CN111588851A

CN111588851A - Tumor-targeted nano-drug system and preparation method thereof

Info

Publication number: CN111588851A
Application number: CN202010087764.4A
Authority: CN
Inventors: 高漓
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-02-12
Filing date: 2020-02-12
Publication date: 2020-08-28
Anticipated expiration: 2040-02-12
Also published as: CN111588851B

Abstract

The invention discloses a nano-drug system for targeting tumors, wherein a carrier of the nano-drug system is Zn-BP-P-Apt NSs, and the characteristic structural formula of the nano-drug system is as follows: Zn-BP-P-Apt/D NSs. The nano-drug system is combined with chemophotothermal therapy to treat the prostate cancer, has high targeting property on cancer cells, realizes the treatment effect of enhancing the tumor, and can reduce the toxic and side effects generated by chemotherapeutic drugs.

Description

Tumor-targeted nano-drug system and preparation method thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a tumor-targeted nano-drug system and a preparation method thereof.

Background

One of the biggest killers threatening human life health is tumor, chemical drugs are indispensable means for treating prostate cancer, but the non-specificity of the chemical drugs to cancerated cells and normal cells of human body causes the chemical drugs to kill not only cancerated cells but also normal cells in the treatment process, thereby causing serious toxic and side effects on the whole body. In recent years, the search for safe and effective targeted drugs is a hot spot in the research of tumor treatment.

The nano targeting drug uses nano particles as a carrier of the drug, and ensures the targeting of the drug to lesion parts such as tumor and the like mainly by changing the distribution and the pharmacokinetic characteristics of the drug in vivo. The nano drug delivery system can realize the control of drug delivery and release at the molecular level, and can realize the passive or active targeting of the drug at the tumor part through the carrier modification, thereby greatly enhancing the treatment effect and obviously improving the toxic and side effects. In addition, some medicines are highly combined with the nano-carrier by a pharmaceutic method, so that the medicines which are originally easy to degrade and damage due to unstable physicochemical properties or are influenced by large adverse reactions are highly dispersed in the medicine carrier by a special method, and the problems that the medicines cannot be orally taken or have large side effects are solved.

Black Phosphorus (BP) is a black, metallic crystal formed by the conversion of white phosphorus at very high pressure and at relatively high temperatures. Black phosphorus is the least reactive among the phosphorus allotropes and it does not ignite in air. The black phosphorus has strippability, a graphite-like material with excellent electron transfer performance is formed, and the stripped black phosphorus nanosheet is unstable in property, can be oxidized when exposed to air and water, and can be sublimated when heated to 400 ℃ in vacuum. In the prior art, the black phosphorus nanosheet/anti-tumor drug composite material prepared by taking the black phosphorus nanosheet as a substrate is used for treating tumors, so that the anti-tumor effect can be improved. However, the instability of the black scale nanometer material affects the overall stability of a drug carrying system, and the black scale nanometer material is difficult to store, so that the practical use is greatly limited.

Disclosure of Invention

The invention aims at the problems and provides a nano-drug system for targeting tumors and a preparation method thereof, the nano-drug system is combined with chemophotothermal therapy to treat prostate cancer, has high targeting property on cancer cells, realizes the effect of enhancing the treatment effect of tumors, and can reduce the toxic and side effects generated by chemotherapeutics.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the carrier of the nano-drug system for targeting the tumor is Zn-BP-P-Apt NSs.

The preparation method of the carrier comprises the following steps:

A. 70 to 130 parts by weight of Apt-SH with the optical density of 20ODAdding 80000-160000 parts by weight of tris buffer solution (tris); wherein the tris content in tris buffer is 10 mM/ml, pH = 7.4; then adding 160-250 parts by weight of NH₂PEG-MAL and 2-6 parts by weight of TCEP, stirring for 2-5 hours in the dark to obtain NH₂-PEG-Apt solution;

wherein Apt-SH is a thiolated aptamer, OD is an optical density value unit, OD = lg (1/trans), and trans is a light transmittance value of a detection object; NH (NH)₂-PEG-MAL is aminopolyethylene glycol maleimide; TCEP is tris (2-carboxyethyl) phosphine;

B. adding 160-240 parts by weight of BP NSs into the NH obtained in the previous step₂Carrying out ultrasonic treatment on the solution of PEG-Apt for 8 to 20 minutes, stirring the solution for 3 to 8 hours, centrifuging the solution to obtain a precipitate to obtain BP-P-Apt, washing the precipitate twice with deionized water, and adding the washed BP-P-Apt into water to prepare a BP-P-Apt nanosheet suspension, wherein the BP-P-Apt content in the suspension is 0.3 to 0.8 mg/mL;

C. adding zinc acetate which is 0.1-0.2% of the BP-P-Apt nanosheet suspension by mass into the BP-P-Apt nanosheet suspension, carrying out ultrasonic treatment for 2-10 minutes, then stirring for 1-5 hours, centrifuging, collecting precipitate, and washing with deionized water for 1-3 times to obtain the BP-P-Apt nanosheet suspension.

The preparation method of the black phosphorus nanosheet comprises the following steps:

dispersing 800 parts by weight of 300-0000 black phosphorus crystal powder in 1600000-2400000 1-methyl-2-pyrrolidone (NMP), then carrying out ultrasonic treatment on the mixture for 5-12 hours under the ice bath condition to obtain a brown suspension, centrifuging for 10-20 minutes at the rotating speed of 2000-4000rpm, collecting the supernatant, centrifuging for more than 10 minutes at the rotating speed of more than 10000rpm, and collecting the precipitate.

The pH value of the tris buffer solution is 7.4, and the concentration is 10 mM.

The black phosphorus crystal powder is dispersed in 1-methyl-2-pyrrolidone (NMP), and the ultrasonic treatment process under the ice bath condition comprises the following steps: performing ultrasonic treatment by using a probe, wherein the ultrasonic power is 600- & lt800W, and the on/off cycle is performed, and the on/off cycle time is as follows: 2-4 s/3-6 s.

The anti-tumor drug is DOX, namely doxorubicin hydrochloride.

The tumor-targeted nano-drug system has the following characteristic structural formula: Zn-BP-P-Apt/D NSs.

The preparation method of the tumor-targeted nano-drug system comprises the following steps:

160-BP-P-Apt/NSs of 260 weight portions are dispersed in 160000-250000 weight portions of DOX (doxorubicin hydrochloride) aqueous solution with the concentration of 1-2 mg/mL, and then the mixture is stirred for 6 hours in the dark, centrifuged, taken out the precipitate, washed by deionized water and then freeze-dried, thus obtaining the zinc oxide.

After stirring for 6 hours in the dark, the conditions for centrifugation were: the rotation speed is more than 10000rpm, and the time is more than 10 minutes.

The application of the nano-drug carrier and the nano-drug system in the preparation of the drugs for treating prostate cancer has a particularly prominent treatment effect on the nano-drugs.

The invention has the beneficial effects that:

as a new administration way, a tumor-targeted nano-drug system (Zn-BP-P-Apt/D NSs) is combined with a chemophotothermal therapy, Zn2+ is introduced to overcome the instability of BP NSs, and the modification of PEG-Apt can well overcome the defect of low targeting of the traditional chemotherapy, thereby realizing the enhancement of the treatment effect of tumors, reducing the toxic and side effects generated by the chemotherapy drugs, having excellent treatment effect on prostate cancer treatment and laying a theoretical foundation for the research and development of new drugs.

The invention constructs a multifunctional system based on BP NSs, and is particularly suitable for treating prostatic cancer by combining chemophotothermal therapy. The black phosphorus nanosheet can be used as a photosensitizer for photodynamic therapy of tumors and can also be used as a nano carrier material of chemotherapeutic drugs; the introduced Zn2+ can not only improve the stability of BP, but also play a synergistic role in inhibiting the proliferation of prostate cancer cells to a certain extent, and the PEG-Apt modification enables the platform to have good tumor targeting, can prolong the blood circulation time, and can be accumulated in the tumor site in a targeted manner.

The nano-drug system provided by the invention is used as a new administration way, combines with a chemical photothermal therapy to treat prostate cancer, lays a theoretical foundation for the research and development of new drugs, has high targeting property on cancer cells compared with the traditional chemotherapy, realizes the treatment effect of enhancing tumors, and reduces the toxic and side effects generated by chemotherapy drugs.

In a tumor-targeting nano-drug system (Zn-BP-P-Apt/D NSs), the black phosphorus nanosheet is used as a novel two-dimensional material, has a good extinction coefficient and high photo-thermal conversion efficiency, and metabolites are non-toxic, cannot cause specific immune reaction and are high in safety.

Drawings

FIG. 1 is a DLS particle size distribution plot;

FIG. 2 is a particle size diagram under TEM observation;

FIG. 3 is X-ray photoelectron spectroscopy (XPS);

FIG. 4 is a Zeta potential change diagram;

FIG. 5 is a pH and temperature dependent drug release profile;

FIG. 6 is an experimental graph of photothermal properties of different NS;

FIG. 7 is a graph of the stability assessment experiment of the nano-drug system;

FIG. 8 is a graph showing the experimental results of cell uptake and in vitro photothermal therapy;

FIG. 9 is a graph of in vitro cytotoxicity assays;

FIG. 10 is an in vivo photothermal therapy and in vivo biodistribution profile;

FIG. 11 is a black and white image of histological analysis and TUNEL immunofluorescence staining;

figure 12 is a color map of histological analysis and TUNEL immunofluorescent stainings.

Detailed Description

The present invention will be described in detail below with reference to specific examples.

Example 1

The nano-drug system has a characteristic structural formula: the preparation method of the Zn-BP-P-Apt/D NSs comprises the following steps:

A. 3mg of black phosphorus crystal powder is dispersed in 16ml of 1-methyl-2-pyrrolidone, and then the mixture is subjected to ultrasonic treatment for 6 hours under ice bath conditions by using a probe, wherein the ultrasonic power is 600W, and the on/off cycle is carried out, and the on/off cycle time is as follows: 3 s/3 s, centrifuging the obtained brown suspension at 2000 rpm for 10 min, collecting supernatant, centrifuging at 10000rpm above for 10 min, and collecting precipitate to obtain BP NSs.

B. 700ug of Apt-SH with optical density of 20OD is dissolved in 0.8ml of tris buffer; wherein the tris content in tris buffer is 10 mM/ml, pH = 7.4; then 1.6mg of NH were added₂PEG-MAL and 20ug of TCEP, stirring for 2 hours in the dark to obtain NH₂-PEG-Apt solution;

C. adding 1.6mg of BP NSs to the NH obtained in the previous step₂Carrying out ultrasonic treatment on the solution in PEG-Apt for 8 minutes, stirring the solution for 3 hours, centrifuging the solution to obtain a precipitate to obtain BP-P-Apt, washing the precipitate twice with deionized water, and adding the washed BP-P-Apt into water to prepare a BP-P-Apt nanosheet suspension, wherein the content of the BP-P-Apt in the suspension is 0.3 mg/mL;

D. and (3) adding zinc acetate which is 0.1 percent of the mass of the BP-P-Apt nanosheet suspension into 5ml of BP-P-Apt nanosheet suspension, carrying out ultrasonic treatment for 2 minutes, then stirring for 1 hour, centrifuging, collecting precipitate, and washing with deionized water for 1 time to obtain the BP-P-Apt nanosheet suspension.

E. Dispersing 1.6mg of Zn-BP-P-Apt/NSs in 1.6mL of DOX aqueous solution with the concentration of 1 mg/mL, stirring for 6 hours in the dark, centrifuging at the rotating speed of more than 10000rpm for more than 10 minutes, taking the precipitate, washing with deionized water, and freeze-drying to obtain the product.

Example 2

A. dispersing 8mg of black phosphorus crystal powder in 24ml of 1-methyl-2-pyrrolidone, and then carrying out ultrasonic treatment on the mixture for 12 hours under the ice bath condition by using a probe, wherein the ultrasonic power is 800W, and the on/off cycle is carried out, and the on/off cycle time is as follows: 4s/6 s, centrifuging the obtained brown suspension at the rotating speed of 4000rpm for 20 minutes, collecting supernatant, centrifuging at the rotating speed of more than 10000rpm for more than 10 minutes, and collecting precipitate to obtain BP NSs for later use.

B. 1300ug of Apt-SH with optical density of 20OD was dissolved in 1.4ml of tris buffer; wherein the tris content in tris buffer is 10 mM/ml, pH = 7.4; then 2.5mg of NH were added₂PEG-MAL and 60ug of TCEP, stirring for 5 hours in the dark to obtain NH₂-PEG-Apt solution;

C. adding 2.4mg of BP NSs to the NH obtained in the previous step₂Carrying out ultrasonic treatment for 20 minutes in a PEG-Apt solution, stirring for 8 hours, centrifuging to obtain a precipitate to obtain BP-P-Apt, washing twice with deionized water, adding the washed BP-P-Apt into water to prepare a BP-P-Apt nanosheet suspension, wherein the content of the BP-P-Apt in the suspension is 0.8 mg/mL;

D. and (3) adding zinc acetate which is 0.2 percent of the mass of the BP-P-Apt nanosheet suspension into 5ml of BP-P-Apt nanosheet suspension, carrying out ultrasonic treatment for 10 minutes, then stirring for 5 hours, centrifuging, collecting precipitate, and washing with deionized water for 3 times to obtain the BP-P-Apt nanosheet suspension.

E. Dispersing 2.6mg of Zn-BP-P-Apt/NSs in 2.2mL of DOX aqueous solution with the concentration of 2 mg/mL, stirring for 6 hours in the dark, centrifuging at the rotating speed of more than 10000rpm for more than 10 minutes, taking the precipitate, washing with deionized water, and freeze-drying to obtain the product.

Example 3

A. 5mg of black phosphorus crystal powder is dispersed in 20ml of 1-methyl-2-pyrrolidone, and then the mixture is subjected to ultrasonic treatment for 8 hours under the ice bath condition by using a probe, the ultrasonic power is 700W, and the on/off cycle is carried out, and the on/off cycle time is as follows: 2s/4 s, centrifuging the obtained brown suspension at the rotation speed of 2000-4000rpm for 10-20 minutes, collecting the supernatant, centrifuging the supernatant at the rotation speed of more than 10000rpm for more than 10 minutes, and collecting the precipitate to prepare BP NSs for later use.

B. 1000ug of Apt-SH with optical density of 20OD was dissolved in 1ml of tris buffer; wherein the tris content in tris buffer is 10 mM/ml, pH = 7.4; then 2mg parts by weight of NH were added₂PEG-MAL and TCEP 40ug, stirring for 2-5 hours in the dark to obtain NH₂-PEG-Apt solution;

C. adding 160-240 parts by weight of BP NSs into the NH obtained in the previous step₂Carrying out ultrasonic treatment for 15 minutes in a PEG-Apt solution, stirring for 5 hours, centrifuging to obtain a precipitate to obtain BP-P-Apt, washing twice with deionized water, adding the washed BP-P-Apt into water to prepare a BP-P-Apt nanosheet suspension, wherein the content of the BP-P-Apt in the suspension is 0.5 mg/mL;

D. adding zinc acetate which is 0.15 percent of the mass of the BP-P-Apt nanosheet suspension into 5ml of the BP-P-Apt nanosheet suspension, carrying out ultrasonic treatment for 8 minutes, then stirring for 3 hours, centrifuging, collecting precipitate, and washing with deionized water for 1-3 times to obtain the product.

E. Dispersing 2mg of Zn-BP-P-Apt/NSs in 2mL of DOX aqueous solution with the concentration of 1.5 mg/mL, stirring for 6 hours in the dark, centrifuging at the rotating speed of more than 10000rpm for more than 10 minutes, taking the precipitate, washing with deionized water, and freeze-drying to obtain the product.

Example 4

A. 7mg of black phosphorus crystal powder was dispersed in 18ml of 1-methyl-2-pyrrolidone, and then the mixture was subjected to ultrasonic treatment for 10 hours under ice bath conditions using a probe with an ultrasonic power of 650W, and on/off cycle was performed for the following on/off cycle time: 3s/5 s, centrifuging the obtained brown suspension at 3000 rpm for 12 min, collecting supernatant, centrifuging at 10000rpm above for 10 min, and collecting precipitate to obtain BP NSs.

B. 900ug of Apt-SH with optical density of 20OD is dissolved in 0.9ml of tris buffer; wherein the tris (hydroxymethyl) aminomethaneTris content in buffer 10 mM/ml, pH = 7.4; then 1.8mg of NH were added₂PEG-MAL and 50ug of TCEP, stirring for 4 hours in the dark to obtain NH₂-PEG-Apt solution;

C. adding 2.1mg of BP NSs to the NH obtained in the previous step₂Carrying out ultrasonic treatment on the solution in PEG-Apt for 16 minutes, stirring the solution for 4 hours, centrifuging the solution to obtain a precipitate to obtain BP-P-Apt, washing the precipitate twice with deionized water, and adding the washed BP-P-Apt into water to prepare a BP-P-Apt nanosheet suspension, wherein the content of the BP-P-Apt in the suspension is 0.6 mg/mL;

D. adding zinc acetate which is 0.13 percent of the mass of the BP-P-Apt nanosheet suspension into 5ml of BP-P-Apt nanosheet suspension, carrying out ultrasonic treatment for 6 minutes, then stirring for 2 hours, centrifuging, collecting precipitate, and washing with deionized water for 2 times to obtain the product.

E. Dispersing 2.1mg of Zn-BP-P-Apt/NSs in 2.3mL of DOX aqueous solution with the concentration of 1.6 mg/mL, stirring for 6 hours in the dark, centrifuging at the rotating speed of more than 10000rpm for more than 10 minutes, taking the precipitate, washing with deionized water, and then freeze-drying to obtain the product.

Example 5

A. 4mg of black phosphorus crystal powder is dispersed in 22ml of 1-methyl-2-pyrrolidone, and then the mixture is subjected to ultrasonic treatment for 11 hours under ice bath conditions by using a probe, wherein the ultrasonic power is 750W, and the on/off cycle is carried out, and the on/off cycle time is as follows: 4s/3 s, centrifuging the obtained brown suspension at 3000 rpm for 16 min, collecting supernatant, centrifuging at 10000rpm above for 10 min, and collecting precipitate to obtain BP NSs.

B. 1100ug of Apt-SH with an optical density of 20OD was dissolved in 1.1ml of tris buffer; wherein the tris content in tris buffer is 10 mM/ml, pH = 7.4; then 1.9mg of NH were added₂PEG-MAL and 45ug of TCEP, stirring for 3 hours in the dark to obtain NH₂-PEG-Apt solutionLiquid;

C. adding 1.9mg of BP NSs to the NH obtained in the previous step₂Carrying out ultrasonic treatment on the mixture for 14 minutes in a PEG-Apt solution, stirring the mixture for 5 hours, centrifuging the mixture to obtain a precipitate to obtain BP-P-Apt, washing the precipitate twice with deionized water, and adding the washed BP-P-Apt into water to prepare a BP-P-Apt nanosheet suspension, wherein the content of the BP-P-Apt in the suspension is 0.7 mg/mL;

D. adding zinc acetate which is 0.11 percent of the mass of the BP-P-Apt nanosheet suspension into 5ml of the BP-P-Apt nanosheet suspension, carrying out ultrasonic treatment for 8 minutes, then stirring for 3 hours, centrifuging, collecting precipitate, and washing with deionized water for 3 times to obtain the product.

E. Dispersing 2.1mg of Zn-BP-P-Apt/NSs in 1.9mL of DOX aqueous solution with the concentration of 1.1 mg/mL, stirring for 6 hours in the dark, centrifuging at the rotating speed of more than 10000rpm for more than 10 minutes, taking the precipitate, washing with deionized water, and freeze-drying to obtain the product.

Example 6

A. 6mg of black phosphorus crystal powder is dispersed in 17ml of 1-methyl-2-pyrrolidone, and then the mixture is subjected to ultrasonic treatment for 9 hours under the ice bath condition by using a probe, the ultrasonic power is 700W, and the on/off cycle is carried out, and the on/off cycle time is as follows: 2 s/3 s, centrifuging the obtained brown suspension at 3000 rpm for 15 min, collecting supernatant, centrifuging at 10000rpm or above for 10 min, and collecting precipitate to obtain BP NSs.

B. 1200ug of Apt-SH with an optical density of 20OD was dissolved in 1ml of tris buffer; wherein the tris content in tris buffer is 10 mM/ml, pH = 7.4; then 2.0mg of NH were added₂PEG-MAL (aminopolyethylene glycol maleimide) and 40ug of TCEP, stirring for 3 hours in the dark to obtain NH₂-PEG-Apt solution;

C. adding 2mg of BP NSs to the NH obtained in the previous step₂-PEG-Apt solution, sonicating for 15 minutes and stirring 5After hours, centrifuging to obtain a precipitate to obtain BP-P-Apt, washing twice with deionized water, and adding the washed BP-P-Apt into water to prepare a BP-P-Apt nanosheet suspension, wherein the BP-P-Apt content in the suspension is 0.5 mg/mL;

D. adding zinc acetate which is 0.17 percent of the mass of the BP-P-Apt nanosheet suspension into 5ml of the BP-P-Apt nanosheet suspension, carrying out ultrasonic treatment for 9 minutes, then stirring for 4 hours, centrifuging, collecting precipitate, and washing with deionized water for 3 times to obtain the product.

E. Dispersing 2.5mg of Zn-BP-P-Apt/NSs in 2mL of DOX aqueous solution with the concentration of 1.5 mg/mL, stirring for 6 hours in the dark, centrifuging at the rotating speed of more than 10000rpm for more than 10 minutes, taking the precipitate, washing with deionized water, and freeze-drying to obtain the product.

Experimental example 1

The particle size distributions of BP NSs and Zn-BP-P-Apt/D NSs were measured by dynamic light scattering Detection (DLS), respectively, and the results are shown in FIG. 1: the part A is a BP NSs particle size distribution diagram, and the part B is a Zn-BP-P-Apt/D. NSs particle size distribution diagram.

Experimental example 2

The morphology was observed with a Transmission Electron Microscope (TEM), and the results are shown in fig. 2: (C) BPs; (D) BP-P-Apt; (E) Zn-BP-P-Apt,; (F) Zn-BP-P-Apt/D NSs. After Apt and Zn2+ were introduced, there was no significant difference in the morphology of BP (D and E). In fig. F, when the drug was loaded onto BP NSs, a rougher surface was clearly observed in the TEM pictures, indicating successful drug loading.

Experimental example 3

X-ray photoelectron spectroscopy (XPS) was performed using a Kratos Axis Ultra DLD spectrometer with AlK alpha radiation (1486.6 eV photons, 150W). The size and zeta potential of the samples were measured on a Malvern Mastersizer 2000. As a result, as shown in FIG. 3, in part A and part B, Zn2P spectra showed that peaks of Zn2P were 1045.3 eV and 1022.2 eV, respectively, observed from Zn-BP-P-Apt and Zn-BP-P-Apt/D NSs, but a peak of Zn2P was not detected from bare BP and BP-P-Apt, confirming successful zinc ion binding. Zn-BP-P-Apt/D NSs have a weaker Zn2P peak intensity compared to BP-P-Apt-Zn, which is due to the loading of DOX.

Experimental example 4

Zeta potential change: x-ray photoelectron spectroscopy (XPS) was performed using a Kratos Axis Ultra DLD spectrometer with AlK alpha radiation (1486.6 eV photons, 150W). The size and zeta potential of the samples were measured on a Malvern Mastersizer 2000. The results are shown in FIG. 4, where C, 1, 2, 3 and 4 represent BP, BP-P-Apt, Zn-BP-P-Apt and Zn-BP-P-Apt/D, respectively, with the original zeta potential of bare BP being approximately-28.3 mV, and subsequently increasing to-24.1 mV after introduction of NH2-PEG-Apt at the BP surface. With the conjugation of Zn2+, the Zeta potential of PB-P-Apt-Zn became-5.3 mV. Finally, it increased to 12.7 mV after loading with DOX. It can be seen that the Zeta potential increases gradually from 1 to 4.

Experimental example 5

pH and temperature dependent drug release experiments: the cumulative amount of DOX released by Zn-BP-P-Apt/DNSs as a drug was measured in PBS buffer solution at different pH values, and as shown in FIG. 5, the cumulative amount of DOX released by Zn-BP-P-Apt/D NSs in 24 hours was about 43.1%. In contrast, only 22.4% of DOX was released at pH 7.4 at the same time period. This indicates that the acidic environment will accelerate the release of loaded DOX, indicating that the acidic environment of tumors in vivo will accelerate the release of anticancer drugs. Under the stimulation of 3 808nm laser on/off treatment cycles, the DOX accumulation rate under the pH value of 5.0 is increased to 56.8 percent after 24 hours, and the photo-thermal induced drug release behavior is proved, and the results show that the pH and temperature response drug release of Zn-BP-P-Apt/D can not only reduce the side effect of the drug, but also improve the utilization rate of the drug.

Experimental example 6

Photothermal property detection experiments of different NS:

BP, Zn-BP-P-Apt/D NSs and pure water are exposed to NIR laser irradiation at 808nm, and the temperature change is monitored and quantified by an infrared thermal imager, and the result is shown in FIG. 6, part A, wherein the temperature of a BP solution (100 mu gmL-1) is increased by about 29.6C, while pure water is hardly heated by irradiation, which indicates that BP can effectively convert NIR light into heat energy.

The temperature profile of the Zn-BP-P-Apt/D suspension at a concentration of 100. mu.g/mL under NIR laser at different powers, as shown in part C of the graph, shows that the temperature profile of the dispersion can still reach about 16.1 ℃ with laser powers as low as 0.5w cm-2.

100 μ g/mL of Zn-BP-P-Apt/DNSs suspension at a power density of 1.0 w/cm^-2Four laser on/off cycles in water were performed with the 808nmNIR laser of (a), as shown in part D of the figure, and the temperature history did not show any significant change after 4 cycles of irradiation with the NIR laser. The above shows that Zn-BP-P-Apt/D NSs has satisfactory light stability.

In the graph, the part B is a temperature rise curve of Zn-BP-P-Apt/D NSs suspensions with different concentrations at the laser power density of 1.0w cm < -2 >, and the Zn-BP-P-Apt/D shows concentration-dependent photothermal properties.

Experimental example 7

And (3) stability evaluation: bare BP and Zn-BP-P-Apt/D NSs with the same BP concentration (100. mu. gmL-1) were dispersed in water and exposed to air for 6 days, irradiated with NIR laser at 808nm, at a power density of 1w cm-2. They were then tested for photothermal performance at predetermined time intervals. The results are shown in fig. 7, parts a and B, with the temperature of the bare BP increasing by about 29.8 ℃ in 10 minutes, but the radiation increased the temperature by only 20 ℃ after 6 days, so the photothermal properties of the BP rapidly decay due to its degradation. In contrast, Zn-BP-P-Apt/D apparently has higher photo-thermal stability. After 6 days, the temperature rise changed only about 3.3C (from 26.4 to 23.1C). Indicating that coordination with Zn2+ can greatly stabilize BP.

Bare BP and Zn-BP NSs were dispersed in water and exposed to air at room temperature, 95% humidity for 2 days. The morphology of the two samples was observed by an ultra-deep three-dimensional microscope, and in fig. 7, in the C-F part, the C part is an image of bare BP at 0 hour, the D part is an image of bare BP at 48 hours, the E part is an image of Zn-BP NSs at 0 hour, and the F part is an image of Zn-BP NSs at 48 hours, so that bare BP degradation, especially at the edge, can be clearly seen. In contrast, the surface of Zn-BP-P-Apt/DNSs is almost unchanged, and the coordination of zinc ions and BP NSs can effectively prevent the oxidation of BP in humid air, thereby improving the stability of the BP.

Experimental example 8

1. The results of laser scanning confocal microscopy images of PC3 cells treated with DOX, Zn-B P-P/D, Zn-BP-P-Apt/D, Zn-BP-P-Apt/D + free Apt, respectively, are shown in FIG. 8, e.g., section A in FIG. 8, the Zn-BP-P-Apt/D group exhibits a stronger fluorescence signal than the Zn-BP-P/D group, indicating that aptamer-modified NPs are able to bind efficiently to PC3 cells. Since a large amount of free AS1411 aptamer binds to nucleolin (Apt receptor) on the plasma membrane, the binding between Zn-BP-P-Apt/D and nucleolin is largely inhibited, resulting in a significantly lower red fluorescence intensity than Zn-BP-P-Apt/D. Due to the small size of DOX molecules, it is free to penetrate the plasma and nuclear membranes rapidly by passive diffusion effects, and the free DOX group shows the strongest red fluorescence intensity.

2. Survival of PC3 cells under only NIR laser irradiation, different concentrations of BP + NIR laser irradiation, BP-P-Apt + NIR laser irradiation, as shown in part B of FIG. 8, Apt-modified BP NSs (BP-P-Apt) exhibited the highest photothermal cytotoxicity, and about 87.8% of PC3 cells were killed at a BP-P-Apt concentration of 50 μ g mL-1, at much higher levels than the BP (70.8%) and BP-P (72.7%) groups at the same concentration. In contrast, NIR irradiation alone was not very cytotoxic to PC3 cells.

Experimental example 9

Zn2+ cytotoxicity to PC 3: referring to section A of FIG. 9, the relative survival of human prostate cancer cells (PC 3) after 24, 48 and 72 hours incubation at various concentrations of Zn2+ (1, 2.5, 5, 7.5, 10, 20 and 30 μ gmL-1), the toxicity of Zn2+ to PC3 cells was time and dose dependent, and significant cytotoxicity began to appear on PC3 cells when the concentration of Zn2+ exceeded 5 μ gmL-1.

Cytotoxicity of DOX on PC3 cells: see FIG. 9 for relative survival of PC3 cells after 24, 48 and 72 hours incubation at different concentrations of DOX (0.05, 0.1, 0.5, 1, 2.5, 5 and 10 μ g mL-1) in part B, which is time and dose dependent on toxicity of DOX to PC3 cells.

And combined cytotoxicity of DOX on PC3 cells: referring to part C of FIG. 9, cell viability of PC3 cells after 48h treatment with different concentrations of DOX and DOX + Zn2+ (1 μ g mL-1 concentration of Zn2 +) respectively, zinc ion enhanced the efficacy of DOX on PC3 cells in vitro to some extent for all DOX concentrations tested.

Combined cytotoxicity of DOX and photothermal therapy on PC3 cells: referring to section D in FIG. 9, cell viability of PC3 cells after 48h with or without laser irradiation for samples of varying concentrations of DOX (0.05, 0.1, 0.5, 1, 2.5, and 5 μ g mL-1), BP NSs (denoted as BP-P/D) loaded with DOX but without Apt and Zn2+ showed moderate cytotoxicity, and cell viability at 5 μ g DOX mL-1 after 48h was about 57.1%. In contrast, after Apt is introduced, BP-P-Apt/D shows stronger toxicity to PC3 cells due to the active tumor targeting ability of Apt. After NIR irradiation, BP-P-Apt/D has better killing toxicity to tumor cells than other formulations without NIR irradiation, indicating that the combination chemo/photothermal therapy has better therapeutic effect than chemotherapy alone. In addition, Zn2+ bound, Zn-BP-P-Apt/D + NIR group showed the best tumor cell killing effect, which indicates the importance of Zn ion enhanced chemo/photothermal therapy in combination for the treatment of prostate cancer.

Experimental example 10

In vivo photothermal imaging: establishing a mouse tumor model, injecting Phosphate Buffered Saline (PBS), Zn-BP-P/D and Zn-BP-P-Apt/D, irradiating for 5 minutes by laser (1.5W cm-2) with 808nm 24 hours after injection, and collecting an in vivo infrared thermograph. Under laser irradiation, as shown in part a of fig. 10, the tumor temperature of the PBS-treated group slightly increased after 5 minutes of laser irradiation. In contrast, in the group treated with Zn-BP-P/D, high temperature was rapidly generated in the tumor region and reached about 47.3 ℃. Zn-BP-P-Apt/D showed better performance than Zn-BP-P/D, with a temperature rise to about 51.2 ℃ within 5 minutes sufficient to kill tumor cells. The result shows that Zn-BP-P-Apt/D can be effectively accumulated at the tumor site and can be used as a photo-thermal agent to generate high heat in vivo so as to effectively kill tumor cells.

In vivo biodistribution: mouse tumor model after injection of 100. mu.L of LDOX, Zn-BP-P/D and Zn-BP-P-Apt/D (100. mu.L, 5mg of DOX mL-1), 3 or 24 hours, the distribution of DOX in heart, liver, spleen, lung, kidney and tumor was measured by Maestro ™ automatic in vivo imaging system, see section B in FIG. 10, and after 3 hours, DOX in Zn-BP-P/D and Zn-BP-P-Apt/D NSs was mainly distributed on tumor, while free DOX group could detect stronger fluorescence intensity not only in tumor but also in liver. For the free DOX group 24 hours after injection, the DOX intensity weakened at the tumor site, meaning that its retention time in blood and tissues was short. In contrast, after 24 hours, much stronger fluorescence signals of DOX in tumors were observed in both groups of BP loaded with DOX. As expected, Zn-BP-P-Apt/D showed the strongest DOX signal in tumors, indicating excellent tumor targeting ability.

Experimental example 11

In vivo treatment: the mouse tumor model is injected with PBS (1), (2) DOX, (3) BP-P/D, (4) BP-P-Apt/D, (5) BP-P-Apt/D + NIR and (6) Zn-BP-P-Apt/D + NIR respectively, after 16 days, tumors and main organs such as heart, liver, spleen, lung and kidney are taken for histological analysis and TUNEL immunofluorescence staining. As shown in fig. 11, sections a and B, (a) tumor images taken from mice on day 16, and (B) tumor growth curves after different treatments, rapid growth of tumors was observed in the PBS-treated group during the 16-day treatment period. The tumor volume of free DOX injected mice can be partially but not significantly reduced compared to the control group, indicating that the dose of DOX administered is not sufficient to effectively kill tumor cells. In contrast, a potent antitumor effect was found in the BP-P/D and BP-P-Apt/D groups compared to free DOX. This is due to the fact that due to the EPR effect, DOX accumulates more at the tumor site. In addition, BP-P-Apt/D shows more effective tumor ablation effect than BP-P/D, which indicates that Apt has good tumor targeting capability. After photothermal treatment, PC3 tumors were further inhibited in the BP-P-Apt/D group. Encouraging, the tumor growth curves of the Zn-BP-P-Apt/D + NIR group showed the lowest growth rate, and three mice in this group were completely cured after 16 days. This result demonstrates the most excellent tumor suppression of Zn-BP-P-Apt/D + NIR, which is attributed to Zn2+ enhanced chemo/photothermal combination therapy.

Section C in fig. 11 is TUNEL fluorescent staining of tumor sections at the end of the in vivo anti-tumor experiment. Few TUNEL positive cells (light colored in black and white) were observed in the BPS and pure DOX groups, while significant TUNEL positive apoptotic cells were observed in the Zn-BP-P-Apt/D NSs group. The original color map is attached as FIG. 12, and TUNEL-positive cells are the green part of the map.

Claims

1. A tumor-targeting nano-drug system, characterized by: the carrier is Zn-BP-P-Apt NSs.

2. The tumor-targeting nano-drug system according to claim 1, characterized in that:

the preparation method of the carrier comprises the following steps:

A. adding 70-130 parts by weight of Apt-SH with the optical density of 20OD into 80000-160000 parts by weight of tris buffer; wherein the tris content in tris buffer is 10 mM/ml, pH = 7.4; then adding 160-250 parts by weight of NH₂PEG-MAL and 2-6 parts by weight of TCEP, stirring for 2-5 hours in the dark to obtain NH₂-PEG-Apt solution;

B. adding 160-240 parts by weight of BP NSs into the NH obtained in the previous step₂Carrying out ultrasonic treatment for 8-20 minutes in a PEG-Apt solution, stirring for 3-8 hours, centrifuging to obtain a precipitate to obtain BP-P-Apt, washing twice with deionized water, adding the washed BP-P-Apt into water to prepare a BP-P-Apt nanosheet suspension, wherein the content of BP-P-Apt in the suspension is 0.3-0.8 mg/mL

3. The tumor-targeting nano-drug system according to claim 2, characterized in that:

dispersing 800 parts by weight of 300-material black phosphorus crystal powder in 1600000-material 2400000-material 1-methyl-2-pyrrolidone, then carrying out ultrasonic treatment on the mixture for 5-12 hours under the ice bath condition to obtain a brown suspension, centrifuging for 10-20 minutes at the rotating speed of 2000-material 4000rpm, collecting the supernatant, centrifuging for more than 10 minutes at the rotating speed of more than 10000rpm, and collecting the precipitate.

4. The tumor-targeting nano-drug system according to claim 3, characterized in that:

5. The tumor-targeting nano-drug system according to claim 3, characterized in that:

the black phosphorus crystal powder is dispersed in 1-methyl-2-pyrrolidone, and the ultrasonic treatment process under the ice bath condition comprises the following steps: performing ultrasonic treatment by using a probe, wherein the ultrasonic power is 600- & lt800W, and the on/off cycle is performed, and the on/off cycle time is as follows: 2-4 s/3-6 s.

6. The tumor-targeting nano-drug system according to claim 1, characterized in that: the anti-tumor drug is DOX.

7. The tumor-targeting nano-drug system according to claim 6, characterized by the following structural formula: Zn-BP-P-Apt/D NSs.

8. The tumor-targeted nano-drug system according to claim 7, wherein the preparation method comprises the following steps:

dispersing 200 parts by weight of Zn-BP-P-Apt/NSs into 200000 parts by weight of DOX aqueous solution with the concentration of 1-2 mg/mL, stirring for 6 hours in the dark, centrifuging, taking precipitate, washing with deionized water, and freeze-drying to obtain the product.

9. The tumor-targeting nano-drug system according to claim 8, characterized in that: after stirring for 6 hours in the dark, the conditions of centrifugation were: the rotation speed is more than 10000rpm, and the time is more than 10 minutes.

10. Use of the tumor-targeted nano-drug system of claims 1-9 for the preparation of a medicament for the treatment of prostate cancer.