Peptide lipid nanoparticle, preparation method, application and cancer combination therapy drug combination

Abstract

The application provides a peptide lipid nanoparticle, a preparation method, an application and a cancer combined treatment drug combination, and belongs to the technical field of biological medicines.The application proposes a novel synergistic anti-tumor strategy based on the combination of a peptide lipid nanoparticle and a low-dose mTOR inhibitor, synergistically inhibits a PI3K / AKT / mTOR signal pathway, co-applies the peptide lipid nanoparticle loaded with p53 circular RNA and the mTOR inhibitor to cancer cells, restores p53 function, reactivates PTEN, blocks the feedback activation of PI3K / AKT, and directly inhibits mTOR by the mTOR inhibitor, thereby producing a persistent cascade double blocking. Moreover, p53 inhibits the evirolimus-induced pro-survival autophagy, converts it into a stress-induced process, and realizes the double inhibition and persistent blocking of the PI3K / AKT / mTOR pathway.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a peptide lipid nanoparticle, its preparation method, its application, and a combination of drugs for cancer treatment. Background Technology

[0002] The phosphatidylinositol 3-kinase / protein kinase B / mammalian target of rapamycin (PI3K / AKT / mTOR) signaling pathway plays a central role in regulating key biological processes such as cell proliferation, survival, metabolism, and protein synthesis. This pathway is aberrantly activated in approximately 50% of human malignancies, becoming a significant driver of tumor development and progression, and is therefore considered a highly promising target for anticancer therapy. Currently, inhibitors targeting this pathway, especially selective inhibitors of the mTORC1 complex such as everolimus, have been approved by the US FDA and regulatory agencies in several other countries for the clinical treatment of various cancers, including renal cell carcinoma, breast cancer, and neuroendocrine tumors. However, despite the preliminary efficacy shown by these targeted drugs in some patients, the overall response rate remains limited by inherent or acquired tumor resistance mechanisms, particularly in non-small cell lung cancer (NSCLC), a highly prevalent and deadly malignant tumor.

[0003] Studies have shown that p53, as a classic "genomic guardian," is a key tumor suppressor protein with irreplaceable roles in maintaining genome stability, inducing cell cycle arrest, promoting DNA repair, and triggering apoptosis. In NSCLC, the incidence of p53 gene deletion or inactivation mutations is as high as approximately 72%, representing a significant molecular basis for tumor progression and treatment resistance. Notably, p53 not only directly regulates multiple cell fate-determining genes but also indirectly inhibits the overactivation of the PI3K / AKT / mTOR pathway by upregulating the expression of negative regulators such as PTEN (a homolog of phosphatase and tensin). When p53 function is lost, PTEN expression is significantly downregulated, leading to persistent activation of the PAM pathway, thereby weakening the anti-tumor effect of mTORC1 inhibitors and forming a typical "pathway escape" mechanism. Therefore, restoring p53 function is considered one of the key strategies for reversing resistance to mTOR inhibitors in p53-deficient NSCLC and improving sensitivity to targeted therapy.

[0004] In recent years, nucleic acid-based gene therapy has provided new insights into p53 functional reconstruction. Early attempts often used adenovirus vectors to deliver wild-type p53 genes (such as Gendicine). Although approved for use in some head and neck cancers, its strong immunogenicity, low transduction efficiency, and uncontrollable expression limited its widespread application. With the development of RNA therapy, linear mRNA has been used for in vivo p53 protein reconstruction due to its advantages such as not requiring genome integration and controllable transient expression. For example, Chinese patent CN201911107061.7 discloses a p53 delivery system based on linear mRNA, which can effectively restore p53 expression in vitro and enhance the sensitivity of tumor cells to targeted drugs. However, linear mRNA has inherent defects: its 5' cap structure and 3' poly(A) tail are easily recognized and degraded by nucleases, resulting in a short half-life and limited duration of protein expression; at the same time, unmodified linear mRNA can activate innate immune pathways such as Toll-like receptors (TLRs), triggering unnecessary inflammatory responses and affecting treatment safety and tolerability.

[0005] Against this backdrop, circular RNA (circRNA), as an emerging non-coding RNA platform, exhibits significantly superior physicochemical and biological properties compared to linear mRNA due to its unique covalently closed circular structure: First, circRNA lacks free ends, is highly resistant to exonucleases, possesses extremely strong molecular stability, and can persist in cells for several days or even longer; second, engineered circRNA can efficiently recruit translation initiation factors, achieving long-term, stable protein expression; third, circRNA naturally has low immunogenicity, significantly reducing the risk of adverse reactions to the delivery system. These advantages make it an ideal vector for delivering tumor suppressor genes (such as p53).

[0006] However, the clinical translation of circRNA still faces significant challenges—an efficient in vivo delivery system. While current mainstream lipid nanoparticles (LNPs) have achieved success in linear mRNA vaccines, they suffer from low encapsulation efficiency and difficulty in intracellular release of larger molecular weight, highly complex secondary structures like circRNA, resulting in insufficient bioavailability in tumor tissues and difficulty in achieving effective therapeutic concentrations. Therefore, there is an urgent need to develop a novel nanocarrier capable of specifically targeting tumor tissues, efficiently loading and protecting p53-circRNA, and achieving controlled release and sustained expression within cells. Summary of the Invention

[0007] The purpose of this invention is to provide peptide lipid nanoparticles, their preparation method, applications, and combination therapies for cancer treatment. It constructs a stable and efficient RNA delivery platform and achieves a new paradigm of anti-tumor treatment that is potent, low in toxicity, and resistant to drug resistance through synergistic mechanisms, showing promising prospects for clinical translation.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing peptide lipid nanoparticles, comprising the following steps: (1) Mix and dissolve peptide lipids and auxiliary lipids to obtain a lipid phase; (2) Dissolve p53-CircRNA to obtain an aqueous phase; (3) The lipid phase and the aqueous phase are mixed in a microfluidic chip and dialyzed to obtain peptide lipid nanoparticles.

[0009] Preferably, in step (1), the auxiliary lipids include two or more of DOPE, cholesterol, and DSPE-PEG2000; the lipid phase concentration is 0.5-2 mg / mL based on peptide lipid concentration; the peptide lipids include CDO, and the chemical structural formula of CDO is as follows: .

[0010] Preferably, in step (2), the concentration of the aqueous phase is 0.05~0.1 mg / mL.

[0011] Preferably, in step (3), the volume ratio of the peptide lipid to p53-CircRNA is 1:2~4.

[0012] The present invention also provides peptide-lipid nanoparticles prepared by the above-mentioned method for preparing peptide-lipid nanoparticles.

[0013] The present invention also provides an application of the above-mentioned peptide lipid nanoparticles in the preparation of cancer treatment drugs.

[0014] The present invention also provides a combination therapy for cancer, the combination therapy comprising an mTOR inhibitor and the above-mentioned peptide-lipid nanoparticles.

[0015] Preferably, the mTOR inhibitor includes everolimus.

[0016] Preferably, the dosage of everolimus is 2.0~2.5 mg / kg.

[0017] Preferably, the cancer for which the combination therapy is applicable includes p53-deficient cancers.

[0018] Preferably, the p53-deficient cancers include non-small cell lung cancer, liver cancer, neuroblastoma, and pancreatic cancer caused by p53 deficiency.

[0019] The beneficial effects of the invention compared to the prior art are as follows: (1) This invention proposes a novel synergistic anti-tumor strategy based on the combined use of peptide-lipid nanoparticles and a low-dose (50% of the clinical dose) mTOR inhibitor. By synergistically inhibiting the PI3K / AKT / mTOR signaling pathway, peptide-lipid nanoparticles loaded with p53 circular RNA and an mTOR inhibitor are co-administered to cancer cells, restoring p53 function, reactivating PTEN, and blocking the feedback activation of PI3K / AKT. Meanwhile, the mTOR inhibitor directly inhibits mTOR, resulting in a durable cascade of dual blockade. Furthermore, p53 inhibits everolimus-induced pro-survival autophagy, transforming it into a stress-induced process, achieving dual and durable blockade of the PI3K / AKT / mTOR pathway. More importantly, the restored p53 function can transform mTOR inhibitor-induced pro-survival autophagy into stress-induced pro-death autophagy, further enhancing the tumor cell killing effect.

[0020] (2) The peptide-lipid nanoparticles prepared in this invention can efficiently encapsulate circular RNA (circRNA-p53) encoding p53 protein, with an RNA encapsulation efficiency of up to 95.8%, and can maintain good particle size stability and RNA integrity even after being stored at 4°C for 90 days. These nanoparticles can precisely deliver circRNA-p53 into tumor cells, restoring the expression of functional p53 protein.

[0021] (3) The combination therapy of the peptide lipid nanoparticles of the present invention with a low dose of mTOR inhibitor exhibits excellent anti-tumor activity. In various tumor models, the tumor inhibition rate of the combination therapy group is as high as 98%, which is close to complete regression. Mechanistic studies have shown that the combination therapy significantly upregulates the pro-apoptotic proteins PUMA and Bax, reduces the Bcl-2 / Bax ratio, and activates Caspase-9 and Caspase-3, indicating that it plays a synergistic role by enhancing the mitochondrial apoptosis pathway.

[0022] (4) In terms of safety, the major organ structures remained intact during the combination therapy, with no inflammatory infiltration or pathological damage, indicating that the recovery of p53 function effectively alleviated the systemic toxicity of mTOR inhibitors. In addition, this combination therapy has the potential to overcome drug resistance by blocking pathway feedback activation and reprogramming autophagy fate. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a flowchart illustrating the preparation process of peptide-lipid nanoparticles in Example 1; Figure 2 The chemical structural formula of the peptide lipid CDO in Example 1 is shown below; Figure 3 Transmission electron microscopy image of the peptide lipid nanoparticles prepared in Example 1; Figure 4 This is an agarose gel electrophoresis image of the peptide-lipid nanoparticles in Experiment Example 1 of this invention; Figure 5 This is a graph showing the cellular uptake efficiency of peptide-lipid nanoparticles in p53-deficient NCI-H1299 cells in Experimental Example 2 of this invention. Figure 6 This is a graph showing the cell transfection efficiency of peptide-lipid nanoparticles in p53-deficient NCI-H1299 cells in Experiment Example 3 of this invention. Figure 7 This is a diagram showing the experimental results of peptide-lipid nanoparticle-induced apoptosis in NCI-H1299 cells in Experiment Example 3 of this invention; Figure 8 This is a diagram illustrating the synergistic tumor-inhibiting effect of peptide-lipid nanoparticles and everolimus in a mouse subcutaneous tumor model in Experiment Example 4 of this invention. Figure 9 This is a diagram illustrating the synergistic tumor-inhibiting effect of peptide-lipid nanoparticles and everolimus in a mouse orthotopic tumor model in Experiment Example 4 of this invention. Figure 10 This is a representative protein blot image of the combined treatment with peptide lipid nanoparticles and everolimus in Experiment Example 4 of this invention. Figure 11 This is a representative immunohistochemical image of peptide lipid nanoparticles and everolimus after combined treatment in Experiment Example 4 of this invention; Figure 12 This is a model diagram of the combined treatment mechanism of peptide lipid nanoparticles and everolimus in Experiment Example 4 of the present invention; Figure 13 This is a representative H&E staining image after combined treatment with peptide lipid nanoparticles and everolimus in Experiment Example 4 of this invention; Figure 14 This is a graph showing serum biochemical indicators after combined treatment with peptide lipid nanoparticles and everolimus in Experiment Example 4 of this invention. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Example 1 Embodiment 1 of the present invention is based on Figure 1 The process shown is used to prepare peptide-lipid nanoparticles. The specific steps are as follows: (1) The peptide lipid CDO (chemical structure formula shown) Figure 2 ), DOPE, cholesterol and DSPE-PEG2000 were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to prepare a lipid phase with a CDO concentration of 1 mg / mL.

[0031] (2) The purified p53-CircRNA (purchased from APExBIO) was dissolved in citrate buffer (pH 4.0) to form an aqueous phase with an RNA concentration of 0.066 mg / mL.

[0032] (3) At a flow rate of 12 mL / min, the lipid phase and aqueous phase were simultaneously pumped into the microfluidic chip at a volume ratio of 1:3 for mixing. The resulting nanoparticle suspension was dialyzed with PBS (pH 7.4) to remove ethanol and replace the buffer solution, finally obtaining peptide-lipid nanoparticles, which were stored at 4℃ for later use. The peptide-lipid nanoparticles were observed under a transmission electron microscope, as shown in the figure. Figure 3 As shown.

[0033] Comparative Example 1 Comparative Example 1 of this invention uses SM-102 as a control to prepare an SM-102-CircRNA-LNP. The specific steps are as follows: (1) SM-102, DOPE, cholesterol and DSPE-PEG2000 were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to prepare a lipid phase with a concentration of 1 mg / mL of SM-102.

[0034] (2) The purified p53-CircRNA was dissolved in citrate buffer (pH 4.0) to form an aqueous phase with an RNA concentration of 0.066 mg / mL.

[0035] (3) The lipid phase and aqueous phase were simultaneously pumped into the microfluidic chip at a flow rate of 12 mL / min in a 1:3 ratio for mixing. The resulting nanoparticle suspension was dialyzed with PBS (pH 7.4) to remove ethanol and replace the buffer, finally obtaining SM-102-CircRNA-LNPs, which were stored at 4℃ for later use.

[0036] Experimental Example 1 Experimental Example 1 of this invention characterizes the performance of nanoparticles prepared in Example 1 and Comparative Example 1. The specific steps are as follows: The encapsulation effect of the peptide-lipid nanoparticles prepared in Example 1 and the SM-102-CircRNA-LNPs prepared in Comparative Example 1 was detected by agarose gel electrophoresis. The agarose gel electrophoresis conditions were as follows: a 1% agarose gel was prepared; the LNP sample containing 0.3 μg of RNA was mixed with the loading buffer; electrophoresis was performed at 100 V and 45 mA for approximately 1 hour, with the endpoint determined by the migration of bromophenol blue indicator to the top of the gel. The results are shown below. Figure 4 As shown.

[0037] Figure 4 The results showed that peptide-lipid nanoparticles have a strong encapsulation effect on nucleic acids, with an RNA encapsulation efficiency of 95.8% and SM-102-CircRNA (88%). After being stored at 4°C for 90 days, the peptide-lipid nanoparticles still maintained good particle size stability and RNA integrity.

[0038] Experimental Example 2 Example 2 of this invention tested the cellular uptake efficiency of the peptide-lipid nanoparticles prepared in Example 1 in vitro. The specific steps are as follows: Using SM-102-CircRNA-LNPs as the control group, NCI-H1299 cells were divided into groups of 1×10⁻⁶. 7 Cells were seeded at a density of 1 / 2 well in 48-well plates and cultured in RPMI-1640 (10% serum) medium for 18 h. When the cell density reached 80%-90%, the medium was replaced with serum-free medium. Cy5-labeled RNA-loaded peptide-lipid nanoparticles were diluted in serum-free medium for cell uptake experiments. LNPs were quantified as RNA (0.3 μg / well) in triplicate. Cells were collected after 2 h, 4 h, and 6 h of culture and centrifuged at 12000 rpm for 5 min at 4 °C. The cell pellet was resuspended in PBS, and LNP uptake was detected by flow cytometry. The results are shown below. Figure 5 As shown.

[0039] Figure 5 The results showed that the cellular uptake rate of peptide-lipid nanoparticles reached 98.9% within 6 hours. Confocal microscopy revealed that LNPs successfully escaped from endosomes / lysosomes into the cytoplasm within 6 hours.

[0040] Experimental Example 3 Experimental Example 3 of this invention tested the cell transfection and apoptosis effects of the peptide-lipid nanoparticles prepared in Example 1 in vitro. The specific steps are as follows: Using SM-102-CircRNA-LNPs as the control group, NCI-H1299 cells were divided into groups of 1×10⁻⁶. 7 The nanoparticles were seeded at a density of / wells in 48-well plates and cultured in RPMI-1640 (10% serum) medium for 18 hours. The medium was then replaced with serum-free medium, and the resulting peptide-lipid nanoparticles were diluted in the serum-free medium and transfected for 6 hours (e.g., ...). Figure 6 As shown in the image, the culture medium containing the peptide-lipid nanoparticles was removed, the cells were washed with PBS, and replaced with complete RPMI-1640 medium containing 10% fetal bovine serum and 1% antibiotics. The cells were then cultured for 48 hours, and the apoptosis of NCI-H1299 cells transfected with the peptide-lipid nanoparticles was observed. The results are as follows: Figure 7 As shown.

[0041] Figure 7 Annexin V / PI double staining revealed significant apoptotic features in cells treated with peptide-lipid nanoparticles, with an apoptosis rate as high as 49.7%, which was 1.18 times that of the SM-102-CircRNA group. Furthermore, strong perinuclear green fluorescence and nuclear fragmentation confirmed the presence of a large number of apoptotic cells.

[0042] Test Example 4 Experimental Example 4 of this invention tested the tumor-suppressive effect of peptide-lipid nanoparticles. The specific steps are as follows: (1) Establishment of mouse model Subcutaneous and in situ non-small cell lung cancer models were constructed using immunodeficient BALB / c nude mice (4-6 weeks old) and NCI-H1299 cells, respectively.

[0043] After expanding the cells to the logarithmic growth phase in vitro, they were then used at a rate of 1×10⁻⁶. 7 The cells were mixed with an equal volume of matrix gel at a density of / mL. A subcutaneous non-small cell lung cancer model was established by injecting 100μL of cell suspension into the right groin for easy observation and measurement.

[0044] An equal volume of suspension was injected into the left lung parenchyma through precise positioning in the left intercostal space, and the needle was left in place for 5 seconds to prevent reflux. After the operation, the lungs were sutured in layers to construct an in situ non-small cell lung cancer model.

[0045] (2) Grouping and administration Two weeks after inoculation, tumor formation was verified by in vivo imaging. After tumor formation, tumor-bearing mice were randomly divided into four groups: saline control group, everolimus monotherapy group (2.5 mg / kg), peptide lipid nanoparticle monotherapy group (700 μg / kg CircRNA), and everolimus (50% of the clinical treatment dose, 2.5 mg / kg) combined with peptide lipid nanoparticle therapy group.

[0046] Administer the medication once every 3 days for a total of 7 times, and record the mouse's weight during the administration period.

[0047] In the in situ model, tumor growth was monitored using bioluminescence imaging, and the results were as follows: Figure 8 , 9 As shown.

[0048] Figure 8 The combined treatment also achieved 90% tumor growth inhibition in the subcutaneous model.

[0049] Figure 9 The results showed that in the in situ model, the combination therapy group exhibited near-complete tumor regression with an inhibition rate as high as 98%, and the efficacy was significantly better than that of each single-drug therapy group.

[0050] (3) Western blot and immunohistochemical analysis Western blot and immunohistochemical analysis were performed on tumor tissues from treated mice, and the results are as follows: Figure 10 , 11 As shown, Figure 10 , 11The results showed that p53 and PTEN protein expression was significantly upregulated in the combination therapy group, while PI3K and AKT activities were inhibited. Everolimus monotherapy effectively inhibited p-mTOR and p-p70S6K, but triggered feedback activation of PI3K / AKT; while combination therapy blocked this feedback activation, achieving dual inhibition of the PAM pathway. Furthermore, everolimus monotherapy induced autophagy (ATG7 and LC3B-II upregulated), while combination therapy significantly downregulated these autophagy markers, indicating that the restoration of p53 inhibited everolimus-induced pro-survival autophagy. Combination therapy also significantly upregulated the pro-apoptotic proteins PUMA and Bax, reduced the Bcl-2 / Bax ratio, and activated Caspase-9 and Caspase-3, indicating that it exerts a synergistic anti-tumor effect by enhancing the mitochondrial apoptosis pathway. The mechanism model of the combination therapy of peptide-lipid nanoparticles and everolimus is as follows: Figure 12 As shown.

[0051] (4) Toxicity Mouse body weight was monitored during treatment; no significant weight loss was observed in the combined treatment group. After treatment, tissue sections from organs such as the heart, liver, spleen, lungs, and kidneys were collected for H&E staining analysis, and the results are as follows. Figure 13 As shown.

[0052] Figure 13 The lung tissue showed intact alveolar structure and regular shape, with no obvious alveolar collapse or adhesion. No pathological damage such as inflammatory cell infiltration was observed in other organs.

[0053] Serum biochemical analysis was performed on each group. Figure 14 The results showed that the everolimus monotherapy group exhibited elevated liver enzymes (ALT, AST), indicating hepatotoxicity; while the liver enzyme levels in the combination therapy group were not significantly different from those in the saline control group, indicating that the combination therapy effectively reduced its systemic toxicity.

[0054] 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 and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing peptide-lipid nanoparticles, characterized in that, Includes the following steps: (1) Mix and dissolve peptide lipids and auxiliary lipids to obtain a lipid phase; (2) Dissolve p53-CircRNA to obtain an aqueous phase; (3) The lipid phase and the aqueous phase are mixed in a microfluidic chip and dialyzed to obtain peptide lipid nanoparticles.

2. The method for preparing peptide-lipid nanoparticles according to claim 1, characterized in that, In step (1), the auxiliary lipids include two or more of DOPE, cholesterol, and DSPE-PEG2000; the lipid phase concentration is 0.5-2 mg / mL based on peptide lipid concentration; the peptide lipids include CDO, and the chemical structural formula of CDO is as follows: 。 3. The method for preparing peptide-lipid nanoparticles according to claim 1, characterized in that, In step (2), the concentration of the aqueous phase is 0.05~0.1 mg / mL.

4. The method for preparing peptide-lipid nanoparticles according to claim 1, characterized in that, In step (3), the volume ratio of the peptide lipid to p53-CircRNA is 1:2~4.

5. Peptide-lipid nanoparticles prepared by the method of any one of claims 1 to 4.

6. The use of the peptide lipid nanoparticles of claim 5 in the preparation of a cancer treatment drug.

7. A combination therapy drug for cancer, characterized in that, The combination therapy for cancer includes an mTOR inhibitor and the peptide-lipid nanoparticles of claim 6.

8. The combination therapy drug for cancer according to claim 7, characterized in that, The mTOR inhibitor includes everolimus, and the dosage of everolimus is 2.0~2.5 mg / kg.

9. The combination therapy drug for cancer according to claim 7, characterized in that, The combination of cancer treatment drugs is applicable to cancers including p53-deficient cancers.

10. The combination therapy for cancer according to claim 9, characterized in that, The p53-deficient cancers include non-small cell lung cancer, liver cancer, neuroblastoma, and pancreatic cancer caused by p53 deficiency.

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