CN112641959A

CN112641959A - Novel multifunctional nano probe and preparation method and application thereof

Info

Publication number: CN112641959A
Application number: CN202011389708.2A
Authority: CN
Inventors: 游蓁
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-12-01
Filing date: 2020-12-01
Publication date: 2021-04-13

Abstract

The invention discloses a novel multifunctional nano probe and a preparation method and application thereof, and relates to the field of nano materials. The nanoprobe prepared by the method for preparing the novel multifunctional nanoprobe comprises a gold nanoparticle core, gadolinium ions and green IR820 of new indole phthalocyanine carried on the surface of the gold nanoparticle core, wherein the particle size of the gold nanoprobe is 50-100nm, the nanoprobe can release drugs with acid response, the aggregation of the drugs at tumor cell parts is improved, the drugs can be dissociated or degraded in the acidic environment of the tumor parts, the excretion efficiency is high, and the retention amount of the nanoprobe in the tumor cells and the toxicity risk caused to a human body are greatly reduced; under the guidance of dual-mode imaging, more accurate photothermal and photodynamic cooperative treatment is carried out, and a more sensitive detection and more efficient treatment integrated multi-mode imaging diagnosis and treatment system is realized.

Description

Novel multifunctional nano probe and preparation method and application thereof

Technical Field

The invention belongs to the field of nano materials, and particularly relates to a novel multifunctional nano probe, and a preparation method and application thereof.

Background

Cancer has become one of the leading causes and major public health problems threatening human life safety. It is reported that 1410 ten thousand new cancer cases are added worldwide in 2015, 820 ten thousand people die of cancer, and the number of new cancer cases is expected to increase by 70% in the next 20 years. Currently, liver cancer treatment advocates different regimens according to different stages, mainly including early surgical treatment, middle and late chemotherapy, radiotherapy and radiotherapy/chemotherapy, and emerging biological treatment. Because the onset symptoms of liver cancer are hidden and the current clinical early diagnosis means is single, many liver cancer patients are difficult to diagnose at an early stage, so that expected treatment effects are difficult to obtain. Therefore, establishing a novel accurate diagnosis and treatment strategy for early cancer, improving the accuracy and sensitivity of early cancer diagnosis and treatment, and effectively reducing the cancer mortality is a major problem related to the national civilization.

With the development of nano technology and industry, the application of nano materials in the biomedical field is more and more extensive, and the nano materials have played an important role in the aspects of multi-modal imaging, early disease diagnosis and detection, drug delivery and tumor treatment in the biomedical field by virtue of the unique properties of the nano materials^[1-4]。

The drug is generally adsorbed by electrostatic force or loaded on the nanoparticle through chemical bond coupling to construct a nanoprobe, when the nanoprobe is used for in vivo cancer treatment, besides photodynamic treatment through the physical and chemical properties of the material, most of all, various drugs are loaded, and the EPR (enhanced permeability and retentivity effect) effect of the nanoprobe in vivo is utilized to carry out targeted delivery on tumor tissues, so that targeted treatment is further carried out. However, after the nanoprobes enter the organism through intravenous injection, if the nanoprobes cannot be timely excreted from the organism and excessively stay in the organism, a great toxicity risk is caused to the human body, and from the toxicological point of view, the toxicity possibly caused by the nanoprobes in the organism is closely related to the element composition, the shape and the size and the physicochemical property of the surface ligand, and an obvious quantity-effect relationship exists^[5,6]To reduce the toxicity risk of the nano-material in vivo, the main solution is to reduce the retention of the nano-probe in vivo.

Ideal nano diagnosis and treatment probe in vivoCan be excreted to outside of the body through the main excretory pathway (liver-feces, kidney-urine)^[7,8]Thereby maximally reducing the toxicity possibly caused by the nanometer material in the body; the largest factor influencing the distribution and excretion of the nanoprobe in vivo is the size, and the current research proves that the nanoparticles with the size of 10-100 nanometers mainly excrete by the liver-feces route, but the excretion rate is extremely low, and most of the nanoparticles are still retained in the organism; while nanoparticles less than 10 nanometers in size can be rapidly excreted from the organism via the kidney-urine pathway and essentially cleared from the organism after 24 hours^[9-11]. However, this also creates a contradiction, and the larger size nanoparticles have better tumor targeting ability due to longer blood circulation time, but are more difficult to escape from the body; the small size of the nanoparticles can rapidly drain from the body, but the tumor targeting ability is poor, the drug targeted release can reduce or avoid the side effect of the drug, and the dose of the drug can be reduced.

Reference documents:

[1]Chen A,Chatterjee S.Nanomaterials based electrochemical sensors for biomedical applications.Chemical Society Reviews.2013；42:5425-38.

[2]Mitragotri S,Anderson DG,Chen X,Chow EK,Ho D,Kabanov AV,et al.Accelerating the Translation of Nanomaterials in Biomedicine.Acs Nano.2015；9:6644-54.

[3]Lopes TS,Alves GG,Pereira MR,Granjeiro JM,Leite PEC.Advances and potential application of gold nanoparticles in nanomedicine.Journal of Cellular Biochemistry.2019.

[4]Doane TL,Burda C.The unique role of nanoparticles in nanomedicine:imaging,drug delivery and therapy.Chemical Society Reviews.2012；41:2885-92.

[5]Singh P,Prasuhn D,Yeh RM,Destito G,Rae CS,Osborn K,et al.Bio-distribution, toxicity and pathology of cowpea mosaic virus nanoparticles in vivo.Journal of Controlled Release.2007；120:41-50.

[6]Chithrani BD,Chan WCW.Elucidating the Mechanism of Cellular Uptake and Removal of Protein-Coated Gold Nanoparticles of Different Sizes and Shapes.Nano letters. 2007；7:1542-50.

[7]Souris JS,Lee C-H,Cheng S-H,Chen C-T,Yang C-S,Ho J-aA,et al.Surface charge-mediated rapid hepatobiliary excretion of mesoporous silica nanoparticles.Biomaterials. 2010；31:5564-74.

[8]Singh R,Pantarotto D,Lacerda L,Pastorin G,Klumpp C,Prato M,et al.Tissue biodistribution and blood clearance rates of intravenously administered carbon nanotube radiotracers.Proc Natl Acad Sci U S A.2006；103:3357-62.

[9]Kim,S.J.Toxicity and Tissue Distribution of Magnetic Nanoparticles in Mice. Toxicological Sciences.2006；89:338-47.

[10]Lankveld DPK,Oomen AG,Krystek P,Neigh A,Jong ATd,Noorlander CW,et al.The kinetics of the tissue distribution of silver nanoparticles of different sizes.Biomaterials. 2010；31:8350-61.

[11]Changhui,Fu,Tianlong,Liu,Linlin,Li,et al.The absorption,distribution,excretion and toxicity of mesoporous silica nanoparticles in mice following different exposure route. Biomaterials.2013；34:2565-75.

disclosure of Invention

The invention aims to provide a novel multifunctional nanoprobe and a preparation method and application thereof, solves the problems of excessive detention of nanoprobe nanoparticles in a living body and great toxicity risk to the human body in the prior art, can be used for nuclear magnetic resonance imaging and fluorescence imaging of tumor cells, provides a bimodal imaging mode, and carries out accurate diagnosis and treatment on the tumor cells.

The technical scheme of the invention is as follows:

in a first aspect, a novel multifunctional nanoprobe, comprising: the gold nanoparticle probe comprises a gold nanoparticle core, gadolinium ions and green indocyanine green IR820 loaded on the surface of the gold nanoparticle core, and the particle size of the gold nanoparticle probe is 50-100 nm.

Preferably, the particle size of the gold nanoprobe is 70 nm.

In a second aspect, the invention also provides a preparation method of the novel multifunctional nanoprobe, which comprises the following steps:

1) preparing a gold seed solution:

under the condition of vigorous stirring, adding a 1% trisodium citrate solution into a boiling 1mmol/L HAuCl4 solution, continuously stirring, and gradually changing the reaction solution from light golden yellow to light purple to obtain a gold seed solution;

2) preparation of gold nanoparticles

2.1) cooling the gold seed solution prepared in step 1) to room temperature, filtering with 0.22 μm nitrocellulose membrane, refrigerating the filtered filtrate at 4 deg.C, adding the refrigerated filtrate to a solution containing 1mol/L HCl and 1.6mmol/L HAuCl under moderate stirring₄Continuously stirring the mixed solution to obtain a mixed solution;

2.2.) freshly prepared 3mol/L AgNO₃Respectively and rapidly adding the solution and 1mol/L ascorbic acid solution into the mixed solution obtained in the step 2.1), rapidly adding 50mg/ml sulfhydryl-PEG-carboxyl solution into the mixed solution under the condition of vigorous stirring when the color of the reaction solution is rapidly changed from light red to deep red, and continuously stirring and reacting to obtain gold nanoparticle suspension;

3) preparing a nano probe:

3.1) dissolving the gold nanoparticle suspension prepared in the step 2) in deionized water, stirring in a water bath kettle at the temperature of 37 ℃, uniformly mixing, adding 45-60mmol/L gadolinium chloride solution, and reacting overnight;

3.2) centrifuging the solution obtained in the step 3.1), discarding the supernatant, washing the precipitate, then suspending the precipitate in deionized water, adding 1mg/mL of a new indocyanine green IR820 solution, continuing stirring and reacting for 24 hours, then centrifuging again, discarding the supernatant, washing the precipitate, and suspending the precipitate in deionized water to obtain the nanoprobe.

Preferably, the trisodium citrate in step 1) is reacted with HAuCl₄The volume ratio of (A) to (B) is as follows: 15-20:100, and more preferably 15: 100.

Preferably, in said step 2)Gold seed solution, HCl solution, HAuCl₄Solution, AgNO₃The volume ratio of the solution, the ascorbic acid solution and the sulfhydryl-PEG-carboxyl solution is as follows: 1:1:100:1:0.5:0.02.

Preferably, the volume ratio of the gold nanoparticle suspension, the green indocyanine IR820 solution and the gadolinium chloride solution in the step 3) is as follows: 10:0.01-0.02:0.1-0.15, and more preferably 10:0.01: 0.1.

Preferably, the stirring speed in the step 1) is 500-1000rpm, and the stirring time is 15 min.

Preferably, the stirring speed in the step 2.1) is 200-300rpm, and the stirring time is 1-5 min; the stirring speed in the step 2.2) is 500-1000rpm, and the stirring time is 10-15 min.

Preferably, the stirring speed in the step 3.1) is 200-300rpm, and the water bath reaction time is 12-18 h; the two centrifugations in the step 3.2) are carried out at room temperature, and the first centrifugation condition is as follows: centrifuging for 10-15min under the environment with the centrifugal force of 8000-9000g, wherein the second centrifugation condition is as follows: centrifuging for 5-10min under the environment with the centrifugal force of 9000-1000 g.

In a third aspect, the invention provides an application of a novel multifunctional nanoprobe, which can be applied to cancer targeted cooperative therapy and multi-modal imaging-guided photothermal/photodynamic cooperative therapy.

The prepared nano probe is characterized by means such as a through-network high-resolution Transmission Electron Microscope (TEM), an ultraviolet spectrophotometer (UV-Vis-NIR), a ZETA potential/particle size analyzer and the like; the uptake condition of the cells to the nano-probe is researched by observing fluorescence which is respectively quantified and oriented by Hoechst instant kit detection and flow cytometry experiment; the safety of the nanoprobe is evaluated by utilizing the detection of an activity detection kit (CCK-8) and a flow cytometry experiment; the distribution of the nano-probe in the tumor cell is evaluated by respectively quantitatively and directionally observing fluorescence through a laser confocal microscope and a flow cytometry; evaluating the photodynamic characteristics of the nanoprobe through the sosg singlet fluorescence spectrum; measuring the photothermal effect and photothermal attenuation of the nano probe by a near-infrared thermal imager; observing the intracellular singlet oxygen content condition treated by the nanoprobe through an ROS fluorescent probe and a Leica laser confocal microscope; after an in vitro light treatment experiment, a calcein/PI cell double-staining method and a cell flow type apoptosis experiment are respectively used for evaluating the in vitro light dynamic treatment effect of the nano probe; finally, the in vivo toxicity, the tumor inhibition effect and the in vivo photodynamic therapy effect of the nanoprobe are evaluated by intravenous administration of the nanoprobe to a tumor-bearing mouse.

Has the advantages that:

1. the nano probe can be dissociated or degraded in an acidic environment of a tumor part, and then is excreted through a liver-excrement way and a kidney-urine way, so that the excretion efficiency is high, the retention of the nano probe in tumor cells is greatly reduced, and the possible chronic/acute reaction caused by excessive retention of nano particles in organisms is avoided;

2. the nano probe of the invention is prepared from Gd³⁺The self-assembly formed by ion induction can perform acid-responsive drug release, and the aggregation of the drug at the tumor cell part is improved;

3. the nano probe can carry out active targeted aggregation on the liver cancer tumor part through the EPR effect, greatly improves the delivery efficiency of the medicament, and has good biocompatibility and targeting property;

4. the gadolinium ions in the nanoprobe can form nuclear magnetic resonance imaging, and the IR820 can form fluorescence imaging, so that a dual-mode imaging mode is provided, and more accurate photo-thermal and photo-dynamic cooperative treatment is carried out under the guidance of dual-mode imaging, so that important preconditions are provided for a subsequent multi-mode imaging diagnosis and treatment system integrating more sensitive detection and more efficient treatment, and the nanoprobe has good application prospect and important practical significance.

Drawings

FIG. 1 is a TEM electron micrograph prepared in example 1;

FIG. 2 is an ultraviolet absorption spectrum of gold nanoparticles, nanoprobes, and IR820 prepared in example 1;

FIG. 3 is a graph showing the analysis of the hydration radius of the nanoprobe prepared in example 1;

FIG. 4 shows fluorescence intensity and distribution of nanoprobes inside liver cancer cells under a confocal laser microscope;

FIG. 5 is a flow quantitative analysis of the uptake of nanoprobes by cells after the nanoprobes and cancer cells are incubated for different periods of time;

FIG. 6 is the apoptosis diagram of the nanoprobes with different concentrations in the liver cancer cell;

FIG. 7 is a graph showing the relative activities of cells in hepatoma cells incubated with Gd-AuNPS @ IR820, PBS, Au and IR820 at different concentrations, respectively;

FIG. 8 is a graph of the fluorescence spectra of SOSG at various time points on a fluorescence spectrophotometer;

FIG. 9 is a near infrared thermal imaging graph of PBS, Gd-AuNPS @ IR820 and IR820 under 808nm laser irradiation, respectively;

FIG. 10 is a graph of irradiation time and temperature rise of PBS, Gd-AuNPS @ IR820 and IR820 under 808nm laser irradiation, respectively;

FIG. 11 is a graph of the temperature change of PBS, Gd-AuNPS @ IR820, and IR820, respectively, after 5 cycles of irradiation with 808nm laser radiation;

FIG. 12 is an observation image of hepatoma carcinoma cells cultured by adding PBS, IR820, Gd-AuNPS @ IR820 suspension, respectively, under a laser confocal microscope;

FIG. 13 is a cell double-staining fluorescence image observed under a laser confocal microscope after staining of hepatoma carcinoma cells which are respectively added with PBS, IR820, Gd-AuNPS and Gd-AuNPS @ IR820 suspension for culture and are subjected to laser (808 nm, 6 minutes, 1W CM-2) mediated combined treatment, and the hepatoma carcinoma cells are stained by a calcein/PI double-staining kit;

FIG. 14 is an analysis chart of hepatoma carcinoma cells after flow cytometry analysis by adding PBS, IR820, Gd-AuNPS @ IR820 suspension culture and laser (808 nm, 6min, 1W CM-2) mediated combination therapy, respectively;

FIG. 15 shows the observed and photographed images of Gd-AuNPS @ IR820 suspension injected intravenously into tumor-bearing mice at various times on a nuclear magnetic imager;

FIG. 16 is an image of a tumor-bearing rat at each time after intravenous injection of Gd-AuNPS @ IR820 suspension, respectively observed and taken on an in vivo imager;

FIG. 17 is a KI-67 and HE staining observation picture of tumor tissues taken out on the 14 th day after the tumor-bearing mice are respectively injected with PBS, AuNPS solution, IR820 solution, Gd-Au @ IR820 solution and Gd-AuNPS @ IR820 suspension in the same volume by vein and then treated with 808 nanometer laser irradiation mediated combination therapy;

FIG. 18 is an HE staining observation chart of main organs of each treatment group taken out on the 14 th day after the completion of 808nm laser irradiation mediated combination treatment after the completion of PBS, AuNPS solution, IR820 solution, Gd-Au @ IR820 solution and Gd-AuNPS @ IR820 suspension which are injected into the tumor-bearing mice respectively in the same volume through veins;

FIG. 19 is a graph showing the change in body weight of tumor-bearing mice after treatment;

FIG. 20 is a graph of tumor-bearing mouse survival after treatment;

FIG. 21 is a graph showing the change in tumor volume in mice with tumor after treatment.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

In the present application, "AuNPS" refers to gold nanoparticles;

"Gd-AuNPS @ IR 820" refers to the nanoprobe;

"Gd-AuNPS" refers to gadolinium ion-loaded gold nanoparticles;

"Saline" refers to normal Saline;

"PBS" refers to phosphate buffered saline;

"IR 820" refers to new indocyanine green IR 820;

the main raw material sources used in the examples are as follows:

the main instrument sources used in the examples are illustrated:

example 1

1) Preparing a gold seed solution:

15mL of 1% trisodium citrate solution was added to 100mL of boiling 1.0mM HAuCl at a stirring rate of 500rpm₄Continuously stirring the solution for 15min, and gradually changing the reaction solution from light golden yellow to light purple to obtain a gold seed solution;

2) preparing gold nanoparticles:

2.1) cooling the Au seed solution prepared in step 1) to room temperature, filtering with a 0.22 μm nitrocellulose membrane, refrigerating the filtered filtrate at 4 deg.C, adding 1mL of the refrigerated filtrate into a mixture of 1mL (1mol/L) of HCl solution and 100mL (1.6mmol/L) of HAuCl₄In the mixed solution composed of the solution, continuously stirring for 1min under the condition that the stirring speed is 200rpm to obtain the mixed solution;

2.1) freshly prepared 1ml (3mol/L) AgNO₃Respectively and rapidly adding the solution and 0.5ml (1mol/L) ascorbic acid solution into the mixed solution obtained in the step 2.1), adjusting the stirring speed of a stirrer to be 500rpm, rapidly changing the color of the reaction solution from light red to deep red, rapidly adding 50mg/ml sulfhydryl-PEG-carboxyl solution, and continuously stirring and reacting for 10min to obtain gold nanoparticle suspension;

3) preparing a nano probe:

3.1) dissolving 10mL of the gold nanoparticle suspension prepared in the step 2) in 10mL of deionized water, stirring in a water bath at 37 ℃, adding 10 mu L (45-60mmol /) L of gadolinium chloride solution after uniformly mixing, and reacting for 12 hours;

3.2) centrifuging the solution obtained in the step 3.2) for 10min in an environment with the centrifugal force of 8000g, discarding the supernatant, washing the precipitate, then suspending the precipitate in 5mL of deionized water, then adding 100 mu L (1mg/mL) of new indocyanine green IR820 solution, stirring and reacting for 24h at room temperature, then centrifuging for 5min in an environment with the centrifugal force of 9000g, discarding the supernatant, washing the precipitate, and suspending the precipitate in 5mL of deionized water to obtain the novel multifunctional nanoprobe.

The characterization of the nanoprobe (Gd-AuNPS @ IR820) prepared in the example is illustrated by means of a through-the-wire high-resolution Transmission Electron Microscope (TEM), an ultraviolet spectrophotometer (UV-Vis-NIR), a ZETA potential/particle size analyzer and the like:

(1) through net high resolution Transmission Electron Microscope (TEM)

The nanoprobe prepared in the example is dropped on a carbon-supported film copper net, naturally air-dried at room temperature, observed on a through-net high-resolution Transmission Electron Microscope (TEM), and the characterization result is shown in a TEM electron micrograph of figure 1, and figure 1 shows that the ultra-small gold nanoparticles have uniform particle size and good dispersibility under a 100nm ruler.

(2) Ultraviolet spectrophotometer (UV-Vis-NIR)

The ultraviolet absorption spectra of the IR820 used in this example and AuNPS, Gd-AuNPS @ IR820 prepared in this example were measured, respectively, and the characterization results are shown in fig. 2, as shown in fig. 2, the ultra-small gold nanoparticles exhibited a characteristic peak of the gold nanoparticles at 520 nm, the IR820 exhibited main and shoulder peaks at 695 nm and 820 nm, the shoulder peak of Gd-AuNPS @ IR820 exhibited a red shift of about 30nm, and a stronger absorption peak at 808nm, further indicating the successful preparation of the nanoprobe.

(3) ZETA potential/particle size instrument

The particle size and particle size distribution of the gold nanoprobe are characterized by the dynamic light scattering of the ZETA potential/particle size analyzer, the characterization result is shown in figure 3, the average particle size of the gold nanoprobe is 72.4 and the particle size distribution coefficient is 0.304 as can be seen from figure 3, the probe is shown to have good particle size and distribution, and the later functional verification is facilitated.

Example 2

1) Preparing a gold seed solution:

20mL of a 1% trisodium citrate solution was added to 150mL of boiling 1.0mM HAuCl at a stirring rate of 1000rpm₄Continuously stirring the solution for 15min, and gradually changing the reaction solution from light golden yellow to light purple to obtain a gold seed solution;

2) preparing gold nanoparticles:

2.1) cooling the Au seed solution prepared in step 1) to room temperature, filtering with a 0.22 μm nitrocellulose membrane, refrigerating the filtered filtrate at 4 deg.C, adding 1mL of the refrigerated filtrate into a mixture of 1mL (1mol/L) of HCl solution and 100mL (1.6mmol/L) of HAuCl₄In the mixed solution composed of the solution, continuously stirring for 5min under the condition that the stirring speed is 300rpm to obtain the mixed solution;

2.1) freshly prepared 1ml (3mol/L) AgNO₃Respectively and rapidly adding the solution and 0.5ml (1mol/L) ascorbic acid solution into the mixed solution obtained in the step 2.1), adjusting the stirring speed of a stirrer to be 500rpm, rapidly changing the color of the reaction solution from light red to deep red, rapidly adding 60mg/ml sulfhydryl-PEG-carboxyl solution, and continuously stirring and reacting for 15min to obtain gold nanoparticle suspension;

3) preparing a nano probe:

3.1) dissolving 10mL of the gold nanoparticle suspension prepared in the step 2) in 10mL of deionized water, stirring in a water bath at 37 ℃, adding 20 mu L (45-60mmol /) L of gadolinium chloride solution after uniformly mixing, and reacting for 18 h;

3.2) centrifuging the solution obtained in the step 3.2) for 15min in an environment with a centrifugal force of 9000g, discarding the supernatant, washing the precipitate, then suspending the precipitate in 5mL of deionized water, then adding 150 muL (1mg/mL) of green IR820 solution of new indole phthalocyanine, stirring and reacting for 24h at room temperature, then centrifuging for 10min in an environment with a centrifugal force of 10000g, discarding the supernatant, washing the precipitate, and suspending the precipitate in 5mL of deionized water to obtain the novel multifunctional nanoprobe.

The characterization results of the obtained nanoprobes are similar to those in example 1.

Example 3

1) Preparing a gold seed solution:

18mL of 1% trisodium citrate solution were added to 125mL of boiling 1.0mM HAuCl with a stirring rate of 750rpm₄Continuously stirring the solution for 15min, and gradually changing the reaction solution from light golden yellow to light purple to obtain a gold seed solution;

2) preparing gold nanoparticles:

2.1) cooling the Au seed solution prepared in step 1) to room temperature, filtering with a 0.22 μm nitrocellulose membrane, refrigerating the filtered filtrate at 4 deg.C, adding 1mL of the refrigerated filtrate into a mixture of 1mL (1mol/L) of HCl solution and 100mL (1.6mmol/L) of HAuCl₄In the mixed solution composed of the solution, continuously stirring for 3min under the condition that the stirring speed is 250rpm to obtain the mixed solution;

2.1) freshly prepared 1ml (3mol/L) AgNO₃Respectively and rapidly adding the solution and 0.5ml (1mol/L) ascorbic acid solution into the mixed solution obtained in the step 2.1), adjusting the stirring speed of a stirrer to 750rpm, rapidly changing the color of the reaction solution from light red to deep red, rapidly adding 45mg/ml sulfhydryl-PEG-carboxyl solution, and continuously stirring and reacting for 13min to obtain gold nanoparticle suspension;

3) preparing a nano probe:

3.1) dissolving 10mL of the gold nanoparticle suspension prepared in the step 2) in 10mL of deionized water, stirring in a water bath at 37 ℃, adding 15 mu L (45-60mmol /) L of gadolinium chloride solution after uniformly mixing, and reacting for 15 h;

3.2) centrifuging the solution obtained in the step 3.2) for 13min under the environment with the centrifugal force of 9000g, discarding the supernatant, washing the precipitate, then suspending the precipitate in 5mL of deionized water, then adding 125 muL (1mg/mL) of green IR820 solution of new indole phthalocyanine, stirring and reacting for 24h at room temperature, then centrifuging for 8min under the environment with the centrifugal force of 10000g, discarding the supernatant, washing the precipitate, and suspending the precipitate in 5mL of deionized water to obtain the novel multifunctional nanoprobe.

Example 4

In the embodiment, the uptake condition of the cells to the nano-probe is researched by observing fluorescence which is respectively quantified and oriented by Hoechst instant kit detection and flow cytometry experiment;

1) cell culture:

the liver cancer HCC-LM3 cells were planted in four-chamber dishes and 24-well plates, respectively, after overnight incubation, the culture medium was changed to fresh DMEM cell culture medium containing PBS, IR820, Gd-AuNPS @ IR820 (from example 1), respectively, and the HCC-LM3 cells were incubated for 2 hours and 8 hours;

2) fixing cells in a four-chamber dish by using paraformaldehyde, observing by using laser confocal method, and observing the fluorescence intensity and distribution in the liver cancer cells under a laser confocal microscope, wherein the result is shown in figure 4, after incubation for 2 hours, fluorescence signals appear in the IR820 and Gd-AuNPS @ IR820 groups, but the fluorescence signal of the Gd-AuNPS @ IR820 group is obviously stronger than that of the IR820 group; after 8 hours of co-incubation, the fluorescence signal of the Gd-AuNPS @ IR820 group was still stronger than that of the IR820 treated group; the cells are proved to have good endocytosis capacity to the nano-probe, and the capacity of the nano-probe to enter the cells is stronger than that of the IR 820.

3) After the cells in the 24 wells are collected, the operation is carried out according to the operation steps of the Hoechst ready-to-use kit, and the uptake of the IR820 by the hepatoma cells is quantitatively analyzed by a flow cytometer, and the result is shown in FIG. 5, wherein the longer the time is, the higher the fluorescence intensity of the cells is, the more the IR820 and Gd-AuNPS @ IR820 enter the cells is proved, and the Gd-AuNPS @ IR820 has stronger ability to enter the cells than the IR820, which is consistent with the result of co-focusing.

This example shows that liver cancer cells can effectively take the nanoprobe prepared by the invention.

Example 5

This example evaluates the safety of nanoprobes using the activity detection kit (CCK-8) assay and the BD apoptosis kit assay.

1) Cell culture

The method comprises the following steps of planting liver cancer HCC-LM3 cells in a 24-well plate, after overnight culture, dividing the cells into an Au control group, a PBS control group, an IR820 control group and a Gd-AuNPS @ IR820 suspension experimental group, and replacing a cell culture medium of the Gd-AuNPS @ IR820 suspension experimental group with a freshly prepared cell culture medium, wherein the cell culture medium respectively contains the following components in concentration: DMEM cell culture medium containing nanoprobe suspensions (from example 1) at 0. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL, and 16. mu.g/mL, PBS solution was added to the cell culture medium of the PBS control group, and gold nanoparticle solution and IR820 solution were added to the corresponding concentrations (0-16. mu.g/mL) of the Au control group and IR820 control group, respectively, and the cells were collected after adding the cells and culturing for 8 hours;

2) BD apoptosis kit detection

Collected cells were subjected to the operation of the BD apoptosis kit, and finally analyzed by a BD flow cytometer, and the result is shown in FIG. 6, and HCC-LM3 cells did not undergo significant apoptosis in the range of incubation concentration (0-16. mu.g/mL).

3) Detection by activity detection kit (CCK-8)

Detecting the collected cells according to an activity detection kit (CCK-8), wherein the result is shown in figure 7, and the activity of HCC-LM3 cells is not obviously reduced within the range of incubation concentration of a control group and a Gd-AuNPS @ IR820 experimental group;

the example shows that the nano probe prepared by the invention has good biocompatibility with liver cancer cells under normal conditions and has no toxicity.

Example 6

The implementation utilizes SOSG as a singlet capture agent to detect the singlet oxygen generation capability of the nano probe, and is used for explaining the photodynamic characteristics of the nano probe;

the experimental method comprises the following steps: the nanoprobe suspension prepared in example 1 and a new indocyanine green IR820 solution with the same concentration were taken, and then a SOSG singlet oxygen probe (10) was added^-12M), measuring under 808nm laser irradiation, and recording the content of singlet oxygen at different time points;

the experimental results are as follows: as shown in figure 8, under the laser irradiation of 808nm, the number of singlet state particles generated by the nanoprobe is rapidly increased, and after 10 minutes, the amount of singlet state oxygen reaches 5.6 times of that of 0.5 minute irradiation, which indicates that the nanoprobe has great potential for enhancing photodynamic therapy.

Example 7

This example illustrates the photothermal effect and photothermal attenuation of nanoprobes;

(1) photothermal effect test

Taking 1ml of Gd-AuNPS @ IR820 suspension prepared in example 1, 1ml of IR820 solution, and 1ml of PBS as blank control groups, wherein the contents of IR820 in the IR820 solution and Gd-AuNPS @ IR820 suspension are the same, placing the solutions in 1.5ml centrifuge tubes respectively, and then performing temperature monitoring under 808nm laser irradiation (1W CM-2) through a near infrared thermal imager (see FIG. 9 and FIG. 10), FIG. 9 is a near infrared thermal imaging graph, FIG. 10 is a graph plotting irradiation time and temperature rise according to data results, as can be seen from FIG. 9, under the near infrared thermal imager, the temperature of the nanoprobe is increased significantly when the IR820 and Gd-AuNPS @ IR820 suspensions are irradiated under 808nm laser irradiation for 120 seconds, the temperature rise is rapid, but as can be seen from FIG. 10, under the same contents of IR820, the temperature of Gd-AuNPS @ IR820 is increased significantly, while the PBS group has no significant temperature rise during the whole irradiation period, therefore, the possibility of temperature rise caused by overlarge laser irradiation power is eliminated, and the nano probe prepared by the invention has a very remarkable photothermal effect.

(2) Photothermal attenuation experiment

1ml of Gd-AuNPS @ IR820 suspension prepared in example 1, 1ml of IR820 solution and 1ml of PBS are taken as blank control groups, wherein the content of IR820 in the IR820 solution and the Gd-AuNPS @ IR820 suspension is the same, 5 cycles of irradiation are respectively carried out under 808nm laser irradiation, the change of temperature is recorded, and the result is shown in figure 11, the thermal attenuation effect of the IR820 is obvious in the later period shown in figure 11, so that compared with the IR820, the synthesized nano probe has more continuous heat generation effect, and the nano probe prepared by the invention has slower thermal attenuation speed, has longer heat generation effect and has protective effect on the IR820 under the photo-thermal condition.

Example 8

This example is presented to illustrate the intracellular singlet oxygen content treated with nanoprobes;

1) cell culture and therapy

Respectively planting the hepatoma carcinoma cells in four-chamber dishes, after overnight culture, respectively adding fresh DMEM cell culture media respectively added with PBS, IR820, Gd-AuNPS and Gd-AuNPS @ IR820 suspensions with the same volume, continuously culturing for 8 hours, irradiating the cells for 6min by using 808nm laser (1W CM-2) to perform laser-mediated combination treatment, and after the treatment is finished, continuously culturing for 4 hours.

2) Experimental methods and results

After the four-chamber dish cells were stained with the ROS fluorescent probe, they were observed by a laser confocal microscope, and the results are shown in FIG. 12: under the laser irradiation of 808nm, cells of the Gd-AuNPS @ IR820 suspension group generate the most singlet oxygen, and as the metabolism of tumor cells is anaerobic respiration, the more singlet oxygen indicates that the toxicity generated in the tumor cells is higher, thereby indicating that the nano probe has obvious cytotoxicity to in-vitro liver cancer cells under the environment of photo-thermal/photodynamic combined treatment.

Example 9

This example is used to demonstrate the photothermal/photodynamic therapy effect of nanoprobes on in vitro liver cancer cells

1) Cell culture and treatment:

respectively planting hepatoma carcinoma cells in four-chamber dishes, culturing overnight, respectively adding fresh cell culture solutions of PBS, IR820, Gd-AuNPS and Gd-AuNPS @ IR820 suspensions with the same volume, wherein the Gd-AuNPS and Gd-AuNPS @ IR820 suspensions are prepared in example 1, continuously culturing for 8 hours, and then using 808nm laser (1W CM) to cultivate^-2) Irradiating the cells for 6min for laser-mediated combination therapy, and culturing for 4 hr.

2) Experimental methods and results

a. The collected cells in the four-chamber dish are stained by a calcein/PI double staining kit, and the observation result is shown in FIG. 13 when observed by a laser confocal microscope, the FIG. 13 is a double staining fluorescence diagram of the cells, when the laser irradiation treatment is not carried out, the cells in the treatment groups basically have no death phenomenon, after the laser irradiation treatment, the PBS group still has no obvious cell death, the IR820 treatment group and Gd-AuNPs have obvious cell death, most of the cells in the Gd-AuNPS @ IR820 group have death, and only a few of the cells survive;

b. the collected four-chamber dish cells are operated according to the operation steps of the BD cell apoptosis kit, and finally analyzed by a BD flow cytometer, the result is shown in FIG. 14, the apoptosis rate of the PBS group is very low after laser irradiation, the IR820 treatment group and the Gd-AuNPS are increased to 15-20%, the apoptosis rate of the Gd-AuNPS @ IR820 group is the highest and is more than twice of the former two groups and reaches more than 40%, and the result is consistent with the result of double staining.

The embodiment shows that the nano probe prepared by the invention can kill in-vitro liver cancer cells in the environment of 808nm laser-mediated photo-thermal/photodynamic combined treatment, has an obvious photo-thermal/photodynamic treatment effect, and has an effect obviously superior to that of single IR820 or Gd-AuNPs.

Example 10

This example serves to illustrate the distribution and metabolism of nanoprobes on tumor-bearing mice, particularly the accumulation at the tumor site, after intravenous administration to the tumor-bearing mice;

the experimental method comprises the following steps:

injecting the nano probe suspension prepared in the embodiment 1 into a tumor-bearing mouse anesthetized with isoflurane in advance through tail vein, and observing and shooting imaging pictures on a nuclear magnetic imager and a living body imager respectively at each time, wherein the parameters shot at different time points are consistent under each imaging mode, and the result is shown in fig. 15 and fig. 16;

the experimental results are as follows: FIG. 15 is a T1-MR imaging micrograph showing that Gd-AuNPS @ IR820 is predominantly distributed in the liver and also significantly aggregated in the tumor after 1 hour of tail vein injection of Gd-AuNPS @ IR820 into tumor-bearing mice; when the observation time point of the 3 rd hour is reached, the number of Gd-AuNPS @ IR820 at the tumor part is further increased, and the signal at the kidney part is obviously enhanced, which proves that the nanoprobe can be excreted through a kidney-urine metabolic pathway after the liver is separated; the result of the T1-MR signal of the tumor part also shows that the aggregation amount of the nanoprobe at the tumor part is gradually increased along with the increase of the observation time, and the aggregation amount of the tumor part reaches the peak value after 12 hours;

FIG. 16 is a live fluorescence imaging real-time observation picture, according to the fluorescence characteristics of the IR820 loaded by the nanoprobe, the real-time fluorescence imaging picture clearly shows that Gd-AuNPS @ IR820 is injected into a tumor-bearing mouse through tail vein, focuses on the liver, the fluorescence signal in the liver gradually weakens with the lapse of time, and the nanoprobe can be free by dissipation from the liver, while the fluorescence signal at the tumor gradually increases, and reaches a peak value 12h after injection;

the results show that the nano probe can be excreted through a liver-feces path and a kidney-urine path, the excretion efficiency is high, the retention amount of the nano probe in tumor cells is reduced, the fluorescence signal of the tumor part is gradually increased, and the tumor targeting capability of the nano probe is strong.

Example 11

This example is presented to demonstrate the in vivo toxicity and tumor suppression efficacy of nanoprobes after intravenous administration in tumor-bearing mice;

1) the experimental method comprises the following steps:

the same volume of PBS, AuNPS solution, IR820 solution, Gd-Au @ IR820 solution and Gd-AuNPS @ IR820 suspension (from example 1) are respectively injected into the tumor-bearing mice anesthetized by isoflurane through tail veins, and then the 808nm laser irradiation mediated combination treatment is carried out; wherein Gd-Au @ IR820 refers to a control material with poorer targeting, and compared with a nano probe, the Gd-Au @ IR820 has wider particle size and poorer targeting.

2) And (4) processing a result:

a. tumor tissue from each group of treated mice was removed on day 14 of completion of treatment for HE and KI-67 staining and observation; and the main viscera of each treatment group are taken out on the 14 th day, HE staining and pathology observation are carried out, the results are shown in fig. 17 and 18, the results are shown in fig. 17 for tumor tissues KI-67 and HE staining observation, the tumor tissues of the normal saline group can not obviously die, but the tumor cells of the AuNPS group, the IR820 group and the Gd-Au @ IR820 group can be observed to vacuole and die, while the tumor cells of the Gd-AuNPS @ IR820 group die most, and the in vivo treatment results fully prove that the nano probe prepared by the invention can effectively kill the tumor cells in laser irradiation mediated photothermal/photodynamic combined treatment and has obvious treatment effect; FIG. 18 shows that the tumor-bearing mice of each treatment group were subjected to HE staining of the organs 14 days after injection of Gd-AuNPS @ IR820 suspension, and it can be clearly seen that no significant pathological damage occurred in the main organs of each treatment group, which proves that the nanoprobe has good in vivo biocompatibility and little toxic and side effects on normal cells and tissues in the laser irradiation-mediated combination treatment environment;

b. the tumor volume, the body weight and the survival rate of the tumor-bearing mice of each treatment group are respectively recorded within 21 days after the treatment is completed, the results are shown in fig. 19, fig. 20 and fig. 21, fig. 19 shows the change of the body weight of the tumor-bearing mice after the treatment, and the change of the body weight among the groups has no significant difference; FIG. 20 is a graph showing the survival curves of tumor-bearing mice after treatment, with significant differences in survival rates, with the highest survival rate for the Gd-AuNPS @ IR820 group at the end of the 21-day observation period; FIG. 21 shows that the tumor volumes of various groups of tumor-bearing mice are changed after treatment, the tumor volumes of Gd-AuNPS @ IR820 groups are not obviously increased, and the tumor volumes of other groups are obviously increased, which shows that the nano-probe has good tumor inhibition effect.

In summary, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited too much, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that the technical solutions described in the foregoing embodiments can be easily deduced, replaced, or substituted for some technical features without departing from the spirit of the present invention, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A novel multifunctional nanoprobe is characterized in that: the novel multifunctional probe includes: the gold nanoparticle probe comprises a gold nanoparticle core, gadolinium ions and green indocyanine green IR820 loaded on the surface of the gold nanoparticle core, and the particle size of the gold nanoparticle probe is 50-100 nm.

2. The method for preparing a novel multifunctional nanoprobe according to claim 1, which comprises the following steps:

1) preparing a gold seed solution:

2) preparation of gold nanoparticles

3) preparing a nano probe:

3. The method for preparing a novel multifunctional nanoprobe according to claim 3, which is characterized in that: the trisodium citrate and HAuCl in the step 1)₄The volume ratio of (A) to (B) is as follows: 15-20:100-150.

4. The method for preparing a novel multifunctional nanoprobe according to claim 3, which is characterized in that: the gold seed solution, the HCl solution and the HAuCl in the step 2)₄Solution, AgNO₃The volume ratio of the solution, the ascorbic acid solution and the sulfhydryl-PEG-carboxyl solution is as follows: 1:1:100:1:0.5:0.02.

5. The method for preparing a novel multifunctional nanoprobe according to claim 3, which is characterized in that: the volume ratio of the gold nanoparticle suspension, the gadolinium chloride solution and the green indocyanine green IR820 solution in the step 3) is as follows: 10:0.01-0.02:0.1-0.15.

6. The method for preparing a novel multifunctional nanoprobe according to claim 3, which is characterized in that: the stirring speed in the step 1) is 500-1000rpm, and the stirring time is 15 min;

7. the method for preparing a novel multifunctional nanoprobe according to claim 3, which is characterized in that: the stirring speed in the step 2.1) is 200-300rpm, and the stirring time is 1-5 min; the stirring speed in the step 2.2) is 500-1000rpm, and the stirring time is 10-15 min.

8. The method for preparing a novel multifunctional nanoprobe according to claim 3, which is characterized in that: the stirring speed in the step 3.1) is 200-300rpm, and the water bath reaction time is 12-18 h; the first centrifugation conditions in step 3.2) are as follows: centrifuging for 10-15min under the environment with the centrifugal force of 8000-9000g, wherein the second centrifugation condition is as follows: centrifuging for 5-10min under the environment with the centrifugal force of 9000-1000 g.

9. The use of the novel multifunctional nanoprobe of claim 1, wherein: the nanoprobe can be used for cancer targeted cooperative therapy and multi-modal imaging-guided photothermal/photodynamic cooperative therapy.