Ultrasound-responsive pickering emulsion and use thereof in the preparation of tumor treatment preparations

Abstract

This invention discloses an ultrasound-responsive Pickering-like emulsion and its application in the preparation of tumor therapeutic agents, relating to the field of tumor therapeutic nanomaterials. The emulsion consists of oxygen-carrying perfluorohexane droplets and copper-coordinated 1 / 4-carboxyphenyl porphyrin nanoparticles, wherein the nanoparticles act as colloidal surfactants, armoring the surface of the perfluorohexane droplets to form a Pickering-like emulsion. The preparation method includes: coordinating 1 / 4-carboxyphenyl porphyrin with copper chloride and co-assembling it with Pluronic F127 to obtain FTC NPs; then emulsifying perfluorohexane with the FTC NPs to obtain PFTC EMs, which can be further oxygen-saturated to obtain an oxygen-carrying emulsion. Under ultrasound, the emulsion undergoes responsive disintegration and simultaneously releases oxygen and FTC NPs, which can both deplete glutathione and alleviate tumor hypoxia, and promote the generation of reactive oxygen species and Cu. + Induced copper death, thereby reshaping the tumor microenvironment and enhancing the synergistic antitumor effect of sonodynamics / copper death.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials for tumor therapy, specifically to an ultrasound-responsive Pickering-like emulsion and its application in the preparation of tumor therapeutic agents. Background Technology

[0002] Sonodynamic therapy (SDT) has shown promising application prospects in the field of tumor treatment due to its advantages such as deep tissue penetration, non-invasiveness, and real-time temperature control. However, the tumor microenvironment barrier, which is prevalent in solid tumors, severely limits its therapeutic effect. In particular, high levels of glutathione and severe hypoxia weaken the effects and generation of reactive oxygen species (ROS), respectively, leading to insufficient efficacy of SDT. Meanwhile, copper death, as a novel cell death mechanism, can synergistically enhance the effects of SDT. Therefore, constructing a combined SDT and copper death therapy system is of great significance.

[0003] Currently, strategies to enhance the synergistic effect of sonodynamic therapy and copper death mainly fall into two categories: the first is to introduce copper-based components to enhance the therapeutic effect by depleting glutathione and inducing copper death; the second is to improve tumor hypoxia by using oxygen-carrying systems such as perfluorocarbons, thereby increasing the generation of reactive oxygen species. Although the former can promote glutathione depletion and copper death activation to some extent, it is still difficult to solve the dual inhibition of sonodynamic therapy and copper death by hypoxia; although the latter has oxygen supply capabilities, when using traditional processes, such as using the surfactant Pluronic F127 to directly emulsify perfluorohexane droplets to prepare PFEMs, the interfacial layer only plays a dispersing and stabilizing role, which is limited in terms of short in vivo circulation time, poor tumor accumulation capacity, and subsequent functional integration.

[0004] Therefore, there is an urgent need to develop a novel tumor treatment system that can both prolong in vivo circulation time and tumor accumulation capacity, and simultaneously achieve oxygen supply, glutathione depletion, and copper death activation under external ultrasound stimulation, thereby effectively reshaping the tumor microenvironment and enhancing the synergistic anti-tumor effect of sonodynamic therapy and copper death. The above effects have been verified in the following examples, comparative examples, and experimental cases. Summary of the Invention

[0005] Existing technologies struggle to maintain the stability of oxygen-carrying emulsions while simultaneously achieving oxygen supply, glutathione depletion, and copper death activation at the tumor site. Consequently, they fail to effectively reshape the tumor microenvironment and fully enhance the synergistic anti-tumor effect of sonodynamics / copper death. Therefore, this invention provides an ultrasound-responsive Pickering-like emulsion and its preparation method, as well as the application of this emulsion in tumor therapeutic formulations to enhance the synergistic tumor treatment effect of sonodynamics / copper death.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing an ultrasonically responsive Pickering-like emulsion, comprising the following steps: S1. Add the solution of tetra(4-carboxyphenyl)porphyrin (TCPP) dropwise to the solution of Pluronic F127, add the solution of copper chloride (CuCl2) under stirring and react at room temperature, and then purify by ultrafiltration to obtain the FTC NPs dispersion; Among them, Pluronic F127, also known as Poloxamer 407, is a triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene ABA type. S2. Perfluorohexane (PFH) was added to the FTC NPs dispersion under ice bath conditions, and after ultrasonic emulsification by probe, it was collected by centrifugation to obtain PFTC EMs emulsion. S3. The PFTC EMs emulsion is saturated with oxygen to obtain an oxygen-carrying emulsion. The emulsion is a pickerine-like emulsion that responds to ultrasound.

[0007] As a further preferred embodiment of the present invention, in step 1), the volume ratio of the neu-tetra(4-carboxyphenyl)porphyrin solution, the Pluronic F127 solution, and the copper chloride solution is 1:(1~3):(0.5~1.5), preferably 1:2:1; the final concentration of neu-tetra(4-carboxyphenyl)porphyrin in the reaction system is 250~350 μM, the final concentration of Pluronic F127 is 0.08~0.12 mM, and the concentration of copper chloride is 0.5~16 w / v%. More preferably, in step 1), the concentration of the neu-tetra(4-carboxyphenyl)porphyrin solution is 1.26 mM, the concentration of the Pluronic F127 solution is 0.2 mM; the final concentration of neu-tetra(4-carboxyphenyl)porphyrin in the reaction system is 315 μM, and the final concentration of Pluronic F127 is 0.1 mM.

[0008] As a further preferred embodiment of the present invention, the solvent used in the preparation of the 4-carboxyphenyl porphyrin solution is a polar organic solvent (such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF)) or a water-organic solvent mixture (such as water-ethanol, water-methanol or water-DMSO mixture); the solvent used in the preparation of the Pluronic F127 solution and the copper chloride solution is water.

[0009] As a further preferred technical solution of the present invention, the ultrafiltration purification is performed multiple times using an ultrafiltration tube with a molecular weight cutoff of 25~35 kDa, such as 3 times.

[0010] As a further preferred technical solution of the present invention, in step 2), the amount of perfluorohexane added is 0.1~0.3 v / v% of the volume of the FTC NPs dispersion, preferably 8~12 μL of PFH is added to 4 mL of FTC NPs dispersion; and / or, the ultrasonic emulsification is carried out under ice bath conditions for 6~12 min; and / or, the centrifugation speed is 10000~12000 rpm for 5~15 min.

[0011] As a further preferred technical solution of the present invention, in step 3), the oxygen saturation treatment time is 15~30 min, and more preferably 30 min.

[0012] According to a second aspect of the present invention, the present invention also provides an ultrasonically responsive Pickering-like emulsion, which is prepared by the above-described preparation method; the emulsion comprises an oxygen-carrying perfluorohexane droplet forming a core, and an interface layer formed by FTC NPs armoring the surface of the droplet, wherein the FTC NPs are co-assembled from neu-tetra(4-carboxyphenyl)porphyrin, copper chloride and Pluronic F127.

[0013] According to a third aspect of the invention, the invention also provides the application of an ultrasound-responsive Pickering-like emulsion in the preparation of tumor therapeutic agents. The Pickering-like emulsion simultaneously releases oxygen and FTC NPs under ultrasound, thereby achieving glutathione depletion, relief of tumor hypoxia, enhanced sonodynamic therapy, activation of copper death, and enhanced antitumor immune response.

[0014] The principle of this invention is as follows: Using tetra(4-carboxyphenyl)porphyrin coordinated with copper chloride and co-assembled with Pluronic F127, FTC NPs with both acoustic and copper-source functions are formed. These nanoparticles are then used as colloidal surfactants to emulsify PFH, constructing an oxygen-carrying emulsion with a Pickering-like structure. This structure allows the nanoparticles to partially embed at the oil-water interface, forming a relatively stable rigid interface, thereby inhibiting droplet aggregation and increasing the circulation time of the emulsion in vivo, which is beneficial for tumor accumulation. Under ultrasonic stimulation, the emulsion disintegrates and simultaneously releases oxygen and FTC NPs. The released oxygen can alleviate tumor hypoxia, and the released FTC NPs can deplete intracellular glutathione and promote Cu. + This generates, thereby amplifying both sonodynamic therapy and copper death effects, achieving a dual reshaping of the tumor microenvironment.

[0015] Compared with the prior art, the present invention can achieve the following beneficial effects: 1) This invention uses FTC NPs to armor perfluorohexane droplets to construct a Pickering-like interface, so that the resulting PFTC EMs have both oxygen-carrying and interface therapy functions, and have good colloidal stability; the experimental example 4 provided by this invention shows that the PFTC EMs have a circulating half-life of about 9.1 h in vivo, and the tumor accumulation is about 2.0 times that of traditional PF EMs.

[0016] 2) The Pickering-like emulsion of the present invention can simultaneously release oxygen and FTC NPs under the action of ultrasound, realizing the spatiotemporal coupling of oxygen supply and functional component release; the experimental example 2 provided by the present invention shows that its glutathione depletion efficiency is about 86%, the oxygen carrying capacity is about 7.29 mg / g, and the dissolved oxygen concentration of the system can be increased to about 4.9 mg / L after ultrasonic stimulation.

[0017] 3) This invention significantly enhances sonodynamic / copper death synergistic therapy through dual tumor microenvironment remodeling via glutathione depletion and hypoxia relief; Experimental Examples 3 and 5 provided by this invention demonstrate that oxygen-carrying... Under ultrasound, it exhibits enhanced reactive oxygen species generation and cell-killing capabilities, achieving a tumor inhibition rate of up to 95.2% in vivo, and can promote dendritic cell maturation and CD8+. + T-cell infiltration. Attached Figure Description

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0019] Figure 1 Characterization results of FTC NPs under different Cu concentrations with TCPP concentration fixed at 315 μM are shown, where: (a) is a transmission electron microscopy image; (b) is a polydispersity index diagram; and (c) is a mean hydrated particle size diagram. Scale bar: 100 nm.

[0020] Figure 2 The following are the characterization results of PETC EMs under different Cu concentrations with TCPP concentration fixed at 315 μM: (a) Transmission electron microscopy image; (b) Polydispersity index diagram; (c) Average hydrated particle size diagram. Scale bar: 100 nm.

[0021] Figure 3 The results show the copper loading and dispersion stability of PFTC EMs under different Cu concentrations, where: (a) is the copper loading diagram; (b) is the Tyndall effect diagram after resuspending in water and FBS.

[0022] Figure 4The surface electrical properties, spectral characteristics, and elemental distribution of PF EMs, FTC NPs, and PFTC EMs are shown, where: (a) is the Zeta potential diagram; (b) is the UV-Vis absorption spectrum; and (c) is the elemental distribution of O, N, and Cu. Scale bar: 100 nm.

[0023] Figure 5 The results of glutathione depletion, oxygen carrying capacity, oxygen release, and sonodynamic performance of PFTC EMs are shown below: (a) Comparison of glutathione depletion capacity (specific groupings are as follows: Conrol: blank group, US: ultrasound-only group, PFTC: PFTC EMs-only group, PFTC + US: PFTC EMs-only group with ultrasound stimulation); (b) Oxygen carrying capacity graph; (c) Oxygen release curve under ultrasound stimulation; (d) Absorbance of ABDA sample at 400 nm after ultrasound treatment under hypoxic conditions.

[0024] Figure 6 The results of in vitro cytotoxicity of PFTC EMs are shown, where: (a) 4T1 cells were subjected to... and Dose-dependent cytotoxicity curves and IC50 after treatment 50 Figure (b) shows the survival rate of 4T1 cells in different treatment groups under hypoxic conditions.

[0025] Figure 7 The following are the in vivo pharmacokinetic and biodistribution results of PFTC EMs, including: (a) in vivo and in vitro fluorescence imaging images; (b) semi-quantitative analysis of tumor fluorescence intensity; (c) semi-quantitative analysis of fluorescence intensity of major organs and tumors in vitro; and (d) plasma pharmacokinetic curves.

[0026] Figure 8 The results show the in vivo anti-tumor effects and immune activation, including: (a) tumor growth curve; (b) CD3+ in the tumor. + CD8 + Quantitative analysis diagram of T cells.

[0027] The objectives, features, and advantages of this invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0028] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0029] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0030] Example 1: Oxygen-carrying Preparation 1) Under magnetic stirring, 1 mL of TCPP solution (1.26 mM, DMSO solvent) was added dropwise to 2 mL of Pluronic F127 aqueous solution (0.2 mM), followed by 1 mL of CuCl2 aqueous solution with a concentration of 1 w / v%. The final concentration of TCPP in the reaction system was 315 μM, and the final concentration of Pluronic F127 was 0.1 mM. The mixture was stirred continuously overnight at room temperature to allow TCPP to coordinate with CuCl2 and co-assemble with Pluronic F127, yielding a crude product dispersion. This dispersion was then purified three times by ultrafiltration in an ultrafiltration tube with a molecular weight cutoff of 30 kDa. The purified dispersion was collected to obtain the FTC NPs dispersion.

[0031] 2) Add 10 μL PFH to 4 mL of FTC NPs dispersion pre-cooled in an ice bath, emulsify for 12 min using an ultrasonic homogenizer under ice bath conditions, then centrifuge at 10000 rpm for 10 min, collect the emulsion, and obtain PFTC EMs emulsion.

[0032] 3) PFC-TC EMs were saturated with oxygen for 30 minutes to obtain oxygen-carrying EMs. .

[0033] To further demonstrate the beneficial technical effects of the present invention, based on the preparation method of Example 1, while keeping the amounts of TCPP and Pluronic F127 constant, CuCl2 with concentrations of 0.5~16 w / v% was used (see details). Figure 1 and Figure 2 The components were assembled to investigate the effects of different copper ion concentrations on FTC NPs and subsequent emulsion properties.

[0034] First, the purified FTC NPs nanoparticles were subjected to transmission electron microscopy (Figure 1a) and dynamic light scattering (FLS) analysis. Figure 1 b and Figure 1 c) Characterization results show that as the CuCl2 concentration in the assembly system increases, the average hydrated particle size of the obtained FTC NPs increases from about 19 nm to about 110 nm.

[0035] The obtained PFTC EMs were then characterized by transmission electron microscopy (Figure 2a) and dynamic light scattering (Figure 2b and Figure 2c). The results showed that the particle size of the PFTC EMs emulsion increased from approximately 157 nm to approximately 465 nm with increasing FTC NP particle size. When the CuCl2 concentration reached 2 w / v%, the copper loading in the PFTC EMs nearly plateaued (Figure 3a), while the sample showed significant aggregation in FBS (Figure 3b). This indicates that while increasing the copper ion concentration can increase the copper ion loading within a certain range, it leads to enhanced protein adsorption capacity of the PFTC EMs and impairs serum stability. Therefore, considering the particle size, copper loading, and stability results, a CuCl2 concentration of 1 w / v% was determined to be the optimal condition.

[0036] Comparative Example 1: Preparation of Traditional PF EMs Take 4 mL of Pluronic F127 solution (0.1 mM), add 10 μL of PFH, emulsify for 12 min under the same ice bath and ultrasonic disruption conditions as in Example 1, then centrifuge at 10000 rpm for 10 min, collect the emulsion, and obtain PF EMs stabilized by conventional surfactants.

[0037] Comparative Example 1 serves as a control experiment for Example 1, used to subsequently compare the pharmacokinetics and biodistribution before and after the construction of the pickerine-like interface.

[0038] The surface electrical properties, spectral characteristics, and elemental distribution of PF EMs (Comparative Example 1), FTC NPs, and PFTC EMs (Example 1) were tested, and the results are as follows: Figure 4 As shown in Figure 4a, the Zeta potential results indicate that the surface potential of PFTC EMs is approximately -2.6 mV, significantly close to that of FTC NPs (approximately -2.4 mV), and different from that of traditional PF EMs (approximately -13.0 mV). This demonstrates that FTC NPs have been successfully distributed at the emulsion interface of PFTC EMs. UV-Vis absorption spectroscopy results (Figure 4b) show that PFTC EMs retain the characteristic Soret and Q band absorption peaks of TCPP. Simultaneously, elemental distribution results show that O, N, and Cu elements are co-located in PFTC EMs (Figure 4c), further confirming that FTC NPs have successfully armored the PFH droplet interface and formed a Pickering-like interface.

[0039] The above results indicate that the obtained product has successfully constructed a nanoparticle armor emulsion with a relatively stable interface layer, which is beneficial for inhibiting droplet aggregation and improving emulsion stability.

[0040] The product of Example 1 was further tested, as follows: Test Example 1: Stability, glutathione depletion, oxygen carrying capacity, and oxygen release performance of the product from Example 1. Using the PFTC EMs obtained in Example 1 as the test object, PFTC EMs with a copper concentration of 20 μg / mL were incubated with 1 mM glutathione at 37 °C for 30 min. The residual glutathione content was determined by the DTNB method, and a control group was set up with and without ultrasonic treatment (Figure 5a). The results showed that the glutathione depletion efficiency under ultrasonic treatment could reach about 86%, which was significantly higher than that of the emulsion group without ultrasonic treatment.

[0041] Furthermore, the oxygen-carrying and oxygen-releasing performance of the final product of Example 1 was evaluated using a dissolved oxygen detection device. Figure 5 b and Figure 5 The results show that the oxygen-carrying capacity of PFTC EMs is approximately 7.29 mg / g; after 300 s of ultrasonic stimulation, the dissolved oxygen concentration in the system can increase to approximately 4.9 mg / L. Further analysis using an ABDA probe was conducted to detect the reactive oxygen species (ROS) generation capacity. Figure 5 The 'd' in the figure indicates oxygen carrier It exhibits a stronger ability to generate reactive oxygen species under ultrasonic irradiation. The above results demonstrate that the final product of this invention can achieve oxygen supply, glutathione depletion, and acoustic sensitization under ultrasonic irradiation.

[0042] Test Example 2: In vitro antitumor effect of the product of Example 1 Using the PFTC EMs obtained in Example 1 as the test subject, 4T1 breast cancer cells were used to evaluate their in vitro biological effects. The antitumor effect of PFTC EMs was assessed by CCK-8 assay under hypoxic conditions. Figure 6 a and Figure 6 As can be seen from b in the text, for 4T1 cells, oxygen-carrying capacity... half-maximal inhibitory concentration (IC50) 50 The concentration decreased from 15.93 μg / mL in the oxygen-free group to 8.39 μg / mL. Figure 6 As shown in 'c', further comparisons were made between different treatment groups under a copper concentration of 10 μg / mL. The group has the strongest cell-killing effect.

[0043] The above results demonstrate that the final product of this invention can significantly enhance the in vitro acoustic dynamics / copper death synergistic antitumor effect under ultrasound.

[0044] Test Example 3: In vivo pharmacokinetics and biodistribution of the product from Example 1 Using the PFTCEMs obtained in Example 1 as the test subjects and the PFEMs obtained in Comparative Example 1 as the control, their in vivo pharmacokinetics and biodistribution were evaluated in a 4T1 subcutaneous tumor-bearing BALB / c mouse model. The DiR-labeled PFEMs and PFTCEMs were injected via the tail vein and their fluorescence was detected.

[0045] Depend on Figure 7 a to Figure 7 In vivo and in vitro fluorescence imaging results and quantitative analysis of c showed that the tumor accumulation of PFTC EMs was approximately 2.0 times that of conventional PF EMs. Furthermore, blood fluorescence detection indicated ( Figure 7 (d) PFTC EMs have a longer cyclic half-life of approximately 9.1 h.

[0046] Test Example 4: In vivo antitumor effect and immune activation of the product of Example 1 When the tumor volume is approximately 100 mm 3 At that time, tumor-bearing mice were randomly divided into groups and injected with the emulsion via the tail vein at a copper dose of 1.25 mg / kg. 24 h after administration, the designated groups were subjected to ultrasound irradiation (1 MHz, 1.0 W / cm²). 2 (50% duty cycle, 5 min), once every other day, for a total of 3 cycles.

[0047] Figure 8a shows that, Group 1 exhibited the most significant tumor-suppressing effect, with a tumor inhibition rate reaching 95.2%. Furthermore, Figure 8b shows that this group significantly improved dendritic cell maturation levels and promoted an increase in tumor-infiltrating T cells, particularly CD3+. + CD8 + The proportion of T cells increased from about 0.8% in the control group to about 7.2%, indicating that in addition to directly inhibiting tumors, the system can also activate anti-tumor immune responses.

[0048] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. The scope of protection of the present invention is defined only by the appended claims.

Claims

1. A method for preparing an ultrasonically responsive Pickering-like emulsion, characterized in that, Includes the following steps: S1. Add the methyl-tetra(4-carboxyphenyl)porphyrin solution dropwise to the Pluronic F127 solution, add copper chloride solution under stirring and react at room temperature, then purify by ultrafiltration to obtain the FTC NPs dispersion; S2. Perfluorohexane was added to the FTC NPs dispersion under ice bath conditions, and after ultrasonic emulsification by probe, it was collected by centrifugation to obtain PFTC EMs emulsion. S3. The PFTC EMs emulsion is saturated with oxygen to obtain an oxygen-carrying emulsion. The emulsion is a pickerine-like emulsion that responds to ultrasound.

2. The method for preparing the ultrasonically responsive Pickering-like emulsion according to claim 1, characterized in that, In step 1), the volume ratio of the methyl-tetra(4-carboxyphenyl)porphyrin solution, the Pluronic F127 solution, and the copper chloride solution is 1:(1~3):(0.5~1.5); And / or, in step 1), the final concentration of tetra(4-carboxyphenyl)porphyrin in the reaction system is 250~350 μM, the final concentration of Pluronic F127 is 0.08~0.12 mM, and the concentration of copper chloride is 0.5~16 w / v.

3. The method for preparing the ultrasonically responsive Pickering-like emulsion according to claim 1, characterized in that, The solvent used in the preparation of the methyl-tetra(4-carboxyphenyl)porphyrin solution is a polar organic solvent or a water-organic solvent mixture.

4. The method for preparing the ultrasonically responsive Pickering-like emulsion according to claim 1, characterized in that, The ultrafiltration purification process involves multiple purification steps using an ultrafiltration tube with a molecular weight cutoff of 25-35 kDa.

5. The method for preparing the ultrasonically responsive Pickering-like emulsion according to claim 1, characterized in that, In step 2), the amount of perfluorohexane added is 0.1~0.3 v / v% of the volume of the FTC NPs dispersion. And / or, the ultrasonic emulsification is performed under ice bath conditions for 6 to 12 minutes; And / or, the centrifugation speed is 10000~12000 rpm, and the time is 5~15 min.

6. The method for preparing the ultrasonically responsive Pickering-like emulsion according to claim 1, characterized in that, In step 3), the oxygen saturation treatment time is 15-30 min.

7. An ultrasound-responsive Pickering-like emulsion, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the ultrasound-responsive pickerling-like emulsion of claim 7 in the preparation of tumor therapeutic agents.

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