Pancreatic cancer tissue penetrating nano-drug delivery system, preparation method and application thereof

Abstract

The present disclosure belongs to the technical field of biological medicine, and provides a pancreatic cancer tissue penetrating nano drug delivery system, a preparation method and application thereof. The system comprises an RNA nanoflower core (siRFC), a Cas protein and a hyaluronic acid (HA) layer coated on the surface of the siRFC; the siRFC is formed by a first and a second RNA chain; the first RNA chain comprises a plurality of crRNA sequence units connected in series, and the second RNA chain comprises a plurality of siRNA sequence units connected in series; and the Cas protein is anchored in the siRFC by being combined with the crRNA sequence units. The preparation method of the system and the application of the system in preparing a drug for treating pancreatic cancer are also disclosed. By coating the HA layer on the surface of the system, the homophilic affinity mechanism is utilized to significantly improve the penetration ability to tumor stroma. The system can also realize high loading and precise delivery of nucleic acid drugs, and has good biological safety and stability.

Description

Technical Field

[0001] This disclosure relates to the field of biomedical technology, and in particular to a pancreatic cancer tissue-penetrating nanodrug delivery system, its preparation method, and its application. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant digestive system tumor characterized by difficulties in early diagnosis, rapid progression, and extremely poor prognosis. Its dense fibrotic tumor microenvironment and complex gene mutation characteristics pose a core challenge to clinical treatment. The physical barrier formed by excessive deposition of the tumor extracellular matrix (ECM), especially the high-abundance hyaluronic acid (HA) network, severely hinders the penetration of therapeutic drugs, making it difficult for conventional chemotherapy, targeted therapy, and immunotherapy drugs to reach the deep areas of the tumor, thus significantly limiting the therapeutic effect.

[0003] Nucleic acid drugs (such as CRISPR / Cas gene editing systems and siRNA for gene silencing) can precisely regulate tumor-related pathways at the gene level, providing a novel strategy for PDAC treatment. Among them, CRISPR / Cas12a ribonucleoprotein (RNP) can directly achieve targeted gene editing, avoiding the risk of DNA vector integration; siRNA can efficiently silence oncogene expression, and the combined application of the two has synergistic anti-tumor potential. However, nucleic acid drugs are easily degraded by nucleases, have low cellular uptake efficiency, and poor in vivo circulation stability, necessitating safe and efficient nanodelivery systems for in vivo delivery.

[0004] Current nucleic acid nanocarriers mainly include liposomes, polymer nanoparticles, and inorganic nanomaterials. They are mostly loaded with nucleic acid drugs through physical encapsulation and electrostatic adsorption. However, they have the following key defects in PDAC treatment: (1) Insufficient tumor tissue penetration: The particle size and surface properties of traditional nanocarriers are difficult to adapt to the dense ECM environment of PDAC, and they cannot break through the HA matrix barrier. The drug is only enriched in the periphery of the tumor and cannot kill deep tumor cells, which easily leads to tumor residue and recurrence. (2) Poor drug loading and stability: The loading capacity of large molecular complexes such as CRISPR / Cas9 RNP is low, and the binding force between the carrier and the drug is weak. Premature drug leakage is likely to occur in the in vivo circulation, which reduces the therapeutic effect and increases the risk of off-target toxicity. (3) Potential risks to biosafety: Some artificially synthesized carrier materials have poor biocompatibility and have problems such as long-term in vivo toxicity and immunogenicity, which restricts clinical translation and application.

[0005] In summary, developing a nanodelivery system that can efficiently penetrate the PDAC tumor matrix, achieve high loading and precise delivery of nucleic acid drugs, and possess good biosafety is a key technological requirement for overcoming the bottleneck of PDAC gene therapy. Summary of the Invention

[0006] This disclosure provides a pancreatic cancer tissue-penetrating nanodrug delivery system, its preparation method, and its application, in order to at least solve the above-mentioned technical problems existing in the prior art.

[0007] According to a first aspect of this disclosure, a pancreatic cancer tissue-penetrating nanodelivery system is provided, comprising an RNA nanoflower core (siRFC), a Cas protein, and a hyaluronic acid layer coating the surface of the RNA nanoflower core; The RNA nanoflower core is formed by the self-assembly of a first RNA chain and a second RNA chain through complementary base pairing; the first RNA chain includes several tandem crRNA (CRISPR RNA) sequence units, and the second RNA chain includes several tandem siRNA sequence units. The Cas protein is anchored within the core of the RNA nanoflower by specifically binding to the crRNA sequence unit.

[0008] Specifically, this disclosure constructs an RNA nanoflower core with both CRISPR gene editing and RNAi gene silencing functions in one step through base complementation self-assembly of a first RNA strand and a second RNA strand. This structure has a high specific surface area and a multi-branched structure, providing ample sites for Cas protein binding and siRNA release, while avoiding the leakage risk of physical embedding in traditional vectors. This achieves precise co-localization, controllable ratio, and structurally stable integrated loading of two nucleic acid drugs, fundamentally solving the stability and synergy problems of dual-drug co-loading. Secondly, this disclosure utilizes the natural specific binding characteristics of Cas protein and crRNA sequence units to directly anchor the Cas protein to the crRNA site of the RNA nanoflower core, without additional chemical modification or in vitro pre-assembly, achieving in-situ, efficient, and activity-preserving loading of the Cas protein, while ensuring the targeted editing activity of the CRISPR system and significantly reducing off-target risks. Furthermore, this disclosure involves coating the core of the RNA nanoflower with a hyaluronic acid layer. Utilizing the homologous affinity interaction between HA and HA in the tumor matrix, this endows the nanodelivery system with the ability to actively penetrate the dense tumor matrix, achieving highly efficient drug delivery to deep pancreatic cancer tissues and fundamentally overcoming the physical barrier bottleneck of pancreatic cancer. The HA layer also possesses tumor targeting properties (targeting the highly expressed CD44 receptor on the surface of tumor cells), further enhancing the drug accumulation efficiency at the tumor site and reducing systemic toxicity.

[0009] This disclosure utilizes an RNA nanoflower core to achieve a synergistic dual-mechanism approach of CRISPR gene editing (Cas-crRNA) and RNAi gene silencing (siRNA), simultaneously targeting the oncogenic pathways, drug resistance pathways, and stromal microenvironment of pancreatic cancer. This precisely inhibits tumor proliferation, invasion, and metastasis at the gene level. Combined with stromal penetration of the HA layer and tumor targeting, it achieves an integrated therapy of penetration-targeting-dual gene regulation, significantly improving the treatment efficacy of pancreatic cancer. The entire system is constructed based on natural nucleic acids and proteins, exhibiting good biocompatibility and low immunogenicity, and possesses excellent clinical translational potential.

[0010] In one embodiment, the siRNA sequence unit is a hairpin structure, and its two ends contain Dicer enzyme-specific cleavage sites.

[0011] Specifically, Dicer enzyme belongs to the ribonuclease III family of endonucleases. It can specifically recognize and cleave double-stranded RNA and RNA precursors with hairpin structures, producing small interfering RNA (siRNA) of about 21-23 nt in length. It is the core functional enzyme of the RNA interference pathway. After entering the cell, siRNA must be cleaved by Dicer enzyme to become mature siRNA, thereby silencing tumor genes and achieving therapeutic effects.

[0012] In one embodiment, the RNA nanoflower core forms magnesium pyrophosphate (Mg2PPi) crystals during self-assembly, which together constitute the nanoflower morphology.

[0013] In one embodiment, the Cas protein is selected from any one of Cas12a protein, Cas9 protein, Cas12b protein, Cas12c protein, and Cas12e protein.

[0014] According to a second aspect of this disclosure, a method for preparing the above-mentioned nano-drug delivery system is provided, comprising the following steps: S1: Prepare the first and second circular DNAs as templates; S2: The first circular DNA and the second circular DNA are placed in the same rolling circle transcription (RCT) reaction system for rolling circle transcription. During the transcription process, Cas protein is added dropwise so that the Cas protein binds to the crRNA sequence unit on the first RNA chain being synthesized, thereby obtaining the RNA nanoflower core loaded with Cas protein. S3: Mix the RNA nanoflower core with hyaluronic acid to coat the surface of the RNA nanoflower core with hyaluronic acid, thereby obtaining the nano-drug delivery system.

[0015] In one embodiment, step S1, which involves preparing the first circular DNA and the second circular DNA, includes: annealing the first linear DNA template and the second linear DNA template with RCT primers, and then circularizing them with T4 DNA ligase to obtain the first circular DNA and the second circular DNA, respectively.

[0016] Specifically, the first linear DNA template contains, from 5' to 3', an RCT primer binding sequence, several tandem crRNA coding sequences, and a sequence region complementary to the second linear DNA template.

[0017] Specifically, the second linear DNA template contains, from 5' to 3', an RCT primer binding sequence, several tandem siRNA coding sequences, and a sequence region complementary to the first linear DNA template; the RNA encoded by the siRNA coding sequence includes a Dicer enzyme-specific cleavage site and the siRNA sequence.

[0018] In one embodiment, step S2, during the rolling circle transcription, also includes the addition of RNA polymerase, an NTP mixture, and Mg. 2+ And pyrophosphatase inhibitors.

[0019] Specifically, in step S2, during the rolling circle transcription catalyzed by RNA polymerase, NTPs continuously polymerize to form RNA chains, releasing one molecule of pyrophosphate (PPi) for each nucleotide linked. 2+ It is an essential cofactor for RNA polymerase, and high concentrations of PPi and Mg 2+ The molecules bind together and spontaneously form insoluble magnesium pyrophosphate crystals. The generated magnesium pyrophosphate crystals co-precipitate and self-assemble with the RNA chain and the in-situ bound Cas protein, ultimately forming a stable RNA nanoflower core loaded with Cas protein.

[0020] In one embodiment, the reaction conditions for rolling circle transcription in step S2 include: a reaction temperature of 36~38℃ and a reaction time of 4~8h.

[0021] In one possible implementation, step S2 involves transcribing a first RNA chain from a first circular DNA and transcribing a second RNA chain from a second circular DNA.

[0022] In one embodiment, the molecular weight of hyaluronic acid is 10~100kDa, and the concentration is 0.5~2mg / mL.

[0023] In one embodiment, the volume ratio of the RNA nanoflower core to the hyaluronic acid in step S3 is 1:0.5~2, and the mixing time is 20~40 min.

[0024] In one embodiment, step S3 involves electrostatic adsorption or physical coating to allow hyaluronic acid to self-assemble on the surface of the RNA nanoflower core.

[0025] According to a third aspect of this disclosure, the above-described nanodelivery system is provided for use in the preparation of drugs for treating pancreatic cancer.

[0026] In one possible implementation, the pancreatic cancer is pancreatic ductal adenocarcinoma.

[0027] According to one possible implementation of this disclosure, at least the following beneficial effects are achieved: 1. This disclosure significantly enhances the penetration ability of a nanomedicine delivery system by coating its surface with a hyaluronic acid (HA) layer and utilizing the high homology affinity of HA with the overexpressed CD44 receptor in the pancreatic cancer tumor matrix. This design overcomes the technical bottleneck of traditional nanomedicines' inability to penetrate the dense matrix of pancreatic ductal adenocarcinoma, enabling drugs to precisely reach deep regions of the tumor and significantly increasing local drug concentration.

[0028] 2. The nanodelivery system disclosed herein co-delivers a CRISPR-Cas system and siRNA. The Cas protein can specifically cleave a particular pathogenic gene via crRNA-guided cleavage, achieving gene knockout; the co-delivered siRNA can exert its interference function under the action of the Dicer enzyme. The two work synergistically to block pathways related to cancer cell proliferation, invasion, and metastasis from different molecular mechanisms, producing a synergistic therapeutic effect of 1+1>2, effectively inhibiting tumor growth and significantly improving anti-tumor efficacy.

[0029] 3. This disclosure employs rolling circle transcription (RCT) technology, using circular DNA as a template, to simultaneously and efficiently synthesize two long-chain polymeric RNAs in a single reaction system, achieving in-situ self-assembly and efficient loading of Cas proteins. Compared with traditional complex processes such as chemical coupling and layer-by-layer self-assembly, this disclosure eliminates the need for complex subsequent modification steps and offers advantages such as mild reaction conditions, simple operation, low cost, and ease of industrial-scale production.

[0030] 4. The nano-drug delivery system disclosed herein is constructed from biocompatible materials such as RNA, protein, and HA, and is biodegradable in vivo, making it unlikely to produce long-term toxicity. Simultaneously, the HA shell effectively protects the internal Cas protein and RNA from degradation by in vivo enzymes, significantly prolonging their in vivo circulation time, further ensuring therapeutic efficacy and reducing toxic side effects.

[0031] 5. Since pancreatic cancer is prone to multidrug resistance, the gene-gene synergistic therapy strategy disclosed in this paper attacks the core signaling pathways of cancer cells through a dual mechanism, which can effectively overcome the drug resistance problem caused by traditional chemotherapy drugs, and provides a new and broad-spectrum solution for the treatment of refractory pancreatic cancer and other refractory tumors.

[0032] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this disclosure, nor is it intended to limit the scope of this disclosure. Other features of this disclosure will become readily apparent from the following description. Attached Figure Description

[0033] The above and other objects, features, and advantages of this disclosure will become readily apparent from the following detailed description of exemplary embodiments, taken in conjunction with the accompanying drawings. Several embodiments of this disclosure are illustrated in the drawings by way of example and not limitation, in which: In the accompanying drawings, the same or corresponding reference numerals indicate the same or corresponding parts.

[0034] Figure 1 A schematic diagram of the process for preparing a pancreatic cancer tissue-penetrating nanodelivery system according to Embodiment 1 of this disclosure is shown; Figure 2 The diagram shows the PAGE gel electrophoresis results of linear DNA template, circular DNA, and RCT products in Example 1 of this disclosure; wherein, Ladder-Marker; T7 promoter; Template-1 - first linear DNA template; Template-2 - second linear DNA template; Circ-DNA-1 - first circular DNA; Circ-DNA-2 - second circular DNA; RCTProduct-1 - RNA synthesized based on the first circular DNA; RCT Product-2 - RNA synthesized based on the second circular DNA; cRCT Product-1&2 - products co-transcribed by adding the above two circular DNAs simultaneously in the same system; Figure 3 A TEM image of the RNA nanoflower core prepared in Example 1 of this disclosure is shown; Figure 4 The elemental analysis results of the RNA nanoflower core prepared in Example 1 of this disclosure are shown in the figure. Figure 5 A TEM image of the nano-drug delivery system prepared according to Example 1 of this disclosure is shown; Figure 6 The diagram shows the hydration particle size test results of the RNA nanoflower core and the nano-drug delivery system prepared in Example 1 of this disclosure; where a is the RNA nanoflower core and b is the nano-drug delivery system. Figure 7 The diagram shows the test results of the in-situ loading efficiency and the ex-situ loading efficiency of Cas12a protein in Embodiment 1 of this disclosure; wherein, RFS@HA represents ex-situ loading and RFC@HA represents in-situ loading. Figure 8 The diagram shows the results of the in vitro three-dimensional tumor sphere penetration experiment in Embodiment 2 of this disclosure; wherein, the yellow arrow indicates the penetration direction from the surface (0μm) of the tumor sphere to the deeper part; Figure 9 The diagram shows the results of an in vivo tumor tissue penetration experiment in Embodiment 2 of this disclosure; the yellow arrows indicate the penetration direction from the tumor surface to its deep interior. Detailed Implementation

[0035] To make the objectives, features, and advantages of this disclosure more apparent and understandable, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0036] Example 1 This embodiment prepared a nano-drug delivery system that inhibits penetration of pancreatic cancer tissue; the preparation process is as follows. Figure 1 The details are as follows: (1) The first linear DNA template (Tem-RCT-1) and the second linear DNA template (Tem-RCT-2) were designed and synthesized. The sequence of Tem-RCT-1, from 5' to 3', contains the RCT primer binding sequence, several tandem crRNA coding sequences, and a sequence region complementary to Tem-RCT-2; the sequence of Tem-RCT-2, from 5' to 3', contains the RCT primer binding sequence, several tandem siRNA coding sequences (the encoded RNA contains a Dicer enzyme-specific cleavage site and the siRNA sequence (a hairpin structure)), and a sequence region complementary to Tem-RCT-2. Tem-RCT-1 and Tem-RCT-2 were annealed with RCT primers (containing the T7 promoter sequence) and circularized under the action of T4 DNA ligase to obtain the first circular DNA (circDNA-1) and the second circular DNA (circDNA-2), respectively. The successful synthesis of the circular DNA template and the RCT product was confirmed by 12% PAGE gel electrophoresis (see [link to PAGE gel electrophoresis]). Figure 2 ).

[0037] (2) Add circDNA-1 and circDNA-2 (100 nM each) to a container containing T7 RNA polymerase (2 U / μL), NTPs (2.5 mM each), and Mg. 2+ The reaction was carried out at 37°C for 6 hours in RCT reaction buffer containing pyrophosphatase. At the 1st, 2nd, and 3rd hours after the start of the reaction, equal amounts of Cas12a protein solution (total protein amount 10 μg, Cas12a protein derived from Streptococcus pyogenes, ~160 kDa) were added dropwise three times to bind the Cas12a protein to the crRNA sequence unit on the synthesized first RNA strand. After the reaction, the Cas12a protein-loaded RNA nanoflower core (siRFC) was obtained by centrifugation purification, in which Mg2PPi crystals were formed. Transmission electron microscopy (TEM) revealed that the siRFC had a typical nanoflower-like structure (see [link to TEM]). Figure 3 Elemental analysis (HAADF-STEM) confirmed the presence of RNA (P element), Mg2PPi (Mg element), and Cas12a protein (S element) (see [link]). Figure 4 ).

[0038] (3) The siRFC obtained in step (2) was mixed with an equal volume of 1 mg / mL hyaluronic acid (HA, molecular weight 100 kDa) solution and incubated at room temperature for 30 min. HA was then self-assembled onto the surface of siRFC by electrostatic adsorption, resulting in a pancreatic cancer tissue-penetrating nanodelivery system (siRFC@HA). TEM showed that its surface exhibited a blurred coating state (see...). Figure 5 ).

[0039] The hydration particle size of the RNA nanoflower core (siRFC) and the nanodelivery system (siRFC@HA) was characterized using dynamic light scattering, and the results are as follows: Figure 6 As shown. Figure 6 The results showed that the hydrated particle size of siRFC was approximately 520.1 nm; after coating with HA, the hydrated particle size of siRFC@HA increased to 548.5 nm, confirming the successful coating with HA.

[0040] The loading efficiency of Cas12a protein was determined using the BCA protein quantification method. The in-situ loading group involved dropwise addition of Cas12a protein; the ex-situ loading group first synthesized RNA nanoflower cores without Cas12a protein, then simply mixed and incubated with Cas12a protein. The initial amount of Cas12a added to both reaction systems was exactly the same. After the reaction, both samples were ultracentrifuged at 4℃ and 12000rpm for 30 min to precipitate the nanocomposite. The clear supernatant containing unbound free Cas12a protein was carefully aspirated. The concentration of free Cas12a in the supernatant was determined using the BCA method. Combined with the initial total amount of Cas12a protein added, the loading efficiency was calculated using the formula: Loading efficiency = (Initial total protein amount - Supernatant free protein amount) / Initial total protein amount × 100%. The results are shown below. Figure 7 As shown. Figure 7 The results showed that the in-situ loading efficiency of Cas12a protein was as high as ~80.9%, significantly higher than the ex-situ loading efficiency (~37.9%) of mixing siRCF with Cas12a. This demonstrates that the in-situ loading strategy disclosed herein can significantly improve the loading efficiency of Cas12a, providing a guarantee for efficient gene therapy.

[0041] Example 2 This embodiment verifies the tumor tissue penetration performance of the nano-drug delivery system prepared in Example 1.

[0042] 1. In vitro three-dimensional tumor spheroid model penetration experiment: (1) Model construction: Multicellular tumor spheroids (MTS) with a diameter of 1.0 mm were constructed using PANC-1 human pancreatic cancer cells. To simulate the HA-rich ECM of pancreatic cancer, a portion of the MTS was pretreated in HA solution (1 mg / mL) for 24 h.

[0043] (2) MTS were divided into four groups: ① HA pretreated MTS + Cy5 labeled siRFC@HA; ② HA pretreated MTS + Cy5 labeled siRFC; ③ Normal MTS + Cy5 labeled siRFC@HA; ④ Normal MTS + Cy5 labeled siRFC.

[0044] (3) Penetration observation: After each group of MTS and corresponding nanomedicine were co-incubated for 4 hours, a confocal laser scanning microscope (CLSM) was used to perform layer scanning from the tumor surface (Z=0μm) inward to observe the fluorescence signal at different depths (20, 40, 60, 80μm).

[0045] The results are as follows Figure 8 As shown. Figure 8The results showed that strong fluorescence signals were observed from the tumor spheroid surface to a depth of 60 μm only in group ① (HA-pretreated MTS + siRFC@HA). In contrast, the fluorescence signal in group ② (HA-pretreated MTS + siRFC) remained only at the surface (Z=0 μm) and could hardly penetrate. In groups ③ and ④, in normal MTS lacking the HA-mimicking matrix, both siRFC and siRFC@HA could penetrate to a certain depth, but the signal intensity was not as high as in group ①. These results clearly demonstrate that the HA shell of siRFC@HA can overcome the matrix barrier and achieve deep tumor tissue penetration by utilizing its homology affinity with HA in the tumor ECM.

[0046] 2. In vivo tumor tissue penetration experiment: (1) Model: Establish PANC-1 tumor-bearing nude mouse model.

[0047] (2) Administration and observation: Cy5-labeled siRFC@HA and Cy5-labeled siRFC were injected via tail vein. After 24 hours, tumor tissue was removed for frozen sectioning, and the fluorescence distribution of the tumor sections was observed using CLSM.

[0048] The results are as follows Figure 9 As shown. Figure 9 The results showed that strong Cy5 fluorescence signals were observed in tumor sections from the surface to deep within the tumor in mice injected with siRFC@HA. In contrast, the fluorescence signal in tumor sections injected with siRFC was weak and mainly limited to the periphery of the tumor. These results strongly confirm the superior tumor penetration performance of siRFC@HA at the in vivo level.

[0049] It should be understood that the various forms of processes shown above can be used to rearrange, add, or delete steps. For example, the steps described in this disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution of this disclosure can be achieved, and this is not limited herein.

[0050] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this disclosure, "a plurality of" means two or more, unless otherwise explicitly specified.

[0051] The above description is merely a specific embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. A pancreatic cancer tissue-penetrating nanomedicine delivery system, characterized in that, The nanodelivery system includes an RNA nanoflower core, a Cas protein, and a hyaluronic acid layer coating the surface of the RNA nanoflower core. The RNA nanoflower core is formed by the self-assembly of a first RNA chain and a second RNA chain through complementary base pairing; the first RNA chain includes several tandem crRNA sequence units, and the second RNA chain includes several tandem siRNA sequence units. The Cas protein is anchored within the core of the RNA nanoflower by specifically binding to the crRNA sequence unit.

2. The nano-drug delivery system according to claim 1, characterized in that, The siRNA sequence unit is a hairpin structure, and its two ends contain Dicer enzyme-specific cleavage sites.

3. The nano-drug delivery system according to claim 1, characterized in that, The Cas protein is selected from any one of Cas12a, Cas9, Cas12b, Cas12c, and Cas12e proteins.

4. The method for preparing the nano-drug delivery system according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1: Prepare the first and second circular DNAs as templates; S2: The first circular DNA and the second circular DNA are placed in the same rolling circle transcription reaction system for rolling circle transcription. During the transcription process, Cas protein is added dropwise so that the Cas protein binds to the crRNA sequence unit on the first RNA chain being synthesized, and the RNA nanoflower core loaded with Cas protein is obtained. S3: Mix the RNA nanoflower core with hyaluronic acid to coat the surface of the RNA nanoflower core with hyaluronic acid, thereby obtaining the nano-drug delivery system.

5. The preparation method according to claim 4, characterized in that, Step S1, which involves preparing the first circular DNA and the second circular DNA, includes annealing the first linear DNA template and the second linear DNA template with RCT primers, and then circularizing them with T4 DNA ligase to obtain the first circular DNA and the second circular DNA, respectively. The first linear DNA template contains, from 5' to 3', an RCT primer binding sequence, several tandem crRNA coding sequences, and a sequence region complementary to the second linear DNA template. The second linear DNA template contains, from 5' to 3', an RCT primer binding sequence, several tandem siRNA coding sequences, and a sequence region complementary to the first linear DNA template; the RNA encoded by the siRNA coding sequence includes a Dicer enzyme-specific cleavage site and the siRNA sequence.

6. The preparation method according to claim 4, characterized in that, In step S2, during the rolling circle transcription, RNA polymerase, an NTP mixture, and Mg are also added. 2+ And pyrophosphatase inhibitors.

7. The preparation method according to claim 4, characterized in that, The reaction conditions for rolling circle transcription in step S2 include: a reaction temperature of 36~38℃ and a reaction time of 4~8h.

8. The preparation method according to claim 4, characterized in that, The hyaluronic acid has a molecular weight of 10~100kDa and a concentration of 0.5~2mg / mL.

9. The preparation method according to claim 4, characterized in that, In step S3, the volume ratio of the RNA nanoflower core to the hyaluronic acid is 1:0.5~2, and the mixing time is 20~40 min.

10. The use of the nanodelivery system according to any one of claims 1 to 3 in the preparation of a drug for treating pancreatic cancer.

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