Temozolomide gene co-loaded targeted ferritin nano delivery system for drug-resistant glioblastoma

Abstract

The invention discloses a gene nano delivery system CendR-Fn (at) T / siRNA which has a tumor targeting effect, is loaded with targeting polypeptide CendR and can be used for treating drug-resistant glioblastoma, discloses a preparation method of a gene co-loaded targeting ferritin nano delivery system for down-regulating drug resistance of temozolomide, discloses a nano structure of the gene co-loaded targeting ferritin nano delivery system, discloses culture of drug resistance of U87MG cells, and further discloses a preparation method of the gene co-loaded targeting ferritin nano delivery system. The invention discloses the efficiency of carrying a medicine temozolomide (TMZ) by the nano delivery system, discloses targeted uptake of CendR-Fn (at) T / siRNA by a U87MG cell, and further discloses a treatment effect of the nano delivery system on glioblastoma and a protection effect on a blood-brain barrier BBB. Therefore, the application of the multifunctional nano-drug delivery system in induction of programmed death of tumor cells and enhancement of gene drug delivery efficiency is clarified.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a temozolomide gene co-loaded targeted ferritin nano-delivery system for drug-resistant glioblastoma. Background Art

[0002] Glioblastoma (GBM) is a malignant tumor located in the brain and spinal cord, clinically referred to as "glioblastoma". The median survival rate of patients with brain glioma is only 14.6 months, and the 5-year survival rate is still less than 10%. This type of tumor usually originates from the mutation and abnormal proliferation of common glial cells in brain tissue, which will form glioblastomas. It has the characteristics of high invasiveness, low survival rate, and high morbidity. In the early stage, the treatment of GBM was mainly surgical resection, adjuvant radiotherapy and chemotherapy. In recent years, immunotherapy, gene therapy, and oncolytic virus therapy have provided new options for GBM treatment.

[0003] At present, temozolomide has become a first-line drug for the treatment of glioblastoma due to its good therapeutic effect and low toxicity. Temozolomide (TMZ) is a prodrug of imidazole tetrazine, which is stable in acidic conditions and can tolerate gastric acid and can be administered orally. Since the prodrug has strong lipophilicity, it can pass through the blood-brain barrier (BBB) ​​smoothly. In the brain with a pH value of about 7, TMZ can spontaneously open the ring and hydrolyze to form the active metabolite 3-methyl (triazaalkenyl) imidazole-4-carboxamide (MTIC). MTIC is stable under alkaline conditions and further hydrolyzes under acidic conditions to form 5-amino-4-carboxamide imidazole (AIC) and methyldiazonium cation. Methyldiazonium cation can react with nucleophilic sites N7-guanine, O6-guanine and N3-adenine on DNA to form methyl adducts. Methylation at O6-guanine and N7-guanine sites can lead to cytotoxic damage, thereby stimulating nucleotide base mispairing during replication, resulting in DNA damage, cell cycle arrest, and further programmed cell death.

[0004] Although TMZ is currently the first-line drug for glioblastoma, most patients will gradually develop resistance to TMZ during treatment. Combining TMZ with other therapies, such as immunotherapy and gene therapy, is an effective strategy to address TMZ resistance. RNAi therapy has broad prospects in the field of tumor treatment. Nano-drug delivery systems can deliver therapeutic RNA to target sites, regulate related gene expression and protein levels, and thus achieve the purpose of treating diseases. Studies have shown that DNA-glycosylases can also remove N7-methylguanine and N3-methyladenine adducts and promote the repair of tumor cell DNA. In addition, the expression of DNA glycosylases in glioma tissues is significantly higher than that in non-tumor brain tissues. In addition to DNA-glycosylases, signal transducer and activator of transcription 3 (STAT 3) can increase the expression of O6-alkylguanine-DNA alkyltransferase (MGMT), thereby stimulating the DNA repair pathway and promoting tumor cell survival. To increase patients' sensitivity to TMZ, STAT 3-siRNA (siSTAT 3) can knock down STAT 3 at the mRNA and protein levels, thereby inhibiting MGMT expression and reducing the occurrence of drug resistance.

[0005] However, TMZ will be hydrolyzed into MTIC in the systemic circulation. MTIC has high polarity and is difficult to pass through the blood-brain barrier (BBB), thus reducing the therapeutic effect. Therefore, how to efficiently target TMZ to GB M and prevent its degradation in the body has become a problem to be solved. Nanotechnology is considered to be one of the most effective methods to improve drug solubility, stability and bioavailability. Among various nanoencapsulation strategies, ferritin (Human H-ferritin, HFn) has always been a hot topic of research. HFn is a cage-shaped structure with a central cavity, and its cavity structure can be loaded with drugs to achieve drug encapsulation and delivery. In addition, ferritin has the characteristics of self-assembly ability, high thermal stability, permeability and retention, so it is widely used in the delivery of chemotherapeutic drugs. Based on the structural characteristics and biological properties of ferritin, physical incubation method, pH-mediated depolymerization / recombination method and urea gradient method are used to achieve ferritin drug loading. At present, ferritin is mainly used as a drug carrier for the delivery of small molecule chemical drugs and large molecule nucleic acid drugs. HFn can bind to transferrin receptor 1 (TfR 1) overexpressed in BBB endothelial cells and deliver drugs across the BBB via receptor-mediated transcytosis. Therefore, ferritin is an excellent drug carrier for the treatment of brain diseases.

[0006] In addition, some studies have successfully used ferritin heavy chain to deliver siRNA, and the drug loading method is the pH method. However, due to the shortcomings of the preparation process, the drug loading of siRNA is not ideal. Therefore, if ferritin is used as a carrier for gene therapy, it is necessary to explore a more efficient drug loading method to increase the drug loading of siRNA. The researchers introduced Ca2+ during the self-assembly of HFn to promote the loading of siRNA. TMZ, as a small molecule drug, can diffuse into the nanocage through the channel on the surface of HFn. In order to improve the targeting of ferritin nanocages to GBM, the researchers modified the polypeptide sequence CendR (RGERPPR) with a high affinity for the transmembrane glycoprotein (NRP-1) overexpressed on the GBM cell membrane to the surface of Fn@T / siRNA, enhancing the permeability of tumor blood vessels and tumor tissues, thereby facilitating drug delivery.

[0007] Based on the above characteristics, the present invention constructs a method using HFn as a delivery carrier and Ca 2+ Assisted siRNA loading and enhanced drug lysosomal escape, delivering TMZ and siRNA at the same time. With the help of NHS-PEG-Mal, the targeting peptide CendR was modified to the surface of Fn@T / siRNA, thereby achieving dual targeting of BBB and GBM, downregulating MGMT expression, and solving current problems. The possibility of ferritin nanocages as a drug delivery system for drugs related to inducing glioblastoma death is expected to develop an efficient and safe tumor-targeted drug.

[0008] The present invention discloses a gene nano-delivery system CendR-Fn@T / siRNA with tumor targeting effect, loaded with targeting polypeptide CendR, and can be used for the treatment of drug-resistant brain glioblastoma, discloses a preparation method of a gene co-loaded targeted ferritin nano-delivery system for down-regulating temozolomide resistance, discloses its nanostructure, discloses the cultivation of U87MG cell resistance, discloses the efficiency of the nano-delivery system carrying the drug temozolomide (TM Z), discloses the targeted uptake of CendR-Fn@T / siRNA by U87MG cells, and further discloses the therapeutic effect of the nano-delivery system on glioblastoma and the protective effect on the blood-brain barrier BBB. Therefore, the application of the ferritin nano-drug delivery system in inducing tumor cell programmed death and enhancing the efficiency of gene drug delivery is clarified. Summary of the invention

[0009] The purpose of the present invention is to invent a gene drug delivery system for inducing glioblastoma cell death.

[0010] CendR-Fn@T / siRNA.

[0011] The gene delivery system of the present invention is prepared by the gene delivery vector HFn which is natural and targets tumor cells, the gene drug siRNA which down-regulates the expression of MGMT and the TMZ which synergistically induces the death of tumor cells.

[0012] The gene delivery system of the present invention is a gene drug delivery system Ce ndR-Fn@T / siRNA for inducing ferroptosis of tumor cells, wherein the molar ratio of each component is about siRNA: HFn: TMZ = 1: 3: 1200.

[0013] The surface of the nano-delivery system of the present invention is modified with a targeting polypeptide CendR, so as to enhance the targeting to drug-resistant glioblastoma.

[0014] The small interfering RNA (siRNA) is an siRNA used for down-regulating the expression of O6-alkylguanine-DNA alkyltransferase (MGM T) to enhance the therapeutic effect of temozolomide on drug-resistant glioblastoma.

[0015] The gene delivery system of the present invention is a nanostructure. The particle size of the nano delivery system is 50-100 nm and has a uniform and round spherical structure.

[0016] Another object of the present invention is to provide a method for preparing a gene delivery system.

[0017] The preparation method of the gene drug delivery system of the present invention comprises the following steps:

[0018] First, HFn@Fn@T / siRNA was prepared by the pH method, and then the CendR polypeptide was modified onto the surface of Fn@T / siRNA with the help of NHS-PEG-Mal to obtain CendR-Fn@T / siRNA.

[0019] Specifically, the preparation method of the gene drug delivery system of the present invention comprises the following steps:

[0020] (1) Prepare CendR solution and NHS-PEG-Mal solution;

[0021] (2) Mix the siRNA solution with CaCl 2 The solutions were mixed and incubated together, and free Ca was removed using an ultrafiltration tube. 2+ ;

[0022] (3) adding HFn solution to the above solution, adjusting the pH value and incubating;

[0023] (4) Add TMZ solution and continue incubation;

[0024] (5) Use ultrafiltration tube to remove free Ca 2+, siRNA, HFn subunit to obtain Fn@T / siRNA solution;

[0025] (6) reacting the CendR polypeptide with NHS-PEG-Mal solution and removing unreacted peptide by dialysis;

[0026] (7) The Fn@T / siRNA solution is mixed with the NHS-PEG-CendR solution, and CendR-Fn@T / siRNA is obtained after incubation.

[0027] The molar ratio of the CendR polypeptide to HFn is about 1:10.

[0028] In more detail, the preparation method of CendR-Fn@T / siRNA includes the following steps:

[0029] 1. Preparation of solution

[0030] 1.1 Prepare CendR solution: Accurately weigh 5 mg of CendR peptide and add 1 mL of ultrapure water to dissolve it to obtain a 5 mg / mL CendR solution, which was stored in a -20°C refrigerator.

[0031] 1.2 Prepare NHS-PEG-Mal solution: Accurately weigh 10 mg NHS-PEG-Mal, add 1 mL 20 mM PB 150 mM NaCl to dissolve, to obtain 10 mg / mL NHS-PEG-Mal solution, and store in a -20°C refrigerator.

[0032] 2. Preparation of CendR-Fn@T / siRNA

[0033] 2.1 Take 2 μL of 20 μmol / L siRNA solution and 20 μL of 20 mM CaCl 2 The solution was placed in a 0.5 mL EP tube and incubated for 20 min (200 rpm, 20 min). Free Ca was removed using a 10 kD ultrafiltration tube. 2+ , ultrafiltration once (8000r, 10min);

[0034] 2.2 Take 10 μL of 0.25 mg / mL HFn solution, add the ultrafiltered Ca-siRNA solution, adjust the system pH to 2 with 0.1 M HCl solution, and incubate for 20 min; then adjust the system pH to 8 with 0.1 M NaOH solution, and incubate for 2 h;

[0035] 2.3 Add 5 μL of 10 mM TMZ solution to the solution system and incubate for 1 h;

[0036] 2.4 Use 50kD ultrafiltration tube to remove free Ca 2+, siRNA, and HFn subunit were ultrafiltered once (8000r, 10min), diluted and concentrated with 20mM PB 150mM NaCl to obtain 100μL Fn@T / siRNA solution, which was stored in a 4°C refrigerator;

[0037] 2.5 Place 200 μL of CendR peptide solution and 1 mL of NHS-PEG-Mal solution in a 1.5 mL EP tube and react at room temperature for 2 h;

[0038] 2.6 Use 2kD dialysis bag to dialyze for 24 hours to remove unreacted peptides;

[0039] 2.7 Pipette 30 μL of Fn@T / siRNA solution and 480 μL of NHS-PEG-CendR solution into a 1.5 mL EP tube and incubate for 2 h. The molar ratio of CendR:HFn is about 1:10.

[0040] Another object of the present invention is to provide an application of a gene delivery system in the preparation of a drug for inhibiting tumors.

[0041] Another object of the present invention is to provide an application of a gene delivery system in the preparation of a drug for inhibiting tumors.

[0042] Another object of the present invention is to provide use of a nano-delivery system in the preparation of a drug for treating drug-resistant glioblastoma.

[0043] Another object of the present invention is to evaluate the efficiency of the gene delivery system CendR-Fn@T / siRNA in down-regulating the expression of siGPX4 at the mRNA and protein levels.

[0044] Another object of the present invention is to evaluate the ability of the gene delivery system CendR-Fn@T / siRNA to penetrate the blood-brain barrier.

[0045] Another object of the present invention is to evaluate the effect of the gene delivery system CendR-Fn@T / siRNA in inhibiting tumor cell proliferation.

[0046] Another object of the present invention is to evaluate the uptake efficiency of the gene delivery system CendR-Fn@T / siRNA in delivering the drug TMZ in tumor cells.

[0047] Another object of the present invention is to use the nano-delivery system in the preparation of a drug capable of down-regulating the expression of MGMT.

[0048] The gene delivery system of the present invention has the following advantages over existing gene delivery systems:

[0049] 1. The gene drug delivery system of the present invention can achieve efficient co-loading of genes and temozolomide drugs, and can achieve coordinated administration of the two, and the encapsulation efficiency is better than that of existing delivery systems;

[0050] 2. The present invention modifies and improves the structure of ferritin, solving the problem that traditional ferritin cannot accurately target glioma cells during gene drug delivery, and can achieve efficient synergistic delivery of genes and chemotherapy drugs to glioma cells.

[0051] Further explanation of the following words appearing in the manual:

[0052] GBM: Glioblastoma multiforme

[0053] HFn:Human ferritin heavy chain

[0054] MGMT: O6-methylguanine-DNA methyltransferase, O6-alkylguanine-DNA alkyltransferase

[0055] RNAi: RNA interference, RNA interference

[0056] siGPX4: small interfering RNA targeting Glutathione Peroxidase 4, small interfering RNA targeting glutathione peroxidase 4

[0057] BBB: blood-brain barrier

[0058] DLS: Dynamic light scattering

[0059] TEM:Transmission electron microscope

[0060] ICP-MS: Inductively Coupled Plasma-Mass Spectrometry

[0061] TMZ: Temozolomide

[0062] RNase A: RNase

[0063] OD value: optical density value

[0064] EX:Excitation Wavelength, excitation wavelength

[0065] EM:Emission Wavelength

[0066] NC: non-cognate-siRNA

[0067] Cy5: cyanine dye fluorescent labeling

[0068] AGE: agarose gel electrophoresis

[0069] NHS: N-hydroxysuccinimide

[0070] PEG: polyethylene glycol and

[0071] Mal: Maleimide

[0072] U87 MG: U87 human glioblastoma

[0073] DMEM: Dulbecco's modified eagle medium

[0074] FBS: fatal bovine serum

[0075] PBS: phosphate buffer saline, phosphate buffered saline

[0076] MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

[0077] DMSO: dimethyl sulfoxide

[0078] NS: Normal saline

[0079] FAM: carboxyfluorescein labeling

[0080] LSCM: laser scanning confocal microscopy, laser scanning confocal fluorescence microscopy

[0081] FCM: Flow CytoMetry, flow cytometry

[0082] MFI: Mean fluorescence intensity, mean fluorescence intensity. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 Schematic diagram of the action mechanism of CendR-Fn@T / siRNA.

[0084] Figure 2 A: Gene encapsulation efficiency of nanoparticles with different molar ratios of Cy5-siGPX4 / HFn (%); Figure 2 B: Encapsulation efficiency of Fn@T / siRNA with different RNA to HFn ratios.

[0085] Figure 3 A: TMZ encapsulation efficiency of nanoparticles with different molar ratios of TMZ / HFn (%); Figure 3 B: UV absorption spectra of Fn@T / siRNA and MTIC; Figure 3 C: Encapsulation efficiency of Fn@T / siRNA with different TMZ to HFn ratios.

[0086] Figure 4 Flow chart of the preparation of CendR-Fn@T / siRNA.

[0087] Figure 5 A: Particle size distribution of Fn@T / siRNA; Figure 5 B: Particle size distribution of CendR-Fn@T / siRNA; Figure 5 C: Zeta potential diagram of Fn@T / siRNA and CendR-Fn@T / siRNA.

[0088] Figure 6 A: TEM image of Fn@T / siRNA; Figure 6 B: TEM image of CendR-Fn@T / siRNA.

[0089] Figure 7 Calcium ion content standard curve.

[0090] Figure 8 AGE detection results after naked siRNA and Fn@T / siRNA were incubated with RNase A at 37°C for different time periods.

[0091] Fig. 9 AGE detection results after naked siRNA and Fn@T / siRNA were incubated with 50% FBS at 37°C for different time periods.

[0092] Fig.10 A Particle size of Fn@T / siRNA after 1, 3, 5, 7, and 14 days of storage; Fig.10 B Zeta potential of Fn@T / siRNA after storage for 1, 3, 5, 7, and 14 days.

[0093] Fig.11 A Particle size of CendR-Fn@T / siRNA after storage for 1, 3, 5, 7, and 14 days; Fig.11B Zeta potential of CendR-Fn@T / siRNA after 1, 3, 5, 7, and 14 days of storage.

[0094] Fig.12 The concentration of drug-resistant cells was determined by MTT method.

[0095] Fig.13 Transwell model.

[0096] Fig.14 Laser confocal scanning microscopy was used to characterize the targeting of CendR-Fn@T / siRNA nanoparticles to U87 MG cells.

[0097] Fig.15 A The number of fluorescent-labeled positive U87 MG cells detected by flow cytometry 4 hours after administration; Fig.15 B. Flow cytometry was used to detect the mean fluorescence intensity of cells in different drug administration groups. DETAILED DESCRIPTION

[0098] In order to further illustrate the present invention, a series of examples are given below. These examples are completely illustrative, and they are only used to specifically describe the present invention and should not be understood as limiting the present invention.

[0099] Example 1 Preparation of Fn@T / siRNA

[0100] Solution preparation

[0101] (1) siRNA solution:

[0102] Take 0.5OD siRNA, centrifuge at low speed (1500r, 1min) before dissolving, add 62.5μL DEPC water to dissolve, obtain 20μmol / L siRNA solution, and store in a -20℃ refrigerator.

[0103] (2) HFn solution:

[0104] The 100 g HFn purchased was in solid powder form. Before dissolution, it was centrifuged at low speed (1500 r, 3 min) and 400 μL ddH 2 Dissolve in 5% HFn and prepare a 0.25 mg / mL HFn solution, which is then divided into 10 portions of 40 μL and stored in a -20°C refrigerator.

[0105] (3) 10 mM temozolomide solution:

[0106] The purchased temozolomide was in the form of solid powder. 0.0019 g of temozolomide was accurately weighed, 1 mL of ultrapure water was added to dissolve it, and the mixture was stored in a refrigerator at 4°C for later use.

[0107] (4) 20 mM CaCl2 Solution:

[0108] Accurately weigh 0.0022g CaCl 2 , add 1 mL of ultrapure water to dissolve, and store at room temperature for later use.

[0109] (5) 0.1M NaOH solution:

[0110] Accurately weigh 0.02 g of NaOH, add 1 mL of ultrapure water to dissolve, and store at room temperature for later use.

[0111] (6) 0.1M HCl solution:

[0112] Pipette 1 mL of 6 mol / L HCl solution, add 60 mL of ultrapure water to dissolve, and store at room temperature for later use.

[0113] (7) 20 mM PB 150 mM NaCl solution:

[0114] Weigh 0.0716g Na2HPO4·12H2O accurately, add 1mL ultrapure water to dissolve, and prepare 0.2M Na2HPO4·12H2O solution. Weigh 0.0312g NaH2PO4·2H2O accurately, add 1mL ultrapure water to dissolve, and prepare 0.2M NaH2PO4·2H2O solution; pipette 190μL 0.2M NaH2PO4·2H2O and 810μL Na2HPO4·12H2O into a 1.5mL EP tube, filter through a 0.22μm filter membrane, and dilute to 10mL with ultrapure water to obtain 20mM PB buffer; weigh 0.08766g NaCl accurately, add it to 20mM PB buffer, and prepare 20mM PB 150mM NaCl solution, and store at room temperature for later use.

[0115] Preparation of Fn@T / siRNA

[0116] (1) 20 μmol / L siRNA solution and 20 mM CaCl2 solution were placed in a 0.5 mL EP tube and incubated for 20 min (200 rpm, 20 min). Free Ca2+ was removed using a 10 kD ultrafiltration tube and ultrafiltration was performed once (8000 r, 10 min).

[0117] (2) Take 0.25 mg / mL HFn solution, add ultrafiltered Ca-siRNA solution, adjust the system pH to 2 with 0.1 M HCl solution, and incubate for 20 min; then adjust the system pH to 8 with 0.1 M NaOH solution, and incubate for 2 h;

[0118] (3) Add 10 mM TMZ solution to the solution system and incubate for 1 h;

[0119] (4) Use a 50 kD ultrafiltration tube to remove free Ca2+, siRNA, and HFn subunits, ultrafilter once (8000 r, 10 min), dilute and concentrate with 20 mM PB 150 mM NaCl to obtain 100 μL Fn@T / siRNA solution, and store it in a 4 °C refrigerator.

[0120] Example 2 Single factor method to investigate the effects of RNA and TMZ addition on Fn@T / siRNA preparation

[0121] Investigate the effect of RNA addition amount on Fn@T / siRNA encapsulation efficiency

[0122] Experimental methods

[0123] (1) The encapsulation efficiency (EE) was calculated using Cy5-siGPX4. Fn@T / siRNA of different ratios were prepared using Cy5-siGPX4 according to the above method to explore the optimal formulation ratio for gene encapsulation.

[0124] Table 1 Cy5-siGPX4 / HFn Fn@T / siRNA at different molar ratios

[0125]

[0126] (2) Sample preparation

[0127] According to the above method, different molar ratios of Fn@T / siRNA were prepared, 20 μL of the liquid filtered by 10KD ultrafiltration tube was taken, and diluted to 1 mL with DEPC water. 20 μL of the liquid filtered by 50KD ultrafiltration tube was taken, and diluted to 1 mL with DEPC water.

[0128] (3) Preparation of standard products

[0129] Pipette 2 μL of 20 μmol / L Cy5-siGPX4 stock solution and dilute it to 100 μL with DEPC water as the standard solution.

[0130] (4) Determination of sample fluorescence intensity using a fluorescence spectrophotometer

[0131] Take 20 μL of the standard solution, dilute it to 1 mL with DEPC water, and measure the fluorescence intensity. The detection slit width is 10 nm, the excitation wavelength (EX) of Cy5 is 646 nm, and the emission wavelength (EM) is 664 nm.

[0132] (5) The single-point method was used to determine the content of Cy5-siGPX4 in the sample and calculate the encapsulation efficiency.

[0133] Experimental Results

[0134] like Figure 2 As shown in (A), when the molar ratio of Cy5-siGPX4 to HFn reached 1:6, the maximum encapsulation efficiency was 84.66±3.81%. Figure 2 (B) It can be seen that when the molar ratio of Cy5-siGPX4 to HFn is 1:3 and 1:6, the RNA encapsulation efficiency is not much different. Therefore, in subsequent experiments, RNA: HFn = 1:3 was selected as the optimal reaction ratio to prepare Fn@T / siRNA to explore the optimal TMZ encapsulation efficiency.

[0135] Investigate the effect of TMZ addition on Fn@T / siRNA encapsulation efficiency

[0136] Experimental methods

[0137] (1) Fn@T / siRNA was prepared according to the above method, and the amount of TMZ added was continuously changed to explore the optimal formulation ratio for encapsulating TMZ.

[0138] Table 2 Different molar ratios of TMZ / HFn Fn@T / siRNA

[0139]

[0140] (2) Sample preparation

[0141] Fn@T / siRNA with different molar ratios were prepared according to the above method, and 30 μL of Fn@T / siRNA solution was taken and diluted to 1.5 mL with ultrapure water.

[0142] (3) Preparation of standard products

[0143] Take 5 μL, 10 μL, and 20 μL of 10 mM TMZ solution, respectively, and dilute to 100 μL with water as standard solutions.

[0144] (4) Determination of TMZ content using the single-point method and calculation of encapsulation efficiency

[0145] Take 30 μL of the standard solution, dilute it to 1.5 mL with ultrapure water, and then measure it. Since TMZ can be rapidly hydrolyzed into the active metabolite MTIC under neutral or alkaline conditions, the absorbance of MTIC at 269 nm is measured to calculate the encapsulation efficiency of TMZ.

[0146] Experimental Results

[0147] In this experiment, the molar ratio of RNA to HFn was controlled at 1:3, and the amount of TMZ added was changed to explore the optimal molar ratio of the preparation for TMZ encapsulation; Figure 3 As shown in (A), when the molar ratio of TMZ to HFn reached 400:1, the encapsulation efficiency of TMZ reached 60.47±6.47%, and Figure 3 (C) It can be seen that as the addition ratio of TMZ increases, the encapsulation efficiency of TMZ gradually decreases.

[0148] Example 3 Preparation of CendR-Fn@T / siRNA

[0149] Solution preparation

[0150] (1) CendR solution:

[0151] Accurately weigh 5 mg of CendR peptide and add 1 mL of ultrapure water to dissolve it to obtain a 5 mg / mL CendR solution, which was stored in a -20°C refrigerator.

[0152] (2) NHS-PEG-Mal solution:

[0153] Accurately weigh 10 mg of NHS-PEG-Mal, add 1 mL of 20 mM PB 150 mM NaCl to dissolve, to obtain a 10 mg / mL NHS-PEG-Mal solution, and store in a -20°C refrigerator.

[0154] Preparation of CendR-Fn@T / siRNA

[0155] (1) Take 2 μL of 20 μmol / L siRNA solution and 20 μL of 20 mM CaCl 2 The solution was placed in a 0.5 mL EP tube and incubated for 20 min (200 rpm, 20 min). Free Ca was removed using a 10 kD ultrafiltration tube. 2+ , ultrafiltration once (8000r, 10min);

[0156] (2) Take 10 μL of 0.25 mg / mL HFn solution, add the ultrafiltered Ca-siRNA solution, adjust the system pH to 2 with 0.1 M HCl solution, and incubate for 20 min; then adjust the system pH to 8 with 0.1 M NaOH solution, and incubate for 2 h;

[0157] (3) Add 5 μL of 10 mM TMZ solution to the solution system and incubate for 1 h;

[0158] (4) Use 50kD ultrafiltration tube to remove free Ca 2+ , siRNA, and HFn subunit were ultrafiltered once (8000r, 10min), diluted and concentrated with 20mM PB 150mM NaCl to obtain 100μL Fn@T / siRNA solution, which was stored in a 4°C refrigerator;

[0159] (5) Place 200 μL of CendR peptide solution and 1 mL of NHS-PEG-Mal solution in a 1.5 mL EP tube and react at room temperature for 2 h;

[0160] (6) Use 2 kD dialysis bag to dialyze for 24 h to remove unreacted peptides;

[0161] (7) Pipette 30 μL of Fn@T / siRNA solution and 480 μL of NHS-PEG-CendR solution into a 1.5 mL EP tube and incubate for 2 h. The molar ratio of CendR:HFn is approximately 1:10.

[0162] Example 4 Characterization of Particle Size and Zeta Potential of Fn@T / siRNA and CendR-Fn@T / siRNA

[0163] Experimental methods

[0164] (1) Dynamic light scattering (DLS) characterization of particle size

[0165] Take 20 μL of Fn@T / siRNA and CendR-Fn@T / siRNA sample solutions and dilute them to 3 mL with ultrapure water. Add the sample solutions to the particle size measurement cup and perform particle size analysis using a dynamic light scattering particle size analyzer.

[0166] (2) Characterization of Zeta potential

[0167] The Fn@T / siRNA and CendR-Fn@T / siRNA sample solutions were placed in a potential measurement cup and the potential was measured using Zetasizer-Nano-ZS90.

[0168] Experimental Results

[0169] like Figure 5 (A) and Figure 5 As shown in (C), the particle size of Fn@T / siRNA was determined by DLS method to be 118.70±4.42nm; the Zeta potential of the nanoparticles at pH=7.4 was -10.18±1.75mV, indicating that the nanoparticles have a certain stability.

[0170] like Figure 5 (B) and Figure 5 As shown in (C), the particle size of CendR-Fn@T / siRNA determined by DLS was 121.93±5.13nm, and the Zeta potential was -30.40±4.25mV; the changes in particle size and potential proved the successful modification of the targeting polypeptide molecule CendR.

[0171] Example 5 Characterization by transmission electron microscopy

[0172] Experimental methods

[0173] (1) Preparation of Fn@T / siRNA samples: 10 μL of Fn@T / siRNA stock solution was dropped onto the electrophoretic lens and placed in a 37°C oven to dry for at least 24 h to allow the sample to be fully deposited, concentrated and dried;

[0174] (2) Preparation of CendR-Fn@T / siRNA samples: The samples were centrifuged and concentrated (8000r, 10min), 10μL of the samples were dropped onto the electrophoresis lens, and placed in a 37°C oven to dry for at least 24h to allow the samples to be fully deposited, concentrated and dried;

[0175] (3) Observe the transmission electron microscope (TEM) images and record photos.

[0176] The experimental results are from Figure 6 TEM images show that both Fn@T / siRNA and CendR-Fn@T / siRNA encapsulate calcium ions and have a uniform, rounded spherical structure with a particle size of approximately 50-100 nm.

[0177] Example 6 Quantitative determination of calcium ion content in Fn@T / siRNA by inductively coupled plasma mass spectrometry (ICP-MS)

[0178] Experimental methods

[0179] (1) Prepare aqua regia: Pipette 4.2 mL of concentrated HCl and 1.4 mL of concentrated nitric acid solution into a centrifuge tube and mix;

[0180] (2) Sample digestion: 40 μL Fn@T / siRNA was taken, and 160 μL aqua regia was added, and digested for 2 days;

[0181] (3) Transfer 200 μL of the solution to a 15 mL centrifuge tube, add ultrapure water to dilute to 10 mL, and wait for measurement.

[0182] Experimental Results

[0183] Figure 7 This is the standard curve of calcium ion content measured by ICP-MS. The calculated calcium content of the sample is 5.45 μg / mL.

[0184] Example 7 Characterization of RNase Stability by Agarose Gel Electrophoresis

[0185] Experimental methods

[0186] (1) Preparation of solution

[0187] 5 mg / mL proteinase K solution: Accurately weigh 0.0050 g of proteinase K solid, add 1 mL of ultrapure water to dissolve, and store in a 4°C refrigerator;

[0188] 0.8 mg / mL heparin sodium solution: accurately weigh 0.0080 g of solid heparin sodium, add 10 mL of ultrapure water to dissolve, and store in a 4°C refrigerator;

[0189] TBE buffer: Measure 10 mL of TBE (10×) boric acid buffer, add 90 mL of ultrapure water to dilute, and store at room temperature for later use.

[0190] (2) Sample preparation

[0191] Take 10 μL of the sample solution, add 5 μL of 10 μg / mL RNase A solution, and incubate at 37°C for 30 min to digest the unencapsulated siRNA;

[0192] At 0, 2, 4, 6, and 8 h, 5 μL of 5 mg / mL proteinase K solution was added and incubated at 37 °C for 30 min to digest RNase A. For samples without RNase A decomposition, 10 μL of DEPC water was added;

[0193] All samples were added with 4 μL of 0.8 mg / mL heparin sodium solution and incubated for 30 min;

[0194] Pipette 10 μL of sample into a centrifuge tube, add 2 μL of loading buffer (5×), mix well, and wait for sample loading.

[0195] (3) Agarose gel electrophoresis

[0196] Weigh 0.5 g of agarose into a beaker, add 50 mL of TBE buffer, stir to evenly disperse the agarose, then place in a microwave oven and heat on high for 2 min;

[0197] Add 4 μL of Gel Red nucleic acid gel dye (10000×) while hot and stir evenly with a glass rod;

[0198] Pour the above solution into the mold, insert the comb, wait for 30 minutes for it to completely solidify, and remove the comb;

[0199] Place the prepared gel into the electrophoresis tank, with one side of the sample well close to the cathode, ready for sample loading;

[0200] Use a pipette to draw 10 μL of solution and add it to the loading wells;

[0201] Turn on the electrophoresis apparatus, set the voltage to 100 V, the time to 30 min, and start electrophoresis;

[0202] After the electrophoresis is completed, the agarose gel is taken out and placed in an electrophoresis imager to observe the experimental results and take photos for record.

[0203] Experimental Results

[0204] like Figure 8 AGE results showed that naked siRNA would be degraded by RNase A, while HFn could successfully load siRNA and protect siRNA from degradation by RNase A, indicating that the above method can achieve RNA encapsulation and ensure that siRNA is not decomposed during in vitro and in vivo circulation.

[0205] Example 8 Fn@T / siRNA in vitro serum stability experiment

[0206] Experimental methods

[0207] (1) Sample preparation:

[0208] Take 10 μL of sample solution, add 2 μL of 50% FBS solution, and incubate at 37°C for 30 min;

[0209] Add 5 μL of 5 mg / mL proteinase K solution and incubate at 37°C for 30 min. For samples without 50% FBS decomposition, add 7 μL of DEPC water. Add 4 μL of 0.8 mg / mL heparin sodium solution at 0, 2, and 24 h, respectively, and incubate for 30 min. Pipette 10 μL of sample into a centrifuge tube, add 2 μL of loading buffer (5×), mix well, and wait for loading.

[0210] (2) The operation steps of AGE are the same as those mentioned above.

[0211] Experimental Results

[0212] like Fig. 9 AGE results showed that Fn@T / siRNA had a certain serum stability and could protect siRNA from degradation.

[0213] Example 9 Storage Stability of Fn@T / siRNA and CendR-Fn@T / siRNA

[0214] Experimental methods

[0215] Fn@T / siRNA and CendR-Fn@T / siRNA were stored at 4°C, and storage stability experiments were performed. The particle size and Zeta potential were measured at 1, 3, 5, 7, and 14 days, respectively.

[0216] Experimental Results

[0217] Fn@T / siRNA and CendR-Fn@T / siRNA were stored at 4°C, and their particle sizes and Zeta potentials were measured at 1, 3, 5, 7, and 14 days. Fig.10 and Fig.11 It can be seen that the particle size and Zeta potential of Fn@T / siRNA and Cend R-Fn@T / siRNA are relatively stable under storage conditions, indicating that the preparations have good stability.

[0218] Example 10 U87 MG drug-resistant cell culture

[0219] Determination of drug-resistant cell culture concentration by MTT assay

[0220] (1) Seed plate: Aspirate the culture medium in the culture bottle, wash with PBS 1-2 times, add 2mL of trypsin to digest for 2 minutes, add 2mL of complete culture medium to stop digestion, aspirate the liquid in the culture bottle into a 15mL centrifuge tube, and centrifuge (1500r, 5min). Add 2mL of complete culture medium to resuspend, pipette and evenly mix, and aspirate 0.05mL of cell suspension to count using a cell counting plate. Dilute with complete culture medium to a cell concentration of 5×10 4 Take a 96-well plate and add 100 μL of cell suspension to each well, keeping each well containing about 5×10 3 cells. Seal the 96-well plate with a circle of PBS buffer solution. Place in a 37°C, 5% CO 2 Culture in a cell culture incubator in an environmental environment for 24 h;

[0221] (2) Drug administration: The group without drug administration was used as the negative control group. 100 μL of TMZ drug administration solution of different concentrations was added to each well of the experimental group and placed at 37°C and 5% CO 2 Incubate in an incubator for 48 h;

[0222] (3) MTT method: Weigh 10 mg of MTT solid on a balance and add 2 mL of PBS buffer. Dissolve it completely under ultrasound and filter it with a 0.22 μm filter membrane to obtain a 5 mg / mL MTT working solution. Add 25 μL of 5 mg / mL MTT working solution to each well and incubate at 37°C and 5% CO. 2 Incubate in the incubator for 4 hours, then carefully remove the supernatant, add 150 μL of DMSO solution to each well, and shake on a shaker for 5 minutes. Use an ELISA reader, set the detection wavelength to 570 nm, measure the OD value of each well of the 96-well plate, calculate the cell viability in each well, and determine the concentration that induces U87 MG resistance. The calculation formula is Cell viability (%) = (OD sample -OD blank ) / (ODcontrol -OD blank )×100, where OD sample OD is the absorbance value of the sample well. control The wells for the control group are the absorbance values, OD bl ank is the absorbance value of the blank DMSO well.

[0223] Inducing drug resistance in U87 MG cells

[0224] (1) U87 MG cells were cultured in MEM medium containing 10% fetal bovine serum at 37°C and 5% CO. 2 Culture in a cell culture incubator, change the culture medium every 24-48 hours, and subculture once every 48 hours;

[0225] (2) Preparation of TMZ solution: Weigh 0.0019 g of TMZ powder into a 1.5 mL EP tube and add 1 mL of complete culture medium (MEM) to dissolve to obtain 10 mM TMZ. Pipette the 10 mM TMZ solution and add complete culture medium to dilute it to prepare the dosing concentration solution;

[0226] Normal complete medium was added on days 1, 2, 6, and 7, and complete medium containing TMZ was added on days 3, 4, 5, 8, and 9. This cycle was repeated 3 times to obtain U87 MG / TMZ cells.

[0227] Results and Analysis

[0228] from Fig.12 It can be seen that when the TMZ concentration is 0.9mmol / L, the cell survival rate is 67.59±2.07%, and this concentration is used as the drug-resistant cell culture concentration.

[0229] Example 11 Fluorescein sodium permeability experiment to investigate the establishment of an in vitro BBB model

[0230] Experimental methods

[0231] (1) Glue laying: Take out Matrigel from a -20°C refrigerator and place it in a 4°C refrigerator for 24 h to melt it. Perform the operation in ice. Dilute the melted Matrigel with 4°C blank DMEM medium at a ratio of 1:8. Place the Transwell chamber in a 24-well plate. The chamber is called the upper chamber and the culture plate is called the lower chamber. Add 80 μL of Matrigel dilution to the upper chamber and place it in a 37°C, 5% CO 2 The mixture was placed in an incubator for 2 h to allow the gel to form;

[0232] (2) Hydration: Add 50 μL of blank culture medium to the upper chamber and place the culture plate in a 37°C, 5% CO 2The cells were placed in an incubator for 30 min to allow the matrix to gel;

[0233] (3) Inoculation of HCMEC / D3 cells: HCMEC / D3 cells were inoculated at 2×10 5 The density of cells / mL (500 μL) was inoculated in the upper chamber, and 1500 μL of the same culture medium was filled in the lower chamber to maintain the osmotic pressure inside and outside the membrane. The cells were replaced every two days.

[0234] (4) Penetration of tracer dye to verify the BBB model:

[0235] ① Use complete culture medium to prepare fluorescein sodium into a 10 μg / mL solution;

[0236] ② Add 0.5 mL of solution containing sodium fluorescein to the upper chamber (AP), and add 1.5 mL of complete medium without sodium fluorescein to the lower chamber (BL). Collect the culture medium from the lower chamber at 1 h, 2 h, 4 h, 6 h, and 8 h of incubation, measure the fluorescence intensity of the samples, and calculate the Papp value. When Papp < ​​15 × 10 6 , it indicates that the model is successfully established;

[0237] ③ Use complete culture medium to dissolve fluorescein sodium to prepare 0.1, 0.2, 0.5, 1, and 5 μg / mL standard solutions, measure the fluorescence intensity, repeat 3 times, and plot the mean fluorescence intensity y against the concentration x to obtain a standard curve.

[0238] ④ Calculate the apparent permeability coefficient (Papp) of the tracer molecule using the following formula:

[0239] Papp = (dC / dt) / C 0 A

[0240] Where dC / dt is the drug transport amount per unit time, A is the effective permeation area, and C 0 is the initial concentration of tracer molecules added to the upper chamber of the transwell.

[0241] Experimental Results

[0242] It can be seen from Table 3 that Papp in each time period is less than 15×10 -6 (cm / s), proving that the barrier permeability of sodium fluorescein is poor, indicating that the blood-brain barrier model was successfully established.

[0243] Table 3 Fluorescein sodium permeability test

[0244]

[0245] Example 12 Confocal imaging to investigate the targeted uptake of CendR-Fn@T / siRNA in U87 MG cells

[0246] Experimental methods

[0247] (1) Seed plate: U87 MG was plated at 1×10 5 The cells were inoculated at a density of 100 / mL (1 mL) in a confocal culture dish. After incubation for 12 h, 1 mL of Cy3, Fn@T / siRNA, and CendR-Fn@T / siRNA solution were added, respectively, and incubated for 4 h. The drug-containing culture medium was discarded and the cells were rinsed twice with PBS.

[0248] (2) Staining: Prepare 4 mL of staining working solution, add 500 μL of working solution respectively, incubate for 15-30 min, and observe under laser confocal scanning microscope.

[0249] The experimental results are from Fig.14 It can be seen that naked siRNA and Fn@T / siRNA were less taken up by human astroglioma (U87MG) cells, while the uptake of CendR-Fn@T / siRNA in U87 MG cells was significantly increased, indicating that HFn-linked CendR peptide can better target U87 MG cells.

[0250] Example 13 Quantitative investigation of targeted uptake of CendR-Fn@T / siRNA in U87 MG cells by flow cytometry

[0251] Experimental methods

[0252] (1) Seed plate: 1×10 5 U87 MG cells were inoculated with 100 μg / mL (2 mL) and incubated for 12 h to allow the cells to adhere;

[0253] (2) Drug administration: discard the culture medium, add drug-containing culture medium (RNA, Fn@T / siRNA, CendR-Fn@T / siRNA), and transfect for 6 h;

[0254] (3) Aspirate the culture medium, save it, add 500 μL of trypsin to digest the cells in a 2 mL centrifuge tube, add 500 μL of complete culture medium to terminate the digestion, and centrifuge (2000 r, 5 min) to obtain a cell pellet;

[0255] (4) Add 500 μL PBS to resuspend the cells and pipette to disperse the cells evenly.

[0256] Experimental Results

[0257] from Fig.15 It can be seen that the average fluorescence intensity of the preparation connected to the CendR polypeptide is stronger, indicating that it is more taken up by U87 MG cells. This shows that CendR-Fn@T / siRNA can well target tumor cells and be successfully taken up by cells.

Claims

1. A gene co-loading targeted ferritin nano-delivery system for drug-resistant glioblastoma, characterized in that: The nano-delivery system is composed of the following components: human ferritin heavy chain (HFn), small interfering RNA (siRNA) and temozolomide (TMZ), and the molar ratio of the components of the nano-delivery system is approximately siRNA: HFn: TMZ = 1:3:1200.

2. The nano delivery system according to claim 1, characterized in that The surface of the nano-delivery system is modified with a targeting polypeptide CendR, so as to enhance the targeting to drug-resistant glioblastoma.

3. The nano delivery system according to claim 1 or 2, characterized in that The small interfering RNA (siRNA) is an siRNA used for down-regulating the expression of O6-alkylguanine-DNA alkyltransferase (MGMT) to enhance the therapeutic effect of temozolomide on drug-resistant glioblastoma.

4. The nano delivery system according to any one of claims 1 to 3, characterized in that The particle size of the nano delivery system is 50-100 nm and has a uniform and round spherical structure.

5. A method for preparing the nano delivery system according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Prepare CendR solution and NHS-PEG-Mal solution; (2) Mix the siRNA solution with the CaCl2 solution and use an ultrafiltration tube to remove free Ca after co-incubation. 2+ ; (3) adding HFn solution to the above solution, adjusting the pH value and incubating; (4) Add TMZ solution and continue incubation; (5) Use ultrafiltration tube to remove free Ca 2+ , siRNA, HFn subunit to obtain Fn@T / siRNA solution; (6) reacting the CendR polypeptide with NHS-PEG-Mal solution and removing unreacted peptide by dialysis; (7) The Fn@T / siRNA solution is mixed with the NHS-PEG-CendR solution, and CendR-Fn@T / siRNA is obtained after incubation.

6. The preparation method according to claim 5, characterized in that: The molar ratio of the CendR polypeptide to HFn is about 1:

10.

7. Use of the nano delivery system according to any one of claims 1 to 4 in the preparation of a drug for treating drug-resistant glioblastoma.

8. Use of the nano delivery system according to any one of claims 1 to 4 in the preparation of a drug capable of penetrating the blood-brain barrier.

9. Use of the nano delivery system according to any one of claims 1 to 4 in the preparation of a drug capable of down-regulating MGMT expression.

10. Use of the nano delivery system according to any one of claims 1 to 4 in the preparation of a drug capable of inhibiting the proliferation of drug-resistant glioblastoma cells.

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