Preparation and application of nano system for reversing drug resistance of liver cancer lenvatinib

Abstract

The invention relates to the technical field of biology, and discloses preparation and application of a nano system for reversing the drug resistance of liver cancer lenvatinib, the nano system for reversing the drug resistance of liver cancer lenvatinib is PLGA-PEG (si-HECTD2 # 3) NPs, the nano system comprises a nano carrier PLGA-PEG and a nano drug si-HECTD2 # 3, and the mass ratio of PLGA-PEG to si-HECTD2 # 3 is 20: 1. According to the present invention, the PLGA-PEG (si-HECTD2 # 3) NPs is utilized to target the HECTD2 so as to resist the drug resistance of the lenvatinib in the HCC treatment, such that the new view angle is provided for the understanding of the drug resistance of the HCC on the lenvatinib, and the promising diagnosis and treatment strategy is possibly provided for the clinical treatment of the HCC.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the preparation and application of a nanosystem for reversing lenvatinib resistance in liver cancer. Background Art

[0002] The in vivo delivery of anti-tumor drugs currently still faces many challenges, such as low tumor uptake and low targeting, which have a huge impact on the therapeutic effect of tumors. Nano-delivery systems can improve the penetration, uptake and accumulation of drugs in tumor sites, and achieve controlled release of drugs in tumor extracellular matrix or tumor cells, thereby improving the therapeutic efficacy of tumors. Our study found that HECTD2 is associated with lenvatinib resistance in liver cancer, confirming that HECTD2 can promote lenvatinib resistance in liver cancer. On this basis, we synthesized a nano drug delivery system that can effectively target HECTD2 in vivo. The constructed nanoparticles have good tumor targeting, sustained drug release, safety and no toxic side effects, can increase the therapeutic sensitivity of liver cancer to lenvatinib, and provide a theoretical and translational application basis for HCC treatment. Summary of the invention

[0003] In order to solve the technical problems existing in the prior art, the present invention provides a preparation and application of a nanosystem for reversing lenvatinib resistance in liver cancer.

[0004] The present invention adopts the following technical scheme to achieve: a nanosystem for reversing lenvatinib resistance in liver cancer, wherein the nanosystem is PLGA-PEG (si-HECTD2#3) NPs, and the nanosystem comprises a nanocarrier PLGA-PEG and a nanodrug si-HECTD2#3, wherein the mass ratio of PLGA-PEG to si-HECTD2#3 is 20:1.

[0005] As a further improvement of the above scheme, the si-HECTD2#3 nucleic acid sequence is shown as SEQ ID NO:1.

[0006] A method for preparing a nanosystem for reversing lenvatinib resistance in liver cancer, characterized in that the preparation steps are:

[0007] Step S1, dissolving si-HECTD2#3 in enzyme-free water, and fully mixing it with spermidine at an N / P ratio of 8:1 to form a mixture, and incubating the resulting mixture at room temperature for 15 minutes to form a si-HECTD2#3 spermidine complex;

[0008] Step S2, dissolving 10 mg of PLGA-PEG-COOH in 500 μl of dichloromethane, adding the dichloromethane solution dropwise to the si-HECTD2#3 thiol complex in an ice bath, and then mixing and emulsifying with a probe sonicator to obtain a primary emulsion;

[0009] Step S3, the obtained primary emulsion was further added dropwise to 4 mL of water containing 2.5% polyvinyl alcohol, ultrasonically emulsified with a probe for 1 minute, stirred at room temperature for 4 hours until the organic solvent evaporated, and then centrifuged for 15 minutes to collect NPs, and washed twice with DEPC water to obtain a nano drug delivery system.

[0010] Application of a nanosystem in reversing lenvatinib resistance in liver cancer.

[0011] As a further improvement of the above scheme, the nanosystem PLGA-PEG (si-HECTD2#3) NPs can effectively target HECTD2 and combat lenvatinib resistance in vivo.

[0012] A biological product for reversing lenvatinib resistance in liver cancer, comprising a nanosystem, wherein the nanosystem is PLGA-PEG (si-HECTD2#3) NPs.

[0013] As a further improvement of the above solution, the biological product includes: reagents, test kits, and chips.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. The present invention uses PLGA-PEG (si-HECTD2#3) NPs to target HECTD2 to combat lenvatinib resistance in the treatment of HCC, providing a new perspective for understanding the resistance of HCC to lenvatinib and potentially providing a promising diagnostic and therapeutic strategy for the clinical treatment of HCC. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The preparation and stability analysis of PLGA-PEG (si-HECTD2#3) NPs; wherein:

[0017] Figure 1 A is the finished product image of PLGA-PEG (si-HECTD2#3) NPs;

[0018] Figure 1 B is a graph showing the particle size of PLGA-PEG (si-HECTD2#3) NPs;

[0019] Figure 1 C is the Zeta potential diagram of PLGA-PEG (si-HECTD2#3) NPs;

[0020] Figure 1 D is the release behavior diagram of si-HECTD2#3 from PLGA-PEG (si-HECTD2#3) NPs;

[0021] Figure 1 E is a graph showing the diameter and PDI of PLGA-PEG (si-HECTD2#3) NPs;

[0022] Figure 1 F is a graph showing the particle size and PDI of PLGA-PEG (si-HECTD2#3) NPs in FBS;

[0023] Figure 1 G is a graph showing the particle size and PDI of PLGA-PEG (si-HECTD2#3) NPs in PBS;

[0024] Figure 1 H is a graph showing the particle size and PDI of PLGA-PEG (si-HECTD2#3) NPs in water;

[0025] Figure 2 The targeting analysis spectrum of PLGA-PEG (si-HECTD2#3) NPs; wherein:

[0026] Figure 2 A is a visualization and quantification analysis of the accumulation of Free-DiR and DiR-NPs in the tumor site;

[0027] Figure 2 B is the analysis of the accumulation of Free-DiR and DiR-NPs in the heart, liver, spleen, lung, kidney and tumor;

[0028] Figure 2 C is an analysis of lysosomal escape experiments in HCCLM3 and Huh7 cells;

[0029] Figure 2 D is an analysis of the effect of NPs on the uptake of siRNAs by HCCLM3 cells;

[0030] Figure 2 E is an analysis of the effect of NPs on the uptake of siRNAs by Huh7 cells;

[0031] Figure 3 To analyze the spectrum of the drug resistance of lenvatinib in vivo by targeting HECTD2 using PLGA-PEG (si-HECTD2#3) NPs; wherein:

[0032] Figure 3 A is the in vivo imaging of xenografts treated with saline, PLGA-PEG (si-NC) NPs, free si-HECTD2#3, and PLGA-PEG (si-HECTD2#3) NPs;

[0033] Figure 3B is a photo of tumors treated with saline, PLGA-PEG (si-NC) NPs, free si-HECTD2#3, and PLGA-PEG (si-HECTD2#3) NPs;

[0034] Figure 3 C is the volume curve of tumors in different groups;

[0035] Figure 3 D is the weight graph of tumors in different groups;

[0036] Figure 4 The analytical spectrum of PLGA-PEG (si-HECTD2#3) NPs with no obvious toxicity to major organs; among them:

[0037] Figure 4 A is an analysis diagram of H&E results of heart, liver, spleen, lung, and kidney;

[0038] Figure 4 B is an analysis chart of the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), and blood urea nitrogen (BUN) in serum;

[0039] Figure 5 The analytical profile of the effects of PLGA-PEG (si-HECTD2#3) NPs in the DEN / CCl4-induced HCC mouse model; wherein:

[0040] Figure 5 A is a diagram of the DEN / CCl4-induced HCC mouse model;

[0041] Figure 5 B is a representative photo of tumors in different groups;

[0042] Figure 5 C is an analysis chart of the ratio of liver to body weight;

[0043] Figure 5 D is the analysis diagram of the maximum diameter of tumors in different groups;

[0044] Figure 5 E is a graph showing the survival rate of mice in different groups. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0046] Embodiment 1:

[0047] A nanosystem for reversing lenvatinib resistance in liver cancer, wherein the nanosystem is PLGA-PEG (si-HECTD2#3) NPs, and the nanosystem comprises a nanocarrier PLGA-PEG and a nanodrug si-HECTD2#3, wherein the mass ratio of PLGA-PEG to si-HECTD2#3 is 20:1.

[0048] The si-HECTD2#3 nucleic acid sequence is shown in SEQ ID NO:1.

[0049] A method for preparing a nanosystem for reversing lenvatinib resistance in liver cancer, characterized in that the preparation steps are:

[0050] Step S1, dissolving si-HECTD2#3 in enzyme-free water, and fully mixing it with spermidine at an N / P ratio (ratio of polyamine molecules to siRNA phosphate molecules) of 8:1 to form a mixture, and incubating the resulting mixture at room temperature for 15 minutes to form a si-HECTD2#3 spermidine complex;

[0051] Step S2, 10 mg of PLGA-PEG-COOH (Daigang Bio, Jinan, China) was dissolved in 500 μL of dichloromethane (Aladdin). The dichloromethane solution was added dropwise to the si-HECTD2#3 thiol complex in an ice bath, and then mixed and emulsified using a probe sonicator to obtain a primary emulsion;

[0052] Step S3, the obtained primary emulsion was further added dropwise to 4 mL of water containing 2.5% polyvinyl alcohol (Aladdin), ultrasonically emulsified with a probe for 1 min, stirred at room temperature for 4 h until the organic solvent evaporated, and then centrifuged for 15 min to collect NPs, and washed twice with DEPC water to obtain a nano drug delivery system.

[0053] Application of a nanosystem in reversing lenvatinib resistance in liver cancer.

[0054] The nanosystem PLGA-PEG (si-HECTD2#3) NPs can effectively target HECTD2 and combat lenvatinib resistance in vivo.

[0055] A biological product for reversing lenvatinib resistance in liver cancer, comprising a nanosystem, wherein the nanosystem is PLGA-PEG (si-HECTD2#3) NPs.

[0056] The biological products include: reagents, test kits, and chips.

[0057] Embodiment 2:

[0058] Verify that PLGA-PEG (si-HECTD2#3) NPs targeting HECTD2 can effectively delay lenvatinib resistance in vivo;

[0059] Since naked siRNA is difficult to penetrate the cell membrane, double emulsion solvent diffusion method was used to prepare PEGylated PLGA NPs loaded with si-HECTD2#3 and arginine, named PLGA-PEG (si-HECTD2#3) NPs; the obtained PLGA-PEG was milky white in solution, as shown in Figure 1 As shown in A, the particle size is 139.9nm. Figure 1 As shown in B, the zeta potential is -7.92mV. Figure 1 As shown in C;

[0060] The biodistribution of NPs in vivo was examined by tail vein injection of free DiR dye and DiR-NPs. Figure 1 D and Figure 1 As shown in Figure E, NPs can increase the absorption of drugs by cells; the study also tested the release ability of si-HECTD2#3 from PLGA-PEG (si-HECTD2#3) NPs, such as Figure 1 As shown in D, after 24 hours, the cumulative release of siRNA in free si-HECTD2#3 and PLGA-PEG (si-HECTD2#3) NPs was approximately 80% and 40%, respectively, and the release time could last for about a week; the diameter and polydispersity index (PDI) of PLGA-PEG (si-HECTD2#3) NPs were also determined, as shown in Figure 1 As shown in E;

[0061] To evaluate the stability of PLGA-PEG(si-HECTD2#3) NPs, their particle size and PDI were assessed in FBS, PBS, and water; the results showed that the particle size and PDI of the NPs remained relatively unchanged for more than eight days, indicating that they have excellent stability in vitro, as Figure 1 F- Figure 1 As shown in H;

[0062] like Figure 2 A and Figure 2 As shown in Figure 2B, it was found that NPs could increase the aggregation of DiR at the tumor site, which confirmed the targeting of PLGA-PEGNPs. Courmarin-6NPs were used to study whether NPs could effectively escape from lysosomes. No co-localization fluorescence of NPs and lysosomes was observed, indicating that the presence of NPs facilitated the escape of loaded drugs from lysosomes. Figure 2 As shown in C;

[0063] To detect the cellular uptake of siRNA in vitro, PLGA-PEG(si-HECTD2#3) NPs were labeled with Courmarin-6 and cultured with HCCLM3-LR and Huh7-LR cells.

[0064] Subsequently, PLGA-PEG(si-HECTD2#3)NPs were evaluated in vivo; PDX models were treated by tail vein injection of saline, PLGA-PEG(si-NC)NPs, free si-HECTD2#3, and PLGA-PEG(si-HECTD2#3)NPs; the results showed that tumor growth was significantly inhibited after treatment with PLGA-PEG(si-HECTD2#3)NPs, as shown in Figure 2. Figure 3 A and Figure 3 As shown in B, the volume and weight of xenograft tumors in mice injected with PLGA-PEG(si-HECTD2#3) NPs were significantly reduced. Figure 3 C and Figure 3 As shown in D;

[0065] Finally, we evaluated whether PLGA-PEG(si-HECTD2#3)NPs would cause toxicity to major organs. H&E results showed that intravenous injection of PLGA-PEG(si-HECTD2#3)NPs had no obvious toxicity to major organs including heart, liver, spleen, lung and kidney. Figure 4 As shown in A; In addition, there were no significant differences in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), and blood urea nitrogen (BUN) levels among different groups. Figure 4 As shown in B.

[0066] In addition, a DEN / CCl4-induced HCC mouse model of PLGA-PEG(si-HECTD2#3)NPs was established. Figure 5 As shown in A; compared with the control group, lenvatinib group and PLGA-PEG (si-HECTD2#3) NPs group, PLGA-PEG (si-HECTD2#3) NPs combined with lenvatinib had the most significant inhibitory effect on HCC growth. Figure 5 B- Figure 5 D; In addition, the survival rate of PLGA-PEG (si-HECTD2#3) NPs combined with lenvatinib was the highest, as shown in Figure 5 As shown in E; In conclusion, PLGA-PEG(si-HECTD2#3) NPs can effectively target HECTD2 and combat lenvatinib resistance in vivo.

[0067] Embodiment 3:

[0068] Preparation of PLGA-PEG nanomaterials:

[0069] PLGA-PEG(si-HECTD2#3)NPs were prepared by double emulsion solvent diffusion method; first, si-HECTD2#3 was dissolved in enzyme-free water and thoroughly mixed with spermidine at an N / P ratio (ratio of polyamine molecules to siRNA phosphate molecules) of 8:1; the resulting mixture was incubated at room temperature for 15 minutes to form si-HECTD2#3 spermidine complex; 10 mg PLGA-PEG-COOH (Daigang Bio, Jinan, China) was dissolved in 500 μL dichloromethane (Aladdin); the above dichloromethane solution was added dropwise to the si-HECTD2#3 subthiol complex in an ice bath, and then mixed and emulsified using a probe sonicator; the obtained primary emulsion was further added dropwise to 4 mL of water containing 2.5% polyvinyl alcohol (Aladdin), and emulsified using a probe sonicator for 1 minute; stirred at room temperature for 4 hours until the organic solvent evaporated; then the NPs were collected by centrifugation for 15 minutes and washed twice with DEPC water to obtain the nanomaterial.

[0070] In the present invention, the FDA-approved nanocarrier PLGA was used to synthesize PEGylated PLGA NPs loaded with HECTD2-targeted siRNA, called PLGA-PEG (si-HECTD2#3) NPs; the PLGA-PEG (si-HECTD2#3) NPs were characterized and analyzed, and their biodistribution in vivo was evaluated. By using DiR-NPs, it was found that the NPs can promote accumulation at the tumor site; Courmarin-6-NPs were used, and it was found that the NPs help the loaded drugs escape from lysosomes; in addition, the NPs can also increase the absorption of drugs by cells; the constructed NPs can ensure sustained release for about one week and remain stable in FBS, PBS and water; in order to explore the effect of PLGA-PEG (si-HECTD2#3) NPs in alleviating lenvatinib resistance in vivo, a PDX mouse model derived from HCC patient tissue was used; compared with the saline group, PLGA-PEG (si-NC) NPs group and The volume and weight of transplanted tumors in mice injected with PLGA-PEG(si-HECTD2#3) NPs were significantly reduced compared with the free si-HECTD2#3 group; the biosafety of PLGA-PEG(si-HECTD2#3) NPs was evaluated by H&E sections of heart, liver, spleen, lung and kidney, as well as ALT, AST, CRE and BUN levels in serum; no obvious toxicity of the constructed NPs was observed compared with other groups; PLGA-PEG(si-HECTD2#3) NPs were demonstrated to be a promising candidate drug for alleviating HCC resistance to lenvatinib.

[0071] The present invention utilizes PLGA-PEG (si-HECTD2#3) NPs to target HECTD2 to combat lenvatinib resistance in the treatment of HCC, provides a new perspective for understanding the resistance of HCC to lenvatinib, and may provide a promising diagnostic and therapeutic strategy for the clinical treatment of HCC.

[0072] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A nanosystem for reversing lenvatinib resistance in liver cancer, characterized in that: The nanosystem is PLGA-PEG (si-HECTD2#3) NPs, and the nanosystem comprises a nanocarrier PLGA-PEG and a nanodrug si-HECTD2#3, wherein the mass ratio of PLGA-PEG to si-HECTD2#3 is 20:

1.

2. A nanosystem for reversing lenvatinib resistance in liver cancer according to claim 1, characterized in that: The si-HECTD2#3 nucleic acid sequence is shown in SEQ ID NO:

1.

3. A method for preparing a nanosystem for reversing lenvatinib resistance in liver cancer, characterized in that: The preparation steps are: Step S1, dissolving si-HECTD2#3 in enzyme-free water, and fully mixing it with spermidine at an N / P ratio of 8:1 to form a mixture, and incubating the resulting mixture at room temperature for 15 minutes to form a si-HECTD2#3 spermidine complex; Step S2, dissolving 10 mg of PLGA-PEG-COOH in 500 μl of dichloromethane, adding the dichloromethane solution dropwise to the si-HECTD2#3 thiol complex in an ice bath, and then mixing and emulsifying with a probe sonicator to obtain a primary emulsion; Step S3, the obtained primary emulsion was further added dropwise to 4 mL of water containing 2.5% polyvinyl alcohol, ultrasonically emulsified with a probe for 1 minute, stirred at room temperature for 4 hours until the organic solvent evaporated, and then centrifuged for 15 minutes to collect NPs, and washed twice with DEPC water to obtain a nano drug delivery system.

4. Application of a nanosystem in reversing lenvatinib resistance in liver cancer.

5. Use of a nanosystem as claimed in claim 4 in reversing lenvatinib resistance in liver cancer, characterized in that: The nanosystem PLGA-PEG (si-HECTD2#3) NPs can effectively target HECTD2 and combat lenvatinib resistance in vivo.

6. A biological product for reversing lenvatinib resistance in liver cancer, characterized in that: The biological product comprises a nanosystem, and the nanosystem is PLGA-PEG (si-HECTD2#3) NPs.

7. The biological product according to claim 6, characterized in that: The biological products include: reagents, test kits, and chips.

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