Lipid nanoparticle and preparation method and application thereof

Abstract

The application belongs to the field of biological medicine and nano delivery technology, and particularly relates to a kind of lipid nanoparticles and its preparation method and application.The pancreas targeted lipid nanoparticles of the application contain lipid components and encapsulated nucleic acids;The lipid components contain ionizable lipids, auxiliary phospholipids, sterol components and PEG-lipids.The pancreas selectivity of the lipid nanoparticles of the application is high, and the pancreas radiation ratio is stably above 90%;And the treatment is widely used, and the treatment effect is verified in three application scenarios of acute pancreatitis treatment, pancreatic cancer gene editing and mRNA tumor vaccine, and has broad clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanodelivery technology, specifically relating to a lipid nanoparticle, its preparation method, and its application. Background Technology

[0002] mRNA therapy holds great promise for treating infectious diseases, inflammation, cancer, and genetic disorders. Its clinical success depends on delivery vectors that can safely and efficiently transport mRNA to target tissues. Lipid nanoparticles (LNPs) are currently the most mature non-viral delivery system in clinical application and have been widely validated in COVID-19 vaccines.

[0003] However, existing LNP technologies have the following core defects, which restrict RNA therapy for pancreatic diseases: (1) Strong liver tropism: After systemic administration (intravenous injection) or intraperitoneal injection, existing LNPs accumulate in the liver due to strong interactions with apolipoprotein E (ApoE), low-density lipoprotein receptor (LDLR), type I scavenger receptor (SR-B1), and Kupffer cells, and cannot reach an effective therapeutic concentration in the pancreas. (2) Difficulty in pancreatic targeting: The pancreas is located deep in anatomy, has a special blood supply, and is surrounded by abundant connective tissue, which constitutes a natural barrier to nanomedicine delivery. Although existing pancreatic-targeting LNP strategies (such as organ membrane filtration mechanisms that rely on particle enlargement) have made breakthroughs, they generally suffer from insufficient specificity, poor cross-species conservation, and complex preparation processes. (3) Lack of receptor targeting mechanism: Most existing LNPs rely on passive accumulation or non-specific uptake and lack an active targeting mechanism mediated by pancreatic cell-specific receptors, resulting in high off-target expression and a narrow therapeutic window. (4) Limited translational potential: Most existing pancreatic targeting strategies have only been validated in mice, and their cross-species extrapolation is unknown, which limits their clinical translation prospects.

[0004] Therefore, there is an urgent need in this field for an mRNA delivery platform with a clear targeting mechanism, high pancreatic selectivity, simple preparation, and cross-species extrapolation capability for the precise RNA therapy of pancreatic diseases such as pancreatitis and pancreatic ductal adenocarcinoma (PDAC). Summary of the Invention

[0005] This invention provides pancreatic-targeting lipid nanoparticles, wherein the lipid nanoparticles comprise lipid components and encapsulated nucleic acids; the lipid components comprise ionizable lipids, cofactor phospholipids, sterol components, and PEG-lipids.

[0006] In some embodiments, the ionizable lipid comprises one or more of SM102, C12-200, CCK-E12, 306Oi10, ALC-0315, and MC3.

[0007] In some embodiments, the auxiliary phospholipid comprises one or more of DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine), DSPC, DOPC, and DPPC.

[0008] In some embodiments, the sterol component includes bile acid derivatives. In some specific embodiments, the bile acid derivatives include one or more of glycodeoxycholic acid (GDCA), CDCA, TCA, and TUDCA.

[0009] In some embodiments, the PEG-lipids include DMG-PEG2000, ALC-0159, and DSPE-PEG2000.

[0010] In some embodiments, the nucleic acid includes mRNA and sgRNA. In some specific embodiments, the mRNA includes therapeutic mRNA, gene-editing mRNA, or vaccine mRNA.

[0011] In some embodiments, the molar ratio of the lipid components is ionizable lipids: co-phospholipids: sterol components: PEG-lipids = 35~50: 10~16: 38~48: 1~3.

[0012] In some specific embodiments, the molar ratio of the lipid components is ionizable lipid: co-phospholipid: sterol component: PEG-lipid = 40:10:48:2, 35:16:46.5:2.5 or 50:10:38.5:1.5.

[0013] In some embodiments, the mass ratio of the lipid component to the nucleic acid is 5–15:1–4. In some specific embodiments, the mass ratio of the lipid component to the nucleic acid is 10:1.

[0014] The present invention also provides a method for preparing the lipid nanoparticles, wherein the lipid nanoparticles are prepared by microfluidic methods.

[0015] In some embodiments, the preparation of the lipid nanoparticles includes the following steps: (1) Preparation of the ethanol phase: Dissolve the lipid components in anhydrous ethanol; (2) Preparation of aqueous phase: Dissolve the nucleic acid in citrate buffer; (3) Hybridization: The aqueous phase and the ethanol phase are rapidly mixed through a microfluidic chip or a channel to form lipid nanoparticles through self-assembly. (4) Dialysis purification: The prepared lipid nanoparticles are dialyzed to remove ethanol and free components.

[0016] In some embodiments, the ratio of the aqueous phase to the ethanol phase in step (3) is 2 to 4:1 (v / v). In some specific embodiments, the ratio of the aqueous phase to the ethanol phase in step (3) is 3:1 (v / v).

[0017] This invention also provides the use of the lipid nanoparticles in the preparation of medicaments for treating diseases. In some embodiments, the diseases include pancreatic diseases. In some specific embodiments, the pancreatic diseases include pancreatitis and pancreatic cancer.

[0018] In some implementations, the use includes any of the following: (1) The nucleic acid is IL-1 receptor antagonist (IL-1RA) mRNA, and the disease is acute pancreatitis; or (2) The nucleic acid is Cas9 mRNA and sgRNA targeting the KrasG12D mutation site, and the disease is pancreatic cancer; or (3) The nucleic acid is a mutated KrasG12D or KrasG12V neoantigen mRNA, and the disease is pancreatic cancer.

[0019] In some embodiments, the method of administration of the drug includes intraperitoneal injection, intravenous injection, or subcutaneous administration.

[0020] Compared with traditional cholesterol LNP, the lipid nanoparticles of the present invention have the following targeting advantages: (1) Rapid pancreatic accumulation: Within 600 seconds after intraperitoneal injection, the LNP signal rapidly concentrates from the peritoneum to the pancreatic tissue, and the percentage of pancreatic injection dose (%ID) exceeds 5% at 10 minutes, which is significantly higher than Chol-LNP (<2.5%); (2) High pancreatic selectivity: The pancreatic radiation intensity accounts for >90% of the total organ radiation (Chol-LNP is about 50%), achieving an increase of about 15 times in the pancreatic / liver expression ratio; (3) Low protein crown: Compared with Chol-LNP, the lipid nanoparticles of the present invention adsorb less total protein and more homogeneous protein (mainly albumin) in the ascites environment, which is beneficial to maintain particle targeting and reduce immune clearance.

[0021] The present invention also provides a pancreas-targeting human or veterinary pharmaceutical composition, the composition comprising the lipid nanoparticles and other excipients.

[0022] In some embodiments, the veterinary drug includes drugs for rats, rabbits, pigs, and non-human primates.

[0023] The beneficial effects of this invention are: (1) The lipid nanoparticles of the present invention have high pancreatic selectivity and the pancreatic radiation ratio is stable at over 90%, which is significantly better than the similar products reported so far (4A2-B8-pH: 78.8%, 4A2-B8-C10: 74.7%, Chol-LNP: about 50%). Moreover, they have a wide range of therapeutic applications. The therapeutic effects have been verified in three application scenarios: acute pancreatitis treatment, pancreatic cancer gene editing, and mRNA tumor vaccine. They have broad clinical application prospects.

[0024] (2) The lipid nanoparticles of the present invention have a wide dose range, and pancreatic selectivity remains stable (>89%) in a wide dose range from 0.125 mg / kg to 1.0 mg / kg, providing sufficient flexibility for clinical dose optimization.

[0025] (3) The lipid nanoparticles of the present invention have good effects in different species: >90% pancreatic delivery specificity was verified in C57BL / 6 mice, BALB / c mice, hamsters, SD rats, rabbits, miniature pigs and cynomolgus monkeys (non-human primates). Functional mRNA expression (hEPO protein) was achieved in cynomolgus monkeys. The safety was good (liver, kidney and pancreatic function indicators were normal). Attached Figure Description

[0026] Figure 1 This study presents a screening scheme for LNP formulations and the chemical structure of the cholic acid used in LNP formulation screening.

[0027] Figure 2 To measure the particle size distribution, zeta potential, and polydispersity index (PDI) of LNPs formulated with bile acids by dynamic light scattering (DLS).

[0028] Figure 3 Encapsulation efficiency of LNPs prepared for bile acids.

[0029] Figure 4 Stability study of GDCA-LNP at 4℃ for different storage times.

[0030] Figure 5 Representative cryo-electron microscopy images of Chol-LNP and GDCA-LNP.

[0031] Figure 6 Representative bioluminescence imaging of LNPs 6 hours after intraperitoneal injection of different bile acids.

[0032] Figure 7 Representative bioluminescence imaging of LNPs 6 hours after intraperitoneal injection of different ionizable lipid replacements.

[0033] Figure 8 Representative bioluminescence imaging of LNPs 6 hours after intraperitoneal injection of different helper phospholipid replacements.

[0034] Figure 9 Representative bioluminescence images of LNPs with different lipid ratios 6 hours after intraperitoneal injection.

[0035] Figure 10 BLI radioactivity analysis 6 hours after intraperitoneal injection of Chol-LNP and GDCA-LNP.

[0036] Figure 11 To analyze the distribution of LNPs encapsulating mCherry mRNA in different cell subpopulations of the mouse pancreas by flow cytometry.

[0037] Figure 12 Serum amylase and lipase levels after LNP treatment with mIL-1RA mRNA-encapsulated protein in a mouse model of acute pancreatitis.

[0038] Figure 13 MPO levels in serum of mice treated with LNPs containing mIL-1RA mRNA in an acute pancreatitis model.

[0039] Figure 14 H&E staining scores of pancreatic tissue pathology after LNP treatment encapsulating mIL-1RA mRNA in a mouse model of acute pancreatitis.

[0040] Figure 15 TUNEL staining score of pancreatic tissue after LNP treatment encapsulating mIL-1RA mRNA in a mouse model of acute pancreatitis.

[0041] Figure 16 Survival rate of mice with acute pancreatitis after treatment with LNP containing mIL-1RA mRNA.

[0042] Figure 17 Evaluation of the safety parameters ALT and AST after LNP treatment encapsulating mIL-1RA mRNA in a mouse model of acute pancreatitis.

[0043] Figure 18 GDCA LNP is used to deliver CRISPR / Cas9 mRNA carrying sgRNA targeting the Kras gene, as well as Kras gene insertion / deletion (indel) mutations detected by T7E1 in different organs.

[0044] Figure 19 Survival curves of KPC mice in the saline group, Chol-LNP group and GDCA-LNP group after receiving specified treatment were obtained by administering the Cas9 editor at P30 and P45 before tumor formation.

[0045] Figure 20 The timeline for KrasG12D mRNA lipid nanoparticle (LNP) vaccine treatment of an orthotopic KPC model is shown, specifying the survival curves of mice carrying KPC tumors after treatment.

[0046] Figure 21 The ELISpot assay is used to quantitatively determine the number of human interferon-γ (IFN-γ) dot cells formed by human spleen cells after in vitro restimulation with mixed tumor antigens.

[0047] Figure 22 Survival curves of humanized pancreatic cancer-bearing mice treated with Chol-LNP and GDCA-LNP.

[0048] Figure 23 The results show the results of Masson's trichrome staining (MTS) and α-smooth muscle actin (α-SMA) immunofluorescence of pancreatic carcinoma in situ (PDAC) tissue from the representative treatment group, as well as the quantification of MTS and α-SMA positive regions in each field of view.

[0049] Figure 24 The results of quantitative analysis of the M1 / M2 ratio, CD4+% and CD8+% in CD3+ T cells, Foxp3+ Treg in CD4+ T cells, GZMB in CD8+ T cells, IFN-γ in CD8+ T cells, and IFN-γ in CD4+ T cells in PDAC mice after treatment.

[0050] Figure 25 Representative BLI images of rabbits treated with GDCA-LNP-mLuc 3 hours later, and quantitative analysis results of mean radioactivity and radioactivity ratio of different organs.

[0051] Figure 26 To conduct a quantitative biosafety analysis of rabbits 24 hours after injection of GDCA-LNP or SM102-LNP (Luc mRNA 0.5 mg / kg), the following indicators were measured: amylase, lipase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin-6 (IL-6), tumor necrosis factor-α (TFN-α), creatinine (CREA), erythrocytes (RBC), hematocrit (HCT), erythrocyte distribution width coefficient of variation (RDW-CV), hemoglobin (HGB), and white blood cells (WBC). Hematoxylin and eosin (H&E) stained sections of different organs were also analyzed 24 hours post-treatment.

[0052] Figure 27 Representative BLI images of miniature pigs treated with GDCA-LNP-mLuc three hours later, along with quantitative analysis of mean radioactivity and radioactivity ratios in different organs.

[0053] Figure 28 Light sheet imaging of a miniature pig pancreas 3 hours after delivery of GDCA-LNPs containing Cy5-tagged mRNA.

[0054] Figure 29 Representative H&E-stained sections of pancreatic tissue 24 hours after delivery of different doses of GDCA-LNP to miniature pigs.

[0055] Figure 30 To conduct quantitative biosafety analysis of ALT, AST, ALP, CRE, amylase and TNF-α in miniature pigs treated with GDCA-LNP.

[0056] Figure 31 This study aimed to conduct quantitative biosafety analyses on hEPO expression and weight changes in cynomolgus monkeys treated with GDCA-LNP-mhEPO for 14 days, as well as on ALT, AST, CRE, amylase, lipase, erythrocyte count, hematocrit, erythrocyte distribution width coefficient of variation, hemoglobin, reticulocytes, leukocytes, neutrophils, lymphocytes, and monocytes.

[0057] Figure 32 To perform BLI organ imaging of cynomolgus monkeys 6 hours after injection of GDCA-LNP-mLuc, and to quantitatively analyze the total radiation and radiation ratio of different organs of cynomolgus monkeys.

[0058] Figure 33 Representative H&E images of the pancreas of cynomolgus monkeys after injection of GDCA-LNP-mLuc.

[0059] Figure 34 Immunohistochemical images of representative Flux cells from different cell populations in the pancreas of cynomolgus monkeys after injection of GDCA-LNP-mLuc.

[0060] Figure 35 Quantitative analysis of FLuc immunohistochemical (IHC) signals in different pancreatic regions. Detailed Implementation

[0061] Example 1: Screening of bile acid derivatives in LNP lipid fractions 1. LNP preparation (1) Ethanol phase: SM102, DOPE, GDCA and DMG-PEG2000 were dissolved in anhydrous ethanol at a molar ratio of SM102 / DOPE / GDCA / DMG-PEG2000 = 40 / 10 / 48 / 2.

[0062] (2) Aqueous phase: Dissolve Luc mRNA in 100 mM citrate buffer (pH 4.0).

[0063] (3) Mixing: The aqueous phase and the ethanol phase are mixed at a ratio of 3:1 (v / v) through a microfluidic chip, so that the total lipid content to mRNA mass ratio is 10:1.

[0064] (4) Dialysis: Dialyze with PBS (1×) for ≥2 hours (MWCO 3.5 kDa).

[0065] (5) Volume adjustment: Dilute with PBS to the working concentration (in vivo experiment: 100 μL / mouse) to prepare GDCA-LNP.

[0066] Using the same method described above, except that glycodeoxycholic acid (GDCA) was replaced with cholesterol (Chol) and other bile acid derivatives: DDCA, GCDCA, TCA, OCAG, GCA, HDCA, 6-EDCA, CA, GUDCA, ACA, DCA, LCA, UDCA, and CDCA, Chol-LNP, DDCA-LNP, GCDCA-LNP, TCA-LNP, OCAG-LNP, GCA-LNP, HDCA-LNP, 6-EDCA-LNP, CA-LNP, GUDCA-LNP, ACA-LNP, DCA-LNP, LCA-LNP, UDCA-LNP, and CDCA-LNP were prepared. The difference in composition between Chol-LNP and GDCA-LNP is as follows: Figure 1 As shown.

[0067] 2. Physicochemical characterization (1) Particle size determination The particle size of the prepared Chol-LNP, GDCA-LNP, DDCA-LNP, GCDCA-LNP, TCA-LNP, OCAG-LNP, GCA-LNP, HDCA-LNP, 6-EDCA-LNP, CA-LNP, GUDCA-LNP, ACA-LNP, DCA-LNP, LCA-LNP, UDCA-LNP, and CDCA-LNP was determined using DLS. Specifically, the prepared LNP samples were subjected to particle size, zeta potential, and PDI measurements at 25°C using a ZetaSizer 7.11 (Nano ZS90, Malvern Instruments) to confirm the uniformity of the prepared LNPs. The results are as follows: Figure 2 As shown, the GDCA-LNP has a particle size of approximately 174 nm, a PDI < 0.2, and a Zeta potential of approximately 5 mV (neutral pH).

[0068] (2) mRNA encapsulation efficiency determination The mRNA encapsulation efficiency was determined using the Ribogreen method, and the results are as follows: Figure 3As shown, the mRNA encapsulation rate in GDCA-LNP is >80%.

[0069] (3) Determination of the stability of nanoparticles The prepared GDCA-LNP was stored at 4°C for at least 6 days, and then administered to mice on days 0, 3, and 6 to confirm the expression of Fluc mRNA in mice. Results are as follows: Figure 4 As shown, GDCA-LNP maintains stable performance for at least 6 days.

[0070] (4) Cryo-electron microscopy (cryo-EM) confirmed the morphology of nanoparticles Cryo-electron microscopy images of nanoparticles were obtained using a cryo-transmission electron microscope (FEI, Talos F200C200kV) at the Cryo-Electron Microscopy Center of Zhejiang University. The results are as follows: Figure 5 As shown, GDCA-LNP particles are uniform in size and exhibit a regular, multi-layered, spherical shape.

[0071] (5) Determination of delivery efficiency and targeting efficiency in mice The LNPs prepared above were administered to mice. Six hours later, the mice were injected intraperitoneally with potassium fluorescein. Five minutes later, the mice were dissected, and major organs were removed. The bioluminescence intensity of the removed organs was collected using a live animal imaging system. The delivery efficiency and targeting efficiency at the pancreas site were calculated based on the bioluminescence intensity. The results are as follows: Figure 6 As shown, GDCA-LNP exhibits superior delivery and targeting efficiency.

[0072] Example 2 Screening of ionizable lipids Using the same method as in Example 1, keeping the components DOPE, GDCA, and PEG-2000 in the LNP lipid fraction unchanged, only the ionizable lipid SM102 was replaced with C12-200, CCK-E12, 5A2-SC8, and FTT5, respectively. The delivery efficiency and targeting efficiency of different LNPs in mice were measured. The results are as follows: Figure 7 As shown, when the ionizable lipid is selected as SM102, both the LNP delivery efficiency and targeting efficiency are superior.

[0073] Example 3: Screening of Assisted Phospholipids Using the same method as in Example 1, while keeping the components SM102, GDCA, and PEG-2000 in the LNP lipid fraction unchanged, the helper phospholipid was changed from DOPE to DSPC. The delivery efficiency and targeting efficiency of different LNPs in mice were measured. The results are as follows: Figure 8 As shown, when the auxiliary phospholipid is DOPE, both the LNP delivery efficiency and targeting efficiency are superior.

[0074] Example 4 Screening of lipid ratios Using the same method as in Example 1, LNPs were prepared with the molar ratios of the components in the LNP lipid fraction being SM102:DOPE:GDCA:DMG-PEG2000 = 40:10:48:2, 35:16:46.5:2.5, or 50:10:38.5:1.5. The delivery efficiency and targeting efficiency of different LNPs in mice were measured. The results are shown in the table below. Figure 9 As shown, the LNP delivery efficiency and targeting efficiency are both superior when the lipid ratio is selected as 35:16:46.5:2.5.

[0075] Example 5: Verification of pancreatic targeting via intraperitoneal administration in mice 1. Experimental Design C57BL / 6 mice (6–8 weeks old) were intraperitoneally injected with either GDCA-LNP or Chol-LNP (both loaded with luciferase mRNA, 0.125 mg / kg), and in vivo bioluminescence imaging (IVIS) was performed 3–6 hours after injection.

[0076] 2. Results like Figure 10 As shown, in the Chol-LNP group, the pancreas was irradiated by approximately 50% and the liver by approximately 30%; in the GDCA-LNP group, the pancreas was irradiated by ≥90%, while the liver, spleen, and other organs were irradiated by <10%. The pancreas / liver expression ratio in the GDCA-LNP group was approximately 15 times that in the Chol-LNP group.

[0077] like Figure 11 As shown, mice were intraperitoneally injected with GDCA-LNP or Chol-LNP (both loaded with mCherry mRNA, 0.125 mg / kg), and the pancreas was dissected 24 h later. The pancreas was prepared into a single-cell suspension and the distribution of mCherry signals in different cell subpopulations of the mouse pancreas was analyzed by flow cytometry. Flow cytometry confirmed that GDCA-LNP preferentially transfected acinar cells (73.3% Cy5+) and ductal cells (64.8% Cy5+).

[0078] Example 6: IL-1RA mRNA Treatment of Acute Pancreatitis 1. Model Building Acute pancreatitis was induced by intraperitoneal injection of L-arginine (L-Arg). L-Arg was prepared as a 20% (w / w) L-Arg solution with physiological saline, and the pH was adjusted to approximately 7 with concentrated hydrochloric acid. Mice were administered 20% L-Arg (4 g / kg) intraperitoneally twice, with a 1-hour interval. Pathological scoring was performed using the modified pancreatitis scoring system of Schmidt et al., with edema, acinar necrosis, hemorrhage / fat necrosis, inflammatory infiltration, and perivascular inflammation scored from 0 to 4 points. A score meeting the pathological criteria indicated successful model establishment.

[0079] 2. Dosing regimen Using the same method as in Example 1, Luc mRNA was replaced with IL-1RA mRNA (Gene ID: 100136091, https: / / www.ncbi.nlm.nih.gov / gene / 100136091) to prepare Chol-LNP or GDCA-LNP loaded with IL-1RA mRNA. Three hours before induction of acute pancreatitis, Chol-LNP or GDCA-LNP (IL-1RA mRNA, 0.5 mg / kg) was administered intraperitoneally.

[0080] 3. Main therapeutic indicators and results (1) Marker detection Seventy-two hours after induction of acute pancreatitis, blood was collected via ocular sampling. Whole blood was allowed to stand for 15 minutes, then centrifuged to obtain serum. Serum amylase was detected using the iodine-starch colorimetric method, lipase (a marker of acinar cell damage) was detected using an enzyme colorimetric method, and serum MPO (a marker of neutrophil inflammation) was detected using an ELISA method. Results are as follows: Figure 12 As shown, serum amylase and lipase levels in the GDCA-LNP group were significantly lower than those in the saline and Chol-LNP groups (P<0.05); Figure 13 As shown, serum MPO was also significantly reduced in the GDCA-LNP group.

[0081] (2) Histopathological score Seventy-two hours after induction of acute pancreatitis, the pancreas of mice with pancreatitis was dissected, and sections were prepared by paraffin embedding and H&E staining. Histopathological scoring was performed by analyzing the degree of tissue damage in the sections. Results are as follows: Figure 14 As shown, edema, acinar necrosis, hemorrhage and inflammatory infiltration were significantly reduced in the GDCA-LNP group (P<0.05).

[0082] (3) TUNEL staining Seventy-two hours after induction of acute pancreatitis, the pancreas of mice with pancreatitis was dissected, and TUNEL staining was performed on paraffin-embedded sections. Fluorescence intensity was analyzed using ImageJ, and the results are as follows: Figure 15 As shown, apoptosis of acinar cells was significantly reduced in the GDCA-LNP group.

[0083] (4) Survival rate The survival status of mice with acute pancreatitis was recorded within 72 hours after induction of acute pancreatitis, and the results are as follows: Figure 16 As shown, the survival rate of the GDCA-LNP group was significantly higher than that of the control group (log-rank, P<0.05).

[0084] (5) Safety Twenty-four hours after drug administration, blood was collected via ocular sampling. After the whole blood was allowed to stand for 15 minutes, it was centrifuged, and the supernatant serum was collected. ALT and AST were measured using ELISA. The results are as follows: Figure 17 As shown, no significant increases in ALT and AST were observed, indicating no obvious liver toxicity.

[0085] Example 7: Kras gene editing and tumor prevention in KPC mice 1. Experimental subjects The KPC mouse (LSL-KrasG12D; LSL-Trp53R172H; Pdx1-Cre, a spontaneous pancreatic cancer model) is a successfully established mouse model of pancreatic ductal adenocarcinoma. The KPC mouse's P53 gene contains a dominant repressive point mutation (TP53R172H), and the KRAS gene contains a conditional activation point mutation (KRASG12D). The upstream of the KRAS mutant gene contains a lox-stop-lox termination sequence and is not expressed in the absence of Cre recombinase. After ligating Cre recombinase to the PDX1 promoter, it is expressed in the acini, islets, and ducts of the pancreas. Therefore, the pancreas of KPC mice gradually expresses KRASG12D protein after birth, leading to tumor development.

[0086] 2. Dosing regimen Using the same method as in Example 1, Luc mRNA was replaced with Cas9 mRNA (SEQ ID NO:1) and sgRNA targeting KrasG12D (SEQ ID NO:2), with a Cas9 / sgRNA mass ratio of 3:2, to prepare GDCA-LNP-Cas9-KrasG12D and Chol-LNP-Cas9-KrasG12D, respectively.

[0087] On days 30 (P30) and 37 (P37) after birth, KPC mice were intraperitoneally injected with GDCA-LNP-Cas9-KrasG12D, Chol-LNP-Cas9-KrasG12D (total RNA 1.0 mg / kg), or physiological saline, respectively.

[0088] 3. Results (1) T7E1 detection Mice administered the drug were dissected on day 37, and genomic DNA was extracted from pancreatic tissue. The KrasG12D sequence was then amplified by PCR. The amplified product was incubated with T7E1 restriction enzyme for 30 min, and after incubation, the DNA product was subjected to gel electrophoresis on a 2% agarose gel. The DNA bands in the gel were visualized and photographed using an Azure Biosystems c150. The cleavage efficiency was quantitatively analyzed using ImageJ, and the results are as follows: Figure 18 As shown, the insertion / deletion efficiency of the pancreatic Kras gene in the GDCA-LNP group reached 29.3%, while that in the Chol-LNP group was only 9.6%; moreover, the off-target effect of the spleen in the GDCA-LNP group was negligible, while the off-target effect of the spleen in the Chol-LNP group was 6.1%.

[0089] (2) Median survival The survival of KPC mice after drug administration was continuously monitored for 240 days, and mortality was recorded. The results are as follows: Figure 19 As shown, the survival time of the GDCA-LNP group was significantly longer than that of the saline group (P<0.0001) and the Chol-LNP group (P=0.013).

[0090] Example 8: mRNA tumor vaccine – in situ KPC model 1. Experimental Design Using the same method as in Example 1, replace Luc mRNA with Kras G12D GDCA-LNP-Kras was prepared from mRNA (SEQ ID NO:3). G12D KPC tumor cells were orally implanted into C57BL / 6 mice, and GDCA-LNP-Kras were injected intraperitoneally on days 10 and 17 post-inoculation. G12D (mRNA 0.25 mg / kg).

[0091] 2. Survival determination The survival of orthotopic KPC mice was continuously monitored for 50 days after drug administration, and mortality was recorded. The results are as follows: Figure 20 As shown, compared with the saline group, GDCA-LNP-Luc group and Chol-LNP-Kras group G12DCompared to the previous group, GDCA-LNP-Kras G12D The survival time of the group was significantly prolonged (P<0.05).

[0092] 3. Treatment efficacy Reverse validation of GDAC-LNP-Kras using antibody blocking method G12D As a measure of the vaccine's therapeutic effect in in situ pancreatic cancer, anti-CD4 and anti-CD8 were injected after administration, and the results were as follows: Figure 20 As shown, double-exhaustion CD4 + / CD8 + The T-cell therapy essentially eliminated the therapeutic effect, and the survival time of mice was significantly reduced, indicating that CD4+... + and CD8 + T cells are involved in the anti-tumor effect, suggesting vaccine-dependent adaptive immunity.

[0093] Example 9: mRNA Tumor Vaccine – Humanized PDX Model 1. Experimental subjects The humanized PDX model was established using huHSC-NCG mice (18 weeks old, male, strain number T057531), purchased from Nanjing Genscript Biotech. NCG mice were first irradiated with a sublethal dose, followed by intravenous injection of CD34. + Artificial hematopoietic stem cells (CD34) + HSC). By analyzing hCD45 in peripheral blood. + Cellular monitoring of immune reconstitution. Patient-derived samples (PDX) were obtained from a 65-year-old male patient with KRAS G12V-mutant pancreatic cancer and used to construct an orthotopic PDAC model. After three passages in NCG mice, the cells were digested into a single-cell suspension and injected into the pancreatic tail of humanized NCG mice on day 14 to establish a humanized orthotopic PDX model. Baseline tumor size (approximately 100 mm³) was assessed by ultrasound before treatment. The committee approved a maximum tumor volume of 2,000 mm³. Euthanasia was performed if the patient experienced a weight loss exceeding 15%, was near death, or exhibited signs of distress.

[0094] 2. Dosing regimen Using the same method as in Example 1, replace Luc mRNA with Kras G12V GDCA-LNP-Kras was prepared from mRNA (sequence shown in SEQ ID NO:4). G12V Two doses of GDCA-LNP-Kras were administered intraperitoneally. G12V (mRNA 0.25 mg / kg / time).

[0095] 3. ELISpot method for determining IFN-γ Single-cell suspensions of treated mouse pancreas were prepared and plated in culture plates that had been activated, coated with capture antibodies, and blocked. After adding stimulants, the plates were incubated for 24 hours. After incubation, the cells were lysed and removed. Detection antibodies and enzyme-linked compounds were then added for color development. Spot counting and analysis were performed using a professional ELISpot plate reader. Results are as follows: Figure 21 As shown in the ELISpot results, IFN-γ secretion in the GDCA-LNP group was significantly higher than that in the Chol-LNP group and the saline group.

[0096] 4. Productivity Measurement The survival of humanized orthotopic KPC mice was continuously monitored for 40 days after drug administration, and mortality was recorded. The results are as follows: Figure 22 As shown, the GDCA-LNP group had a 100% survival rate within the 40-day observation window.

[0097] 5. Tumor tissue staining analysis On day 25 of treatment in humanized orthotopic KPC mice, pancreatic cancer tissue was harvested from the mice through dissection. MTS and α-SMA staining was performed on paraffin-embedded sections, and fluorescence intensity was analyzed using ImageJ. The results are as follows: Figure 23 As shown, Masson trichrome staining and α-SMA staining of tumor tissue indicated that fibrosis was significantly reduced in the GDCA-LNP group.

[0098] 6. Flow cytometry was used to determine the proportions of various immune cells in mouse tumors. On day 12 of treatment in humanized orthotopic KPC mice, pancreatic cancer tissue was harvested from the mice. The pancreatic cancer cells were prepared into a single-cell suspension, stained with immunomarker antibodies, and then the distribution of antibody signals in the mouse pancreatic cancer was analyzed by flow cytometry. The results are as follows: Figure 24 As shown, tumor immune microenvironment remodeling: the M1 / M2TAM ratio was increased in the GDCA-LNP group, CD4 + / CD8 + Increased T cell infiltration, Foxp3 + Regulatory T cells decreased, while granzyme B and IFN-γ expression increased.

[0099] Example 10: Multi-species verification 1. Rabbit Rabbits were intraperitoneally injected with GDCA-LNP-Luc at doses of 0.02 mg / kg and 0.05 mg / kg, respectively. The results were as follows: Figure 25 As shown, all samples exhibited highly selective pancreatic expression, and the BLI signal was positively correlated with the dose.

[0100] To conduct a safety analysis, rabbits were intraperitoneally injected with either GDCA-LNP or SM102-LNP (formulation: SM102 / DSPC / CHOL / PEG2000). The injection dose for both LNPs was 0.5 mg / kg of Luc mRNA. Samples were collected 24 hours post-injection. Whole blood was used for three complete blood counts, and serum was used to detect amylase, lipase, ALT, AST, IL-6, TFN-α, and CREA. Hematologic and epithelial (H&E) stained sections from different organs were analyzed 24 hours post-treatment. Results are as follows: Figure 26 As shown, the high dose of 0.5 mg / kg was well tolerated, and blood routine and liver, kidney and pancreatic function indicators were normal.

[0101] 2. Miniature pigs Miniature pigs were intraperitoneally injected with 0.01 and 0.02 mg / kg GDCA-LNP-Luc, respectively. Bioluminescent signals were detected 3 hours later using a live animal imaging system. Results are as follows: Figure 27 As shown, the specificity of pancreatic BLI is >90%.

[0102] The distribution of GDCA-LNP-Cy5 in the pancreas of miniature pigs was determined using light-sheet imaging. The specific steps were as follows: Miniature pig pancreas samples were collected after drug administration and then placed in Cubic-L (10% n-butyl diethanolamine, 10% Triton-100, 80% deionized water) for one week for clearing, with the medium changed every 3 days. Subsequently, the tissue was treated with 50% Cubic-RA (45% antipyrine, 30% N-methylnicotinamide, 25% deionized water, 0.5% n-butyl diethanolamine), and the cell nuclei were stained with YO-PRO-1. The medium was changed after 24 hours. Hyaluronic acid was then treated with 100% Cubic-RA for one week, with the medium changed every 3 days. Finally, the samples were embedded in agarose-CUBIC reagent. After complete embedding, 3D images were captured using light-sheet imaging (LiTone XL light-sheet microscope, Photogenic Technology Ltd.), and the data were analyzed using Imaris. Results are as follows: Figure 28 As shown, light slice imaging revealed that Cy5-mRNA was mainly distributed in the pancreatic duct, with some distribution in the acinar tissue; After drug administration, miniature pig pancreas was paraffin-embedded, sectioned, stained with H&E, and scanned. Results are as follows: Figure 29 As shown, HE staining revealed no pancreatic tissue damage.

[0103] To conduct a safety analysis, blood was collected via cardiac puncture after intraperitoneal injection of GDCA-LNP into miniature pigs. Serum was then extracted and tested for amylase, ALT, AST, ALP, TFN-α, and CREA. Results are as follows: Figure 30 As shown, there were no significant abnormalities in blood biochemistry.

[0104] 3. Crab-eating macaques (non-human primates) (1) hEPO mRNA verification Serum hEPO levels were monitored within 14 days after intraperitoneal injection of 0.2 and 0.4 mg / kg GDCA-LNP-Luc into cynomolgus monkeys. Results are as follows: Figure 31 As shown, serum hEPO levels steadily increased over 14 days; ALT, AST, CRE, amylase, and lipase showed no significant changes; and routine blood tests were normal.

[0105] (2) Biodistribution of Luc mRNA Following an intraperitoneal injection of 0.2 mg / kg GDCA-LNP-Luc, cynomolgus monkeys underwent bioluminescence signal acquisition from various organs using a live animal imaging system. The specific procedure involved intraperitoneal injection of 0.2 mg / kg GDCA-LNP-Luc, followed by intraperitoneal injection of potassium fluorescein 6 hours later. The monkeys were then euthanized, and their organs were dissected and harvested. Bioluminescence signals from the harvested organs were immediately acquired using a live animal imaging system. Results are as follows: Figure 32 As shown, BLI confirmed that >90% of the signal accumulated in the pancreas; the pancreatic tissue samples from cynomolgus monkeys were paraffin-embedded, sectioned, stained with H&E, and scanned, as shown... Figure 33 As shown, no pathological changes were observed in the pancreas after HE staining.

[0106] To analyze the specific expression and expression region of Luc mRNA in the pancreas of cynomolgus monkeys, pancreatic tissue samples were paraffin-embedded, sectioned, and subjected to Fluc immunohistochemistry and scanning. The results are as follows: Figure 34 , Figure 35 As shown, FLuc immunohistochemistry confirmed that expression was mainly located in the acini and duct regions.

Claims

1. A pancreas-targeting lipid nanoparticle, characterized in that, The lipid nanoparticles comprise lipid components and encapsulated nucleic acids; The lipid component includes ionizable lipids, cofactor phospholipids, sterol components, and PEG-lipids; Preferably, the ionizable lipid comprises one or more of SM102, C12-200, CCK-E12, 306Oi10, ALC-0315 and MC3; Preferably, the auxiliary phospholipid comprises one or more of DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine), DSPC, DOPC, and DPPC; Preferably, the sterol component comprises a bile acid derivative; more preferably, the bile acid derivative comprises one or more of glycodeoxycholic acid (GDCA), CDCA, TCA, and TUDCA. Preferably, the PEG-lipids include DMG-PEG2000, ALC-0159, and DSPE-PEG2000.

2. The lipid nanoparticles according to claim 1, characterized in that, The nucleic acid includes mRNA and sgRNA; Preferably, the mRNA includes therapeutic mRNA, gene-editing mRNA, or vaccine mRNA.

3. The lipid nanoparticles according to claim 1, characterized in that, The molar ratio of the lipid components is ionizable lipids: co-phospholipids: sterol components: PEG-lipids = 35~50: 10~16: 38~48: 1~3; Preferably, the molar ratio of the lipid components is ionizable lipid: co-phospholipid: sterol component: PEG-lipid = 40:10:48:2, 35:16:46.5:2.5 or 50:10:38.5:1.

5.

4. The lipid nanoparticles according to claim 1, characterized in that, The mass ratio of the lipid component to the nucleic acid is 5-15:1-4; Preferably, the mass ratio of the lipid component to the nucleic acid is 10:

1.

5. The method for preparing lipid nanoparticles according to any one of claims 1-4, characterized in that, The lipid nanoparticles were prepared using a microfluidic method. Preferably, the preparation of the lipid nanoparticles includes the following steps: (1) Preparation of the ethanol phase: Dissolve the lipid components in anhydrous ethanol; (2) Preparation of aqueous phase: Dissolve the nucleic acid in citrate buffer; (3) Hybridization: The aqueous phase and the ethanol phase are rapidly mixed through a microfluidic chip or a channel to form lipid nanoparticles through self-assembly. (4) Dialysis purification: The prepared lipid nanoparticles are dialyzed to remove ethanol and free components.

6. The preparation method according to claim 5, characterized in that, The ratio of the aqueous phase to the ethanol phase in step (3) is 2 to 4:1 (v / v). Preferably, the ratio of the aqueous phase to the ethanol phase in step (3) is 3:1 (v / v).

7. Use of the lipid nanoparticles according to any one of claims 1-4 in the preparation of medicaments for treating diseases; Preferably, the disease includes pancreatic diseases; More preferably, the pancreatic diseases include pancreatitis and pancreatic cancer.

8. The use according to claim 7, characterized in that, Including any of the following: (1) The nucleic acid is IL-1 receptor antagonist (IL-1RA) mRNA, and the disease is acute pancreatitis; or (2) The nucleic acid is Cas9 mRNA and sgRNA targeting the KrasG12D mutation site, and the disease is pancreatic cancer; or (3) The nucleic acid is a mutated KrasG12D or KrasG12V neoantigen mRNA, and the disease is pancreatic cancer.

9. The use according to claim 7, characterized in that, The methods of administration of the drug include intraperitoneal injection, intravenous injection, or subcutaneous administration.

10. A pancreas-targeting human or veterinary pharmaceutical composition, characterized in that, The composition comprises the lipid nanoparticles according to any one of claims 1-4 and other excipients; Preferably, the veterinary drug includes drugs for rats, rabbits, pigs, and non-human primates.

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