An iRGD-targeted ultrasound-responsive phase-transition liposome as well as a preparation method and application thereof

Abstract

The present application relates to an iRGD-targeted ultrasound-responsive phase transition liposome and a preparation method and application thereof, comprising: a phospholipid bilayer membrane; a surface modifier, which is embedded in the phospholipid bilayer membrane with a hydrophobic end and carries an iRGD peptide segment exposed on the surface of the liposome with a hydrophilic end; an inner aqueous phase, which is contained in the inner cavity formed by the phospholipid bilayer membrane and co-loaded with doxorubicin, catalase and liquid perfluoropentane; and the liquid perfluoropentane is dispersed in the form of droplets in the inner aqueous phase or adsorbed / attached to the inner surface of the phospholipid bilayer membrane. The present application organically integrates four functions of "targeted delivery", "phase transition imaging", "drug burst release" and "hypoxia improvement" through the triggering of LIFU. iRGD is responsible for accurately transporting drugs to tumor sites; LIFU triggers PFP phase transition, which on the one hand generates an ultrasound signal for imaging, and on the other hand physically destroys the liposome membrane to promote DOX release; the released CAT improves hypoxia, further amplifying the cytotoxicity of DOX.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an iRGD-targeted ultrasound-responsive phase change liposome, its preparation method, and its application. Background Technology

[0002] Laryngeal cancer is one of the most common malignant tumors of the head and neck. In addition to threatening the patient's survival, its treatment often affects functions such as speech and swallowing, significantly impacting quality of life. Chemotherapy, represented by doxorubicin, remains an important component of comprehensive treatment for laryngeal cancer. However, systemic chemotherapy has common limitations common to solid tumors, such as poor tumor selectivity, strong cytotoxicity, and insufficient effective drug concentration within the lesion.

[0003] In recent years, targeted chemotherapy based on nanocarriers has been extensively studied. Systems such as liposomes and polymer micelles can improve the dispersibility of poorly soluble drugs, prolong circulation time, and increase tumor deposition through the EPR effect. However, traditional nanocarriers still face multiple obstacles: passive enrichment alone cannot guarantee penetration across vascular endothelium and deep tumor layers; premature drug leakage during circulation reduces the amount of active drug within the tumor; after entering the tumor stroma, cell internalization, intracellular release, and subcellular target accessibility are still insufficient; most carriers only have transport functions and lack in vivo real-time tracking and site activation capabilities.

[0004] Ultrasound, due to its non-invasive nature, deep tissue penetration, real-time imaging capabilities, and ability to serve as an external trigger, is used as an external stimulation method for controlled drug release. Low-boiling-point fluorocarbon liquids such as perfluoropentane (PFP) undergo a liquid-gas phase transition and generate microbubbles under ultrasound irradiation. This physical phenomenon has been disclosed for enhancing ultrasound contrast or assisting drug release. For example, Chinese invention patent application CN202210280294.2 (publication number CN114699541A) discloses a PFP-containing silicone lipid nanoemulsion for ultrasound imaging; Chinese invention patent application CN201510466249.6 (publication number CN105106977A) discloses cationic ultrasound microbubbles carrying iRGD membrane-penetrating peptides, using iRGD to target integrins and combining ultrasound-mediated gene delivery. The above disclosures respectively address the acoustic response core or iRGD surface modification, but do not locate the PFP phase transition core in the aqueous phase of the liposome and couple it with the drug-carrying space.

[0005] Regarding active targeted modification, the mechanism by which iRGD peptides are anchored to the carrier surface via PEG-phospholipids, first binding to αvβ3 and then exposing the CendR motif through protease cleavage to promote penetration has been disclosed. For example, Chinese invention patent application CN202311016741.4 (publication number CN117017918A) discloses the preparation of a DSPE-PEG2000-iRGD conjugate and membrane-fused nanoliposomes for the regulation of the tumor immune microenvironment. In terms of microenvironment regulation, the decomposition of intratumoral H2O2 by catalase (CAT) to produce oxygen and alleviate hypoxia has also been disclosed. Chinese invention patent application CN202110628617.8 (publication number CN115702881A) discloses a liposome simultaneously encapsulating doxorubicin and catalase, which releases DOX in response to H2O2 stimulation, but does not involve ultrasound triggering, iRGD penetration, or the PFP phase transition nucleus.

[0006] Traditional drug-loaded liposomes, such as PEGylated doxorubicin liposomes, can reduce cardiotoxicity to some extent through long circulation, but they still generally have the following problems in solid tumors such as laryngeal cancer: lack of active penetrating peptide modification leading to shallow matrix penetration; lack of CAT integration leading to hypoxia and unresolved chemotherapy resistance; release dependent on membrane leakage or single stimulation, without synergistic controlled release from exogenous ultrasound and endogenous acidic microenvironment; and spatial separation of PFP-type acoustic core from the drug-loaded aqueous phase leading to phase transition-drug release decoupling.

[0007] In summary, no publicly available studies have disclosed liposome delivery methods for laryngeal cancer that utilize iRGD membrane embedding targeting, PFP phase-change nuclei placed in the inner aqueous phase or on the inner side of the membrane, DOX and CAT co-loaded in the same intracavitary aqueous phase, and synergistic ultrasound-triggered acidic microenvironment. Furthermore, the publicly known disclosures of a single element do not provide a clear explanation of how to achieve this. "Technological insights from chain-like links." Summary of the Invention

[0008] The first technical problem to be solved by the present invention is to provide an iRGD-targeted ultrasound-responsive phase change liposome that is iRGD-embedded and targeted, with an internal aqueous phase co-loaded with doxorubicin, catalase, and liquid perfluoropentane, in order to achieve targeted accumulation at the laryngeal cancer site, ultrasound-triggered drug release, and synergistic regulation of the hypoxic microenvironment.

[0009] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned iRGD-targeted ultrasound-responsive phase change liposomes, in view of the current state of the technology.

[0010] The third technical problem to be solved by the present invention is to provide an application of the above-mentioned iRGD-targeted ultrasound-responsive phase change liposomes in light of the current state of the technology.

[0011] The technical solution adopted by the present invention to solve the first technical problem mentioned above is: an iRGD-targeted ultrasound-responsive phase change liposome, characterized in that it comprises:

[0012] Phospholipid bilayer membrane;

[0013] Surface modification, for Its hydrophobic end is embedded in the phospholipid bilayer membrane, while its hydrophilic end carries the iRGD peptide and is exposed on the liposome surface;

[0014] An inner aqueous phase is contained within the cavity encapsulated by the phospholipid bilayer membrane and is loaded with doxorubicin, catalase, and liquid perfluoropentane. The liquid perfluoropentane is dispersed in the inner aqueous phase in the form of droplets or adsorbed / attached to the inner surface of the phospholipid bilayer membrane.

[0015] Furthermore, the phospholipid bilayer membrane is composed of dipalmitoylphosphatidylcholine and cholesterol, and the dipalmitoylphosphatidylcholine, cholesterol, and... The molar ratio is 65:30:1.

[0016] Furthermore, the average hydrated particle size of the ultrasound-responsive phase change liposomes is 236.10±9.28 nm, the polydispersity index is 0.21±0.03, and the zeta potential is −31.42±0.69 mV. This particle size range (<250 nm) meets the ideal scale for tumor EPR effect, which is beneficial for long-term retention in tumor tissue and avoids capillary interception caused by excessively large particle size. At the same time, the negative potential effectively inhibits particle aggregation, enabling the liposomes to be stably preserved for more than 7 days in DMEM medium containing 10% FBS and PBS, which is significantly better than unmodified traditional liposomes.

[0017] The technical solution adopted to further solve the second technical problem mentioned above is: a method for preparing iRGD-targeted ultrasound-responsive phase change liposomes as described above, characterized by comprising the following steps:

[0018] (1) Phospholipids, cholesterol and It dissolves in an organic solvent, and the solvent is removed by rotary evaporation to form a lipid film;

[0019] (2) Add buffer solution to hydrate the lipid membrane to obtain a lipid suspension;

[0020] (3) Mix doxorubicin solution, catalase solution and perfluoropentane, and perform pre-emulsification treatment to obtain a pre-emulsified solution;

[0021] (4) Add the pre-emulsion to the lipid suspension and perform ultrasonic emulsification to obtain the initial product;

[0022] (5) Centrifuge and wash to remove unencapsulated material, collect the precipitate and resuspend it to obtain the iRGD-targeted ultrasound-responsive phase change liposomes.

[0023] Furthermore, the power of the pre-emulsification process in step (3) is 100W, using pulse mode, with a working time to intermittent time ratio of 1:1 and a total duration of 3 minutes;

[0024] The ultrasonic emulsification process in step (4) has a power of 125W, uses pulse mode, has a working time to intermittent time ratio of 1:1, and a total duration of 5min.

[0025] Furthermore, the organic solvent in step (1) is chloroform;

[0026] The buffer solution in step (2) is a phosphate buffer solution with a pH of 7.4;

[0027] The centrifugation in step (5) is performed at a speed of 8000 rpm for 5 minutes.

[0028] The technical solution adopted to further solve the third technical problem mentioned above is: the application of iRGD-targeted ultrasound-responsive phase change liposomes as described above in the preparation of combination therapy drugs for tumors.

[0029] Furthermore, the combined treatment is an ultrasound-triggered combined treatment, in which the ultrasound-responsive phase change liposomes undergo a liquid-gas phase change under ultrasound irradiation, achieving a sudden release of the drug. At the same time, catalase catalyzes the decomposition of hydrogen peroxide in the tumor microenvironment to generate oxygen, thereby alleviating tumor hypoxia and enhancing the therapeutic effect.

[0030] Furthermore, the tumor is a solid carcinoma, specifically laryngeal cancer.

[0031] Furthermore, the ultrasonic irradiation is low-intensity focused ultrasound irradiation with a frequency of 1.0 MHz and a sound intensity of 2.5. .

[0032] Compared with the prior art, the advantages of the present invention are: the iRGD-targeted ultrasound-responsive phase change liposomes of the present invention are modified on their phospholipid bilayer membrane. The phospholipid bilayer membrane is loaded with doxorubicin and catalase in its aqueous phase, while liquid perfluoropentane is dispersed in the aqueous phase in the form of droplets or adsorbed / attached to the inner surface of the membrane.

[0033] in, Embedded modification allows liposomes to first target laryngeal cancer blood vessels via integrin αvβ3, followed by protease cleavage to expose the CendR motif, achieving deep tissue penetration and cellular uptake. This overcomes the shortcomings of traditional carriers, which only passively accumulate and have shallow penetration. The triple targeting synergistically enhances intratumoral accumulation and penetration. Furthermore, the embedded liquid perfluoropentane undergoes a liquid-gas phase transition under ultrasound irradiation, generating microbubbles that enhance cavitation and promote membrane perturbation for drug release. Simultaneously, the weakly acidic tumor microenvironment assists in drug release within the aqueous phase. Exogenous ultrasound triggering and endogenous acidic microenvironment triggering are complementary, avoiding the instability of single triggering. Furthermore, CAT and DOX are co-encapsulated in the same aqueous phase, allowing CAT to catalyze in situ upon reaching the tumor. The decomposition process generates oxygen, alleviating tumor hypoxia and enhancing DOX chemosensitivity. Both are delivered and take effect simultaneously, avoiding temporal misalignment of compartmentalized loading. Furthermore, the phase transition occurs in the region adjacent to the drug-loaded lumen, and the microbubble expansion directly disturbs the phospholipid bilayer membrane, improving the instantaneous release efficiency of DOX. At the same time, the phase transition microbubbles enhance the ultrasound contrast signal.

[0034] In summary, this invention organically integrates four major functions—targeted delivery, phase transition imaging, drug burst release, and hypoxia improvement—through LIFU triggering. Specifically, iRGD is responsible for precisely delivering drugs to the tumor site; LIFU triggers the PFP phase transition, generating ultrasound signals for imaging and physically disrupting liposome membranes to promote DOX release; the released CAT improves hypoxia, further amplifying the cytotoxicity of DOX. This spatiotemporally controllable synergistic mechanism provides a novel "therapeutic integration" strategy to address the challenge of ineffective traditional chemotherapy for laryngeal cancer. Attached Figure Description

[0035] Figure 1 For the present invention Physicochemical characterization diagram: Figure 1 a is and Particle size distribution diagram; Figure 1 b is and Zeta potential diagram; Figure 1 c is Particle size stability curves in PBS and serum-containing DMEM over 7 days; Figure 1 d is and Comparison chart of DOX drug loading in China;

[0036] Figure 2 For the present invention In vitro ultrasound phase transition and drug release behavior diagram: Figure 2 a is Optical microscope images before and after LIFU irradiation; Figure 2 b represents the two-dimensional grayscale pattern and contrast-enhanced image (CEUS) during LIFU irradiation; Figure 2 c is a quantitative graph of CEUS signal intensity under different irradiation times; Figure 2 d represents the presence or absence of LIFU at pH 5.0. and The cumulative release curve of DOX; Figure 2 e represents the presence or absence of LIFU at pH 7.4. and The cumulative release curve of DOX;

[0037] Figure 3 Figures related to cell experiments: Figure 3 a represents the effect of TU686 cells on free DOX, and Intracellular uptake CLSM images and quantitative fluorescence collection analysis (scale bar = 100 μm); Figure 3 b is CLSM images of the carrier (FITC channel), DOX (red), and cell nucleus (DAPI) with and without LIFU irradiation after cell treatment;

[0038] Figure 4 Figure for in vitro cytotoxicity evaluation: Figure 4 a is a bar chart showing the survival rate of TU686 cells under different concentrations and with or without LIFU; Figure 4 b is and The dose-response curve and IC50 value; Figure 4 c is Live and dead double-stained CLSM images (scale bar = 100 μm);

[0039] Figure 5 In vivo ultrasound imaging: Figure 5 a is a two-dimensional grayscale and CEUS image of the tumor area in tumor-bearing mice after tail vein administration (white dashed circles represent tumor areas). Figure 5 b is a quantitative analysis diagram of ultrasound signal intensity in the tumor region;

[0040] Figure 6 Evaluation chart for improvement in tumor hypoxia: Figure 6 a represents tumor slices from different treatment groups treated with pipemnidazole (hypoxia probe) and... Immunofluorescence image (green indicates hypoxic regions, red indicates hypoxia-related protein expression, blue represents cell nuclei, scale bar = 100 μm); Figure 6 b is the quantitative fluorescence intensity diagram of pipemnidazole; Figure 6 c is Quantitative fluorescence intensity graph;

[0041] Figure 7 In vivo anti-tumor efficacy evaluation chart: Figure 7 a is a schematic diagram of an animal experimental treatment plan; Figure 7 b shows photos of mice in each group at days 0, 9, and 18. Figure 7 c represents the mouse body weight change curve during treatment; Figure 7 d represents the tumor growth curve;

[0042] Figure 8 Immunohistochemical staining image of PCNA in tumor tissue on day 18 (scale bar = 100 μm).

[0043] Figure 9 For TU686 cells and CLSM images after 1h, 2h, and 4h of incubation (blue represents cell nuclei, red represents DOX fluorescence, scale bar = 100μm).

[0044] Figure 10 for Cells treated with different concentrations ± LIFU Staining image (green indicates positive calcium alginate-AM surviving cells, red indicates positive PI dead cells, scale bar = 100 μm);

[0045] Figure 11 for Different concentrations Cells after treatment Staining image (green indicates positive calcium alginate-AM surviving cells, red indicates positive PI dead cells, scale bar = 100 μm);

[0046] Figure 12 Photos of tumor specimens removed from mice in each group on day 18;

[0047] Figure 13 Semi-quantitative bar chart of PCNA-positive area ratio in tumor sections;

[0048] Figure 14 for A schematic diagram illustrating ultrasound-guided chemotherapy and hypoxia regulation for laryngeal cancer. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0050] The materials and pharmaceuticals used in each embodiment are as follows:

[0051] Dipalmitoylphosphatidylcholine (DPPC) and Purchased from Avanti (USA);

[0052] Cholesterol was purchased from Xi'an Ruixi Biotechnology Co., Ltd. (Xi'an, China);

[0053] (Distearylphosphatidylethanolamine-polyethylene glycol 2000-iRGD coupling) was purchased from Guangzhou Carbon Technology Co., Ltd. (Guangzhou, China);

[0054] DOX (98%) and CAT (3500U / mg) were purchased from Shanghai Maclean Biotech Co., Ltd. (Shanghai, China).

[0055] (DAPI) was purchased from Beyotime Biotechnology (Shanghai, China);

[0056] (PI) was purchased from Wuhan Baiqiandu Biotechnology Co., Ltd. (Wuhan, China);

[0057] RPMI-1640 medium, fetal bovine serum (FBS) and Purchased from Wuhan Saiweier Biotechnology Co., Ltd. (Wuhan, China);

[0058] Anti-hypoxia inducible factor-1α ( Rabbit polyclonal antibody and proliferating cell nuclear antigen (PCNA) antibody (goat anti-rabbit) were purchased from Beijing Bio-Antibody Co., Ltd. (Beijing, China).

[0059] The Hypoxyprobe Plus Kit was purchased from Shanghai Mokang Biotechnology Co., Ltd. (Shanghai, China).

[0060] Perfluoropentane (PFP, 98% purity) was purchased from Shanghai Haohong Biomedical Technology Co., Ltd. (Shanghai, China).

[0061] Human laryngeal carcinoma cell line TU686 was purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM supplemented with 10% (v / v) FBS, 100 U / mL penicillin, and 100 U / mL streptomycin. Cells were cultured in a humidified incubator at 37°C with 5% CO2.

[0062] Specific pathogen-free (SPF) male BALB / c nude mice (BALB / c-nu, 5-8 weeks old) were purchased from Guangdong Provincial Laboratory Animal Center (Guangzhou, China); all animal procedures were approved by the Ethics Committee of Guangxi Zhuang Autonomous Region People's Hospital (Approval No.: Mice were anesthetized with isoflurane / O2 (4-5% induction, 0.5-1.5% maintenance) for all in vivo procedures, following institutional guidelines for laboratory animal care and use. At the end of the experiment, animals were euthanized by CO2 inhalation, followed by cervical dislocation to confirm death, in accordance with the American Veterinary Medical Association (AVMA) guidelines for animal euthanasia.

[0063] Statistical analysis:

[0064] Data are expressed as mean ± standard deviation (SD). Independent samples t-tests were used to analyze differences between two groups, and one-way ANOVA was used to analyze differences among multiple groups. A p-value < 0.05 was considered statistically significant. Statistical differences are expressed as follows: .

[0065] Example 1: Preparation and physicochemical characterization

[0066] DPPC, cholesterol (Chol) and Dissolve in 5 L of chloroform at a molar ratio of 65:30:1. Evaporate at 42 °C and 100 rpm for 2 h to remove the organic solvent and form a uniform lipid film on the flask wall.

[0067] The membrane was hydrated with 5 mL of PBS (pH 7.4) to obtain a lipid suspension. Separately, doxorubicin solution (DOX, 200 μL, 10 mg / mL), catalase solution (CAT, 200 μL, 5 mg / mL), and perfluoropentane (PFP, 200 μL) were mixed and pre-emulsified using a probe via ultrasonication (100 W, 3 min, 5 s on / 5 s off).

[0068] The pre-emulsion was added to the above lipid suspension, and sonication was continued at 125W for 5 minutes (5 seconds on, 5 seconds off) to obtain... The dispersion (initial product) was then centrifuged at 8000 rpm for 5 min, washed three times with deionized water to remove unencapsulated drug and free PFP, and the final formulation was stored at 4°C for later use.

[0069] The control formulations include: (1) (Without PFP); (2) (Without CAT); (3) (by Alternative ).

[0070] The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the liposomes were determined using a Malvern Zetasizer Nano ZS particle size analyzer (Malvern, USA) at 25°C via dynamic light scattering (DLS) technology. The colloidal stability of the nanoparticles in PBS (0.01 M, pH 7.4) and DMEM medium containing 10% fetal bovine serum (FBS) was evaluated over 7 days. DOX content was quantified by absorbance at 480 nm using a microplate reader (Lambda25, PerkinElmer, USA). Drug loading (DL, %) was calculated using the following formula:

[0071]

[0072] in, This indicates the amount of DO loaded into the nanoparticles. This indicates the total amount of nanoparticles.

[0073] Dynamic light scattering (DLS) results show that The average hydrated particle size was 236.10 ± 9.28 nm, the polydispersity index (PDI) was 0.21 ± 0.03, and the zeta potential was −31.42 ± 0.69 mV. Figure 1 a, Figure 1 b). Control formulation The particle size was 223.05±6.09 nm, the PDI was 0.17±0.03, and the Zeta potential was −30.22±0.06 mV, indicating that the introduction of PFP did not significantly change the basic colloidal properties of liposomes, but led to a slight increase in particle size and dispersion.

[0074] Stability tests showed that the nanoparticle system exhibited no significant change in particle size after 7 days of storage in PBS (pH 7.4) and DMEM containing 10% fetal bovine serum. Figure 1 c) It exhibits good colloidal stability. The DOX drug loading was 4.95% ± 0.98%, lower than that of the group without PFP (7.35% ± 0.55%). Figure 1 d) It is speculated that the competition for internal loading space with PFP and the influence of the ultrasonic emulsification process on liposome structure are related.

[0075] The invention obtained The particle size is in the <250 nm range, which is beneficial for tumor accumulation and transport. Simultaneously, polyethylene glycolation further reduces uptake by the mononuclear phagocyte system by increasing surface hydrophilicity and providing spatial shielding, thereby prolonging in vivo circulation time. Furthermore, the zeta potential of −31 mV can maintain dispersion stability by limiting particle aggregation. These physicochemical characteristics collectively support its suitability as a targeted nanocarrier for laryngeal cancer.

[0076] Example 2: Evaluation of in vitro ultrasound-triggered phase transition behavior and ultrasound imaging performance

[0077] PFP is a low-boiling-point perfluorinated carbon that undergoes a liquid-gas phase transition when exposed to ultrasound above its vaporization threshold. Therefore, PFP-loaded nanoparticles can be transformed into echo-generating microbubbles via acoustic droplet vaporization (ADV), thereby enhancing ultrasonic contrast. In addition to signal enhancement, this phase transition can also improve local retention by increasing particle size and acoustic responsiveness at the irradiated site.

[0078] To visually observe the phase transition process, take The sample was placed on a glass slide, and a set of low-intensity focused ultrasound (LIFU, sound intensity 2.5) was applied. The group was irradiated for 5 min (frequency: 1.0 MHz, 2 s working / 2 s intermittent) while the other group was not irradiated. Morphological observation was then performed under an optical microscope. Simultaneously, a 3% agarose gel model was constructed to evaluate its in vitro ultrasound-triggered phase transition and imaging stability. The diluted... The solution was injected into the agarose gel channels, and LIFU irradiation was applied for 1 min, 2 min, 3 min, and 4 min, respectively. B-mode and contrast-enhanced ultrasound (CEUS) images were acquired at each time point using an ultrasound diagnostic instrument, and the ultrasound signal intensity was quantitatively analyzed using DFY-II software (developed by the Institute of Ultrasound Imaging, Second Affiliated Hospital of Chongqing Medical University, see Zheng Yuanyi et al., Journal of Clinical Ultrasound Medicine, 2005, 7(5):338-340).

[0079] Optical microscopy revealed that LIFU irradiation significantly increased the size of the nanoparticles, accompanied by the formation of numerous microbubbles. Figure 2 a) This conforms to the liquid-gas phase transition characteristics induced by acoustic droplet vaporization (ADV). Since the phase-change droplet shows almost no signal enhancement before activation but becomes highly scattered after vaporization, this direct visual evidence provides direct evidence that the encapsulated PFP still exhibits acoustic responsiveness within the liposome structure. Ultrasound imaging results show that both the B-mode signal and the CEUS signal increase with increasing irradiation time. Figure 2 b), and as the irradiation time increases, the Mode B signal reaches its peak at 2 minutes, and the CEUS signal reaches its peak at 3 minutes. Figure 2 c) Subsequently, the signal showed a downward trend, indicating that the PFP underwent a controllable phase transition under ultrasonic triggering, with enhanced scattering in the early stage and signal attenuation in the later stage due to bubble destruction or droplet depletion.

[0080] Example 3: DOX in vitro release behavior regulated by both pH and LIFU

[0081] The newly prepared or Dispersed in PBS buffer containing 10 g / L serum, the pH was adjusted to 5.0 or 7.4. A control group (without ultrasound) and a LIFU-treated group were set up for each pH condition. Samples were taken at 0, 1h, 4h, 6h, 12h, and 24h. After centrifugation at 8000rpm, 100μL of supernatant was collected, and an equal volume of fresh release medium under the same conditions was immediately added to maintain a constant system volume. The absorbance of DOX was measured at 480nm using a microplate reader, and the cumulative release rate was calculated according to the formula.

[0082]

[0083] like Figure 2 d and Figure 2As shown in Figure e, under both pH conditions, the DOX release of the PFP-containing formulation was higher than that of the control group without PFP, and the difference was more significant after LIFU irradiation, indicating that PFP plays an important role in promoting drug release.

[0084] In vitro release assays showed that the introduction of PFP significantly altered the release behavior of DOX. Thanks to the low boiling point of PFP, even under non-ultrasonic conditions, some encapsulated droplets may undergo slight vaporization, increasing membrane tension and promoting DOX leakage from the liposome core. Building upon this, LIFU irradiation further significantly improved the release of DOX. The DOX release level was higher than that of the control formulation. The difference was most significant, especially at pH 5.0. Figure 2 d). This phenomenon conforms to the acoustic droplet vaporization (ADV) mechanism: acoustic stimulation triggers a liquid-gas phase transition in the PFP, leading to a sudden increase in internal pressure and driving liposome expansion; the resulting mechanical stress can damage the phospholipid shell, forming transient membrane defects or even causing shell rupture, thereby accelerating the diffusion of DOX into the external medium. The above release behavior is consistent with... Figure 2 a- Figure 2 The phase transition and imaging results shown in c are in high agreement, confirming that LIFU-induced vaporization enhances ultrasonic contrast and also verifies the acoustic responsiveness of nanodroplets.

[0085] Furthermore, the release profiles exhibited a clear pH dependence: regardless of the application of ultrasound, the release rates of both formulations were significantly higher at pH 5.0 than at pH 7.4. This is primarily attributed to the distribution characteristics of DOX within liposomes and its relationship with the transmembrane pH gradient. As a water-soluble drug, the dissolution of DOX in the liposome's water core lowers the internal pH. When the external medium is near neutral, the resulting pH gradient favors DOX retention in the acidic core, thereby inhibiting diffusion-mediated release. Under weakly acidic conditions, this pH gradient is weakened, reducing the DOX retention effect and promoting drug release. Given that the tumor microenvironment is often weakly acidic due to high glycolysis and lactic acid accumulation, this pH-responsive characteristic has significant biological implications.

[0086] In summary, external pH, PFP incorporation, and LIFU irradiation work synergistically to regulate [the system]. The DOX release behavior endows it with the dual functions of ultrasound-triggered release and imaging enhancement, providing a mechanistic basis for the construction of an ultrasound-activated diagnostic and therapeutic platform.

[0087] Example 4: Cell-targeted uptake and LIFU-triggered intracellular DOX release

[0088] TU686 laryngeal cancer cells were used Inoculate with cells / mL at a density of 1,000 cells / mL in confocal culture dishes, and after 24 hours of incubation, replace with a culture dish containing 1,000 cells / mL. , or Serum-free culture medium was incubated at 37℃ for 1 h, 2 h, and 4 h, respectively, after which the medium was discarded. The cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 min, and stained with DAPI for 5 min. The distribution of DOX red fluorescence was observed using a confocal laser scanning microscope (CLSM, A1R-Si, Nikon). The excitation wavelengths of the CLSM were set to DAPI 405 nm, FITC 488 nm, and TRITC 561 nm; the objective magnification was 10×.

[0089] To monitor LIFU-triggered release, FITC-labeled iRGD was used to synthesize nanoparticles with the same structure instead of ordinary iRGD. After 4 hours of cell incubation, one group was treated with LIFU (2.5 g / L). The first group was treated for 5 minutes, while the second group was left untreated. Subsequently, DAPI staining and CLSM were used to observe the intracellular localization of FITC (vector) and DOX.

[0090] CLSM results confirmed that iRGD modification significantly enhances the targeted uptake of nanoparticles. In time-gradient experiments, TU686 cells and The intracellular DOX red fluorescence was strongest after 4 hours of co-incubation. Figure 9 This indicates that this time point is most conducive to cell accumulation.

[0091] like Figure 3 As shown in a, further comparisons are presented. and All groups showed strong intracellular DOX fluorescence, indicating effective cell association and internalization. Free DOX and the non-targeted control groups, however, showed different results. The group exhibited weak fluorescence, suggesting that the nanocarrier promotes cellular uptake through endocytosis, while free DOX is susceptible to efflux by membrane transport proteins, resulting in low retention. Notably, the PFP-containing group ( The intracellular DOX fluorescence of the group was stronger than that of the group without PFP. Consistent with the in vitro release trend, this suggests that PFP may enhance intracellular drug concentration by promoting DOX release and endosome escape.

[0092] Based on this, the effect of LIFU on intracellular DOX delivery was further investigated. The results are as follows: Figure 3 As shown in b. DOX accumulates extensively around the cell periphery; without LIFU, DOX fluorescence is mainly confined to the carrier region, with limited release; after LIFU irradiation, intranuclear DOX fluorescence is significantly enhanced, indicating rapid drug release and effective nuclear entry. This effect is attributed to LIFU-triggered PFP droplet vaporization (ADV): ​​the phase transition leads to increased intraparticle pressure, inducing liposome expansion or shell rupture, accelerating DOX release. Since the nanoparticles are pre-accumulated around the target cells, the LIFU-triggered localized burst release significantly increases perinuclear DOX concentration, thereby enhancing cytotoxic delivery.

[0093] Example 5: Evaluation of in vitro antitumor activity ( (and live / dead cell staining)

[0094] TU686 cells were used One sample per well was inoculated into a 96-well plate, and after 24 hours of incubation, [the following was added]... or The final DOX concentrations were adjusted to 1.5 μg / mL, 3 μg / mL, 6 μg / mL, and 12 μg / mL. Subsequently, one group immediately received LIFU (2.5 μg / mL). The first group was incubated for 5 minutes, while the second group received no treatment and continued incubation for 24 hours. Add [amount missing] to each well. 10 μL of reagent, incubate Cell viability was then calculated by measuring absorbance at 450 nm using a Synergy4 (BioTek) microplate reader. The cell viability calculation is as follows:

[0095]

[0096] in To process the absorbance of the cells in the treatment group, This is the absorbance of the control group cells. It is the absorbance of light containing culture medium and CCK-8 but without cells.

[0097] In addition, the cells were... Cells were seeded per well in a 24-well plate and treated in the same way. After 30 min of Calcein-AM / PI double staining, surviving (green) and dead (red) cells were observed by CLSM.

[0098] like Figure 4 As shown in a The results show that, under LIFU-free conditions, Its cytotoxicity is slightly higher than This indicates that PFP incorporation promotes higher levels of basal intracellular DOX release, and... Figure 2 The release behavior observed was consistent with that observed in [the study / study].

[0099] LIFU irradiation significantly increased The difference in efficacy between the control and the treatment. The tests showed that at concentrations of 6 μg / mL and 12 μg / mL, The group receiving the combined LIFU treatment exhibited the lowest cell viability, and this advantage was concentration-dependent. Dose-response curves were calculated to show that... The IC50 value was 3.27 μg / mL, which is much lower than... Group 11.27 μg / mL ( Figure 4 b). Live / dead cell staining ( The results were consistent with the above data: without LIFU, both groups of cells exhibited predominantly green live-cell fluorescence, with only a small number of red dead cells, indicating weak basic toxicity; however, after LIFU irradiation... The red fluorescence (dead cells) group showed a significant increase, while the green fluorescence (live cells) group showed a significant decrease, which was even more pronounced at high concentrations. Figure 4 c. Figure 10 , Figure 11 This directly demonstrates that the LIFU-triggered PFP phase transition effectively enhances the cytotoxic effect of nanodroplets by promoting intracellular DOX release.

[0100] Example 6: In vivo ultrasound contrast imaging verification

[0101] Establishing a laryngeal cancer xenograft model in SPF-grade BALB / c nude mice by subcutaneous inoculation with TU686 cells: One TU686 cell was suspended in 100 μL of cell suspension and subcutaneously injected into the flank of the back of each mouse. Tumor growth was monitored every 3 days by measuring the longest diameter (a) and shortest diameter (b) with calipers. Tumor volume was calculated using the following formula:

[0102]

[0103] When the tumor reaches a size of approximately 100 mm 3 Then, injected via tail vein. or (DOX dose 6 mg / kg). 6 hours after injection, LIFU (2.5 mg / kg) was applied to the tumor area. (10 min), followed by CEUS imaging using a Mindray ultrasound system (L3-12 probe, contrast mode). The tumor ROI was manually delineated, and the changes in ultrasound signal grayscale values ​​before and after LIFU irradiation were analyzed using system software.

[0104] Each group consisted of 5 mice. In the LIFU experiment, the probe was held by hand and directly coupled to the mouse skin. The mouse skin was coated with coupling agent to isolate it from the air.

[0105] injection Following LIFU irradiation, the tumor region showed obvious patchy hyperechoic signals in both B-mode and CEUS images, and ROI analysis showed a significant increase in signal intensity; while simple LIFU irradiation... Group or those not irradiated by LIFU No similar enhancement phenomenon was observed in any of the groups, confirming that the liposome can achieve LIFU-triggered phase transition enhancement imaging in vivo.

[0106] The results are as follows Figure 5 As shown in a, i is injected via the tail vein. Six hours later, the CEUS signal in the tumor area was weak, and LIFU irradiation showed no significant enhancement. (Administered alone) Without LIFU, contrast is also limited. Only... P combined with LIFU (2.5) The group (10 min) showed clear sheet-like hyperechoic areas in both grayscale and CEUS images. This indicates that iRGD nanodroplets can accumulate in tumors, and that PFP retains acoustic responsiveness in vivo. LIFU triggers the PFP phase transition to generate microbubbles, achieving imaging through enhanced local acoustic scattering. No similar enhancement was observed in the non-PFP control group, ruling out non-specific ultrasound effects. ROI quantitative analysis results ( Figure 5 b) Further confirmation, It can effectively induce phase transitions in vivo, generating stable and enhanced signals.

[0107] Example 7: Evaluation of CAT-mediated tumor hypoxia improvement effect

[0108] Tumor-bearing nude mice were randomly divided into 5 groups (n=5 in each group): PBS control group, free DOX group, etc. Group, Group, Group. Administered every 3 days along with LIFU irradiation (2.5). Mice were administered the drug intraperitoneally (10 min) for 15 consecutive days. Ninety minutes after the last administration, mice were given an intraperitoneal injection of pimonidazole hydrochloride (30 mg / kg). Tumor tissue was then collected and frozen sections were prepared. The sections were compared with exogenous hypoxia probe antibodies and anti-inflammatory drugs. Primary antibody incubation, secondary antibody labeling with CoraLite Plus 555 for staining, and fluorescence signal observation using CLSM. The proportion of pimonidazole- and HIF-1α-positive stained areas to the total tumor area was calculated using ImageJ software, as shown below:

[0109]

[0110] in, Indicates the area of ​​positive fluorescent staining. This indicates the total area of ​​the tumor slice.

[0111] Tumor hypoxia assessment results showed ( Figure 6 a) In the control group, pimonidazole and HIF-1α showed the strongest fluorescence, indicating significant hypoxia. Free DOX and The fluorescence in the control group was weaker than that in the control group, indicating partial improvement in oxygenation; however, The hypoxia signal in this group remained relatively strong. In contrast, Piperamidazole showed the weakest green fluorescence and the lowest quantitative signal. Figure 6 a, Figure 6 b), and The expression is also the lowest ( Figure 6 a, Figure 6 c), confirming its optimal inhibitory effect on hypoxia. It is noteworthy that CAT-free... The group remains at a high level This indicates that ultrasound-triggered drug release alone is insufficient to alleviate hypoxia. The effect is attributed to CAT releasing endogenous... Decomposed into It increases local oxygen concentration; at the same time, iRGD targeting and LIFU activation synergistically promote the accumulation and release of CAT in the tumor, thereby effectively improving the hypoxic microenvironment of the tumor.

[0112] Example 8: Evaluation of in vivo antitumor efficacy and biosafety

[0113] TU686 tumor-bearing nude mice were randomly divided into 8 groups (n=5 per group): PBS group, free DOX group, etc. Group, Group, Group, Group, Group, Group. The corresponding preparation (DOX 6 mg / kg) was injected via tail vein, and LIFU (2.5 mg / kg) was applied to the tumor area 6 hours later. Treatment was administered every 3 days for 18 days (10 minutes). During this period, the tumor's long diameter (a) and short diameter (b) were measured every 3 days, and analyzed according to the formula... Tumor volume was calculated, and body weight changes were monitored simultaneously. Mice were sacrificed on day 18, and tumor tissue was collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and subjected to PCNA immunohistochemical staining to assess cell proliferation activity.

[0114] The results show that ( Figure 7 a- Figure 7 d and Figure 12 In the control group, tumor growth was the fastest, and free DOX only had a mild tumor-suppressing effect. The nano-formulation group without LIFU showed moderate tumor-suppressing activity, weaker than the corresponding LIFU-activated group. LIFU significantly enhanced... and The therapeutic effect confirms the importance of ultrasound-triggered release. Among them, The group with the slowest tumor growth and smallest terminal volume was superior to the group without PFP. The study demonstrated the synergistic effects of iRGD targeting, CAT oxygen production, PFP phase transition, and LIFU activation. No significant weight loss was observed in any group during treatment. Figure 7 c), indicating good treatment safety. PCNA immunohistochemical analysis ( Figure 8 , Figure 13 The results showed that the control group had the highest proliferative activity, followed by the free DOX and no-ultrasound groups; PCNA expression was significantly reduced in the ultrasound treatment group, especially in The lowest value was observed in the group, consistent with the trend of the tumor growth curve, confirming that this combined strategy can effectively inhibit tumor cell proliferation.

[0115] In summary, the liposomes of the present invention use a phospholipid bilayer membrane as the shell, with the surface intercalated with... The targeted peptide, with doxorubicin and catalase co-loaded in the aqueous phase and liquid perfluoropentane phase transition core dispersed in the lumen, integrates tumor targeting, hypoxia regulation, phase transition response, and controlled drug release into a single nanoformulation. Figure 14 As shown.

[0116] These liposomes exhibit suitable particle size and good colloidal stability, demonstrating effective cellular uptake in TU686 human laryngeal squamous cell carcinoma cells. Under low-intensity focused ultrasound (LIFU) irradiation, the encapsulated liquid perfluoropentane undergoes an aerosol phase transition (ADV), generating microbubbles in situ. This enhances ultrasound contrast for imaging guidance and, through cavitation, promotes rapid release of doxorubicin. Simultaneously, catalase co-loaded in the internal aqueous phase catalyzes tumor-endogenous... This liposome decomposes oxygen, alleviating the hypoxic microenvironment of tumors and enhancing chemosensitivity. In vitro cell experiments and in vivo tumor-bearing animal experiments have confirmed that this liposome possesses multifunctional diagnostic and therapeutic efficacy, combining ultrasound imaging and chemotherapy. It not only improves the local drug delivery efficiency and therapeutic effect of laryngeal cancer lesions but also provides real-time ultrasound imaging feedback during treatment. This strategy offers a feasible technical approach for non-invasive, precise, and image-guided treatment of laryngeal cancer.

Claims

1. An iRGD-targeted ultrasound-responsive phase change liposome, characterized in that, include: Phospholipid bilayer membrane; Surface modification, for Its hydrophobic end is embedded in the phospholipid bilayer membrane, while its hydrophilic end carries the iRGD peptide and is exposed on the liposome surface; The aqueous phase is contained within the cavity encapsulated by the phospholipid bilayer membrane and is co-loaded with doxorubicin, catalase, and liquid perfluoropentane. The liquid perfluoropentane is dispersed in the inner aqueous phase in the form of droplets, or adsorbed / attached to the inner surface of the phospholipid bilayer membrane.

2. The iRGD-targeted ultrasound-responsive phase change liposome as described in claim 1, characterized in that, The phospholipid bilayer is composed of dipalmitoylphosphatidylcholine and cholesterol, and the dipalmitoylphosphatidylcholine, cholesterol, and The molar ratio is 65:30:

1.

3. The iRGD-targeted ultrasound-responsive phase change liposome as described in claim 1, characterized in that, The average hydrated particle size of the ultrasound-responsive phase change liposomes was 236.10±9.28 nm, the polydispersity index was 0.21±0.03, and the zeta potential was −31.42±0.69 mV.

4. A method for preparing iRGD-targeted ultrasound-responsive phase change liposomes as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Phospholipids, cholesterol and It dissolves in an organic solvent, and the solvent is removed by rotary evaporation to form a lipid film; (2) Add buffer solution to hydrate the lipid membrane to obtain a lipid suspension; (3) Mix doxorubicin solution, catalase solution and perfluoropentane, and perform pre-emulsification treatment to obtain a pre-emulsified solution; (4) Add the pre-emulsion to the lipid suspension and perform ultrasonic emulsification to obtain the initial product; (5) Centrifuge and wash to remove unencapsulated material, collect the precipitate and resuspend it to obtain the iRGD-targeted ultrasound-responsive phase change liposomes.

5. The method for preparing iRGD-targeted ultrasound-responsive phase change liposomes as described in claim 4, characterized in that, The power of the pre-emulsification process in step (3) is 100W, using pulse mode, with a working time to intermittent time ratio of 1:1 and a total duration of 3 minutes; The ultrasonic emulsification process in step (4) has a power of 125W, uses pulse mode, has a working time to intermittent time ratio of 1:1, and a total duration of 5min.

6. The method for preparing iRGD-targeted ultrasound-responsive phase change liposomes as described in claim 4, characterized in that, The organic solvent in step (1) is chloroform; The buffer solution in step (2) is a phosphate buffer solution with a pH of 7.4; The centrifugation in step (5) is performed at a speed of 8000 rpm for 5 minutes.

7. The use of an iRGD-targeted ultrasound-responsive phase change liposome as described in any one of claims 1 to 3 in the preparation of a combination therapy drug for tumors.

8. The application as described in claim 7, characterized in that, The combined treatment is an ultrasound-triggered combined treatment. The ultrasound-responsive phase change liposomes undergo a liquid-gas phase change under ultrasound irradiation, realizing the sudden release of drugs. At the same time, catalase catalyzes the decomposition of hydrogen peroxide in the tumor microenvironment to produce oxygen, thereby alleviating tumor hypoxia and enhancing the therapeutic effect.

9. The application as described in claim 8, characterized in that, The tumor is a solid tumor, specifically laryngeal cancer.

10. The application as described in claim 8, characterized in that, The ultrasonic irradiation was low-intensity focused ultrasound irradiation with a frequency of 1.0 MHz and a sound intensity of 2.

5. .

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