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CN-122005456-A - Preparation method and application of Wen Minzhi mass nanoparticles for double-targeting tumor

CN122005456ACN 122005456 ACN122005456 ACN 122005456ACN-122005456-A

Abstract

The invention discloses a multifunctional nano delivery system for tumor cooperative treatment. The system is based on a double-targeting Wen Minzhi plastid, the skeleton of which consists of Wen Minzhi mass, cholesterol and the like, and the surface of which is modified with ginsenoside and AS1411 aptamer to realize targeted delivery to tumors. The liposome internally co-encapsulates an LDH-targeting CRISPR-Cas9 system, a photothermal agent IR-1048 and a phase change agent PFP. Under the irradiation of near infrared light, the photo-thermal effect mediated by IR-1048 triggers PFP phase change, thereby realizing the controllable release of the therapeutic drugs. The released CRISPR-Cas9 system enhances the anti-tumor immune response by editing LDHA genes to remodel the immunosuppressive microenvironment and in conjunction with photothermal therapy-induced Immunogenic Cell Death (ICD). In vivo and in vitro experiments show that the nanoparticle realizes efficient tumor cooperative treatment through the dual functions of 'local physical ablation and metabolic intervention' and 'systemic immune activation'. The research provides a potential technical platform for developing a new generation of tumor accurate immunotherapy strategy.

Inventors

  • LIU YUJIAO
  • CHEN ZIQUN
  • DU HAOTIAN

Assignees

  • 天津工业大学

Dates

Publication Date
20260512
Application Date
20260120

Claims (9)

  1. 1. A tumor microenvironment double-targeting Wen Minzhi liposome is characterized by comprising the following components of (1) Wen Minzhi mass, (2) cationic lipid, (3) cholesterol, (4) polyethylene glycol modified lipid, (5) surface modified ginsenoside, (6) surface modified AS1411 nucleic acid aptamer, (7) CRISPR-Cas9 system for specifically editing lactate dehydrogenase A (LDHA) gene, and (8) near infrared photothermal agent IR-1048 and (9) perfluoropentane (PFP) internally encapsulated.
  2. 2. The liposome according to claim 1, wherein the Wen Minzhi mass is DPPC, DSPC, HSPC or a combination thereof, the cationic lipid is DOTAP, DOTMA or a combination thereof, the polyethylene glycol modified lipid is DSPE-PEG2000, DMG-PEG2000 or a combination thereof, and the mass ratio of the components is Wen Minzhi mass, namely cationic lipid, cholesterol/ginsenoside and polyethylene glycol modified lipid=2-7:0.5-5:0.5-5/0.5-5:0.5-5.
  3. 3. The liposome according to claim 1, wherein the ginsenoside is selected from ginsenoside Rg3, rh2 or a combination thereof, and the aptamer is an AS1411 aptamer targeting nucleolin.
  4. 4. The liposome of claim 1, wherein the CRISPR-Cas9 system comprises a Cas9 protein or its encoding mRNA, and a single stranded guide RNA (sgRNA) that specifically targets an LDHA or LDHB gene, the sgRNA targeting a catalytically active region of the LDHA gene.
  5. 5. The liposome according to claim 1, wherein the liposome is stable at normal temperature, and undergoes a phase change to release the content when the local temperature is raised to 30-70 ℃ under the irradiation of near infrared light, wherein the wavelength of near infrared light is 780-2500nm, and the power density is 0.1-5.0W/cm 2 .
  6. 6. A pharmaceutical composition comprising the tumor microenvironment dual-targeting Wen Minzhi plastid of any one of claims 1-5 and a pharmaceutically acceptable carrier.
  7. 7. A method for preparing a tumor microenvironment double-targeting Wen Minzhi liposome according to any one of claims 1-5, which is characterized by comprising the following steps of (1) dissolving Wen Minzhi lipid, cationic lipid, cholesterol/ginsenoside, polyethylene glycol modified lipid and IR-1048 in an organic solvent to form a lipid/IR-1048 mixture, (2) evaporating the lipid mixture obtained in the step (1) to form a lipid film, preparing a basic liposome by ultrasonic and extrusion, (3) loading a CRISPR-Cas9 system into the aqueous core of the basic liposome obtained in the step (2) by adopting a microfluidic technology or ultrasonic treatment, (4) loading perfluoropentane into the liposome obtained in the step (3) by adopting ultrasonic treatment, and (5) modifying a nucleic acid aptamer on the surface of the liposome obtained in the step (4) by adopting a chemical coupling method.
  8. 8. Use of a tumor microenvironment dual-targeting Wen Minzhi plastid according to any one of claims 1-5 in the manufacture of a medicament for the treatment of a tumor, wherein the tumor is selected from breast cancer, colon cancer, pancreatic cancer, lung cancer, liver cancer, melanoma or other solid tumors.
  9. 9. The use according to claim 8, wherein the synergistic treatment of tumors is achieved by (1) dual targeted delivery mediated by ginsenoside and AS1411 aptamer, enhancing enrichment of liposomes in tumor microenvironments, (2) photo-thermal effect of IR-1048 under near infrared light irradiation, increasing temperature, triggering perfluoropentane phase transition, (3) volume expansion of perfluoropentane phase transition, disrupting liposome structure, releasing CRISPR-Cas9 system and IR-1048, (4) editing LDHA gene by the released CRISPR-Cas9 system, inhibiting lactic acid production, reversing tumor immunosuppression microenvironment, (5) direct killing of tumor cells by IR-1048 mediated photo-thermal effect, and inducing immunogenic tumor cell death, (6) synergistic activation of anti-tumor immune response by LDHA gene editing and photo-thermal treatment.

Description

Preparation method and application of Wen Minzhi mass nanoparticles for double-targeting tumor Technical Field The invention relates to the technical field of biological medicine, in particular to a multifunctional nano-drug delivery system for tumor treatment. More specifically, a multifunctional Wen Minzhi plastid nanocarrier is presented herein for use in the synergistic treatment of tumors. The vector integrates targeted delivery, gene editing, photothermal effect and immunoregulation functions. The strategy aims at effectively activating and enhancing the anti-tumor immune response of the organism through the synergistic effect of photothermal therapy and metabolic intervention. The method can not only directly remove the tumor, but also radically remodel the tumor immune microenvironment, and provides a key tool and an innovative strategy for improving the response rate and durability of the existing immunotherapy. Background A major challenge in modern tumor therapy is the immunosuppressive Tumor Microenvironment (TME), which results in therapeutic resistance. One very promising strategy for disrupting immunosuppression of TMEs is to combine immunotherapy with tumor metabolic reprogramming. The core of this approach is to target the key enzyme in the "warburg effect", the Lactate Dehydrogenase (LDHA). Excess lactate produced by LDHA catalysis acidifies TME, a key driver that impairs immune cell function, causing immunosuppression. Therefore, inhibiting LDHA is expected to remodel TME, and the treatment effect is improved. Studies have shown that high concentration lactic acid and low pH environments in tumor microenvironments severely inhibit T cell and Natural Killer (NK) cell function, while promoting infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), together constituting a powerful immunosuppressive barrier. Therefore, the targeting of the LDHA gene to reduce lactic acid generation is expected to improve the tumor microenvironment and enhance the anti-tumor immune response, thereby providing a new idea for tumor immunotherapy. CRISPR-Cas9 is taken as a revolutionary gene editing tool, and provides unprecedented opportunities for accurately regulating and controlling LDHA gene expression and remodelling tumor metabolism with high specificity and high efficiency. However, despite its great potential, the clinical application of CRISPR-Cas9 systems is still limited by a series of severe delivery bottlenecks. These challenges include their poor biological stability in vivo, insufficient targeting efficiency to tumor tissue, and difficulty in achieving efficient endosomal escape after endocytosis by cells, which together severely hamper their clinical transformation progression. Photothermal therapy (PTT) has shown great potential in the field of tumor therapy as a spatially and temporally controllable non-invasive physical ablation technique. The core advantage is the ability to induce Immunogenic Cell Death (ICD), by releasing Tumor Associated Antigens (TAAs) and lesion associated molecular patterns (DAMPs), to effectively transform immunologically "cold" tumors into "hot" tumors that are recognized by the immune system, thereby initiating an adaptive anti-tumor immune response. However, as a local therapy, single PTT is limited by the tissue penetration depth of near infrared light, often making it difficult to completely clear the tumor, and more unable to inhibit distant metastasis, resulting in a higher risk of tumor recurrence. Although nano delivery systems have been widely used in gene therapy and photothermal therapy, an intelligent platform that can synergistically integrate three strategies, tumor metabolic reprogramming, photothermal therapy and immune activation, is currently still left blank. Particularly, the multifunctional liposome system capable of realizing dual targeting, photo-thermal triggering and controlled release of tumor microenvironment and synergistic effect of gene editing and physical ablation at the same time has not been reported yet. The invention designs and constructs a novel multifunctional nano delivery system, which can precisely target tumors, remodel an immunosuppression microenvironment through CRISPR-Cas9 mediated LDHA gene editing, and cooperate with photothermal therapy to effectively activate systemic anti-tumor immunity. This work provides an innovative and viable strategy for developing next generation accurate cancer immunotherapy. Disclosure of Invention In order to solve the limitations of the prior art, the invention provides a novel multifunctional Wen Minzhi plastid. The nano platform integrates four core functions of (1) dual-targeting delivery of tumor microenvironment, (2) photo-thermal triggered temperature-sensitive controlled release, (3) CRISPR-Cas9 mediated LDHA gene editing and (4) photo-thermal ablation. Through deep synergy of metabolic reprogramming and photothermal immunotherapy, the system finall