Ultrasonic response type siRNA nano delivery system as well as preparation method and application thereof

Abstract

The invention relates to an ultrasonic response type siRNA nano-delivery system as well as a preparation method and application thereof. The nano-delivery system comprises a nano-carrier G5PBA, a sound-sensitive agent and siRNA for specifically silencing CD300ld, the nano carrier G5PBA is a fifth generation PAMAM (Polyamidoamine) dendritic polymer modified by 4-(bromomethyl) phenylboronic acid. Under ultrasonic irradiation, the nano system positions an endoplasmic reticulum in a targeted manner and induces local generation of high-intensity reactive oxygen species (ROS), so that endoplasmic reticulum stress is remarkably amplified, and further immunogenic cell death (ICD) is triggered; meanwhile, the release of siRNA and the efficient silencing of the CD300ld gene are realized, so that PMN-MDSC infiltration is inhibited, the T cell activation is promoted, and the anti-tumor immune response is enhanced. Compared with the prior art, the invention shows good targeting, biocompatibility and synergistic immune regulation effect in breast cancer, and has wide clinical transformation application prospect.

Description

Technical Field

[0001] This invention relates to the field of biology, and in particular to an ultrasound-responsive siRNA nanodelivery system, its preparation method, and its applications. Background Technology

[0002] Tumor immunotherapy, as a new generation of cancer treatment strategies, has made significant progress in clinical practice, mainly including immune checkpoint blockades (ICBs), chimeric antigen receptor T-cell (CAR-T) therapy, and tumor vaccines. However, the response rates of these therapies remain low in some immune "cold tumors," especially in triple-negative breast cancer (TNBC), where the objective response rate is less than 20%. The core obstacles lie in low antigen presentation efficiency, insufficient T-cell infiltration, and the formation of an immunosuppressive tumor microenvironment (TME).

[0003] Immunogenic cell death (ICD), as an important strategy for activating anti-tumor immunity, can activate dendritic cell and T cell immune responses by releasing damage-related molecules such as calreticulin (CRT), high-mobility group box 1 (HMGB1), and ATP. Among these, endoplasmic reticulum (ER) stress is a key driving mechanism for inducing ICD, which can enhance cellular immunogenicity by activating the unfolded protein response (UPR).

[0004] In recent years, sonodynamic therapy (SDT), as a physically activated ICD induction strategy, has attracted widespread attention due to its non-invasiveness, good deep tissue penetration, and high safety. Studies have shown that when sonosensitizers are localized to the endoplasmic reticulum (ER) and activated under ultrasound irradiation, they can generate reactive oxygen species (ROS), thereby effectively amplifying the ER stress response and enhancing the ICD effect. However, traditional sonosensitizers often suffer from poor water solubility, weak targeting, and low ROS-generating activity, significantly limiting their practical application in immunotherapy. Therefore, constructing nanosystems with endoplasmic reticulum targeting, high ROS production capacity, and excellent ultrasound responsiveness has become a current research hotspot in this field.

[0005] Patent CN118542839A discloses a US-stimuli-responsive silica nanoparticle modulator for regulating tumor-infiltrating Tregs and mediating tumor immunogenic death through sonodynamic therapy, as well as its preparation method and application. The silica particle comprises a drug core and a US-stimuli-responsive silica shell. The drug core is desmethylcanthalassemia, a drug that can be used to downregulate tumor-infiltrating Tregs. The outer shell, composed of silica-formed lipids, unsaturated phospholipids, pyromethesphalophylloside lipids, and other commercially available phospholipids, is stable under physiological cycling conditions, preventing leakage of the carried DMC. However, when stimulated by US, reactive oxygen species (ROS) generate reactive oxygen species (ROS), which can cause DOPC peroxidation in the shell, leading to system instability and release of DMC. Simultaneously, ROS can induce ICD in tumor cells.

[0006] Furthermore, although ICDs can effectively activate anti-tumor immune responses, their induction process may be accompanied by side effects such as adenosine accumulation and hypoxia, thereby promoting the recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and creating a therapeutic negative feedback loop. PMN-MDSCs, as an important immunosuppressive cell population, can inhibit CD8+. + T cell activation and killing function further enhance the immunosuppressive state in the tumor microenvironment (TME), thereby reducing the immune activation effect induced by ICD. Previous studies have shown that CD300ld is highly expressed in various malignant tumors, mainly involved in regulating the chemotactic migration and immunosuppressive activity of PMN-MDSCs. It is a key molecule mediating the enrichment of PMN-MDSCs in tumor tissue and is closely related to poor prognosis in various tumors. Therefore, targeted silencing of CD300ld holds promise for effectively blocking the abnormal recruitment of PMN-MDSCs, alleviating their mediated immunosuppression, synergistically amplifying the ICD effect, and thus significantly improving the tumor's response to immunotherapy.

[0007] Patent WO2023088464A1 provides a CD300LD inhibitor that specifically and efficiently targets and regulates PMN-MDSCs, offering a new target and approach for tumor immunotherapy by targeting and regulating PMN-MDSCs. It can significantly inhibit the development of various tumors and exhibits a significant anti-tumor synergistic effect with PD1 antibodies, providing a targeted drug for tumor immunotherapy.

[0008] Fifth-generation polyamide dendritic polymers (G5 PAMAM) are widely used in siRNA delivery due to their cationic properties, controllable structure, and good biocompatibility. Their surface can be functionalized with various targeted modification molecules, making them suitable for constructing organelle-directed delivery systems. Studies have shown that G5 PAMAM can stably load siRNA, improving its transport efficiency and preventing in vivo degradation.

[0009] Currently, there is no technology that can combine ICD induction with CD300ld silencing. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of traditional sonication agents, such as poor water solubility, weak targeting, and low ROS generation activity. Furthermore, when ICDs are effectively activated to induce anti-tumor immune responses, they may be accompanied by side effects such as adenosine accumulation and hypoxia, forming a negative feedback loop in treatment. Therefore, this invention provides an ultrasound-responsive siRNA nanodelivery system, its preparation method, and its application.

[0011] The ultrasound-responsive siRNA nanodelivery system provided by this invention is an endoplasmic reticulum-targeted CD300ld gene silencing nanodelivery system based on ultrasound response, that is, an siRNA nanodelivery system with endoplasmic reticulum targeting, ultrasound responsiveness and synergistic immune regulation functions.

[0012] The objective of this invention can be achieved through the following technical solutions: The first aspect of this invention provides an ultrasound-responsive siRNA nanodelivery system, namely an endoplasmic reticulum-targeted CD300ld gene silencing nanodelivery system based on ultrasound response, which can synergistically induce immunogenic cell death (ICD) and silence the immunosuppressive gene CD300ld, significantly improving the efficacy of tumor immunotherapy.

[0013] The nanodelivery system includes: a nanocarrier G5PBA, a somatosensitizer, and siRNA (siCD300ld) for specifically silencing CD300ld.

[0014] In one embodiment of the present invention, the nanocarrier G5PBA is a fifth-generation PAMAM dendritic polymer modified with 4-(bromomethyl)phenylboronic acid (PBA), which has good cationicity and surface modifiability, and is suitable for the synergistic loading of nucleic acids and small molecule drugs.

[0015] In one embodiment of the present invention, the sound-sensing agent is selected from hematoporphyrin monomethyl ether (HMME).

[0016] In one embodiment of the present invention, the sequence of the siRNA (siCD300ld) used for specifically silencing CD300ld is as follows: Justice Chain: GAUGGUCUUUGUGGAGUUA (dT) (dT); Antonym chain: UAACUCCACAAAGACCAUC (dT) (dT).

[0017] In one embodiment of the present invention, the ultrasound-responsive siRNA nanodelivery system is a Pardaxin-modified ultrasound-responsive siRNA nanodelivery system, also referred to as a Pardaxin-modified endoplasmic reticulum-targeted ultrasound-responsive siRNA nanodelivery system. The Pardaxin-modified ultrasound-responsive siRNA nanodelivery system refers to an ultrasound-responsive siRNA nanodelivery system with an endoplasmic reticulum-targeting peptide Pardaxin (Par) modified on its surface, with the sequence (H-GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE-OH). Par peptide, as a natural membrane-active peptide, possesses lipophilicity and endoplasmic reticulum localization characteristics. After modification on the surface of nanoparticles, it can effectively guide their accumulation in the endoplasmic reticulum, enhancing the release efficiency of ROS within the endoplasmic reticulum. The Pardaxin is used to enhance the endoplasmic reticulum accumulation capacity of the nanodelivery system, achieving targeted delivery and action at the subcellular level.

[0018] In this invention, the unmodified Pardaxin-based ultrasound-responsive siRNA nanodelivery system is referred to as the GH@siRNA complex or GH@siRNA system, while the Pardaxin-modified ultrasound-responsive siRNA nanodelivery system is referred to as the PGH@siRNA system.

[0019] Preferably, the particle size of the system is controlled at around 150 nm, the zeta potential is suitable, and it has good tumor targeting ability and cell uptake efficiency.

[0020] The GH@siRNA system or PGH@siRNA system provided by this invention can induce local reactive oxygen species (ROS) production under ultrasound irradiation, thereby amplifying endoplasmic reticulum stress and enhancing ICD; simultaneously, it triggers siRNA release and silences CD300ld, alleviating PMN-MDSC-mediated immunosuppression, enhancing T cell infiltration, and achieving a synergistic anti-tumor effect. In a second aspect, this invention provides a method for preparing the ultrasound-responsive siRNA nanodelivery system, which has simple steps, mild conditions, and good prospects for industrial transformation. The specific preparation method is as follows: S1. G5PBA nanocarriers and sound-sensitive agents are self-assembled to form GH; S2. The GH is incubated with siRNA (siCD300ld) for specifically silencing CD300ld to form a GH@siRNA complex, i.e., an ultrasound-responsive siRNA nanodelivery system.

[0021] In one embodiment of the present invention, the nanocarrier G5PBA is prepared by reacting fifth-generation PAMAM dendrimer (G5 PAMAM) with 4-(bromomethyl)phenylboronic acid (PBA) in anhydrous dimethyl sulfoxide (DMSO) to obtain the nanocarrier G5PBA.

[0022] Preferably, the preparation method of the nanocarrier G5PBA is as follows: fifth-generation PAMAM dendrimer (G5PAMAM) and 4-(bromomethyl)phenylboronic acid (PBA) are dissolved in anhydrous dimethyl sulfoxide (DMSO) at a molar ratio of 1:256, and reacted at 80°C for 48 hours. After the reaction is completed, the reaction solution is dialyzed in deionized water (MWCO: 8,000–14,000 Da) for 72 hours, and then freeze-dried to obtain PBA-modified G5PAMAM (G5PBA).

[0023] In one embodiment of the present invention, in step S1, when the nanocarrier G5PBA and the sound sensitizer are self-assembled to form GH, G5PBA and the sound sensitizer hematoporphyrin monomethyl ether (HMME) are dissolved in DMF at a mass ratio of 8:1, deionized water is slowly added dropwise under ultrasound, and the mixture is sonicated, dialyzed, and then freeze-dried to obtain G5PBA@HMME nanoparticles (hereinafter referred to as GH).

[0024] Preferably, when the nanocarrier G5PBA is self-assembled with the acoustic sensitizer to form GH, the ultrasonic treatment time is 10 min and the dialysis time is 4 hours.

[0025] In one embodiment of the present invention, in step S2, GH and siCD300ld are incubated at room temperature at a mass ratio of 10:1 to form GH@siRNA. The GH complex forms GH@siRNA by electrostatic adsorption of siRNA.

[0026] In one embodiment of the present invention, the preparation method of the Pardaxin-modified ultrasound-responsive siRNA nanodelivery system (hereinafter referred to as the PGH@siRNA system) is as follows: (a) Pardaxin peptide was modified onto Cat-PEG via EDC / NHS activation to form Par-PEG polymer; (b) GH@siRNA was incubated with Par-PEG under stirring conditions to form a Pardaxin-modified ultrasound-responsive siRNA nanodelivery system, namely PGH@siRNA.

[0027] In one embodiment of the present invention, in step (a), under ice-dark conditions, the amino group of Pardaxin is first protected with (BOC)2O at a molar ratio of 1:5.2, Pardaxin is activated with EDC and NHS, and then a Cat-PEG reaction is introduced. After the reaction is completed, the protection is removed and the mixture is purified by dialysis to obtain Par-PEG.

[0028] In one embodiment of the present invention, in step (b), Par-PEG and GH@siRNA are mixed at a mass ratio of 1:5 and incubated slowly with stirring at room temperature to obtain the final PGH@siRNA system.

[0029] The preparation method of the GH@siRNA system or PGH@siRNA system provided by this invention can be completed under light-protected, room temperature or ice-dark conditions, and the proportion of each component, pH value and time parameters can be optimized and adjusted according to needs to improve loading efficiency and delivery stability, and has endoplasmic reticulum targeting capability.

[0030] The present invention achieves a subcellular level endoplasmic reticulum targeting and gene silencing synergistic function through structural design.

[0031] This invention found that when only the PGH system (containing only GH and Par, without siRNA) is constructed, the system can activate HMME and induce a certain degree of ICD under ultrasound irradiation; however, due to the lack of silencing intervention on CD300ld, it cannot effectively inhibit PMN-MDSC recruitment, thus limiting the anti-tumor immune effect.

[0032] In contrast, the PGH@siRNA system can synergistically induce ICD and CD300ld silencing, enhancing DC activation and T cell infiltration while alleviating negative immune feedback inhibition, thus achieving dual-pathway immune regulation.

[0033] A third aspect of the present invention is to propose the application of the nanosystem in the preparation of tumor immunotherapy drugs or pharmaceutical compositions, wherein the tumor immunotherapy drugs or pharmaceutical compositions are used to induce immunogenic cell death (ICD), enhance antigen presentation, silence CD300ld expression and reduce PMN-MDSC recruitment, thereby being used for tumor immunotherapy.

[0034] Its application in various malignant tumors has established a broad-spectrum immunotherapy strategy based on the synergistic mechanism of CD300ld inhibiting PMN-MDSCs and ICD amplification.

[0035] The applicable cancer types include, but are not limited to, breast cancer, liver cancer, colorectal cancer, ovarian cancer, kidney cancer, glioma, melanoma, and lung cancer. The PGH@siRNA system, activated by ultrasound, can be administered intravenously, followed by local ultrasound activation of the tumor site. This enables in situ ROS burst in ER, CD300ld silencing, ICD amplification, and synergistic PMN-MDSC intervention, exhibiting excellent targeting, safety, and immunotherapy enhancement effects. It also demonstrates good clinical translational potential while avoiding damage to normal tissues.

[0036] A fourth aspect of the present invention is to provide a pharmaceutical composition comprising the ultrasound-responsive siRNA nanodelivery system.

[0037] This composition can be a pharmaceutical composition or a research reagent composition, used in applications such as tumor treatment or gene function research.

[0038] Preferably, the composition can be formulated as: an injection (intravenous or local injection), sustained-release microspheres, a gel, a solution, a lyophilized powder, etc.

[0039] Furthermore, the composition may contain pharmaceutically acceptable carriers or excipients, such as phosphate buffer, physiological saline, polyethylene glycol, etc., to meet clinical needs.

[0040] In one embodiment of the present invention, the nanosystem can be used in combination with immune checkpoint inhibitors (such as anti-PD-1 / PD-L1) to form a combined immunotherapy regimen.

[0041] The fifth aspect of this invention provides an ultrasound-activated assisted treatment strategy.

[0042] In one embodiment, the ultrasound-responsive siRNA nanodelivery system is activated by low-intensity focused ultrasound at a frequency of 1.0 MHz after injection, initiating the synergistic release of HMME-mediated ROS and siRNA.

[0043] Preferably, the ultrasound irradiation can also promote lysosomal escape, endoplasmic reticulum localization, and membrane permeability of the nanosystem, thereby improving the bioavailability of the drug in tumor cells.

[0044] Furthermore, in another embodiment, the nanosystem can be combined with a multi-channel ultrasound device, theoretically possessing the potential to achieve time-, location-, and dose-controlled activation, providing a technological foundation for future image-guided precision treatment.

[0045] A sixth aspect of the present invention provides a method for tumor treatment and gene expression regulation, including but not limited to: (a) The PGH@siRNA system was applied to experimental animals or lesion areas; activated by local or surface ultrasound irradiation; (b) Achieving efficient release of siCD300ld and silencing of the CD300ld gene; (c) Combined induction of ICD to enhance antigen presentation; (d) Remodeling the tumor immune microenvironment.

[0046] In this application, an ultrasound-responsive siRNA nanodelivery system is used in a tumor model to release siRNA and specifically silence the CD300ld gene through ultrasound activation, thereby inducing immunogenic cell death and regulating the tumor immune microenvironment.

[0047] Preferably, this method is applicable to in vitro and in vivo experiments on humans or non-human mammals, and is particularly suitable for exploring the prospects of novel ICD and MDSC combined targets in clinical immunotherapy.

[0048] This invention provides an ultrasound-responsive siRNA nanodelivery system, which is based on G5 PAMAM and combines ER targeting, ultrasound responsiveness and synergistic immune regulation functions. It achieves ICD induction and CD300ld silencing in combination, which can enhance the response of tumor immunotherapy. It is especially suitable for immune "cold tumors" such as TNBC, and has important scientific research significance and clinical application potential.

[0049] The system provided by this invention utilizes a fifth-generation PAMAM dendritic polymer modified with Pardaxin peptide as a carrier, synergistically loading small interfering RNA (siRNA) and the sonosensitive agent hematoporphyrin monomethyl ether (HMME) to construct a nanoplatform with ultrasound responsiveness and subcellular endoplasmic reticulum targeting capability. Under ultrasound irradiation, it induces local high-intensity ROS generation, thereby amplifying endoplasmic reticulum stress and enhancing immunogenic cell death (ICD); simultaneously, it achieves siRNA release and efficient silencing of the CD300ld gene, thereby inhibiting PMN-MDSC infiltration, promoting T cell activation, and enhancing anti-tumor immune responses. This nanosystem exhibits good targeting, biocompatibility, and synergistic immunomodulatory effects in breast cancer tumor models, and has broad prospects for clinical translational applications.

[0050] Compared with the prior art, the present invention has the following beneficial effects: 1. Achieved synergistic delivery of siRNA via endoplasmic reticulum targeting and ultrasound response: The PGH@siRNA nanosystem constructed in this invention, combining G5 PAMAM dendritic polymer, hematoporphyrin monomethyl ether (HMME), and Pardaxin peptide, achieves stable siRNA loading, lysosomal escape, and endoplasmic reticulum-directed delivery. Under low-intensity ultrasound irradiation, the system can efficiently release ROS and induce immunogenic cell death (ICD), enhancing antigen presentation and T cell activation.

[0051] 2. Synergistic silencing of CD300ld to alleviate the immunosuppressive microenvironment: Silencing the highly expressed immunosuppressive gene CD300ld in breast cancer tissue via specific siRNA effectively blocks the recruitment and immunosuppressive function of PMN-MDSCs, significantly alleviating the ICD-induced immune negative feedback mechanism. This synergistic effect promotes the expression of CD8+ in tumor tissue. + T-cell infiltration and functional recovery comprehensively enhance the intensity of the immune response in the tumor microenvironment.

[0052] 3. The preparation process is simple and the parameters are adjustable, showing good prospects for transformation and application: The nanosystem provided by this invention has a simple and mild preparation process, uniform particle size distribution, and strong targeting, making it suitable for systemic delivery and ultrasound-activated therapy of solid tumors such as breast cancer. It has good prospects for clinical application and industrialization. Attached Figure Description

[0053] Figure 1 This image shows the fabrication of an endoplasmic reticulum-targeted CD300ld gene silencing nanodelivery system based on ultrasound response, along with schematic diagrams of TEM and AFM.

[0054] Figure 2 A schematic diagram illustrating the tumor targeting capability of the ultrasound-responsive endoplasmic reticulum-targeted CD300ld gene silencing nanodelivery system for in vivo imaging verification.

[0055] Figure 3 This is a schematic diagram illustrating the cellular phagocytosis effect of PGH@siRNA at different time points observed using CLSM.

[0056] Figure 4 This is a schematic diagram illustrating the efficacy of the PGH@siRNA nanosystem in inducing tumor cell apoptosis and immunogenic cell death (ICD) in vitro.

[0057] Figure 5 This is a schematic diagram illustrating the in vivo antitumor efficacy evaluation of the PGH@siRNA nanosystem.

[0058] Figure 6 This diagram illustrates the efficacy and immune memory assessment of the PGH@siRNA nanosystem in treating lung metastasis. Detailed Implementation

[0059] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0060] the term As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only open-ended definitions but also semi-closed and closed definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0061] PGH@siRNA Nanosystem and its preparation method This invention develops an endoplasmic reticulum-targeted CD300ld gene silencing nanodelivery system (PGH@siRNA) based on ultrasound response. The PGH@siRNA nanosystem includes a nanocarrier G5PBA; a sonosensitive agent hematoporphyrin monomethyl ether (HMME); siRNA for specifically silencing CD300ld (siCD300ld); and an endoplasmic reticulum-targeting peptide Pardaxin (Par). The sequence of the siRNA (siCD300ld) used for specific silencing of CD300ld is as follows: Justice Chain: GAUGGUCUUUGUGGAGUUA (dT) (dT) (SEQ ID NO.1); Antonym chain: UAACUCCACAAAGACCAUC (dT) (dT) (SEQ ID NO.2).

[0062] The carrier is loaded with a sound-sensitive agent and siCD300ld.

[0063] This invention provides a method for preparing the PGH@siRNA nanosystem described herein, the method comprising: (1) Synthesis of G5PBA A fifth-generation polyamide-amine resin (G5 PAMAM) was dissolved in anhydrous dimethyl sulfoxide (DMSO) at a molar ratio of 1:256 with 4-(bromomethyl)phenylboronic acid (PBA), and the mixture was reacted at 80 °C for 48 hours. After the reaction was completed, the reaction solution was dialyzed against deionized water (MWCO: 8,000–14,000 Da) for 72 hours, followed by freeze-drying to obtain PBA-modified G5PAMAM (G5PBA).

[0064] Preferably, the reaction system is kept in a light-protected state to reduce the photosensitive degradation of PBA.

[0065] (2) Construction of GH nanoparticles G5PBA (16 mg) and the sonosensitive agent hematoporphyrin monomethyl ether (HMME, 2 mg) were dissolved together in 2 mL of anhydrous DMF. 5 mL of deionized water was slowly added dropwise under sonication, and sonication continued for 10 minutes. The resulting mixture was dialyzed for 4 hours and then freeze-dried to obtain G5PBA@HMME nanoparticles (GH).

[0066] (3) siRNA complex GH and siCD300ld were mixed at a mass ratio of 10:1 and incubated at room temperature for 20 minutes to form a GH@siRNA complex.

[0067] (4) Synthesis and modification of Par-PEG The endoplasmic reticulum-targeting peptide Pardaxin (Par) was coupled with Cat-PEG. Specifically, under ice-dark conditions, the amino group of Pardaxin was first protected with (BOC)₂O (1:5.2 molar ratio), then activated with EDC / NHS, followed by the introduction of Cat-PEG. After the reaction, the protection was removed and the mixture was purified by dialysis to obtain Par-PEG.

[0068] Preferably, the reaction is carried out under ice-dark conditions to avoid Par peptide degradation, with the pH value controlled between 7.0 and 7.4.

[0069] Preferably, the Pardaxin peptide sequence used for modification is: H-GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE-OH (SEQ ID NO.3).

[0070] (5) Construction of the PGH@siRNA system The GH@siRNA obtained in step (3) was mixed with Par-PEG in step (4) at a mass ratio of 5:1 and incubated slowly with stirring at room temperature to obtain the final endoplasmic reticulum-targeted PGH@siRNA nanodelivery system.

[0071] Furthermore, the obtained nanoparticles have a diameter of approximately 150 nm and a zeta potential of approximately +25 mV, exhibiting good dispersibility and stability, making them suitable for in vivo delivery via intravenous injection or other routes.

[0072] use This invention provides a PGH@siRNA system that can be used to prepare pharmaceutical or research compositions for inducing ICD, silencing CD300ld, remodeling the tumor immune microenvironment, and enhancing the efficacy of breast cancer immunotherapy.

[0073] Preferably, after applying the PGH@siRNA nanosystem as described in the first aspect of the present invention, the target site for gene silencing (such as target cells or target organs) is subjected to ultrasonic irradiation to activate the sonosensitive agent and promote siRNA release.

[0074] This invention provides an application of an ultrasound device for preparing an instrument, said instrument being used for one or more applications selected from the group consisting of: an ultrasound-activated assisted therapeutic strategy.

[0075] In one embodiment, the system is irradiated with low-intensity focused ultrasound at a frequency of 1.0 MHz after injection to initiate HMME-mediated ROS release and simultaneously promote siRNA release.

[0076] Preferably, the ultrasound irradiation can also promote lysosomal escape, endoplasmic reticulum localization, and membrane permeability of the nanosystem, thereby improving the bioavailability of the drug in tumor cells.

[0077] Furthermore, in another embodiment, the nanosystem can be combined with a multi-channel ultrasound device, theoretically possessing the potential to achieve time-, location-, and dose-controlled activation, providing a technological foundation for future image-guided precision treatment.

[0078] method This invention provides a method for silencing genes in cells, the method comprising: contacting the PGH@siRNA nanosystem described in this invention with cells, thereby silencing genes in the cells.

[0079] In a preferred embodiment of the present invention, the method is an in vitro method.

[0080] In a preferred embodiment of the present invention, the method is a non-diagnostic and non-therapeutic method.

[0081] In a preferred embodiment of the invention, the contact is an external contact.

[0082] In a preferred embodiment of the invention, the contact includes contact under ultrasonic irradiation.

[0083] Preferably, the cells are subjected to ultrasonic irradiation after the application of the PGH@siRNA nanosystem as described in the first aspect of the present invention.

[0084] The present invention also provides a method for gene silencing, the method comprising: applying the PGH@siRNA nanosystem described in the present invention to a desired subject to thereby perform gene silencing.

[0085] In a preferred embodiment of the invention, the object includes a human or a non-human mammal.

[0086] Preferably, the non-human mammals include cattle, horses, sheep, dogs, cats, or rats.

[0087] In a preferred embodiment of the invention, the application is oral or injectable.

[0088] Preferably, the injection is administered intravenously.

[0089] In a preferred embodiment of the present invention, the target site for gene silencing (such as target cells or target organs) is subjected to ultrasound irradiation.

[0090] In a preferred embodiment of the present invention, after applying the PGH@siRNA nanosystem as described in claim 3, the gene silencing target site (such as target cells or target organs) is subjected to ultrasonic irradiation.

[0091] Device This invention provides a device for gene silencing, the device comprising the PGH@siRNA nanosystem described in this invention; and an ultrasound device.

[0092] Preferably, the system or device further includes a manual or label, which states: The PGH@siRNA nanosystem described in this invention is applied to the target object to perform ultrasonic irradiation treatment on the gene silencing target site (such as target cells or target organs).

[0093] Composition The compositions described in this invention are preferably pharmaceutical compositions or reagent compositions, and may include pharmaceutically or reagent-acceptable carriers.

[0094] As used herein, "pharmaceutically acceptable carrier" refers to one or more compatible solid, semi-solid, liquid, or gel fillers that are suitable for human or animal use and must have sufficient purity and sufficiently low toxicity. "Compatibility" refers to the ability of the components and active ingredients in a pharmaceutical composition, as well as their intermingling, to not significantly reduce the efficacy of the drug.

[0095] It should be understood that there are no particular limitations on the pharmaceutically acceptable carriers described in this invention. Commonly used materials in the art can be selected, or they can be prepared using conventional methods or purchased from the market. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), buffers, chelating agents, thickeners, pH adjusters, transdermal penetration enhancers, colorants, flavoring agents, stabilizers, antioxidants, preservatives, antibacterial agents, pyrogen-free water, etc.

[0096] In a preferred embodiment of the present invention, the dosage form of the composition is a solid dosage form, a liquid dosage form, or a semi-solid dosage form.

[0097] In a preferred embodiment of the present invention, the dosage form of the composition is an oral formulation, a topical formulation, or an injectable formulation. Preferably, the dosage form of the composition is a tablet, injection, infusion, ointment, gel, solution, microsphere, or film.

[0098] Preferably, the injectable preparation is an intravenous injection preparation.

[0099] The pharmaceutical formulation should be matched with the route of administration. The pharmaceutical products of this invention can also be used with other synergistic therapeutic agents (including before, during, or after administration). When using the pharmaceutical composition or formulation, a safe and effective amount of the drug is administered to the desired subject (e.g., human or non-human mammal), typically at least about 10 micrograms per kilogram of body weight, and in most cases not exceeding about 10 milligrams per kilogram of body weight; preferably, the dose is about 10 micrograms per kilogram of body weight to about 5 milligrams per kilogram of body weight. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, which are within the scope of a skilled physician's expertise.

[0100] Example 1 1. Preparation of an endoplasmic reticulum-targeted CD300ld silencing delivery system (PGH@siRNA) (1) Preparation of GH 100 mg of fifth-generation polyamide-amine dendritic polymer (G5PAMAM) and 191 mg of 4-(bromomethyl)phenylboronic acid (PBA) were weighed and dissolved in 5 mL of anhydrous dimethyl sulfoxide (DMSO) at a molar ratio of 1:256. The mixture was reacted at 80°C for 48 hours. The reaction solution was dialyzed nine times with 2 L of distilled water (molecular weight cutoff: 8000–14000 Da), and then lyophilized to obtain PBA-modified G5PAMAM (G5PBA). Subsequently, to achieve encapsulation with hematoporphyrin monomethyl ether (HMME), 16 mg of G5PBA and 2 mg of HMME were dissolved in 2 mL of dimethylformamide (DMF), and 5 mL of deionized water was slowly added dropwise under sonication. The sonication was continued for 10 minutes. The mixture was dialyzed for 4 hours (MWCO: 8000–14000 Da) and then lyophilized to obtain GH nanoparticles.

[0101] (2) Synthesis of Par-PEG Par-PEG was constructed by coupling Cat-PEG with Pardaxin peptide. The specific steps are as follows: Under light-protected ice bath conditions, the amino group of the Pardaxin peptide was protected with di-tert-butyl carbonate anhydride ((BOC)₂O) at a molar ratio of 1:5.2 for 12 hours. Subsequently, carboxyl activation was performed using EDC / NHS (Par:EDC:NHS = 1:5:10) for 2 hours, followed by the addition of Cat-PEG (1:1 molar ratio) and a further reaction for 24 hours. After the reaction was complete, 12 M HCl was added for deprotection, 3 M NaOH was added to neutralize the system, and the mixture was dialyzed for 48 hours to remove impurities. Finally, the mixture was lyophilized to obtain Par-PEG.

[0102] (3) Assembly of PGH@siRNA The GH prepared above was incubated with siCD300ld at a mass ratio of 10:1 at room temperature for 20 minutes to form a GH@siRNA complex. Then, GH@siRNA was mixed with Par-PEG (mass ratio 5:1), stirred continuously at room temperature for 24 hours, dialyzed, and then lyophilized to obtain the final endoplasmic reticulum-targeted CD300ld gene silencing nanodelivery system PGH@siRNA.

[0103] (4) Characterization The preparation process of PGH@siRNA and the results of transmission electron microscopy (TEM) are as follows: Figure 1 As shown, from Figure 1 It can be seen that the composite nanoparticles of GH, GH@siRNA and PGH@siRNA all exhibit a uniform and regular microstructure.

[0104] 2. Tumor targeting capability of the endoplasmic reticulum-targeted CD300ld gene silencing nanodelivery system (PGH@siRNA) Six-week-old female Balb / c mice (purchased from LifeRiver Laboratory Animal Technology) were selected, and 1 × 10 6 One 4T1 cell was injected subcutaneously into the second pair of mammary fat pads on the right side of mice to establish a unilateral primary 4T1 tumor-bearing model. The tumor volume reached 150–200 mm. 3 Mice were randomly selected for intravenous injection of Cy5.5 fluorescently labeled PGH@siRNA (the fluorescent labeling product was generated by the reaction of Cy5.5-NHS and PGH@siRNA composite nanoparticles at room temperature with stirring for 24 h). The injection dose was 5 mg / kg, and the injection volume was 150 μL. Mice were anesthetized by isoflurane inhalation before imaging.

[0105] Dynamic imaging was performed at 0, 2, 4, 8, 12, 24, and 48 hours post-administration using the VISQUE InVivo Smart-LF in vivo fluorescence imaging system to evaluate the distribution of PGH@siRNA in vivo and its targeting ability to tumor tissue. Experimental results showed that PGH@siRNA significantly accumulated at the tumor site after injection, exhibiting good active tumor targeting ability. Related imaging results are shown below. Figure 2 As shown.

[0106] 3. Investigate the in vitro phagocytic effect of the endoplasmic reticulum-targeted CD300ld silencing delivery system (PGH@siRNA). To evaluate the cellular uptake capacity of PGH@siRNA, Cy3-PGH@siRNA and Cy3-GH@siRNA control systems were constructed using Cy3-labeled siRNA. The fluorescently labeled products were generated by reacting Cy3 with PGH@siRNA and GH@siRNA composite nanoparticles, respectively, at room temperature with stirring for 24 h. 4T1 cells were seeded in glass slides and, after adhesion, co-incubated with the two complexes at 37°C and 5% CO2 for 2, 4, and 8 hours, respectively. After incubation, cells were washed three times with PBS, stained with DAPI, and fixed with 4% paraformaldehyde.

[0107] Cellular uptake was observed using a laser confocal microscope (ZEISS LSM900). Results showed that Cy3-PGH@siRNA exhibited significant intracellular fluorescence signal within 4 hours, and its intracellular distribution further increased after 8 hours, indicating that this system possesses good cellular uptake capacity and rapid endocytosis kinetics. Related fluorescence images are shown below. Figure 3 As shown.

[0108] 4. Evaluation of the endoplasmic reticulum targeting ability and in vitro immunogenic cell death (ICD) induction efficacy of the PGH@siRNA system To verify the endoplasmic reticulum targeting characteristics of the PGH@siRNA system at the cellular level and its ability to induce ICD, experiments were conducted on three aspects: fluorescence colocalization, apoptosis, and DC maturation.

[0109] First, Cy3-labeled PGH@siRNA and GH@siRNA were constructed. After adherent culture of control 4T1 cells, these materials were added and incubated for 6 hours. Subsequently, the endoplasmic reticulum was stained with ER-Tracker dye for 30 min, and the nuclei were stained with DAPI. The co-localization of Cy3 and ER signals was observed using confocal laser microscopy (CLSM). Results are as follows: Figure 4 a, Figure 4 As shown in b, the ER co-localization fluorescence signal of the PGH@siRNA + US group was significantly enhanced, indicating that the system has good endoplasmic reticulum targeting ability under sonication activation.

[0110] Secondly, to evaluate its cell death-inducing effect, the treated cells were incubated for another 12 hours, then stained with calcein-AM and propidium iodide (PI) for 15 minutes. After washing with PBS, the apoptosis of 4T1 cells was observed under an inverted fluorescence microscope. The results are as follows: Figure 4 As shown in c, the PGH@siRNA + US group significantly induced cell death.

[0111] Finally, 4T1 cells (1 × 10⁻⁶) under different treatments were collected. 5 ) and immature dendritic cells (DCs) (1 × 10 6 The cells were cultured in the Transwell system for 24 hours, and the expression of maturation markers on the surface of DCs was detected by flow cytometry to evaluate their maturation status.

[0112] Experimental results are as follows Figure 4 As shown in d, PGH@siRNA, when activated by ultrasound, can target the endoplasmic reticulum, enhance endoplasmic reticulum stress, induce apoptosis, and significantly promote the release of ICD-related signals and DC maturation, confirming its ability to synergistically induce immunogenic cell death in vitro.

[0113] 5. Evaluation of the in vivo antitumor therapeutic effect and immune memory function of the PGH@siRNA system To evaluate the in vivo therapeutic effect of the PGH@siRNA nanosystem, a 4T1 breast cancer xenograft mouse model was constructed. 1 × 10⁻⁶ cells were used. 6 4T1 cells were injected into the second pair of mammary fat pads on the right side of 6-week-old female Balb / c mice until the tumor volume reached 150–200 mm.3 Afterwards, they were randomly divided into 6 groups (n=6): control group, US group, GH@siRNA+US group, PGH+US group, PGH@siRNA group, and PGH@siRNA+US group.

[0114] like Figure 5 a. Each treatment group received a tail vein injection of the nano-formula, once every two days, for a total of four times. The ultrasound processing parameters were 1.0 MHz and 1.0 W / cm². 2 A 50% duty cycle, 5-minute irradiation, with the first US irradiation scheduled the day after the first drug administration, followed by irradiation every two days. Tumor volume is measured every two days, calculated using the formula: (length × width) 2 ) / 2. Tumor growth curves are shown in [reference needed]. Figure 5 b. After the experiment, the tumor weight in the mice was as follows: Figure 5 c, Ki-67 staining of the tumor as shown Figure 5 d. Based on Figure 5 It can be seen that the PGH@siRNA + US group exhibited a significant tumor-suppressive effect.

[0115] To further evaluate the anti-metastasis and immune memory functions of this system, such as Figure 6 As shown in Figure a, a tumor model was established in another group of mice by subcutaneous inoculation with Luc+4T1 cells. These mice were divided into a control group and a PGH@siRNA + US group, receiving the same treatment. The primary tumor was surgically removed on day 21, and lung tissue was harvested on day 42. Lung metastasis was assessed using a fluorescence imaging system, and the results are as follows: Figure 6 b. The lung metastatic nodules were fixed with Brunner's solution and counted under a microscope. The results are as follows: Figure 6 c, based on Figure 6 b, 6c, show that the number of lung metastases was significantly reduced in the PGH@siRNA +US group.

[0116] In addition, to analyze whether the immune memory response was activated after treatment, spleen single-cell suspensions were collected and stained with anti-CD8a-APC, anti-CD3-FITC, anti-CD44-PE, and anti-CD62L-APC / Cy7 antibodies. CD8+ was then detected by flow cytometry. + and CD4 + Effector memory T cells (TEM, phenotype CD3) in the T cell population + CD8 + CD44 + CD62L - ) and central memory T cells (TCM, phenotype CD3) + CD4 + CD44 + CD62L + ) proportion.

[0117] The results are as follows Figure 6 As shown in the figure, compared with the control group, the proportion of TEM cells in CD8+ T cells and CD4+ T cells in mice treated with PGH@siRNA + US was significantly increased, while the proportion of TCM cells was decreased, indicating that the TCM subset underwent a significant transformation to the TEM phenotype. These results indicate that PGH@siRNA + US treatment not only inhibits tumor growth and metastasis but also significantly increases the proportion of memory T cells, suggesting that this system can induce durable immune protection and has the potential to prevent tumor recurrence.

Claims

1. An ultrasound-responsive siRNA nanodelivery system, characterized in that, The nanodelivery system includes: a nanocarrier G5PBA, a sonosensitive agent, and siRNA for specifically silencing CD300ld; The nanocarrier G5PBA is a fifth-generation PAMAM dendritic polymer modified with 4-(bromomethyl)phenylboronic acid. The sequence of the siRNA used for specifically silencing CD300ld is as follows: Justice Chain: GAUGGUCUUUGUGGAGUUA (dT) (dT); Antonym chain: UAACUCCACAAAGACCAUC (dT) (dT).

2. The ultrasound-responsive siRNA nanodelivery system according to claim 1, characterized in that, The sound-sensitizing agent is selected from hematoporphyrin monomethyl ether.

3. The ultrasound-responsive siRNA nanodelivery system according to claim 1, characterized in that, The ultrasound-responsive siRNA nanodelivery system is a Pardaxin-modified ultrasound-responsive siRNA nanodelivery system; The sonicated siRNA nanodelivery system modified with Pardaxin refers to a sonicated siRNA nanodelivery system with an endoplasmic reticulum-targeting peptide, Pardaxin, modified on its surface. The Pardaxin peptide sequence used for modification is as follows: H-GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE-OH; Pardaxin is used to enhance the endoplasmic reticulum enrichment capacity of nanodelivery systems, enabling targeted delivery and action at the subcellular level.

4. The method for preparing the ultrasound-responsive siRNA nanodelivery system according to claim 1, characterized in that, Includes the following steps: S1. G5PBA nanocarriers and sound-sensitive agents are self-assembled to form GH; S2. The GH is incubated with siRNA for specifically silencing CD300ld to form a GH@siRNA complex, i.e., an ultrasound-responsive siRNA nanodelivery system.

5. The method for preparing the ultrasound-responsive siRNA nanodelivery system according to claim 4, characterized in that, The preparation method of the nanocarrier G5PBA is as follows: the fifth-generation PAMAM dendritic polymer is reacted with 4-(bromomethyl)phenylboronic acid in anhydrous dimethyl sulfoxide to obtain the nanocarrier G5PBA.

6. The method for preparing the ultrasound-responsive siRNA nanodelivery system according to claim 4, characterized in that, In step S1, when the nanocarrier G5PBA and the sound sensitizer are self-assembled to form GH, G5PBA and the sound sensitizer hematoporphyrin monomethyl ether (HMME) are dissolved in DMF at a mass ratio of 8:

1. Deionized water is slowly added dropwise under ultrasound, and the mixture is sonicated. After dialysis, it is lyophilized to obtain G5PBA@HMME nanoparticles (hereinafter referred to as GH).

7. The method for preparing the ultrasound-responsive siRNA nanodelivery system according to claim 4, characterized in that, In step S2, GH and siCD300ld are incubated at room temperature at a mass ratio of 10:1 to form GH@siRNA; the GH complex forms GH@siRNA by electrostatic adsorption of siRNA.

8. The method for preparing the ultrasound-responsive siRNA nanodelivery system according to claim 4, characterized in that, The preparation method of the Pardaxin-modified ultrasound-responsive siRNA nanodelivery system is as follows: (a) Pardaxin peptide was modified onto Cat-PEG via EDC / NHS activation to form Par-PEG polymer; (b) GH@siRNA was incubated with Par-PEG under stirring conditions to form a Pardaxin-modified ultrasound-responsive siRNA nanodelivery system, namely PGH@siRNA.

9. The use of the ultrasound-responsive siRNA nanodelivery system according to claim 1 in the preparation of tumor immunotherapy drugs or pharmaceutical compositions, characterized in that, The aforementioned tumor immunotherapy drug or drug composition is used to induce immunogenic cell death, enhance antigen presentation, silence CD300ld expression, and reduce PMN-MDSC recruitment, thereby being used for tumor immunotherapy.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the ultrasound-responsive siRNA nanodelivery system of claim 1.

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