Ultrasonic label guided and targeted nano liquid drop as well as preparation method and application thereof

Abstract

The invention discloses an ultrasonic labeling guided targeting nano liquid drop. The nano liquid drop comprises an inner core and a polymer layer coating the surface of the inner core, the inner core is perfluorinated carbon; the polymer layer comprises fluorine-containing polycaprolactone, a copolymer as shown in a formula I, an ultrasonic activation prodrug and a sound-sensitive agent; the perfluorinated carbon can be dissolved by the fluorine-containing polycaprolactone. According to the application, the tumor tissue is precisely treated by utilizing ultrasound to induce the tumor tissue to highly express the heat shock protein, so that the nano liquid drops modified with the heat shock protein targeting polypeptide are efficiently enriched at the tumor part, and the difference between the drug concentration in the tumor tissue and the drug concentration in other normal tissues is remarkably increased; the drug targeting selectivity and the drug release accuracy are improved.

Description

An ultrasonically labeled and targeted nanodroplet, its preparation method and application Technical Field

[0001] This application relates to the field of pharmaceutical technology, specifically to an ultrasound-labeled and targeted nanodroplet, its preparation method, and its application. Background Technology

[0002] Cancer treatment is a major challenge facing medicine today. While traditional chemotherapy can kill cancer cells, the lack of specificity in the distribution of chemotherapy drugs in the body can damage normal tissues and produce severe toxic effects, limiting the dosage and therapeutic efficacy of chemotherapy drugs. To address this, the prodrug strategy has emerged. Prodrugs have low or no activity in vitro, but can be activated and release drugs through specific mechanisms after entering the body, thereby improving tumor selectivity.

[0003] Current methods primarily rely on endogenous stimulation of the tumor microenvironment to activate prodrugs, but their clinical efficacy is limited by the heterogeneity of the tumor microenvironment and insufficient activation specificity. Therefore, the development of an active targeting system that can improve targeting selectivity while also possessing efficient enrichment and precise drug release capabilities is urgently needed. Summary of the Invention

[0004] In view of this, the technical problem to be solved by this application is to provide an ultrasound-labeled and guided targeted nanodroplet, its preparation method and application. The nanodroplet provided by this application can effectively achieve high-efficiency enrichment at the tumor site, thereby improving drug targeting selectivity and drug release accuracy.

[0005] This application provides an ultrasound-labeled and targeted nanodroplet carrier, comprising a core and a polymer layer coating the surface of the core; the core is perfluorinated carbon; the polymer layer comprises fluorinated polycaprolactone and a copolymer represented by Formula I; Formula I; wherein, peptide is a heat shock protein targeting polypeptide containing thiol, n is the degree of polymerization, which is selected from an integer between 1 and 111, m is the degree of polymerization, which is selected from an integer between 1 and 51; the fluorinated polycaprolactone can dissolve perfluorinated carbon.

[0006] In some specific implementations, the perfluorocarbon includes one or more of perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoropropane, perfluorobutane, and perfluoro-15-crown-5-ether; the fluorinated polycaprolactone has a structure of formula II; Formula II; wherein R is a perfluoroalkyl group, a is the degree of polymerization, and a is an integer selected from 1 to 51; the mass ratio of the perfluorocarbon, the fluorinated polycaprolactone and the copolymer shown in Formula I is 10 to 80: 5 to 40: 1 to 8.

[0007] This application also provides an ultrasound-labeled and guided targeted nanodroplet, comprising a core and a polymer layer coated on the surface of the core; the core is perfluorinated carbon; the polymer layer comprises fluorinated polycaprolactone, a copolymer of Formula I, an ultrasound-activated prodrug, and a sonosensitive agent; Formula I; wherein, peptide is a heat shock protein targeting polypeptide containing thiol, n is the degree of polymerization, which is selected from an integer between 1 and 111, m is the degree of polymerization, which is selected from an integer between 1 and 51; the fluorinated polycaprolactone can dissolve perfluorinated carbon.

[0008] In some specific implementations, the perfluorocarbon includes one or more of perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoropropane, perfluorobutane, and perfluoro-15-crown-5-ether; the fluorinated polycaprolactone has the structure shown in Formula II; Formula II; wherein R is a perfluoroalkyl group, a is the degree of polymerization, and a is an integer selected from 1 to 51; the ultrasound-activated prodrug includes one or more of DOX-N3, CPT-N3, 6-MP-N3, R837-N3, R848-N3 and IMDQ-N3; ​​the sonic sensitizer includes one or more of riboflavin tetrabutyrate, riboflavin, hematoporphyrin and its derivatives, curcumin, zinc phthalocyanine and IR-780; the mass ratio of perfluorinated carbon, fluorinated polycaprolactone, copolymer shown in Formula I, ultrasound-activated prodrug and sonic sensitizer is 10~80:5~40:1~8:0.7~9.6:1.4~19.2.

[0009] This application also provides the above-mentioned nanodroplet carrier and the application of nanodroplets in an ultrasonic labeling-guided targeted nanodelivery system.

[0010] In some specific implementations, the ultrasound-labeled guided targeted nanodelivery system includes heat shock protein targets, which include one or more of HSP27, HSP60, HSP70, HSP90α, HSP90β, HSP105, and HSP40.

[0011] This application also provides the above-mentioned nanodroplet carrier and the application of nanodroplets in the preparation of drugs for treating tumors.

[0012] In some specific implementations, the tumor is one or more of CT26 colon cancer, MC38 colon cancer, 4T1 breast cancer, and H22 liver cancer.

[0013] This application also provides a method for preparing the above-mentioned ultrasound-labeled and targeted nanodroplets, comprising the following steps: performing a click chemical reaction between a heat shock protein targeting peptide and the copolymer shown in Formula III to obtain the copolymer shown in Formula I; Formula III; where n is the degree of polymerization, selected from an integer between 1 and 111, and m is the degree of polymerization, selected from an integer between 1 and 51; fluorinated polycaprolactone, the copolymer shown in Formula I, an ultrasonically activated prodrug, a sonicator, and an organic solvent are mixed to obtain an organic phase; the organic phase, perfluorinated carbon, and water are mixed to obtain ultrasonically labeled and targeted nanodroplets.

[0014] In some specific implementations, the copolymer represented by Formula III is prepared by the following method: maleimide-polyethylene glycol-hydroxyl and caprolactone undergo ring-opening polymerization to obtain the copolymer represented by Formula III.

[0015] This application provides an ultrasound-labeled, targeted nanodroplet carrier, comprising a core and a polymer layer coating the surface of the core; the core is perfluorinated carbon; the polymer layer comprises fluorinated polycaprolactone and a copolymer represented by Formula I; the fluorinated polycaprolactone can dissolve the perfluorinated carbon. The fluorinated segments introduced into the fluorinated polycaprolactone molecular chain exhibit good "fluorine-fluorine interaction" and compatibility with the perfluorinated carbon, enabling the core to be efficiently and stably embedded, thereby significantly improving the structural stability of the perfluorinated carbon and the nucleation stability of the droplets, avoiding the problems of easy leakage, aggregation, and poor storage stability of gas nuclei in traditional nanodroplets. Simultaneously, the copolymer represented by Formula I is terminally linked to a heat shock protein targeting peptide, enabling the nanodroplets to actively recognize heat shock proteins highly expressed in tumor cells under stress, thereby improving enrichment efficiency at tumor sites and reducing uptake by normal tissues, achieving higher delivery selectivity. Therefore, this nanodroplet carrier possesses excellent perfluorinated carbon loading stability, good blood circulation stability, and active tumor targeting capability, providing a highly stable and selective delivery platform for subsequent drug loading.

[0016] This application also provides an ultrasound-labeled, targeted nanodroplet. The nanodroplet, based on the aforementioned nanodroplet carrier structure, is further loaded with an ultrasound-activated prodrug and a sonosensitive agent, forming a multifunctional structure with perfluorinated carbon as the core, a fluoropolymer as the stable shell, and both targeting and ultrasound-responsive therapeutic properties. Under the action of external focused ultrasound, the perfluorinated carbon core undergoes a liquid-gas phase transition, leading to responsive release. This amplifies the difference in prodrug accumulation between the tumor and other tissues. Simultaneously, the large number of free radicals instantaneously generated by ultrasound and perfluorinated carbon further activates the prodrug, inducing a reduction reaction and achieving high-level enrichment, release, and activation of the original drug at the tumor site. Because the outer fluorinated polycaprolactone layer has a dissolving and stabilizing effect on the perfluorinated carbon, the nanodroplet is less prone to premature vaporization or drug leakage during blood circulation, thus significantly reducing systemic toxicity. Furthermore, ultrasound triggering ensures drug activation only in the tumor region, further improving the spatial and temporal controllability of the treatment. Compared to existing nanomedicines that rely on passive EPR effects, the nanodroplets provided in this application possess multiple superior properties, including structural stability, targeted delivery, ultrasonic responsiveness, and therapeutic safety. These properties significantly increase the effective drug concentration at the tumor site while reducing damage to normal tissues, thereby enhancing anti-tumor efficacy and improving treatment safety. This provides a novel, precise, and effective treatment approach for cancer therapy, potentially significantly improving the efficacy and safety of cancer treatment and bringing new hope and breakthroughs to the clinical treatment of cancer patients. Attached Figure Description

[0017] Figure 1 is a diagram illustrating the preparation process of the ultrasonically labeled and targeted nanodroplets of this application; Figure 2 is a 1H NMR spectrum of MAL-PEG-PCL prepared in Example 1 of this application; Figure 3 is a 1H NMR spectrum of Pep-PEG-PCL prepared in Example 1 of this application; Figure 4 is a 1H NMR spectrum of F-PCL prepared in Example 1 of this application; Figure 5 is a TEM image of Pep-PEG-PCL@P / D / T-NPs prepared in Example 2 of this application; Figure 6 is an elemental mapping analysis diagram of Pep-PEG-PCL@P / D / T-NPs prepared in Example 2 of this application; Figure 7 is a TEM image of Pep-PEG-PCL@P / D / T-NPs prepared in Example 2 of this application. Figure 8 shows the dynamic light scattering pattern of Pep-PEG-PCL@P / D / T-NPs prepared in Example 2 of this application; Figure 9 shows the Zeta potential measurement of Pep-PEG-PCL@P / D / T-NPs prepared in Example 2 of this application; Figure 10 shows the effect of ultrasound stimulation and GRP78 targeting peptide on GRP78 protein expression in this application; Figure 11 shows the effect of ultrasound stimulation and GRP78 targeting peptide on GRP78 protein expression in this application; Figure 12 shows the flow cytometry verification of the change of GRP78 protein over time after ultrasound; Figure 13 shows the flow cytometry verification of GRP78 protein expression at different ultrasound times in this application. Figure 14 shows the changes in GRP78 under different ultrasound intensities as verified by flow cytometry in this application; Figure 15 shows the changes in the expression of different heat shock proteins under ultrasound as verified by flow cytometry in this application; Figure 16 shows the changes in the ability of Pep-PEG-PCL@P-NPs to generate hydroxyl radicals under different ultrasound conditions in this application; Figure 17 shows the cytotoxicity verification of Pep-PEG-PCL@P / D / T-NPs in this application; Figure 18 shows the cellular endocytosis assessment of Pep-PEG-PCL@P / D / T-NPs in this application; Figure 19 shows the cellular endocytosis assessment of Pep-PEG-PCL@P / D / T-NPs in this application. Figure 20 shows the co-localization of Pep-PEG-PCL@P / D / T-NPs and heat shock protein GRP78 in this application; Figure 21 shows the cell viability and mortality staining after ultrasound treatment with Pep-PEG-PCL@P / D / T-NPs in this application; Figure 22 shows the ability of Pep-PEG-PCL@P / D / T-NPs in this application to induce ICD effect in CT26 cells after ultrasound treatment; Figure 22A shows the expression changes of calreticulin (CRT) in different experimental groups; Figure 22B shows the changes in the content of high-mobility group box 1 (HMGB-1) efflux in different experimental groups; Figure 22C shows the changes in the content of adenosine triphosphate (ATP) in different experimental groups.Figure 23 shows the tumor suppression effect of Pep-PEG-PCL@P / D / T-NPs combined with focused ultrasound in this application. Figure 23A is a schematic diagram of the drug administration regimen in the tumor suppression experiment. Figure 23B is a curve of tumor volume change. Figure 23C is a survival observation diagram. Figure 24 shows the changes in the level of immune cells in the tumor induced by Pep-PEG-PCL@P / D / T-NPs combined with focused ultrasound in this application. Figure 24A shows CD3. + Statistical graph of T cell content changes. Figure 24B shows CD3. + CD4 + Statistical graph of T cell content changes. Figure 24C shows CD3. + CD8 + Figure 24D shows the statistical chart of changes in T cell content; Figure 25 shows the statistical chart of changes in M1 macrophage content; Figure 25A shows the statistical chart of changes in intratumoral cytokine levels induced by Pep-PEG-PCL@P / D / T-NPs combined with focused ultrasound in this application; Figure 25B shows the statistical chart of changes in cell necrosis factor-α (TNF-α) content; Figure 25C shows the statistical chart of changes in interferon-γ (IFN-γ) content; Figure 25D shows the statistical chart of changes in interleukin-2 (IL-2) content; Figure 26 shows the distribution of active drugs in various tissues after Pep-PEG-PCL@P / D / T-NPs combined with focused ultrasound in this application. Detailed Implementation

[0018] This application provides an ultrasonically labeled and guided targeted nanodroplet, its preparation method, and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. The method and application of this application have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this application to realize and apply the technology of this application.

[0019] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0020] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0021] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0022] The use of any and all instances or exemplary language such as “e.g.” or “including” herein is merely intended to better illustrate the application and does not constitute a limitation on the scope of the application unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.

[0023] Furthermore, the numerical ranges and parameters used to define this application are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0024] This application provides an ultrasound-labeled and targeted nanodroplet carrier, comprising a core and a polymer layer coating the surface of the core; the core is perfluorinated carbon; the polymer layer comprises fluorinated polycaprolactone (PCL) and a copolymer of Formula I; Formula I; wherein, peptide is a heat shock protein targeting peptide containing active thiol groups, preferably a heat shock protein GRP78 targeting peptide containing active thiol groups, the sequence number of the heat shock protein GRP78 targeting peptide is preferably WIFPWIQLC, C in the sequence number WIFPWIQLC is Cys (thiol group), the thiol group in the heat shock protein targeting peptide can undergo click chemical reaction with maleimide; n is the degree of polymerization of the PEG segment, the n is selected from an integer between 1 and 111, preferably an integer between 22 and 111, more preferably an integer between 45 and 111; m is the degree of polymerization of the PCL segment, the m is selected from an integer between 1 and 51, preferably an integer between 10 and 51, more preferably an integer between 20 and 51; the fluorinated polycaprolactone can dissolve perfluorinated carbon.

[0025] The ultrasound-labeled, targeted nanodroplet carrier provided in this application includes a core, which is perfluorocarbon, used to undergo a liquid-gas phase transition under ultrasound, thereby triggering drug release. In some specific implementations, the perfluorocarbon is preferably one or more of perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoropropane, perfluorobutane, and perfluoro-15-crown-5-ether, more preferably one or more of perfluoropentane, perfluorohexane, and perfluoroheptane. Perfluorocarbon possesses excellent bioinertness and phase transition response characteristics, making it a key functional core for achieving ultrasound-responsive release.

[0026] The ultrasonically labeled and targeted nanodroplet carrier provided in this application comprises a polymer layer coated on the surface of the perfluorinated carbon core. The polymer layer comprises fluorinated polycaprolactone (F-PCL) and a copolymer shown in Formula I. In the nanodroplet, the mass ratio of the perfluorinated carbon, the fluorinated polycaprolactone, and the copolymer shown in Formula I is preferably 10~80:5~40:1~8, more preferably 30~50:20~30:4~7.5. The fluorinated polycaprolactone is capable of dissolving the perfluorinated carbon, and its structure is shown in Formula II. The F-PCL is a polycaprolactone derivative with fluorinated alkyl groups introduced into its side chains or end groups. The fluorinated segments are perfluoroalkyl segments, preferably one or more of perfluoropentyl, perfluorohexyl, perfluoroheptyl, perfluorooctyl, perfluoropropyl, and perfluorobutyl, more preferably one or more of perfluoropentyl, perfluorohexyl, and perfluoroheptyl. The fluorine element in the F-PCL molecular chain has a strong fluorine-fluorine interaction with perfluorinated carbon, which significantly improves the solubility and stability of perfluorinated carbon in the polymer layer and prevents the perfluorinated carbon from leaking out prematurely in the body. Formula II; wherein, R is a fluorinated segment, preferably one or more of perfluoropentyl, perfluorohexyl, perfluoroheptyl, perfluorooctyl, perfluoropropyl and perfluorobutyl, more preferably one or more of perfluoropentyl, perfluorohexyl and perfluoroheptyl; a is the degree of polymerization of the PCL segment, wherein a is selected from an integer between 1 and 51, preferably an integer between 10 and 51, more preferably an integer between 20 and 51.

[0027] The copolymer represented by Formula I is an amphiphilic block copolymer composed of hydrophilic polyethylene glycol (PEG) and hydrophobic polycaprolactone (PCL) segments, wherein a heat shock protein targeting peptide (Pep) is covalently linked to the PEG end. The PEG segments provide a hydrophilic protective layer, prolonging the circulation time of the nanodroplets in vivo; the PCL segments are hydrophobic, participating in the construction of a stable polymer shell and used to encapsulate hydrophobic drug molecules; the heat shock protein targeting peptide is a targeting peptide that specifically recognizes heat shock proteins highly expressed on the surface or intracellularly of tumor cells. In some specific implementations, the heat shock protein targeting peptide Pep is preferably the HSP70 family targeting peptide GRP78. The heat shock protein targeting peptide Pep endows the nanodroplets with the ability to actively target ultrasound-induced tumor cells highly expressing heat shock proteins.

[0028] This application also provides an ultrasound-labeled and guided targeted nanodroplet, comprising a core and a polymer layer coating the surface of the core; the core is perfluorinated carbon; the polymer layer is fluorinated polycaprolactone, a copolymer of Formula I, an ultrasound-activated prodrug, and a sonicator; Formula I; wherein, peptide is a heat shock protein targeting peptide containing active thiol groups, preferably a heat shock protein GRP78 targeting peptide containing active thiol groups, the sequence number of the heat shock protein GRP78 targeting peptide is preferably WIFPWIQLC, C in the sequence number WIFPWIQLC is Cys (thiol group), the thiol group in the heat shock protein targeting peptide can undergo click chemical reaction with maleimide; n is the degree of polymerization of the PEG segment, the n is selected from an integer between 1 and 111, preferably an integer between 22 and 111, more preferably an integer between 45 and 111; m is the degree of polymerization of the PCL segment, the m is selected from an integer between 1 and 51, preferably an integer between 10 and 51, more preferably an integer between 20 and 51; the fluorinated polycaprolactone can dissolve perfluorinated carbon.

[0029] The nanodroplets provided in this application are further loaded with ultrasonically activated prodrugs and sonic sensitizers based on the above-mentioned nanodroplet carrier structure, forming a stable structure with perfluorinated carbon as the core and fluoropolymer as the stable shell.

[0030] The nanodroplet provided in this application comprises a core and a polymer layer coating the surface of the core. The polymer layer coats the surface of the perfluorinated carbon core and comprises fluorinated polycaprolactone, a copolymer of Formula I, an ultrasound-activated prodrug, and a sonosensitizer. The perfluorinated carbon and the fluorinated polycaprolactone and the copolymer of Formula I in the polymer layer are as described above. In the nanodroplet, the mass ratio of the perfluorinated carbon, fluorinated polycaprolactone, copolymer of Formula I, ultrasound-activated prodrug, and sonosensitizer is preferably 10~80:5~40:1~8:0.7~9.6:1.4~19.2, more preferably 20~40:10~20:2~4:2.4~4.8:4.8~9.6.

[0031] In some specific implementations, the ultrasound-activated prodrug is an azide-containing drug prodrug, preferably one or more of DOX-N3, CPT-N3, 6-MP-N3, R837-N3, R848-N3, and IMDQ-N3, more preferably an azide-containing doxorubicin prodrug (DOX-N3). The DOX-N3 remains in a low-toxicity or non-toxic state under normal physiological conditions, but can be converted into cytotoxic doxorubicin (DOX) in a free radical or reducing environment, thereby achieving spatially controllable drug activation. In some specific implementations, the sonosensitive agent is preferably one or more of riboflavin tetrabutyrate (TBR), riboflavin, hematoporphyrin and its derivatives, curcumin, zinc phthalocyanine, and IR-780, more preferably TBR. Under ultrasound, the TBR can generate reactive oxygen species and free radicals, which can enhance oxidative damage to tumor cells and promote the conversion of DOX-N3 to doxorubicin, improving the prodrug activation efficiency.

[0032] This application also provides a method for preparing the above-mentioned ultrasonically labeled and guided targeted nanodroplets. The preparation process is shown in Figure 1. Figure 1 is a diagram of the preparation process of the ultrasonically labeled and guided targeted nanodroplets of this application, which includes the following steps: performing a click chemical reaction between the heat shock protein targeting peptide and the copolymer shown in Formula III to obtain the copolymer shown in Formula I; Formula III; wherein, n is the degree of polymerization of the PEG segment, which is selected from an integer between 1 and 111, preferably an integer between 22 and 111, and more preferably an integer between 45 and 111; m is the degree of polymerization of the PCL segment, which is selected from an integer between 1 and 51, preferably an integer between 10 and 51, and more preferably an integer between 20 and 51; fluorinated polycaprolactone, the copolymer shown in Formula I, and an organic solvent are mixed to obtain an organic phase; the organic phase, perfluorinated carbon, and water are mixed to obtain ultrasonically labeled and targeted nanodroplets.

[0033] This application uses a heat shock protein targeting peptide and a copolymer (MAL-PEG-PCL) as raw materials, and prepares a copolymer (Pep-PEG-PCL) with targeting function as shown in Formula I through a thiol-maleimide click chemistry reaction. The heat shock protein targeting peptide is a heat shock protein targeting peptide containing active thiol groups. The thiol groups in the heat shock protein targeting peptide can undergo a Michael addition click chemistry reaction with the maleimide double bond to form a stable thioether bond under mild conditions, thereby covalently linking the targeting peptide to the PEG chain end to obtain the copolymer shown in Formula I.

[0034] Specifically, the click chemistry reaction is preferably carried out at room temperature, with a reaction time preferably of 12h to 72h, more preferably 24h to 48h. In some specific implementations, the heat shock protein targeting peptide and MAL-PEG-PCL are reacted in an organic solvent and a weak base system. The organic solvent is preferably one or more of N,N-dimethylformamide (DMF), tetrahydrofuran (THF), and dimethyl sulfoxide (DMSO), more preferably N,N-dimethylformamide; the weak base is preferably one or more of triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), more preferably triethylamine. The click chemistry reaction has the characteristics of high specificity, high reaction efficiency, and few side reactions, which can ensure the integrity of peptide structure and biological activity, and directionally expose Pep-PEG-PCL on the surface of nanodroplets, providing a molecular basis for subsequent tumor-targeted recognition.

[0035] In some specific implementations, the mass ratio of the heat shock protein targeting peptide to the copolymer shown in Formula III is preferably 1:3~7, more preferably 1:4~6. The heat shock protein targeting peptide is a functional peptide molecule containing free thiol groups, capable of specifically recognizing heat shock protein receptors highly expressed on the surface of tumor cells. In some specific implementations, the heat shock protein targeting peptide is preferably the HSP70 family targeting peptide GRP78, and the serial number of the targeting peptide GRP78 is preferably WIFPWIQLC, where C in WIFPWIQLC represents Cys (thiol group). The copolymer shown in Formula III is an amphiphilic block polymer with a maleimide active group at one end of a PEG segment and a hydrophobic PCL segment at the other end. The copolymer shown in Formula III is prepared by ring-opening polymerization of maleimide-polyethylene glycol-hydroxy (MAL-PEG-OH) with caprolactone.

[0036] The copolymer represented by Formula III in this application is prepared using MAL-PEG-OH as a macromolecular initiator and caprolactone as a monomer, and undergoes a ring-opening polymerization reaction. Specifically, the ring-opening polymerization reaction is preferably carried out under heating conditions, with a reaction temperature preferably between 100°C and 130°C, more preferably between 110°C and 120°C, and a reaction time preferably between 4 h and 48 h, more preferably between 12 h and 24 h.

[0037] In some specific implementations, the MAL-PEG-OH reacts with caprolactone in an organic solvent system, preferably one or more of anhydrous toluene, xylene, and chlorobenzene, more preferably anhydrous toluene. In this reaction, the terminal hydroxyl groups of the PEG undergo a nucleophilic reaction with the caprolactone monomer under the catalysis of a catalyst, initiating ester bond ring opening and gradual chain growth, forming polycaprolactone segments at the PEG end, thereby obtaining a block copolymer MAL-PEG-PCL with an amphiphilic structure. The catalyst is preferably one or more of stannous isooctanoate, dibutyltin dilaurate, tetrabutyl titanate, and aluminum isopropoxide, more preferably stannous isooctanoate, and the amount of catalyst used is preferably 0.05wt%~0.5wt% of the monomer mass, more preferably 0.1wt%~0.3wt%. By adjusting the feed ratio of caprolactone to MAL-PEG-OH, the degree of polymerization of the PCL segments can be effectively controlled, so that the resulting polymer has both good hydrophobic drug loading capacity and maintains suitable self-assembly properties. In some specific implementations, the mass ratio of caprolactone to MAL-PEG-PCL is preferably 1~5:1, more preferably 1~2:1.

[0038] The fluorinated polycaprolactone (F-PCL) provided in this application is prepared by ring-opening polymerization of caprolactone as a monomer using fluorinated small molecule alcohols or fluorinated small molecule amines as initiators. The initiator is preferably a perfluoroalkyl alcohol and / or a perfluoroalkyl amine. Under the action of a catalyst, the hydroxyl and / or amino groups in the initiator also initiate ring-opening of caprolactone, resulting in a fluorinated alkyl structure at one end and / or on the side chain of the polycaprolactone chain. In some specific implementations, the mass ratio of caprolactone to initiator is preferably 10-20:1, more preferably 16-18:1. The catalyst is preferably one or more of stannous isooctanoate, dibutyltin dilaurate, tetrabutyl titanate, and aluminum isopropoxide, more preferably stannous isooctanoate. The amount of catalyst used is preferably 0.05wt%-0.5wt% of the monomer mass, more preferably 0.1wt%-0.3wt%.

[0039] In some specific implementations, the initiator reacts with the monomer in an organic solvent system, preferably one or more of anhydrous toluene, xylene, and chlorobenzene, more preferably anhydrous toluene. The reaction is preferably carried out under heating conditions, with a reaction temperature preferably between 100°C and 130°C, more preferably between 110°C and 120°C, and a reaction time preferably between 4 h and 48 h, more preferably between 12 h and 24 h. Due to the strong fluorine-fluorine interaction between the fluorinated segments and the perfluorinated carbon molecules, this structure can significantly improve the solubility and compatibility of perfluorinated carbon in polymer systems, thereby enhancing the encapsulation stability of the perfluorinated carbon core by nanodroplets and reducing the risk of leakage of the perfluorinated carbon during bulk cycling.

[0040] After obtaining Pep-PEG-PCL and F-PCL, they are dissolved together with an ultrasound-activated prodrug and a sonosensitizer in an organic solvent to form a homogeneous organic phase. In some specific implementations, the mass ratio of F-PCL, Pep-PEG-PCL, ultrasound-activated prodrug, and sonosensitizer is preferably 5~40:1~8:0.7~9.6:1.4~19.6, more preferably 10~20:2~4:2.4~4.8:4.8~9.6. The organic solvent is preferably one or more of N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, and acetonitrile, more preferably N,N-dimethylformamide, and the amount of organic solvent used is preferably 0.25mL~2mL, more preferably 0.5mL~1mL. At this point, the hydrophobic segments are in an extended state in the solvent. When perfluorinated carbon liquid is added to the system and stirring is continued, the fluorinated segments preferentially interact with the perfluorinated carbon, encapsulating it and forming a preliminary fluoropolymer-perfluorinated carbon composite core. Subsequently, an aqueous phase is slowly added dropwise to this organic phase. As the solvent polarity changes rapidly, the organic solvent and water are replaced, and the amphiphilic polymer begins to self-assemble: the polycaprolactone segments and fluorinated segments aggregate inward due to hydrophobic interactions, encapsulating the perfluorinated carbon and hydrophobic drug molecules, while the polyethylene glycol segments extend outward to form a hydrophilic shell. Simultaneously, the targeting peptide grafted to the PEG ends is exposed on the surface of the nanodroplet. Ultimately, a core-shell nanodroplet structure is formed, with a liquid core containing perfluorinated carbon at the center, surrounded by a fluoropolymer stabilizing layer, a hydrophobic drug-encapsulated layer, and a hydrophilic PEG-peptide shell. In some specific implementations, the volume ratio of the organic phase, perfluorinated carbon and water is preferably 0.1~4:0.001~0.1:0.4~16, more preferably 1~2:0.01~0.02:4~8.

[0041] This application also provides an application of the aforementioned ultrasound-labeled and guided targeted nanodroplet carrier and the ultrasound-labeled and guided targeted nanodroplets, which can be used in an ultrasound-labeled and guided targeted nanodelivery system. The nanodroplets provided in this application have a surface modified with heat shock protein-targeting peptides and are internally co-loaded with perfluorinated carbon, an ultrasound-activated prodrug, and a sonosensitive agent. These nanodroplets exhibit excellent ultrasound response characteristics and molecular targeting recognition capabilities.

[0042] In the application of the ultrasound-labeled guided targeted nanodelivery system, ultrasound treatment of the target tissue region generates controllable acoustic-thermal and mechanical effects, thereby inducing abnormally high expression of heat shock proteins (HSPs) on the cell surface or inside the region, forming an ultrasound-labeled microenvironment. The nanodroplets, through surface-modified heat shock protein targeting peptides, specifically bind to the ultrasound-induced highly expressed heat shock protein targets (or promoters), thereby achieving active recognition and enrichment of the ultrasound-treated area and improving the spatial positioning accuracy and tissue selectivity of the nanodelivery system. In some specific implementations, the heat shock protein targets include one or more of all heat shock proteins and related proteins, such as HSP60, HSP70, HSP90α, and HSP90β, preferably one or more of HSP27, HSP60, HSP70, HSP90α, HSP90β, HSP105, and HSP40.

[0043] Furthermore, ultrasound can trigger a liquid-gas phase transition in the perfluorinated carbon within the nanodroplets, enhancing the local acoustic response signal and enabling guided imaging. Simultaneously, the microjets and cavitation effects generated by the phase transition can increase cell membrane permeability, promoting the delivery of nanocarriers into cells. The acoustic sensitizer generates an acoustic dynamic effect under ultrasound, further enhancing the local biological response. Therefore, the nanodroplets not only serve as drug delivery units but also function as both ultrasound-responsive labeling units and target recognition units, constructing an ultrasound-labeled guided targeted nanodelivery system with ultrasound labeling, active recognition, response enhancement, and precise delivery.

[0044] This application also provides the above-mentioned ultrasound-labeled and guided targeted nanodroplet carrier and its application in the preparation of drugs for treating tumors. In some specific implementations, the tumor is one or more of CT26 colon cancer, MC38 colon cancer, 4T1 breast cancer, and H22 liver cancer. The nanodroplets provided in this application are modified with heat shock protein targeting peptides and co-loaded with perfluorinated carbon, ultrasound-activated prodrugs, and sonosensitive agents. The nanodroplets are ultrasound-responsive. By using ultrasound to precisely treat the tumor site, abnormal expression of heat shock proteins in tumor cells can be induced, thereby enabling the nanodroplets to be efficiently enriched in the tumor region with the help of the targeting peptides. Ultrasound further triggers the liquid-gas phase transition of perfluorinated carbon, promoting the responsive release of the drug. While expanding the difference in prodrug accumulation between the tumor and other tissues, the large number of free radicals generated instantaneously by ultrasound and perfluorinated carbon further activate the prodrug, inducing a reduction reaction, thereby achieving high-level enrichment, release, and activation of the original drug at the tumor site.

[0045] Specifically, the ultrasound-labeled and targeted nanodroplets provided in this application achieve precise treatment in vivo through two-step ultrasound regulation: First, ultrasound pre-stimulation of the tumor region induces high expression of heat shock proteins in tumor cells, thereby enhancing the active targeting and enrichment of Pep-PEG-PCL on tumor tissue; then, after the nanodroplets are enriched at the tumor site, a second ultrasound is applied to trigger a liquid-gas phase transition of perfluorinated carbon and generate a cavitation effect, promoting polymer shell rupture and drug release. At the same time, the sonosensitive agent TBR generates active free radicals under the action of ultrasound. These free radicals further convert DOX-N3 into doxorubicin, achieving in-situ activation and efficient killing of the prodrug at the tumor site.

[0046] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0047] The present application will be further described below with reference to the embodiments.

[0048] Unless otherwise specified, all raw materials used in the embodiments of this application are obtained through commercial purchases; unless otherwise specified, room temperature or ambient temperature in the embodiments of this application refers to 25±3℃.

[0049] In the embodiments of this application, the symbols are: US for therapeutic ultrasound and FUS for focused ultrasound.

[0050] Example 1

[0051] Synthesis of Pep-PEG-PCL@P-NPs: A) Synthesis of MAL-PEG-PCL: 5.0 g of 1.0 mmol / L maleimide-polyethylene glycol 5000-hydroxyl (Mal-PEG) was added. 5k 4.9 g of 42.93 mmol of caprolactone was dissolved in 100 mL of dry toluene, and 30 μL of 1.25 g / mL stannous isooctanoate was added. The mixture was refluxed at 115 °C for 12 h. After the toluene was dried by rotary evaporation, the mixture was precipitated with 500 mL of diethyl ether to obtain the white solid MAL-PEG-PCL shown in Formula III.

[0052] Formula III; where n is the degree of polymerization of the PEG segment, n is 114, and m is the degree of polymerization of the PCL segment, m is 51.

[0053] The structure of the MAL-PEG-PCL was characterized by 1H NMR spectroscopy (500M, solvent: deuterated chloroform). The results are shown in Figure 2. Figure 2 is the 1H NMR spectrum of the MAL-PEG-PCL prepared in Example 1 of this application. As can be seen from Figure 2, the position of the characteristic peak is consistent with the expected structure, indicating the successful synthesis of the MAL-PEG-PCL structure of Formula III copolymer.

[0054] B) Synthesis of Pep-PEG-PCL: 100.0 mg MAL-PEG-PCL and 18.0 mg GRP78 targeting peptide (serial number WIFPWIQLC) prepared in step A) were dissolved in 10 mL N,N-dimethylformamide (DMF) and stirred. 4.2 μL of triethylamine (TEA) was added dropwise, and the reaction was carried out at room temperature for 48 h. After the reaction, the product was dialyzed three times sequentially with DMF and double-distilled water using a 3500 Da dialysis bag. The product was then lyophilized to obtain 106.3 mg of the white solid Pep-PEG-PCL shown in Formula I. The reaction process is as follows: The structure of the Pep-PEG-PCL polymer was characterized by 1H NMR spectroscopy (500M, solvent: deuterated chloroform). The results are shown in Figure 3. Figure 3 is the 1H NMR spectrum of the Pep-PEG-PCL prepared in Example 1 of this application. As can be seen from Figure 3, the characteristic peak of maleimide disappeared, which proves the successful bonding of the polypeptide in the structure of the copolymer Pep-PEG-PCL of Formula I. Formula I; where, peptide is a GRP78-targeting polypeptide (serial number WIFPWIQLC) chain segment.

[0055] C) Synthesis of F-PCL: 0.25 g of 0.72 mmol of 2,2,3,3,4,4,5,5,6,6,7,7,7-tridecylfluoroheptane-1-amine and 4.3 g of 37.67 mmol of caprolactone were dissolved in 50 mL of dry toluene. 30 μL of 1.25 g / mL stannous isooctanoate was added, and the mixture was refluxed at 115 °C for 12 h. After the toluene was dried by rotary evaporation, the mixture was precipitated with 200 mL of diethyl ether to obtain the white solid F-PCL shown in Formula II. Formula II; wherein R is tridecylfluoroheptyl, a is the degree of polymerization, and a is 51.

[0056] The structure of F-PCL was characterized by 1H NMR (500M, solvent: deuterated chloroform). The results are shown in Figure 4. Figure 4 is the 1H NMR spectrum of F-PCL prepared in Example 1 of this application. As can be seen from Figure 4, the position of the characteristic peak is consistent with the target molecular structure, indicating the successful synthesis of the F-PCL structure shown in Formula II.

[0057] D) Synthesis of Pep-PEG-PCL@P-NPs: The weighed F-PCL (20.0 mg) prepared in step C) and Pep-PEG-PCL (4.0 mg) prepared in step B) were dissolved in 1 mL of N,N-dimethylformamide and stirred until homogeneous at room temperature in the dark. Then, perfluorohexane liquid (0.02 mL, 2%, v / v) was added and stirring continued for 10 min. 4 mL of double-distilled water was gradually added and stirred for 20 min. After centrifugation at 3000 rpm for 5 min, the mixture was further concentrated. Subsequently, the mixture was dialyzed twice using a 3500 Da dialysis bag to remove residual organic solvents, yielding Pep-PEG-PCL@P-NPs.

[0058] Example 2

[0059] Synthesis of Pep-PEG-PCL@P / D / T-NPs nanodroplets: Pep-PEG-PCL@P / D / T-NPs were prepared according to the same steps A), B), and C) as in Example 1, except that DOX-N3 and TBR were added in step D). Step D) of Example 2 is as follows: Step D) Synthesis of Pep-PEG-PCL@P / D / T-NPs: The weighed F-PCL (20.0 mg) prepared in step C), Pep-PEG-PCL (4.0 mg) prepared in step B), doxorubicin prodrug containing azide group (DOX-N3, 4.8 mg) and riboflavin tetrabutyrate (TBR, 9.6 mg) were dissolved in 1 mL of N,N-dimethylformamide and stirred evenly at room temperature in the dark. Then, perfluorinated carbon liquid (0.02 mL, 2%, v / v) was added and stirring was continued for 10 min. 4 mL of double-distilled water was gradually added and stirred for 20 min. After centrifugation at 3000 rpm for 5 min, the solution was further concentrated. Then, the solution was dialyzed twice using a 3500 Da dialysis bag to remove residual organic solvents, yielding Pep-PEG-PCL@P / D / T-NPs nanodroplets. In these Pep-PEG-PCL@P / D / T-NPs nanodroplets, P, D, and T represent perfluorinated carbon, the ultrasound-activated prodrug DOX-N3, and the sonosensitive agent riboflavin tetrabutyrate (TBR), respectively.

[0060] The Pep-PEG-PCL@P / D / T-NPs nanodroplets were characterized, and the results are shown in Figures 5-9. Figure 5 is a TEM image of the Pep-PEG-PCL@P / D / T-NPs prepared in Example 2 of this application; Figure 6 is an elemental mapping analysis diagram of the Pep-PEG-PCL@P / D / T-NPs prepared in Example 2 of this application; Figure 7 is a dynamic light scattering diagram of the Pep-PEG-PCL@P / D / T-NPs prepared in Example 2 of this application; Figure 8 is a UV-Vis absorption spectrum of the Pep-PEG-PCL@P / D / T-NPs prepared in Example 2 of this application; and Figure 9 is a Zeta potential measurement diagram of the Pep-PEG-PCL@P / D / T-NPs prepared in Example 2 of this application. Combined with the characterization results in Figures 5-9, the successful synthesis of Pep-PEG-PCL@P / D / T-NPs nanodroplets is demonstrated, and the average size of the nanodroplets is 139.1 ± 1.0 nm.

[0061] Example 3

[0062] Synthesis of PEG-PCL@P / D / T-NPs: The difference between the PEG-PCL@P / D / T-NPs prepared in Example 3 and the Pep-PEG-PCL@P-NPs prepared in Example 2 is that the outer coating polymer of the PEG-PCL@P / D / T-NPs is PEG-PCL, a polymer without peptide targets. The PEG-PCL was commercially available.

[0063] PEG-PCL@P / D / T-NPs were prepared according to the same steps A) and C) as in Example 2, except that the PEG-PCL obtained in step B) was commercially available. Step D) is as follows: Synthesis of PEG-PCL@P / D / T-NPs in step D): The weighed F-PCL (20.0 mg) prepared in step C), the commercially available PEG-PCL (4.0 mg), the doxorubicin prodrug containing an azide group (DOX-N3, 4.8 mg) and riboflavin tetrabutyrate (TBR, 9.6 mg) were dissolved in 1 mL of N,N-dimethylformamide and stirred evenly at room temperature in the dark. Then, perfluorocarbon liquid (0.02 mL, 2%, v / v) was added and stirring was continued for 10 min. 4 mL of double-distilled water was gradually added and stirred for 20 min. After centrifugation at 3000 rpm for 5 min, the solution was further concentrated. Then, the solution was dialyzed twice using a 3500 Da dialysis bag to remove residual organic solvent, yielding PEG-PCL@P / D / T-NPs nanodroplets. In the PEG-PCL@P / D / T-NPs nanodroplets, P, D, and T represent perfluorinated carbon, the ultrasound-activated prodrug DOX-N3, and the sonosensitive agent riboflavin tetrabutyrate (TBR), respectively.

[0064] Example 4

[0065] Synthesis of Pep-PEG-PCL@D / T-NPs: Pep-PEG-PCL@D / T-NPs were prepared according to the same steps A), B), and C) as in Example 2, except that perfluorinated carbon liquid was not added in step D). Step D) of Example 4 is as follows: Step D) Synthesis of Pep-PEG-PCL@D / T-NPs: The weighed F-PCL (20.0 mg) prepared in step C), Pep-PEG-PCL (4.0 mg) prepared in step B), doxorubicin prodrug containing azide group (DOX-N3, 4.8 mg) and riboflavin tetrabutyrate (TBR, 9.6 mg) were dissolved in 1 mL of N,N-dimethylformamide and stirred evenly at room temperature in the dark. 4 mL of double-distilled water was gradually added and stirred for 20 min. After centrifugation at 3000 rpm for 5 min, the mixture was further concentrated. Subsequently, it was dialyzed twice using a 3500 Da dialysis bag to remove residual organic solvents from the system, yielding Pep-PEG-PCL@D / T-NPs.

[0066] Example 5

[0067] Proof-of-concept ultrasound-guided targeting: The expression levels of the heat shock protein GRP78 in CT26 cells after ultrasound were characterized using immunofluorescence staining, flow cytometry, and Western blotting. For immunofluorescence staining, CT26 cells were seeded in glass-bottom cell culture dishes and cultured overnight at 37°C. Cells were then divided into four groups: control group (no heat shock protein targeting peptide and no therapeutic ultrasound treatment), US group (therapeutic ultrasound treatment only, no heat shock protein targeting peptide), Peptide group (heat shock protein targeting peptide only, no therapeutic ultrasound treatment), and Peptide+US group (heat shock protein targeting peptide and therapeutic ultrasound treatment). Prior to therapeutic ultrasound treatment, the Peptide-containing treatment groups (Peptide group and Peptide+US group) were co-incubated with the GRP78 targeting peptide (50 μM) for 2 hours. Subsequently, the US group and Peptide+US group underwent therapeutic ultrasound treatment (ultrasound conditions: 1.5 W / cm²). 2(1.0 MHz, 50% Duty, 5 min). After 8 h, cells were gently washed with PBS and stained with GRP78 mouse polyclonal antibody (1:100 dilution according to the instructions) at 4°C for 1 h. Subsequently, cells were washed and stained with FITC-labeled goat anti-mouse IgG (1:500 dilution according to the instructions) for 30 min. Finally, cells were fixed with 4% PFA after washing and directly observed under a confocal microscope. Quantitative results were obtained by Image J processing. See Figures 10 and 11 for the results. Figures 10 and 11 show the effect of ultrasound stimulation and GRP78-targeting peptide on GRP78 protein expression. The results in Figures 10 and 11 show that the expression level of GRP78 was significantly increased after ultrasound treatment, and the peptide could efficiently bind to the expressed GRP78, verifying its targeting ability.

[0068] Example 6

[0069] Optimal conditions for ultrasound-guided targeted therapy were screened: Flow cytometry was used to characterize the changes in the expression level of the heat shock protein GRP78 in CT26 cells over time after ultrasound. Specifically, CT26 cells were seeded in 12-well plates and cultured overnight at 37°C, then divided into groups: Control, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h. The Control group received neither drug treatment nor ultrasound therapy, while the experimental groups (excluding the Control group) received only ultrasound therapy (ultrasound conditions: 1.5 W / cm²). 2 Cells were subjected to flow cytometry at 1.0 MHz, 50% Duty, for 3 min, without drug administration. After the corresponding time, cells were collected with PBS (pH=7.4) and stained with GRP78 mouse polyclonal antibody (diluted 1:100 according to the manufacturer's instructions) at 4°C for 1 hour. Subsequently, the cells were washed and stained with FITC-labeled goat anti-mouse IgG (diluted 1:500 according to the manufacturer's instructions) for 30 min. Finally, after washing, the cells were fixed with 4% PFA and directly used for flow cytometry analysis. Quantitative results were obtained using FlowJo. The results are shown in Figure 12. Figure 12 shows the change in GRP78 protein over time after sonication as verified by flow cytometry in this application. As shown in Figure 12, the GRP78 expression level was close to its peak 8 h after sonication treatment, providing strong evidence for subsequent drug administration.

[0070] Furthermore, flow cytometry was used to characterize the expression levels of the heat shock protein GRP78 in CT26 cells under different ultrasound conditions. Specifically, CT26 cells were seeded in 12-well plates and cultured overnight at 37°C. Cells were then grouped by controlling different variables, as follows: ① The duration of the therapeutic ultrasound was varied to ensure an ultrasound intensity of 1.5 W / cm². 2① The frequency is 1.0MHz, and the duty cycle is kept constant at 50%. The grouping is: Control, FITC, 1min, 2min, 3min, 4min, 5min; ② The intensity of the therapeutic ultrasound is changed, while ensuring the ultrasound time is 3min, the frequency is 1.0MHz, and the duty cycle is kept constant at 50%. The grouping is: Control, FITC, 0.5W / cm 2 1.0W / cm 2 1.5W / cm 2 2.0W / cm 2 2.5W / cm 2 It should be noted that the Control and FITC groups received neither medication nor therapeutic ultrasound treatment. The difference lies in that the Control group did not use any antibody incubation. Subsequently, the experimental groups other than the Control and FITC groups underwent therapeutic ultrasound treatment. After 8 hours, cells were collected using PBS (pH=7.4), and the experimental groups other than the Control group were stained with GRP78 mouse polyclonal antibody (diluted 1:100 according to the instructions) at 4°C for 1 hour. Afterward, the cells were washed, and the experimental groups other than the Control group were stained with FITC-labeled goat anti-mouse IgG (diluted 1:500 according to the instructions) for 30 minutes. Finally, after washing, the cells were fixed with 4% paraformaldehyde (PFA) fixative for 30 minutes and directly used for flow cytometry testing. Quantitative results were obtained using FlowJo. The results are shown in Figures 13 and 14. Figure 13 shows the changes in GRP78 at different ultrasound times as verified by flow cytometry in this application, and Figure 14 shows the changes in GRP78 at different ultrasound intensities as verified by flow cytometry in this application. As shown in Figures 13 and 14, increasing the ultrasound intensity and prolonging the ultrasound time both promote the expression of GRP78 protein. Subsequently, an intensity of 1.5 W / cm was selected. 2 The optimal ultrasound labeling conditions were 1.0 MHz, 3 min, and 50% duty cycle, which were used for subsequent verification.

[0071] The expression changes of different types of heat shock proteins after ultrasound were evaluated using the same method. The experimental methods were basically the same as described above, except that after cell collection, the corresponding mouse polyclonal antibodies (HSP27, HSP60, HSP70, HSP90α, and HSP90β, diluted 1:100 according to the instructions) were used to stain the experimental groups (excluding the Control group) at 4°C for 1 hour. Other experimental methods were consistent with the previous descriptions. The results are shown in Figure 15, which illustrates the expression changes of different heat shock proteins under ultrasound as verified by flow cytometry. As shown in Figure 15, the expression of heat shock protein targets was significantly increased after physiotherapy ultrasound, demonstrating the broad applicability of this ultrasound-labeled targeted strategy.

[0072] Example 7

[0073] Verification of the level of free radicals induced by ultrasound in Pep-PEG-PCL@P-NPs: The level of free radicals directly affects the reduction efficiency of ultrasound-activated prodrugs (the prodrug DOX-N3 used in this application), especially the efficiency of free radical-mediated self-elimination reactions. Therefore, terephthalic acid (TA) was used to detect the level of hydroxyl radicals generated by unloaded Pep-PEG-PCL@P-NPs under different therapeutic ultrasound conditions to indirectly characterize their ability to generate reduced free radicals. The specific groupings are shown in Figure 16. Figure 16 shows the changes in the ability of Pep-PEG-PCL@P-NPs to generate hydroxyl radicals under different ultrasound conditions. Group G1 (Control group) received neither physiotherapy ultrasound treatment nor Pep-PEG-PCL@P-NPs, only containing solvent. Groups G2 to G11 all received Pep-PEG-PCL@P-NPs (at a polymer concentration of 1.0 mM) and TA (1.0 mM). Group G12 received Pep-PEG-PCL@P-NPs (at a polymer concentration of 1.0 mM) and TA (2.0 mM). Groups G2 to G12 were treated with different physiotherapy ultrasound conditions, specifically: Group G2 ultrasound condition: 0.5 W / cm 2 1.0MHz, 50% Duty, 5min; G3 group ultrasound conditions: 1.0W / cm 2 1.0MHz, 50% Duty, 5min; G4 group ultrasound conditions: 1.5W / cm 2 1.0MHz, 50% Duty, 5min; G5 group ultrasound conditions: 2.0W / cm 2 1.0MHz, 50% Duty, 5min; G6 group ultrasound conditions: 2.0W / cm 2 1.0MHz, 50% Duty, 1min; G7 group ultrasound conditions: 2.0W / cm 2 1.0MHz, 50% Duty, 2min; G8 group ultrasound conditions: 2.0W / cm 2 1.0MHz, 50% Duty, 3min; G9 group ultrasound conditions: 2.0W / cm 2 1.0MHz, 50% Duty, 4min; G10 group ultrasound conditions: 2.0W / cm 2 1.0MHz, 50% Duty, 5min; G11 group ultrasound conditions: 2.0W / cm 2 3.0MHz, 50% Duty, 5min; G12 group ultrasound conditions: 2.0W / cm 21.0 MHz, 50% Duty, 5 min. After physiotherapy ultrasound treatment, the solution was diluted with double-distilled water and tested using a fluorescence spectrophotometer. The emission wavelength was set to 310 nm, and the excitation wavelength range was set to 330 nm to 700 nm. The results are shown in Figure 16. As can be seen from Figure 16, Pep-PEG-PCL@P-NPs combined with TBR significantly amplified the level of free radicals.

[0074] Example 8

[0075] Cytotoxicity validation of nanodroplets Pep-PEG-PCL@P / D / T-NPs: The cytotoxicity of Pep-PEG-PCL@P / D / T-NPs in CT26 cells and its cytotoxicity under combined sonication were characterized using the CCK-8 assay. Specifically, CT26 cells were seeded in 96-well plates and cultured overnight in a 37°C incubator. They were then divided into the following groups: DOX group (only doxorubicin was administered, without physiotherapy or sonication), DOX-N3 group (only DOX-N3 was administered, without physiotherapy or sonication), Pep-PEG-PCL@P / D / T-NPs group (loaded with DOX-N3 and TBR sonication agent, without physiotherapy or sonication), Pep-PEG-PCL@P-NPs group (without DOX-N3 or TBR sonication agent, without physiotherapy or sonication), and Pep-PEG-PCL@P / D / T-NPs+US group (loaded with DOX-N3 and TBR sonication agent and subjected to physiotherapy or sonication). DOX-N3 (final concentrations of 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, and 1 mM), Pep-PEG-PCL@P / D / T-NPs (final concentrations based on DOX-N3 dosages of 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, and 1 mM), and Pep-PEG-PCL@P-NPs (final concentration consistent with the corresponding polymer carrier concentration in the Pep-PEG-PCL@P / D / T-NPs group) were added according to the corresponding groups. After administration, the corresponding experimental groups were subjected to sonication (ultrasonic conditions: 2.0 W / cm²). 2 (1.0 MHz, 50% Duty, 5 min). After incubation for 24 hours, the supernatant was removed, and culture medium containing 10% CCK-8 solution was added to cover the cells. The absorbance was measured at 450 nm using a microplate reader to characterize cytotoxicity. The results are shown in Figure 17, which is a cytotoxicity verification diagram of Pep-PEG-PCL@P / D / T-NPs. As shown in Figure 17, Pep-PEG-PCL@P / D / T-NPs exhibited 76-fold reduced toxicity compared to DOX, and its efficacy was reduced under therapeutic ultrasound, verifying its excellent safety and selectivity.

[0076] Example 9

[0077] Validation of the endocytosis capacity and co-localization of heat shock proteins of Pep-PEG-PCL@P / D / T-NP nanodroplets: Immunofluorescence staining was used to characterize the endocytosis capacity and co-localization of heat shock proteins of Pep-PEG-PCL@P / D / T-NP nanodroplets.

[0078] For the endocytosis experiment, CT26 cells were seeded in glass-bottomed cell culture dishes and cultured overnight in a 37°C incubator. They were then divided into four groups: DOX (only the original drug doxorubicin was administered, without sonication), DOX-N3 (only the prodrug DOX-N3 was administered, without sonication), Pep-PEG-PCL@P / D / T-NPs (loaded with DOX-N3 and TBR, without sonication), and Pep-PEG-PCL@P / D / T-NPs+US (loaded with DOX-N3 and TBR and subjected to sonication). The treatment group (Pep-PEG-PCL@P / D / T-NPs+US) underwent sonication (ultrasound conditions: 2.0 W / cm²). 2 Two hours after administration (1.0 MHz, 50% Duty, 5 min), cells were co-incubated with Pep-PEG-PCL@P / D / T-NPs (at a dose equivalent to 50 μM DOX). Four hours after administration, cells were gently washed with PBS and stained with Hoechst 33342 dye (1:1000 dilution according to the instructions) at room temperature for 20 minutes. After washing, cells were fixed with 4% PFA for 30 minutes and directly observed under a confocal microscope. Quantitative results were obtained using ImageJ. The results are shown in Figures 18 and 19. Figures 18 and 19 are assessments of the endocytic capacity of Pep-PEG-PCL@P / D / T-NPs in this application. As shown in Figures 18 and 19, the nanomedicine Pep-PEG-PCL@P / D / T-NPs is more readily endocytosed by tumor cells compared to small molecule drugs. Furthermore, the modification of the peptides after ultrasound labeling further enhanced the endocytosis of the nanomedicine by cells.

[0079] For the co-localization experiment, CT26 cells were seeded in glass-bottomed cell culture dishes and cultured overnight in a 37°C incubator. They were then divided into three groups: a Control group (no drug administration and no sonication), a Pep-PEG-PCL@P / D / T-NPs group (loaded with the prodrug DOX-N3 and the sonosensitive agent TBR, without sonication), and a Pep-PEG-PCL@P / D / T-NPs+US group (loaded with the prodrug DOX-N3 and the sonosensitive agent TBR and subjected to sonication). Each group underwent sonication treatment (ultrasound conditions: 1.5 W / cm²) 8 hours before drug administration.2 The treatment group (Pep-PEG-PCL@P / D / T-NPs+US group), which included physiotherapy ultrasound treatment (1.0MHz, 3min, 50% Duty), required physiotherapy ultrasound treatment 4 hours after drug administration (ultrasound conditions: 2.0W / cm²). 2 (1.0 MHz, 50% Duty, 5 min). Four hours after therapeutic ultrasound treatment, cells were gently washed with PBS and stained with GRP78 mouse polyclonal antibody (1:100 dilution according to the instructions) at 4°C for 1 hour. Afterward, cells were washed and stained with FITC-labeled goat anti-mouse IgG (1:500 dilution according to the instructions) for 30 min. After washing, cells were stained with Hoechst 33342 dye (1:1000 dilution according to the instructions) at room temperature for 20 min. Finally, after washing, cells were fixed with 4% PFA for 30 min and directly observed under a confocal microscope. The results are shown in Figure 20. Figure 20 shows the co-localization of Pep-PEG-PCL@P / D / T-NPs and heat shock protein GRP78 in this application, where heat shock protein GRP78 is FITC-labeled. As shown in Figure 20, ultrasound simultaneously increased heat shock protein expression and drug endocytosis, which is beneficial for the efficient enrichment and targeting of nanomedicines in vivo.

[0080] Example 10

[0081] Cell viability and death verification of nanodroplets Pep-PEG-PCL@P / D / T-NPs: Immunofluorescence staining was used to characterize Pep-PEG-PCL@P / D / T-NPs and their killing effect on CT26 cells under combined ultrasound. Specifically, CT26 cells were seeded in 12-well plates and cultured overnight at 37°C. They were then divided into several groups: Control group (no drug administration or sonication), US group (sonication only, no drug administration), DOX-N3 group (only DOX-N3 prodrug administered, no sonication), DOX-N3+TBR+US group (DOX-N3 prodrug and sonication agent TBR administered, plus sonication), Pep-PEG-PCL@P / D / T-NPs group (loaded with DOX-N3 prodrug and sonication agent TBR, no sonication), and Pep-PEG-PCL@P / D / T-NPs+US group (loaded with DOX-N3 prodrug and sonication agent TBR, plus sonication). All nanoparticle (NP) drug administration groups underwent sonication treatment (ultrasound conditions: 1.5 W / cm²) 8 hours prior to drug administration. 2Treatment groups receiving physiotherapy ultrasound treatment (US group, DOX-N3+TBR+US group, Pep-PEG-PCL@P / D / T-NPs+US group) were required to undergo physiotherapy ultrasound treatment 4 hours after drug administration (ultrasound conditions: 2.0 W / cm²). 2 (1.0 MHz, 50% Duty, 5 min). Cells were then incubated at 37°C for 24 hours, the supernatant was removed, and the cells were gently washed twice with PBS. Subsequently, cells were stained at 37°C with calcitonin-AM (Calcein-AM, 1:1000 dilution) and propylene glycol iodide (PI, 1:1000 dilution) for 30 minutes, and immediately observed using a fluorescence microscope. The results are shown in Figure 21. Figure 21 shows the cell viability and mortality staining after sonication with Pep-PEG-PCL@P / D / T-NPs of this application. In Figure 21, green represents Calcitonin-AM, and red represents PI. As shown in Figure 21, compared with other control groups, Pep-PEG-PCL@P / D / T-NPs combined with sonication significantly killed tumor cells.

[0082] Example 11

[0083] Validation of the immunogenic cell death (ICD) effect of nanodroplets Pep-PEG-PCL@P / D / T-NPs at the cellular level: The ability of Pep-PEG-PCL@P / D / T-NPs combined with ultrasound to induce ICD effects on the CT26 cell model was characterized by flow cytometry and ELISA kits, respectively. For calreticulin (CRT) assay, specifically, CT26 cells were seeded in 12-well plates and cultured overnight at 37°C. Cells were then divided into the following groups: PBS group (no drug administration or sonication), US group (sonication only, no drug administration), DOX-N3 group (only DOX-N3 administered, no sonication), DOX-N3+TBR+US group (DOX-N3 and TBR administered, sonication), Pep-PEG-PCL@P / D / T-NPs group (DOX-N3 and TBR loaded, no sonication), and Pep-PEG-PCL@P / D / T-NPs+US group (DOX-N3 and TBR loaded, sonication). Among them, the nanoparticle (NP) administration groups (Pep-PEG-PCL@P / D / T-NPs group and Pep-PEG-PCL@P / D / T-NPs+US group) all required physical therapy ultrasound treatment (ultrasound conditions: 1.5W / cm) 8 hours before administration. 2Treatment groups receiving physiotherapy ultrasound treatment (US group, DOX-N3+TBR+US group, and Pep-PEG-PCL@P / D / T-NPs+US group) were required to undergo physiotherapy ultrasound treatment 4 hours after drug administration (ultrasound conditions: 2.0 W / cm²). 2 The cells were incubated at 1.0 MHz, 50% Duty, for 5 min. After incubation at 37°C for 24 hours, cells were collected with PBS (pH=7.4), stained with AF647-labeled CRT antibody at room temperature for 30 minutes, and finally fixed with 4% PFA after washing. The cells were then directly used for flow cytometry analysis, and quantitative results were obtained using FlowJo. The results are shown in Figure 22. Figure 22 illustrates the ability of Pep-PEG-PCL@P / D / T-NPs combined with ultrasound to induce ICD effects in CT26 cells. Figure 22A shows the expression changes of calreticulin (CRT) in different experimental groups; Figure 22B shows the changes in the content of high-mobility group box 1 (HMGB-1) efflux in different experimental groups; and Figure 22C shows the changes in the content of adenosine triphosphate (ATP) in different experimental groups. As shown in Figure 22, compared with the control group, Pep-PEG-PCL@P / D / T-NPs combined with ultrasound significantly induced increased CRT expression.

[0084] For the detection of high-mobility group box 1 (HMGB-1) and adenosine triphosphate (ATP), the tests were performed according to the instructions of the respective kits. The results are shown in Figure 22. As can be seen from Figure 22, compared with the control group, Pep-PEG-PCL@P / D / T-NPs combined with ultrasound significantly induced an increase in HMGB-1 protein and extracellular ATP levels, strongly demonstrating that this ultrasound-labeled targeted therapy strategy can effectively induce ICD effects.

[0085] Example 12

[0086] Tumor suppression experiment validation of Pep-PEG-PCL@P / D / T-NPs nanodroplets combined with focused ultrasound: To establish a CT26 colon cancer model, 6-8 week old female BALB / c mice were subcutaneously injected with CT26 cells (1.0 × 10⁻⁶ cells per mouse). 6 (each cell). When the tumor volume reaches approximately 100 mm... 3At that time, the mice were randomly divided into nine groups (n = 5): PBS group (Group 1, control group, neither drug nor focused ultrasound treatment), FUS group (Group 2, focused ultrasound treatment only), DOX-N3+TBR+FUS group (Group 3, DOX-N3 prodrug and TBR sonostatin administered, focused ultrasound treatment, DOX-N3 dose 5.0 mg / kg, TBR dose 10.0 mg / kg), Pep-PEG-PCL@P-NPs group (Group 4, no DOX-N3 prodrug and TBR sonostatin loaded, perfluorinated carbon loaded, no focused ultrasound treatment, dose calculated based on total polymer mass, 25.0 mg / kg), Pep-PEG-PCL@P-NPs+FUS group (Group 5, no DOX-N3 prodrug and TBR sonostatin loaded, perfluorinated carbon loaded, focused ultrasound treatment, dose calculated based on total polymer mass, 25.0 mg / kg), Pep-PEG-PCL@P / D / T-NPs group (Group 6, PBS group ... TBR prodrug and TBR loaded, perfluorinated carbon loaded, focused ultrasound treatment, dose calculated based on total polymer mass, 25.0 mg / kg), Pep-PEG-PCL@P / D / T-NPs group (Group 6, PBS group, no TBR prodrug and TBR loaded, perfluorinated carbon loaded, focused ultrasound treatment, dose calculated based on total polymer mass, 25.0 mg / kg), Pep-PEG-PCL@P / D / T-NPs group (Group 6, P / D / T-NPs group, P / D / T / T-NPs group, P / D / T / T-NPs group, P / D / T / T / T-NPs group, P / D / T / T / Group 7 (loaded with prodrug DOX-N3, sonosensitive agent TBR, and perfluorocarbon, without focused ultrasound treatment, dose calculated based on DOX-N3 dose of 5.0 mg / kg), PEG-PCL@P / D / T-NPs+FUS group (Group 7, loaded with prodrug DOX-N3, sonosensitive agent TBR, and perfluorocarbon, without modification of heat shock protein targeting peptide, treated with focused ultrasound, dose calculated based on DOX-N3 dose of 5.0 mg / kg), Pep-PEG-PCL The @D / T-NPs+FUS group (group 8, loaded with the prodrug DOX-N3 and the sonosensitive agent TBR, without perfluorocarbon loading, and subjected to focused ultrasound treatment, with the dose calculated based on a DOX-N3 dose of 5.0 mg / kg) and Pep-PEG-PCL@P / D / T-NPs+FUS (group 9, loaded with the prodrug DOX-N3, the sonosensitive agent TBR, and perfluorocarbon, and subjected to focused ultrasound treatment, with the dose calculated based on a DOX-N3 dose of 5.0 mg / kg) were used. Mice received the corresponding drug treatment and ultrasound treatment on days 0 and 3, respectively, and were observed for 60 days. When the tumor volume of the mice reached 1500 mm², the treatment was continued. 3 The tumor suppression endpoint was considered reached in mice. The small molecule prodrug DOX-N3 and other nanomedicines were administered intravenously, while TBR was administered intraperitoneally. It is important to note that all experimental groups (groups 4, 5, 6, 8, and 9) receiving the peptide-targeted (Pep) nanomedicine underwent focused ultrasound treatment (ultrasound conditions: 1.5 W / cm²) 8 hours prior to administration. 2 The ultrasound was performed at 1.0 MHz for 3 minutes at 50% Duty. All experimental groups requiring combined focused ultrasound (FUS) (Groups 2, 3, 5, 7, 8, and 9) used the same ultrasound conditions (2.0 W / cm²). 2(1.0 MHz, 50% Duty, 5 min). The results are shown in Figure 23. Figure 23 illustrates the tumor-suppressing effect of Pep-PEG-PCL@P / D / T-NPs combined with focused ultrasound in this application. Figure 23A is a schematic diagram of the drug administration regimen for the tumor suppression experiment, Figure 23B is a curve showing the tumor volume change, and Figure 23C is a survival observation graph. As shown in Figure 23, compared with other experimental groups, the Pep-PEG-PCL@P / D / T-NPs+FUS (G9) group achieved complete tumor cure with no recurrence within 60 days, and the survival time of mice was significantly prolonged, demonstrating the superiority and safety of this treatment strategy.

[0087] Example 13

[0088] Validation of changes in intracellular immune cells in tumor cells induced by Pep-PEG-PCL@P / D / T-NPs combined with focused ultrasound: The experimental design and grouping were the same as in Example 12, except that mice were euthanized on day 6 of treatment, and tumor tissue was extracted. Tumor cells were extracted by digestion and grinding with tumor homogenate. After washing with PBS, immune cells were stained with corresponding fluorescent antibodies for 30 min, including FITC-conjugated anti-CD3, APC / Cy7-conjugated anti-CD45, APC-conjugated anti-CD8a, PE-conjugated anti-CD4, APC-conjugated anti-CD11b, APC-conjugated anti-F4 / 80, PE / Cy7-conjugated anti-CD80, etc. After further washing, the cells were fixed with 4% PFA solution for 30 min and then used for flow cytometry. The quantitative results were obtained by FlowJo processing. The results are shown in Figure 24. Figure 24 shows the changes in immune cell levels induced by Pep-PEG-PCL@P / D / T-NPs combined with focused ultrasound in this application, where Figure 24A shows CD3. + Statistical graph of T cell content changes. Figure 24B shows CD3. + CD4 + Statistical graph of T cell content changes. Figure 24C shows CD3. + CD8 + Figure 24D shows the statistical chart of changes in T cell content, and Figure 24D shows the statistical chart of changes in M1 macrophage content. As shown in Figure 24, compared to the control group, Pep-PEG-PCL@P / D / T-NPs combined with focused ultrasound can significantly upregulate CD3+, etc. + T cells, CD4 + T cells, CD8 + Levels of immune cells, including T cells and M1 macrophages.

[0089] Example 14

[0090] Changes in intratumoral cytokines induced by Pep-PEG-PCL@P / D / T-NPs combined with focused ultrasound: The experimental design and grouping were the same as in Example 12, except that mice were euthanized on day 6 of treatment, and tumor tissue was extracted. Tumor homogenate was extracted using tumor homogenate solution. The samples were processed according to the ELISA kit instructions and tested at 450 nm using a microplate reader. The results are shown in Figure 25. Figure 25 shows the changes in intratumoral cytokine levels induced by Pep-PEG-PCL@P / D / T-NPs combined with focused ultrasound. Figure 25A shows the statistical changes in interleukin-6 (IL-6) content, Figure 25B shows the statistical changes in necrosis factor-α (TNF-α) content, Figure 25C shows the statistical changes in interferon-γ (IFN-γ) content, and Figure 25D shows the statistical changes in interleukin-2 (IL-2) content. As shown in Figure 25, compared with the control group, Pep-PEG-PCL@P / D / T-NPs combined with focused ultrasound can significantly upregulate the level of intratumoral cytokines, proving that it effectively enhances the immune response of mice.

[0091] Example 15

[0092] Validation of tissue distribution of active drug after combined focused ultrasound with Pep-PEG-PCL@P / D / T-NPs nanodroplets: To establish a CT26 colon cancer model, 6-8 week old female BALB / c mice were subcutaneously injected with CT26 cells (1.0 × 10⁻⁶ cells per mouse). 6 (each cell). When the tumor volume reaches approximately 100 mm... 3Mice were randomly divided into four groups (n = 4): the DOX-N3+TBR+FUS group (only the prodrug DOX-N3 and the sonosensitive agent TBR were administered, followed by focused ultrasound treatment; the DOX-N3 dose was 25.0 mg / kg, and the TBR dose was 50.0 mg / kg); the Pep-PEG-PCL@P / D / T-NPs group (loaded with the prodrug DOX-N3, the sonosensitive agent TBR, and perfluorocarbon, without focused ultrasound treatment; the dose was calculated based on the DOX-N3 dose of 25.0 mg / kg); and the Pep-PEG-PCL@P / D / T-NPs group. The PEG-PCL@D / T-NPs+FUS group (loaded with the prodrug DOX-N3 and the sonosensitive agent TBR, without perfluorinated carbon, and subjected to focused ultrasound treatment, with the dose calculated based on a DOX-N3 dose of 25.0 mg / kg) and the Pep-PEG-PCL@P / D / T-NPs+FUS group (loaded with the prodrug DOX-N3, the sonosensitive agent TBR, and perfluorinated carbon, and subjected to focused ultrasound treatment, with the dose calculated based on a DOX-N3 dose of 25.0 mg / kg). The small molecule prodrug DOX-N3 and other nanomedicines were administered intravenously, while TBR was administered intraperitoneally. It is important to note that all experimental groups receiving the peptide-targeted (Pep) nanomedicine (Pep-PEG-PCL@P / D / T-NPs, Pep-PEG-PCL@D / T-NPs+FUS, and Pep-PEG-PCL@P / D / T-NPs+FUS) required focused ultrasound treatment (ultrasound conditions: 1.5 W / cm²) 8 hours prior to administration. 2 (1.0 MHz, 3 min, 50% Duty), and all experimental groups requiring combined focused ultrasound (FUS) used the same ultrasound conditions (2.0 W / cm²). 2 (1.0 MHz, 50% Duty, 5 min). Mice were euthanized at 12 h, and tumor, heart, liver, spleen, lung, kidney, and muscle tissues were extracted. PBS was added, and the tissues were ground and centrifuged to extract the supernatant. An equal volume of acetonitrile was added for extraction, followed by centrifugation. The supernatant was used for high-performance liquid chromatography (HPLC) to determine the content of active DOX. The results are shown in Figure 26. Figure 26 shows the distribution of active drugs in various tissues after Pep-PEG-PCL@P / D / T-NPs combined with focused ultrasound. As shown in Figure 26, the level of active drugs in the Pep-PEG-PCL@P / D / T-NPs+FUS treatment group was significantly higher than that in other treatment groups, indicating that this ultrasound-labeled targeted strategy can effectively improve the enrichment level of active drugs in tumors, showing significant differences compared to other tissues.

[0093] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this invention should be included within the scope of protection of this invention. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. An ultrasonically labeled and guided targeted nanodroplet carrier, characterized in that, It includes a core and a polymer layer covering the surface of the core; the core is perfluorinated carbon; the polymer layer includes fluorinated polycaprolactone and a copolymer of Formula I; Formula I; wherein, peptide is a heat shock protein targeting polypeptide containing thiol, n is the degree of polymerization, which is selected from an integer between 1 and 111, m is the degree of polymerization, which is selected from an integer between 1 and 51; the fluorinated polycaprolactone can dissolve perfluorinated carbon.

2. The nanodroplet carrier according to claim 1, characterized in that, The perfluorocarbon includes one or more of perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoropropane, perfluorobutane, and perfluoro-15-crown-5-ether; the fluorinated polycaprolactone has the structure shown in Formula II. Formula II; wherein R is a perfluoroalkyl group, a is the degree of polymerization, and a is an integer selected from 1 to 51; the mass ratio of the perfluorocarbon, the fluorinated polycaprolactone and the copolymer shown in Formula I is 10 to 80: 5 to 40: 1 to 8.

3. A type of ultrasonically labeled and guided targeted nanodroplet, characterized in that, It includes a core and a polymer layer covering the surface of the core; the core is perfluorinated carbon; the polymer layer includes fluorinated polycaprolactone, a copolymer of Formula I, an ultrasound-activated prodrug, and a sonication agent; Formula I; wherein, peptide is a heat shock protein targeting polypeptide containing thiol, n is the degree of polymerization, which is selected from an integer between 1 and 111, m is the degree of polymerization, which is selected from an integer between 1 and 51; the fluorinated polycaprolactone can dissolve perfluorinated carbon.

4. The nanodroplet according to claim 3, characterized in that, The perfluorocarbon includes one or more of perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluoropropane, perfluorobutane, and perfluoro-15-crown-5-ether; the fluorinated polycaprolactone has the structure shown in Formula II. Formula II; wherein R is a perfluoroalkyl group, a is the degree of polymerization, and a is an integer selected from 1 to 51; the ultrasound-activated prodrug includes one or more of DOX-N3, CPT-N3, 6-MP-N3, R837-N3, R848-N3 and IMDQ-N3; ​​the sonic sensitizer includes one or more of riboflavin tetrabutyrate, riboflavin, hematoporphyrin and its derivatives, curcumin, zinc phthalocyanine and IR-780; the mass ratio of perfluorinated carbon, fluorinated polycaprolactone, copolymer shown in Formula I, ultrasound-activated prodrug and sonic sensitizer is 10~80:5~40:1~8:0.7~9.6:1.4~19.

2.

5. The application of the nanodroplet carrier as described in claim 1 or 2, or the nanodroplet as described in claim 3 or 4, in an ultrasonically labeled and guided targeted nanodelivery system.

6. The application according to claim 5, characterized in that, The ultrasound-labeled guided targeted nanodelivery system includes heat shock protein targets, which include one or more of HSP27, HSP60, HSP70, HSP90α, HSP90β, HSP105, and HSP40.

7. The use of the nanodroplet carrier as described in claim 1 or 2, or the nanodroplets as described in claim 3 or 4, in the preparation of medicaments for treating tumors.

8. The application according to claim 7, characterized in that, The tumor is one or more of the following: CT26 colon cancer, MC38 colon cancer, 4T1 breast cancer, and H22 liver cancer.

9. The method for preparing ultrasonically labeled and targeted nanodroplets according to claim 3 or 4, characterized in that, Includes the following steps: A click chemistry reaction was performed between the heat shock protein targeting peptide and the copolymer shown in Formula III to obtain the copolymer shown in Formula I. Formula III; where n is the degree of polymerization, selected from an integer between 1 and 111, and m is the degree of polymerization, selected from an integer between 1 and 51; fluorinated polycaprolactone, the copolymer shown in Formula I, an ultrasonically activated prodrug, a sonicator, and an organic solvent are mixed to obtain an organic phase; the organic phase, perfluorinated carbon, and water are mixed to obtain ultrasonically labeled and targeted nanodroplets.

10. The preparation method according to claim 9, characterized in that, The copolymer represented by Formula III is prepared by the following method: maleimide-polyethylene glycol-hydroxyl and caprolactone are subjected to ring-opening polymerization to obtain the copolymer represented by Formula III.

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