Novel nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a novel nano-drug as well as a preparation method and application thereof, and co-delivery of siRNA and 3BP is realized on an exosome nano-platform, so that a novel nano-drug Exo (at) siRNA / 3BP-CTX (ERBC) engineering exosome is generated. The surface-modified cetuximab (CTX) is targeted to tumors, and shows high targeting and low toxicity in imaging analysis. CCK-8, western blot and immunofluorescence detection are used for detecting the tumor killing effect and the synergistic effect. The ERBC finds that the occurrence of death-promoting autophagy in KRAS mutation tumors is promoted on the transmission electron microscope (TEM) and molecular level.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a novel nano drug for realizing co-delivery of siRNA and 3BP, and a preparation method and application thereof. Background Art

[0002] The KRAS gene is the most common mutation in the MAPK pathway, accounting for 30% of all human cancers. KRAS mutations are routinely tested in clinical practice in up to 52% of cancer (CRC) cases. These mutations are associated with low sensitivity to chemotherapy and resistance to treatment with targeted EGFR inhibitors (cetuximab), often with a poor prognosis. In addition, patients with wild-type KRAS may develop subsequent mutations during anti-EGFR treatment, resulting in resistance to EGFR inhibitors and treatment failure. Currently, there are no drugs that can effectively treat KRAS mutations.

[0003] The ability of tumors to maintain proliferative signaling depends on the continued activation of driver oncogenes such as KRAS. Small interfering RNA (siRNA) can recognize specific messenger RNA (mRNA) sequences for silencing and is a potential therapeutic approach for the treatment of drug-resistant tumors and refractory mutant oncogenes. Mutation-specific KRAS siRNA or broad-spectrum KRAS siRNA has been shown to specifically inhibit tumors that rely on KRAS mutations for survival with minimal associated toxicity. However, naked siRNA is easily degraded by nucleases in vivo and has the risk of inducing off-target effects. Therefore, the exploration of advanced siRNA-based therapies continues, with a focus on improving stability and on-target efficacy.

[0004] The activity of oncogenes is closely related to abnormal tumor metabolism and changes in the tumor microenvironment. Notably, even under aerobic conditions, tumor cells prefer glycolysis rather than oxidative phosphorylation as a metabolic mode of energy supply, an abnormal phenomenon known as the "Warburg effect". A large number of intermediate metabolites produced during glycolysis provide raw materials for the vigorous proliferation of cancer cells. Therefore, targeting glycolysis has become an innovative approach for cancer treatment. The oncogenic KRAS gene has been shown to mediate metabolic reprogramming by upregulating glucose transporter-1 (GLUT1), which enhances glucose uptake and glycolytic metabolism, allowing cells to survive under low glucose conditions. 3BP, a small molecule pyruvate mimetic and anticancer alkylating agent, is also reported to be a biocompatible antitumor agent that will block the mitochondrial respiratory chain. 3BP has a significant inhibitory effect on multiple metabolic interactions, especially on excessive glycolysis mediated by overexpressed hexokinase type II (HK-II) in tumor cells, which will lead to a collapse in adenosine triphosphate (ATP) production. This perturbation leads to reduced oxygen consumption and restores oxygen levels to combat hypoxia. Due to the enhanced permeability and retention (EPR) effect, the small molecular size of 3BP is difficult to be retained in tumor tissues, a phenomenon that limits the application of 3BP in anti-tumor therapy.

[0005] Exosomes (Exo) are nanoscale lipid bilayer membrane vesicles with a diameter of 30 to 200 nm, which are secreted by almost all cell types. They contain many proteins, coding and non-coding RNAs, and lipids that play an important role in intercellular communication and transport. As carriers, exosomes have the characteristics of low immunogenicity, biocompatibility, non-toxicity, strong cargo capacity, and cargo protection. Using exosomes as carriers for transporting siRNA can protect the stability of siRNA and provide sufficient space for carrying 3BP at the same time. However, tumor-derived exosomes that homologously target tumors may not be the best choice. Increasing evidence shows that nucleic acids (RNA / DNA) and proteins can promote tumor development and metastasis. Selective membrane modification can overcome the disadvantage of low targeting ability of natural exosomes. EGFR is one of the key factors driving cell proliferation, differentiation, and survival. It is overexpressed in most colorectal cancers and is the theoretical basis for using EGFR inhibitors as targeted modifications (cetuximab, CTX). CTX can be modified into targeting antibodies on the exosome membrane, and antibody-mediated endocytosis is used to unload drugs to obtain therapeutic effects. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a new type of nano drug and its preparation method and application.

[0007] In order to solve the above problems, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing a novel nano drug comprises the following steps:

[0009] (1) Collect, isolate and purify exosomes;

[0010] (2) Loading siRNA and 3BP into exosomes by electroporation: siRNA, 3BP and exosomes were mixed in 400 μL electroporation buffer and electroporated on a Bio-Rad Gene electroporation device in a 0.4 cm cuvette at a working voltage of 400 m V and a capacitance of 125 μF (pulse time 10-15 ms);

[0011] (3) After electroporation, the exosomes were treated with RNase to remove any siRNA that might be bound to the exosome membrane. The exosomes were then diluted with pre-cooled PBS and centrifuged at 100,000 g for 70 min to remove unbound siRNA and 3BP.

[0012] In certain embodiments, the step of unlinking cetuximab to the surface of exosomes is further included: CTX and DSPE-PEG-NHS are mixed in a MES buffer (PH=8) at a mass ratio of 4:1, stirred at room temperature at 80 rpm for 4 hours for reaction connection, DSPE-PEG-CTX is dissolved in DMSO, and then mixed with the exosomes prepared in step (3) at a weight ratio of DSPE-PEG-CTX: exosomes of 1:1, incubated at 37°C for 2 hours, and then ultrafiltered in a 300K Nanosep centrifuge to remove free DSPE-PEG-CTX and CTX.

[0013] In certain embodiments, the exosomes are obtained by culturing HEK293T cells, collecting the cell supernatant, performing multi-step gradient centrifugation, and resuspending.

[0014] In certain embodiments, the multi-step gradient centrifugation comprises the following steps: (1) centrifugation at 1000 g for 10 min to remove dead cells and collect the supernatant;

[0015] (2) centrifugation at 2000 g for 20 min to remove cell debris;

[0016] (3) Centrifuge at 10,000 g for 30 min to remove large vesicles and small cell debris, collect the supernatant and discard the precipitate;

[0017] (4) Centrifuge at 100,000 g for 70 min to precipitate exosomes.

[0018] In certain embodiments, before perforation, the weight ratio of exosomes: siRNA is 8-11:1, and the weight ratio of exosomes: 3BP is 1:5-6.

[0019] In certain embodiments, the weight ratio of exosomes:siRNA is 10:1, and the weight ratio of exosomes:3BP is 1:4.

[0020] In certain embodiments, the final concentration of exosomes in the electroporation mixture does not exceed 0.5 μg / μL.

[0021] In certain embodiments, reagents and consumables are treated with DEPC before electroporation to reduce RNA degradation.

[0022] The present invention also provides a novel nano drug prepared by the above method.

[0023] In certain embodiments, the drug can be used to prepare an anti-tumor drug with high biocompatibility.

[0024] The present invention designs and synthesizes Exo@siRNA / 3BP-CTX engineered exosomes (ERBC), which selectively target tumor tissues overexpressing EGFR. The exosome siRNA delivery platform adopted effectively alleviates the instability and nonspecific targeting associated with unmodified siRNA, thereby ensuring stable gene silencing efficacy. And in the KRAS-mutant colorectal cancer model, the therapeutic sensitivity of CTX was reestablished after oncogene silencing. This gene silencing method not only reduces the proliferation and viability of mutant cancer cells, but also inhibits the overexpression of the GLUT1 gene. Thereby weakening cellular glucose uptake. In addition, enhancing 3BP synergistically enhances starvation therapy through direct energy inhibition. It also prolongs the survival time of animal model mice, which is attributed to enhanced targeting ability to reduce treatment side effects.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. This nanomedicine contains both gene therapy and starvation therapy;

[0027] 2. It has specific targeting for KRAS mutated tumors, and its accumulation in normal tissues is much smaller than that in tumor tissues, with little side effects outside the tumor.

[0028] 3. Encapsulated by exosomes, the compatibility in organisms is higher than that of inorganic nanomaterials;

[0029] 4. Compared with inorganic nanomaterials, materials modified by exosomes can be completely decomposed in the body without the possibility of cumulative toxicity.

[0030] 5. The drug is highly stable and maintains its original biological efficacy after being stored in different buffers for 48 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1TEM images and Coomassie brilliant blue protein staining of HEK293T exosomes of the present invention;

[0032] Figure 2 is the particle size of the exosomes of the present invention;

[0033] Figure 3 Western blot and FACS analysis of the extracted exosomes and cell membranes for the present invention;

[0034] Figure 4 The fluorescence signal absorption of Cy3 and the UV-visible spectrum of 3BP in the ERBC prepared by the present invention;

[0035] Figure 5 The RFU value of Cy3-siRNA and the UV absorption spectrum of 3BP in the supernatant after electroporation;

[0036] Figure 6 The linear curves are between siRNA concentration and Cy3 RFU and the linear relationship between 3BP concentration and 280 nm UV-Vis absorbance;

[0037] Figure 7 The gel electrophoresis diagram of siRNA before and after siRNA electroporation;

[0038] Figure 8 This is a Coomassie brilliant blue protein staining image. Free CTX: naked CTX; CTX-PEG-DSPE: CTX and DSPE-PEG-NHS were mixed in a ratio of 1:4 and stirred for 4 hours to obtain CTX-PEG-DSPE;

[0039] Exosomes: HEK293T exosome protein; ERBC: Exo@siRNA / 3BP-CTX NPs protein; LBSU: lower buffer solution after the first ultrafiltration. CTX is reduced to heavy chain (55kDa) and light chain (25kDa);

[0040] Fig. 9 TEM images of ERBC NPs;

[0041] Fig.10 (A) shows the particle size analysis of Exosomes and ERBC NPs in Nano-ZetaView; (B) shows the Zeta analysis of Exosomes and ERBC NPs in Nano-ZetaView.

[0042] Fig.11(A) shows the particle size analysis of ERBC NPs stored in 10% FBS, MES buffer, and PBS at 4°C for 4 hours, 24 hours, and 48 hours. (B) shows the Zeta analysis of ERBC NPs stored in 10% FBS, MES buffer, and PBS at 4°C for 4 hours, 24 hours, and 48 hours. (N=3);

[0043] Fig.12 Real-time confocal images of SW480 cells and SW620 cells treated with Dil-labeled ERCB NPs and Exosomes for 4 hours. Red fluorescence represents the Dil signal (Dil labels the exosome membrane), and the cell nucleus is stained with DAPI (blue). Scale bar = 20 μm. It can be seen that ERBCs are obviously aggregated in EGFR-overexpressing SW480 cells.

[0044] Fig.13 To observe the in vivo biodistribution of DiR-labeled ERBC NPs or Exosomes NPs injected into the tail vein of SW480 and SW620 cell nude mice models at the time points of 8h 24h 48h, it was used to observe the obvious aggregation of ERBC in EGFR-overexpressing SW480 model mice;

[0045] Fig.14 The cytotoxicity of SW480 cells was detected by treating them with Exos-CTX NPs, Exo@3BP-CTX NPs, ERBC NPs, ERBC NPs+Free CTX, Exo@siRNA-CTX NPs, Exo@siRNA-CTX NPs+Free CTX, and Exosoms; the ERBC group had the strongest cytotoxic effect and the lowest cell survival rate;

[0046] Fig.15 SW480 cells were treated with Exos-CTX NPs, Exo@3BP-CTX NPs, Exo@siRNA-CTX NPs, Exo@siRNA-CTX NPs+Free CTX or ERBC+Free CTX for 48 h, and apoptotic cells were analyzed by flow cytometry using Annexin V-FITC and PI assays; the ERBC+Free CTX group had the most apoptosis;

[0047] Fig.16Tumor volume curves during the growth of tumor-bearing nude mice after treatment with (1) PBS, (2) Exo@siRNA-CTX NPs, (3) Exo@siRNA-CTX NPs+CTX Free, (4) Exo@3BP-CTX NPs, or (5) ERBC NPs+CTX Free. The tumor in the ERBC+Free CTX group had the best therapeutic effect and its volume became smaller.

[0048] Fig.17 Representative images of H&E, KRAS, and KI67-stained tumor tissues of tumor-bearing mice 21 days after treatment with PBS, Exo@siRNA-CTX NPs, Exo@siRNA-CTX NPs+Free CTX, Exo@3BP-CTXNPs, and ERBC NPs+Free CTX; Ki67-positive proliferating cells and KRAS-positive cells were stained brown. DETAILED DESCRIPTION

[0049] Example 1, Preparation of Exo@siRNA / 3BP-CTX engineered exosomes (ERBC)

[0050] 1. Cell culture

[0051] HEK293T tool cells were used as parental cells for exosome extraction.

[0052] (1) Cell recovery

[0053] Prepare a clean bench for UV disinfection for 30 minutes, and rewarm the culture medium for later use. When reviving frozen cells, a stable temperature of 37°C is required to quickly dissolve the frozen cryopreservation solution. Preheat the water bath to 37°C in advance. Wear anti-freeze gloves and take out the cells from the liquid nitrogen tank or -80°C ultra-low temperature refrigerator. The cells need to be placed in a preheated 37°C water bath to dissolve in the shortest possible time. Next, collect the cell suspension in a centrifuge tube, mix it with the culture medium in a 1:2 ratio, and place it in a low-speed centrifuge at 700rpm / min for 2 minutes. The precipitate is cells, discard the supernatant directly, and gently blow and mix with the preheated complete culture medium (1% penicillin / streptomycin and 10% FBS) with a pipette tip. Add appropriate culture medium to a new culture dish, transfer the cell mixture and shake well. Continue to place in the incubator (5% CO 2 Cryopreserved cells need to be observed and processed as soon as possible, and the cell adhesion and status should be observed for 8 hours.

[0054] (2) Cell passaging

[0055] After cell passage or resuscitation, observe the cell density and cell morphology under a microscope to see if they are normal and whether there is any contamination. When the density of adherent cells is 80%-85%, they can be processed for passage or plating. Discard the supernatant liquid directly into the culture dish. Before trypsin digestion, add 2ml of PBS and shake well in the dish to wash. After discarding PBS, cover the culture dish with 0.5-2ml of trypsin and place it in the incubator for digestion for 2-5 minutes depending on the cells. Observe under the microscope that the cells become round or floating. Immediately add trypsin and complete culture medium in a ratio of 1:2 to stop digestion and shake well. Use a pipette to gently blow off the cells and transfer them to an 8ml centrifuge tube. Centrifuge for 2 minutes at 700rpm / min, room temperature. Discard the supernatant and add new complete culture medium with a pipette to resuspend the cells evenly. Choose a ratio of 1:3 to pass the cells into a new culture dish and place it in a 37℃ constant temperature incubator for culture.

[0056] (3) Cell cryopreservation

[0057] The cell freezing solution needs to be prepared in advance, 10ml DMEM medium + 80ml FBS + 10ml DMSO, and stored at 4℃ away from light. When the cells grow to a density of about 65%, the cells are in good condition under the microscope. Replace the fresh complete medium the night before freezing, and the cells can be frozen the next day. The previous steps are synchronized with cell passaging. Finally, pour out the supernatant and add 1ml cell freezing solution to resuspend the cells, transfer to a programmed cooling freezing box, place in a -80℃ ultra-low temperature refrigerator for 24 hours, and then move the cryopreservation tube to a liquid nitrogen tank for long-term storage.

[0058] 2. Exosome isolation, purification and characterization

[0059] Exosome-free FBS was prepared by ultracentrifugation at 120,000g for 16 hours. This can avoid contamination of exosomes with bovine serum. For exosome collection, the exosomes were collected by replacing the exosome-free serum medium after the cell density reached 80%, and the supernatant was collected after 48 hours of continuous culture. The supernatant of HEK293T cells was harvested and subjected to multi-step gradient centrifugation to separate exosomes. As follows:

[0060] (1) 1000g for 10 min to remove dead cells. Then collect the supernatant;

[0061] (2) 2000 g for 20 min to remove cell debris;

[0062] (3) 10,000 g for 30 min to remove large vesicles and small cell fragments, then collect the supernatant and discard the precipitate. Be careful not to aspirate the precipitate to avoid contamination;

[0063] (4) Centrifuge the exosomes at 100,000 g for 70 min. The exosomes are precipitated;

[0064] Resuspend the exosome pellet in 200ul PBS and store it in a -80℃ ultra-low temperature freezer and use it as soon as possible for up to 2 weeks. To detect the concentration, hydrated diameter and zeta potential of exosomes, 100μl of exosomes were diluted to 1ml and analyzed using NTAZetaView. The protein content of the exosome suspension was quantified in the corresponding buffer using the Bradford reagent / BCA assay and BSA standards for quantification in subsequent experiments. Collecting 500ml of cell supernatant can extract 400-600ul, 3-5μg / μl exosomes. Characterization of exosomes The morphology of exosomes was observed under TEM. CD63, Alix, CD9, TSG101 and Pan cadherin were verified as biomarkers of exosomes and cell membranes by Western blot. Exosome surface proteins were identified using flow cytometry: Bead-exosome analysis.

[0065] The extracted mother exosomes were observed to be round or biconcave membrane structures in TEM. TEM showed that the size of the exosomes was about 110nm, with a clear membrane structure and uniform size distribution ( Figure 1 A). Coomassie brilliant blue protein staining results show that there is a significant difference in the expression of exosome membrane proteins and cell membrane proteins ( Figure 1 B). NTA showed that the average particle size of exosomes was 122.7±6.5nm ( Figure 2 ). Western blot results showed that exosomes highly expressed Alix, CD63, TSG101, CD9 ( Figure 3 A). The same results can be obtained by flow cytometry detection of CD63 and CD9 ( Figure 3 B), while Pancadherin, one of the cell membrane surface molecules, is not expressed in exosomes ( Figure 3 A).

[0066] 3. Preparation and characterization of EXO@siRNA / 3BP-CTX(ERBC)

[0067] siRNA and 3BP were loaded by electroporation. The oligonucleotide of siRNA used was 5'-3': GGACUCUGAAGAUGUACCU. The reagents and consumables were treated with DEPC before perforation to reduce RNA degradation. In order to load siRNA, 3BP and both into exosomes at a suitable concentration ratio, we conducted preliminary experiments to explore the optimal reaction parameters and component ratios in the reaction system. It was found that the weight ratio of Exosomes:siRNA before perforation was close to 10:1, and the ratio of Exosomes:3BP was 1:4, which could meet the therapeutic dose of the subsequent exosome platform. The final concentration of exosomes in the electroporation mixture should not exceed 0.5μg / μl. The above ratio mixture was mixed into 400μL electroporation buffer and implemented on a Bio-Rad Gene electroporation device, and electroporation was performed in a 0.4cm cuvette at a working voltage of 400mV and a capacitance (pulse time 10-15ms) of 125μF. After electroporation, the exosomes were treated with RNase to remove any siRNA that might be bound to the exosome membrane. The exosomes were then diluted with pre-chilled PBS and centrifuged at 100,000 g for 70 min to remove unbound siRNA and 3BP.

[0068] In order to determine the amount of siRNA electroporated into exosomes, Cy3-labeled siRNA was used to evaluate the relative fluorescence units (RFU) of the Cy3 fluorescence signal using a microplate reader. The RFU value and siRNA concentration relationship were determined according to different concentrations of naked Cy3-siRNA to obtain an RFU-concentration calibration curve. The absorption peaks of free 3BP at different concentrations were measured in the UV-Vis visible spectrum to obtain a concentration-absorption peak calibration curve. It was used to measure the unloaded 3BP in the supernatant. The encapsulation efficiency and drug loading rate of ERBCNPs were calculated: Encapsulation efficiency (EE) = (weight of siRNA or 3BP loaded in ERBC NPs / total siRNA or 3BP weight added) × 100%.

[0069] CTX and DSPE-PEG-NHS were mixed in MES buffer (PH=8) at a mass ratio of 4:1 and stirred at 80 rpm for 4 hours at room temperature for reaction connection. DSPE-PEG-CTX was dissolved in DMSO and then mixed with Exosomes at a DSPE-PEG-CTX: Exosomes protein weight ratio of 1:1. After incubation at 37°C for 2 hours, unincorporated (free) DSPE-PEG-CTX was removed by ultrafiltration in a 300K Nanosep centrifuge device. Coomassie brilliant blue protein staining was used to verify the successful self-loading of CTX on exosomes. ERBC NPs were suspended in PBS, 10% FBS and MES (PH8.8) buffer, and their size was detected by NTA and their potential was analyzed by Zete at 4h, 24h, and 48h for stability analysis. Exo-CTX NPs are empty exosomes with CTX attached to the surface, Exo@siRNA-CTX NPs are exosomes encapsulating siRNA and modified with CTX on the surface, and Exo@3BP-CTX are exosomes encapsulating 3BP with CTX modified on the surface.

[0070] The fluorescence signal absorption peak of Cy3 siRNA at 570 nm was detected in the exosomes after electroporation, verifying the successful loading of siRNA (Figure 2 Figure 4 A). The UV-Vis spectrum curve shows that the characteristic absorption peak of 3BP is at 280nm as shown in Figure (4B). In order to determine the encapsulation efficiency of siRNA or 3BP in exosomes, the unloaded substances in the supernatant were analyzed in the fluorescence intensity or UV absorption measurement experiment. Cy3 siRNA and 3BP can be detected in the supernatant after perforation ( Figure 5 A, 5B). The drug loading was calculated from the calibration curve of the fluorescence RFU value and concentration of CY3-siRNA and the UV-Vis spectrum concentration calibration curve of 3BP ( Figure 6 A, 6B). The loading rates of siRNA and 3BP were 21.4% and 19.49%, respectively. The integrity of siRNA after perforation was verified by gel electrophoresis, and it was found that electroporation did not destroy the integrity of siRNA ( Figure 7 ). In order to attach cetuximab to the surface of exosomes and increase the tumor targeting of exosomes, PEG non-immunogenic and safe compounds were selected as bridges. DSPE-PEG-NHS is an excellent amphiphilic affinity. Cetuximab is linked to PEG by forming an amide bond, while the DSPE end is linked to the lipid membrane structure of the exosomes. After centrifugation, the assembly was analyzed by Coomassie Brilliant Blue protein staining. ERBC NPs found characteristic bands of CTX at 25kDa and 55kDa ( Figure 8 ), proving that CTX was successfully linked to the surface of exosomes through PEG modification.

[0071] Stability analysis of ERBC NPs

[0072] ERBC NPs were characterized by TEM, Western blot and Nano-ZetaView. TEM showed that ERBC NPs ( Fig. 9 ) The appearance and structure of the exosomes were the same as those of the unmodified control exosomes, and there was no significant change. Electroporation and modification had no effect on the morphology of the exosomes. Western blot showed that the exosome marker proteins of ERBC NPs and Exosomes were consistent ( Figure 3 A). When comparing Exosomes and ERBC NPs, it was found that the size of NTA did not change significantly after self-assembly ( Fig.10 A). Zeta of ERBC NPs increased due to CTX modification ( Fig.10 B). After electroporation and drug loading, surface modification of exosomes with antibodies is a bio-friendly strategy that does not interfere with the biological activity of engineered exosomes. Placing ERBC NPs in different buffers at 4°C for 4h, 24h, and 48h does not affect the size change and charge change. ERBC NPs have high biostability ( Fig.11 ).

[0073] Example 2, in vitro targeting verification

[0074] In order to achieve maximum tumor cell killing and reduce toxic side effects, it is necessary to ensure high enrichment of the drug in the tumor site. EGFR is an ideal targeting site, with an expression level 20 times higher in most tumor cells than in normal cells. Cetuximab is modified on the surface of exosomes to target tumor cells with high EGFR expression, and the antibody can mediate the endocytosis of exosomes into tumor cells. To this end, we validated Dil-stained ERBC NPs and Exosomes in SW620 and SW480 cell lines, respectively. The exosomes and cells were cultured together and incubated for 4 hours, and then the biodistribution of the stained exosomes in the cells was monitored under real-time confocal microscopy ( Fig.12 The results showed that the internalization ability of SW480 cell line was significantly enhanced after cetuximab modification. Since SW620 cell line does not express EGFR, the internalization of ERBC NPs in SW620 cells was different from that of Exosomes. ERBC NPs exerted a tumor targeting effect to ensure that sufficient drugs were enriched in the tumor site.

[0075] Example 3, in vivo targeting verification

[0076] Based on in vitro validation, it was found that ERBC NPs target tumors with high EGFR expression. The in vivo biodistribution of DiR-labeled ERBC NPs and Exosomes was further tested in SW480 and SW620 cell subcutaneous nude mouse tumor models. The four groups of mice ( Fig.13 As shown). It is worth noting that the ERBC NPs group continued to accumulate in the subcutaneous tumor model of SW480 nude mice, while the Exosomes group had no obvious aggregation effect. This proves that exosomes have specific EGFR-positive tumor targeting ability. SW620 model nude mice can have a certain degree of ERBC NPs aggregation effect. After PEG modification, exosomes can increase the circulation time of engineered exosomes in vivo. The enhanced permeability and retention (EPR) effect of nanomaterials is more obvious than that of naked exosomes.

[0077] Example 4: Combination therapy promotes death-promoting autophagy and enhances sensitivity to cetuximab therapy

[0078] In most cases, the autophagic response of cells is a cytoprotective mechanism. Unlike selective autophagy, the reduction in total autophagic protein levels induced by starvation disrupts the internal balance of autophagy regulation. The protective mechanism is transformed into tumor destruction. The highest goal of tumor treatment is to specifically kill tumor cells and reduce nonspecific damage. CTX-modified exosomes carrying siRNA and 3BP can be highly enriched in tumor sites and play the role of siRNA gene silencing and starvation-promoting autophagy. CCK-8 was used in vitro to detect the cytotoxicity of SW480 cells treated with PBS, Exos-CTX NPs, Exo@3BP-CTX NPs, ERBC NPs, ERBC NPs+CTX Free, Exo@siRNA-CTX NPs, Exo@siRNA-CTX NPs+CTX Free, and Exosomes for 24h and 48h. In the ERBC NPs+CTX Free group, the survival rate of SW480 was only 27.06%, which was greater than the sum of the cell survival rates of Exo@siRNA-CTX NPs+CTXFree and Exo@3BP-CTX NPs alone ( Fig.14As shown). This further verifies that combined energy deprivation under combined action promotes cell death rather than survival. In contrast, no cell activity inhibition was observed in PBS, Exosomes, and Exo-CTX NPs, indicating good biosafety in vitro. The survival rate of cells in the Exo@siRNA-CTX NPs group was about 66.66%, while the survival rate of Exo@siRNA-CTX NPs+Free CTX was reduced to about 47.76%. CTX exhibited toxic effects after KRAS gene silencing and restored the therapeutic effect of cetuximab. To further verify the cell death pathway, FCM was used to determine whether the cells exerted tumor killing effects through the apoptosis pathway. The apoptosis of SW480 cells after 48h of treatment with PBS, Exos-CTX NPs, Exo@3BP-CTX NPs, Exo@siRNA-CTX NPs, Exo@siRNA-CTX NPs+Free CTX or ERBC NPs+Free CTX was evaluated by Annexin V-FITC and Propidium lodide (PI) staining kit. It can be seen that in Exo@3BP-CTX NPs and Exo@siRNA-CTX NPs+Free CTX, the apoptosis rates were 39.56% and 50.50%, respectively, while the ERBC NPs+Free CTX group showed a significant enhancement effect, with a cell apoptosis rate of 75.90% ( Fig.15 The ERBC NPs+Free CTX group can have a synergistic effect to better kill tumors and achieve the effect of targeted treatment of refractory KRAS mutant tumors.

[0079] Example 5, in vivo anti-tumor and safety evaluation

[0080] The in vivo anti-tumor evaluation of CTX-modified exosomes co-delivering KRAS siRNA and 3BP nanoparticles was evaluated using nude mice subcutaneous tumor SW480 colorectal cancer cells. The in vivo therapeutic effect was evaluated by tracking the changes in tumor volume in the nude mouse subcutaneous tumor model in different treatment groups. KRAS gene silencing based on the Exo@siRNA-CTX NPs group inhibited tumor proliferation. Compared with the PBS group, the tumor volume increased by 9.73 times, while the volume of the Exo@siRNA-CTX NPs group increased by only 3.86 times. Compared with the Exo@siRNA-CTX NPs group, the Exo@siRNA-CTX NPs+Free CTX group showed similar toxicity results with in vitro cells, and tumor proliferation was significantly inhibited. The anti-tumor effect of the Exo@siRNA-CTX NPs+Free CTX group was better than that of the Exo@siRNA-CTX NPs group, and CTX showed cell therapy toxicity. This is attributed to the restoration of CTX anti-tumor therapeutic sensitivity in colorectal cancer cells with silencing KRAS expression. In addition, the volume of ERBC NPs+Free CTX was reduced by 2.19 times compared with Exo@3BP-CTX NPs. The results showed that KRAS-siRNA and 3BP acted synergistically ( Fig.16 shown).

[0081] Example 6: Anti-tumor and apoptosis-promoting effects of ERBC NPs in vivo

[0082] To further evaluate the therapeutic effects of different treatment groups, immunohistochemical analysis (including H&E, Ki67 and KRAS) was used to further investigate the apoptosis, proliferation level and KRAS silencing in tumor tissues after different treatments. Fig.17 As shown). In the ERBC NPs+Free CTX group, immunohistochemistry showed that the brown color of KRAS and Ki67 was significantly reduced. KRAS immunohistochemistry analysis showed that exosomes carrying KRAS siRNA successfully silenced the KRAS gene in vivo. H&E staining in the ERBC NPs+Free CTX treatment group showed tumor tissue necrosis, reduced Ki67 expression, and significant proliferation inhibition.

[0083] The experimental steps involved in the present invention can be found in the following description:

[0084] In vitro cellular uptake assay

[0085] The membrane was labeled with the cell membrane fluorescent probe Dil, and ERBC NPs and blank exosomes were incubated at 37°C for 2 hours, and free dyes were removed by 300K Nanosep centrifugal ultrafiltration three times. Exo Free NPs and Exo@siRNA / 3BP-CTX NPs nanocarriers with a concentration of 100μg / mL were incubated with SW480 and SW620 cells for 4 hours, respectively, and detected by confocal laser scanning microscopy (CLSM) scanning and flow cytometry measurement (FCM). After SW480 and SW620 cells were seeded in glass-bottomed culture dishes and stabilized, Exo Free NPs and Exo@siRNA / 3BP-CTX NPs were incubated in a carbon dioxide incubator for 4 hours at a concentration of 100μg / ml Exo (equivalent to 20nM siRNA, 10μg / ml 3BP). Next, cells were fixed with 4% paraformaldehyde and stained with DAPI. Subsequently, all plates were collected and imaged by Leica confocal microscope. For FCM experiments, cells were digested and resuspended in PBS before being tested.

[0086] Animal fluorescence imaging

[0087] In vivo biodistribution studies were performed in a subcutaneous SW480 / nude mouse tumor model, with the SW620 model as a control (n=3). Exosomes were labeled with 5 μM DiR for 30 min at 37°C and ultrafiltered three times in a 300K Nanosep centrifuge to remove unincorporated (free) DiR. When the tumor volume reached 150-200 mm 3 , 3 mg / ml DiR-labeled ERBCNPs or Exo were injected intravenously into mice (without self-loading of CTX). The in vivo fluorescence signal of exosomes was observed at the designated time points by Xenogen IVIS Spectrum. Mice were killed 48 h later. Major organ tissues were collected and in vivo biodistribution was measured by IVIS Spectrum.

[0088] Cell viability assay

[0089] Cell Counting Kit-8 (CCK-8) was used to examine the cytotoxicity of the cells after treatment. NCM460 and SW480 were used at 1×10 4Inoculated into 96-well plates, and treated with corresponding concentrations after 24 hours of attachment. In NCM460 cells. 3BP concentrations of 5μg / ml, 10μg / ml, 15μg / ml, 20μg / ml, 30μg / ml, 40μg / ml, 50μg / ml, 60μg / ml, 80μg / ml, and 100μg / ml were selected for treatment. SW480 was grouped into Exosomes NPs, Exo-CTX NPs, Exo@siRNA-CTXNPs, Exo@3BP-CTX NPs, Exo@siRNA-CTX NPs+Free CTX, ERBC NPs, and ERBC+Free-CTX NPs. The concentration of exosomes was 100μg / ml, and Free-CTX was 250μg / ml. The absorbance value at 450 was measured with an enzyme reader to calculate the cell survival rate. Survival rate = [(As: absorbance of experimental well - Ab: absorbance of blank well)) / (Ac: absorbance of control well - Ab: absorbance of blank well)] x 100%.

[0090] SW480 cells were plated in 24-well plates at 5 × 10 5 cells / well at 37°C and 5% CO 2 Incubate for 24 hours, and then incubate for another 48 hours with different treatment groups. According to the instructions for the Annexin V-FITC / PI apoptosis detection kit, 100,000 cells in each group were selected for staining for subsequent experiments. Cell apoptosis was detected by flow cytometry, and the sample size for each test was 10,000 cells.

[0091] Detection of cell starvation and autophagy

[0092] The autophagic process and starvation during treatment were investigated using MDC staining assays, TEM images of autophagosomes, and determination of intracellular lactate and ATP concentrations. SW480 cells were incubated with Exo@siRNA-CTX NPs, Exo@3BP-CTX NPs, or ERBC NPs for 48 h. SW480 cells were then incubated with the MDC staining kit for 30 min according to the instructions. After washing three times with PBS, specific labeled autophagosomes were monitored by fluorescence microscopy. In order to observe autophagosomes by TEM, cells were fixed with 2.5% glutaraldehyde for 2 h and then submitted for examination. Finally, transmission electron microscopy was used to observe autophagosomes in cell sections. Intracellular lactate concentration was calculated by counting 1×10 after SW480 cells were treated with different nanomaterials according to the quality standards. 6The cells were used for subsequent experiments. According to the standard of the lactate assay kit, the absorbance was measured at 570 nm using a UV spectrophotometer. The lactate concentration was calculated with reference to the obtained standard curve. For the detection of ATP, the intracellular ATP level was detected by the ATP luminescence assay kit. The luminescence signal was measured by the luminometer function of the microplate reader, and the intracellular ATP content was calculated according to the calibration curve obtained in the instructions.

[0093] Cell membrane extraction

[0094] Cultivate about 20-50 million cells, place on ice and wash three times with PBS to remove the culture medium. Use PBS as a buffer, scrape the cells with a cell scraper and collect them in a 2ml centrifuge tube. Collect the cell precipitate by centrifugation at 1000g for 5min, add solution A according to the instructions of the cell membrane protein and cytoplasm protein extraction kit, place it in an ice bath for 10-15 minutes, and then freeze and thaw it repeatedly in liquid nitrogen for 3 times to break the cells. Centrifuge at 4℃, 700g for 10 minutes to remove the nucleus and unbroken cells. Collect the supernatant and transfer it to a new EP tube. Do not touch the precipitate when aspirating the supernatant! Centrifuge at 4℃, 14000g for 30 minutes to precipitate cell membrane fragments. Collect the precipitate and do your best to aspirate the supernatant. Add 100 microliters of membrane protein extraction reagent B, vortex at the highest speed for 5 seconds to resuspend the precipitate, and ice bath for 5-10 minutes. Subsequently, centrifuge at 4℃, 14000g for 5 minutes, and collect the supernatant as the cell membrane protein solution.

[0095] Exosome flow cytometry analysis

[0096] 4 μM aldehyde / sulfate latex beads were prepared for passive adsorption according to the manufacturer's instructions. Because exosomes (<100 nm) are too small to be identified by FACS. By using latex beads, multiple exosomes are bound to each bead, and then the overall size of the particles basically exceeds the lower limit of FACS detection, i.e., 500 nm. 30 μg of exosomes were incubated in 100 μl volume of beads at room temperature for 15 min. 900 μl PBS was added, and the mixture was incubated for another 2 h at room temperature. The coupling reaction was terminated by adding 100 mM glycine. Centrifuge at 3000 g for 10 minutes at room temperature to precipitate the exosome-coated beads. The beads were resuspended in 5 ml PBS and washed three times and settled at 3000 g for 10 min. The beads were stained with a 100 μl volume of fluorescent secondary antibody conjugate at the antibody concentration recommended by the manufacturer to detect exosome membrane markers CD63 and CD9. Incubate at 4 ° C for 1 h. Wash the microbeads three times with PBS. The stained beads were resuspended in 100 μl FACS buffer and analyzed by flow cytometry using appropriate isotype control stained beads.

[0097] Western blot experiments

[0098] Total exosomal protein, cellular protein, and tissue protein were prepared by resuspending the samples in RIPA lysis buffer. The concentration of the obtained protein was evaluated according to the instructions of the BCA protein assay kit. 10-30 μg of total protein was taken from each supernatant, electrophoresed on SDS-PAGE gel, and transferred to PVDF membrane. Then it was blocked in 5% skim milk for 1.5 hours. And incubated with antibodies against Alix, CD 63, CD 9, TSG101, P62, EGFR, HIF-1α, pERK, KRAS, HK-II, ERK, GAPDH, β-Actin, Pan cadherin, LC3-I / II, Caspase-3, GLUT1 for 16 hours at 4°. Then incubated with horseradish peroxidase-conjugated secondary antibody for 2 hours. Chemiluminescence was detected using ECL detection solution. Band intensity was quantified using Image software, and protein expression was calculated with β-Actin as the normalized expression.

[0099] Animal Experiment Section

[0100] The animal experiments were approved by the Animal Ethics Review Committee of Chongqing Medical University. Female nude mice aged 4-5 weeks were inoculated with human colorectal cancer cells (5×10 6 / 100μl) was injected into the lateral abdominal wall of nude mice. Tumor growth was measured every 3 days using a digital caliper. The width (W) and length (L) of the tumor were measured, and the tumor volume was calculated as (L×W×W) / 2. When the tumor size reached approximately 75mm 3 At the same time, the mice were randomly divided into 5 groups (PBS, Exo@siRNA-CTX NPs, Exo@siRNA-CTX NPs+Free CTX, Exo@siRNA / 3BP-CTX NPs, ERBC NPs+Free CTX). Exosomes were injected via the tail vein, and in order to further study the sensitivity of cetuximab, cetuximab (5 mg / kg) was added to Exo@siRNA-CTX NPs+Free CTX and ERBC NPs+Free CTX for treatment. The treatment was repeated every other day for a total of 10 injections. Body weight was measured every 3 days within 21 days. Tumor tissues and major organs were sliced ​​and further pathologically analyzed using hematoxylin-eosin (H&E) and immunohistochemical analysis. After 21 days of treatment, the clinical indicators of liver and kidney function, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine (CRE), were measured.

Claims

1. A method for preparing a novel nano drug, characterized in that: The following steps are involved: (1) Collect, isolate and purify maternal exosomes; (2) Loading siRNA and 3BP into exosomes by electroporation: siRNA, 3BP and exosomes were mixed in 400 μL electroporation buffer and electroporated on a Bio-Rad Gene electroporation device in a 0.4 cm cuvette at a working voltage of 400 mV, a capacitance of 125 μF, and a pulse time of 10 to 15 ms; (3) After electroporation, the exosomes were treated with RNase to remove any siRNA that might be bound to the exosome membrane. The exosomes were then diluted with pre-cooled PBS and centrifuged at 100,000 g for 70 min to remove unbound siRNA and 3BP.

2. The method for preparing a novel nano drug according to claim 1, characterized in that: The method further includes the step of uncoupling cetuximab on the surface of exosomes, mixing CTX and DSPE-PEG-NHS in a mass ratio of 4:1 in MES buffer (PH=8), stirring at room temperature at 80 rpm for 4 hours to react and connect, dissolving DSPE-PEG-CTX in DMSO, and then mixing with the exosomes prepared in step (3) at a weight ratio of DSPE-PEG-CTX:exosomes of 1:1, incubating at 37°C for 2 hours, and ultrafiltration in a 300K Nanosep centrifuge to remove free DSPE-PEG-CTX and CTX.

3. The preparation method of the novel nano drug according to claim 1, characterized in that: The exosomes are obtained by culturing HEK293T cells, then performing multi-step gradient centrifugation and resuspension.

4. The method for preparing the novel nano drug according to claim 1, characterized in that: The multi-step gradient centrifugation comprises the following steps: (1) Centrifuge at 1000 g for 10 min to remove dead cells and collect the supernatant; (2) centrifugation at 2000 g for 20 min to remove cell debris; (3) Centrifuge at 10,000 g for 30 min to remove large vesicles and small cell debris, collect the supernatant and discard the precipitate; (4) Centrifuge at 100,000 g for 70 min to precipitate exosomes.

5. The preparation method of the novel nano drug according to claim 1, characterized in that: Before perforation, the weight ratio of exosomes: siRNA was 8-11:1, and the weight ratio of exosomes: 3BP was 1:5-6.

6. The method for preparing the novel nano drug according to claim 5, characterized in that: The weight ratio of exosomes: siRNA is 10:1, and the weight ratio of exosomes: 3BP is 1:

4.

7. A method for preparing a novel nano drug according to any one of claims 1 to 6, characterized in that: The final concentration of exosomes in the electroporation mixture did not exceed 0.5 μg / μL.

8. The method for preparing the novel nano drug according to claim 7, characterized in that: Treat reagents and consumables with DEPC before electroporation to reduce RNA degradation.

9. A novel nano drug, characterized in that: The method is prepared by any one of claims 1 to 8.

10. Use of the drug according to claim 9 in the preparation of anti-tumor drugs with high biocompatibility.

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