CN112641942A

CN112641942A - Near-red light-control nano gas diagnosis and treatment agent and preparation method and application thereof

Info

Publication number: CN112641942A
Application number: CN202011572952.2A
Authority: CN
Inventors: 何前军; 金召奎; 朵燕红; 赵鹏赫; 温艳媛
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2021-04-13
Anticipated expiration: 2040-12-25
Also published as: CN112641942B

Abstract

The invention provides a near-red light control nano gas diagnosis and treatment agent as well as a preparation method and application thereof, wherein the near-red light control nano gas diagnosis and treatment agent comprises a two-dimensional nano sheet and a load loaded on the two-dimensional nano sheet, the load comprises a transition metal carbonyl compound or a nucleophilic NO donor, the preparation method of the near-red light control nano gas diagnosis and treatment agent is simple, CO or NO gas is released under the response of near-infrared light, the ingenious combination of low-toxicity and high-efficiency tumor photothermal therapy and gas therapy can be realized, the treatment efficiency is greatly improved, and the development of clinical application is facilitated.

Description

Near-red light-control nano gas diagnosis and treatment agent and preparation method and application thereof

Technical Field

The invention relates to the technical field of nano-drugs, in particular to a near-red light-control nano-gas diagnosis and treatment agent and a preparation method and application thereof.

Background

Chemotherapy is the main method for clinically treating cancer, especially middle and late stage cancer, and is an effective systemic treatment means. However, most of the current chemotherapy drugs have no specific recognition, are easy to damage normal cells and tissues, even cause systemic poisoning, have low drug intake efficiency of tumor cells, and easily cause drug resistance of the tumor cells due to repeated administration. Currently, a nano-drug delivery system is adopted to deliver chemotherapeutic drugs to tumor cells in a targeted manner, so that efficient and directional delivery of the chemotherapeutic drugs is achieved. However, chemotherapy drugs still leak during delivery and the toxic side effects of chemotherapy are often not eliminated at all.

Photothermal therapy has advantages of short time, significant efficacy, low toxic and side effects, etc., and is further widely noted and studied, and has been partially used in clinical applications. However, their therapeutic efficacy is limited by the availability of highly effective photothermal agents.

Gas therapy, an emerging tumor therapy technology, mainly utilizes therapeutic gases (nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H)₂S), oxygen (O)₂) Etc.) to replace traditional chemotherapeutic drugs. The CO and NO therapeutic gas medicine can selectively induce the apoptosis of tumor cells and protect normal cells under proper concentration, so that the CO and NO therapeutic gas medicine is regarded as a green therapeutic technology. However, both modes of administration of clinical gas therapy, direct inhalation of therapeutic gases and injection of gas-releasing molecular compounds (NO/modifying molecules, NORMs/CORMs), are difficult to control in blood levels of gas and effective levels in the focal zone.

Currently, the main development technologies for targeted delivery and controlled release of gaseous drugs include: (1) the development of pH or temperature responsive gas prodrugs to achieve tumor microenvironment and temperature responsive gas release, but still suffers from the disadvantage that gas prodrugs will spontaneously release; (2) the light-responsive gas prodrug is developed to realize light-controlled release of CO or NO gas, but the light-responsive bands of the prodrugs reported in some current researches are mainly in ultraviolet and visible light regions, and near-infrared light-responsive drugs with tissue penetration depth and low phototoxicity are still difficult to prepare. (3) Based on nano engineering, the nano carrier is used for loading the gas prodrug to realize tumor targeted transportation and responsive release. The photoresponse CORMs represented by transition metal carbonyl compounds can release CO gas under the triggering of in vitro ultraviolet light, visible light and even near infrared light, for example, graphene oxide-manganese carbonyl compounds (MnCO-GO) with a cage structure can release CO gas on demand in vivo through in vitro near infrared light switches. However, most of these nano-drugs require external stimulation, and have high toxicity, lack of release specificity, and complicated synthesis process.

Disclosure of Invention

The invention provides a near-red light-controlled nano gas diagnosis and treatment agent, a preparation method and application thereof, aiming at realizing the ingenious combination of low-toxicity and high-efficiency tumor photothermal therapy and gas therapy, improving the treatment efficiency and promoting the development of clinical application.

In order to achieve the above purpose, the present invention provides a near-red light control nano gas diagnosis and treatment agent, which comprises a two-dimensional nanosheet, a load loaded on the two-dimensional nanosheet, and a lipid membrane wrapping the two-dimensional nanosheet and the load, wherein the load comprises a transition metal carbonyl compound or a nucleophilic NO donor.

Preferably, the loading of the load is 80-150%.

Preferably, the two-dimensional nanoplatelets comprise black phosphorus nanoplatelets, arsine nanoplatelets or stibine nanoplatelets;

the length and the width of the two-dimensional nano sheet are both less than or equal to 150nm, and the thickness of the two-dimensional nano sheet is less than or equal to 2 nm.

Preferably, the transition metal carbonyl compound is a manganese carbonyl compound or an iron carbonyl compound.

Preferably, the manganese carbonyl compound comprises a manganese carbonyl compound and a manganese carbonyl bromide compound, and the iron carbonyl compound comprises an iron pentacarbonyl compound and an iron dodecacarbonyl compound.

Preferably, the nucleophilic NO donor has an [ N (O) NO ] functional group.

Preferably, the near-red light-controlled nano gas diagnosis and treatment agent responds to release CO or NO gas under the irradiation of near-infrared light, and generates a photothermal effect at the same time.

In addition, the invention also provides a preparation method of the near-red light control nano gas diagnosis and treatment agent, which comprises the following steps:

dispersing the two-dimensional nanosheets in isopropanol solution, adding the load, stirring at room temperature under the protection of nitrogen for a preset time, and centrifuging to obtain two-dimensional nanosheets loaded with transition metal carbonyl compounds or nucleophilic NO donors;

dispersing the two-dimensional nanosheets loaded with the transition metal carbonyl compound or the nucleophilic NO donor into chloroform, adding 1, 2-distearoyl-sn-propanetriyl-3-phosphorylcholine, cholesterol and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, uniformly dissolving, then drying a solvent, and adding deionized water for redissolution and dispersion to obtain the near-red light control nano gas diagnosis and treatment agent.

Preferably, the mass ratio of the two-dimensional nanosheet to the transition metal carbonyl compound or the nucleophilic NO donor is 1: 1.5 to 6.

In addition, the invention also provides application of the near-red light control nano gas diagnosis and treatment agent, and the near-red light control nano gas diagnosis and treatment agent is applied to preparation of nano medicines for inhibiting tumor cells

Compared with the prior art, the invention provides a near-red light control nano gas diagnosis and treatment agent and a preparation method and application thereof, wherein the near-red light control nano gas diagnosis and treatment agent comprises a two-dimensional nano sheet and a load loaded on the two-dimensional nano sheet, the load comprises a transition metal carbonyl compound or a nucleophilic NO donor, the preparation method of the near-red light control nano gas diagnosis and treatment agent is simple, CO or NO gas is released under the response of near-infrared light, the ingenious combination of low-toxicity and high-efficiency tumor photothermal therapy and gas therapy can be realized, the treatment efficiency is greatly improved, and the development of clinical application is facilitated.

Drawings

FIG. 1 is a diagram showing the structure and performance of the near-red light-controlled nano-gas medical agent of the present invention;

FIG. 2 is a graph showing the photo-thermal effect and the light-controlled CO release performance of the near-red light-controlled nano-gas diagnostic agent according to the present invention;

FIG. 3 is a graph showing the results of a simulation experiment of the near-red light-controlled nano-gas therapeutic agent of the present invention at the cellular level;

FIG. 4 is a graph showing the results of photoacoustic imaging and photothermal imaging experiments performed at the model animal level by the near-red light-controlled nano-gas diagnostic agent according to the present invention;

FIG. 5 is a graph showing the animal-level anticancer effect of the near-red light-controlled nano-gas therapeutic agent model of the present invention;

fig. 6 is a schematic flow chart of the method for preparing the near-red light-controlled nano-gas medical treatment agent of the present invention.

The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.

Detailed Description

It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The embodiment of the invention provides a near-red light-controlled nano gas diagnosis and treatment agent. The near-red light-controlled nano gas diagnosis and treatment agent comprises a two-dimensional nano sheet, a load loaded on the two-dimensional nano sheet and a lipid membrane wrapping the two-dimensional nano sheet and the load, wherein the load comprises a transition metal carbonyl compound or a nucleophilic NO donor.

Wherein the loading capacity of the load is 80-150%.

Specifically, the two-dimensional nanoplatelets include Black Phosphorus Nanoplatelets (BPN), arsene nanoplatelets (Arsenene nanoplatelets), or stibene nanoplatelets (antistonene nanoplatelets); the length and the width of the two-dimensional nano sheet are both less than or equal to 150nm, and the thickness of the two-dimensional nano sheet is less than or equal to 2 nm.

The nucleophilic NO donor has an [ N (O) NO ] functional group. For example, the nucleophilic monomer may be O2-vinyl diazeniumdiolates, O2-glycosylated diazeniumdiolates. The diazeniumdiolates functional group is a resonant structure formed by one N atom and two O atoms. Where diazen in diazeniumdiolates represents the "N ═ N" structure, "ium" represents a positive charge on the N atom, and "diolate" represents two negatively charged O atoms in the molecule. The nucleophilic NO donor releases NO in response to infrared light. Furthermore, the nucleophilic NO donor may also be another compound comprising an [ N (O) NO ] functional group.

In this example, the lipid membrane is a Lipid Membrane (LM) formed by dispersing two-dimensional nanoplatelets loaded with a transition metal carbonyl compound or a nucleophilic NO donor into chloroform, and adding 1, 2-distearoyl-sn-propanetriyl-3-phosphorylcholine (DSPC), cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).

Specifically, the near-red light-controlled nano gas diagnosis and treatment agent can be a compound of a two-dimensional nano sheet, a transition metal carbonyl compound and a lipid membrane, or a compound of a two-dimensional nano sheet, a nucleophilic NO donor and a lipid membrane. For example, the composite BPN-MnCO @ LM formed by the black phosphorus nanosheet, the manganese carbonyl compound and the lipid membrane, and the composite BPN-N (O) NO @ LM formed by the black phosphorus nanosheet, the nucleophilic NO donor and the lipid membrane.

In the embodiment, the near-red light control nano gas diagnosis and treatment agent has a good photo-thermal effect. Specifically, BPN-MnCO @ LM is taken as an example for detailed description. The near-red light-control nano gas diagnosis and treatment agent BPN-MnCO @ LM comprises a black scale nano sheet, a carbonyl manganese compound and a lipid film. As shown in fig. 1, fig. 1 is a structural and performance characterization diagram of the near-red light-controlled nano-gas medical treatment agent of the present invention. FIG. 1a is a plot of the size and thickness profile of the nano-drug, FIGS. 1a and 1b are a plot of the thickness profile and diameter profile, respectively, of BPN-MnCO @ LM, FIGS. 1a and 1b clearly show that BPN-MnCO @ LM is a well dispersed nanosheet, with a thickness of 1.5-3nm being abundant, preferably less than 2 nm; the length and the width of the two-dimensional nano sheet are both less than or equal to 150nm, and most of the length and the width of the two-dimensional nano sheet are less than 100 nm.

Further, with continued reference to FIG. 1, FIG. 1c is an infrared spectrum of BPN-MnCO @ LM with wavenumber on the abscissa and transmittance on the ordinate; FIG. 1d is a Raman spectrum of BPN-MnCO @ LM with Raman shift on the abscissa and intensity on the ordinate; FIG. 1e is an XPS spectrum of BPN-MnCO @ LM with binding energy on the abscissa and intensity on the ordinate); fig. 1f is a transient spectrum, with delay time on the abscissa and optical density difference on the ordinate. The infrared spectrum shows that the BPN-MnCO @ LM nano diagnosis and treatment agent consists of C/O/Mn/P elements; the Raman spectrum shows that the BPN-MnCO @ LM nano diagnosis and treatment agent contains a functional group of carbonyl manganese, MnCO is well loaded on the BPN, and the XPS spectrum and the transient spectrum show that the BPN and the MnCO have a coordination bonding effect and have energy transfer performance.

Further, referring to fig. 2, fig. 2 is a graph showing the photo-thermal effect and the photo-controlled CO release performance of the near-red light-controlled nano-gas medical agent of the present invention. Specifically, FIG. 2a shows the photothermal stability of BPN-MnCO @ LM and FIG. 2b shows the photothermal effect of BPN-MnCO @ LM, which has better photothermal properties and more excellent photothermal stability than BPN, as shown in FIGS. 2a and 2 b. FIG. 2c shows the photothermal effect of BPN-MnCO @ LM, the abscissa shows the near-infrared laser irradiation time, and the ordinate shows the CO release amount, and FIG. 2c shows that the BPN-MnCO @ LM nano-diagnostic agent optically releases CO in a concentration-dependent manner, and the higher the concentration, the greater the CO release amount.

In order to verify whether the BPN-MnCO @ LM can carry out effective light-controlled release in cells, a cloning experiment, an anti-migration experiment and a DNA damage experiment at a cell level are carried out. Specifically, fig. 3 is a graph showing the simulation experiment result of the near-red light-controlled nano-gas therapeutic agent at the cellular level. Wherein, FIG. 3a is the result of quantitative analysis of cell cloning experiments, and FIG. 3a shows that when BPN-MnCO @ LM is illuminated, it can obviously inhibit the cloning proliferation of tumor cells; FIG. 3b is the result of quantitative analysis of cell migration assay, and FIG. 3b shows that BPN-MnCO @ LM significantly inhibits tumor cell migration when irradiated with light; FIG. 3c is the result of quantitative analysis of DNA damage experiments on cells, and FIG. 3c shows that when BPN-MnCO @ LM is irradiated, it can cause significant DNA damage to cancer cells.

To study the photothermal effect of the BPN-MnCO @ LM at the animal level, photothermal experiments were performed in vivo in tumor mice, which were injected with a nano diagnostic agent via the tail vein and then irradiated with near-infrared laser at the tumor area. FIG. 4 is a graph showing the results of photoacoustic imaging and photothermal imaging experiments at model animal level of the near-red light-controlled nano-gas diagnostic agent of the present invention. Figure 4 shows that with prolonged irradiation time, the temperature of the tumor area increases significantly, showing good passive targeting of the tumor and photothermal effects.

Further, to study the anti-cancer effect of the BPN-MnCO @ LM on animals, the anti-cancer effect of tumor mice injected with the BPN-MnCO @ LM was recorded. Specifically, as shown in fig. 5, fig. 5 is a graph showing the animal-level anticancer effect of the near-red light-controlled nano gas therapeutic agent of the present invention. Wherein figure 5a is a flow chart of animal experiments: tumor cells are inoculated in mice in advance (generally 7 days before the injection of the BPN-MnCO @ LM nano-drug), then the BPN-MnCO @ LM nano-drug is injected in the mice, the irradiation is carried out by infrared light, and various indexes of the mice are tested and recorded at 2 days, 4 days and 14 days. FIG. 5b is a graph showing the change in tumor volume of mice, with the time taken to raise the mice on the abscissa and the tumor volume on the ordinate. The results show that the nano diagnosis and treatment agent and laser group has excellent capacity of inhibiting the growth of mouse tumors; fig. 5c is a photograph of the tumor site obtained by dissecting the corresponding experimental mouse after 14 days of treatment, and the result shows that the tumor of the nano diagnosis and treatment agent plus laser group is the smallest and the inhibition effect is the best; fig. 5d is the corresponding tumor mass statistics, which also verifies the best antitumor effect of the nano-diagnostic agent plus laser group.

The embodiment provides a near-red light control nano gas diagnosis and treatment agent, and a preparation method and application thereof, wherein the near-red light control nano gas diagnosis and treatment agent comprises a two-dimensional nano sheet and a load loaded on the two-dimensional nano sheet, the load comprises a transition metal carbonyl compound or a nucleophilic NO donor, CO or NO gas is released under the response of near-infrared light, the ingenious combination of low-toxicity and high-efficiency tumor photothermal therapy and gas therapy can be realized, the treatment efficiency is greatly improved, and the development of clinical application is facilitated.

Further, the present invention also provides a method for preparing a near-red light-controlled nano-gas diagnostic agent, specifically, as shown in fig. 6, fig. 6 is a schematic flow chart of the method for preparing a near-red light-controlled nano-gas diagnostic agent of the present invention, and the method includes:

step S101: dispersing the two-dimensional nanosheets in isopropanol solution, adding the load, stirring at room temperature under the protection of nitrogen for a preset time, and centrifuging to obtain two-dimensional nanosheets loaded with transition metal carbonyl compounds or nucleophilic NO donors;

step S102: dispersing the two-dimensional nanosheets loaded with the transition metal carbonyl compound or the nucleophilic NO donor into chloroform, adding 1, 2-distearoyl-sn-propanetriyl-3-phosphorylcholine, cholesterol and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, uniformly dissolving, then drying a solvent, and adding deionized water for redissolution and dispersion to obtain the near-red light control nano gas diagnosis and treatment agent.

And preparing the two-dimensional black phosphorus nanosheet by adopting a liquid-phase mechanical crushing method in advance. Specifically, 25mg of black phosphorus powder was added to 50mL of isopropyl alcohol, argon gas was introduced to remove oxygen in the solvent, and then the mixed solution was placed in constant-temperature ice water and ultrasonicated with an ultrasonicator (power: 25%; ON/OFF time: 45s/15s) for 12 hours. Removing uncrushed black phosphorus from the product by low-speed centrifugation (1000rpm, 10min), collecting the nanosheets by ultrafiltration (MWCO 100kDa, 3750rpm,30min), washing for 2 times by using deoxidized deionized water, cleaning to remove additional products of the reaction, obtaining the two-dimensional black scale nanosheets, and storing at 4 ℃ after packaging. The size of the Black Phosphorus Nanosheet (BPN) is within 150nm, and the thickness is within 2 nm.

And then loading a transition metal carbonyl compound or a nucleophilic carbonyl compound on the two-dimensional nanosheet through in-situ reaction so as to perform coordination with the transition metal or a nucleophilic group through a metal empty orbit of the two-dimensional nanosheet. Specifically, dispersing the two-dimensional nanosheets in an isopropanol solution treated by nitrogen, adding a certain proportion of the transition metal carbonyl compound or the nucleophilic NO donor, dissolving, dispersing, and then continuing stirring at room temperature for reaction for 24 hours under the protection of nitrogen; after the reaction is finished, collecting the precipitate by high-speed centrifugation (12000rpm), and washing the precipitate by ethanol and deionized water to obtain the drug-loaded nanosheet. The feeding mass ratio of the two-dimensional nanosheet to the transition metal carbonyl compound or the nucleophilic NO donor is 1: 1.5 to 6.

Then, dispersing the two-dimensional nanosheet loaded with the transition metal carbonyl compound or the nucleophilic NO donor into chloroform, adding 1, 2-distearoyl-sn-propanetriyl-3-phosphorylcholine, cholesterol and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to form a lipid membrane on the surface of the two-dimensional nanosheet loaded with the transition metal carbonyl compound or the nucleophilic NO donor, uniformly dissolving, then spin-drying the solvent, and adding deionized water for redissolving and dispersing to obtain the near-red light control nano gas diagnosis and treatment agent. Wherein, DSPC: cholesterol: the preferable mass ratio of the DSPE-PEG2000 is 1: 0.326: 0.178.

furthermore, the invention also provides application of the near-red light control nano gas diagnosis and treatment agent, and the near-red light control nano gas diagnosis and treatment agent is applied to preparation of nano medicines for inhibiting tumor cells.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or system. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a process, method, article, or system that comprises the element.

The above-mentioned serial numbers of the embodiments of the present invention are merely for description and do not represent the merits of the embodiments.

Through the above description of the embodiments, those skilled in the art will clearly understand that the method of the above embodiments can be implemented by software plus a necessary general hardware platform, and certainly can also be implemented by hardware, but in many cases, the former is a better implementation manner. Based on such understanding, the technical solution of the present invention may be embodied in the form of a software product, which is stored in a storage medium (e.g., ROM/RAM, magnetic disk, optical disk) as described above and includes instructions for causing a terminal device to execute the method according to the embodiments of the present invention.

The above description is only for the preferred embodiment of the present invention and is not intended to limit the scope of the present invention, and all equivalent structures or flow transformations made by the present specification and drawings, or applied directly or indirectly to other related arts, are included in the scope of the present invention.

Claims

1. The near-red light-controlled nano gas diagnosis and treatment agent is characterized by comprising a two-dimensional nano sheet, a load loaded on the two-dimensional nano sheet and a lipid membrane wrapping the two-dimensional nano sheet and the load, wherein the load comprises a transition metal carbonyl compound or a nucleophilic NO donor.

2. The near-red light control nano-gas medical treatment agent according to claim 1, wherein the loading amount of the loading substance is 80-150%.

3. The near-red photo-controlled nano-gas diagnostic agent according to claim 1, wherein the two-dimensional nanosheets comprise black phosphorus nanosheets, arsenic ene nanosheets, or antimony ene nanosheets;

4. The near-red light-controlled nano-gas diagnostic agent according to claim 1, wherein the transition metal carbonyl compound is a manganese carbonyl compound or an iron carbonyl compound.

5. The near-red photo-controlled nano-gas diagnosis and treatment agent according to claim 4, wherein the manganese carbonyl compounds comprise manganese carbonyl compounds and manganese carbonyl bromide compounds, and the iron carbonyl compounds comprise iron pentacarbonyl compounds and iron dodecacarbonyl compounds.

6. The near-red light-controlled nano-gas diagnostic agent according to claim 1, wherein the nucleophilic NO donor has a [ N (O) NO ] functional group.

7. The near-red light-controlled nano-gas diagnosis and treatment agent according to claim 1, wherein the near-red light-controlled nano-gas diagnosis and treatment agent responsively releases CO or NO gas under irradiation of near-infrared light and simultaneously generates photothermal effect.

8. A preparation method of a near-red light control nano-gas diagnosis and treatment agent is characterized by comprising the following steps:

9. The method according to claim 8, wherein the mass ratio of the charge of the two-dimensional nanoplatelets to the transition metal carbonyl compound or the nucleophilic NO donor is 1: 1.5 to 6.

10. The application of the near-red light control nano gas diagnosis and treatment agent is characterized in that the near-red light control nano gas diagnosis and treatment agent is applied to the preparation of nano medicines for inhibiting tumor cells.