Acid-responsive nanoprobe as well as preparation and anti-tumor application thereof

Abstract

The invention discloses an acid responsive nanoprobe as well as preparation and anti-tumor application thereof. The nanoprobe has a core-shell structure, takes a nanoparticle self-assembled by a polycation aggregation-induced emission (AIE) photosensitizer and functional nucleic acid as a core, and takes a hyaluronic acid modified metal polyphenol network as a shell. The nanoprobe utilizes the acid responsiveness and broad-spectrum absorption performance of the metal polyphenol network and the diagnosis and treatment advantages of the polycation AIE photosensitizer to realize the acid responsiveness'switch 'controllable diagnosis and treatment of the tumor nanoprobe. In a neutral environment, the fluorescence is weak, the generation of ROS is less, and the background noise signal of the nanoprobe and the toxicity of non-targeted tissues are reduced; in an acid environment, fluorescence is enhanced, the ROS yield is improved, and the treatment efficiency and the curative effect monitoring accuracy are improved. The nano diagnosis and treatment probe is ingenious in design and simple to prepare, and can realize PDT and gene therapy synergistic anti-tumor, in-situ real-time report of early tumor treatment effect and assistance in precise cancer treatment.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to a nanoprobe for tumor treatment and efficacy diagnosis, and specifically to an acid-responsive nanoprobe, a preparation method thereof, and an anti-tumor application thereof. Background Art

[0002] Malignant tumors seriously threaten human health. Due to the diversity, complexity, and heterogeneity of tumors, there are significant differences in the responses of different patients to the same treatment strategy. This makes it crucial to monitor the anti-tumor efficacy in real time for achieving precise tumor treatment and prognosis. Unfortunately, in current clinical practice, cancer diagnosis and treatment are often separated, which not only hinders doctors from accurately grasping the best treatment timing but also leads to an increase in the side effects of multiple and fixed-dose administrations, thereby seriously affecting the quality of life of patients. Therefore, constructing a tumor diagnosis and treatment integrated probe that combines a new treatment method with low toxicity and a real-time efficacy monitoring function will be beneficial to precise cancer treatment.

[0003] Compared with traditional tumor therapies, gene therapy and photodynamic therapy (PDT) have shown good application prospects in precise tumor treatment due to their high selectivity, low drug resistance, and low toxic side effects. However, during the delivery process of functional nucleic acids, problems such as the stability and targeting of nucleic acid carriers, as well as the effective release and expression of functional nucleic acids in tumor cells, have greatly hindered the clinical application of gene therapy. Some research reports have shown that after photosensitizer molecules are activated by absorbing photons of a specific wavelength, they can not only generate ROS to kill tumor cells and achieve PDT but also promote photochemical internalization and enhance the effect of gene therapy. At the same time, the changes in related proteins in tumor cells caused by gene therapy, such as reducing the expression of vascular endothelial growth factor, monocarboxylate transporter 1, etc., and promoting the expression of arachidonic acid-12-lipoxygenase (ALOX12), cyclooxygenase, etc., can further enhance the effect of PDT, thereby achieving synergistic anti-tumor effects. In addition, the activated photosensitizer molecules can also release energy in the form of fluorescence to return to the ground state from the excited state, realizing fluorescence imaging (diagnosis) of tumors. Therefore, a tumor diagnosis and treatment probe constructed based on photosensitizers and siRNA can simultaneously achieve photocatalytic-induced tumor diagnosis and combined treatment, which is beneficial to improving the efficiency of precise treatment of malignant tumors and reducing toxic side effects.

[0004] However, existing diagnostic and therapeutic probes based on photosensitizers and functional nucleic acids still have some problems that need to be solved. On the one hand, traditional photosensitizers are prone to unexpected aggregation, which not only leads to the quenching of fluorescence signals, affecting the sensitivity and accuracy of tumor diagnosis, but also reduces the generation of ROS in PDT, reducing the therapeutic effect; on the other hand, most diagnostic and therapeutic probes are always in an activated state, and there are side effects caused by off-target effects, making it difficult to achieve accurate diagnosis and treatment. In addition, real-time monitoring of the effect of tumor treatment is also a major challenge in current clinical treatment. Existing diagnostic and therapeutic probes cannot accurately and real-timely feedback the treatment effect in situ on the tumor.

[0005] Metal-polyphenol networks are supramolecular network structures formed by the interaction of natural polyphenols and metal ions through coordination bonds. They are not only safe, stable, biodegradable, and low-cost, but are also widely used in various drug delivery systems. Importantly, metal-polyphenol networks have a broad absorption spectrum, which can lead to quenching of the activity of the photosensitizers they encapsulate. In addition, metal-polyphenol networks are uniquely responsive to the acidic conditions in the tumor microenvironment and can disintegrate under acidic conditions, thereby achieving precise release and activation of the loaded photosensitizers and realizing precise tumor diagnosis and treatment. Although metal-polyphenol networks have shown certain application potential in the field of tumor diagnosis and treatment, the current nanodiagnostic probes constructed based on metal-polyphenol networks still have some shortcomings. For example, how to further improve their targeting to tumor cells, how to achieve synergistic anti-tumor effects of gene therapy and PDT, and how to construct a system that can report tumor treatment effects in real time and in situ are all issues that need to be further studied and resolved.

[0006] In summary, the development of an acid-responsive nanoprobe with efficient tumor targeting, capable of synergistic PDT and gene therapy, and capable of in situ, real-time self-reporting of early tumor treatment efficacy has important practical significance and clinical application value for improving tumor diagnosis and treatment. It is against this backdrop that the present invention proposes a novel acid-responsive nanoprobe for tumor diagnosis and treatment and a method for its preparation, aiming to address the aforementioned issues existing in the prior art. Summary of the Invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to address the deficiencies of the existing technology and provide an acid-responsive nanoprobe for tumor diagnosis and treatment, as well as a preparation method and application thereof.

[0008] Invention concept: The present invention utilizes a metal-polyphenol network to encapsulate polycationic AIE photosensitizer-functional nucleic acid nanoparticles, and modifies the tumor-targeting group HA on the surface of the metal-polyphenol network to construct a "switch-type" tumor-targeted nano-diagnostic and therapeutic probe, thereby achieving synergistic anti-tumor effects of PDT combined with gene therapy, and in situ, real-time self-reporting of early tumor treatment effects.

[0009] In the first aspect of the present invention, there is provided an acid-responsive nanoprobe for tumor diagnosis and treatment.

[0010] Among them, the nano-probe has a core-shell structure. The core is a nanoparticle self-assembled from a multi-cationic AIE photosensitizer and functional nucleic acid, and the shell is a metal polyphenol network modified with hyaluronic acid. The hyaluronic acid is a tumor-targeting molecule. The nano-probe utilizes the acid responsiveness, broad-spectrum absorption performance of the metal polyphenol network, and the diagnosis and treatment advantages of the multi-cationic AIE photosensitizer to achieve acid-responsive "switch" controllable diagnosis and treatment of tumor nano-probes.

[0011] Among them, the multi-cationic AIE photosensitizer is composed of a triphenylamine derivative and four cations, and its structural formula is shown in Formula I:

[0012]

[0013] Among them,

[0014] R1 is selected from 2,2’,2”-(benzene-1,3,5-triacyl)triacetonitrile group, 3-cyanobenzyl cyanide group or 2,2’-(benzo[1,2-D:4,5-D’]bis(thiazole)-2,6-diyl)diacetonitrile group shown in Formula II;

[0015]

[0016] R2 is a cation, preferably at least one of pyridinium salt cation, quinolinium salt cation and acridinium salt cation, such as to improve the binding ability with nucleic acid.

[0017] In some embodiments, the structure of the multi-cationic AIE photosensitizer is shown as follows:

[0018]

[0019] Among them, the functional nucleic acid is at least one of plasmid DNA, siRNA, peptide nucleic acid, mRNA, miRNA, ribozyme, such as the ALOX12-mCherry fusion expression recombinant plasmid (for the specific construction method, see Wang X, Chen Y, Yang X, Cheng L, He Z, Xin Y, Huang S, Meng F, Zhang P, Luo L. Activation of ALOX12 by a multi-organelle-orienting photosensitizer drives ACSL4-independent cell ferroptosis. Cell Death Dis. 2022, 13(12):1040.), and it self-assembles with the multi-cationic AIE photosensitizer through non-covalent interactions such as electrostatic interactions to form nanoparticles, improving the stability of the functional nucleic acid.

[0020] Among them, the metal polyphenol network is formed by the coordination of metal ions and natural polyphenols. The metal ions are at least one of iron ions, copper ions, and zinc ions, and the natural polyphenols are one or more of tannic acid, quercetin, and epigallocatechin gallate (EGCG). The metal polyphenol network is stable in the physiological pH environment and degrades in the acidic environment of tumors, realizing the acid-responsive "switch" of the diagnostic and therapeutic functions of the nanoprobe.

[0021] Among them, the molecular weight range of the HA is 1-150 kDa, such as 30 kDa, 35 kDa, 40 kDa, and it binds to the metal polyphenol network through non-covalent interactions such as electrostatic interactions.

[0022] In the second aspect of the present invention, there is provided a preparation method of the acid-responsive nanoprobe described in the first aspect above.

[0023] Among them, the preparation method includes the following steps:

[0024] (1) Prepare the multi-cationic AIE photosensitizer

[0025] (2) Prepare the nanoparticles formed by the self-assembly of the functional nucleic acid and the multi-cationic AIE photosensitizer

[0026] In the aqueous solution of the multi-cationic AIE photosensitizer, slowly add the functional nucleic acid solution and continuously stir. Through non-covalent interactions such as electrostatic interactions, obtain the nanoparticles formed by the self-assembly of the functional nucleic acid and the multi-cationic AIE photosensitizer;

[0027] (3) Wrap the nanoparticles in step (2) with the metal polyphenol network

[0028] Under stirring conditions, the nanoparticles described in step (2) are slowly added to the aqueous solution of natural polyphenols, and then the metal ion solution is quickly added, followed by continuous stirring. After centrifugation at low temperature and washing, an acid-responsive nanotheranostic probe without tumor-targeting group modification is obtained.

[0029] (4) HA modification

[0030] The HA aqueous solution is added to the theranostic probe prepared in step (3), and continuous stirring is carried out. After stirring for a period of time, free HA is removed by ultrafiltration centrifugation to obtain the acid-responsive nanosensor for tumor diagnosis and treatment.

[0031] In steps (2), (3) and (4), the addition method includes dropwise addition.

[0032] In step (2), the molar ratio of the functional nucleic acid to the polycationic AIE photosensitizer is 1:5 to 1:40, such as 1:15, 1:20, 1:25; the concentration of the aqueous solution of the polycationic AIE photosensitizer is 0.5 to 3 mM, such as 1 mM, 1.5 mM, 2 mM; the stirring time is 10 to 120 min, such as 20 min, 30 min, 40 min.

[0033] In step (3), the concentration of the aqueous solution of natural polyphenols is 0.5 to 6 mg / mL, such as 1 mg / mL, 2 mg / mL, 4 mg / mL; the metal ion solution is an aqueous solution of metal ions; the concentration of the metal ion solution is 8 to 12 mg / mL, such as 10 mg / mL.

[0034] In step (3), the molar ratio of natural polyphenols to metal ions is 1:2 to 4:1, such as 3:2; the mass ratio of natural polyphenols to nanoparticles is 5:2 to 6, such as 5:4.

[0035] In step (3), the slow addition of the nanoparticles described in step (2) is carried out in about 5 min, such as 4 - 6 min; the rapid addition is carried out in about 1 min, such as 0.5 - 1 min.

[0036] In step (3), during the stirring state and continuous stirring, the stirring rate is independently selected from 500 - 2000 rpm; the continuous stirring time is 1 - 6 h, such as 2 h; the continuous stirring is the stirring time after the addition of the metal ion solution is completed; the centrifugation conditions are 3 - 5 °C, 11000 - 13000 rpm, 0.5 - 1.5 h, such as 4 °C, 12000 rpm, 1 h.

[0037] In step (4), the mass ratio of the HA to the diagnostic probe is 1:100 - 200, preferably 1:150; the concentration of the HA aqueous solution is 0.5 - 3 mg / mL, such as 1 mg / mL, 2 mg / mL; the average molecular weight of the HA is 30 - 40 kD, such as 35 kD.

[0038] In step (4), the rate of continuous stirring is 500 - 2000 rpm, such as 1000 rpm; the time of continuous stirring is 2 - 8 h, such as 6 h; the cut-off molecular weight of the ultrafiltration tube is 80 - 120 kD, such as 100 kD; the conditions of centrifugation are 3 - 5 °C, 2000 - 6000 rpm, 0.2 - 1.5 h, such as 4 °C, 4000 rpm, 0.5 h.

[0039] In the third aspect of the present invention, there is provided an anti-tumor product.

[0040] Among them, the anti-tumor product includes the acid-responsive nanoprobe described in the first aspect above.

[0041] In the fourth aspect of the present invention, there is provided the application of the acid-responsive nanoprobe described in the first aspect above in the preparation of an anti-tumor product.

[0042] In the third and fourth aspects,

[0043] The acid-responsive nanoprobe is used in combination with PDT and / or gene therapy to treat cancer; the cancer includes melanoma, cervical cancer and breast cancer.

[0044] Before being triggered by the acidic microenvironment of the tumor, it is in an inactivated state and non-toxic. After the metal polyphenol network of the probe shell is disintegrated by acid, the activity of the photosensitizer is restored, and a large amount of ROS is generated under light conditions. These ROS can not only kill tumor cells through PDT, but also promote the lysosomal escape of functional nucleic acids through photochemical internalization, enhancing the effect of gene therapy; in addition, the changes in the expression of tumor cell-related genes induced by functional nucleic acids further enhance the effect of PDT, thus realizing the synergistic anti-tumor effect of PDT and gene therapy.

[0045] In the fifth aspect of the present invention, there is provided an imaging agent for tumor efficacy diagnosis.

[0046] Among them, the imaging agent includes the acid-responsive nanoprobe described in the first aspect above.

[0047] In the sixth aspect of the present invention, there is provided the application of the acid-responsive nanoprobe described in the first aspect above in the preparation of an anti-tumor efficacy diagnosis product.

[0048] In the fifth and sixth aspects,

[0049] Among them, the efficacy diagnosis includes tumor efficacy diagnosis, especially early tumor efficacy diagnosis.

[0050] Among them, it remains inactive before being triggered by the tumor acidic microenvironment, and the probe has extremely low fluorescence signals. When the outer metal polyphenol network is degraded by acid, the diagnostic and therapeutic activity of the probe is activated. That is, the multi-cationic AIE photosensitizer has very weak fluorescence in living cells, but under light illumination, during the process of cell death, the photosensitizer synchronously enters the dead cell nucleus, binds to nuclear DNA, undergoes AIE, and the fluorescence increases significantly, self-reporting cell death and realizing in-situ and real-time reporting of the early anti-tumor efficacy.

[0051] In summary, the present invention solves the technical problem in the prior art that the activity of the nano-probe that can achieve the combined treatment of gene therapy and PDT has always been in the "on" state and cannot effectively achieve precise tumor treatment. The provided acid-responsive nano-probe for tumor diagnosis and treatment has a core-shell structure. The core is a self-assembled nanoparticle of a multi-cationic AIE photosensitizer and functional nucleic acid, and the shell is a metal polyphenol network modified with hyaluronic acid. The AIE photosensitizer is composed of a triphenylamine derivative and four cations. The nano-probe of the present invention targets tumor tissues, is non-toxic to normal tissues, and under the tumor acidic microenvironment, without the need to additionally add an imaging contrast agent, and utilizes the effect of the photosensitizer to light up the cell nucleus of dead cells, thereby enabling in-situ and real-time reporting of the effect of synergistic anti-tumor.

[0052] Beneficial effects:

[0053] (1) The acid-responsive nano-probe for tumor diagnosis and treatment of the present invention will only activate its diagnostic and therapeutic activity under the action of the tumor acidic microenvironment. In normal tissues, the diagnostic and therapeutic activity is always in the "off" state and will not produce cytotoxicity. This not only realizes the precise diagnosis and treatment of tumors but also ensures high biosafety.

[0054] (2) The acid-responsive nano-probe for tumor diagnosis and treatment of the present invention, after precisely targeting tumor tissues, the acidic microenvironment inside the tumor will cause the disintegration of the probe's outer metal polyphenol network, restoring the diagnostic and therapeutic activity of the probe. Under light illumination, the photosensitizer is activated to generate a large amount of ROS. These ROS can not only directly kill tumor cells through the PDT effect but also promote the escape of functional nucleic acid from lysosomes through photochemical internalization, enhancing the effect of gene therapy.

[0055] (3) The acid-responsive nano-probe for tumor diagnosis and treatment of the present invention has its fluorescence quenched due to the broad-spectrum absorption of the metal polyphenol network. In normal tissues and living cells, the fluorescence signal is hardly detectable, and the background fluorescence of the probe is extremely weak. However, during the process of cell death, the fluorescence signal will gradually increase, and its brightness can increase by dozens of times, having a high signal-to-noise ratio. This change can reflect the effect of anti-tumor treatment in real time and can be judged without relying on other contrast agents. This characteristic provides a convenient and direct means for evaluating the synergistic anti-tumor efficacy of PDT and gene therapy of the probe.

[0056] (4) The acid-responsive nanoprobe for tumor diagnosis and treatment of the present invention can evaluate the treatment effect when tumor cells die but the tumor volume has not changed significantly, achieving early diagnosis and treatment, which is superior to the traditional diagnosis and treatment techniques that only rely on the change of tumor volume. The reason is that: the multi-cationic AIE photosensitizer has a high affinity for nucleic acids. When the permeability of the nuclear membrane changes due to the death of tumor cells, the multi-cationic AIE photosensitizer easily transfers from the cytoplasm to the nucleus, and after binding to intracellular nucleic acids, the internal movement of the photosensitizer molecules is blocked, resulting in AIE and a significant increase in fluorescence.

[0057] (5) The acid-responsive nanoprobe for tumor diagnosis and treatment of the present invention has a relatively wide range of applications. Regarding cancer treatment, it has good synergistic anti-tumor effects on melanoma, cervical cancer, and breast cancer.

[0058] (6) The preparation process of the acid-responsive nanoprobe in the present invention is simple, the production and raw material costs are low, and it is easy to industrialize production with broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0060] Figure 1 Variations of the hydrodynamic diameter (A) and PDI (B) of the aqueous solution of the acid-responsive nanoprobe for tumor diagnosis and treatment of the present invention after being placed at 4 °C for different times (n = 3).

[0061] Figure 2 Fluorescence emission spectra (A) and fluorescence intensities (B) of the acid-responsive nanoprobe for tumor diagnosis and treatment and free photosensitizer 1 aqueous solution (λex = 430 nm; λem = 640 nm).

[0062] Figure 3 Fluorescence emission spectra (A) and changes in fluorescence intensities (B) of the acid-responsive nanoprobe for tumor diagnosis and treatment in HEPES buffer solutions with different pH values (λex = 430 nm; λem = 630 nm).

[0063] Figure 4 ROS generation of the acid-responsive nanoprobe for tumor diagnosis and treatment in HEPES buffer solutions with different pH values under certain light conditions.

[0064] Figure 5 Fluorescence images of the acid-responsive nanoprobe for tumor diagnosis and treatment transfected with the recombinant plasmid of the fusion expression of ALOX12 and mCherry at the cellular level, and the scale bar is 100 μm.

[0065] Figure 6 Dark toxicity (A) and phototoxicity (B) of acid-responsive nanoprobes for tumor diagnosis and treatment on 4T1 cells, and phototoxicity (C) on B16F10 cells.

[0066] Figure 7 These are fluorescence microscope images of 4T1 cells after 24 hours of pretreatment with acid-responsive nanoprobes for tumor diagnosis and treatment, and after irradiation with blue light under a fluorescence microscope for different times. The scale bar is 25 μm. DETAILED DESCRIPTION

[0067] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0068] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0069] Example 1

[0070] This embodiment relates to a method for preparing an acid-responsive nanoprobe for tumor diagnosis and treatment, and the specific steps are as follows:

[0071] (1) Preparation of polycationic AIE photosensitizers

[0072] Synthesis route:

[0073]

[0074] 4-(Bis(4-(pyridin-4-yl)phenyl)amino)benzaldehyde (427.1 mg), 2,2'-(benzo[C][1,2,5]thiadiazole-4,7-diylbis(4,1-phenylene))diacetonitrile (120.6 mg), and potassium tert-butoxide (561.7 mg) were dissolved in 30 mL of methanol and heated to 70°C with stirring under reflux for 24 hours under nitrogen. After cooling to room temperature, the solvent was removed under reduced pressure, and the mixture was extracted three times with dichloromethane and water. The organic layer was collected and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography. The dried residue was dissolved in dichloromethane and loaded onto the sample. The mobile phase consisted of dichloromethane:anhydrous ethanol in a ratio of 17:1 to obtain AIE photosensitizer 1. 11H NMR (400 MHz, CDCl3) δ 8.71–8.64 (m, 8H), 8.10 (d, J = 8.3 Hz, 4H), 7.92 (d, J = 8.6 Hz, 4H), 7.87 (d, J = 8.3 Hz, 5H), 7.69–7.62 (m, 8H), 7.62–7.54 (m, 10H), 7.31 (d, J = 6.8 Hz, 8H), 7.24 (d, J = 8.5 Hz, 5H).

[0075] AIE photosensitizer 1 (100 mg) was added to a flask containing 20 mL of acetonitrile. After stirring and dissolving, 1 mL of methyl iodide was added. After reacting at 40 °C for 1 h, the temperature was raised to 80 °C and the reaction was refluxed for 12 h. After the reaction was completed, it was cooled to room temperature, an appropriate amount of anhydrous ether was added, allowed to stand for 20 min, and then filtered to obtain an orange powder solid, which was the polycationic AIE photosensitizer 1. 1 1H NMR (400 MHz, DMSO) δ = 8.97 (d, J = 6.9, 8H), 8.49 (d, J = 6.9, 8H), 8.32–8.03 (m, 21H), 8.03–7.85 (m, 4H), 7.36 (d, J = 8.7, 11H), 4.31 (d, J = 9.4, 12H), 3.39 (s, 29H), 3.10 (s, 5H), 2.89 (s, 2H), 2.81–2.70 (m, 2H), 2.59–2.45 (m, 22H).

[0076] (2) Obtaining the nanoparticles formed by the self-assembly of the functional nucleic acid and the polycationic AIE photosensitizer

[0077] Into the aqueous solution (1 mM) of the polycationic AIE photosensitizer, the solution of the ALOX12-mCherry fusion expression recombinant plasmid (10 mM) was slowly added dropwise, and stirring was continued for 30 min. The two interacted through non-covalent bonds such as electrostatic interaction, and the formation of colloidal nanoparticles by their self-assembly could be judged by the Tyndall effect. The molar ratio of the functional nucleic acid to the polycationic AIE photosensitizer was 1:20.

[0078] (3) Coating the nanoparticles in step (2) with a metal polyphenol network

[0079] The molar ratio of natural polyphenol tannic acid to ferric ions is controlled to be 3:2, and the mass ratio of natural polyphenol tannic acid to nanoparticles is 5:4. The stirring rate is 1500 rpm. Under the condition of stirring at room temperature, the nanoparticles in step (2) are slowly added dropwise to the aqueous solution of tannic acid (1 mg / mL) within 5 min, and then the aqueous solution of FeCl3 (10 mg / mL) is quickly added dropwise within 1 min; the stirring rate is changed to 800 rpm, and the continuous stirring time is 2 h. The color of the solution changes from yellow to black, indicating the successful formation of metal polyphenol-coated nanoparticles. Finally, centrifugation is carried out, and it is washed 3 times with ultrapure water. The black precipitate is taken, and the centrifugation condition for each time is 4 °C, 12,000 rpm, and 1 h. After being wrapped by the metal polyphenol network, the Zeta potential of the nanoparticles undergoes a polarity conversion, changing from the initial positive potential state to the negative potential state, showing completely different surface charge properties, that is, an acid-responsive nano-theranostic probe without tumor-targeting group modification is obtained.

[0080] (4) HA modification

[0081] The mass ratio of HA to the probe is controlled to be 1:150. The aqueous solution of HA with an average molecular weight of 35 kD (1 mg / mL, 5 μL) is added dropwise to the theranostic probe prepared in step (3), and continuous stirring is carried out. The stirring rate is 1000 rpm, and the time is 6 h. Then the mixed solution is placed in an ultrafiltration tube with a molecular weight cut-off of 100 kD, and free HA is removed by ultrafiltration centrifugation. The centrifugation condition is 4 °C, 4000 rpm, and 0.5 h, and the precipitate is taken. After three rounds of ultrafiltration centrifugation purification, it is measured that compared with before HA modification, the Zeta potential of the nano-preparation is significantly reduced, the absolute value increases, and the negative charge increases. It shows that HA modification significantly changes the charge properties of the nanoparticle surface, significantly improving its surface charge density, that is, the acid-responsive nano-probe for tumor diagnosis and treatment as described is obtained.

[0082] Example 2

[0083] This example relates to the stability test of the acid-responsive nano-theranostic probe for tumor diagnosis and treatment prepared in Example 1

[0084] The acid-responsive nano-theranostic probe solution prepared in Example 1 is stored at 4 °C and observed continuously for 7 days. It is measured once every 24 h. It is diluted to a concentration of 100 μg / mL with ultrapure water and ultrasonicated for 1 min to disperse it. Then 1 mL is taken and added to the measuring cell, and the particle size and distribution are detected using a dynamic light scattering particle size analyzer. The preset temperature is 25 °C, the angle is 90°, and it is continuously detected three times. The average value and standard deviation are calculated through the three values. The results are as Figure 1 shown. The particle size of the nano-probe is about 100 nm. There is no obvious change in the particle size within one week, and the PDI values are all less than 0.2, indicating good dispersibility. It shows that the nano-probe has good stability under this storage condition.

[0085] Example 3

[0086] This example relates to the acid-responsive performance test of the acid-responsive nanoprobe for tumor diagnosis and treatment prepared in Example 1

[0087] 1. The fluorescence of the nanoprobe is quenched

[0088] The nanoprobe prepared in Example 1 was diluted to a concentration of 400 μg / mL with ultrapure water, and the concentration of photosensitizer 1 was 70 μM. An aqueous solution of free photosensitizer 1 (70 μM) was used as a control. It was added to a quartz cuvette, and then the fluorescence emission spectra of different solutions were measured using a fluorescence spectrophotometer. The excitation light wavelength was set to 430 nm, and the emission wavelength collection range was set to 450 nm to 800 nm. The results are as Figure 2 shown. From Figure 2 it can be seen that for photosensitizer 1 with the same concentration and under the same measurement conditions (excitation wavelength of 430 nm), the fluorescence intensity of free photosensitizer 1 at 640 nm is 37 times that of the nanoprobe, indicating that the metal polyphenol shell can efficiently quench the fluorescence of photosensitizer 1

[0089] 2. The fluorescence signal of the nanoprobe has the property of being turned on in response to acid

[0090] The nanoprobe prepared in Example 1 was diluted to a concentration of 100 μg / mL with HEPES buffer solution of different pH values, and after standing at room temperature for 24 h, it was sonicated for 1 min and then added to a quartz cuvette. Then, the fluorescence emission spectra of different solutions were measured using a fluorescence spectrophotometer. The excitation light wavelength was set to 430 nm, and the emission wavelength collection range was set to 450 nm to 800 nm. The results are as Figure 3 shown. From Figure 3 it can be seen that the fluorescence of the nanoprobe in the HEPES solution increases with the decrease of pH. It shows that the nanoprobe has good characteristics of turning on the fluorescence signal in response to acid

[0091] 3. The ROS generation ability of the nanoprobe has the property of being turned on in response to acid

[0092] 2',7'-Dichlorodihydrofluorescein (DCFH) was used as a fluorescent probe for ROS detection. In the presence of ROS, DCFH is converted into 2,7-dichlorofluorescein (DCF) with a high quantum yield (the maximum excitation wavelength is 488 nm and the maximum emission wavelength is 524 nm). First, 50 μL of ethanol solution of 2,7-dichlorofluorescein diacetate (DCFH-DA, 10 mM) was added to 450 μL of ethanol for dilution, and then NaOH aqueous solution (0.01 M, 2 mL) was added and left to stand in the dark at room temperature for 30 min for activation. Finally, PBS (10 mM, pH = 7.4) was added to terminate the reaction, and the mixture was evenly mixed to obtain a DCFH solution with a final concentration of 80 μM and placed on ice for use.

[0093] The nano-probe prepared in Example 1 was diluted to a concentration of 100 μg / mL with HEPES buffer solutions of different pH values, and after standing at room temperature for 24 h, it was sonicated for 1 min to disperse. Then, 0.4 mL of the dispersed nano-probe was added to a quartz cuvette containing 0.4 mL of DCFH solution (80 μM), and after mixing evenly, the solution was irradiated with blue light (450 nm, 2 mW / cm 2 ) for 5 min. The fluorescence emission spectra of different solutions were recorded in the wavelength range of 500 - 650 nm under the condition that the excitation wavelength was set at 488 nm, and the results are as Figure 4 shown. As can be seen from Figure 4 the figure, the fluorescence of DCF in the HEPES solution of the nano-probe increases with the decrease of pH. It shows that the nano-probe has a good characteristic of opening the ROS generation ability to acid.

[0094] Example 4

[0095] This example relates to the promotion of gene transfection test of the acid-responsive nano-probe for tumor diagnosis and treatment prepared in Example 1

[0096] The nano-probe prepared in Example 1 was diluted to a concentration of 100 μg / mL with complete medium, and then HeLa cells were incubated in the dark for 24 h. For the non-irradiation group, incubation in the dark was continued for 24 h; for the irradiated group of cells, after irradiation with blue light (450 nm, 2 mW / cm 2 , 5 min), incubation in the dark was continued for 24 h. Finally, the intensity of the red fluorescence signal in the cells was observed under a fluorescence microscope to evaluate the transfection efficiency of the recombinant plasmid expressing the fusion of ALOX12 and mCherry, and the results are as Figure 5 shown. According to Figure 5 the figure, the red fluorescence intensity of the irradiated group of cells was significantly higher than that of the non-irradiated group, indicating that under weak light conditions, the ROS generated by the nano-probe mainly promoted the cellular uptake and lysosomal escape of the nano-probe through photochemical internalization, improving the transfection efficiency.

[0097] Example 5

[0098] This example involves the cytotoxicity test of the acid-responsive nanoprobe for tumor diagnosis and treatment prepared in Example 1

[0099] Investigation of dark toxicity: 4T1 in the logarithmic growth phase was inoculated into a 96-well plate at an inoculation amount of 5000 cells / well. After overnight incubation and adherence, the old culture medium was discarded, and different concentrations of nanoprobes diluted with complete medium were added. The concentrations of the polycationic AIE photosensitizer 1 were 2 - 32 μM respectively. After 24 h of light avoidance, the cells were washed twice with sterile PBS, and then 100 μL of freshly prepared MTT solution (0.5 mg / mL) was added to each well. After continued incubation for 3 h, the MTT solution was discarded, and 150 μL of DMSO was added to each well to completely dissolve the formazan. Finally, the absorbance value of each well solution at 570 nm was measured by a multifunctional microplate reader, and the cell survival rate was calculated. According to Figure 6 A, under the condition of no light irradiation, the viability of 4T1 cells decreased with the increase of the nanoprobe concentration. This phenomenon may be attributed to the metal polyphenol network composed of tannic acid-Fe 3+ promoting the Fenton reaction in cells, resulting in the overproduction of intracellular reactive oxygen species (ROS).

[0100] Investigation of phototoxicity: 4T1 and B16F10 in the logarithmic growth phase were inoculated into a 96-well plate at an inoculation amount of 4000 cells / well. After overnight incubation and adherence, the old culture medium was discarded, and different concentrations of nanoprobes diluted with complete medium were added. The concentrations of the polycationic AIE photosensitizer 1 were 1 - 16 μM respectively. After 24 h of light avoidance, the old culture solution was discarded, 100 μL of complete medium was added to each well, and then the plate was placed under a blue light for irradiation (450 nm, 2 mW / cm 2 , 30 min). After the irradiation ended, the cells were returned to the cell culture incubator and continued to be cultured in the dark. After 6 h, the old culture solution was discarded, and 100 μL of freshly prepared MTT solution (0.5 mg / mL) was added to each well. After continued incubation for 3 h, the MTT solution was discarded, and 150 μL of DMSO was added to each well to completely dissolve the formazan. Finally, the absorbance value of each well solution at 570 nm was measured by a multifunctional microplate reader, and the cell survival rate was calculated. According to Figure 6 B and 6C, under the condition of light irradiation, the viability of 4T1 and B16F10 cells decreased with the increase of the nanoprobe concentration. And, according to Figure 6 A and 6B, at the same nanoprobe concentration, the cytotoxicity of 4T1 cells in the light irradiation group was significantly higher than that in the non-irradiation group, indicating that the nanoprobe has good combined anti-tumor efficacy.

[0101] Example 6

[0102] This example relates to the self-reporting cell death test of the acid-responsive nanoprobe for tumor diagnosis and treatment prepared in Example 1

[0103] 4T1 cells in the logarithmic growth phase were seeded into a 12-well plate at an inoculation density of 20,000 cells / well. After overnight incubation for adhesion, the old culture medium was discarded, and the nanoprobe diluted with complete medium was added, where the concentration of the polycationic AIE photosensitizer 1 was 10 μM. After 24 h of light avoidance, the old culture solution was discarded, 300 μL of PBS was added to each well, and then it was placed under a fluorescence microscope for observation. The bright-field and fluorescence images of the cells were recorded after irradiation with blue light as the excitation light for different times (0 min or 2 min). The experimental results are as Figure 7 shown. According to Figure 7 it can be seen that without light irradiation, the cell membrane structure of 4T1 cells is intact and there is almost no fluorescence signal inside the cells; however, after 2 min of blue light irradiation under the fluorescence microscope, 4T1 cells will gradually die, and the main morphological characteristics are: the cell membrane surface swells, forms bubbles, and the volume of vesicles increases from small to large until they detach from the cell membrane. At the same time, for cells with severely damaged cell membrane structures, the fluorescence intensity of their cell nuclei shows an increasing trend. This phenomenon indicates that as the cell death process progresses, the amount of the polycationic aggregation-induced emission (AIE) photosensitizer 1 that can penetrate the nuclear membrane and specifically bind to nuclear nucleic acids increases continuously. Since the enhancement process of the fluorescence intensity of dead cell nuclei is synchronous with the cell death process, this property can be used to monitor and report the cell death process in real time, thereby making an accurate evaluation of the treatment effect.

[0104] The nanoprobe provided by the present invention utilizes the acid responsiveness, broad-spectrum absorption performance of the metal polyphenol network, and the diagnosis and treatment advantages of the polycationic AIE photosensitizer to achieve acid-responsive "switch" controllable diagnosis and treatment of tumor nanoprobes. In a neutral environment, the fluorescence is weak and the generation of ROS is less, reducing the background noise signal of the nanoprobe and the toxicity of non-target tissues; in an acidic environment (such as tumor tissue), the fluorescence is enhanced and the ROS yield is increased, improving the treatment efficiency and the accuracy of efficacy monitoring. The nano-diagnosis and treatment probe of the present invention is ingeniously designed and simply prepared, can realize the synergistic anti-tumor effect of PDT and gene therapy, in-situ and real-time report the early treatment effect of tumors, and contribute to the precise treatment of cancer.

[0105] The above-described embodiments merely represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. An acid-responsive nanoprobe, characterized in that, With the nanoparticles self-assembled from a multi-cationic AIE photosensitizer and functional nucleic acid as the core and a metal polyphenol network modified with hyaluronic acid as the shell.

2. The acid-responsive nanoprobe according to claim 1, wherein The multi-cationic AIE photosensitizer consists of a triphenylamine derivative and four cations, and its structural formula is shown in Formula I: Among them, R1 is selected from 2,2’,2”-(benzene-1,3,5-triacyl)triacetonitrile group, 3-cyanobenzyl cyanide group or 2,2’-(benzo[1,2-D:4,5-D’]bis(thiazole)-2,6-diyl)diacetonitrile group; R2 is a cation; preferably, the cation is at least one of pyridinium salt cation, quinolinium salt cation and acridinium salt cation.

3. The acid-responsive nanoprobe according to claim 1, wherein The functional nucleic acid is at least one of plasmid DNA, siRNA, peptide nucleic acid, mRNA, miRNA, ribozyme.

4. The acid-responsive nanoprobe according to claim 1, wherein The metal polyphenol network is formed by the coordination of metal ions and natural polyphenols. The metal ions are at least one of iron ions, copper ions and zinc ions; the natural polyphenols are one or more of tannic acid, quercetin and epigallocatechin gallate; Preferably, the molecular weight range of the hyaluronic acid is 1-150 kDa, and it is combined with the metal polyphenol network through non-covalent bonds such as electrostatic interaction.

5. The preparation method of the acid-responsive nanoprobe according to any one of claims 1 to 4, characterized in that, It includes the following steps: (1) Prepare the nanoparticles self-assembled from a multi-cationic AIE photosensitizer and functional nucleic acid In the aqueous solution of the multi-cationic AIE photosensitizer, add the functional nucleic acid solution, and after stirring, obtain the nanoparticles self-assembled from the multi-cationic AIE photosensitizer and the functional nucleic acid; (2) Wrap the nanoparticles in step (1) with the metal polyphenol network Under stirring, add the nanoparticles in step (1) to the aqueous solution of the natural polyphenol, then add the metal ion solution, continuously stir, centrifuge and wash to obtain an acid-responsive nano-theranostic probe without tumor targeting group modification; (3) Hyaluronic acid modification Add the aqueous solution of hyaluronic acid to the nano-theranostic probe prepared in step (2), stir, and remove the free hyaluronic acid to obtain the acid-responsive nano-probe for tumor diagnosis and treatment.

6. The preparation method of the acid-responsive nanoprobe according to claim 5, characterized in that, In step (1), the molar ratio of the functional nucleic acid to the multi-cationic AIE photosensitizer is 1:5-1:40, and the stirring time is 10-120 min; Preferably, in step (2), the molar ratio of the natural polyphenol to the metal ion is 1:2-4:1, and the mass ratio of the natural polyphenol to the nanoparticles is 5:2-6; Under the stirring rate of 500-2000 rpm, add the nanoparticles in step (1) to the aqueous solution of the natural polyphenol, then add the metal ion solution, and continuously stir for 1-6 h; The centrifugation conditions are 3-5 °C, 11000-13000 rpm, 0.5-1.5 h; Preferably, in step (3), the mass ratio of the hyaluronic acid to the nano-theranostic probe is 1:100-200; the concentration of the aqueous solution of hyaluronic acid is 0.5-3 mg / mL; the average molecular weight of the hyaluronic acid is 30-40 kD; The stirring rate is 500-2000 rpm; the stirring time is 2-8 h.

7. An anti-tumor product, characterized in that, It includes the acid-responsive nano-probe according to any one of claims 1-4; Preferably, the tumor includes melanoma, cervical cancer and breast cancer.

8. An imaging agent for early efficacy diagnosis of tumors, characterized in that, Comprising the acid-responsive nanoprobe according to any one of claims 1 to 4.

9. Use of the acid-responsive nanoprobe according to any one of claims 1 to 4 in the preparation of an anti-tumor product; preferably, the acid-responsive nanoprobe is used in combination with photodynamic therapy and / or gene therapy for anti-tumor; preferably, the tumor includes melanoma, cervical cancer and breast cancer.

10. Use of the acid-responsive nanoprobe according to any one of claims 1 to 4 in the preparation of an imaging agent for early efficacy diagnosis of tumors.

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