CN114522238A

CN114522238A - Lanthanide-based oxyfluoride nanocrystal-based miRNA vector and application thereof

Info

Publication number: CN114522238A
Application number: CN202210179094.8A
Authority: CN
Inventors: 闫瑾; 尤伟名
Original assignee: Xian Jiaotong University
Current assignee: Shaanxi Wanjia State Yuan Biotechnology Co ltd
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2022-05-24
Anticipated expiration: 2042-02-25
Also published as: CN114522238B

Abstract

The invention provides a lanthanide-based oxyfluoride nanocrystal miRNA vector and application thereof. The invention discloses a lanthanide oxyfluoride nanocrystal-based miRNA vector and application of the vector-loaded anti-tumor miRNA as miRNA therapeutic drugs. The miRNA carrier based on the lanthanide oxyfluoride nanocrystals provided by the invention can realize the efficient loading of miRNA, and can protect miR-30c in cells for more than 24 hours. The miRNA with the anti-tumor load as miRNA treatment medicine has good biocompatibility while the function of the miRNA is kept.

Description

Lanthanide-based oxygen-fluorine nanocrystal miRNA vector and application thereof

Technical Field

The invention belongs to the technical field of nano composite materials, relates to a drug carrier material, and particularly relates to a lanthanide oxy-fluoro nanocrystal-based miRNA carrier and application thereof.

Background

The Wnt signaling pathway plays a crucial role in embryonic development, tissue homeostasis, and stem cell proliferation and differentiation in all animals. However, the abnormal activation of the signal is the basis of various human cancers, and the deregulated Wnt signal pathway causes the accumulation of the transcription activator beta-catenin in cytoplasm and nucleus, thereby promoting the expression of genes related to the proliferation and migration of tumor cells. In the process, a beta-catenin coactivator (comprising Bcl9) is used as a part of the abnormal activation of the Wnt signal pathway, is over-expressed in various malignant tumors, and forms a stable complex with the beta-catenin to promote the expression of the oncogenes. Dysregulation of the Wnt signaling pathway reduces infiltration of chemotherapeutic drugs and T lymphocytes, leading to resistance to chemotherapy and PD-1/PD-L1 checkpoint blockade immunotherapy. The inhibition of the inherent activity of the tumor beta-catenin signal channel not only can inhibit the development of the tumor, but also has the sensitization effect on chemotherapy and immunotherapy.

The activated Wnt pathway is a common element in the regulation of stem/progenitor cell regeneration and maintenance in non-cancerous tissues and organs. Therefore, the Wnt/beta-catenin inhibitor always has targeted toxicity, and no drug is approved to be applied to clinic at present. To address this problem, Bcl9, which is highly expressed in tumors but not in cells of tumor origin, has received considerable attention. The oncogenic effects of Bcl9 were reported to be rescued by siRNA/shRNA-induced gene knock-out or treatment with modified Bcl9 peptides, both of which reduced proliferation, metastasis and resistance to therapy, underscoring the importance of Bcl9 in targeted tumor therapy.

MicroRNAs (miRNA) are gene expression regulation factors involved in various tumor pathogenesis, and are expected to create a new treatment way for anticancer treatment. Among them, a family of mirnas named miR-30s, which are expressed at low levels in a large fraction of cancers, can regulate the expression of Bcl 9. More importantly, miR-30c has been proved to be effective in inhibiting the abnormal expression of Wnt/beta-catenin signals in malignant tumor cells. However, like other mirnas, miR-30s also has two major pharmacological drawbacks: poor nuclease stability and low membrane permeability severely limit its clinical application. Various fine chemicals for miRNA modification and delivery vectors have been developed and significant progress has been made in improving the pharmacological properties of clinical therapeutic drugs for mirnas. However, chemically modified nuclease-resistant mirnas invariably carry the risk of potentially off-target effects and clearance of the particle system. In addition, the commonly used miRNAs carrier lipids and biodegradable polymers are rapidly cleared by the liver and spleen due to the high positive charge, and non-specific uptake by the cells. These drawbacks necessarily prevent widespread clinical use of miRNAs therapy. Thus, there is a need for novel and clinically viable delivery systems to advance the discovery and development of miRNAs.

Nanoparticle-mediated delivery of miRNAs holds promise in overcoming many of the limitations of traditional delivery systems. The appropriate loading of miRNAs onto nanoparticles can significantly improve their resistance to nucleases, membrane permeability and bioavailability. In fact, nanoparticle-based drug delivery systems are particularly attractive in the treatment of solid tumors, since nanoparticles can actively accumulate through endothelial leakage, known as nanol. Recent researches show that the rare earth doped nanoparticles (LDNp) have good surface compatibility and biocompatibility and can be used as a biomolecule drug carrier.

Disclosure of Invention

The invention aims to provide a lanthanide oxyfluoride nanocrystal-based miRNA carrier, which is used for loading miRNA so as to solve the problems of poor biocompatibility, insufficient miRNA binding capacity, insufficient transport capacity in miRNA cells, poor actual treatment effect and the like of clinical miRNA treatment medicines.

In view of the above, the present application addresses this need in the art by providing a miRNA vector based on lanthanide oxyfluoride nanocrystals and applications thereof.

In one aspect, the invention relates to a lanthanide oxyfluoride nanocrystal-based miRNA vector, which comprises a rare earth doped nanocrystal and poly-L-lysine, wherein the poly-L-lysine is coated on the surface of the rare earth doped nanocrystal through a coordination reaction, and the rare earth doped nanocrystal is GdOF: 45% Ce, 15% Tb.

Further, in the miRNA vector based on lanthanide oxyfluoride nanocrystals provided by the present invention, the poly-L-lysine forms a coordination bond with the rare earth-doped nanocrystals.

Further, the diameter of the miRNA carrier particle in the lanthanide oxyfluoride nanocrystal-based miRNA carrier provided by the invention is 5.2 +/-0.4 nm.

In another aspect, the present invention provides a method for preparing a miRNA vector based on lanthanide oxyfluoride nanocrystals, comprising: synthesizing a rare earth doped nanocrystal by adopting an oleylamine assisted hydrothermal method, removing oleylamine on the surface of the rare earth doped nanocrystal by adopting hydrochloric acid with pH of 4.0, and wrapping poly L-lysine on the surface of the nanocrystal; the rare earth doped nanocrystal is GdOF: 45% Ce, 15% Tb.

In another aspect, the present invention provides a composite comprising: loading a complex formed by miRNA on the surface of a lanthanide oxyfluoride nanocrystal-based miRNA carrier; the miRNA and the miRNA carrier based on the lanthanide oxyfluoride nanocrystals are in a ratio of 50: 1-400: 1 by mass; preferably, the ratio of the miRNA to the lanthanide oxyfluoride nanocrystal-based miRNA vector is 300: 1.

In another aspect, the present invention provides a method of preparing a complex, comprising: carrying out electrostatic adsorption on miRNA for 30min at the temperature of 37 ℃, loading the miRNA on the surface of a miRNA carrier based on lanthanide oxyfluoride nanocrystals, wherein the ratio of the miRNA to the miRNA carrier based on the lanthanide oxyfluoride nanocrystals is 50: 1-400: 1 by mass; preferably, the ratio of the miRNA to the lanthanide oxyfluoride nanocrystal-based miRNA vector is 300:1 by mass.

Further, the above-mentioned miRNA vector includes miR-30 c.

According to the invention, through a mode of coupling cation polylysine and miRNA carrier electrostatic bonding, the potential treatment effect of lanthanide fluorine oxide nanocrystals (GdOF: Ce, Tb) as miRNA carriers on tumors is explored. In this proof of concept study, exemplary miR-30c, a member of the miR-30s family, specifically targets Bcl9 in colorectal cancer, is delivered to MC38 and HCT116 cells in vitro and in vivo to inhibit the Wnt/β -catenin pathway. Experiments prove that after the miRNA carrier based on the lanthanide oxyfluoride nanocrystals provided by the invention loads miR-30c, the development of tumors is effectively inhibited in vivo, but more importantly, the miRNA carrier effectively enhances the effects of chemotherapy and immunotherapy in vivo. Thus, the present invention further claims the use of the miRNA vector in a medicament for the treatment of cancer, including colon cancer; the invention further claims a medicament for treating cancer, which acts in a way of inhibiting or blocking the Wnt/beta-catenin pathway; the invention further claims a chemo-or immunotherapy drug sensitizer.

Compared with the prior art, the invention has the following beneficial effects or advantages:

compared with the prior art, the main contribution of the invention is to provide the miRNA carrier based on the lanthanide oxyfluoride nanocrystals, which can realize the high-efficiency loading of miRNA and can protect miR-30c in cells for more than 24 h. The miRNA with the anti-tumor load carrier as the miRNA therapeutic drug has good biocompatibility while the function of the miRNA is kept. After the carrier is loaded with the anti-tumor miRNA, chemotherapy and immunotherapy are effectively enhanced in vivo.

Drawings

FIG. 1 is a schematic diagram of the design and application of LnP nanoparticles.

FIG. 2 is a representation of Ln, PLL and LnP nanoparticles; (A) infrared spectra of Ln, PLL, and LnP; (B) LnP and Ln are turbidity after polymer modification; (C) and (D) typical TEM images showing morphology and size of Ln and LnP; (E) particle sizes of LnP and Ln at different times; (F) cell viability at

days

1, 2 and 3 for LnP nanoparticles at different concentrations (100, 200, 300 and 400 μ g/mL); PEI25K was at a concentration of 12.5ug/mL, PEI25K was a positive control.

FIG. 3 is an evaluation LnP of the gene binding ability of nanoparticles by Agarose Gel Electrophoresis (AGE); (A) agarose gel electrophoresis results of the binding capacity of the complex of LnP/miR-30c and PEI25K/miR-30c under different W/W ratios; (B) quantitative binding capacity for miRNA (n-4); (C) agarose gel electrophoresis results of miR-30c and LnP/miR-30c and PEI25K/miR-30c complex dissociation when the W/W ratio is 12.5: 1; (D) quantitative dissociation result of miRNA capability (n-4); (E) agarose gel electrophoresis results of serum stability of naked miR-30c, LnP/miR-30c and PEI25K/miR-30c complexes (w/w is 12.5:1) at different time points; (F) quantitative miRNA detection serum stability (n ═ 4), assay data were quantitatively analyzed by ImageJ software, P <0.05 and P < 0.01.

FIG. 4 shows the transfection efficiency of miR-30c analyzed by confocal laser scanning microscopy. miR-30c is labeled with FAM (green), and the nucleus is labeled with DAPI (blue); (A) fluorescence confocal microscopy images (scale bar: 20 μm) after incubation of different weight ratios and different polymer-gene complexes with miR-30c for 24 h; (B-C) relative fluorescence intensity of fluorescence confocal microscopy images. PEI25K and Lipo2000 were positive controls; p <0.05, P <0.01, all experiments were repeated three times (n-4 per group).

FIG. 5 shows the delivery efficiency of LnP nanoparticles transfected for 24h and miR-30c in MC38 cells at different weight ratios; (A) expression of miR-30c after transfection of different complexes; (B) expression of Bcl9 following transfection of the different complexes; (C) expression of C-myc after transfection of the different complexes; (D) expression of Cyclin D following transfection of the different complexes; (E) western blot analysis of Cyclin D and beta-catenin; (F) carrying out protein gray level analysis by using Image J software; lipo2000 is a positive control, and beta-actin is used as an internal reference; p <0.05, P < 0.01.

FIG. 6 is the delivery efficiency of intracellular transfection of 24h and miR-30c in HCT116 cells using LnP nanoparticles in different weight ratios; (A) expression of miR-30c after transfection of different complexes; (B) expression of Bcl9 following transfection of the different complexes; (C) expression of C-myc after transfection of the different complexes; (D) expression of Cyclin D following transfection of the different complexes; (E) western blot analysis of Cyclin D and beta-catenin; (F) image J software performs protein grayscale analysis. Lipo2000 is a positive control; beta-actin as an internal reference protein; p <0.05, P < 0.01.

FIG. 7 is the tumor suppression effect of LnP/miR-30c nanoparticles in vivo; (A) LnP/miR-30C nanoparticles treat the tumor growth curve of MC38 cell transplantation tumor C57 mice after different times; (B) photographs of MC38 tumors obtained 12 days after treatment; (C) tumor weight after 12 days of treatment; (D) h & E staining of tumor tissue after 12 days of treatment; (E) relative protein expression of beta-catenin and Cyclin D in subcutaneous tumors; (F) TUNEL staining of tumor tissue after 12 days of treatment; p < 0.001; a scale: 20 μm.

FIG. 8 is the in vivo tumor suppressive effect of LnP/miR-30c nanoparticles and the tumor suppressive effect of chemotherapy; (A) tumor volume in MC38 cell transplantable tumor C57 mice after various times of treatment with 5-fluorouracil and LnP/miR-30C nanoparticles; (B) photographs of MC38 tumors obtained 12 days after treatment; (C) tumor weight at 12 days, tumor tissues were analyzed for H & e (d) and tunel (e) 12 days after treatment with 5-fluorouracil and LnP/miR-30c nanoparticles; (F) relative protein expression of beta-catenin and Cyclin D in subcutaneous tumors; p < 0.001; a scale: 20 μm.

FIG. 9 is an in vivo LnP/miR-30c nanoparticle and the tumor suppression effect of immunotherapy; (A) the tumor volume of MC38 cell transplantation tumor C57 mice after different time of treatment with PD-L1 and LnP/miR-30C nanoparticles; (B) photographs of MC38 tumors obtained 12 days after treatment; (C) tumor weight at day 12, H & e (d) and tunel (e) image analysis of tumor tissue 12 days after PD-L1 and LnP/miR-30c treatment; (F) relative protein expression of β -catenin and Cyclin D in subcutaneous tumors, <0.05, <0.01, < 0.001; a scale: 20 μm.

FIG. 10 is an evaluation of biocompatibility of major organs in vivo; the mice in the administration group are subjected to HE staining of the histology of heart, liver, spleen, lung and kidney, and the proportion scale is as follows: 50 μm.

FIG. 11 is a photograph of bands of β -catenin and cyclinD in subcutaneous tumors measured by immunoblotting; different treatments (1: control; 2: PD-L1; 3: 5-fluorouracil (5-FU); 4: LnP/miR-30 c; 5: 5-FU + LnP/miR-30 c; 6: PD-L1+ LnP/miR-30 c).

FIG. 12 is the biocompatibility of various complexes for in vivo treatment; (A) body weight of mice at different time points; (B) body weight of mice at the end of the experiment.

Wherein Ln is rare earth doped nanocrystal (GdOF: 45% Ce, 15% Tb), LnP is Ln with poly-L-lysine coated on the surface, LnP/miR-30c is a compound formed after LnP loads miR-30c, and PLL is poly-L-lysine.

Detailed Description

The following examples are given to illustrate the technical means of the present invention, but the present invention is not limited to the following examples.

In the following examples, unless otherwise specified, Ln is a rare earth doped nanocrystal (GdOF: 45% Ce, 15% Tb), LnP is Ln with poly-L-lysine coated on the surface, LnP/miR-30c is a complex formed after LnP supports miR-30c, and PLL is poly-L-lysine.

Example 1

This example provides LnP preparation and characterization.

LnP preparation: synthesis of Ln is free oleylamine GdOF: 45% Ce, 15% Tb, prepared Ln was redispersed in PBS (pH7.4) buffer salt. PLL and LDN are coupled by coordination bonds through amino groups in the polymer. 5mg of Ln was dissolved in 10mL of PBS buffer containing 10mg of PLL. Incubating for 60min under stirring at 30 ℃, centrifuging at 10000g, and collecting LnP.

LnP characterization: the chemical structures of Ln, PLL and LNP were analyzed using an infrared spectrometer. The morphology was observed with a transmission electron microscope. Zeta potential and hydrodynamic size were measured with a laser particle sizer. Fourier transform Infrared Spectroscopy (FTIR) confirmed the coordination bond of PLL to Ln at 1659cm^-1This is also evidenced by the significant reduction in the characteristic infrared absorption peak at the amino group (see FIG. 2A). This example then used a standard method based on 350nm turbidity measurements to compare the water solubility of ligand-free ln (ln) and PLL-coated ln (lnp) (see fig. 2B). The PBS solution gradually became cloudy with increasing nanoparticle concentration at a pH of 7.4 and a temperature of 37 ℃. Higher turbidity values were measured in Ln samples without ligand, indicating that PLL imparts optimal hydrophilicity to Ln (see fig. 2B). To further support the advantages of PLL coating, this example uses Transmission Electron Microscopy (TEM) to observe morphology and monodispersity of Ln and LnP, with particle aggregation in the Ln sample (see fig. 2C). Whereas LnP nanoparticles showed uniform monodisperse nanostructures, the diameter was calculated to be 5.2 + -0.4 nm from 100 randomly sampled particles (see FIG. 2D). In addition, LnP was also tested in comparison to Ln for colloidal stability, where LnP remained monodisperse after 1h incubation in PBS (pH7.4) at 37 ℃ and Ln appeared to aggregate after 1h in PBS solution (see FIG. 2E). Optimized hydrophilicity and monodispersity of LnP demonstrated better biocompatibility compared to commercial non-viral vectors PEI25K and Lipo2000 as found by viability testing of MC38 cells. LnP cultured at high concentration of 400 μ g/mL for 3d, the cell survival rate can reach over 100%; in sharp contrast, PEI25K and Lipo2000 at a concentration of 15. mu.g/mL had cell viability around 30% and 70%, respectively, and the biocompatibility of LNP was superior to that of the commercial non-viral vectors PEI25K and Lipo 2000.

Example 2

This example provides preparation and characterization of LnP-loaded miR-30 c.

And (3) carrying out electrostatic adsorption on the miR-30c for 30min at the temperature of 37 ℃, and loading the miR-30c on the nanoparticles LnP to form a nanoparticle/miR 30c compound.

Preparing a miR-30c compound: before preparing the miR-30c complex, miR-30c is centrifuged at 12000rpm for 10min and dissolved in fresh DEPC water. LnP and PLL solution were added to an equal volume of miR-30c solution (0.264mg/mL) to calculate miR-30c complexes of different specific gravities. The miR-30c complex was prepared in a HEPES environment at 37 ℃ (PH 6.84). The particle size and Zeta potential of various miR-30c complexes were measured by hydrodynamic particle size distribution DLS. All complexes were prepared in 1mL ultra pure water containing 2.6 μ g mir-30c (12.5 w/w) for differential scanning calorimetry analysis.

LnP characterization of miR-30c Supported: the decrease in Zeta potential confirms the successful loading of miR-30 onto LnP (see table 1).

TABLE 1 polydispersity index of polymers and Polymer/miRNA complexes

Notably, the positive charge of LnP/miR30c indicates that the complex has an innate advantage of transport across membranes. Next, this example assesses LnP miRNA loading on PEI25K by agarose gel electrophoresis contrast, with PEI25K being the most robust commercial non-viral vector on the market (see fig. 3A). The results show that LnP showed similar miRNA loading as PEI25K, with differences on the same order of magnitude (see figure 3B). Next, to assess whether miR-30C can be released from the LnP/miR-30C complex, competition experiments were performed with heparin, a negatively charged polyanion that mimics intracellular biomacromolecules for miR-30C disproportionation (see FIG. 3C). As the PEI25K/miR-30c complex, more than 50% of the miRNA was released from LnP/miR-30c at a heparin concentration of 60 μ g/μ L, indicating that LnP/miR-30c has a similar miRNA release capacity as PEI25K/miR-30c (see FIG. 3D). Naked mirnas are readily degraded by nucleases both in vitro and in vivo, so it is necessary to verify the resistance of LnP/miR-30c to degradation of mirnas. Incubation in serum at 37 ℃ degraded more than half of the naked miR-30c within 1 hour, while the miR-30c in LnP/miR-30c remained intact (see FIG. 3E). Importantly, LNP can protect miR-30c for more than 24 hours when at least 75% of miR-30c is intact (see fig. 3F).

Example 3

This example provides a validation experiment of LnP efficient transport of MiR-30c in cells.

LnP/miR-30c, PEI25K/miR-30c and Lipo2000/miR-30c cellular uptake was assessed on a confocal laser microscope (CLSM, FV1200, Olympus) by monitoring the green fluorescence of the FAM-labeled miR-30c (see FIG. 4). To screen LnP: optimal weight ratio of miR-30c to efficient internalization of cells, the present example first tested the cellular uptake of LnP/miR-30c at different weight ratios (50: 1-400: 1). Laser confocal images (see FIG. 4A) and green fluorescence intensity analysis (see FIG. 4B) showed that LnP/miR-30c, at a weight ratio of 300:1, had the strongest cellular internalization. In addition, LNP/miR-30C (weight ratio 300:1) also showed the brightest green fluorescence in MC38 cells compared to Lipo2000/miR-30C and LNP/miR-30C (see FIG. 4C). These results indicate that LnP is a potent miR-30c vector and is the best alternative to commercial non-viral gene vectors.

Example 4

The embodiment provides a verification test that LnP/miR-30c can effectively inhibit the Wnt/beta-catenin pathway.

Bcl9 is a transcription coactivator of Wnt signals, plays a role in beta-catenin nuclear translocation, and is always over-expressed in tumor cells, thereby promoting tumor progression and treating drug resistance. According to the structural design of the invention, the intracellular miR-30c can inhibit the expression of Bcl9, so that the beta-catenin nuclear translocation disorder is caused, and the wnt/beta-catenin pathway is blocked.

To confirm this, this example first semi-quantitatively detected the expression of miR-30c in MC38 cells treated with LnP/miR-30c and Lipo2000/miR-30c using RT-PCR. Consistent with the laser confocal results (see fig. 5), the weight ratio was 300:1 LNP/miR-30c the amount of intracellular miR-30c was the greatest, even exceeding one of the most effective commercial non-viral vectors Lipo2000 (see figure 5A). In LnP/miR-30C-treated cells, the mRNA level of Bcl9 was statistically significantly down-regulated (see FIG. 5B), and as a result, the gene expression of C-myc (see FIG. 5C) and CyclinD (see FIG. 5D) was correspondingly reduced in the Wnt downstream signaling pathway. Furthermore, downregulated β -catenin and Cyclin D at the protein level fully demonstrated inhibition of the Wnt/β -catenin signaling pathway (see fig. 5E and F). Notably, LnP/miR-30c showed stronger Wnt blocking than Lipo2000/miR-30c (see FIGS. 5B-F), which is consistent with LnP-enhanced miR-30c delivery (see FIG. 5A). Furthermore, it can be concluded from the results associated with HCT116 cells that HCT116 cells are a Wnt-hyperactive human colon cancer cell (see FIG. 6). In conclusion, the results provide verification that LnP/miR-30c effectively inhibits the Wnt/beta-catenin pathway.

Example 5

This example provides a validation experiment of LnP/miR-30c for inhibiting Wnt/beta-catenin pathway and tumor growth in vivo.

To verify the excellent performance of LnP/miR-30c in inhibiting Wnt/beta-catenin, this example further tested its in vivo activity. To this end, MC 38-tolerant tumors (50-100 mm) will be used³) The C57BL/6 mice were randomly divided into two groups: PBS (control) and LnP/miR-30c, two weeks after treatment. As shown in FIG. 7A, LnP/miR-30 c-treated mice had significant tumor suppression compared to the PBS-treated control group. At the end of the experiment, the tumor bodies were detached and weighed (see FIGS. 7B and C). The mean tumor weight of PBS-treated mice was 4-fold that of LnP/miR-30C-treated mice, indicating that LnP/miR-30C has a strong tumor-inhibiting effect (see FIG. 7C). In addition, H of tumor tissue&The E staining again confirmed this result (see fig. 7D). Furthermore, the decrease in β -catenin and Cyclin D protein levels indicates that this tumor inhibition is due to blockade of the Wnt/β -catenin pathway (see fig. 7E), which is again evidenced by TUNEL staining showing increased apoptosis (see fig. 7F).

Example 6

This example provides a validation experiment of the anti-tumor effect of LnP/miR-30c in vivo sensitization chemotherapy.

Chemotherapy is currently the most widely used treatment as the standard treatment for locally advanced and metastatic cancers. However, in some cases, the clinical efficacy of chemotherapy is unsatisfactory, as tumors often develop chemotherapy resistance. 5-fluorouracil is a first-line chemotherapeutic drug for treating colon cancer, and more evidence indicates that inhibition of Wnt/beta-catenin signaling pathway can inhibit drug resistance of 5-fluorouracil (5-FU) chemotherapy.

To confirm this, 5-FU and 5-FU/LNP/miR-30c were applied in combination, again using the MC38 transplantation model. LnP/miR-30C significantly increased the antitumor effect of 5-FU (see FIGS. 8A-C). LnP/miR-30c can obviously improve the anti-tumor activity of 5-FU. This result was further confirmed by HE staining and TUNEL staining, histology (see fig. 8D and E). In addition, western blot analysis was also performed in this example to investigate changes in protein levels. As shown in FIG. 8F, the protein levels of the LnP/miR30c-5-FU combination group β -catenin and Cyclin D were significantly reduced compared to the 5-FU only group. LnP/miR-30c and 5-FU have synergistic antitumor effect.

Example 7

This example provides the antitumor effect of LnP/miR-30c in vivo sensitization PD-L1 checkpoint blockade immunotherapy

Binding of PD1 to PD-L1 immunologically inhibited the anti-cancer activity of Cytotoxic T Lymphocytes (CTLs), leading to tumor immune escape and subsequent tumor progression in the tumor microenvironment. Recent studies have shown that immune checkpoint blockade of the inhibitory immune receptors PD-L1 and PD-1 has become a successful therapeutic strategy to increase the efficiency of anti-tumor immunity by releasing T lymphocytes. However, many cancer patients do not respond to PD-1/PD-L1 checkpoint blockade, which is often due to T cell rejection, a key feature of Wnt activation. Thus, it is contemplated that effective blockade of the Wnt/β -catenin signaling pathway by LnP/miR-30c could abrogate this resistance and act synergistically with PD-L1 checkpoint blockade.

To verify this hypothesis, C57 mice received PD1/PDL1 inhibitor (PPI) alone or PPI-LnP/miR-30C combination therapy, respectively, in allograft tumors of MC38 colon cancer. As shown in FIGS. 9A-C, after two weeks of treatment, PPI-LnP/miR-30C combination-treated mice showed greater tumor suppression compared to PPI-treated mice. In addition, HE and TUNEL measurements showed a decrease in tumor tissue density and significant apoptosis in the co-treated group (see fig. 9D and E). Furthermore, Westernblot grey value analysis at the protein level showed a significant decrease in β -catenin and Cyclin D levels in the synergistic treatment group (see fig. 9F and fig. 11). The results show that LnP/miR-30c has a synergistic effect with PD1/PD-L1 checkpoint blockade immunotherapy.

Example 8

This example provides an in vivo safety assessment assay of LnP/miR-30 c.

Xenograft tumor model: female C57BL/6 mice were purchased from the school of medicine of Western Ann university of transportation and used at 3 weeks of age. The experimental procedure was carried out according to the guidelines of the animal care committee of the university of transport of west ampere. MC38 cells (3X 10)⁶) The injection is injected into the groin of the mouse subcutaneously to form a xenograft tumor model. The shortest and longest diameters of the tumor were measured with a vernier caliper and calculated as: volume (mm3) 1/2 x length x width²

Tumor inhibition experiment: the tumor volume reaches 50mm³After left and right, mice were randomly divided into 6 groups and administered intraperitoneally, once every two days. Mouse body weight and tumor volume were measured. At the last time point, tumors were collected for weighing, measurement and immunohistochemical analysis.

To verify LnP/miR-30 c's biosafety in vivo, this example was performed by comprehensive evaluation of H & E of major internal organs (see FIG. 10). And mouse body weight after drug treatment (see figure 12). After 2 weeks of administration, the mice of the LnP/miR-30c single-dose group, LnP/miR-30c + 5-fluorouracil or PD1/PDL1 inhibitor group had a significant increase in body weight compared to the control group (PBS), while the body weight of the 5-fluorouracil-treated group was significantly reduced (P <0.05) (see FIG. 12). No morphological or pathological changes were observed in the histology of the heart, liver, spleen, lung, and kidney of the mice in the control group, LnP/miR-30c group, PD/PDL1 inhibitor group, LnP/miR-30c + 5-fluorouracil, or PD1/PDL1 inhibitor group (see FIG. 10). Only the 5-fluorouracil group showed common side effects of chemotherapeutic drugs, such as liver necrosis, steatosis, kidney necrosis, inflammation, alveolar wall thickening, etc. Taken together, these results further demonstrate that LnP/miR-30c has good biocompatibility and biosafety as a potentiator for chemotherapy or immune-adjuvant therapy.

As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.

Claims

1. A lanthanide oxyfluoride nanocrystal-based miRNA carrier comprises rare earth doped nanocrystals and poly-L-lysine, wherein the poly-L-lysine is coated on the surfaces of the rare earth doped nanocrystals through a coordination reaction, and the rare earth doped nanocrystals are GdOF: 45% Ce, 15% Tb.

2. The miRNA vector of claim 1, wherein the poly-L-lysine forms a coordinate bond with the rare-earth doped nanocrystal.

3. The miRNA vector of claim 1, wherein the miRNA vector particle is 5.2 ± 0.4nm in diameter.

4. A preparation method of a miRNA vector based on lanthanide oxyfluoride nanocrystals is characterized by comprising the following steps: synthesizing a rare earth doped nanocrystal by adopting an oleylamine assisted hydrothermal method, removing oleylamine on the surface of the rare earth doped nanocrystal by adopting hydrochloric acid with pH of 4.0, and wrapping poly L-lysine on the surface of the nanocrystal; the rare earth doped nanocrystal is GdOF: 45% Ce, 15% Tb.

5. A complex comprising the miRNA vector of claim 1 surface-loaded with miRNA; the ratio of the miRNA to the miRNA vector of claim 1 is 50: 1-400: 1 by mass.

6. The complex of claim 5, wherein the ratio of the miRNA to the miRNA vector of claim 1 is 300:1 by mass.

7. The miRNA vector of any one of claims 1 to 6, wherein the miRNA comprises miR-30 c.

8. Use of a miRNA vector of any one of claims 1 to 6 in a medicament for the treatment of cancer, including colon cancer.

9. A medicament for the treatment of cancer comprising a complex according to claim 5 in a manner that inhibits or blocks the Wnt/β -catenin pathway.

10. A chemo-or immunotherapeutic drug sensitizer comprising the complex of claim 5.