CN112641942B

CN112641942B - Near-infrared light control nano gas diagnosis and treatment agent and preparation method and application thereof

Info

Publication number: CN112641942B
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: 2023-05-19
Anticipated expiration: 2040-12-25
Also published as: CN112641942A

Abstract

The invention provides a near-infrared light control nano-gas diagnosis and treatment agent and a preparation method and application thereof, wherein the near-infrared 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-infrared light control nano-gas diagnosis and treatment agent is simple, CO or NO gas is released under near-infrared light response, low-toxicity and high-efficiency ingenious combination of tumor phototherapy and gas treatment can be realized, the treatment efficiency is greatly improved, and the clinical application is facilitated to be unfolded.

Description

Near-infrared 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 medicines, in particular to a near-infrared light control nano gas diagnosis and treatment agent and a preparation method and application thereof.

Background

Chemotherapy is a main method for clinically treating cancers, especially middle and late stage cancers, and is an effective systemic treatment means. However, most of the chemotherapy drugs at present have the defects of no specific recognition, easy damage to normal cells and tissues, even systemic poisoning, low drug intake efficiency of tumor cells, easy drug resistance of tumor cells caused by repeated administration, and the like. Currently, a nano-drug delivery system is adopted to target and deliver the chemotherapeutic drugs into tumor cells, thereby solving the problem of high-efficiency directional delivery of the chemotherapeutic drugs. However, chemotherapeutic agents still have drug leakage during delivery and often cannot eliminate the toxic side effects of chemotherapy at all.

Phototherapy has the advantages of short time, remarkable curative effect, small toxic and side effects and the like, is further widely focused and studied, and is partially applied to clinic. However, its therapeutic effect is limited by the availability of highly effective photothermal agents.

Gas therapy is an emerging tumor treatment technique, mainly using therapeutic gases (nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H) ₂ S), oxygen (O) ₂ ) Etc.) to replace traditional chemotherapeutic agents. The CO and NO therapeutic gas drugs are considered as "green" therapeutic techniques in that they selectively induce apoptosis in tumor cells at appropriate concentrations while protecting normal cells. However, both modes of administration of gas therapy in the clinic, i.e., direct inhalation of therapeutic gas and injection of molecular compounds capable of releasing gas (NO/COreleasing moleculars, norm/CORMs), have difficulty in controlling the plasma concentration of gas and the effective concentration at the focal zone.

Currently, the main development technologies for targeted delivery and controlled release of gaseous drugs include: (1) Development of pH or temperature responsive gas prodrugs to achieve tumor microenvironment and temperature responsive gas release, but still suffer from the disadvantage that the gas prodrug will spontaneously release; (2) The luminescent response type gas prodrug is opened to realize light-controlled release of CO or NO gas, but the light response wave band of the prodrug reported by some current researches is mainly in the ultraviolet and visible light regions, and the near infrared light response type drug with more tissue penetration depth and low phototoxicity still has preparation difficulty. (3) Based on nano engineering, the nano carrier is utilized to load the gas prodrug, so that tumor targeted transportation and responsive release are realized. The light-responsive CORMs represented by the transition metal carbonyl compound can release CO gas under the triggering of ultraviolet light, visible light and even near infrared light in vitro, such as graphene oxide-manganese carbonyl compound (MnCO-GO) with a cage structure, and can realize the on-demand CO gas release in vivo through a near infrared switch in vitro. However, most of these nano-drugs require exogenous stimulation, are relatively toxic, lack release specificity, and have relatively complex synthesis processes.

Disclosure of Invention

The invention provides a near-infrared light control nano-gas diagnosis and treatment agent and a preparation method and application thereof, and aims to realize ingenious combination of low-toxicity and high-efficiency tumor photothermal therapy and gas therapy, improve treatment efficiency and promote development of clinical application.

In order to achieve the above object, the present invention provides a near-red light controlled nano-gas medical agent, which comprises a two-dimensional nano-sheet, a loading substance loaded on the two-dimensional nano-sheet, and a lipid membrane coating the two-dimensional nano-sheet and the loading substance thereof, wherein the loading substance comprises a transition metal carbonyl compound or a nucleophilic NO donor.

Preferably, the load is 80-150%.

Preferably, the two-dimensional nanosheets include black phosphorus nanosheets, arsene nanosheets or antimene nanosheets;

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

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 a 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 releases CO or NO gas in a responsive way under the irradiation of near-infrared light, and simultaneously generates a photo-thermal effect.

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

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

dispersing the two-dimensional nano sheet 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, dissolving uniformly, spin-drying the solvent, adding deionized water, and re-dissolving and dispersing to obtain the near-red light controlled nano gas diagnosis and treatment agent.

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

In addition, the invention also provides application of the near-infrared light control nano gas diagnosis and treatment agent, and the near-infrared 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 near-infrared light control nano-gas diagnosis and treatment agent and the preparation method and application thereof provided by the invention, wherein the near-infrared 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-infrared light control nano-gas diagnosis and treatment agent is simple, CO or NO gas is released under the near-infrared light response, the ingenious combination of low-toxicity and high-efficiency tumor phototherapy and gas treatment can be realized, the treatment efficiency is greatly improved, and the clinical application is facilitated to be developed.

Drawings

FIG. 1 is a diagram showing the structure and performance characteristics of the near-infrared light-controlled nano-gas diagnostic and therapeutic agent of the present invention;

FIG. 2 is a graph showing the photo-thermal effect and photo-controlled CO release performance of the near-infrared photo-controlled nano-gas diagnostic and treatment agent of the invention;

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

FIG. 4 is a graph of experimental results of photoacoustic imaging and photothermal imaging of the near-infrared light-controlled nano-gas diagnostic and treatment agent of the invention at the animal model level;

FIG. 5 is a graph of the anti-cancer effect of the near-infrared light controlled nano-gas diagnostic agent model animal level of the present invention;

fig. 6 is a schematic flow chart of a method for preparing the near-infrared light control nano-gas diagnosis and treatment agent.

The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.

Detailed Description

It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The embodiment of the invention provides a near-infrared light control 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 thereof, wherein the load comprises a transition metal carbonyl compound or a nucleophilic NO donor.

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

In particular, the two-dimensional nanoplatelets include black phosphorous nanoplatelets (Black phosphorus nanosheets, BPN), arsene nanoplatelets (Arsenene nanosheets), or antimene nanoplatelets (Antimonene nanosheets); the length and the width of the two-dimensional nano sheet are smaller than or equal to 150nm, and the thickness of the two-dimensional nano sheet is smaller than or equal to 2nm.

The nucleophilic NO donor has an [ N (O) NO ] functional group. For example, the nucleophilic donor may be O2-vinyl diazeniumdiolates, O2-glycosylated diazeniumdiolates. The diazeniumdiolate functional group is a resonant structure formed by one N atom and two O atoms. Wherein diazen in diazenium dioxide represents an "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 be other compounds that include an [ N (O) NO ] functional group.

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

In particular, the near-red light controlled nano-gas diagnostic and therapeutic agent can be a composite of a two-dimensional nano-sheet, a transition metal carbonyl compound and a lipid membrane, or a composite 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 nano-sheet, the carbonyl manganese compound and the lipid film can be further exemplified by the composite BPN-N (O) NO@LM formed by the black phosphorus nano-sheet, the nucleophilic NO donor and the lipid film.

In this embodiment, the near-red light controlled nano-gas medical agent has a good photo-thermal effect. Specifically, BPN-MnCO@LM will be described in detail. The near-infrared light control nano-gas diagnosis and treatment agent BPN-MnCO@LM comprises black scale nano-sheets, a manganese carbonyl compound and a lipid membrane. As shown in fig. 1, fig. 1 is a diagram showing the structure and performance characteristics of the near-infrared light control nano-gas diagnosis and treatment agent. FIG. 1a is a graph of size and thickness distribution of a nano-drug, FIGS. 1a and 1b are a graph of thickness distribution and diameter distribution of BPN-MnCO@LM, respectively, FIGS. 1a and 1b clearly show that BPN-MnCO@LM is a well-dispersed nano-sheet, most of which is 1.5-3nm thick, preferably less than 2nm thick; the length and the width of the two-dimensional nano sheet are smaller than or equal to 150nm, and most of the two-dimensional nano sheet is smaller than 100nm.

Further, with continued reference to FIG. 1, FIG. 1c is an infrared spectrum of BPN-MnCO@LM, with the abscissa being wave number and the ordinate being transmittance; 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 the abscissa being the delay time and the ordinate being the optical density difference. 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 manganese carbonyl, the MnCO is well loaded on the BPN, and the XPS spectrum and the transient spectrum show that the BPN and the MnCO have coordination bonding effect and have energy transmission performance.

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

To verify whether BPN-mnco@lm can be effectively optically released in cells, cloning experiments, anti-migration experiments and DNA damage experiments at the cellular level were performed. Specifically, referring to fig. 3, a graph of the results of a simulation experiment of the near-infrared light control nano-gas diagnosis and treatment agent of the present invention at the cellular level is shown. Wherein, FIG. 3a is the quantitative analysis result of the cloning experiment of the cells, and FIG. 3a shows that the proliferation of the tumor cells can be obviously inhibited after BPN-MnCO@LM is added with light; FIG. 3b is a quantitative analysis result of a cell migration experiment, FIG. 3b shows that migration of tumor cells can be significantly inhibited after BPN-MnCO@LM is irradiated, FIG. 3c is a quantitative analysis result of a DNA damage experiment of cells, and FIG. 3c shows that DNA damage of cancer cells can be significantly induced under illumination after BPN-MnCO@LM is irradiated.

In order to study the photo-thermal effect of BPN-mnco@lm at animal level, photo-thermal experiments were performed in vivo in tumor mice, which were injected with a nano-therapeutic agent through the tail vein, and then the tumor area was irradiated with near infrared laser. Fig. 4 is a graph of experimental results of photoacoustic imaging and photothermal imaging of the near-infrared light-controlled nano-gas diagnosis and treatment agent of the present invention at the model animal level. Fig. 4 shows that with prolonged irradiation time, the temperature of the tumor area increases significantly, showing good passive targeting tumor effect and photothermal effect.

Further, in order to study the anticancer effect of BPN-mnco@lm on animals, the anticancer effect of tumor mice after injection of BPN-mnco@lm was recorded. Specifically, as shown in fig. 5, fig. 5 is a graph of the anti-cancer effect of the near-infrared light control nano-gas diagnosis and treatment agent model animal level according to the invention. Wherein fig. 5a is a flow of animal experiments: tumor cells are inoculated in the body of the mice in advance (generally 7 days before BPN-MnCO@LM nano-drug is injected), then the BPN-MnCO@LM nano-drug is injected into the body of the mice, the mice are irradiated by infrared light, and various indexes of the mice are inspected and recorded at 2 days, 4 days and 14 days. FIG. 5b is a graph showing tumor volume change in mice, with the abscissa showing the time of feeding mice and the ordinate showing tumor volume. The result shows that the nanometer diagnosis and treatment agent and laser group has excellent capability of inhibiting the growth of the tumor of the mice; FIG. 5c is a photograph of the tumor site of the corresponding experimental mice dissected after 14 days of treatment, and the result shows that the tumor of the nano-medical agent plus laser group is the smallest and the inhibition effect is the best; fig. 5d shows the corresponding tumor mass statistics, and also verifies that the anti-tumor effect of the nano-medical plus laser group is best.

The embodiment provides a near-infrared light control nano-gas diagnosis and treatment agent and a preparation method and application thereof, wherein the near-infrared 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 near-infrared light response, low-toxicity and high-efficiency tumor phototherapy and gas treatment are skillfully combined, the treatment efficiency is greatly improved, and the clinical application is facilitated to be developed.

Further, the invention also provides a preparation method of the near-infrared light control nano-gas diagnosis and treatment agent, specifically, as shown in fig. 6, fig. 6 is a schematic flow chart of the preparation method of the near-infrared light control nano-gas diagnosis and treatment agent, and the method comprises the following steps:

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

step S102: dispersing the two-dimensional nano sheet 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, dissolving uniformly, spin-drying the solvent, adding deionized water, and re-dissolving and dispersing to obtain the near-red light controlled nano gas diagnosis and treatment agent.

The liquid phase mechanical crushing method is adopted in advance to prepare the two-dimensional black phosphorus nano-sheet. Specifically, the black phosphorus powder of 25 mg was added to 50 mL isopropyl alcohol, argon was introduced to remove oxygen in the solvent, and then the mixed solution was placed in ice water at constant temperature, and subjected to ultrasonic crushing using an ultrasonic crusher (power: 25%; on/off time: 45s/15 s) for 12 h. The product is firstly subjected to low-speed centrifugation (1000 rpm,10 min) to remove uncrushed black phosphorus, finally, the nanosheets are collected by ultrafiltration (MWCO 100 kDa, 3750 rpm, 30 min), and the two-dimensional black scale nanosheets can be obtained after washing with deoxidized deionized water for 2 times to remove the additional products of the reaction, and are stored under the condition of 4 ℃ after being packaged. The black phosphorus nano-sheet (BPN) has a size of 150-nm and a thickness of 2nm.

And loading transition metal carbonyl compounds or nucleophilic carbonyl compounds on the two-dimensional nano-sheets through in-situ reaction so as to coordinate with transition metal or nucleophilic groups through metal empty orbitals of the two-dimensional nano-sheets. Specifically, dispersing the two-dimensional nano-sheets in isopropanol solution after nitrogen treatment, adding a certain proportion of transition metal carbonyl compound or nucleophilic NO donor, dissolving, dispersing, and continuing stirring at room temperature under the protection of nitrogen for reaction for 24 hours; after the reaction, the precipitate was collected by high-speed centrifugation (12000 rpm), and washed with ethanol and deionized water to obtain the drug-loaded nanoplatelets. The feeding mass ratio of the two-dimensional nano-sheet to the transition metal carbonyl compound or the nucleophilic NO donor is 1: 1.5-6.

And then dispersing the two-dimensional nano-sheet loaded with the transition metal carbonyl compound or the nucleophilic NO donor into chloroform, adding 1, 2-distearoyl-sn-propyltri-3-phosphorylcholine, cholesterol and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to form a lipid film on the surface of the two-dimensional nano-sheet loaded with the transition metal carbonyl compound or the nucleophilic NO donor, dissolving uniformly, spin-drying the solvent, and adding deionized water for re-dissolving and dispersing to obtain the near-red light controlled nano-gas diagnosis and treatment agent. Wherein, DSPC: cholesterol: the preferred mass ratio of 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, which is applied to preparation of nano-drugs 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 one … …" does not exclude the presence of other like elements in a process, method, article, or system that comprises the element.

The foregoing embodiment numbers of the present invention are merely for the purpose of description, and do not represent the advantages or disadvantages of the embodiments.

The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or modifications in the structures or processes described in the specification and drawings, or the direct or indirect application of the present invention to other related technical fields, are included in the scope of the present invention.

Claims

1. The near-infrared light control nano gas diagnosis and treatment agent is characterized by comprising two-dimensional nano sheets, a load and a lipid membrane, wherein the load is loaded on the two-dimensional nano sheets, the lipid membrane wraps the two-dimensional nano sheets and the load, the load is manganese carbonyl, the two-dimensional nano sheets are black phosphorus nano sheets, and the black phosphorus nano sheets are loaded with manganese carbonyl through in-situ reaction so as to coordinate with transition metal through metal empty orbitals of the black phosphorus nano sheets;

the near-red light controlled nano gas diagnosis and treatment agent can release the gas with CO therapeutic action in a responsive way under the irradiation of near-infrared light and simultaneously generate a photo-thermal effect,

wherein, the feeding mass ratio of the black phosphorus nano-sheet to the manganese carbonyl is 1:1.5 to 6 parts of the total weight of the composite,

dispersing the black phosphorus nano-sheets in isopropanol solution, adding the load, stirring at room temperature under the protection of nitrogen for a preset period of time, and centrifuging to obtain the black phosphorus nano-sheets loaded with manganese carbonyl;

dispersing the black phosphorus nano sheet loaded with manganese carbonyl into chloroform, adding 1, 2-distearoyl-sn-propyltri-3-phosphorylcholine, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, dissolving uniformly, spin-drying a solvent, adding deionized water for re-dissolving and dispersing to obtain the near-red light controlled nano gas diagnosis and treatment agent, wherein the mass ratio of the 1, 2-distearoyl-sn-propyltri-3-phosphorylcholine, cholesterol and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 is 1:0.326:0.178.

2. the near-infrared light-controlled nanogas diagnostic reagent of claim 1, wherein the loading of the cargo is 80-150%.

3. The near-infrared light-controlled nano-gas diagnosis and treatment agent according to claim 1, wherein the length and width of the two-dimensional nano-sheet are less than or equal to 150nm, and the thickness of the two-dimensional nano-sheet is less than or equal to 2nm.

4. The near-red controlled nano-gas diagnostic and therapeutic agent according to claim 1, wherein the near-red controlled nano-gas diagnostic and therapeutic agent is applied to prepare a nano-drug for inhibiting tumor cells, the near-red controlled nano-gas diagnostic and therapeutic agent can be delivered to a tumor site in a targeted manner to generate therapeutic gas and a photo-thermal effect, the therapeutic gas comprises CO, the near-red controlled nano-gas diagnostic and therapeutic agent comprises two-dimensional nano-sheets and a load loaded on the two-dimensional nano-sheets, and a lipid film wrapping the two-dimensional nano-sheets and the load thereof, wherein the load is manganese carbonyl, and the two-dimensional nano-sheets are black phosphorus nano-sheets.