Gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe, preparation method and application thereof

Abstract

The present invention provides a gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe, a preparation method and application thereof. The probe comprises a gold nanomaterial and a carbon quantum dot; the surface of the gold nanomaterial is modified with an ATP nucleic acid aptamer and FA-modified polyethylene glycol; the surface of the carbon quantum dot is modified with a single-stranded DNA; the single-stranded DNA is complementary to the bases of part or all of the sequence in the ATP nucleic acid aptamer. The probe can be applied to biological imaging and the preparation of photothermal therapeutic agents. During the photothermal treatment process, it can realize nucleolus imaging, monitor nucleolus morphology, increase the specificity of targeting nucleoli, improve the thermal killing ability of tumor cells, reduce damage to surrounding normal tissues, and can also specifically detect ATP content, thereby improving the diagnostic and therapeutic effect of photothermal therapy on tumor cells.

Description

Technical Field

[0001] The invention relates to the technical field of nano-biomaterial synthesis, and in particular to a gold nanorod / carbon quantum dot composite plasma fluorescent nano-probe, a preparation method and application thereof. Background Art

[0002] At present, photothermal therapy has been widely used in anti-tumor treatment worldwide, and its efficacy is remarkable. Photothermal therapy is based on the accumulation of plasma nanoparticles in cancerous sites in the body, and uses the thermal effect of local surface plasmon resonance under infrared light stimulation to kill cancer cells, thereby achieving the purpose of killing tumors. Compared with other combined adjuvant methods, photothermal therapy has the advantages of less trauma, fewer side effects and high selectivity. In addition, studies have found that cancer cells are more sensitive to thermal stimulation than normal cells. Therefore, this treatment method brings hope to the treatment of cancer patients.

[0003] Among them, gold nanorods, as effective photothermal nanoagents, have unique LSPR photothermal effects, excellent biocompatibility and nanosurface energy transfer properties, and have a longer action distance compared to fluorescence resonance energy transfer, and have been widely used in bioimaging, photothermal therapy, drug delivery and molecular detection. However, due to the complexity of the molecular characteristics of cancer biological systems, the specific potential of gold nanorods in clinical diagnosis and treatment applications has not been fully utilized, so it is necessary to modify gold nanorods to optimize the diagnosis and treatment effects.

[0004] The nucleolus, a membrane-less structure in the nucleus of a eukaryotic cell, is the site of assembly of ribosomal RNA (rRNA) and ribosomal subunits, and the size and number of nucleoli have long been used as diagnostic parameters for the prognosis of malignant tumors. In recent years, studies have found that the nucleolus, as the main "cellular stress receptor", bears a heavy metabolic burden, is extremely sensitive to external cellular stimuli, and is the hub of the stress response that drives cancer development. Therefore, the nucleolus is more likely to become a target for anti-cancer strategies in the field of photothermal therapy.

[0005] Adenosine triphosphate (ATP) is an important substance for energy storage and supply in human cells. It is the structural and functional basis of organisms and life phenomena. When the human body encounters strong stimulation, such as invasion by pathogens or dying, ATP will quickly convert into adenosine diphosphate and release huge amounts of energy. Therefore, rapid and specific determination of ATP content is an important means of monitoring tumor cell growth.

[0006] In the prior art, most of the gold nanomaterials are modified with stabilizing molecules and targeting molecules to improve the photothermal stability and targeting of the gold nanomaterials. For example, CN109701017A discloses a targeted gold nanocone composite material, including gold nanocones, the surface of which is coated with pretreated bovine serum albumin, and each gold nanocone surface is modified with targeting molecules. The gold nanocone composite material has good stability and high biocompatibility, and can achieve photothermal treatment of tumors. However, the gold nanocone composite material cannot monitor nucleolar morphology and nucleolar imaging, and specifically measure the intracellular ATP content to improve the diagnosis and treatment effect of tumors. At present, there is no relevant report on the development of gold nanocomposites that monitor nucleolar morphology and measure ATP content at the same time. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe, a preparation method and application thereof. The gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe can realize nucleolus imaging, monitor nucleolus morphology, increase the specificity of targeting nucleolus, improve the thermal killing ability of tumor cells, reduce damage to surrounding normal tissues, and can also specifically detect ATP content, thereby improving the diagnostic and therapeutic effect of photothermal therapy on tumor cells.

[0008] In a first aspect, the present invention provides a gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe, comprising gold nanomaterial and carbon quantum dots;

[0009] The surface of the gold nanomaterial is modified with ATP nucleic acid aptamer and FA-modified polyethylene glycol;

[0010] The surface of the carbon quantum dots is modified with single-stranded DNA;

[0011] The single-stranded DNA is complementary to a part or all of the base sequences in the ATP nucleic acid aptamer.

[0012] Preferably, the molar ratio of the gold nanomaterial, FA-modified polyethylene glycol and ATP nucleic acid aptamer is 1:(1-1.5):(1.8-2.2).

[0013] Preferably, the FA-modified polyethylene glycol is FA-modified thiol polyethylene glycol.

[0014] Preferably, the gold nanomaterial comprises gold nanorods.

[0015] Preferably, the aspect ratio of the gold nanorods is 3.5-4.

[0016] Preferably, the sequence of the ATP nucleic acid aptamer is as shown in SEQ ID No: 1: 5SH-C6-TCGATTCCCTGGGGG AGTA TTG CGGAGGAGG-cy5.

[0017] Preferably, the sequence of the single-stranded DNA is as shown in SEQ ID No: 2: NH2-C6-ACT CCC CCA GGT AAT.

[0018] In a second aspect, the present invention provides a method for preparing the gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe, comprising the following steps:

[0019] The carbon quantum dot solution modified with single-stranded DNA on the surface is mixed with the gold nanomaterial solution modified with ATP nucleic acid aptamer and FA-modified polyethylene glycol on the surface, and then cultured in a 30-40° C. water bath for 10-14 hours to obtain a gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe.

[0020] Preferably, the carbon quantum dot solution with single-stranded DNA modified on the surface is prepared by a preparation method comprising the following steps:

[0021] The coupling agent, carbon quantum dot solution and single-stranded DNA were mixed and reacted at 2-6°C for 8-15h.

[0022] Preferably, the gold nanomaterial solution with ATP nucleic acid aptamers and FA-modified polyethylene glycol on the surface is prepared by a preparation method comprising the following steps:

[0023] (1) mixing a reducing agent and an ATP nucleic acid aptamer and performing a reaction in the dark to obtain a reduced ATP nucleic acid aptamer;

[0024] (2) mixing the reduced ATP nucleic acid aptamer with the gold nanomaterial solution and reacting the mixture to obtain a gold nanomaterial solution modified with the ATP nucleic acid aptamer;

[0025] (3) The FA-modified polyethylene glycol and the ATP nucleic acid aptamer-modified gold nanomaterial solution are mixed and reacted to obtain a gold nanomaterial solution whose surface is modified with the ATP nucleic acid aptamer and the FA-modified polyethylene glycol.

[0026] Preferably, the temperature of the light-shielding reaction in step (1) is 25-35° C., and the time of the light-shielding reaction is 2-5 h.

[0027] Preferably, the reaction temperature in step (2) is 25-35° C., and the reaction time is 8-15 h.

[0028] Preferably, the reaction temperature in step (3) is 25-35° C., and the reaction time is 8-15 h.

[0029] In a third aspect, the present invention provides an application of the gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe or the gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe prepared by the preparation method in biological imaging, preparation of photothermal therapeutic agents and / or ATP detection.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention modifies the surface of gold nanomaterials with ATP nucleic acid aptamers and FA-modified polyethylene glycol, modifies the surface of carbon quantum dots with single-stranded DNA, and composites the carbon quantum dots on the gold nanomaterials by means of the base complementary pairing principle of ATP nucleic acid aptamers and single-stranded DNA, thereby obtaining a gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe;

[0032] (2) The gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe can be used for biological imaging, preparation of photothermal therapeutic agents and / or ATP detection. During the photothermal therapy process, it can not only realize nucleolus imaging, monitor nucleolus morphology, increase the specificity of targeting nucleoli, improve the thermal killing ability of tumor cells, and reduce damage to surrounding normal tissues, but also specifically detect ATP content, thereby improving the diagnostic and therapeutic effect of photothermal therapy on tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the length distribution diagram of AuNRs;

[0034] Figure 2 is the width distribution diagram of AuNRs;

[0035] Figure 3 TEM image of AuNRs;

[0036] Figure 4 TEM images of CDs;

[0037] Figure 5 TEM images of AuNRs-CDs;

[0038] Figure 6 UV-visible spectra of AuNRs, AuNRs-ATP and FA-AuNRs-ATP;

[0039] Figure 7 UV-visible spectra of AuNRs, CDs and AuNRs-CDs;

[0040] Figure 8Zeta potential results of AuNRs, AuNRs-ATP, FA-AuNRs-ATP, CDs, CDs-ss-DNA and AuNRs-CDs;

[0041] Fig. 9 The fluorescence spectra of CDs at different excitation wavelengths;

[0042] Fig.10 The fluorescence spectra of CDs at different concentrations;

[0043] Fig.11 The graph shows the content of AuNRs-CDs absorbed by HepG2 (human liver cancer cells) and HL-7702 (human normal liver cells);

[0044] Fig.12 The temperature of the AuNRs-CDs probe solution changes with time under different laser irradiation intensities;

[0045] Fig.13 The temperature response of AuNRs-CDs and the changes of repeated on / off irradiation cycles;

[0046] Fig.14 The graph shows the absorbance change of AuNRs-CDs probe solution before and after laser irradiation;

[0047] Fig.15 The toxicity results of AuNRs-CDs on HepG2 cells and HL-7702 cells are shown in FIG.

[0048] Fig.16 The figure shows the cell viability results of AuNRs-CDs in HepG2 cells;

[0049] Fig.17 The figure shows the cell viability results of AuNRs-CDs in HL-7702 cells;

[0050] Fig.18 The results of in vitro photothermal performance in HepG2 and HL-7702 cells are shown in Figure 2.

[0051] Fig.19 The fluorescence spectra of AuNRs-CDs at different ATP concentrations;

[0052] Fig. 20 The fluorescence intensity results of AuNRs-CDs in the presence of CTP, GTP, UTP and ATP;

[0053] Fig.21 The fluorescence image results of AuNR-CDs, AuNR-CDs+CaCl2 and AuNRs-ranCDs;

[0054] Fig. 22 The fluorescence intensity change diagram of AuNRs-CDs under different irradiation time;

[0055] Fig.23 The results of co-staining DNase and RNase-treated HepG2 living cells with Hoechst 33342 and AuNRs-CDs for fluorescence imaging;

[0056] Fig.24 The figure shows the changes in nucleolus size in HepG2 cells and HL-7702 cells as the photothermal treatment time increases. DETAILED DESCRIPTION

[0057] The present invention has no special restrictions on the sources of all raw materials involved, and they can be purchased from the market or prepared according to conventional preparation methods well known to those skilled in the art.

[0058] The present invention provides a gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe, comprising gold nanomaterial and carbon quantum dots;

[0059] The surface of the gold nanomaterial is modified with ATP nucleic acid aptamer and FA-modified polyethylene glycol;

[0060] The surface of the carbon quantum dots is modified with single-stranded DNA;

[0061] The single-stranded DNA is complementary to a part or all of the base sequences in the ATP nucleic acid aptamer.

[0062] In the present invention, the gold nanomaterial preferably includes gold nanorods (AuNRs), and the aspect ratio of the gold nanorods is preferably 3.5-4. The FA-modified polyethylene glycol (FA-PEG) is preferably FA-modified thiol polyethylene glycol. The present invention has no particular limitation on the molecular weight of polyethylene glycol, and is preferably polyethylene glycol 5000.

[0063] The present invention obtains FA-AuNRs-ATP by modifying the surface of gold nanorods with ATP nucleic acid aptamers and FA-modified polyethylene glycol by means of gold-sulfur bonds. The ATP nucleic acid aptamer can specifically recognize ATP, and the FA group can distinguish FR positive (FR+) and FR negative (FR-) cells. Therefore, the obtained FA-AuNRs-ATP can effectively enhance the endocytosis of FR positive cancer cells. In addition, single-stranded DNA (ss-DNA) is modified on the surface of carbon quantum dots (CDs) to obtain CDs-ss-DNA, and the carbon quantum dots are compounded on the gold nanomaterial through the base complementarity of the single-stranded DNA and the partial or whole sequence in the ATP nucleic acid aptamer to obtain the gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe (AuNRs-CDs).

[0064] The gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe, CDs that emit stable green fluorescence will initially be quenched by AuNRs through NSET (nano surface energy transfer). However, in the presence of ATP in the cell, NSET between CDs and AuNRs disappears. After the ATP nucleic acid aptamer on AuNRs-CDs specifically recognizes ATP, CDs will separate from AuNRs, and the fluorescence signal will be restored to a certain extent, thereby achieving in situ imaging of the nucleolus. The gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe can be used for biological imaging, preparation of photothermal therapeutic agents and / or ATP detection. During the photothermal treatment process, it can not only achieve nucleolus imaging, monitor nucleolus morphology, increase the specificity of targeted nucleoli, improve the thermal killing ability of tumor cells, and reduce damage to surrounding normal tissues, but also specifically detect ATP content, thereby improving the diagnostic and therapeutic effect of photothermal therapy on tumor cells.

[0065] In the present invention, the molar ratio of the gold nanomaterial, FA-modified polyethylene glycol and ATP nucleic acid aptamer is 1:(1-1.5):(1.8-2.2).

[0066] In the present invention, the sequence of the ATP nucleic acid aptamer is shown in SEQ ID No: 1: 5SH-C6-TCGATTCCCTGGGGG AGTA TTG CGGAGGAGG-cy5. The sequence of the single-stranded DNA is shown in SEQ ID No: 2: NH2-C6-ACTCCC CCA GGT AAT.

[0067] The present invention also provides a method for preparing the gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe, comprising the following steps:

[0068] The carbon quantum dot solution modified with single-stranded DNA on the surface is mixed with the gold nanomaterial solution modified with ATP nucleic acid aptamer and FA-modified polyethylene glycol on the surface, and then cultured in a 30-40° C. water bath for 10-14 hours to obtain a gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe.

[0069] The present invention has no particular restriction on the order of adding the carbon quantum dot solution with single-stranded DNA modified on the surface and the gold nanomaterial solution with ATP nucleic acid aptamer and FA-modified polyethylene glycol modified on the surface. After the obtained mixed solution is cultured in a constant temperature water bath at 30-40°C for 10-14 hours, the gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe can be obtained. In the present invention, it is preferred to co-culture the mixed solution in a constant temperature water bath at 37°C for 12 hours. In the present invention, in order to remove excess ligands, after co-culturing in a constant temperature water bath, it is preferred to further include washing the gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe with HEPES buffer, and the number of washings is not particularly limited, preferably not less than 3 times.

[0070] In the present invention, the carbon quantum dot solution can be purchased from the market or prepared according to a conventional preparation method known to those skilled in the art. Exemplarily, the carbon quantum dot solution is prepared by a preparation method comprising the following steps:

[0071] The m-phenylenediamine and tannic acid aqueous solution are mixed, subjected to hydrothermal reaction, cooled to room temperature, centrifuged, and the supernatant is dialyzed to obtain a carbon quantum dot solution.

[0072] In the present invention, the temperature of the hydrothermal reaction is preferably 160-200°C, and the time of the hydrothermal reaction is preferably 8-12h. The centrifugation is preferably performed at 10000-15000rpm for 10-20min. The dialysis time is preferably 20-30h, and the water is preferably changed every 2h. In the present invention, the molecular weight cutoff of the dialysis is preferably 1000Da.

[0073] In the present invention, the carbon quantum dot solution with single-stranded DNA modified on the surface is prepared by a preparation method comprising the following steps:

[0074] The coupling agent, carbon quantum dot solution and single-stranded DNA were mixed and reacted at 2-6°C for 8-15h.

[0075] The present invention has no particular restrictions on the order of adding the coupling agent, the carbon quantum dot solution and the single-stranded DNA. In the present invention, the coupling agent preferably includes NHS and EDC, and the coupling agent is preferably added to the carbon quantum dot solution, mixed for 15 minutes, and then the single-stranded DNA is added to the obtained mixed solution, and the reaction is carried out at 2-6°C for 8-15 hours, more preferably after adding the single-stranded DNA, mixed at 4°C for 12 hours, and the mixing is preferably carried out under stirring conditions. In the present invention, the reaction preferably also includes dialysis to remove unreacted sequences. The molecular weight of the dialysis is preferably 3500Da.

[0076] The method for modifying single-stranded DNA on the surface of carbon quantum dots is simple, easy and low in cost.

[0077] In the present invention, the gold nanomaterial solution with ATP nucleic acid aptamers and FA-modified polyethylene glycol on the surface is prepared by a preparation method comprising the following steps:

[0078] (1) mixing a reducing agent and an ATP nucleic acid aptamer and performing a reaction in the dark to obtain a reduced ATP nucleic acid aptamer;

[0079] (2) mixing the reduced ATP nucleic acid aptamer with the gold nanomaterial solution and reacting the mixture to obtain a gold nanomaterial solution modified with the ATP nucleic acid aptamer;

[0080] (3) The FA-modified polyethylene glycol and the ATP nucleic acid aptamer-modified gold nanomaterial solution are mixed and reacted to obtain a gold nanomaterial solution whose surface is modified with the ATP nucleic acid aptamer and the FA-modified polyethylene glycol.

[0081] The present invention has no particular restrictions on the order of adding the reducing agent and the ATP nucleic acid aptamer. Preferably, the reducing agent is added to the ATP nucleic acid aptamer for reduction reaction. The temperature of the light-proof reaction is preferably 25-35°C, and the time of the light-proof reaction is preferably 2-5h. The temperature of the reaction after the reduced ATP nucleic acid aptamer is mixed with the gold nanomaterial solution is preferably 25-35°C, and the reaction time is preferably 8-15h. After obtaining the gold nanomaterial solution modified with the ATP nucleic acid aptamer, it is preferably further included to centrifuge and resuspend to remove excess ATP nucleic acid aptamer. In the present invention, specifically: the gold nanomaterial solution modified with the ATP nucleic acid aptamer is centrifuged at 9500rpm for 15min, and the precipitate is resuspended in 10mL of water. In the present invention, the temperature of the reaction in step (3) is preferably 25-35°C, and the reaction time is preferably 8-15h.

[0082] The method for modifying the surface of a gold nanomaterial with an ATP nucleic acid aptamer and FA-modified polyethylene glycol is simple and easy to operate with low difficulty.

[0083] The present invention also provides an application of the gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe or the gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe prepared by the preparation method in biological imaging, preparation of photothermal therapeutic agents and / or ATP detection.

[0084] When the gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe is stimulated by an external 808nm laser, the single-stranded DNA and the ATP nucleic acid aptamer will be disconnected, resulting in free carbon quantum dots that can emit fluorescence, thereby being able to be used for biological imaging, preparing photothermal therapeutic agents or detecting ATP content.

[0085] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0086] In order to further illustrate the present invention, the following examples are provided for detailed description. The experimental raw materials used in the following examples of the present invention can be purchased from the market or prepared according to conventional preparation methods well known to those skilled in the art.

[0087] Preparation Example 1

[0088] This preparation example provides a gold nanorod (AuNRs), and the preparation method thereof is as follows:

[0089] (1) HAuCl4 (0.25 mL, 0.01 M) and CTAB (5 mL, 0.2 M) were mixed to obtain a mixed solution;

[0090] (2) Add freshly prepared glacial NaBH4 (0.25 mL, 0.025 M) to the above mixed solution and stir for 2 min. The solution changes from yellow to brownish yellow. Place the obtained solution in a 30°C water bath for 3 h to obtain a gold nanorod seed solution.

[0091] (3) After mixing HCl (0.4 mL, 2 M) and CTAB (40 mL, 0.1 M), HAuCl4 (2 mL, 0.01 M) and AgNO3 (0.365 mL, 0.01 M) were added, and the resulting solution was placed in a 30 °C water bath for 15 min, and then ascorbic acid (0.32 mL, 0.1 M) was added and shaken until the solution became transparent;

[0092] (4) Add 0.096 mL of gold nanorod seed solution to the solution obtained in step (3), stir for 30 seconds, and place in a 30°C water bath for 12 hours. Centrifuge the obtained solution twice at 9500 r / min for 15 minutes. Take the precipitate and disperse it in water (10 mL) to obtain a gold nanorod solution.

[0093] The length and width of the gold nanorods obtained in Preparation Example 1 were measured. The measurement results are as follows: Figure 1-2 As shown, Figure 1 is the length distribution of gold nanorods, Figure 2 This is the width distribution diagram of gold nanorods. After calculation, the average length of gold nanorods is 51.20nm, the average width is 14.15nm, and the aspect ratio is 3.73.

[0094] Preparation Example 2

[0095] This preparation example provides a carbon quantum dot (CDs), and the preparation method thereof is as follows:

[0096] Take the m-phenylenediamine (10mL, 30mg / mL) aqueous solution and tannic acid aqueous solution (20mL, 33.75mg / mL) and mix them into a hydrothermal reactor. After treating at 180℃ for 10h, cool to room temperature and centrifuge the solution at 15000rpm for 15min. Take the supernatant and dialyze it at room temperature for 24h (MWCO: 1000Da), change the water every 2h, and store the obtained CDs solution at 4℃ for future use.

[0097] Preparation Example 3

[0098] This preparation example provides a CDs-ss-DNA, and the preparation method thereof is as follows:

[0099] NHS (500 μL, 25 mM) and EDC (500 μL, 25 mM) were mixed and added to 1.5 mL of CDs solution. After mixing at room temperature for 15 min, ss-DNA sequence (10 μL, 100 μM) was added, stirred at 4°C for 12 h, and then dialyzed for 24 h (MWCO: 3500 Da) to obtain a CDs-ss-DNA solution.

[0100] Example 1

[0101] This embodiment provides an ATP nucleic acid aptamer and a FA-modified thiol polyethylene glycol-modified gold nanorod probe, and the preparation method thereof is as follows:

[0102] (1) TCEP and ATP aptamer (HS-ABA, final concentration 10 μM) were mixed at a molar ratio of 200:1 and protected from light for 3 h to obtain the reduced ATP aptamer;

[0103] (2) The reduced ATP aptamer DNA (5SH-C6-TCGATTC CCTGGGGGAGTA TTGCGGAGGAGG-cy5) (10 μL, 100 μM) was mixed with 10 mL of the synthesized gold nanorod solution and stirred at 30 °C for 12 h;

[0104] (3) The mixed solution was centrifuged at 9500 rpm for 15 min, and the precipitate was resuspended in 10 mL of water to obtain an ATP-modified gold nanorod solution, namely, AuNRs-ATP;

[0105] (4) FA-PEG5000-SH (100 μL, 1 mM) was mixed with ATP-modified gold nanorod solution (10 mL), stirred and allowed to stand for 12 h to obtain ATP nucleic acid aptamer and FA-modified polyethylene glycol-modified gold nanorod probe, namely FA-AuNRs-ATP.

[0106] Example 2

[0107] This embodiment provides a gold nanorod / carbon quantum dot composite plasma fluorescent nanoprobe (AuNRs-CDs), and the preparation method thereof is as follows:

[0108] (1) The gold nanorod probe solution obtained in Example 1 and the CDs-ss-DNA solution obtained in Preparation Example 3 were mixed in a volume ratio of 1:2, and the obtained mixed solution was co-cultured in a constant temperature water bath (37° C.) for 12 h to obtain an assembled probe;

[0109] (2) Mix the assembled probe with 40 mM HEPES buffer in a volume ratio of 1:1, centrifuge at 9500 rpm for 15 min, repeat 3 times, discard the supernatant, resuspend with 20 mM HEPES buffer, and store at 4°C until use.

[0110] Performance Testing

[0111] The surface morphology of AuNRs, CDs and AuNRs-CDs was characterized by transmission electron microscopy. Figure 3-5 As shown, Figure 3 TEM image of AuNRs. Figure 4 TEM image of CDs. Figure 5 TEM image of AuNRs-CDs. As can be seen from the figure, the gold nanorods have regular morphology and a good rod-like structure. The synthesized CDs have good dispersibility, and the uniform particle size is calculated to be ~2.7±0.44nm.

[0112] The UV-visible spectra of AuNRs, AuNRs-ATP and FA-AuNRs-ATP were tested. The test results are as follows: Figure 6 As shown, it can be seen that the blue shift of the mass spectrometry peak indicates that HS-ATP nucleic acid aptamer and FA-PEG-SH are successfully modified on the surface of AuNRs.

[0113] The UV-visible spectra of AuNRs, CDs and AuNRs-CDs were tested. The test results are as follows: Figure 7 As shown, it can be seen that the UV absorption spectrum of CDs shows two absorption peaks at 263nm and 410nm. The peak at 263nm is attributed to the π-π* transition of the aromatic ring, and the peak at 410nm indicates the existence of n-π* transition of the surface state of CDs.

[0114] The Zeta potential of AuNRs, AuNRs-ATP, FA-AuNRs-ATP, CDs, CDs-ss-DNA and AuNRs-CDs was tested using Nano ZS zetasizer.

[0115] Test results such as Figure 8 As shown, it can be seen that after AuNRs are coupled with ATP-bound aptamers, the Zeta potential of the nanoprobe changes from 30.26 mV to 14 mV, indicating that the ATP-bound aptamer is successfully covalently linked to AuNRs. Then AuNRs-ATP is further modified with FA-PEG-SH to obtain better biocompatibility; the corresponding Zeta potential decreases from 14.00 mV to -2.09 mV, which means that a large amount of CTAB on the surface of AuNRs is replaced by FA-PEG-SH; after ss-DNA is conjugated with CDs, the zeta potential of CDs increases from -20.73 mV to -8.74 mV, indicating that ss-DNA is successfully modified on the surface of CDs by covalent bonds. Finally, AuNRs-CDs are assembled by FA-AuNRs-ATP and CDs-ss-DNA through complementary base pairing. After assembly, the zeta potential of FA-AuNRs-ATP changes from -2.09 mV to -4.43 mV.

[0116] The fluorescence intensity of CDs was tested as follows:

[0117] The fluorescence spectra of CDs were tested using different excitation wavelengths from 300 nm to 500 nm. -1 ~10.4mg mL -1 The CDs were tested by fluorescence spectroscopy.

[0118] Test results such as Figure 9-10 As shown, Fig. 9 The fluorescence spectra of CDs at different excitation wavelengths are shown in Figure 2. Fig.10 The fluorescence spectra of CDs at different concentrations are shown in Figure 2. Fig. 9 It can be seen that the fluorescence emission spectra of CDs show different peak positions and intensities at different excitation wavelengths, showing excitation-dependent behavior, which is due to the surface state formed by the hybridization of π-conjugated aromatic domains and connecting chemical groups. Fig.10 It can be seen that the fluorescence intensity increases with the increase of CDs concentration, which indicates that the synthesis of CDs is successful.

[0119] The AuNRs-CDs absorbed by human liver cancer cells and normal human liver cells were measured using an inductively coupled plasma emission spectrometer (ICP). The specific method is as follows:

[0120] HepG2 cells (high FA expression) and HL-7702 cells (low FA expression) were plated in 6-well plates at 2×10 5 The cells were cultured at 37°C for 12 h at a density of 10 cells / well. Then, AuNRs-CDs (32 μg mL -1) These cells were cultured for 2 h, carefully washed and digested with PBS. All collected cells were subjected to ICP measurement to quantitatively analyze the content of nanoprobes.

[0121] Test results such as Fig.11 As shown, after 2 h of treatment with AuNRs-CDs probe, the intracellular gold contents of HepG2 (human liver cancer cells) and HL-7702 (human normal liver cells) cells were 266.7 ± 51.9 ng / mL and 121.0 ± 2.4 ng / mL, respectively, indicating that FA-modified AuNRs-CDs can enhance the endocytosis of HepG2 cells and will serve as an excellent method for ATP tracking in FR-positive cancer cells.

[0122] The photothermal stability of the AuNRs-CDs probe was tested as follows:

[0123] Take 32 μg mL -1 The AuNRs-CDs were dispersed in 1 mL of ultrapure water, and the solution was recorded at 808 nm laser (2 W cm -2 ) temperature changes under irradiation, record the temperature rise changes for 10 minutes, then turn off the laser, and continue to record the temperature drop changes for 10 minutes. This is one cycle, and a total of three cycles are recorded.

[0124] Test results such as Figure 12-14 As shown, Fig.12 The temperature rise curve of the AuNRs-CDs probe solution under different laser irradiation intensities over time. Fig.13 Figure 3 shows the temperature response of AuNRs-CDs to repeated on / off irradiation cycles (808 nm laser, 2 W cm -2 ), Fig.14 The graph shows the absorbance change of AuNRs-CDs probe solution before and after laser irradiation. Fig.12 It can be seen that at 2W cm -2 Under laser irradiation, AuNRs-CDs (32 μg mL -1 ) The temperature of the solution increased to 55.2±0.2℃ within 5min. Fig.13 It can be seen that the change in the photothermal properties of AuNRs-CDs can be ignored during repeated on / off irradiation cycles. Fig.14 It can be seen that after continuous irradiation for 2 h, the proton peak only slightly decayed. The above results show that the AuNRs-CDs composite probe has good photothermal stability.

[0125] The biocompatibility test of AuNRs-CDs probe is carried out as follows:

[0126] The biocompatibility of AuNRs-CDs was tested by MTT assay. HepG2 and HL-7702 cells (1×10 4 Cells) were seeded in a 96-well plate and cultured at 37°C for 24h. Then, 10μL of AuNRs-CDs of different concentrations were added to each well, supplemented with 90μL of complete medium, and cultured at 37°C for 24h. The plate was then replaced with 90μL of fresh complete medium, after which 10μL of MTT was added to each well and cultured in a CO2 incubator at 37°C for another 4h. Finally, 150μL of DMSO was added to the plate. The absorbance was tested on a microplate reader with a measuring wavelength of 490nm. Cells incubated without nanomaterials were used as a control group.

[0127] Test results such as Fig.15 As shown, it can be seen that both HepG2 and HL-7702 cells maintained a viability of more than 90%, and AuNRs-CDs exhibited excellent biocompatibility.

[0128] The cell viability of the AuNRs-CDs probe was tested under 808nm laser irradiation. The test method is as follows:

[0129] The 808 nm laser was used at different time intervals (1 min, 3 min, 5 min) and different densities (0 W cm -2 , 0.5W cm -2 , 1W cm -2 , 1.5W cm -2 , 2W cm -2 ) -1 ) were incubated with HepG2 and HL-7702 cells for 2 h to test cell viability.

[0130] Test results such as Figure 16-17 As shown, Fig.16 The cell viability results of HepG2 cells at different time intervals and densities using 808nm laser. Fig.17 The cell viability results of HL-7702 cells irradiated with 808nm laser at different time intervals and densities can be seen. After being treated with AuNRs-CDs for 2h, HepG2 cells and HL-7702 cells were irradiated with 808nm laser at different laser intensities. The cell viability results were different at different irradiation times. When the laser intensity was greater than 1Wcm -2 When the treatment time was prolonged, the cell viability of the cancerous HepG2 cells decreased significantly, while the cell viability of the normal HL-7702 cells treated with the same method continued to 1.5 W cm -2 .

[0131] The in vitro photothermal performance of the AuNRs-CDs probe was tested as follows:

[0132] Calcein AM / PI mixed fluorescent dye was used to verify the photothermal effect. All cells were plated in 96-well plates at 1×10 4 Then, the cell culture medium was removed, the cells were washed three times with phosphate buffered saline (PBS, 10 mM, pH = 7.4), and nucleolar-targeted AuNRS-CDs (32 μg mL -1 ) with a certain amount of complete medium. After incubation for 2 h, the cells were washed three times with PBS to remove free nanoprobes. Photothermal ablation under different conditions (power density of 12 W cm -2 、1.52W cm -2 , 2W cm -2 The photothermal efficiency of AuNRs-CDs was recorded for 1 min, 3 min, and 5 min, and the wells without any treatment were set as the control group. Finally, the cells were stained with calcein AM / PI mixed dye for 15 min, washed twice with PBS, and fluorescence images were collected under a Leica microscope.

[0133] Test results such as Fig.18 As shown, Fig.18 Live / dead staining images of HepG2 and HL-7702 cells after laser irradiation at different time intervals and intensities (scale bar = 100 μm). For both cells, at 1.5 W cm -2 After 5 min of 808 nm laser irradiation, most cells died, while at different irradiation times of 1 W cm -2 The cells still had good viability even after being treated with 808 nm of irradiation. The results showed that AuNRs-CDs had good photothermal properties in photothermal therapy applications. Therefore, in order to better explore the intrinsic relationship between ATP energy metabolism and nucleolar morphological transformation and photothermal stimulation, we used 1 W cm -2 The following experiments were carried out with laser intensities of 1.

[0134] The AuNRs-CDs probe was tested for ATP content detection specificity. The test method is as follows:

[0135] Before intracellular ATP sensing, we first measured the response of the nanoprobe to different concentrations (0 nM to 10 mM) of ATP in solution by irradiating it with 808 nm laser for 10 min at a power of 2 W cm -2 We recorded the fluorescence spectra of the above solutions. Next, the solution was diluted to a concentration of 32 μg mL in HEPES containing 2 mM MgCl2. -1The AuNRs-CDs solution was then mixed with different concentrations of ATP or its analogs. The solutions were incubated in a constant temperature water bath for 1 h (37 ° C), and the changes in fluorescence spectra were tested by a spectrofluorimeter (Ex = 460 nm, Em = 480-650 nm) for ATP and analogs. Finally, in order to further prove the feasibility of AuNRs-CDs in monitoring ATP content fluctuations in living cells, 5mmol / L CaCl2 or DNA random sequence (NH2-C6-GCAGCAGCAGCA, AuNRs-ranCDs) was used as a control group to pre-treat HepG2 cells for 2 h, and then co-incubated with AuNRs-CDs for 2 h for subsequent fluorescence imaging (AuNRs-CDs co-incubated with HepG2 cells was the experimental group).

[0136] Test results such as Figure 19-22 As shown, Fig.19 The fluorescence spectrum of AuNRs-CDs in solutions with increasing ATP concentrations from 0 to 10 mM is shown in Figure 2. Fig. 20 The fluorescence intensity results of AuNRs-CDs in CTP, GTP, UTP and ATP are shown in Figure 2. Fig.21 Fluorescence images of HepG2 cells cultured with AuNR-CDs, AuNRs-CDs+CaCl2, and AuNRs-ranCDs. Fig. 22 The fluorescence intensity change diagram of AuNRs-CDs under different irradiation time. Fig.19 It can be seen that the intensity increases with the increase of ATP concentration from 0 to 10 mM, which is the result of ATP-induced release of CDs from the surface of AuNRs. Fig. 20 It can be seen that due to the specificity of nucleic acid aptamers to ATP, the addition of ATP analogs, including UTP, GTP, and CTP, did not show obvious fluorescence intensity enhancement compared with the blank sample. These results show that AuNRs-CDs respond well to ATP in solution. Fig.21 It can be seen that cells treated with 5mM CaCl2 resulted in a significant increase in the fluorescence emission of the CDs of the nanoprobe compared to the control group treated with AuNRs-CDs alone. When the ATP molecules of the nanoprobe were replaced by random DNA sequences, there was no obvious fluorescence. Under the same experimental conditions, no obvious fluorescence signal was observed. This confirms that only ATP can lead to the fluorescence recovery of CDs. Fig. 22 It can be seen that the fluorescence intensity of the AuNRs-CDs probe did not decrease substantially after being irradiated with 808 nm laser for 15 min.

[0137] The test method for monitoring nucleolar morphology during photothermal therapy using AuNRs-CDs probe is as follows:

[0138] First, to explore the nuclear targeting mechanism (DNase and RNase digestion assays), we subsequently treated the cells with ribonuclease (RNase) or deoxyribonuclease (DNase) before CDs staining. First, HepG2 cells were treated with 1% Triton X-100 for 1 min at room temperature and then washed twice with PBS. Then, PBS solution (control experiment), 25 μg mL - 1 DNase and 25 μg mL -1 The cells were treated with RNase for 1 h at 37°C and 5% CO2. After washing twice with PBS, AuNRs-CDs (32 μg mL -1 ) and Hoechst 33342 (5 μg mL -1 ) were used to co-stain the cells. Finally, fluorescence images of co-stained live cells were taken with a fluorescence microscope at an excitation wavelength of 405 nm. Finally, HepG2 and HL-7702 cells were co-stained with AuNR-CDs (32 μg mL -1 ) and incubated for 2 h. Under the same conditions (power density of 1 W cm -2 , lasting 1min, 3min, 5min, 7min, 9min, and 11min), and the changes in the morphology and number of cell nucleoli were observed through photothermal therapy. The wells without any treatment were set as the control group.

[0139] Test results such as Figure 23-24 As shown, Fig.23 Hoechst 33342 and AuNRs-CDs were used to co-stain DNase and RNase treated HepG2 living cells for fluorescence imaging. Fig.24 The following is a diagram showing the changes in nucleolus size in HepG2 cells and HL-7702 cells as the photothermal treatment time increases. Fig.23 It can be seen that CDs can specifically illuminate the nucleolus, which can be clearly seen in the bright images of living HepG2 cells, indicating that AuNRs-CDs can achieve live cell nucleolus imaging. Fig.24 It can be seen that as the treatment time of photothermal therapy increases, the nucleoli of the two cell lines incubated with AuNRs-CDs become smaller. However, compared with HL-7702 normal cells, the nucleolar changes of HepG2 cancer cells are more obvious. The results show that cancer cells have higher thermal sensitivity than normal cells, and AuNRs-CDs can effectively monitor the dynamic changes of cancer cell nucleolar morphology during photothermal therapy.

[0140] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe, comprising gold nanomaterial and carbon quantum dots; The surface of the gold nanomaterial is modified with ATP nucleic acid aptamer and FA-modified polyethylene glycol; The surface of the carbon quantum dots is modified with single-stranded DNA; The sequence of the ATP nucleic acid aptamer is shown in SEQ ID No: 1; The sequence of the single-stranded DNA is shown in SEQ ID No: 2; The single-stranded DNA is complementary to a portion or all of the base sequences in the ATP nucleic acid aptamer; The gold nanomaterial includes gold nanorods; The aspect ratio of the gold nanorods is 3.5-4.

2. The gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe according to claim 1, characterized in that: The molar ratio of the gold nanomaterial, FA-modified polyethylene glycol and ATP nucleic acid aptamer is 1:(1-1.5):(1.8-2.2).

3. The gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe according to claim 1, characterized in that: The FA-modified polyethylene glycol is FA-modified thiol polyethylene glycol.

4. The method for preparing the gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe according to any one of claims 1 to 3, characterized in that: The steps include: The carbon quantum dot solution modified with single-stranded DNA was mixed with the gold nanomaterial solution modified with ATP nucleic acid aptamer and FA-modified polyethylene glycol, and then cultured in a 30-40°C water bath for 10-14 h to obtain a gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe.

5. The preparation method according to claim 4, characterized in that: The carbon quantum dot solution with single-stranded DNA modified on the surface is prepared by a preparation method comprising the following steps: Mix the coupling agent, carbon quantum dot solution and single-stranded DNA and react at 2-6°C for 8-15 h; The coupling agents include NHS and EDC.

6. The preparation method according to claim 4, characterized in that: The gold nanomaterial solution with ATP nucleic acid aptamers and FA-modified polyethylene glycol on the surface is prepared by a preparation method comprising the following steps: (1) mixing a reducing agent and an ATP nucleic acid aptamer and reacting them in the dark to obtain a reduced ATP nucleic acid aptamer; the reducing agent is selected from TCEP; (2) mixing the reduced ATP nucleic acid aptamer with the gold nanomaterial solution and reacting the mixture to obtain a gold nanomaterial solution modified with the ATP nucleic acid aptamer; (3) The FA-modified polyethylene glycol and the ATP-aptamer-modified gold nanomaterial solution are mixed and reacted to obtain a gold nanomaterial solution whose surface is modified with the ATP-aptamer and the FA-modified polyethylene glycol.

7. The preparation method according to claim 6, characterized in that: The temperature of the light-proof reaction in step (1) is 25-35°C, and the time of the light-proof reaction is 2-5 h; The reaction temperature in step (2) is 25-35°C and the reaction time is 8-15 h; The reaction temperature in step (3) is 25-35°C and the reaction time is 8-15 h.

8. Use of the gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe according to any one of claims 1 to 3 or the gold nanomaterial / carbon quantum dot composite plasma fluorescent nanoprobe prepared by the preparation method according to any one of claims 4 to 7 in biological imaging, preparation of photothermal therapeutic agents and / or ATP detection.

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