] The standard MTT method was used to detect the in vitro antitumor activity of different treatment groups on HepG2 cells. Specifically, 6-well plates were selected and 1 mL of HepG2 cells were cultured in the plates to a cell concentration of 1×10 4 The nanoparticles were added to the serum-free culture medium and replaced the culture medium of the original cells. The serum-free culture medium group was used as the control group. Fig.13 As shown in the figure, NBs represents MPDA-GdPMo@CS nanoparticles, and NBs+DOX represents MPDA-GdPMo@CS nanoparticles loaded with DOX. As the concentration increases, the cell viability decreases, indicating that different treatment groups show concentration dependence on cell death. In comparison, under the same concentration conditions, the MPDA-GdPMo@CS nanoparticles loaded with DOX+H2O2+light irradiation group showed better anti-tumor activity.

[0114] Cell imaging after Calcein AM / PI staining was observed under a fluorescence microscope. In the Calcein AM / PI double staining experiment, HepG2 cells treated with MPDA-GdPMo@CS nanoparticles alone showed a bright green color, indicating that our nanoparticles were not toxic to cells. At the same time, after treatment with MPDA-GdPMo@CS nanoparticles loaded with DOX + H2O2 + light, the number of surviving cells was significantly reduced, such as Fig.14 As shown. This indicates that MPDA-GdPMo@CS nanoparticles can be used as ideal agents for photothermal-chemodynamic-chemotherapy synergistic treatment of tumors.

[0115] Example 2

[0116] A composite nanoparticle loaded with doxorubicin is prepared according to the following steps:

[0117] (1) Synthesis of MPDA

[0118] 0.3 g F127 and 0.3 g TMB were dissolved in 100 mL of a mixed solution of water and ethanol in a volume ratio of 1:1, stirred for 30 min, 10 mL of a 90% TRIS aqueous solution and 55 mg of dopamine hydrochloride were added to the solution, stirred at room temperature for 20 h, centrifuged, and washed with ethanol and acetone to obtain mesoporous polydopamine (MPDA) nanospheres;

[0119] (2) Synthesis of GdPMo

[0120] 100 g Na2MoO4·2H2O was dissolved in 400 mL water, 12 mL 85% H3PO4 solution and 70 mL concentrated HCl were added, the mixed solution was boiled and refluxed for 6 h, cooled, 100 g NH4Cl was added, the precipitate was filtered, the precipitate was dissolved in water, concentrated HCl and ether were added for extraction, and H6[P2Mo 18 O 62 ] ether compound. Take 50 mL of the ether compound and add water to 120 mL, remove the ether with cold air, add 7g Gd(NO3)3·6H2O, adjust the pH to 3.8, place it under stirring for 2 h, then add 25 g KCl to the solution, and a precipitate will form under constant stirring. After cooling in an ice bath for 20 min, filter, wash, and recrystallize three times with a sulfuric acid aqueous solution with a pH of 3.7. After vacuum drying, the product GdPMo is obtained;

[0121] (3) Synthesis of r-GdPMo

[0122] Take 20 mg of GdPMo and dissolve it in 12 mL of water, add 110 mg of glutathione (GSH), stir at room temperature in the dark for 10 h, then add anhydrous ethanol to precipitate a blue precipitate, centrifuge, wash with water and ethanol, and dry in vacuum;

[0123] (4) Synthesis of MPDA-GdPMo nanoparticles

[0124] 10 mg of MPDA nanoparticles were dispersed in 24 mL of water, and then 10 mg of r-GdPMo was added. After stirring for 22 h, the mixture was centrifuged and washed three times with water to obtain MPDA-GdPMo nanoparticles.

[0125] (5) Synthesis of MPDA-GdPMo@CS-DOX nanoparticles

[0126] Take 12 mL of 2% chitosan (CS) solution, adjust its pH to 6.2, and add 12 mg of MPDA-GdPMo NPs and 8 mL, 1.2 mg mL -1 The doxorubicin (DOX) aqueous solution was stirred for 22 h, centrifuged, and washed three times with water to obtain MPDA-GdPMo@CS-DOX nanoparticles.

[0127] In the composite nanoparticles prepared in this example, the drug loading of DOX was about 41.4%. The standard MTT method was used to detect the in vitro antitumor activity of different treatment groups on HepG2 cells. The results are as follows: Fig.15 As shown in the figure, with the increase of concentration of MPDA-GdPMo@CS nanoparticles loaded with DOX, cell viability decreased, indicating that different treatment groups showed concentration dependence on cell death. Under the same concentration conditions, the MPDA-GdPMo@CS nanoparticles loaded with DOX + H2O2 + light irradiation group showed better anti-tumor activity. The nanoparticles effectively exerted photothermal-chemodynamic-chemotherapy synergistic treatment and improved the therapeutic effect on tumors.

[0128] Example 3

[0129] A composite nanoparticle loaded with doxorubicin is prepared according to the following steps:

[0130] (1) Synthesis of MPDA

[0131] 0.4 g F127 and 0.4 g TMB were dissolved in a mixed solution of 136 mL water and ethanol in a volume ratio of 1:1, stirred for 30 minutes, 8 mL of 90% TRIS aqueous solution and 65 mg dopamine hydrochloride were added to the above solution, stirred at room temperature for 24 h, centrifuged, and washed with ethanol and acetone to obtain mesoporous polydopamine (MPDA) nanospheres;

[0132] (2) Synthesis of GdPMo

[0133] 100 g Na2MoO4·2H2O was dissolved in 500 mL water, 18 mL 85% H3PO4 solution and 90 mL concentrated HCl were added, the mixed solution was boiled and refluxed for 9 h, cooled, 105 g NH4Cl was added, the precipitate was filtered, the precipitate was dissolved in water, concentrated HCl and ether were added for extraction, and H6[P2Mo 18 O 62] etherate. Take 50 mL of the etherate and add water to 110 mL, remove the ether with cold air, add 9g Gd(NO3)3·6H2O, adjust the pH to near 4.2, place under stirring for 2 h, then add 25g KCl to the solution, and a precipitate will form under constant stirring. After cooling in an ice bath for 20 min, filter, wash, and recrystallize three times with a sulfuric acid aqueous solution with a pH of 3.7, and obtain the product GdPMo after vacuum drying;

[0134] (3) Synthesis of r-GdPMo

[0135] Take 20 mg of GdPMo and dissolve it in 10 mL of water, add 90 mg of glutathione (GSH), stir at room temperature in the dark for 10 h, then add anhydrous ethanol to precipitate a blue precipitate, centrifuge, wash with water and ethanol, and dry in vacuum;

[0136] (4) Synthesis of MPDA-GdPMo nanoparticles

[0137] 8 mg of MPDA nanoparticles were dispersed in 18 mL of water, and then 8 mg of r-GdPMo was added. After stirring for 20 h, the mixture was centrifuged and washed three times with water to obtain MPDA-GdPMo nanoparticles.

[0138] (5) Synthesis of MPDA-GdPMo@CS-DOX nanoparticles

[0139] Take 8 mL of 2% chitosan (CS) solution, adjust its pH to 5.8, and add 10 mg of MPDA-GdPMo NPs and 12 mL, 0.8 mg mL -1 The doxorubicin (DOX) aqueous solution was stirred for 20 h, centrifuged, and washed three times with water to obtain MPDA-GdPMo@CS-DOX nanoparticles.

[0140] According to calculation, the drug loading of DOX in the composite nanoparticles prepared in this example is about 42.2%. The standard MTT method is used to detect the in vitro antitumor activity of different treatment groups on HepG2 cells. The results are as follows: Fig.16 As shown, with the increase of concentration, cell viability decreased, indicating that different treatment groups showed concentration-dependency on cell death.

Claims

1. A composite nanoparticle loaded with doxorubicin, characterized in that: The composite nanoparticles are mesoporous dopamine as a carrier MPDA, and reduced gadolinium phosphomolybdate r-GdPMo is modified on its surface to form MPDA-GdPMo nanoparticles, and chitosan is further coated on the surface, and finally the drug doxorubicin DOX is loaded to prepare MPDA-GdPMo@CS-DOX composite nanoparticles. The reduced gadolinium phosphomolybdate is prepared by dissolving gadolinium phosphomolybdate in water, adding glutathione GSH, stirring at room temperature in the dark for 10-12 hours, then adding anhydrous ethanol to precipitate a blue precipitate, centrifuging, washing with water and ethanol, and vacuum drying. The dosage ratio of the gadolinium phosphomolybdate, water, and GSH is 20 mg: 10-12 mL: 90-110 mL. mg, the mesoporous polydopamine is prepared by dissolving F127 and 1,3,5-trimethylbenzene TMB in a mixed solvent of water and ethanol to form a mixed solution, adding trimethylamine hydrochloride TRIS aqueous solution and dopamine hydrochloride to the mixed solution, stirring at room temperature for 20-24 hours, centrifuging and washing to obtain nanospheres, the amount ratio of F127, TMB and the mixed solvent is 1g:1g:330-340mL, the volume ratio of water and ethanol in the mixed solvent is 1:1, the amount ratio of the mixed solution, TRIS aqueous solution and dopamine hydrochloride is 12-14mL:0.8-1mL:5.5-6.5mg, and the mass concentration of the TRIS aqueous solution is 90%.

2. The composite nanoparticle loaded with doxorubicin according to claim 1, characterized in that: The MPDA-GdPMo nanoparticles are prepared by dispersing MPDA nanoparticles in water, then adding r-GdPMo, stirring for 20-24 hours, centrifuging, and washing to obtain the MPDA-GdPMo nanoparticles, wherein the dosage ratio of the MPDA nanoparticles, water, and r-GdPMo is 8-10 mg:18-24 mL:8-10 mg.

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