Construction method of mitochondrial nano vesicles Pep-1 (at) TMZm (at) MitoNVs for enhancing penetration of blood brain barrier

Abstract

The invention discloses a construction method of mitochondrial nano vesicles Pep-1 (at) TMZm (at) MitoNVs capable of enhancing penetration of blood brain barriers, belongs to the field of preparation of nano drug delivery systems of targeted glioblastoma, and relates to a construction method of nano particles modified and linked with modified temozolomide and Pep-1. According to the mitochondrial nano-vesicle of the Pep-1 (at) TMZm (at) MitoNVs, provided by the invention, the mitochondrial nano-vesicle which can carry temozolomide and has relatively high bioavailability, relatively good histocompatibility, considerable drug loading ratio and encapsulation efficiency and relatively light toxic and side effects is developed; the mitochondrial nano-vesicle based on TMZ modification and Pep-1 is an effective carrier capable of improving the blood brain barrier penetrating capability of a clinical first-line chemotherapeutic drug TMZ and the tumor targeting property and enhancing the anti-tumor activity of the clinical first-line chemotherapeutic drug TMZ.

Description

Technical Field

[0001] This invention belongs to the field of preparation of nanomedicine delivery systems targeting glioblastoma, specifically a Pep-1@TMZ mitochondrial nanovesicle enhanced for penetrating the blood-brain barrier. m The construction method of @MitoNVs. Background Technology

[0002] Glioblastoma (GBM) is the most common and most malignant primary brain tumor in adults, classified as a WHO grade IV glioma. GBM is characterized by rapid growth, high invasiveness, and extremely poor prognosis. Its pathogenesis is extremely complex, and the specific mechanism is not yet fully understood. It may be associated with multiple factors, including genetic factors, environmental factors, and ionizing radiation.

[0003] Currently, the primary treatment for GBM is surgical resection, supplemented by a comprehensive treatment strategy including radiotherapy, chemotherapy, and targeted therapy. The median survival is approximately 14-16 months, with a 5-year survival rate of less than 5%. The goal of surgery is to achieve maximum safe tumor resection, thereby reducing the tumor burden, alleviating symptoms, and creating favorable conditions for subsequent radiotherapy and chemotherapy. Postoperative concurrent chemoradiotherapy is the standard treatment regimen and can significantly prolong patient survival. Temozolomide (TMZ) is currently the most commonly used chemotherapy drug. Although it has improved patient survival to some extent, its efficacy still falls short of clinical expectations. The root cause is that chemotherapy drugs cannot completely penetrate the blood-brain barrier (BBB), preventing them from achieving the desired therapeutic effect.

[0004] In recent years, with the development of molecular biology and genomics, some progress has been made in the molecular subtyping and targeted therapy research of glioblastoma; however, the results of most trials have not yet met expectations. With the continuous advancement of nanotechnology and ongoing research by scholars, targeted therapy to the tumor microenvironment has brought new opportunities for the treatment of glioblastoma. Summary of the Invention

[0005] To address the above problems, this invention prepares a mitochondrial nanovesicle, Pep-1@TMZ, that can enhance penetration of the blood-brain barrier. m @MitoNVs, a method for constructing nanoparticles modified with temozolomide and Pep-1. Based on the characteristics of nanocarriers, this invention develops mitochondrial nanovesicles that can carry temozolomide and exhibit high bioavailability, good tissue compatibility, considerable drug loading and encapsulation efficiency, and relatively mild toxicity. The Pep-1@TMZ of this invention... m@MitoNVs' mitochondrial nanovesicles, based on TMZ modification and Pep-1, are effective carriers for enhancing the ability of the first-line clinical chemotherapy drug TMZ to penetrate the blood-brain barrier and target tumors, thereby increasing its anti-tumor activity.

[0006] This invention is implemented as follows: The blood-brain barrier (BBB) ​​is a key factor limiting the successful development of central nervous system (CNS) drugs. Its selectivity and permeability prevent nearly 100% of large molecule drugs and over 98% of small molecule drugs from effectively penetrating it. Therefore, ideal CNS drugs, in addition to possessing good pharmacological activity, suitable metabolic characteristics, and low toxicity, must also be able to effectively penetrate the BBB and reach the required therapeutic exposure level in the central nervous system. This is a decisive prerequisite for the successful development of such drugs. Based on a deep understanding of the characteristics of CNS drugs and the BBB transport mechanism, systematic structural optimization can significantly improve the ability of compounds to penetrate the BBB.

[0007] Centrally acting drugs generally have fewer hydrogen bond donors, and many compounds with exposed NH₂ exhibit significant P-glycoprotein efflux. Reducing the number of hydrogen bond donors is a crucial modification strategy for optimizing centrally acting drugs. Common methods for reducing hydrogen bond donors include: blocking hydrogen bond donors, replacing hydrogen bond donors with bioisosteres, and forming intramolecular hydrogen bonds. Based on these considerations and the molecular structure characteristics of temozolomide, a commonly used chemotherapy drug for gliomas, we adopted a "blocking hydrogen bond donors" strategy, replacing one H atom in the NH₂ group of temozolomide with a propargyl group, resulting in a temozolomide analog containing an alkyne group. This temozolomide analog reduces hydrogen bond donors, thereby improving blood-brain barrier permeability. Simultaneously, the introduction of the alkyne group facilitates the linkage of the modified temozolomide with the cell-penetrating peptide (Pep-1) via a click reaction.

[0008] Linking drug molecules to the cell-penetrating peptide Pep-1 is a common strategy for constructing efficient drug delivery systems. Click chemistry is a modern, efficient, and highly selective linking method, particularly suitable for complex systems. Its basic principle involves modifying Pep-1 with an azide group (-N3) and modifying TMZ with an alkyne group (-C≡CH) (resulting in the novel TMZ drug TMZ, which enhances its blood-brain barrier penetration). m The modified temozolomide was catalyzed by Cu(I) to form 1,2,3-triazole bonds. Based on click chemistry, after obtaining the modified temozolomide, we further linked the modified temozolomide to the cell-penetrating peptide Pep-1 via click chemistry.

[0009] The reason Pep-1 is used as the functional navigation nanocarrier in this invention is that Pep-1 possesses excellent cell penetration ability and low cytotoxicity, effectively improving the efficiency of drug transmembrane transport. Simultaneously, Pep-1 has a stable structure, is easily chemically modified, and exhibits good compatibility with click chemistry, ensuring the high efficiency and specificity of the conjugation reaction, thereby achieving precise delivery of drug molecules. Furthermore, studies have shown that Pep-1 can specifically bind to the IL-13α2 receptor highly expressed in glioblastoma (normal brain tissue does not express IL-13α2), further enhancing drug accumulation and targeting at the tumor site. This conjugate exhibited significantly superior penetration ability compared to the prototype drug in an in vitro blood-brain barrier model, and its cytotoxic activity against glioma cells was nearly three times higher. Animal experiments showed that the drug concentration in brain tumor tissue was 4.6 times higher than that of free temozolomide, significantly prolonging the survival of tumor-bearing mice, while no significant neurotoxicity or immunogenicity was observed. This design based on a synergistic strategy of structural optimization and targeted delivery provides a new pathway for the treatment of central nervous system tumors that combines high efficiency and safety. This strategy not only overcomes the bottleneck of traditional chemotherapy drugs' inability to cross the blood-brain barrier, but also achieves the dual goals of enhanced efficacy and reduced toxic side effects through the synergistic effect of precise molecular modification and targeted delivery.

[0010] Mitochondria are used as delivery carriers for chemotherapy drugs because mitochondrial nanovesicles possess natural membrane structural stability and biocompatibility, and can effectively evade immune recognition, prolonging drug circulation time in vivo. Their surface is rich in phospholipid bilayers and membrane proteins, which can mediate efficient fusion with target cells, promoting direct drug release into the cytoplasm, and subsequently accumulating in the mitochondria—the energy center of tumor cells. This carrier not only enhances the sensitivity of drugs to drug-resistant tumors but also synergistically disrupts the energy metabolism network of tumor cells, amplifying chemotherapy-induced apoptosis signals. Combined with the aforementioned Pep-1-mediated targeted delivery system, mitochondrial nanovesicles can achieve a "dual-targeting, stepwise amplification" therapeutic effect, precisely targeting tumor cells while increasing intracranial drug concentration, providing a more promising treatment option for refractory brain tumors such as glioblastoma. Through dual-targeting synergy, drugs achieve efficient accumulation in the mitochondrial compartments of tumor cells, significantly inhibiting respiratory chain complex activity, inducing a sharp drop in ATP and a ROS burst, triggering mitochondrial membrane potential collapse, and ultimately activating the caspase cascade reaction, inducing tumor cell apoptosis. Meanwhile, this system demonstrated potent killing ability in various drug-resistant glioma models, overcoming the limitation of traditional temozolomide being susceptible to interference from MGMT repair mechanisms. Crucially, this dual-modal delivery system significantly reduced off-target toxicity to normal neurons while maintaining the integrity of the blood-brain barrier, exhibiting a favorable therapeutic window. Preclinical studies further confirmed that… Finally, combining the "hydrogen bond donor blocking" technology, TMZ is modified by blocking the hydrogen bond donor sites on its imidazole ring without altering its efficacy. This significantly reduces the polar surface area of ​​the drug in the physiological environment, thereby improving its lipophilicity and transmembrane capacity. This modification does not affect the DNA alkylation activity of TMZ, but it exhibits superior brain tissue distribution characteristics in pharmacokinetics. Combined with Pep-1 targeting and mitochondrial delivery systems, the modified TMZ is enzymatically restored to its original form within tumor cells, achieving precise activation and long-term retention in the lesion area. The overall strategy balances penetration efficiency, target specificity, and biosafety, providing a transformable new paradigm for overcoming the bottleneck in glioma treatment. This integrated delivery system significantly prolonged the survival of tumor-bearing mice in animal models without significant neurotoxicity or abnormal liver and kidney function, demonstrating superior safety and efficacy. Through multi-mechanism synergy, it not only enhances drug accumulation at the tumor site but also more effectively inhibits the recurrence process, providing new treatment hope for glioma patients.

[0011] Pep-1@TMZ m The construction method for @MitoNVs is as follows: TMZ m Preparation of @MitoNVs After cardiac perfusion of healthy rats, brain tissue was harvested, and mitochondria were extracted using a tissue mitochondrial isolation kit (C3606, Bayes Biotech). Protein concentration was determined using a BCA protein assay kit (SK1070, Coolaber). The mitochondrial mixture (1 mg / ml) and TMZ (1 mg / ml) were repeatedly passed through polycarbonate filters (0.4 μm and 0.2 μm) more than 10 times using a micro-squeezer. TMZ was obtained after centrifugation at 3500 rpm for 10 minutes at 4°C. m @MitoNVs are treated as sediment and then resuspended for use in subsequent operations.

[0012] Preparation of Pep-1@TMZ@MitoNVs use Figure 1 The method can prepare Pep-1@TMZ m @MitoNVs, specifically: First, utilize the obtained TMZ m @MitoNVs reacted with Pep-1 in the same manner as step two. The principle was as follows: Pep-1 was modified with an azide group (-N3), and TMZ was modified with an alkynyl group (-C≡CH), resulting in the formation of 1,2,3-triazole bonds under Cu(I) catalysis. Based on click chemistry, we obtained the modified TMZ. m Subsequently, the modified TMZ was further processed using click chemistry.m It is linked to the cell-penetrating peptide Pep-1. Attached Figure Description

[0013] Figure 1 TMZ in the embodiments of the present invention m A diagram illustrating the preparation process of @MitoNVs; Figure 2 Pep-1@TMZ in this embodiment of the invention m A diagram illustrating the preparation process of @MitoNVs; Figure 3 This is a potential characterization diagram in an embodiment of the present invention; Figure 4 Pep-1@TMZ in this embodiment of the invention m @MitoNVs particle size chart; Figure 5 This is a biocompatibility diagram from an embodiment of the present invention; Figure 6 Pep-1@TMZ in this embodiment of the invention m @MitoNVs kernel colocalization image; Detailed Implementation

[0014] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following examples provide a more detailed description of the invention. It should be noted that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0015] like Figure 1 As shown, temozolomide and temozolomide have the same antitumor activity. We used click chemistry to modify the alkyne group (-C≡CH) of temozolomide to enhance its blood-brain barrier penetration. Then, by modifying Pep-1 with an azide group (-N3), we synthesized a new drug combination, Pep-1@TMZ, which enhances blood-brain barrier penetration. m like Figure 2 As shown, artificial mitochondria MitoNVs extracted from mouse brains and the obtained Pep-1@TMZ m A mitochondrial nanovesicle, Pep-1@TMZ, with enhanced penetration of the blood-brain barrier was obtained by micro-extrusion, passing through polycarbonate membranes (0.4 μm and 0.2 μm), and centrifugation. m @MitoNVs.

[0016] like Figure 3 As shown, MitoNVs and TMZ were obtained by characterizing their size, charge, and morphology using dynamic light scattering and transmission electron microscopy. m @MitoNVs and Pep-1@TMZ mThe potentials of @MitoNVs are: -24.6mV, -35.1mV, and -37.2mV.

[0017] like Figure 4 As shown, by characterizing Pep-1@TMZ m The particle size of @MitoNVs is approximately 175nm.

[0018] like Figure 5 As shown, MitoNVs and TMZ were verified through cell experiments. m @MitoNVs and Pep-1@TMZ m @MitoNVs have good biocompatibility.

[0019] like Figure 6 As shown, the endocytosis experiment verified the efficacy of Pep-1@TMZ. m @MitoNVs exhibit good penetrability, and fluorescence indicates endocytosis with localization to the cell nucleus.

[0020] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A mitochondria nanovesicle Pep-1@TMZ for enhancing penetration of blood brain barrier m @The method for constructing MitoNVs is characterized in that, The method is: Step one, TMZ m @Preparation of MitoNVs; Step two, Pep-1@TMZ m @Preparation of MitoNVs.

2. The construction method according to claim 1, characterized in that, The step one is specifically: After heart perfusion of healthy rats, the brain tissue was taken out, the mitochondria in the brain was extracted using a tissue mitochondria isolation kit, and the protein concentration was determined using a BCA protein concentration determination kit; The mixture of mitochondria (1 mg / ml,) and TMZ (1 mg / ml) was repeatedly passed through a polycarbonate filter membrane (0.4 μm and 0.2 μm) more than 10 times using a micro-extruder; After centrifugation at 3500 rpm for 10 minutes at 4 DEG C, TMZ@MitoNVs were obtained as precipitates, which were then resuspended for subsequent operation.

3. The construction method of claim 1, wherein, The step two is specifically: After mixing Pep-1 (1 mg) with TMZ@MitoNVs at room temperature, Schiff base reaction and covalent self-assembly principles were carried out through the carbonyl, amino and sulfhydryl groups of Pep-1 and TMZ, and the reaction was carried out at 25 DEG C for 5 h under oscillation at 500 r / min, and after centrifugation (14000 rpm) and washing, Pep-1@TMZ was finally obtained by centrifugation (14000 rpm) again. m @MitoNVs.

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