Development of spermatogonium-targeting lipid nanomaterial and application of spermatogonium-targeting lipid nanomaterial in treatment of male infertility

Abstract

The invention provides development of a spermatogonial-targeting lipid nanomaterial and application of the spermatogonial-targeting lipid nanomaterial in treatment of male infertility, relates to the technical field of biomedicine, and has the technical key points that the lipid nanomaterial and a preparation method thereof are provided, mRNA is delivered to spermatogonial by using the lipid nanomaterial, and the spermatogonial-targeting lipid nanomaterial is prepared by editing the spermatogonial. And edited sperm cells and sperm cells can be obtained. The invention provides an ionizable phospholipid nanoparticle LNP65, which can be injected through a testicular net to effectively transfer mRNA (messenger ribonucleic acid) to spermatogonium in a mouse testis. The spermatogonium has been successfully edited by this method. In one spermatogenic cycle, a modified sperm from a spermatogonial cell edited by LNP65 is obtained. And after three spermatogenesis cycles, observing that the spermatogonium cells edited by the LNP65 are cleared from the testis. The invention develops a lipid nanomaterial delivery mRNA (messenger Ribonucleic Acid) system of targeted spermatogonium, and provides an applicable carrier for treating azoospermia caused by single gene deletion.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the development of lipid nanoparticles targeting spermatogonia and their application in the treatment of male infertility. Background Art

[0002] Male infertility affects men worldwide, impacts national development and stability, and has become a scientific problem that urgently needs to be solved. Among them, genetic factors account for at least 15% of male infertility cases. Together with environmental impacts, infections, social stress, and economic burdens, they play a crucial role. The gene map of male infertility has found that at least 2,000 genes are related to spermatogenesis.

[0003] The monogenic causes of human male infertility have a moderate, strong, or clear association with 120 genes related to 104 male infertility phenotypes. Many of these genes are particularly related to azoospermia, which is characterized by the absence of sperm in semen. In male infertility cases, gene mutations leading to meiotic arrest and azoospermia include mutations in synaptonemal complex protein 3 (Sycp3) and testis-expressed gene 11 (TEX11). Unfortunately, there are no effective treatment methods for these disorders. Therefore, the development of a gene delivery system targeting germ cells is crucial.

[0004] Existing delivery systems include virus-mediated gene delivery systems such as lentiviruses, adenoviruses, and adeno-associated viruses, which are effective due to their high gene transfer efficiency. However, they also pose significant biological hazard risks, are difficult to handle, and may cause harmful effects such as uncontrolled infections or inflammation. On the other hand, non-viral systems such as electroporation and nanoparticles are easier and safer to use. However, they are less effective in efficiently and stably integrating transgenes into the genomes of germ stem cells or somatic cells supporting the testis. To date, an effective, safe, and targeted drug delivery system has not been developed in the male testis. Summary of the Invention

[0005] The purpose of the present invention is to solve the problem that an effective, safe, and targeted drug delivery system has not been developed in the male testis in the prior art.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A lipid nanoparticle LNP65, characterized in that: the lipid nanoparticle LNP65 is prepared from L65, DOPE, Cholestrol, DMG-PEG2000, and a cationic compound.

[0008] Preferably, the ratio of L65, DOPE, Cholestrol, DMG-PEG2000 and the cationic compound is 50:40:20:0.75:20.

[0009] The present application also provides the use of the lipid nanomaterial LNP65 as a delivery carrier system in the preparation of a medicament for treating male infertility, and the lipid nanomaterial LNP65 is the lipid nanomaterial LNP65 as described above.

[0010] Preferably, the lipid nanomaterial LNP65 is used to deliver mRNA, and the lipid nanomaterial targets spermatogonia.

[0011] Preferably, the mRNA is saRNA.

[0012] Preferably, the saRNA is Rik08 self-replicating RNA, and the male infertility is non-obstructive azoospermia with spermatocyte arrest.

[0013] Preferably, the lipid nanoparticles rapidly edit testicular genes by delivering Cre mRNA.

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

[0015] The present application uses lipid nanoparticles to deliver mRNA to spermatogonia, and by editing spermatogonia, the application of obtaining edited spermatocytes and sperm cells can be achieved. The present application provides an ionizable phospholipid nanomaterial LNP65, which can effectively deliver mRNA into spermatogonia in the mouse testis through rete testis injection. In the present application, spermatogonia are successfully edited by this method, and through specific verification experiments, it is verified that within one spermatogenic cycle, modified sperm from LNP65-edited spermatogonia can be obtained. After three spermatogenic cycles, we observed the clearance of LNP65-edited spermatogonia from the testis; the present application develops a lipid nanomaterial delivery mRNA system targeting spermatogonia, providing an application-type carrier for the treatment of azoospermia caused by single-gene deletion, and having broad application prospects. Description of the Drawings

[0016] Figure 1 . Synthesis of ionizable phospholipids. a Synthesis scheme of ionizable phospholipids. b Structures of ionizable phospholipids L1 to L70, including some ionizable phospholipids in our previous paper 1.

[0017] Figure 2.Screen lipid nanoparticles (LNPs) in the spermatogonial cell line. Taking the LNP1 formulation as an example, LNP1@GFP is prepared from L1:DOPE:cholesterol:DMG-PEG2000:cationic compound = 50:40:20:0.75:20 (dissolved in ethanol) and GFP mRNA (dissolved in ddH2O). Panel a shows the particle sizes of LNP1@GFP to LNP70@GFP, n = 3, and the data are presented as mean ± standard deviation. Panel b shows the zeta potentials of LNP1@GFP to LNP70@GFP, n = 3, and the data are presented as mean ± standard deviation. Panel c shows the percentage of GFP-positive cells in spermatogonial cell line GC-1 cells treated with LNP1@GFP to LNP70@GFP, detected by flow cytometry, n = 3, and the data are presented as mean ± standard deviation. Figure 3 For the synthesis of 2-(dioctylamino)ethyl octyl-2-yl phosphate and its analogous compounds.

[0018] Figure 3 .LNP65 delivers mRNA to spermatogonia and Sertoli cells and edits spermatogonia and Sertoli cells in the mouse testis. Panel a shows the treatment schematic diagram of injecting LNP65@Cre mRNA into the rete testis of tdTomato male mice. Three days later, the mice were examined. Panel b shows representative testicular images of the control group and the LNP65@Cre mRNA group, n = 3, and the scale bar is 1 cm. Panel c shows the testicular indices (%) of the control group and the LNP65@Cre mRNA group. The testicular index = testicular weight / body weight × 100, here for one testis, n = 3. The data are presented as mean ± standard deviation. Statistical analysis was performed using a two-tailed Student's t-test, P < 0.05. Panel d shows the genotype analysis of the testes of the control group and the LNP65@Cre mRNA group (by PCR), n = 3. Panel e shows the HE staining of the testes of the control group and the LNP65@Cre mRNA group, n = 3. Panel f shows representative tdTomato fluorescence images of the testes of the control group and the LNP65@Cre mRNA group, n = 3. Panel g shows the statistical analysis of the percentage of tdTomato-positive seminiferous tubules in the total seminiferous tubules, n = 3. The data are presented as mean ± standard deviation. Statistical analysis was performed using a two-tailed Student's t-test, ***P < 0.0001. Panel h shows the immunofluorescence co-staining images of tdTomato (red) and the spermatogonial differentiation marker c-kit (green) in the control group and the LNP65@Cre mRNA group, n = 3. Panel i shows the immunofluorescence co-staining images of tdTomato (red) and the Sertoli cell marker Sox9 (green) in the control group and the LNP65@Cre mRNA group, n = 3.

[0019] Figure 4. Modified sperm were obtained from spermatogonia edited by LNP65 within one spermatogenic cycle. Panel a shows a schematic of the treatment. LNP65@Cre mRNA was injected into the rete testis of tdTomato mouse testes for 24 hours, which is equivalent to one spermatogenic cycle. Panel b shows representative testicular images of the control group and the LNP65@Cre mRNA group, n = 3, scale bar = 1 cm. Panel c shows the testicular index (%) of the control group and the LNP65@Cre mRNA group. Testicular index = testicular weight / body weight × 100, here for one testis, n = 3. Data are presented as mean ± standard deviation. Panel d shows genotype analysis (by PCR) of the testes of the control group and the LNP65@Cre mRNA group, n = 3. Panel e shows HE staining of the testes of the control group and the LNP65@Cre mRNA group, n = 3. Panel f shows representative tdTomato fluorescence images of the testes of the control group and the LNP65@Cre group, n = 3. Panel g shows the one-step acquisition of modified sperm from spermatogonia edited by LNP65 within one spermatogenic cycle.

[0020] Figure 5 . LNP65-edited spermatogonia were eliminated within three spermatogenic cycles. Panel a shows injection of LNP65@Cre mRNA into the rete testis of tdTomato mouse testes for 100 days, which is equivalent to three spermatogenic cycles. Panel b shows testicular images of the control group and the LNP65@Cre mRNA group, n = 3, scale bar = 1 cm. Panel c shows the testicular index (%) of the control group and the LNP65@Cre mRNA group. Testicular index = testicular weight / body weight × 100, here for one testis, n = 3. Data are presented as mean ± standard deviation. Panel d shows genotype analysis (by PCR) of the testes of the control group and the LNP65@Cre mRNA group, n = 4 / 3. Panel e shows HE staining of the testes of the control group and the LNP65@Cre mRNA group, n = 3. Panel f shows representative tdTomato fluorescence images of the testes of the control group and the LNP65@Cre mRNA group, n = 3. Panel g shows that after three spermatogenic cycles, LNP65-edited spermatogonia were eliminated, leaving only edited Sertoli cells in the testis.

[0021] Figure 6 . Schematic strategy of the rapid gene editing technology for lipid nanoparticle LNP65 delivering Cre mRNA (LNP65@Cre mRNA).

[0022] Figure 7 . Delivery of LNP65@Cre mRNA by rete testis injection knocked out ZNF143 in the testes of male mice 3 days later. (a) ZNF143 floxp / floxp Group and ZNF143 KO group schematic for obtaining ZNF143 knockout (KO) by delivering LNP65@Cre mRNA. (b) ZNF143floxp / floxp Testicular hematoxylin-eosin (HE) staining of the ZNF143 group and ZNF143 KO group 3 days after injection (n = 4). (c) WB analysis of ZNF143 floxp / floxp Expression of ZNF143 in the testes of the ZNF143 group and ZNF143 KO group 3 days after testicular injection, n = 4. β-actin was used as an internal control. The intensity was calculated by ImageJ.

[0023] Figure 8 . Delivery of LNP65@Cre mRNA by testicular rete injection to knockout ZNF143 in the testes of male mice 24 days later. (a) ZNF143 floxp / floxp Testicular images of the ZNF143 group and ZNF143 KO group mice 24 days after testicular injection, n = 3. (b) ZNF143 floxp / floxp Testicular index (%) of the ZNF143 group and ZNF143 KO group knockout mice 24 days after testicular injection. Testicular index = testicular weight / body weight * 100, here for one testis, n = 3. Data are expressed as mean ± standard deviation. Statistical significance was analyzed by two-tailed t-test, ***P < 0.001. (c) ZNF143 floxp / floxp and testicular hematoxylin-eosin (HE) staining of ZNF143 knockout mice 24 days after testicular injection, n = 3.

[0024] Figure 9 . Verification of the ZNF143 knockout phenotype. (a) Traditional spermatogonial knockout tool mice Stra8-Cre were used for spermatogonial KO of ZNF143 in the testes. (b) After mating with Stra8-Cre mice for 3 months, the testes of ZNF143 floxp / floxp and ZNF143 KO were subjected to hematoxylin-eosin (HE) staining.

[0025] Figure 10 . Schematic diagram of injecting LNP65@Rik08saRNA into the testes of Rik08KO mice. Nine days later, the mice were investigated.

[0026] Figure 11 . Representative fluorescence images of Rik08 protein expression in the Rik08KO and Rik08KO + LNP65@Rik08saRNA groups, n = 4.

[0027] Figure 12 . HE staining of the testes of WT (wild type), Rik08KO, and Rik08KO + LNP65@Rik08saRNA 9 days after rete infusion, n = 4.

[0028] Figure 13. Representative fluorescence images of PNA-lectin-labeled haploid sperm in WT, Rik08KO, and Rik08KO+LNP65@Rik08saRNA, n = 4.

[0029] Figure 14 . Epididymal smear sperm of Rik08KO and Rik08KO+LNP65@Rik08saRNA are shown, with arrows indicating sperm, n = 4. Detailed implementation manners

[0030] The present invention will be further described in detail below in conjunction with specific embodiments.

[0031] Experimental materials and related sources applied in this application:

[0032] GC-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China);

[0033] Both Cre mRNA (EZ Cap Cre mRNA (m1Ψ), R1030, sequence number: 17108ES60) and Rik08saRNA (EZCap1Rik08-3XFlag asRNA (5mC)) were purchased from Shanghai Weihuan Biotechnology Co., Ltd.;

[0034] Rosa-LSL-tdTomato mice were purchased from Cyagen Biosciences Inc. (C001476);

[0035] ZNF 143 mice were purchased from Nanjing Cyagen Biotechnology Co., Ltd. (S-CKO-05327);

[0036] Rik08KO mice were obtained by CRISPR-Cas9 gene editing technology;

[0037] All mice were raised at Nantong University. The mouse experimental procedures were approved by the Animal Protection and Utilization Committee of Nantong Medical College, Nantong University, China. All drugs and solvents used in the small experiment part were purchased through the reagent management system of Nanjing University. Drug weighing was carried out using an electronic analytical balance. Post-reaction treatment and product purification were mainly carried out by silica gel column chromatography.

[0038] Example 1: Synthesis of ionizable phospholipid compounds

[0039] 2-Chloro-1,3,2-dioxaphospholane (1.1 eq) was added to a beaker together with an alcohol (1.0 eq) and Et3N (1.1 eq), and the mixture was stirred at -15 °C for 5 minutes. Stirring was continued at 25 °C for 12 hours. The precipitate in the mixture was filtered and rinsed three times with ether to remove triethylammonium chloride. Finally, the solvent was removed by rotary evaporation to concentrate and obtain the alkylated phosphorus oxide molecule. The characterization of these ionizable phospholipids was carried out 1 by 1H NMR.

[0040] See Figure 1 , the synthesis and 1H NMR characterization of the ionizable phospholipids are shown below:

[0041] L1: 1H NMR (400 MHz, CDCl3) δ 4.25 - 3.54 (m, 4H), 2.77 - 2.72 (m, 4H), 2.55 - 2.53 (m, 2H), 1.75 - 1.58 (m, 4H), 1.46 (m, 2H), 1.24 (m, 40H), 0.88 - 0.85 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C31H67NO4P, 548.4808 [M+H]+; found: 548.4996.

[0042] L2: 1H NMR (400 MHz, CDCl3) δ 3.97 - 3.59 (m, 4H), 2.68 - 2.64 (m, 4H), 2.46 - 2.37 (m, 2H), 1.66 - 1.50 (m, 4H), 1.35 - 1.30 (m, 2H), 1.18 - 1.11 (m, 48H), 0.80 - 0.76 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C35H75NO4P, 604.5434 [M+H]+; found: 604.5441.

[0043] L3: 1H NMR (400 MHz, CDCl3) δ 4.20 - 3.98 (m, 2H), 3.66 - 3.44 (m, 4H), 2.82 - 2.80 (m, 4H), 1.87 - 1.56 (m, 6H), 1.50 - 1.08 (m, 30H), 0.92 - 0.84 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C26H57NO6P, 510.3924 [M+H]+; found: 510.3932.

[0044] L4: 1H NMR (400 MHz, CDCl3) δ 4.08 - 3.90 (m, 2H), 3.67 - 3.43 (m, 4H), 2.81 - 2.73 (m, 4H), 1.91 - 1.45 (m, 6H), 1.44 - 1.15 (m, 38H), 0.92 - 0.84 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C30H65NO6P, 566.4550 [M+H]+; found: 566.4558.

[0045] L5: 1H NMR (400 MHz, CDCl3) δ 4.54 - 3.36 (m, 4H), 2.86 - 2.82 (m, 4H), 2.35 - 2.24 (m, 1H), 1.85 - 1.57 (m, 7H), 1.32 - 1.22 (m, 52H), 0.87 - 0.84 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C38H78NO6PNa, 698.5464 [M+Na]+; found: 698.5459.

[0046] L6: 1H NMR (400 MHz, CDCl3) δ 4.21 - 3.66 (m, 4H), 2.81 - 2.72 (m, 4H), 2.33 - 2.40 (m, 1H), 1.78 - 1.56 (m, 7H), 1.35 - 1.12 (m, 60H), 0.88 - 0.85 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C42H86NO6PNa, 754.6090 [M+Na]+; found: 754.6045.

[0047] L7: 1H NMR (400 MHz, CDCl3) δ 4.28 - 4.04 (m, 4H), 2.84 - 2.80 (m, 4H), 1.84 - 1.78 (m, 6H), 1.32 - 1.25 (m, 28H), 1.00 - 0.81 (m, 15H). HRMS (ESI-TOF): m / z calcd for: C28H58NO4PNa, 526.4001 [M+Na]+; found: 526.4005.

[0048] L8ZP8(2AP4): 1H NMR (400 MHz, CDCl3) δ 4.55 - 3.82 (m, 4H), 2.85 - 2.81 (m, 4H), 1.85 - 1.80 (m, 6H), 1.29 - 1.23 (m, 36H), 0.99 - 0.83 (m, 15H). HRMS (ESI-TOF): m / z calcd for: C32H67NO4P, 560.4808 [M + H]+; found: 560.4805.

[0049] L9: 1H NMR (400 MHz, CDCl3) δ 4.25 - 3.63 (m, 3H), 2.88 - 2.84 (s, 4H), 2.08 - 2.01 (m, 1H), 1.90 - 1.56 (m, 7H), 1.43 - 0.97 (m, 46H), 0.89 - 0.86 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C35H74NO4PNa, 626.5253 [M + Na]+; found: 626.5236.

[0050] L10: 1H NMR (400 MHz, CDCl3) δ 4.21 - 3.62 (m, 3H), 3.30 - 3.28 (m, 1H), 2.84 - 2.79 (m, 4H), 2.34 - 1.36 (m, 7H), 1.35 - 0.98 (m, 54H), 0.88 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C39H83NO4P, 660.6060 [M + H]+; found: 660.6031.

[0051] L11: 1H NMR (400 MHz, CDCl3) δ 4.41 - 3.65 (m, 4H), 3.17 - 2.67 (m, 5H), 1.81 - 1.57 (m, 4H), 1.29 - 1.26 (m, 22H), 0.89 - 0.85 (m, 6H). HRMS (ESI-TOF): m / z calcd for: C27H43F16NO4P, 780.2674 [M + H]+; found: 780.2643.

[0052] L12: 1H NMR (400 MHz, CDCl3) δ 4.36 - 3.66 (m, 4H), 3.03 - 2.76 (m, 5H), 1.7 - 1.57 (m, 4H), 1.34 - 1.25 (m, 30H), 0.89 - 0.86 (m, 6H). HRMS (ESI-TOF): m / z calcd for: C31H51F16NO4P, 836.3300 [M+H]+; found: 836.3269.

[0053] L13: 1H NMR (400 MHz, CDCl3) δ 4.46 - 3.59 (m, 4H), 2.83 - 2.79 (m, 4H), 2.69 - 2.47 (m, 1H), 1.84 - 1.76 (m, 6H), 1.29 - 1.22 (m, 26H), 0.93 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C26H55NO5P, 492.3818 [M+H]+; found: 492.3798.1

[0054] L14: 1H NMR (400 MHz, CDCl3) δ 4.46 - 3.63 (m, 4H), 2.82 - 2.78 (m, 4H), 2.73 - 2.44 (m, 1H), 1.81 - 1.76 (m, 6H), 1.28 - 1.23 (m, 34H), 0.88 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C30H63NO5P, 548.4444 [M+H]+; found: 548.4419.

[0055] L15: 1H NMR (400 MHz, CDCl3) δ 4.25 - 3.61 (m, 4H), 2.87 - 2.83 (m, 4H), 1.86 - 1.80 (m, 8H), 1.36 - 1.19 (m, 68H), 0.89 - 0.85 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C42H86NO6PNa, 754.6090 [M+Na]+; found: 754.6091.

[0056] L16: 1H NMR (400 MHz, CDCl3) δ 4.19 - 3.64 (m, 6H), 2.83 - 2.77 (m, 4H), 1.78 - 1.59 (m, 6H), 1.28 - 1.23 (m, 32H), 0.86 - 0.83 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C27H59NO4P, 492.4182 [M + H]+; found: 492.4167.

[0057] L17: 1H NMR (400 MHz, CDCl3) δ 4.19 - 3.59 (m, 6H), 2.82 - 2.71 (m, 4H), 1.81 - 1.56 (m, 6H), 1.45 - 1.06 (m, 60H), 0.86 - 0.82 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C41H87NO4P, 688.6373 [M + H]+; found: 688.6339.

[0058] L18: 1H NMR (400 MHz, CDCl3) δ 3.71 - 3.53 (m, 6H), 2.81 - 2.77 (m, 4H), 1.77 - 1.74 (m, 4H), 1.54 - 1.50 (m, 2H), 1.35 - 1.21 (m, 22H), 0.87 - 0.83 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C22H49NO6P, 454.3297 [M + H]+; found: 454.3303.

[0059] L19: 1H NMR (400 MHz, CDCl3) δ 3.64 - 3.54 (m, 6H), 2.81 - 2.73 (m, 4H), 1.73 - 1.49 (m, 6H), 1.34 - 1.03 (m, 50H), 0.84 - 0.80 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C36H77NO6P, 650.5489 [M + H]+; found: 650.5466.

[0060] L20: 1H NMR (400 MHz, CDCl3) δ 3.69 - 3.51 (m, 6H), 2.80 - 2.75 (m, 4H), 1.77 - 1.51 (m, 6H), 1.32 - 1.17 (m, 34H), 0.86 - 0.82 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C28H61NO6P, 538.4237 [M + H]+; found: 538.4242.

[0061] L21: 1H NMR (400 MHz, CDCl3) δ 3.71 - 3.53 (m, 6H), 2.81 - 2.75 (m, 4H), 1.77 - 1.47 (m, 6H), 1.30 - 1.19 (m, 42H), 0.87 - 0.84 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C32H69NO6P, 594.4863 [M+H]+; found: 594.4850.

[0062] L22: 1H NMR (400 MHz, CDCl3) δ 5.33 - 5.24 (m, 2H), 4.31 - 3.60 (m, 6H), 2.80 - 2.75 (m, 4H), 1.77 - 1.53 (m, 6H), 1.25 - 1.22 (m, 28H), 0.86 - 0.77 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C27H57NO4P, 490.4025 [M+H]+; found: 490.4002.

[0063] L23: 1H NMR (400 MHz, CDCl3) δ 5.33 - 5.22 (m, 2H), 4.19 - 3.59 (m, 6H), 2.77 - 2.73 (m, 4H), 1.73 - 1.56 (m, 6H), 1.24 - 1.20 (m, 36H), 0.84 - 0.81 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C31H65NO4P, 546.4651 [M+H]+; found: 546.4622.

[0064] L24: 1H NMR (400 MHz, CDCl3) δ 5.74 - 5.44 (m, 2H), 4.31 - 3.96 (m, 6H), 2.80 - 2.76 (m, 4H), 1.76 - 1.51 (m, 6H), 1.28 - 1.24 (m, 42H), 0.88 - 0.85 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C34H71NO4P, 588.5121 [M+H]+; found: 588.5088.

[0065] L25: 1H NMR (400 MHz, CDCl3) δ 5.48 - 5.21 (m, 2H), 4.81 - 4.62 (m, 1H), 4.15 - 3.95 (m, 4H), 2.80 - 2.76 (m, 4H), 1.74 - 1.55 (m, 6H), 1.25 - 1.21 (m, 32H), 0.85 - 0.82 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C31H63NO6P, 576.4393 [M+H]+; found: 576.4360.

[0066] L26: 1H NMR (400 MHz, CDCl3) δ 4.28 - 3.61 (m, 5H), 2.82 - 2.78 (m, 4H), 1.80 - 1.53 (m, 6H), 1.28 - 1.24 (m, 46H), 0.88 - 0.85 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C35H75NO4P, 604.5434 [M+H]+; found: 604.5402.

[0067] L27: 1H NMR (400 MHz, CDCl3) δ 4.22 - 3.61 (m, 3H), 2.85 - 2.81 (m, 4H), 1.84 - 1.53 (m, 8H), 1.28 - 1.24 (m, 34H), 0.87 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C29H63NO4P, 520.4495 [M+H]+; found: 520.4469.

[0068] L28: 1H NMR (400 MHz, CDCl3) δ 4.24 - 3.61 (m, 3H), 2.85 - 2.81 (m, 4H), 1.85 - 1.55 (m, 8H), 1.29 - 1.24 (m, 42H), 0.89 - 0.85 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C33H71NO4P, 576.5121 [M+H]+; found: 576.5092.

[0069] L29: 1H NMR (400 MHz, CDCl3) δ 4.42 - 3.42 (m, 7H), 3.38 - 3.28 (m, 3H), 2.83 - 2.79 (m, 4H), 1.82 - 1.76 (m, 4H), 1.33 - 1.24 (m, 26H), 1.12 - 1.07 (m, 9H), 0.87 - 0.84 (m, 6H). HRMS (ESI - TOF): m / z calcd for: C28H61NO7P, 554.4186 [M + H]+; found: 554.4185.

[0070] L30: 1H NMR (400 MHz, CDCl3) δ 4.26 - 3.45 (m, 7H), 3.36 - 3.34 (m, 3H), 2.86 - 2.78 (m, 4H), 1.81 - 1.76 (m, 4H), 1.28 - 1.23 (m, 34H), 1.13 - 1.11 (m, 9H), 0.88 - 0.84 (m, 6H). HRMS (ESI - TOF): m / z calcd for: C32H69NO7P, 610.4812 [M + H]+; found: 610.4811.

[0071] L31: 1H NMR (400 MHz, CDCl3) δ 5.34 - 3.27 (m, 5H), 2.85 - 2.81 (m, 4H), 1.83 - 1.80 (m, 4H), 1.29 - 1.22 (m, 28H), 0.87 - 0.84 (m, 12H). HRMS (ESI - TOF): m / z calcd for: C27H55NO8P, 552.3665 [M + H]+; found: 552.3661.

[0072] L32: 1H NMR (400 MHz, CDCl3) δ 4.26 - 4.13 (m, 5H), 2.83 - 2.79 (m, 4H), 1.80 - 1.74 (m, 4H), 1.28 - 1.23 (s, 36H), 0.87 - 0.84 (m, 12H). HRMS (ESI - TOF): m / z calcd for: C31H63NO8P, 608.4291 [M + H]+; found: 608.4261.

[0073] L33: 1H NMR (400 MHz, CDCl3) δ 4.72 - 3.47 (m, 4H), 2.88 - 2.84 (s, 4H), 1.89 - 1.81 (m, 4H), 1.29 - 1.24 (m, 32H), 0.88 - 0.84 (m, 15H). HRMS (ESI-TOF): m / z calcd for: C31H60NO10PNa, 660.3853 [M+Na]+; found: 660.3608.

[0074] L34: 1H NMR (400 MHz, CDCl3) δ 4.49 - 3.91 (m, 4H), 2.86 - 2.82 (m, 4H), 1.86 - 1.80 (m, 4H), 1.29 - 1.23 (m, 40H), 0.88 - 0.85 (m, 15H). HRMS (ESI-TOF): m / z calcd for: C35H68NO10PNa, 716.4479 [M+Na]+; found: 716.4226.

[0075] L35: 1H NMR (400 MHz, CDCl3) δ 4.90 - 3.35 (m, 4H), 2.77 - 2.73 (m, 4H), 1.73 - 1.69 (m, 4H), 1.28 - 1.24 (m, 30H), 0.89 - 0.85 (m, 6H). HRMS (ESI-TOF): m / z calcd for: C31H50F17NO4P, 854.3206 [M+H]+; found: 854.3175.

[0076] L36: 1H NMR (400 MHz, CDCl3) δ 4.20 - 3.78 (m, 6H), 2.80 - 2.75 (m, 4H), 1.77 - 1.62 (m, 6H), 1.26 - 1.20 (m, 28H), 0.86 - 0.83 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C26H55NO6P, 508.3767 [M+H]+; found: 508.3749.

[0077] L37: 1H NMR (400 MHz, CDCl3) δ 4.13 - 3.82 (m, 6H), 2.80 - 2.76 (m, 4H), 1.75 - 1.62 (m, 6H), 1.27 - 1.22 (m, 36H), 0.88 - 0.84 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C30H63NO6P, 564.4393 [M+H]+; found: 564.4369.

[0078] L38: 1H NMR (400 MHz, CDCl3) δ 3.97 - 3.42 (m, 6H), 2.79 - 2.75 (m, 4H), 1.76 - 1.52 (m, 6H), 1.27 - 1.23 (m, 48H), 0.87 - 0.84 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C35H75NO5P, 620.5383 [M + H]+; found: 620.5356.

[0079] L39: 1H NMR (400 MHz, CDCl3) δ 4.23 - 3.62 (m, 4H), 2.76 - 2.72 (m, 4H), 2.48 - 2.42 (m, 1H), 1.71 - 1.54 (m, 6H), 1.27 - 1.24 (m, 60H), 0.88 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C42H89NO4P, 702.6529 [M + H]+; found: 702.6495.

[0080] L40: 1H NMR (400 MHz, CDCl3) δ 4.03 - 3.61 (m, 4H), 2.77 - 2.73 (m, 4H), 2.55 - 2.37 (m, 1H), 1.72 - 1.52 (m, 6H), 1.25 - 1.22 (m, 68H), 0.86 - 0.82 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C46H97NO4P, 758.7155 [M + H]+; found: 758.7140.

[0081] L41: 1H NMR (400 MHz, CDCl3) δ 4.18 - 3.56 (m, 4H), 2.80 - 2.76 (m, 4H), 2.53 - 2.44 (m, 1H), 1.79 - 1.73 (m, 6H), 1.28 - 1.24 (m, 34H), 0.87 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C29H63NO4P, 520.4495 [M + H]+; found: 520.4466.1

[0082] L42: 1H NMR (400 MHz, CDCl3) δ 4.19 - 3.44 (m, 4H), 3.00 - 2.88 (m, 1H), 2.83 - 2.79 (m, 4H), 1.82 - 1.77 (m, 6H), 1.35 - 1.23 (m, 42H), 0.88 - 0.84 (m, 12H). HRMS (ESI - TOF): m / z calcd for: C33H71NO4P, 576.5121 [M + H]+; found: 576.5089.

[0083] L43: 1H NMR (400 MHz, CDCl3) δ 4.19 - 3.58 (m, 4H), 3.04 - 2.91 (m, 1H), 2.83 - 2.74 (m, 4H), 1.81 - 1.79 (m, 6H), 1.28 - 1.24 (m, 48H), 0.88 - 0.85 (m, 12H). HRMS (ESI - TOF): m / z calcd for: C36H77NO4P, 618.5590 [M + H]+; found: 618.5555.

[0084] L44: 1H NMR (400 MHz, CDCl3) δ 5.44 - 5.29 (m, 2H), 4.08 - 3.71 (m, 4H), 2.80 - 2.72 (m, 4H), 2.14 - 1.93 (m, 4H), 1.82 - 1.51 (m, 6H), 1.27 - 1.22 (m, 36H), 0.88 - 0.83 (m, 9H). HRMS (ESI - TOF): m / z calcd for: C32H67NO4P, 560.4808 [M + H]+; found: 560.4774.

[0085] L45: 1H NMR (400 MHz, CDCl3) δ 5.54 - 5.29 (m, 2H), 4.05 - 3.59 (m, 4H), 2.90 - 2.63 (m, 4H), 2.49 - 2.28 (m, 2H), 2.10 - 1.91 (m, 2H), 1.76 - 1.49 (m, 4H), 1.35 - 1.22 (m, 36H), 0.88 - 0.83 (m, 9H). HRMS (ESI - TOF): m / z calcd for: C31H65NO4P, 546.4651 [M + H]+; found: 546.4624.

[0086] L46: 1H NMR (400 MHz, CDCl3) δ 6.03 - 5.93 (m, 2H), 5.58 - 5.48 (m, 2H), 3.99 - 3.73 (m, 4H), 2.84 - 2.76 (m, 4H), 2.06 - 1.98 (m, 2H), 1.76 - 1.72 (m, 6H), 1.71 (d, J = 6.8 Hz, 3H), 1.28 - 1.24 (m, 38H), 0.88 - 0.85 (m, 6H). HRMS (ESI-TOF): m / z calcd for: C34H69NO4P, 586.4964 [M+H]+; found: 586.4944.

[0087] L47: 1H NMR (400 MHz, CDCl3) δ 5.76 - 5.20 (m, 4H), 4.30 - 3.74 (m, 4H), 2.80 - 2.57 (m, 4H), 2.28 - 1.93 (m, 6H), 1.76 - 1.52 (m, 4H), 1.27 - 1.23 (m, 30H), 0.88 - 0.84 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C31H63NO4P, 544.4495 [M+H]+; found: 544.4464.

[0088] L48: 1H NMR (400 MHz, CDCl3) δ 6.20 - 5.91 (m, 1H), 4.40 - 3.97 (m, 6H), 2.81 - 2.77 (m, 4H), 1.77 - 1.75 (m, 4H), 1.28 - 1.24 (m, 28H), 0.88 - 0.85 (m, 6H). HRMS (ESI-TOF): m / z calcd for: C33H51F20NO4P, 936.3236 [M+H]+; found: 936.3189.

[0089] L49: 1H NMR (400 MHz, CDCl3) δ 4.81 - 4.69 (m, 1H), 4.09 - 3.93 (m, 4H), 3.65 - 3.62 (m, 1H), 2.82 - 2.78 (m, 4H), 1.78 - 1.73 (m, 4H), 1.47 - 1.44 (m, 2H), 1.28 - 1.24 (m, 28H), 0.87 - 0.83 (m, 15H). HRMS (ESI-TOF): m / z calcd for: C29H61NO6P, 550.4237 [M+H]+; found: 550.4208.

[0090] L50: 1H NMR (400 MHz, CDCl3) δ 4.78 - 4.72 (m, 1H), 4.12 - 3.90 (m, 4H), 3.65 - 3.62 (m, 1H), 2.82 - 2.78 (m, 4H), 1.78 - 1.77 (m, 4H), 1.49 - 1.45 (m, 2H), 1.28 - 1.23 (m, 36H), 0.88 - 0.85 (m, 15H). HRMS (ESI-TOF): m / z calcd for: C33H69NO6P, 606.4863 [M+H]+; found: 606.4830.

[0091] L51: 1H NMR (400 MHz, CDCl3) δ 6.18 - 5.42 (m, 1H), 5.32 - 4.61 (m, 1H), 4.24 - 3.72 (m, 4H), 2.86 - 2.81 (m, 4H), 1.86 - 1.81 (m, 6H), 1.30 - 1.13 (m, 34H), 0.90 - 0.86 (m, 15H). HRMS (ESI-TOF): m / z calcd for: C32H67NO4P, 560.4808 [M+H]+; found: 560.4798.

[0092] L52 ZP66(2AP28): 1H NMR (400 MHz, CDCl3) δ 4.12 - 3.66 (m, 6H), 2.81 - 2.76 (m, 4H), 2.21 - 2.15 (m, 2H), 1.94 - 1.92 (m, 1H), 1.77 (s, 6H), 1.32 - 1.24 (m, 42H), 0.88 - 0.85 (m, 6H). HRMS (ESI-TOF): m / z calcd for: C34H69NO4P, 586.4964 [M+H]+; found: 586.4962.

[0093] L53: 1H NMR (400 MHz, CDCl3) δ 3.92 - 3.55 (m, 6H), 2.79 - 2.75 (m, 4H), 1.77 - 1.75 (m, 6H), 1.31 - 1.23 (m, 34H), 0.89 - 0.84 (m, 15H), 0.07 - 0.02 (m, 6H). HRMS (ESI-TOF): m / z calcd for: C33H73NO5PSi, 622.4996 [M+H]+; found: 622.4967.

[0094] L54: 1H NMR (400 MHz, CDCl3) δ 4.21 - 3.99 (m, 4H), 3.70 - 3.62 (m, 1H), 2.90 - 2.82 (m, 4H), 1.85 - 1.81 (m, 8H), 1.27 - 1.24 (m, 32H), 0.87 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C29H63NO4P, 520.4495 [M+H]+; found: 520.4470.

[0095] L55: 1H NMR (400 MHz, CDCl3) δ 4.25 - 4.00 (m, 6H), 3.71 - 3.62 (m, 1H), 2.86 - 2.82 (m, 4H), 1.85 - 1.79 (m, 8H), 1.28 - 1.23 (m, 40H), 0.88 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C33H71NO4P, 576.5121 [M+H]+; found: 576.5093.

[0096] L56: 1H NMR (400 MHz, CDCl3) δ 4.45 - 3.58 (m, 4H), 2.86 - 2.82 (m, 4H), 1.87 - 1.79 (m, 6H), 1.65 - 1.49 (m, 1H), 1.28 - 1.23 (m, 26H), 0.86 - 0.83 (m, 18H). HRMS (ESI-TOF): m / z calcd for: C28H61NO4P, 506.4338 [M+H]+; found: 506.4337.1

[0097] L57: 1H NMR (400 MHz, CDCl3) δ 4.48 - 4.16 (m, 4H), 2.87 - 2.83 (m, 4H), 1.86 - 1.81 (m, 6H), 1.54 - 1.47 (m, 1H), 1.30 - 1.24 (m, 34H), 0.88 - 0.85 (m, 18H). HRMS (ESI-TOF): m / z calcd for: C32H69NO4P, 562.4964 [M+H]+; found: 562.4944.

[0098] L58: 1H NMR (400 MHz, CDCl3) δ 5.86 - 5.72 (m, 1H), 5.32 - 5.00 (m, 2H), 4.71 - 4.39 (m, 1H), 4.16 - 3.42 (m, 4H), 2.81 - 2.77 (m, 4H), 1.79 - 1.74 (m, 6H), 1.27 - 1.23 (m, 26H), 0.86 - 0.83 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C26H55NO4P, 476.3869 [M+H]+; found: 476.3846.1

[0099] L59: 1H NMR (400 MHz, CDCl3) δ 5.85 - 5.76 (m, 1H), 5.24 - 5.03 (m, 2H), 4.55 - 4.49 (m, 1H), 4.02 - 3.58 (m, 4H), 2.82 - 2.78 (m, 4H), 1.82 - 1.76 (m, 6H), 1.28 - 1.23 (m, 34H), 0.87 - 0.84 (m, 9H). HRMS (ESI-TOF): m / z calcd for: C30H63NO4P, 532.4495 [M+H]+; found: 532.4468.

[0100] L60: 1H NMR (400 MHz, CDCl3) δ 4.21 - 4.18 (m, 4H), 2.82 - 2.73 (m, 4H), 1.82 - 1.76 (m, 6H), 1.64 - 1.39 (m, 1H), 1.28 - 1.20 (m, 30H), 0.87 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C27H59NO4P, 492.4182 [M+H]+; found: 492.4180.

[0101] L61: 1H NMR (400 MHz, CDCl3) δ 4.22 - 4.19 (m, 4H), 2.89 - 2.76 (m, 4H), 1.84 - 1.80 (m, 6H), 1.70 - 1.42 (m, 1H), 1.29 - 1.24 (m, 38H), 0.88 - 0.85 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C31H67NO4P, 548.4808 [M+H]+; found: 548.4810.

[0102] L62: 1H NMR (400 MHz, CDCl3) δ 4.16 - 3.56 (m, 6H), 2.82 - 2.76 (m, 4H), 1.79 - 1.50 (m, 5H), 1.28 - 1.21 (m, 38H), 0.88 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C30H65NO4P, 534.4651 [M+H]+; found: 534.4624.

[0103] L63: 1H NMR (400 MHz, CDCl3) δ 4.42 - 3.59 (m, 5H), 2.82 - 2.73 (m, 4H), 1.81 - 1.60 (m, 6H), 1.28 - 1.23 (m, 26H), 0.87 - 0.83 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C25H55NO4P, 464.3869 [M+H]+; found: 464.3846.

[0104] L64: 1H NMR (400 MHz, CDCl3) δ 4.39 - 3.50 (m, 5H), 2.82 - 2.78 (m, 4H), 1.82 - 1.80 (m, 6H), 1.27 - 1.22 (m, 34H), 0.87 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C29H63NO4P, 520.4495 [M+H]+; found: 520.4471.

[0105] L65: 1H NMR (400 MHz, CDCl3) δ 4.21 - 3.59 (m, 5H), 2.82 - 2.78 (m, 4H), 1.82 - 1.59 (m, 6H), 1.28 - 1.24 (m, 28H), 0.87 - 0.83 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C26H57NO4P, 478.4025 [M+H]+; found: 478.4008.

[0106] L66: 1H NMR (400 MHz, CDCl3) δ 4.75 - 3.58 (m, 5H), 2.82 - 2.78 (m, 4H), 1.82 - 1.62 (m, 6H), 1.28 - 1.23 (m, 36H), 0.88 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C30H65NO4P, 534.4651 [M+H]+; found: 534.4624.

[0107] L67: 1H NMR (400 MHz, CDCl3) δ 4.03 - 3.65 (m, 6H), 2.81 - 2.77 (m, 4H), 1.78 - 1.62 (m, 5H), 1.27 - 1.23 (m, 40H), 0.87 - 0.54 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C32H69NO4P, 562.4964 [M+H]+; found: 562.4935.

[0108] L68: 1H NMR (400 MHz, CDCl3) δ 4.21 - 3.65 (m, 4H), 2.96 - 2.93 (m, 1H), 2.88 - 2.80 (m, 4H), 1.81 - 1.59 (m, 6H), 1.28 - 1.23 (m, 30H), 0.86 - 2.82 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C27H59NO4P, 492.4182 [M+H]+; found: 492.4155.1

[0109] L69: 1H NMR (400 MHz, CDCl3) δ 4.22 - 3.64 (m, 4H), 3.00 - 2.93 (m, 1H), 2.84 - 2.80 (m, 4H), 1.82 - 1.54 (m, 6H), 1.28 - 1.24 (m, 38H), 0.87 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C31H67NO4P, 548.4808 [M+H]+; found: 548.4779.

[0110] L70: 1H NMR (400 MHz, CDCl3) δ 4.73 - 3.08 (m, 5H), 2.85 - 2.80 (m, 4H), 1.84 - 1.67 (m, 6H), 1.28 - 1.23 (m, 32H), 0.87 - 0.84 (m, 12H). HRMS (ESI-TOF): m / z calcd for: C28H61NO4P, 506.4338 [M+H]+; found: 506.4319.

[0111] Example 2: Synthesis of Lipid Nanomaterial Delivery System

[0112] 1H NMR was carried out using the characterization of the ionizable phospholipid in Example 1. A five-component lipid nanoparticle (LNP) was formulated. 1 1H NMR. A five-component lipid nanoparticle (LNP) was formulated.

[0113] For example, LNP1 is composed of L1, DOPE, cholesterol, DMG-PEG2000 and a cationic compound in a ratio of 50:40:20:0.75:20.

[0114] LNP65 is formulated with L65:DOPE:Cholestrol:DMG-PEG2000:cationic compound = 50:40:20:0.75:20.

[0115] The following are the characterization data of the 1H NMR spectrum of the obtained cationic compound: 1H NMR (400 MHz, CDCl3) δ 4.05 (t, J = 6.4 Hz, 12H), 2.88 - 2.36 (m, 46H), 1.65 - 1.57 (m, 12H), 1.30 - 1.26 (m, 156H), 0.88 (t, J = 6.8 Hz, 21H). 13C NMR (100 MHz, CDCl3) δ 172.70, 172.60, 172.52, 64.54, 62.67, 52.76, 49.68, 32.84, 32.64, 32.53, 32.46, 31.91, 29.68, 29.65, 29.55, 29.49, 29.35, 29.30, 28.62, 25.92, 25.82, 22.67, 14.09. HRMS (ESI-TOF): m / z calcd for: C127H248N5O14, 2067.8848 [M+Na]+; found: 2067.8862.

[0116] Example 3: Screening the efficiency of LNP1-LNP70 in delivering GFP mRNA in the GC-1 spermatogonial cell line

[0117] 1. Screening spermatogonial cell line LNPs

[0118] Using the five-component lipid nanoparticles synthesized in Example 2 and GFP mRNA (lipid / mRNA = 10 / 1, mass ratio), nanoparticles were prepared in a ratio of 1:3 by pipetting or a microfluidic device (Precision NanoSystems, Canada).

[0119] GC-1 cells were cultured in DMEM + 10% FBS + 1% PS (penicillin-streptomycin) at 37 °C and 5% CO₂. GC-1 cells were seeded into 24-well plates at 50,000 cells per well. After 24 h, GC-1 cells were treated with PBS (control), LNP1@GFPmRNA - LNP70@GFPmRNA at a treatment concentration of 500 ng / mL GFPmRNA. After 24 h, the cells were collected and analyzed by flow cytometry (CytoFLEX, Beckman, America).

[0120] The size and zeta potential of the phospholipid nanoparticles were measured using a NanoZS Zetasizer (Malvern, USA).

[0121] Please refer to Figure 2 , the size of LNP1@GFP to LNP70@GFP was approximately 120 nm( Figure 2 a). The potential ranged from 10 to 20 mV( Figure 2 b). 17 kinds of LNPs showed similar GFP-positive percentages in GC-1 cells, and the delivery efficiency of LNP65@GFP was approximately 50%( Figure 2 c). Therefore, LNP65 was used in the in vivo experiments in the following examples.

[0122] 2. Localization of LNP65-delivered Cre mRNA in mouse testes

[0123] Please refer to Figure 3 a, in this application, the tdTomato mouse model was used to study the effect of LNP65 delivering Cre mRNA by testicular injection.

[0124] The effect of LNP65 delivering Cre mRNA by rete testis injection was studied using the tdTomato mouse model. The concentration of Cre mRNA was 0.2 mg / mL, and the volume was 10 μL / testis. LNP@Cre mRNA was administered by rete testis injection. After 3 d of treatment, the testes were collected for subsequent experiments. Testicular index = testis weight / body weight × 100. Genotype analysis of the testes (by PCR technique). HE staining of testicular tissues. tdTomato fluorescence images.

[0125] Please refer to Figure 3 b, c, three days after rete testis injection of LNP@Cre mRNA, the size of the testes increased slightly compared with the control group. Genotype analysis of the testes by PCR found that the DNA of loxp-STOP-loxp-tdTomato was cleaved under the treatment of LNP65@Cre mRNA Figure 3d). Slight morphological changes were observed in the testes 3 days after injection of LNP65@Cre mRNA( Figure 3 e). After injection into the ureter, tdTomato expression was detected in the seminiferous tubules of the testes in 80% of the mice Figure 3 f, 3g).

[0126] To clarify the cellular localization of tdTomato during the development of the male germline lineage, it was found in this application that it was mainly expressed in differentiating spermatogonia (labeled by c-Kit) and Sertoli cells (labeled by Sox9). The results in this application indicate that LNP65 effectively delivered Cre mRNA to differentiating spermatogonia Figure 3 h) and Sertoli cells Figure 3 i), and edited spermatogonia and Sertoli cells. This suggests the possibility of obtaining edited spermatocytes, spermatids, and spermatozoa from the edited spermatogonia

[0127] 3. Obtain modified sperm from LNP65-edited spermatogonia within one spermatogenic cycle

[0128] Please refer to Figure 4 a. In this application, LNP65@Cre mRNA was used to edit spermatogonia in the testes of tdTomato mice, resulting in the production of sperm expressing the tdTomato gene during a complete 24-day spermatogenic cycle

[0129] Specifically, the tdTomato mouse model was used to study the effect of delivering Cre mRNA by rete testis injection with LNP65. The concentration of Cre mRNA was 0.2 mg / mL, and the volume was 10 μL / testis. LNP@Cre mRNA was administered by rete testis injection. After 24 days of treatment, the testes were collected for subsequent experiments. Testis index = testis weight / body weight × 100. Genotype analysis of the testes (by PCR technique). HE staining of testis tissue. tdTomato fluorescence images

[0130] Twenty-four days after intravenous injection of LNP65@Cre mRNA, the testes appeared red compared to the control group Figure 4 b. There was no significant difference in the testis weight between the control group and the group receiving LNP65@Cre mRNA injection Figure 4 c. Genotype analysis of the testes by PCR showed that the DNA of the loxP-STOP-loxP-tdTomato construct was successfully cleaved after treatment with LNP65@Cre mRNA Figure 4 d), and the number of cleaved cells increased compared to 3 days. In addition, no morphological changes were observed in the testes 3 days after injection Figure 4e). After 24 days, tdTomato expression was detected in spermatocytes, spermatozoa, and sperm ( Figure 4 f). These findings indicate that modified sperm were successfully obtained in one step by using lipid nanoparticles to deliver mRNA within a single spermatogenic cycle Figure 4 g).

[0131] 4. LNP65-edited spermatogonia were cleared within three spermatogenic cycles.

[0132] Please refer to Figure 5 a. After 100 days of testicular rete injection with LNP@Cre mRNA, the testes were collected for subsequent experiments. Testis index = testis weight / body weight × 100. Genotype analysis of the testes (by PCR technique). HE staining of testicular tissues. tdTomato fluorescence images.

[0133] After one cycle, compared with the control group, the testes showed an obvious red hue after intravenous injection of LNP65@Cre mRNA Figure 5 b. There was no significant difference in testis weight between the control group and the LNP65@Cre mRNA injection group Figure 5 c. PCR-based genotype analysis of the testes showed that the DNA of loxp-STOP-loxp-tdTomato was cleaved after treatment with LNP65@Cre mRNA Figure 5 d. In addition, compared with day 3 and day 24, the number of cleaved DNAs of loxp-STOP-loxp-tdTomato was less on day 100, indicating that LNP65-edited spermatogonia might be cleared from the testes. There were no obvious changes in testicular morphology 3 days after injection of LNP65@Cre mRNA Figure 5 e. However, 100 days after re-injection of LNP65@Cre mRNA, we observed tdTomato expression in Sertoli cells Figure 5 f. These results indicate that after three spermatogenic cycles, LNP65-edited spermatogonia were cleared from the testes, leaving only the edited Sertoli cells Figure 5 g.

[0134] In summary, in the above embodiments, lipid nanomaterials were used to deliver mRNA to spermatogonia, and spermatogonia were edited to obtain edited spermatocytes and sperm cells. First, in this embodiment, ionizable phospholipid nanoparticles LNP65 were developed to deliver mRNA to spermatogonia. Second, LNP65 successfully edited spermatogonia. Third, within one spermatogenic cycle, we generated modified sperm from LNP65-edited spermatogonia. Finally, after three spermatogenic cycles, we observed that LNP65-edited spermatogonia had been cleared from the testis.

[0135] Example 4: Verification of Delivery of Cre mRNA by Lipid Nanomaterial LNP65 to Knock Out ZNF143 Gene in Testis

[0136] Please refer to Figure 6 , male ZNF143 mice were divided into two groups: ZNF143 floxp / floxp group and ZNF143KO group. The ZNF143 floxp / floxp group was not treated, and the ZNF143KO group was injected with LNP@Cre mRNA through the rete testis. Testis tissues were obtained 3 days after treatment for HE histological staining and WB analysis.

[0137] The results were as Figure 7 shown. When Cre mRNA encapsulated with LNP65 was delivered to spermatogonia through rete testis injection, 3 days after injection, the testis tissue was damaged and the ZNF143 gene in the testis decreased.

[0138] 2. Testis tissues were obtained 24 days after the above treatment for testicular index statistics and HE histological staining studies. One-way ANOVA was used to analyze the statistical significance of the differences. The log-rank (Mantel-Cox) test was used to analyze the statistical significance. *P < 0.05; ***P < 0.001; ****P < 0.001.

[0139] Please refer to Figure 8 . 24 days after injection, the testis of the experimental group with the ZNF143 gene in the testis was significantly smaller than that of the control group, showing a phenotype of germ cell arrest / absence, and the ZNF143 knockout phenotype was successfully obtained in one step.

[0140] 3. Detection of ZNF143 Knockout Phenotype:

[0141] After ZNF143KO mice were mated with Stra8-Cre mice for 3 months, the testes of ZNF143 floxp / floxp and ZNF143KO were stained with hematoxylin and eosin (HE).

[0142] The results were as Figure 9As shown, the phenotype of spermatogonial cells with ZNF143 knockout obtained from traditional mating experiments is consistent with the above experiments.

[0143] In summary, in this embodiment, a method for one-step editing of the ZNF 143 gene is achieved. First, lipid nanoparticles were synthesized for mRNA delivery. Second, LNP65 effectively delivered Cre mRNA to achieve knockout of the ZNF 143 gene in the testes of male mice, providing a rapid and effective means for studying the gene function of mice.

[0144] Example 5: Verification of the treatment of spermatocyte arrest non-obstructive azoospermia by LNP65 delivering Rik08saRNA

[0145] Please refer to Figure 10 , in this example set, the Rik08KO mouse model with spermatocyte arrest was used to study the therapeutic effect of LNP65 delivering Rik08saRNA by rete testis injection. The concentration of Rik08saRNA was 0.2 mg / mL and the volume was 10 μL / testis. LNP65@Rik08saRNA was administered by rete testis injection. After 9 days of treatment, the testes and epididymides were collected for subsequent experiments.

[0146] After collecting the testes, paraffin sections were made, and the expression of Rik08 protein in the testes of Rik08KO mice was detected by immunofluorescence staining of Rik08 protein; the recovery of spermatogenesis in Rik08KO male mice was detected by HE staining of testicular tissues; the production of sperm in the testes of Rik08KO male mice treated with LNP65@Rik08saRNA was verified by immunofluorescence staining of PNA-lectin-labeled sperm.

[0147] Fluorescence images showed that Rik08 was expressed in spermatocytes in the Rik08KO + LNP65@Rik08saRNA group 9 days after testicular vein injection ( Figure 11 ).

[0148] Please refer to Figure 12 , in this example, sperm were observed in the seminiferous tubules of the Rik08KO + LNP65@Rik08saRNA group, while no sperm were found in the Rik08KO group ( Figure 12 ).

[0149] In addition, in this example, testicular sections were stained with peanut agglutinin (PNA)-lectin that binds to the acrosome of sperm. The results showed that approximately 60% of the seminiferous tubules in the Rik08KO + LNP65@Rik08saRNA group were positive for PNA-lectin staining ( Figure 13 ).

[0150] The epididymides were collected, images of the epididymides were made, and hematoxylin staining was performed. The results are as Figure 14 shown. Some sperm were found in the smears taken from the epididymides of the Rik08KO+LNP65@Rik08saRNA group. These findings indicate that LNP65 has the potential to rescue male infertility.

[0151] In summary, in this example, LNP65 was used to deliver Rik08saRNA to spermatogonial cells in the testes of Rik08KO male mice, achieving the restoration of the spermatogenesis process in Rik08KO male mice and treating the spermatocyte arrest caused by Rik08KO gene deficiency.

[0152] In summary, in this application, lipid nanoparticles are used to deliver mRNA to spermatogonial cells, and by editing spermatogonial cells, the application of obtaining edited spermatocytes and sperm cells can be achieved. In this application, an ionizable phospholipid nanomaterial LNP65 is provided, which can effectively deliver mRNA into spermatogonial cells in the testes of mice through rete testis injection. In this application, spermatogonial cells were successfully edited by this method, and through specific verification experiments, it was verified that within one spermatogenic cycle, modified sperm from LNP65-edited spermatogonial cells can be obtained. After three spermatogenic cycles, we observed the clearance of LNP65-edited spermatogonial cells from the testes; this application developed a lipid nanomaterial delivery mRNA system targeting spermatogonial cells, providing an application vector for the treatment of azoospermia caused by single-gene deletion, with broad application prospects.

Claims

1. A lipid nanoparticle material LNP65, characterized in that: The lipid nanoparticle LNP65 is prepared from L65, DOPE, Cholestrol, DMG-PEG2000 and a cationic compound.

2. A lipid nanoparticle material LNP65 according to claim 1, characterized in that: The ratio of L65, DOPE, Cholestrol, DMG-PEG2000 and the cationic compound is 50:40:20:0.75:

20.

3. Use of lipid nanoparticle LNP65 as a delivery carrier system in the preparation of a drug for treating male infertility, characterized in that: The lipid nanoparticle LNP65 is the lipid nanoparticle LNP65 described in claim 1 or 2.

4. Use of the lipid nanoparticle LNP65 according to claim 3 as a delivery carrier system in the preparation of a medicament for treating male infertility, characterized in that: The lipid nanoparticle LNP65 is used for delivering mRNA, and the lipid nanoparticle targets spermatogonia.

5. Use of the lipid nanoparticle LNP65 according to claim 4 as a delivery carrier system in the preparation of a medicament for treating male infertility, characterized in that: The mRNA is saRNA.

6. Use of the lipid nanoparticle LNP65 according to claim 5 as a delivery carrier system in the preparation of a medicament for treating male infertility, characterized in that: The saRNA is Rik08 self-replicating RNA, and the male infertility is non-obstructive azoospermia with spermatocyte arrest.

7. Use of lipid nanoparticle LNP65 as a delivery carrier system in the study of gene function products in mouse testis, characterized in that: The lipid nanoparticle rapidly edits testicular genes by delivering Cre mRNA.

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