An ultrasound-responsive nanoprobe and its preparation method and application

Abstract

The present invention relates to an ultrasound-responsive nanoprobe and its preparation method and application. The nanoprobe has a core-shell structure, with poly(lactic-co-glycolic acid) and a photosensitizer as the outer shell, and fluorocarbon and tirapazamine as the core. The photosensitizer is a porphyrin derivative, and the fluorocarbon is a low-boiling-point liquid fluorocarbon. Compared with the prior art, the present invention designs an ultrasound-responsive nanoprobe to sensitize hypoxia-specific anti-tumor prodrugs and improve the tumor treatment effect. Among them, the photosensitizer generates ROS under ultrasound triggering while consuming oxygen in the near-vascular oxygen-rich region, thereby deteriorating the hypoxia state of the tumor microenvironment and activating hypoxia-responsive drugs. The huge mechanical force generated by the liquid-gas phase change of the low-boiling-point liquid fluorocarbon under ultrasound triggering can destroy the tumor stromal tissue, promote the penetration depth of the hypoxia-responsive drug at the tumor site, and enable the hypoxia-responsive drug to exert an anti-tumor effect on hypoxic cells far from blood vessels.

Description

Technical Field

[0001] The present invention relates to the field of biomedical nanomaterials, and particularly to an ultrasound-responsive nanoprobe and its preparation method and application. Background Art

[0002] Hypoxia-specific antitumor prodrug - tirapazamine (TPZ) is an antitumor drug with selective killing effect on hypoxic cells. Its toxicity is extremely low under aerobic conditions, while under hypoxic conditions, it can be reduced to generate a double-electron product with strong cytotoxicity.

[0003] However, the antitumor effect of TPZ alone is poor, which is related to the limited penetration depth of TPZ and its inability to act on hypoxic tumor cells far from blood vessels. The existence of dense extracellular matrix (such as collagen fibers, reticular fibers) and high tissue interstitial fluid pressure in the tumor microenvironment limits the penetration depth of nanoparticles. Most of the nano-drug delivery systems accumulate in the oxygen-rich area near blood vessels. Tumor cells within this range can be effectively exposed to various antitumor factors and killed, but in the hypoxic area outside this range, the drug concentration is lower in the area farther from blood vessels, resulting in residual tumor cells.

[0004] Therefore, there is an urgent need to find a nano-system that can consume oxygen in the area near blood vessels, sensitize the action of TPZ on non-hypoxic cells, and promote the penetration depth of the drug, so as to also play an antitumor role on hypoxic cells and CSCs far from blood vessels, thereby improving the tumor treatment effect of TPZ. Summary of the Invention

[0005] The purpose of the present invention is to provide an ultrasound-responsive nanoprobe and its preparation method and application in order to overcome at least one of the defects existing in the above-mentioned prior art. This nanoprobe can sensitize hypoxia-specific antitumor prodrugs and improve the tumor treatment effect.

[0006] The inventive concept of the present invention is as follows: As a mechanical wave, ultrasound has various biological effects, including sonodynamic effect and cavitation effect. The sonodynamic effect (SDT) of ultrasound refers to the process in which, after ultrasound irradiation, the photosensitizer in the ground state absorbs energy and jumps to the excited state, and the photosensitizer in the excited state is extremely unstable and reacts with oxygen to produce ROS. Therefore, the oxygen-dependent SDT treatment acts on the oxygen-rich area near blood vessels, and the ROS generated can directly kill tumor cells; at the same time, oxygen is consumed to cause hypoxia in this area, thereby activating TPZ to achieve a synergistic anti-tumor effect. The cavitation effect of ultrasound can be further enhanced by triggering the liquid-gas phase change of low-boiling liquid fluorocarbons - perfluoropentane (PFP). The huge mechanical shear force generated by the liquid-gas phase change of PFP can directly damage the tumor tissue stroma, and the pressure difference formed during the propagation of ultrasound in the medium can promote the generated bubbles after phase change to move towards the deep part of the tumor, further increasing the permeability of the tumor tissue stroma, thereby increasing the penetration depth of the released drug and also playing a role in the hypoxic tumor area far from blood vessels and with low perfusion. Therefore, the designer constructs an ultrasound-responsive phase-changing nanoprobe to sensitize hypoxia-specific anti-tumor prodrugs and improve the tumor treatment effect.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] One object of the present invention lies in an ultrasound-responsive nanoprobe, which has a core-shell structure, with poly(lactic-co-glycolic acid) copolymer and a photosensitizer as the shell and fluorocarbon and tirapazamine as the core.

[0009] Furthermore, the photosensitizer is a porphyrin derivative, preferably protoporphyrin; the fluorocarbon is a low-boiling liquid fluorocarbon, preferably perfluoropentane.

[0010] More specifically, this is because protoporphyrin (PpIX) consumes oxygen in the oxygen-rich area near blood vessels while generating ROS under ultrasound triggering, thereby deteriorating the hypoxic state of the tumor microenvironment and activating TPZ. This is because the huge mechanical force generated by the liquid-gas phase change of perfluoropentane under ultrasound triggering can damage the tumor stroma tissue, promote the penetration depth of TPZ at the tumor site, and enable TPZ to play an anti-tumor role on hypoxic cells far from blood vessels.

[0011] Another object of the present invention lies in a preparation method of the ultrasound-responsive nanoprobe as described above, and this preparation method includes the following steps:

[0012] Dissolve poly(lactic-co-glycolic acid) copolymer and a photosensitizer in a solvent to obtain a first mixed solution, and the solvent is preferably dichloromethane;

[0013] Dissolve tirapazamine in water, then add fluorocarbon and perform emulsification to obtain a second mixed solution;

[0014] Fully mix the first mixture and the second mixture, add polyvinyl alcohol for self-assembly to obtain a third mixture;

[0015] Separate and purify the third mixture to obtain an ultrasound-responsive nanoprobe, namely PpIX-TPZ-PFP@PLGA nano-droplets, denoted as PTP@PLGA.

[0016] More specifically, the steps of the separation and purification are as follows: add an aqueous isopropanol solution to the third mixture, stir in an ice bath for 4 - 6 h to remove dichloromethane; then remove the free photosensitizer and tirapazamine by centrifugation, and finally resuspend with deionized water, and repeat this washing process three times.

[0017] Furthermore, the emulsification method is ultrasonic emulsification.

[0018] Furthermore, the mass ratio of the poly(lactic-co-glycolic acid), photosensitizer, and tirapazamine is 10:(0.75 - 1.25):(1.5 - 2.5), preferably 10:1:2.

[0019] The third object of the present invention lies in the application of an ultrasound-responsive nanoprobe as described above, and this nanoprobe is used as an anti-tumor prodrug for the precise treatment of hypoxic tumor cells.

[0020] Furthermore, under 5 cycles of ultrasonic irradiation, at least 60% of TPZ is released from the nanoprobe. In some embodiments of the present invention, under 5 cycles of ultrasonic irradiation, 61% of TPZ is released, while only about 20% is released without ultrasonic irradiation.

[0021] Furthermore, the photosensitizer in the nanoprobe consumes oxygen and generates reactive oxygen species under ultrasonic irradiation. In some embodiments of the present invention, in vivo and in vitro experiments both prove that PTP@PLGA nano-droplets consume oxygen and generate reactive oxygen species under ultrasonic irradiation, directly killing tumor cells while deteriorating the hypoxic microenvironment, thereby sensitizing the effect of TPZ.

[0022] Furthermore, after ultrasonic irradiation, the tumor tissue penetration depth of tirapazamine is increased by the nanoprobe. In some embodiments of the present invention, the red fluorescence of PTP@PLGA nano-droplets is enhanced and the distribution range becomes wider, proving that the sono-induced phase transition effect of ultrasound combined with PFP can not only promote drug release but also promote the drug penetration depth, acting on more hypoxic cells far from blood vessels.

[0023] Furthermore, after the nano-probe is irradiated by ultrasound, the number of collagen fibers and reticular fibers in the tumor tissue decreases, and the hardness of the tumor tissue decreases. In some embodiments of the present invention, the Gordon-Sweets and Masson staining results also show that only in the PTP@PLGA+US group, the number of collagen fibers and reticular fibers decreases, and the structural integrity is damaged, which is beneficial to the penetration of nano-droplets.

[0024] Compared with the prior art, the present invention designs an ultrasound-responsive nano-probe to sensitize hypoxia-specific anti-tumor prodrugs and improve the tumor treatment effect. Among them, the photosensitizer generates ROS under ultrasound triggering while consuming oxygen in the near-vascular oxygen-rich region, thereby worsening the hypoxia state of the tumor microenvironment and activating hypoxia-responsive drugs. The huge mechanical force generated by the liquid-gas phase change of low-boiling-point liquid fluorocarbon under ultrasound triggering can damage the tumor stromal tissue, promote the penetration depth of hypoxia-responsive drugs in the tumor site, and enable hypoxia-responsive drugs to exert anti-tumor effects on hypoxic cells far from blood vessels. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the transmission electron microscope image of PTP@PLGA nano-droplets in the example;

[0026] Figure 2 It is the particle size distribution diagram of PTP@PLGA nano-droplets in the example;

[0027] Figure 3 It is the PpIX standard curve (Figure A) and TPZ standard curve (Figure B) in the example;

[0028] Figure 4 It is the TPZ release curve of PTP@PLGA (Figure A) and PTP@PLGA+US group (Figure B) in the example;

[0029] Figure 5 It is the reactive oxygen species production of PTP@PLGA nano-droplets under irradiation with different ultrasound irradiation times (Figure A) and different ultrasound powers (Figure B);

[0030] Figure 6 It is the penetration depth of PTP@PLGA nano-droplets in tumor tissue under the conditions of no ultrasound irradiation and with ultrasound irradiation;

[0031] Figure 7 It is the SWE images of tumors of tumor-bearing mice in the control group, US group and PTP@PLGA+US group before and after ultrasound irradiation;

[0032] Figure 8 It is the expression of tumor collagen fibers and reticular fibers in the tumor tissues of the control group, US group, PTP@PLGA and PTP@PLGA+US groups. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0034] An ultrasound-responsive nanoprobe, which has a core-shell structure, with poly(lactic-co-glycolic acid) and a photosensitizer as the shell, and fluorocarbon and hypoxia-responsive drug as the core. The photosensitizer is a porphyrin derivative. The fluorocarbon is a low-boiling-point liquid fluorocarbon. The hypoxia-responsive drug is tirapazamine.

[0035] Example An Ultrasound-Responsive Nanoprobe and Its Preparation Method and Application

[0036] 1. Preparation of the Ultrasound-Responsive Nanoprobe

[0037] Weigh 10 mg of poly(lactic-co-glycolic acid) (PLGA) and 1 mg of protoporphyrin (PpIX), and dissolve them in 2 mL of dichloromethane to obtain a first mixture.

[0038] Take 2 mg of tirapazamine (TPZ) and dissolve it in 200 μg of deionized water, and then ultrasonically emulsify it with 200 μL of perfluoropentane to obtain a second mixture.

[0039] Mix the first mixture and the second mixture, add 10 mL of 4% (w / v) polyvinyl alcohol solution, and emulsify again; then add 10 mL of 2% (w / v) isopropyl alcohol aqueous solution, and stir in an ice bath for 4 - 6 hours to remove dichloromethane.

[0040] Finally, the obtained emulsion is centrifuged (10000 rpm, 5 min) to remove free protoporphyrin and tirapazamine, and then resuspended with deionized water, and this process is repeated and washed three times to obtain the ultrasound-responsive nanoprobe, that is, PpIX-TPZ-PFP@PLGA nanodroplets, denoted as PTP@PLGA.

[0041] 2. Basic Characterization of PTP@PLGA Nanodroplets

[0042] Dilute PTP@PLGA by a certain multiple, and use a Malvern nanoparticle size and zeta potential analyzer to detect the particle size distribution and potential, use a high-resolution transmission electron microscope to detect the morphology and internal structure, use an ultraviolet-visible spectrophotometer to detect the absorption spectrum, and measure the standard curves of PpIX and TPZ, so as to calculate the encapsulation efficiency and drug loading amount. The calculation formulas are as follows:

[0043] Encapsulation efficiency (%) = (mass of total TPZ - mass of free TPZ) / mass of total TPZ;

[0044] Drug loading (%) = (mass of total TPZ - mass of free TPZ) / mass of total PTP@PLGA nano-droplets.

[0045] Figure 1 Figure shows the transmission electron microscopy (TEM) image of PTP@PLGA nano-droplets. It can be seen that the PTP@PLGA nano-droplets are uniform spheres. Figure 2 Figure

[0045] shows the particle size distribution of PTP@PLGA nano-droplets. It can be seen that the hydrodynamic diameter of PTP@PLGA nano-droplets is about 302 ± 88.06 nm. Through the PpIX standard curve ( Figure 3 A) and the TPZ standard curve ( Figure 3 B), the encapsulation efficiency and drug loading of TPZ and PpIX are calculated to be 28.3%, 4.9 wt% and 92.5%, 8 wt%, respectively.

[0046] 3. Release study of TPZ from PTP@PLGA nano-droplets

[0047] 1 mL of PTP@PLGA nano-droplets with a concentration of 10 mg / mL was placed in a dialysis bag with a molecular weight cut-off of 1000 Da, and 50 mL of phosphate buffer solution (PBS) was used as the release medium. The mixture was shaken in the dark at 37 °C and 100 rpm. It was divided into two groups: the PTP@PLGA group and the PTP@PLGA + US (ultrasound) group. In the PTP@PLGA + US group, periodic ultrasound irradiation was given, that is, ultrasound irradiation was given for 5 min every hour with a power of 1 W / cm 2 . 1 mL of the release medium was taken out before and after ultrasound irradiation at four time points: 0, 60, 120, and 180 min to measure the concentration of TPZ. In the PTP@PLGA group, no ultrasound irradiation was given, and the TPZ concentration was measured at four time points: 0, 60, 120, and 180 min.

[0048] The boiling point of PFP is 29 °C. Under ultrasound irradiation, liquid-vapor phase change occurs, generating a large mechanical shear force, which is called acoustic droplet vaporization (ADV). This mechanical force can disrupt the nano-droplets and promote the release of TPZ. Under 5 cycles of periodic ultrasound irradiation, 61% of TPZ was released ( Figure 4 B), while only about 20% was released without ultrasound irradiation ( Figure 4 A).

[0049] 4. In vitro sonodynamic study of PTP@PLGA nano-droplets

[0050] PTP@PLGA nano-droplets were prepared at a certain concentration according to the PpIX concentration, and different ultrasound powers (0.5, 1, 1.5, 2 W / cm 2 , 60 s) and different ultrasound irradiation times (1 W / cm 2, 0, 30, 60, and 120 s). The production of reactive oxygen species was detected using DPBF (10 μL, 10 mM).

[0051] DPB is a singlet oxygen indicator fluorescent probe that can specifically bind to singlet oxygen, and the generated product reduces the ultraviolet absorption peak at 410 nm. It can be seen from Figure 5 that under ultrasonic irradiation, with the increase of ultrasonic power and the prolongation of ultrasonic irradiation time, the peak at 410 nm of PTP@PLGA nanodroplets gradually decreases, indicating that the encapsulated PpIX can generate reactive oxygen species under ultrasonic irradiation. The generation of reactive oxygen species requires the consumption of oxygen. By checking the oxygen concentration in the solution with a dissolved oxygen meter, it is found that with the increase of ultrasonic power and the prolongation of ultrasonic irradiation time, the oxygen concentration gradually decreases. It is proved that PTP@PLGA nanodroplets consume oxygen to generate reactive oxygen species under ultrasonic irradiation, directly killing tumor cells while deteriorating the hypoxic microenvironment, thus sensitizing the effect of TPZ.

[0052] 5. Study on the penetration depth of PTP@PLGA nanodroplets

[0053] Six tumor-bearing mice were randomly divided into two groups: the PTP@PLGA group and the PTP@PLGA+US group. PTP@PLGA was intravenously injected, and ultrasonic irradiation (1 W / cm 2 2, 10 min) was given 5 minutes later. After 24 hours, tumor tissues were taken for CD31 staining to label blood vessels, and the distribution of PTP@PLGA nanodroplets was observed.

[0054] Figure 6 shows the penetration depth of PTP@PLGA nanodroplets in tumor tissues under the conditions of no ultrasonic irradiation and with ultrasonic irradiation (blue is the cell nucleus, green is the blood vessel, and red is the PTP@PLGA nanodroplet). By staining blood vessels with CD31, the distribution of PTP@PLGA nanodroplets was observed. For the PTP@PLGA group, it can be observed that the PTP@PLGA nanodroplets with self-emitted red fluorescence are mainly distributed along the blood vessels marked by green fluorescence, and the penetration depth is relatively shallow. After ultrasonic irradiation, the integrity of the blood vessels is damaged, the red fluorescence of the PTP@PLGA nanodroplets is enhanced and the distribution range becomes wider, indicating that the sono-induced phase transition effect of ultrasound combined with PFP can not only promote the release of drugs, but also promote the penetration depth of drugs and act on more hypoxic cells far from blood vessels.

[0055] 6. Study on the effect of PTP@PLGA nanodroplets on the extracellular matrix of tumor cells

[0056] Nine tumor-bearing mice were randomly divided into three groups: the control group, the US group, and the PTP@PLGA+US group. PTP@PLGA was intravenously injected, and ultrasonic irradiation (1 W / cm 2, 10 min), and the treatment was repeated 3 times. Before and after ultrasonic irradiation, the change in the hardness of the mouse tumor was observed using the real-time ultrasonic shear wave elastography (SWE) mode. After 24 hours, tumor tissues were taken for Gordon-Sweets and Masson staining to label collagen fibers and reticular fibers respectively.

[0057] By observing the changes in the hardness of tumor tissues and the structures of collagen fibers and reticular fibers before and after ultrasonic irradiation in each group. As Figure 7 shown, in the SWE imaging, the color represents the magnitude of hardness, and the redder the color, the higher the hardness. The results showed that only in the PTP@PLGA + ultrasound group, the hardness decreased after ultrasonic irradiation. This may be related to the cavitation effect of ultrasound destroying the interstitial tissue, reducing the interstitial fluid pressure, and the decrease in tension after tumor cell death. The results of Gordon-Sweets and Masson staining also showed that only in the PTP@PLGA + US group, the number of collagen fibers and reticular fibers decreased, and the structural integrity was damaged, which was beneficial to the penetration of nano-droplets ( Figure 8 ).

[0058] In summary, the present invention designs an ultrasound-responsive nano-probe to sensitize hypoxia-specific anti-tumor prodrugs and improve the tumor treatment effect.

[0059] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. An ultrasound-responsive nanoprobe, characterized in that, The nano-probe has a core-shell structure, with poly(lactic-co-glycolic acid) and a sonosensitizer as the shell and fluorocarbon and tirapazamine as the core; The sonosensitizer is a porphyrin derivative; the fluorocarbon is a low-boiling-point liquid fluorocarbon; The mass ratio of poly(lactic-co-glycolic acid), sonosensitizer and tirapazamine is 10:(0.75 - 1.25):(1.5 - 2.5); The preparation method of the ultrasound-responsive nano-probe comprises the following steps: Dissolve poly(lactic-co-glycolic acid) and the sonosensitizer in a solvent to obtain a first mixed solution; Dissolve tirapazamine in water, add fluorocarbon and perform emulsification to obtain a second mixed solution; Fully mix the first mixed solution and the second mixed solution, add polyvinyl alcohol for self-assembly to obtain a third mixed solution; Separate and purify the third mixed solution to obtain the ultrasound-responsive nano-probe.

2. The ultrasound-responsive nanoprobe according to claim 1, wherein The emulsification method is ultrasonic emulsification.

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