CN114522238B

CN114522238B - miRNA carrier based on lanthanide series oxyfluoride nanocrystals and application thereof

Info

Publication number: CN114522238B
Application number: CN202210179094.8A
Authority: CN
Inventors: 闫瑾; 尤伟名
Original assignee: Shaanxi Wanjia State Yuan Biotechnology Co ltd
Current assignee: Shaanxi Wanjia State Yuan Biotechnology Co ltd
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2023-07-07
Anticipated expiration: 2042-02-25
Also published as: CN114522238A

Abstract

The invention provides a miRNA carrier based on lanthanide series oxyfluoride nanocrystals and application thereof. The invention discloses a miRNA carrier based on lanthanide series oxyfluoride nanocrystals and application of the carrier loaded with anti-tumor miRNA as a miRNA therapeutic drug. The miRNA carrier based on the lanthanide series oxyfluoride nanocrystals can realize high-efficiency load of miRNA and can protect miR-30c from being in cells for more than 24 hours. The anti-tumor miRNA loaded by the carrier is used as an miRNA therapeutic drug, and has good biocompatibility while the functions of miRNA are reserved.

Description

miRNA carrier based on lanthanide series oxyfluoride nanocrystals and application thereof

Technical Field

The invention belongs to the technical field of nanocomposite materials, relates to a drug carrier material, and in particular relates to a miRNA carrier based on lanthanide series oxyfluoride nanocrystals and application thereof.

Background

The Wnt signaling pathway plays a critical role in embryonic development, tissue homeostasis, and stem cell proliferation and differentiation in all animals. However, aberrant activation of this signal is the basis for a variety of human cancers, and deregulating the Wnt signaling pathway causes accumulation of the transcriptional activator β -catenin in the cytoplasm and nucleus, thereby promoting expression of genes associated with tumor cell proliferation and migration. In this process, beta-catenin coactivator (including Bcl 9) is used as part of abnormal activation of Wnt signaling pathway, is over-expressed in various malignant tumors, forms stable complex with beta-catenin, and promotes expression of these oncogenes. Deregulation of the Wnt signaling pathway reduces infiltration of chemotherapeutic drugs and T lymphocytes, resulting in resistance to chemotherapy and PD-1/PD-L1 checkpoint blocked immunotherapy. Inhibiting the inherent activity beta-catenin signal channel of tumor can inhibit the development of tumor, and has sensitization effect on chemotherapy and immunotherapy.

The activated Wnt pathway is a common element that regulates stem/progenitor cell regeneration and maintenance in non-cancerous tissues and organs. Thus, wnt/β -catenin inhibitors are always targeted to toxicity and no drug is currently approved for clinical use. To solve this problem, bcl9, which is highly expressed in tumors but not in cells of tumor origin, has received considerable attention. It has been reported that Bcl9 carcinogenesis can be rescued by siRNA/shRNA-induced gene knockout or treatment with modified Bcl9 peptides, both of which can reduce proliferation, metastasis and resistance to treatment, underscores the importance of Bcl9 in targeted tumor therapy.

MicroRNAs (miRNA) is a gene expression regulating factor involved in the pathogenesis of various tumors, and is expected to create a new therapeutic approach for anticancer therapy. Wherein, a miRNA family named miR-30s is expressed at low level in a large part of cancers, and can regulate the expression of Bcl 9. More importantly, miR-30c has been shown to be effective in inhibiting aberrant expression of Wnt/beta-catenin signaling in malignant cells. However, as with other mirnas, miR-30s also has two major pharmacological drawbacks: the nuclease has poor stability and low membrane permeability, which severely limits its clinical application. Various fine chemicals for miRNA modification and delivery vehicles have been developed and significant progress has been made in improving the pharmacological properties of miRNA clinical therapeutic drugs. However, chemically modified anti-nuclease mirnas are always accompanied by a risk of potentially off-target effects and clearance of the particle system. In addition, commonly used miRNAs carrier lipids and biodegradable polymers are rapidly cleared by the liver and spleen due to the high positive charge, and nonspecific uptake by cells. These drawbacks necessarily prevent the 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. Proper 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, because nanoparticles can actively accumulate through endothelial leakage, known as NanoEL. Recent researches show that rare earth doped nano particles (LDNp) have good surface compatibility and biocompatibility, and can be used as a carrier of a biological molecular medicament.

Disclosure of Invention

The invention aims to provide a miRNA carrier based on lanthanide series oxyfluoride nanocrystals, which is used for loading miRNA so as to solve the problems of poor biocompatibility, insufficient miRNA binding capacity, insufficient intracellular transport capacity of miRNA, poor actual treatment effect and the like of miRNA clinical treatment drugs.

Based on the above-mentioned objects, the present application addresses this need in the art by providing a lanthanide oxyfluoride nanocrystals-based miRNA vector and its use.

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

Furthermore, in the miRNA carrier based on lanthanide series oxyfluoride nanocrystals provided by the invention, the poly-L-lysine and the rare earth doped nanocrystals form coordination bonds.

Further, the diameter of the miRNA carrier particles in the miRNA carrier based on the lanthanide series oxyfluoride nano crystal is 5.2+/-0.4 nm.

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

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

In another aspect, the present invention provides a method of preparing a complex comprising: electrostatically adsorbing miRNA for 30min at 37 ℃ and loading the miRNA on the surface of a miRNA carrier based on lanthanide series oxyfluoride nanocrystals, wherein the ratio of the miRNA to the miRNA carrier based on lanthanide series oxyfluoride nanocrystals is 50:1-400:1 in terms of mass ratio; preferably, the ratio of the miRNA to the miRNA carrier based on the lanthanide series oxyfluoride nanocrystals is 300:1 in terms of mass ratio.

Further, the above-mentioned miRNA vectors include miR-30c.

The invention explores the potential therapeutic effect of lanthanide oxyfluoride nanocrystals (GdOF: ce, tb) as miRNA carriers on tumors by coupling cationic polylysine with the mode of electrostatic bonding of the miRNA carriers. In this proof of concept study, illustratively, miR-30c, a member of the miR-30s family, specifically targets Bcl9 in colorectal cancer, and 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 series oxyfluoride nanocrystals provided by the invention loads miR-30c, the development of tumors is effectively inhibited in vivo, but more importantly, the effect of chemotherapy and immunotherapy is effectively enhanced in vivo. Thus, the 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 has the action mode of inhibiting or blocking Wnt/beta-catenin pathway; the invention further claims a chemotherapeutic or immunotherapeutic agent sensitizer.

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

compared with the prior art, the invention mainly contributes to providing the miRNA carrier based on lanthanide series oxyfluoride nanocrystals, which can realize high-efficiency load of miRNA and protect miR-30c from exceeding 24 hours in cells. The anti-tumor miRNA loaded by the carrier is used as an miRNA therapeutic drug, and has good biocompatibility while the functions of miRNA are reserved. After the carrier is loaded with the anti-tumor miRNA, chemotherapy and immunotherapy are effectively enhanced in vivo.

Drawings

Fig. 1 is a schematic design and application of LnP nanoparticles.

FIG. 2 is Ln, PLL, and LnP nanoparticle characterization; (a) infrared spectrograms of Ln, PLL, and LnP; (B) turbidity of LnP and Ln after polymer modification; (C) And (D) a typical TEM image showing the 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 of different concentrations (100, 200, 300 and 400 μg/mL) of LnP nanoparticles; PEI25K was used as a positive control at a concentration of 12.5ug/mL PEI 25K.

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

FIG. 4 is a laser confocal scanning microscope analysis of transfection efficiency of miR-30c. miR-30c is marked with FAM (green), and the nucleus is marked with DAPI (blue); (A) Fluorescence confocal microscopy images (scale: 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 image. PEI25K and Lipo2000 were positive controls; * P <0.05, P <0.01, all experiments were repeated three times (n=4 per group).

FIG. 5 is the delivery efficiency of LnP nanoparticles at different weight ratios for 24h and miR-30c transfection in MC38 cells; (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 following 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 for protein gray scale analysis; lipo2000 as positive control, beta-actin as 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 different weight ratios of LnP nanoparticles; (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 following 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 protein grayscale analysis. Lipo2000 was a positive control; beta-actin is used as an internal reference protein; * P <0.05, P <0.01.

FIG. 7 is an in vivo tumor inhibiting effect of LnP/miR-30c nanoparticles; (A) Tumor growth curves of MC38 cell engrafted tumor C57 mice after different times of LnP/miR-30C nanoparticle treatment; (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 β -catenin and Cyclin D in subcutaneous tumors; (F) tumor tissue TUNEL staining after 12 days of treatment; * P <0.001; ruler: 20 μm.

FIG. 8 is an in vivo tumor-inhibiting effect of LnP/miR-30c nanoparticles and a tumor-inhibiting effect of chemotherapy; (A) Tumor volumes of MC38 cell engrafted tumor C57 mice after different 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 after 12 days treatment with 5-fluorouracil and LnP/miR-30c nanoparticles, tumor tissue was subjected to H & E (D) and TUNEL (E) analyses; (F) Relative protein expression of β -catenin and Cyclin D in subcutaneous tumors; * P <0.001; ruler: 20 μm.

FIG. 9 is an in vivo tumor-inhibiting effect of LnP/miR-30c nanoparticles and immunotherapy; (A) Tumor volumes of MC38 cell engrafted tumor C57 mice after different times of treatment with PD-L1 and LnP/miR-30C nanoparticles; (B) photographs of MC38 tumors obtained 12 days after treatment; (C) Tumor weight at 12 days, H & E (D) and TUNEL (E) image analysis of tumor tissue after 12 days of PD-L1 and LnP/miR-30c treatment; (F) Relative protein expression of β -catenin and Cyclin D in subcutaneous tumors, < P <0.05, < P <0.01, < P <0.001; ruler: 20 μm.

FIG. 10 is an in vivo biocompatibility evaluation of major organs; mice of the dosing group were stained for heart, liver, spleen, lung, kidney histology HE, scale: 50 μm.

FIG. 11 is a diagram showing the determination of β -catenin and CyclinD bands in subcutaneous tumors by immunoblotting experiments; different treatments (1: control; 2: PD-L1;3: 5-fluorouracil (5-FU) group; 4: lnP/miR-30c;5:5-FU+ LnP/miR-30c;6: PD-L1+ LnP/miR-30 c).

FIG. 12 is a graph of biocompatibility of various complex in vivo treatments; (a) mouse body weight at different time points; (B) body weight of mice at the end of the experiment.

Wherein Ln is rare earth doped nano crystal (GdOF: 45% Ce,15% Tb), lnP is Ln with poly-L-lysine wrapped on the surface, lnP/miR-30c is a compound formed by loading miR-30c with LnP, and PLL is poly-L-lysine.

Detailed Description

The following describes the technical aspects of the present invention with reference to examples, 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 a complex formed by coating poly-L-lysine on the surface of Ln, lnP/miR-30c is LnP and miR-30c is loaded, and PLL is poly-L-lysine.

Example 1

This example provides for the preparation and characterization of LnP.

Preparation of LnP: the synthesis of Ln is the free oleylamine GdOF:45% ce,15% tb, the Ln prepared was redispersed in PBS (ph=7.4) buffer salt. PLL and LDN are coupled in coordination bond via amino groups in the polymer. 5mg of Ln was dissolved in 10mL of PBS buffer containing 10mg of PLL. After incubation for 60min under stirring at 30 ℃, lnP can be collected by centrifugation at 10000 g.

Characterization of LnP: the chemical structures of Ln, PLL and LNP were analyzed by infrared spectroscopy. 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 and Ln, 1659cm ^-1 This is also demonstrated by the significant decrease in the peak infrared absorption characteristic of the amino group at this point (see FIG. 2A). The present example then uses a standard method based on 350nm turbidity measurement for comparative detectionLigand-free Ln (Ln) and PLL-coated Ln (LnP) are water soluble (see fig. 2B). When the pH was 7.4 and the temperature was 37 ℃, the PBS solution became cloudy with increasing nanoparticle concentration. 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, the present example uses Transmission Electron Microscopy (TEM) to observe morphology and monodispersion of Ln and LnP, and particle aggregation phenomenon exists in Ln samples (see fig. 2C). While LnP nanoparticles showed a uniform monodisperse nanostructure, calculated from 100 randomly sampled particles to a diameter of 5.2.+ -. 0.4nm (see FIG. 2D). In addition, the colloidal stability of LnP was also compared to Ln, where LnP remained monodisperse after incubation in PBS (ph 7.4) at 37 ℃ for 1h, whereas Ln appeared to aggregate after 1h in PBS solution (see fig. 2E). Compared to the commercial non-viral vectors PEI25K and Lipo2000, the viability test of MC38 cells found that LnP, which was optimized for hydrophilicity and monodispersity, exhibited better biocompatibility. LnP is cultured for 3d under high concentration of 400 mug/mL, and the cell survival rate can reach more than 100%; in sharp contrast, the cell viability of PEI25K and Lipo2000 at concentrations of 15. Mu.g/mL was around 30% and 70%, respectively, and the biocompatibility of LNP was superior to that of the commercial non-viral vectors PEI25K and Lipo2000.

Example 2

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

And (3) electrostatically adsorbing miR-30c for 30min at 37 ℃, and loading miR-30c onto the nano particles LnP to form a nano particle/miR 30c complex.

Preparation of miR-30c complex: before preparation of miR-30c complex, miR-30c was centrifuged at 12000rpm for 10min and dissolved in fresh DEPC water. LnP and PLL solutions were added to an equal volume of miR-30c solution (0.264 mg/mL), and miR-30c complexes of different specific gravities were calculated. miR-30c complexes were prepared in HEPES environment at 37 ℃ (ph=6.84). Particle sizes and Zeta potentials of various miR-30c complexes were measured by hydrodynamic particle size distribution DLS. All complexes were prepared in 1mL of ultrapure water containing 2.6 μg mir-30c (w/w=12.5 w/w) for differential scanning calorimetry analysis.

Characterization of LnP load miR-30 c: the decrease in Zeta potential confirms successful load of miR-30 onto LnP (see Table 1).

TABLE 1 polydispersity index of polymer and polymer/miRNA complexes

Notably, the positive charge of LnP/miR30c suggests that the complex has an innate advantage of transmembrane transport. Next, this example compares the miRNA load of LnP on PEI25K, which is the strongest commercial non-viral vector for miRNA load on the market, by agarose gel electrophoresis (see fig. 3A). The results showed that LnP showed similar miRNA loadings to PEI25K, differing by the same order of magnitude (see fig. 3B). Next, in order to assess whether miR-30C can be released from LnP/miR-30C complex, competition experiments were performed with heparin, a negatively charged polyanion that mimics intracellular biomacromolecules for miR-30C disproportionation (see figure 3C). As a PEI25K/miR-30c complex, more than 50% of miRNA was released from LnP/miR-30c at a heparin concentration of 60. Mu.g/. Mu.L, indicating that LnP/miR-30c has a similar miRNA release capacity as PEI25K/miR-30c (see FIG. 3D). Naked miRNA is easily degraded by nuclease in vitro and in vivo, so that the degradation resistance of LnP/miR-30c to miRNA is necessary to be verified. More than half of the naked miR-30c was degraded within 1 hour when incubated in serum at 37℃while the miR-30c in LnP/miR-30c remained intact (see FIG. 3E). Importantly, LNP can protect miR-30c for more than 24 hours, at least 75% of miR-30c is intact (see FIG. 3F).

Example 3

This example provides a validation test for the efficient transport of MiR-30c in cells by LnP.

Cell uptake of LnP/miR-30c, PEI25K/miR-30c and Lipo2000/miR-30c was assessed by monitoring the green fluorescence of FAM-labeled miR-30c on a laser confocal microscope (CLSM, FV1200, olympus) (see FIG. 4). To screen LnP: optimal weight ratio of miR-30c to effective internalization of cells, cell uptake of LnP/miR-30c at different weight ratios (50:1-400:1) was first tested in this example. As shown by the laser confocal images (see fig. 4A) and the green fluorescence intensity analysis (see fig. 4B), lnP/miR-30c with the weight ratio of 300:1 had the strongest cell internalization. In addition, LNP/miR-30C (weight ratio 300:1) also showed brightest green fluorescence in MC38 cells, compared to Lipo2000/miR-30C and LNP/miR-30C (see FIG. 4C). These results indicate that LnP is an effective 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 Wnt/beta-catenin passage.

Bcl9 is a transcriptional coactivator of Wnt signaling, playing a role in β -catenin nuclear translocation, and is always overexpressed in tumor cells, thereby promoting tumor progression and therapeutic resistance. According to the structural design of the invention, intracellular miR-30c can inhibit the expression of Bcl9, so that beta-catenin nuclear translocation disorder is caused, and a wnt/beta-catenin channel is blocked later.

To confirm this, the present example first semi-quantitatively examined the expression of miR-30c in LnP/miR-30c and Lipo2000/miR-30c treated MC38 cells using RT-PCR. Consistent with the laser confocal results (see fig. 5), the weight ratio is 300:1, the amount of miR-30c in the LNP/miR-30c cell was the greatest, even exceeding Lipo2000, one of the most potent commercial non-viral vectors (see figure 5A). In LnP/miR-30C-treated cells, bcl9 mRNA levels were statistically significantly down-regulated (see FIG. 5B), with a corresponding decrease in gene expression of C-myc (see FIG. 5C) and CyclinD (see FIG. 5D) in the Wnt downstream signaling pathway. Furthermore, at the protein level, down-regulated β -catenin and Cyclin D fully demonstrated inhibition of Wnt/β -catenin signaling pathway (see fig. 5E and F). Notably, lnP/miR-30c showed a stronger Wnt blocking effect 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 Wnt-highly active human colon cancer cells (see fig. 6). Taken together, these results provide verification that LnP/miR-30c effectively inhibits the Wnt/beta-catenin pathway.

Example 5

The embodiment provides a verification test for inhibiting the Wnt/beta-catenin pathway and tumor growth in LnP/miR-30c bodies.

To verify the excellent performance of LnP/miR-30c in inhibiting Wnt/beta-catenin, the in vivo activity of the compound is further tested in the embodiment. For this purpose, MC38 tumors (50-100 mm ³ ) C57BL/6 mice of (C) were randomly divided into two groups: PBS (control group) and LnP/miR-30c group, two weeks after treatment. As shown in fig. 7A, lnP/miR-30c treated mice had significant tumor inhibition compared to PBS treated control. At the end of the experiment, the tumor mass was dissected and weighed (see fig. 7B and C). The average tumor weight of PBS-treated mice was 4 times that of LnP/miR-30C-treated mice, indicating that LnP/miR-30C had a strong tumor inhibition 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 suggests that this tumor inhibition is due to blockade of Wnt/β -catenin pathway (see fig. 7E), which is again demonstrated by increased apoptosis demonstrated by TUNEL staining (see fig. 7F).

Example 6

The embodiment provides a verification test of the anti-tumor effect of the in vivo sensitization therapy of LnP/miR-30 c.

Chemotherapy is currently the most widely used treatment as a standard treatment for locally advanced and metastatic cancers. However, in some cases, the clinical outcome of chemotherapy is not satisfactory, as tumors often develop chemotherapy resistance. 5-fluorouracil is a first-line chemotherapeutic agent for the treatment of colon cancer, and there is increasing evidence that inhibition of the Wnt/β -catenin signaling pathway can inhibit 5-fluorouracil (5-FU) chemotherapy resistance.

To confirm this, the MC38 transplantation model was again used to combine 5-FU with 5-FU/LNP/miR-30 c. LnP/miR-30C significantly improves the antitumor effect of 5-FU (see FIG. 8A-C). LnP/miR-30c can obviously improve the antitumor 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 performed to investigate changes in protein levels. As shown in FIG. 8F, the protein levels of β -catenin and Cyclin D were significantly reduced in the LnP/miR30c-5-FU combination compared to the 5-FU alone group. LnP/miR-30c combined 5-FU has synergistic antitumor effect.

Example 7

The example provides the anti-tumor effect of LnP/miR-30c in vivo sensitized PD-L1 checkpoint blocked immunotherapy

Binding of PD1 to PD-L1 immunologically inhibits 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 blocking suppressive immune receptors PD-L1 and PD-1 have become a successful therapeutic strategy to increase anti-tumor immune efficiency by releasing T lymphocytes. However, many cancer patients do not respond to PD-1/PD-L1 checkpoint blockade, often due to T cell rejection, a key feature of Wnt activation. Thus, it is contemplated that an effective blockade of the Wnt/β -catenin signaling pathway by LnP/miR-30c may 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 fig. 9A-C, PPI-LnP/miR-30C combination treated mice showed greater tumor inhibition than PPI treated mice after two weeks of treatment. In addition, HE and TUNEL detection results showed that the tumor tissue density was reduced in the co-treatment group and apoptosis was evident (see fig. 9D and E). Furthermore, at the protein level, westernblot gray value analysis showed that β -catenin and Cyclin D levels were significantly reduced in the co-therapy group (see fig. 9F and 11). The results show that LnP/miR-30c has a synergistic effect with PD1/PD-L1 checkpoint blocking immunotherapy.

Example 8

The present example provides an in vivo safety assessment test of LnP/miR-30 c.

Xenograft tumor model: female C57BL/6 mice were purchased from the university of Western An traffic medical school and used at 3 weeks of age. Experimental procedure according to the university of traffic of western America animal Care CommitteeGuidelines of the staff meeting. MC38 cells (3X 10) ⁶ ) Subcutaneous injection into the groin of mice resulted in xenograft tumor models. The shortest and longest diameters of the tumor were measured with a vernier caliper, and the calculation formula was: volume (mm 3) =1/2×length×width ²

Tumor inhibition experiment: tumor volume up to 50mm ³ After left and right, mice were randomly divided into 6 groups and given intraperitoneally, once every two days. Mice body weight and tumor volume were measured. At the last time point, tumors were collected for weighing, measurement and immunohistochemical analysis.

To verify the biosafety of LnP/miR-30c in vivo, the present example performed a comprehensive evaluation of H & E of the major internal organs (see FIG. 10). And body weight of mice after drug treatment (see fig. 12). After 2 weeks of administration, the mice in the LnP/miR-30c alone group, lnP/miR-30c+5-fluorouracil or PD1/PDL1 inhibitor group had significantly increased body weight compared to the control group (PBS), while the 5-fluorouracil treatment group had significantly decreased body weight (P < 0.05) (see FIG. 12). The heart, liver, spleen, lung, kidney histology of the control, lnP/miR-30c, PD/PDL1 inhibitor, lnP/miR-30c+5-fluorouracil or PD1/PDL1 inhibitor group mice were not subject to morphological or pathological changes (see FIG. 10). Only the 5-fluorouracil group has common side effects of chemotherapeutics, 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 chemotherapeutic or immune-assisted therapeutic potentiator.

The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the present invention without departing from the spirit of the design of the present invention.

Claims

1. A method for preparing a lanthanide series oxyfluoride nanocrystal-based miRNA carrier, which is characterized by comprising the following steps: synthesizing rare earth doped nanocrystals by an oleylamine assisted hydrothermal method, removing oleylamine on the surfaces of the rare earth doped nanocrystals by hydrochloric acid with pH of 4.0, and wrapping poly-L-lysine on the surfaces of the rare earth doped nanocrystals;

the poly-L-lysine forms coordination bonds with the rare earth doped nanocrystals;

the rare earth doped nanocrystals are GdOF:45% Ce,15% Tb;

the diameter of the miRNA carrier particles is 5.2+/-0.4 nm.

2. A lanthanide oxyfluoride nanocrystal-based miRNA vector prepared by the preparation method of claim 1;

the rare earth doped nano-crystal comprises a rare earth doped nano-crystal and poly-L-lysine, wherein the poly-L-lysine is wrapped on the surface of the rare earth doped nano-crystal through coordination reaction, and the rare earth doped nano-crystal is GdOF:45% Ce,15% Tb.

3. A complex, wherein the miRNA carrier surface of claim 2 is loaded with miRNA;

the ratio of the miRNA to the miRNA carrier of claim 2 is 50:1-400:1 in terms of mass ratio.

4. A complex according to claim 3, wherein the ratio of miRNA to miRNA vector according to claim 2 is 300:1 in mass ratio.

5. The complex of claim 3, wherein the miRNA comprises miR-30c.

6. A medicament for the treatment of cancer comprising a complex according to claim 3 in a manner that inhibits or blocks the Wnt/β -catenin pathway.

7. A chemotherapeutic or immunotherapeutic agent comprising the complex of claim 3.