A calcium / iron-based nanomotor loaded with dihydrochlorin e6 and its preparation method and application

Abstract

The present invention discloses a calcium / iron-based nanomotor loaded with chlorin e6, as well as its preparation method and application, belonging to the field of biomaterials for tumor treatment. The nanomotor of the present invention is prepared as follows: an iron ion / 3,3'-dithiodipropionylhydrazide complex is formed on the surface of nano-CaO2 particles to obtain core-shell calcium / iron nanoparticles; and chlorin e6 and hydrazide-modified hyaluronic acid are loaded onto the calcium / iron nanoparticles to obtain the chlorin e6-loaded calcium / iron-based nanomotor. This nanomotor exhibits light-driven motility, oxygen supply, glutathione scavenging, photodynamic / chemodynamic effects, and tumor targeting, and can be used in the preparation of highly effective anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and specifically relates to a calcium / iron-based nanomotor loaded with dihydrochlorin e6, a preparation method and an application thereof. Background Art

[0002] To date, cancer remains a major risk factor for human health, and the onset of malignant tumors brings heavy losses to countless families. Traditional cancer treatments, including surgery, radiotherapy, and chemotherapy, while effective, inevitably lead to problems such as residual tumor tissue, damage to normal tissue, and multidrug resistance in tumor cells. With the advancement of science and technology, new treatment modalities such as molecular targeted therapy and immunotherapy are increasingly being applied. However, their narrow applicable populations and the development of multidrug resistance after long-term use pose significant challenges to their development. Compared with free drugs, the use of nanocarriers to load anti-tumor drugs can not only achieve specific accumulation of drugs at the tumor site and effectively reduce toxic side effects on normal tissues, but also increase the duration of drug action by prolonging the half-life of drugs in the body; however, for solid tumors, the abnormal vascular structure, high interstitial pressure and dense extracellular matrix network in the tumor microenvironment in the tumor tissue largely inhibit the penetration and cellular internalization of nanodrugs in the tumor. At the same time, the acidic environment inside the lysosomes poses a risk of nanodrug degradation. Nanomotors with autonomous movement capabilities can overcome these shortcomings and increase the permeability of tumors at the micro-nano scale.

[0003] Nanomotors can be driven by magnetic fields, ultrasound or light and are highly controllable. Although existing nanomotors have achieved efficient tumor interstitial penetration and lysosomal escape, their single structural design results in poor therapeutic effects in tumor treatment. Summary of the Invention

[0004] In order to solve the problems of poor tumor targeting of nanomedicines, insufficient tumor tissue penetration ability, and low efficiency of chemodynamic / photodynamic therapy, the purpose of the present invention is to provide a calcium / iron-based nanomotor loaded with dihydrochlorin e6, a preparation method and application. The nanomotor prepared by this method can specifically move to the tumor cell site under the stimulation of 808nm near-infrared light, has long-term blood circulation stability, self-oxygenation and glutathione scavenging ability, and can effectively kill tumor cells through calcium / iron ion overload, chemodynamic effects and photodynamic effects.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a calcium / iron-based nanomotor loaded with dihydrochlorin e6 comprises the following steps:

[0007] Iron ion / 3,3'-dithiodipropionyl hydrazide coordination complexes are formed on the surface of nano-CaO2 particles to obtain core-shell structured calcium / iron nanoparticles;

[0008] Chlorin e6 and hydrazide-modified hyaluronic acid were loaded on calcium / iron nanoparticles to obtain chlorin e6-loaded calcium / iron-based nanomotors.

[0009] Furthermore, core-shell calcium / iron nanoparticles are prepared by the following process: ferrous chloride ethanol solution and 3,3'-dithiodipropionylhydrazine ethanol solution are evenly mixed, and then CaO2 nanoparticle ethanol suspension is added and reacted under stirring to obtain core-shell calcium / iron nanoparticles.

[0010] Furthermore, the particle size of the core-shell structured calcium / iron nanoparticles is 100 to 300 nm.

[0011] Furthermore, the concentration of the ferrous chloride ethanol solution is 80 mg / mL, the concentration of the 3,3'-dithiodipropionylhydrazide ethanol solution is 2-3 mg / mL, and the volume ratio of the ferrous chloride ethanol solution to the 3,3'-dithiodipropionylhydrazide ethanol solution is 0.015 mL:1-3 mL;

[0012] The concentration of the ferrous chloride ethanol solution is 80 mg / mL, the concentration of the CaO2 nanoparticle ethanol suspension is 3-5 mg / mL, the volume of the ferrous chloride ethanol solution and the CaO2 nanoparticle ethanol suspension is 0.015 mL:0.3 mL; the reaction time is 12-16 h.

[0013] Furthermore, a calcium / iron-based nanomotor loaded with dihydrochlorin e6 was prepared by the following process: mixing a calcium / iron nanoparticle ethanol suspension with dihydrochlorin e6, and stirring once in a dark environment to obtain calcium / iron nanoparticles loaded with dihydrochlorin e6;

[0014] The calcium / iron nanoparticle suspension loaded with dihydrochlorin e6 was mixed with an aqueous solution of hydrazide hyaluronic acid and stirred a second time in a dark environment to obtain a calcium / iron-based nanomotor loaded with dihydrochlorin e6.

[0015] Furthermore, the mass ratio of calcium / iron nanoparticles to dihydrochlorin e6 is 1.5-3.5 mg:4.5-6.5 mg;

[0016] The mass ratio of calcium / iron-based nanoparticles loaded with dihydrochlorin e6 to hydrazide hyaluronic acid is 1:(1.5-2).

[0017] Furthermore, the first stirring time is 12 to 24 hours, and the second stirring time is 30 to 40 minutes.

[0018] Furthermore, CaO2 nanoparticles are prepared by the following process: a mixture of a calcium chloride aqueous solution and a polyvinyl pyrrolidone aqueous solution is mixed with a sodium hydroxide solution, and then a hydrogen peroxide solution is added and reacted for 10 to 30 minutes to obtain CaO2 nanoparticles.

[0019] Furthermore, the concentration of the calcium chloride aqueous solution is 15-30 mg / mL, the concentration of the polyvinyl pyrrolidone aqueous solution is 40-80 mg / mL, the volume ratio of the calcium chloride aqueous solution to the polyvinyl pyrrolidone aqueous solution is 1:1, the mass percentage concentration of the hydrogen peroxide solution is 30%, and the volume ratio of the calcium chloride aqueous solution to the hydrogen peroxide solution is 2.5 mL:0.4 mL.

[0020] A calcium / iron-based nanomotor loaded with dihydrochlorin e6, wherein the particle size of the calcium / iron-based nanomotor loaded with dihydrochlorin e6 is 200-300 nm and the zeta potential is -18 mV.

[0021] Application of a calcium / iron-based nanomotor loaded with dihydrochlorin e6 in the preparation of anti-tumor drugs.

[0022] Furthermore, the anti-tumor drug is a highly effective nanomedicine for treating pancreatic cancer.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The calcium / iron-based nanomotor loaded with dihydrochlorin e6 has an asymmetric structure and can specifically self-move to the tumor cell site under near-infrared light stimulation, improving the tumor tissue penetration and lysosomal escape ability of traditional nanomedicines, which is conducive to the efficient cure of tumors;

[0025] (2) CaO2 is acid-responsive. The tumor microenvironment can accelerate the decomposition of CaO2 and further generate hydrogen peroxide, oxygen, and calcium ions. While enriching calcium overload tumor treatment, it also lays the foundation for improving the efficacy of iron-mediated Fenton-like reactions, chemodynamic effects, and photodynamic effects in treating tumors.

[0026] (3) The iron ligand coating containing disulfide bonds formed on the surface of nano-CaO2 particles can react with endogenous glutathione in tumor cells, depleting glutathione and reducing the consumption rate of intracellular reactive oxygen species, which is beneficial to improving the therapeutic efficiency of photodynamic therapy;

[0027] (4) The calcium / iron-based nanomotor of the present invention is equipped with the photosensitizer molecule dihydrochlorin e6, which can generate reactive oxygen species and a large amount of heat under near-infrared light stimulation, which is beneficial for the combined photodynamic and photothermal treatment of tumors.

[0028] (5) The hydrazide-modified hyaluronic acid loaded by the calcium / iron-based nanomotor not only has excellent tumor targeting, but also has excellent biocompatibility and negative charge. The nanomotor modified by it has long circulation characteristics, laying the foundation for improving the efficiency of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a transmission electron microscopy image of the CaO2 nanoparticles in Example 1;

[0030] Figure 2 The transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of the CaO2@Fe-TPH(AT) nanoparticles in Example 1 are shown; (a) is a transmission electron microscopy image, and (b) is a scanning electron microscopy image;

[0031] Figure 3 This is a transmission electron microscopy image of the CaO2@Fe-TPH@Ce6@HHA(ATCH) nanomotor in Example 1;

[0032] Figure 4 Dynamic light scattering images of CaO2, AT, CaO2@Fe-TPH@Ce6 (ATC) and ATCH nanomotors in Example 1;

[0033] Figure 5 The UV-vis spectra of various parts during the preparation of ATCH in Example 1 are shown;

[0034] Figure 6 is the proportion of each element in the ATCH nanomotor in Example 1 measured by ICG-MS;

[0035] Figure 7 This is the glutathione consumption capacity curve of the ATCH nanomotor in Example 1;

[0036] Figure 8 The ability of the ATCH nanomotor in Example 1 to generate ·OH under the action of H2O2; (a) represents different times, and (b) represents different concentrations;

[0037] Figure 9 This is the self-driving trajectory diagram of the ATCH nanomotor in Example 1 under near-infrared light stimulation;

[0038] Figure 10 The photothermal capacity of the ATCH nanomotor in Example 1; (a) represents different laser powers, and (b) represents different ATCH concentrations;

[0039] Figure 11 Fluorescence imaging of the ATCH nanomotor actively targeting tumor cells in mice in Example 1;

[0040] Figure 12This is a diagram showing the effect of the ATCH nanomotor in Example 1 killing pancreatic cancer cells;

[0041] Figure 13 This is a diagram showing the effect of the ATCH nanomotor in Example 1 killing pancreatic cancer cells under different conditions. DETAILED DESCRIPTION

[0042] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present invention. However, the present invention may be implemented in a variety of different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0043] The present invention discloses a calcium / iron-based nanomotor loaded with dihydrochlorin e6, which is composed of a core CaO2, an outer shell iron ion / 3,3'-dithiodipropionyl hydrazide coordination compound, a loaded photosensitizer dihydrochlorin e6, and hydrazide hyaluronic acid; the preparation method comprises the following steps: in situ self-assembly on the surface of nano-CaO2 particles to form an iron ion / 3,3'-dithiodipropionyl hydrazide shell, and then loading the dihydrochlorin e6 and hydrazide hyaluronic acid through coordination.

[0044] Specifically, a preparation method of a calcium / iron-based nanomotor loaded with dihydrochlorin e6 is as follows:

[0045] First, CaO2 nanoparticles were synthesized by preparing a calcium chloride aqueous solution with a concentration of 15 to 30 mg / mL and a polyvinylpyrrolidone aqueous solution with a concentration of 40 to 80 mg / mL, taking 2.5 mL of each solution and mixing them under magnetic stirring for 5 to 10 minutes at a stirring speed of 600 to 1200 rpm; then, 4 mL of a 0.25 mol / L sodium hydroxide solution was added dropwise, and stirring was continued for 5 to 10 minutes. Finally, 0.4 mL of a 30% by mass hydrogen peroxide solution was added dropwise, and the stirring reaction was continued for 10 to 30 minutes; finally, the product was centrifuged and washed with ethanol three times to obtain CaO2 nanoparticles with an average particle size of about 100 nm.

[0046] Then, an iron ion / 3,3'-dithiodipropionyl hydrazide coordination complex is formed on the surface of the nano-CaO2 particles to obtain core-shell calcium / iron nanoparticles, and the core-shell calcium / iron nanoparticles form an iron coordination coating. The specific steps are as follows: 0.015 mL of an 80 mg / mL ferrous chloride ethanol solution and 1 to 3 mL of a 2 to 3 mg / mL 3,3'-dithiodipropionyl hydrazide ethanol solution are evenly mixed, and then 0.3 mL of a 3 to 5 mg / mL CaO2 nanoparticle ethanol suspension is added, and the mixture is reacted under magnetic stirring at 5000 rpm for 12 to 16 hours, and then centrifuged at 5000 rpm / 10 min and washed with ethanol 2 to 3 times to obtain core-shell calcium / iron nanoparticles with a particle size of 100 to 300 nm.

[0047] Finally, chlorin e6 and hydrazide hyaluronic acid were loaded on the calcium / iron nanoparticles to obtain calcium / iron-based nanomotors loaded with chlorin e6. The specific steps were as follows: 1.5-3.5 mg of calcium / iron nanoparticles were ultrasonically suspended in 6-10 mL of ethanol and placed in a dark place. Then, 4.5-6.5 mg of chlorin e6 was added under magnetic stirring, and stirring was continued for 12-24 hours. The mixture was centrifuged at 13000 rpm for 10 minutes and washed with ethanol 2-3 times to obtain calcium / iron nanomotors loaded with chlorin e6. Particles; the nanoparticle suspension is added to a 1 mg / mL hydrazide hyaluronic acid aqueous solution, magnetically stirred for 30 minutes in a dark environment, and centrifuged and washed 1 to 2 times to obtain a calcium / iron-based nanomotor loaded with dihydrochlorin e6, which has a particle size of 200 to 300 nm and a zeta potential of -18 mV. It has the characteristics of light-driven motility, oxygen supply, glutathione scavenging, suspension stability and tumor targeting, and can kill tumor cells through calcium / iron ion overload, chemokinetic effect and photodynamic effect.

[0048] The mass ratio of calcium / iron-based nanoparticles loaded with dihydrochlorin e6 to hydrazide-modified hyaluronic acid is 1:(1.5-2).

[0049] The molecular weight of the hydrazide hyaluronic acid in the present invention is 8-10 kDa, the degree of hydrazide is 10%-20%, and the preparation method of the hydrazide hyaluronic acid adopts the method in patent CN118436846A.

[0050] The following are specific examples.

[0051] Example 1: A method for preparing a calcium / iron-based nanomotor loaded with dihydrochlorin e6 is carried out according to the following steps:

[0052] Step 1: Synthesis of CaO2 nanoparticles:

[0053] A 30 mg / mL aqueous solution of calcium chloride and an 80 mg / mL aqueous solution of polyvinyl pyrrolidone were prepared, 2.5 mL of each solution was taken and mixed under magnetic stirring for 10 minutes at a stirring speed of 600 to 1200 rpm. Then, 4 mL of a 0.25 mol / L sodium hydroxide solution was added dropwise, and stirring was continued for 5 to 10 minutes. Finally, 0.4 mL of a 30% by mass hydrogen peroxide solution was added dropwise, and stirring was continued for another 30 minutes. Finally, the product was centrifuged and washed with ethanol three times to obtain CaO2 nanoparticles with an average particle size of approximately 100 nm.

[0054] Step 2: Synthesis of core-shell calcium / iron nanoparticles (AT):

[0055] 0.015 mL of 80 mg / mL ferrous chloride ethanol solution and 2 mL of 2-3 mg / mL 3,3'-dithiodipropionylhydrazide ethanol solution were mixed evenly, and then 0.3 mL of 3 mg / mL nano-CaO2 ethanol suspension was added. The mixture was reacted under magnetic stirring at 5000 rpm for 12 hours, and then centrifuged at 5000 rpm / 10 min and washed with ethanol 2-3 times to obtain core-shell calcium / iron nanoparticles with a particle size of 100-300 nm.

[0056] Step 3: Synthesis of calcium / iron-based nanomotors (ATCH) loaded with chlorin e6 and hydrazide hyaluronic acid:

[0057] 2.5 mg of calcium / iron nanoparticles were ultrasonically suspended in 10 mL of ethanol and placed in the dark. Then, 6.5 mg of dihydrochlorin e6 was added under magnetic stirring and stirring was continued for 12 to 24 hours. The mixture was centrifuged at 13,000 rpm for 10 minutes and washed with ethanol 2 to 3 times to obtain calcium / iron nanoparticles loaded with dihydrochlorin e6. The nanoparticle suspension was added to a 1 mg / mL aqueous solution of hydrazide-modified hyaluronic acid, magnetically stirred for 30 minutes in the dark, and centrifuged and washed 1 to 2 times to obtain calcium / iron-based nanomotors loaded with dihydrochlorin e6 with a particle size of 200 to 300 nm and a zeta potential of -18 mV.

[0058] Figure 1 TEM image of the CaO2 nanoparticles in Example 1. Figure 1 As shown, the CaO2 nanoparticles are nearly spherical and evenly distributed, with a particle size distribution of around 100 nm, which meets the particle size requirements of nano-drug carriers and provides a prerequisite for the subsequent loading of various molecules.

[0059] Figure 2 Figure 1 is a transmission electron microscope image and a scanning electron microscope image of the AT nanoparticles in Example 1. Figure 2As shown in (a) and (b), AT presents an asymmetric structure with a particle size distribution around 200 nm, and Fe-TPH nanoparticles are attached to the surface of CaO2 nanoparticles, which lays the foundation for its light-driven behavior under near-infrared light stimulation.

[0060] Figure 3 : is a transmission electron microscope image of ATCH nanoparticles in Example 1. Figure 3 As shown, the asymmetric structure of the ATCH nanomotor is more obvious, and its particle size distribution is around 270nm, which meets the particle size requirements of nanomedicines.

[0061] Figure 4 Figure 2 is a dynamic light scattering diagram of CaO2, AT and ATCH nanoparticles in Example 1. Figure 4 As shown, the particle size distribution measured by the nanoparticle size analyzer is consistent with the particle size distribution in the transmission electron microscope image, and the particle size distribution of the nanoparticles obtained in each stage is uniform, which provides a prerequisite for them to enter the tumor cell site through intravenous injection.

[0062] Figure 5 The UV-vis spectra of various parts during the preparation process of the calcium / iron-based nanomotor loaded with dihydrochlorin e6 in Example 1 are shown. Figure 5 As shown, Fe-TPH (iron coordination coating) has an obvious ultraviolet absorption peak near 600nm, CaO2 has no obvious ultraviolet absorption peak, and the ultraviolet characteristic absorption peaks of photosensitizer Ce6 are located near 402nm, 502nm and 663nm. The ultraviolet characteristic absorption peaks of Fe-TPH and Ce6 are shown in AT (CaO2@Fe-TPH, core-shell structured calcium / iron nanoparticles) and ATC (CaO2@Fe-TPH@Ce6, core-shell structured calcium / iron nanoparticles loaded with dihydrochlorin e6), which indicates that the Fe-TPH coordination layer and the loading of photosensitizer Ce6 are successful. The ultraviolet characteristic absorption peak of the photosensitizer Ce6 was displayed in the ATCH (CaO2@Fe-TPH@Ce6@HHA, calcium / iron-based nanomotors loaded with dihydrochlorin e6 and hydrazide hyaluronic acid) particles, while the ultraviolet characteristic absorption peak of Fe-TPH was not revealed, which was attributed to the masking effect after HHA (hydrazide hyaluronic acid) modification.

[0063] Figure 6 is the proportion of each element in the ATCH nanomotor in Example 1 measured by ICP-MS. Figure 6 As shown, the calcium element accounts for 42.0% and the iron element accounts for 22.0% in the ATCH nanomotor, indicating that the nanomotor has the ability to kill tumor cells by calcium / iron overload.

[0064] Figure 7This is the glutathione consumption curve of the ATCH nanomotor in Example 1. Glutathione can react with DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid)) to produce yellow 5-thio-2-nitrobenzoic acid, which has a maximum UV absorption peak at 412nm. Figure 7 As shown in the figure, as the concentration of ATCH nanomotor increases, the ultraviolet absorption peak of 5-thio-2-nitrobenzoic acid at 412nm gradually decreases until it disappears, indicating that the nanomotor can effectively clear glutathione in tumor cells, which is beneficial to improve the efficiency of tumor cell treatment using photodynamic effect.

[0065] Figure 8 The ability of the ATCH nanomotor in Example 1 to generate ·OH under the action of H2O2. ·OH was detected by the TMB method. It can be oxidized by ·OH to an oxidized state and show a blue color with a maximum ultraviolet absorption peak at 650nm. Figure 8 As shown in (a) and (b), after the addition of exogenous H2O2, the UV absorption peak at 650nm gradually increases with time. Figure 8 (a), as well as at different H2O2 concentrations, different degrees of absorption peak enhancement are shown, indicating that the nanomotor can generate reactive oxygen species through a Fenton-like reaction under the action of H2O2, which is beneficial to improving the efficiency of tumor treatment using chemical dynamic effects as a means.

[0066] Figure 9 This is the motion trajectory of the ATCH nanomotor in Example 1 under 808nm near-infrared light stimulation. Figure 9 As shown, with the increase of laser power, the movement distance of the nanomotor gradually increases, which confirms the light-driven ability of the nanomotor, which is beneficial to improving its penetration in tumor tissue and lysosomal escape ability, providing a prerequisite for improving the efficiency of tumor treatment.

[0067] Figure 10 is the photothermal capacity of the ATCH nanomotor in Example 1. Figure 10 As shown in (a), when the concentration of ATCH is 500 μg / mL, the temperature gradually increases with the increase of laser power, reaching a maximum of 50°C; Figure 10 In (b), when the laser power is 2W / cm 2 As the concentration of ATCH increases, the temperature also increases accordingly, reaching a maximum of 60°C. ATCH's excellent photothermal conversion ability lays the foundation for the combined photodynamic and photothermal treatment of tumors.

[0068] Figure 11 This is the fluorescence imaging of the ATCH nanomotor actively targeting tumor cells in mice in Example 1. Hydrazide-modified hyaluronic acid has the ability to specifically target tumor cells, such as Figure 11 As shown, after the nanoparticles were injected intravenously into mice, they gradually accumulated at the tumor site over time. Compared with traditional non-targeted drugs, this specific targeting function can effectively reduce the toxic side effects of drugs on normal tissues and improve the efficiency of tumor treatment.

[0069] Figure 12 and Figure 13 The effect of ATCH nanomotor killing pancreatic cancer cells in Example 1. Figure 12 As shown in Figure 2, ATCH nanomotors exhibited good tumor-killing effects and showed a concentration-dependent relationship; Figure 13 As shown in the figure, when the concentration of the nanomotor remains unchanged, the effect of killing pancreatic cancer cells under the conditions of H2O2, light and H2O2+light is shown. Under the condition of H2O2+light, the nanomotor shows the strongest anti-tumor ability, indicating that this method of combining calcium / iron ion overload, chemokinetic effect and photodynamic effect can effectively improve the efficiency of tumor treatment. Compared with traditional single-mode treatment, this multi-mode combined treatment can effectively inhibit the growth of tumor cells. This nanomotor is of great significance in the field of improving the efficiency of tumor treatment.

[0070] Example 2

[0071] Step 1: Synthesis of CaO2 nanoparticles:

[0072] A 15 mg / mL aqueous solution of calcium chloride and a 50 mg / mL aqueous solution of polyvinyl pyrrolidone were prepared, 2.5 mL of each solution was mixed under magnetic stirring at 1200 rpm for 5 minutes. Then, 4 mL of a 0.25 mol / L sodium hydroxide solution was added dropwise, stirring continued for 5 minutes, and 0.4 mL of a 30% by mass hydrogen peroxide solution was added dropwise. The mixture was stirred for an additional 20 minutes. Finally, the product was centrifuged and washed with ethanol three times to obtain CaO2 nanoparticles with an average particle size of approximately 100 nm.

[0073] Step 2: Synthesis of core-shell calcium / iron nanoparticles (AT):

[0074] 0.015 mL of 80 mg / mL ferrous chloride ethanol solution and 1 mL of 2 mg / mL 3,3'-dithiodipropionylhydrazide ethanol solution were mixed evenly, and then 0.3 mL of 4 mg / mL nano-CaO2 ethanol suspension was added. The mixture was reacted under magnetic stirring at 5000 rpm for 16 h, and then centrifuged at 5000 rpm / 10 min and washed twice with ethanol to obtain core-shell calcium / iron nanoparticles with a particle size of 100 to 300 nm.

[0075] Step 3: Synthesis of calcium / iron-based nanomotors (ATCH) loaded with chlorin e6 and hydrazide hyaluronic acid:

[0076] 1.5 mg of calcium / iron nanoparticles were ultrasonically suspended in 6 mL of ethanol and placed in the dark. Then, 4.5 mg of dihydrochlorin e6 was added under magnetic stirring and stirring was continued for 12 hours. The mixture was centrifuged at 13,000 rpm for 10 minutes and washed with ethanol twice to obtain calcium / iron nanoparticles loaded with dihydrochlorin e6. The nanoparticle suspension was added to a 1 mg / mL aqueous solution of hydrazide-modified hyaluronic acid, magnetically stirred for 30 minutes in the dark, and centrifuged and washed once to obtain calcium / iron-based nanomotors loaded with dihydrochlorin e6.

[0077] Example 3

[0078] Step 1: Synthesis of CaO2 nanoparticles:

[0079] A 20 mg / mL aqueous solution of calcium chloride and a 40 mg / mL aqueous solution of polyvinyl pyrrolidone were prepared, 2.5 mL of each solution was mixed under magnetic stirring at 1000 rpm for 7 minutes. Then, 4 mL of a 0.25 mol / L sodium hydroxide solution was added dropwise, and stirring was continued for 7 minutes. Finally, 0.4 mL of a 30% by mass hydrogen peroxide solution was added dropwise, and the mixture was stirred for an additional 10 minutes. Finally, the product was centrifuged and washed with ethanol three times to obtain CaO2 nanoparticles with an average particle size of approximately 100 nm.

[0080] Step 2: Synthesis of core-shell calcium / iron nanoparticles (AT):

[0081] 0.015 mL of 80 mg / mL ferrous chloride ethanol solution and 3 mL of 3 mg / mL 3,3'-dithiodipropionylhydrazide ethanol solution were mixed evenly, and then 0.3 mL of 5 mg / mL nano-CaO2 ethanol suspension was added. The mixture was reacted under magnetic stirring at 5000 rpm for 14 h, and then centrifuged at 5000 rpm / 10 min and washed with ethanol three times to obtain core-shell calcium / iron nanoparticles with a particle size of 100 to 300 nm.

[0082] Step 3: Synthesis of calcium / iron-based nanomotors (ATCH) loaded with chlorin e6 and hydrazide hyaluronic acid:

[0083] 2 mg of calcium / iron nanoparticles were ultrasonically suspended in 8 mL of ethanol and placed in the dark. Then, 6 mg of dihydrochlorin e6 was added under magnetic stirring and stirring was continued for 17 hours. The mixture was centrifuged at 13,000 rpm for 10 minutes and washed with ethanol three times to obtain calcium / iron nanoparticles loaded with dihydrochlorin e6. The nanoparticle suspension was added to a 1 mg / mL aqueous solution of hydrazide-modified hyaluronic acid, magnetically stirred for 30 minutes in the dark, and centrifuged and washed twice to obtain calcium / iron-based nanomotors loaded with dihydrochlorin e6.

[0084] Example 4

[0085] Step 1: Synthesis of CaO2 nanoparticles:

[0086] A 25 mg / mL aqueous solution of calcium chloride and a 60 mg / mL aqueous solution of polyvinyl pyrrolidone were prepared, 2.5 mL of each solution was mixed under magnetic stirring at 800 rpm for 8 minutes. Then, 4 mL of a 0.25 mol / L sodium hydroxide solution was added dropwise, and stirring was continued for 10 minutes. Finally, 0.4 mL of a 30% by mass hydrogen peroxide solution was added dropwise, and stirring was continued for another 30 minutes. Finally, the product was centrifuged and washed with ethanol three times to obtain CaO2 nanoparticles with an average particle size of approximately 100 nm.

[0087] Step 2: Synthesis of core-shell calcium / iron nanoparticles (AT):

[0088] 0.015 mL of 80 mg / mL ferrous chloride ethanol solution and 2 mL of 2 mg / mL 3,3'-dithiodipropionylhydrazide ethanol solution were mixed evenly, and then 0.3 mL of 3 mg / mL nano-CaO2 ethanol suspension was added. The mixture was reacted under magnetic stirring at 5000 rpm for 13 h, and then centrifuged at 5000 rpm / 10 min and washed twice with ethanol to obtain core-shell calcium / iron nanoparticles with a particle size of 100 to 300 nm.

[0089] Step 3: Synthesis of calcium / iron-based nanomotors (ATCH) loaded with chlorin e6 and hydrazide hyaluronic acid:

[0090] 3.5 mg of calcium / iron nanoparticles were ultrasonically suspended in 9 mL of ethanol and placed in the dark. Then, 5 mg of dihydrochlorin e6 was added under magnetic stirring and stirring was continued for 24 hours. The mixture was centrifuged at 13,000 rpm for 10 minutes and washed with ethanol twice to obtain calcium / iron nanoparticles loaded with dihydrochlorin e6. The nanoparticle suspension was added to a 1 mg / mL aqueous solution of hydrazide-modified hyaluronic acid, magnetically stirred for 30 minutes in the dark, and centrifuged and washed once to obtain calcium / iron-based nanomotors loaded with dihydrochlorin e6.

[0091] The above description is merely a description of the preferred embodiment of the present invention and is not to be construed as limiting the claims. The present invention is not limited to the above embodiment, and variations in the specific structure are permitted. Any variations made within the scope of the independent claims of the present invention are also within the scope of protection of the present invention.

[0092] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

Claims

1. A method for preparing a calcium / iron-based nanomotor loaded with dihydrochlorin e6, characterized in that: The following steps are involved: Iron ion / 3,3'-dithiodipropionyl hydrazide coordination complexes are formed on the surface of nano-CaO2 particles to obtain core-shell structured calcium / iron nanoparticles; Chlorin e6 and hydrazide-modified hyaluronic acid were loaded on calcium / iron nanoparticles to obtain chlorin e6-loaded calcium / iron-based nanomotors.

2. The method for preparing a calcium / iron-based nanomotor loaded with chlorin e6 according to claim 1, characterized in that: Core-shell calcium / iron nanoparticles are prepared by the following process: ferrous chloride ethanol solution and 3,3'-dithiodipropionylhydrazine ethanol solution are evenly mixed, and then CaO2 nanoparticle ethanol suspension is added and reacted under stirring to obtain core-shell calcium / iron nanoparticles.

3. The method for preparing a calcium / iron-based nanomotor loaded with chlorin e6 according to claim 1, wherein: The particle size of core-shell calcium / iron nanoparticles is 100~300 nm.

4. The method for preparing a calcium / iron-based nanomotor loaded with chlorin e6 according to claim 2, wherein: The concentration of the ferrous chloride ethanol solution is 80 mg / mL, the concentration of the 3,3'-dithiodipropionylhydrazide ethanol solution is 2-3 mg / mL, and the volume ratio of the ferrous chloride ethanol solution to the 3,3'-dithiodipropionylhydrazide ethanol solution is 0.015 mL:1-3 mL; The concentration of the CaO2 nanoparticle ethanol suspension is 3~5 mg / mL, the volume of the ferrous chloride ethanol solution and the CaO2 nanoparticle ethanol suspension is 0.015 mL:0.3 mL; the reaction time is 12~16 h.

5. The method for preparing a calcium / iron-based nanomotor loaded with chlorin e6 according to claim 1, wherein: The calcium / iron-based nanomotor loaded with dihydrochlorin e6 was prepared by the following process: mixing an ethanol suspension of calcium / iron nanoparticles with dihydrochlorin e6 and stirring once in a dark environment to obtain calcium / iron nanoparticles loaded with dihydrochlorin e6; The calcium / iron nanoparticle suspension loaded with dihydrochlorin e6 was mixed with an aqueous solution of hydrazide hyaluronic acid and stirred a second time in a dark environment to obtain a calcium / iron-based nanomotor loaded with dihydrochlorin e6.

6. The method for preparing a calcium / iron-based nanomotor loaded with chlorin e6 according to claim 5, wherein: The mass ratio of calcium / iron nanoparticles to dihydrochlorin e6 was 1.5-3.5 mg:4.5-6.5 mg; The mass ratio of calcium / iron-based nanoparticles loaded with dihydrochlorin e6 to hydrazide-modified hyaluronic acid is 1:(1.5~2).

7. The method for preparing a calcium / iron-based nanomotor loaded with chlorin e6 according to claim 5, wherein: The first stirring time is 12~24 h, and the second stirring time is 30-40 min.

8. The method for preparing a calcium / iron-based nanomotor loaded with chlorin e6 according to claim 1, wherein: The CaO2 nanoparticles are prepared by the following process: a mixture of a calcium chloride aqueous solution and a polyvinyl pyrrolidone aqueous solution is mixed with a sodium hydroxide solution, and then a hydrogen peroxide solution is added and reacted for 10 to 30 minutes to obtain CaO2 nanoparticles.

9. A calcium / iron-based nanomotor loaded with chlorin e6 prepared according to the method of any one of claims 1 to 8, characterized in that: The calcium / iron-based nanomotor loaded with dihydrochlorin e6 has a particle size of 200-300 nm and a zeta potential of -18 mV.

10. Use of a calcium / iron-based nanomotor loaded with chlorin e6 prepared according to the method of any one of claims 1 to 8 in the preparation of anti-tumor drugs.

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