Chlorin e6-loaded calcium / iron-based nano motor and preparation method and application thereof

Abstract

The invention discloses a chlorin e6-loaded calcium / iron-based nano motor as well as a preparation method and application thereof, and belongs to the field of biological materials for tumor treatment. The preparation method of the nano motor comprises the following steps: forming an iron ion / 3, 3 '-dithiodipropionyl hydrazide coordination compound on the surface of a nano CaO2 particle to obtain a core-shell structure type calcium / iron nano particle; the chlorin e6 and hydrazide hyaluronic acid are loaded on the calcium / iron nano-particles, so that the chlorin e6 loaded calcium / iron-based nano-motor is obtained. The nano motor has the functions of light-driven motility, oxygen supply, glutathione removal, photodynamic / chemical dynamic effect, tumor targeting and the like, and can be used for preparing efficient anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular 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 the main risk factor threatening human health. The onset of malignant tumors has brought heavy blows to countless families. Traditional cancer treatments mainly include surgery, radiotherapy and chemotherapy. Although they have certain therapeutic effects, they inevitably lead to problems such as residual tumor tissue, damage to normal tissues and multidrug resistance of tumor cells. With the development of science and technology, new treatment methods such as molecular targeted therapy and immunotherapy are also being used continuously, but their narrow applicable population range and multidrug resistance after long-term use bring huge 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 lysosome 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 nano-drugs, insufficient tumor tissue penetration, 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 an 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-oxygen supply and glutathione clearance ability, and can efficiently kill tumor cells through calcium / iron ion overloading, chemodynamic effect and photodynamic effect.

[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 complex is formed on the surface of nano-CaO2 particles to obtain core-shell structured calcium / iron nano-particles;

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

[0009] Furthermore, core-shell calcium / iron nanoparticles are prepared by the following process: ferrous chloride ethanol solution and 3,3'-dithiodipropionylhydrazide ethanol solution are evenly mixed, and then CaO2 nanoparticle ethanol suspension is added, and the mixture is 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] Further, the concentration of the ferrous chloride ethanol solution is 80 mg / mL, the concentration of the 3,3'-dithiodipropionyl hydrazide ethanol solution is 2-3 mg / mL, and the volume ratio of the ferrous chloride ethanol solution to the 3,3'-dithiodipropionyl hydrazide 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, the calcium / iron-based nanomotor loaded with dihydrochlorin e6 is prepared by the following process: mixing the calcium / iron nanoparticle ethanol suspension with dihydrochlorin e6, and stirring once in a dark environment to obtain the calcium / iron nanoparticle loaded with dihydrochlorin e6;

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

[0015] Further, the mass ratio of calcium / iron nanoparticles to chlorin 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 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 move specifically 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-coordination 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 and a large amount of heat under the stimulation of near-infrared light, which is beneficial to 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, which lays the foundation for improving the efficiency of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0030] Figure 2 The transmission electron microscope image and scanning electron microscope image of the CaO2@Fe-TPH(AT) nanoparticles in Example 1; wherein (a) is a transmission electron microscope image, and (b) is a scanning electron microscope 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 in the ATCH preparation process in Example 1;

[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 ability curve of the ATCH nanomotor to consume glutathione in Example 1;

[0036] Figure 8 The ability of the ATCH nanomotor in Example 1 to produce ·OH under the action of H2O2; wherein (a) is different time, and (b) is different concentration;

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

[0038] Fig.10 : is the photothermal capacity of the ATCH nanomotor in Example 1; wherein (a) is different laser powers, and (b) is different ATCH concentrations;

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

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

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

[0042] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly and comprehensively understood.

[0043] The calcium / iron-based nanomotor loaded with dihydrochlorin e6 of the present invention is composed of an inner core CaO2, an outer shell iron ion / 3,3'-dithiodipropionyl hydrazide coordination compound, loaded photosensitizer dihydrochlorin e6 and hydrazide hyaluronic acid; the preparation method thereof is that the iron ion / 3,3'-dithiodipropionyl hydrazide shell is formed by in-situ self-assembly on the surface of nano-CaO2 particles, and then the dihydrochlorin e6 and hydrazide hyaluronic acid are loaded through coordination.

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

[0045] First, synthesize CaO2 nanoparticles: prepare a calcium chloride aqueous solution with a concentration of 15-30 mg / mL and a polyvinyl pyrrolidone aqueous solution with a concentration of 40-80 mg / mL, take 2.5 mL of each and mix them under magnetic stirring for 5-10 minutes at a stirring speed of 600-1200 rpm; then add 4 mL of a sodium hydroxide solution with a concentration of 0.25 mol / L, continue stirring for 5-10 minutes, then add 0.4 mL of a hydrogen peroxide solution with a mass percentage concentration of 30% dropwise, and continue stirring the reaction for 10-30 minutes; finally, the product is 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 80 mg / mL ferrous chloride ethanol solution and 1-3 mL of 2-3 mg / mL 3,3'-dithiodipropionyl hydrazide ethanol solution are mixed evenly, and then 0.3 mL of 3-5 mg / mL CaO2 nanoparticle ethanol suspension is added, and the mixture is reacted under magnetic stirring at 5000 rpm for 12-16 hours, and then centrifuged at 5000 rpm / 10 min and washed with ethanol for 2-3 times to obtain core-shell calcium / iron nanoparticles with a particle size of 100-300 nm.

[0047] Finally, chlorin e6 and acylhydrazine 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, and 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 for 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 overloading, chemokinetic effect and photodynamic effect.

[0048] The mass ratio of calcium / iron-based nanoparticles loaded with dihydrochlorin e6 to hydrazide 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 is prepared by the method in patent CN118436846A.

[0050] The following are specific embodiments.

[0051] Example 1: A method for preparing a calcium / iron-based nanomotor loaded with dihydrochlorin e6, comprising the following steps:

[0052] Step 1: Synthesis of CaO2 nanoparticles:

[0053] A 30 mg / mL calcium chloride aqueous solution and an 80 mg / mL polyvinyl pyrrolidone aqueous solution were prepared, 2.5 mL of each solution was taken and mixed under magnetic stirring for 10 minutes at a stirring speed of 600-1200 rpm; then 4 mL of a 0.25 mol / L sodium hydroxide solution was added dropwise, and stirring was continued for 5-10 minutes, and then 0.4 mL of a 30% hydrogen peroxide solution was added dropwise, and stirring was continued for 30 minutes; finally, the product was centrifuged and washed with ethanol for 3 times to obtain CaO2 nanoparticles with an average particle size of about 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, and the mixture was reacted for 12 h under magnetic stirring at 5000 rpm, and then centrifuged at 5000 rpm / 10 min and washed with ethanol for 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 dihydrochlorin e6 and hydrazide hyaluronic acid:

[0057] 2.5 mg of calcium / iron nanoparticles were ultrasonically suspended in 10 mL of ethanol and placed in a dark place, and then 6.5 mg of dihydrochlorin e6 was added under magnetic stirring, and stirring was continued for 12 to 24 hours. The calcium / iron nanoparticles loaded with dihydrochlorin e6 were obtained by centrifugation at 13000 rpm for 10 minutes and ethanol washing 2 to 3 times; the nanoparticle suspension was 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, with a particle size of 200 to 300 nm and a zeta potential of -18 mV.

[0058] Figure 1 1 is a transmission electron microscope 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 of 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 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 of the calcium / iron-based nanomotor loaded with dihydrochlorin e6 in Example 1. 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 photosensitizer Ce6 are loaded successfully. The characteristic ultraviolet absorption peak of the photosensitizer Ce6 was shown in the ATCH (CaO2@Fe-TPH@Ce6@HHA, calcium / iron-based nanomotors loaded with dihydrochlorin e6 and hydrazide hyaluronic acid) particles, while the characteristic ultraviolet 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 7The curve of glutathione consumption by the ATCH nanomotor in Example 1. Glutathione can react with DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid)) to generate yellow 5-thio-2-nitrobenzoic acid, which has a maximum ultraviolet absorption peak at 412nm. Figure 7 As shown, with the increase of the concentration of ATCH nanomotor, the ultraviolet absorption peak of 5-thio-2-nitrobenzoic acid at 412nm gradually decreased until it disappeared, indicating that the nanomotor can effectively remove glutathione in tumor cells, which is beneficial to improve the efficiency of tumor cell therapy using photodynamic effect.

[0065] Figure 8 The ability of the ATCH nanomotor in Example 1 to generate ·OH under the action of H2O2. ·OH 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 adding exogenous H2O2, the UV absorption peak at 650nm gradually increases with time. Figure 8 In (a), as well as at different H2O2 concentrations, different degrees of absorption peak enhancement are shown, indicating that the nanomotor can generate reactive oxygen through a Fenton-like reaction under the action of H2O2, which is beneficial to improving the efficiency of tumor treatment by means of chemical dynamic effect.

[0066] Fig. 9 FIG. 1 is a motion trajectory diagram of the ATCH nanomotor in Example 1 under the stimulation of 808 nm near-infrared light. Fig. 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] Fig.10 is the photothermal capacity of the ATCH nanomotor in Example 1. Fig.10 As shown in (a), when the concentration of ATCH is 500 μg / mL, as the laser power increases, the temperature gradually increases and can reach a maximum of 50°C. Fig.10 In (b), when the laser power is 2W / cm 2 When the concentration of ATCH increases, the temperature also increases accordingly, up to 60°C. The good photothermal conversion ability of ATCH lays the foundation for the combined photodynamic and photothermal treatment of tumors.

[0068] Fig.11 This is the fluorescence imaging of the ATCH nanomotor actively targeting tumor cells in mice in Example 1. Hydrazide hyaluronic acid has the ability to specifically target tumor cells, such as Fig.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] Fig.12 and Fig.13 The effect of ATCH nanomotor in Example 1 in killing pancreatic cancer cells. Fig.12 As shown, ATCH nanomotors exhibited good tumor killing effects and showed a concentration-dependent relationship; Fig.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 respectively. Under the condition of H2O2 + light, the nanomotor showed the strongest anti-tumor ability, indicating that this combined method of calcium / iron ion overloading, 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 calcium chloride aqueous solution and a 50 mg / mL polyvinyl pyrrolidone aqueous solution were prepared, and 2.5 mL of each solution was mixed under magnetic stirring for 5 min at a stirring speed of 1200 rpm; then 4 mL of a 0.25 mol / L sodium hydroxide solution was added dropwise, and stirring was continued for 5 min, and then 0.4 mL of a 30% mass percent hydrogen peroxide solution was added dropwise, and stirring was continued for 20 min; finally, the product was centrifuged and washed with ethanol for 3 times to obtain CaO2 nanoparticles with an average particle size of about 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, and the mixture was reacted for 16 h under magnetic stirring at 5000 rpm, 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 dihydrochlorin e6 and hydrazide hyaluronic acid:

[0076] 1.5 mg of calcium / iron nanoparticles were ultrasonically suspended in 6 mL of ethanol and placed in a dark place. Then, 4.5 mg of dihydrochlorin e6 was added under magnetic stirring and stirring was continued for 12 h. The calcium / iron nanoparticles loaded with dihydrochlorin e6 were obtained by centrifugation at 13000 rpm for 10 min and ethanol washing twice. The nanoparticle suspension was added to a 1 mg / mL hydrazide hyaluronic acid aqueous solution, magnetically stirred for 30 min in a dark environment, centrifuged and washed once to obtain a calcium / iron-based nanomotor loaded with dihydrochlorin e6.

[0077] Example 3

[0078] Step 1: Synthesis of CaO2 nanoparticles:

[0079] A 20 mg / mL calcium chloride aqueous solution and a 40 mg / mL polyvinyl pyrrolidone aqueous solution were prepared, 2.5 mL of each solution was mixed under magnetic stirring for 7 min at a stirring speed of 1000 rpm; then 4 mL of a 0.25 mol / L sodium hydroxide solution was added dropwise, stirring was continued for 7 min, and then 0.4 mL of a 30% hydrogen peroxide solution was added dropwise, and stirring was continued for 10 min; finally, the product was centrifuged and washed with ethanol 3 times to obtain CaO2 nanoparticles with an average particle size of about 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, and the mixture was reacted for 14 h under magnetic stirring at 5000 rpm, and then centrifuged at 5000 rpm / 10 min and washed with ethanol for 3 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 dihydrochlorin e6 and hydrazide hyaluronic acid:

[0083] 2 mg of calcium / iron nanoparticles were ultrasonically suspended in 8 mL of ethanol and placed in a dark place. Then, 6 mg of dihydrochlorin e6 was added under magnetic stirring and stirring was continued for 17 h. The calcium / iron nanoparticles loaded with dihydrochlorin e6 were obtained by centrifugation at 13000 rpm for 10 min and ethanol washing three times. The nanoparticle suspension was added to a 1 mg / mL hydrazide hyaluronic acid aqueous solution, magnetically stirred for 30 min in a dark environment, 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 calcium chloride aqueous solution and a 60 mg / mL polyvinyl pyrrolidone aqueous solution were prepared, 2.5 mL of each solution was mixed under magnetic stirring for 8 min at a stirring speed of 800 rpm; then 4 mL of a 0.25 mol / L sodium hydroxide solution was added dropwise, and stirring was continued for 10 min, and then 0.4 mL of a 30% hydrogen peroxide solution was added dropwise, and stirring was continued for 30 min; finally, the product was centrifuged and washed with ethanol for 3 times to obtain CaO2 nanoparticles with an average particle size of about 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, and the mixture was reacted for 13 h under magnetic stirring at 5000 rpm, 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 dihydrochlorin e6 and hydrazide hyaluronic acid:

[0090] 3.5 mg of calcium / iron nanoparticles were ultrasonically suspended in 9 mL of ethanol and placed in a dark place. Then, 5 mg of dihydrochlorin e6 was added under magnetic stirring and stirring was continued for 24 h. The calcium / iron nanoparticles loaded with dihydrochlorin e6 were obtained by centrifugation at 13000 rpm for 10 min and ethanol washing twice. The nanoparticle suspension was added to a 1 mg / mL hydrazide hyaluronic acid aqueous solution, magnetically stirred for 30 min in a dark environment, centrifuged and washed once to obtain a calcium / iron-based nanomotor loaded with dihydrochlorin e6.

[0091] The above description is only for the best embodiment of the present invention, but it should not be understood as limiting the claims. The present invention is not limited to the above embodiments, and its specific structure is allowed to be changed. However, all changes made within the protection scope of the independent claims of the present invention are within the protection scope of the present invention.

[0092] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related 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 complex is formed on the surface of nano-CaO2 particles to obtain core-shell structured calcium / iron nano-particles; Chlorin e6 and hydrazide hyaluronic acid were loaded on calcium / iron nanoparticles to obtain calcium / iron-based nanomotors loaded with chlorin e6.

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

3. The method for preparing the calcium / iron-based nanomotor loaded with dihydrochlorin e6 according to claim 1, characterized in that: The particle size of the core-shell structured calcium / iron nanoparticles is 100 to 300 nm.

4. The method for preparing the calcium / iron-based nanomotor loaded with dihydrochlorin e6 according to claim 1, characterized in that: The concentration of the ferrous chloride ethanol solution is 80 mg / mL, the concentration of the 3,3'-dithiodipropionyl hydrazide ethanol solution is 2-3 mg / mL, and the volume ratio of the ferrous chloride ethanol solution to the 3,3'-dithiodipropionyl hydrazide ethanol solution is 0.015 mL: 1-3 mL; 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.

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

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

7. The method for preparing the calcium / iron-based nanomotor loaded with dihydrochlorin e6 according to claim 1, characterized in that: The first stirring time is 12 to 24 hours, and the second stirring time is 30 to 40 minutes.

8. The method for preparing the calcium / iron-based nanomotor loaded with dihydrochlorin e6 according to claim 1, characterized in that: 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 the 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 particle size of the calcium / iron-based nanomotor loaded with dihydrochlorin e6 is 200-300 nm, and the zeta potential is -18 mV.

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

Images