Polypeptide, polypeptide / siRNA compound and application of polypeptide / siRNA compound

Abstract

The invention discloses a polypeptide, a polypeptide / siRNA compound and application thereof, and belongs to the field of biological medicine preparations. The amino acid sequence of the polypeptide disclosed by the invention is as follows: Nap-FFKYGFRRGHHHHRHHHR or Nap-FFKYGFRRGHHHHKHHK. A polypeptide / siRNA compound formed by the polypeptide and siRNA is uniform in particle size and good in stability, the hydrophobic end of the polypeptide / siRNA compound can respond to cathepsin B in lysosome to be assembled into nanofibers, a membrane of the lysosome is damaged, and then escape of the lysosome of the siRNA is promoted; after the siRNA / cationic peptide part is released into cytoplasm, the cationic peptide responds to the change of cytoplasm pH to reduce the electropositivity of the siRNA molecule, so that efficient cytoplasm delivery of the siRNA molecule is realized, and the siRNA molecule is promoted to play a silencing effect. The embodiment of the invention shows that the Pep / siRNA compound disclosed by the invention has relatively strong in-tumor retention capacity in a mouse body and can effectively inhibit the growth of breast cancer tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical preparations, and specifically relates to a polypeptide, a polypeptide / siRNA complex and applications thereof. Background Art

[0002] The research of siRNA drugs is of great significance for the treatment of many diseases. With the advancement of science and technology, siRNA drugs have gradually been used in the research and treatment of diseases that pose a greater threat to human health, such as cancer and cardiovascular diseases. As a nucleic acid drug, siRNA is a double-stranded RNA molecule composed of 21-23 nucleotides, which can specifically bind to the target mRNA, degrade mRNA by forming an RNA-induced silencing complex (RISC), and thus knock out the target gene to achieve the purpose of treatment. Compared with other types of drugs, siRNA can regulate protein expression at the mRNA level, and has the advantages of strong targeting, high efficacy and low toxicity. However, the structure of siRNA double-stranded nucleic acid molecules causes some difficulties in its drug development, such as poor stability and easy clearance in the body; large molecular weight and difficulty in entering cells in the body; immunogenicity and easy to cause side effects, etc. Therefore, research on siRNA delivery systems has become key.

[0003] Due to their good safety, non-viral vectors are increasingly being used for siRNA delivery. Among them, lipid-based nanoparticle delivery systems (LNPs) are the fastest growing and most mature drug delivery systems. However, LNPs still have limitations in clinical applications, such as immune-related adverse reactions caused by lipid-related components, side effects of PEG, and heterogeneity of particle composition.

[0004] The siRNA delivery system can protect siRNA from damage by various enzymes and microenvironments during the in vivo circulation process, and efficiently deliver it to target organs and target cells to exert silencing effects. Among them, promoting the endosomal / lysosomal escape of nucleic acid drugs is a key step in their therapeutic effects. However, the endosomal / lysosomal escape ability of siRNA drug carriers is currently limited. Studies have shown that only 1-2% of internalized siRNA can escape from the endosomes, and this only occurs within a short period of time after entering the cell. Moreover, even after the carrier is released into the cytoplasm, whether siRNA can be effectively released from the cytoplasm is another key to its effectiveness. If the binding ability of the carrier and siRNA is too weak, a relatively stable nanocarrier will not be formed, affecting the in vivo stability of the delivery process; if the binding ability of the carrier and siRNA is too strong, siRNA will not be effectively released in the cytoplasm, affecting its silencing effect. Therefore, it is of great significance to construct a safe and efficient polypeptide system with endosomal / lysosomal escape ability and intracellular siRNA release ability. Summary of the invention

[0005] In view of the problems existing in the siRNA delivery process in the prior art, the purpose of the present invention is to provide a polypeptide, a polypeptide / siRNA complex and its application. The polypeptide / siRNA complex, as a delivery carrier of nucleic acid drugs, can stably and efficiently enter cells during systemic circulation, has efficient endosomal / lysosomal escape ability, intracellular siRNA release ability and good in vivo biocompatibility, and is a convenient and promising anti-tumor strategy.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A polypeptide comprises a hydrophobic end (Nap), a self-assembling peptide (FFKY), a cathepsin B responsive peptide (FRRG) and a cationic peptide, wherein the amino acid sequence of the polypeptide is: Nap-FFKYGFRRGHHHRHHHR or Nap-FFKYGFRRGHHHKHHHK.

[0008] Among them, F is phenylalanine, Y is lysine, G is glycine, K is lysine, H is histidine, R is arginine, and Nap is 2-naphthylacetic acid.

[0009] The present invention also provides a polypeptide / siRNA complex, which consists of the above polypeptide and siRNA.

[0010] Preferably, the particle size of the polypeptide / siRNA complex is 70-150 nm.

[0011] Preferably, the N / P ratio of the polypeptide to the siRNA in the polypeptide / siRNA complex is (10-30):1; more preferably 20:1.

[0012] The present invention also provides a pharmaceutical composition, which comprises the above polypeptide / siRNA complex; the pharmaceutical composition is used for treating breast cancer.

[0013] The present invention also provides the use of the polypeptide / siRNA complex in preparing a product for delivering siRNA to cells or tissues.

[0014] Preferably, the cell is a breast cancer cell or the tissue is a breast cancer tissue.

[0015] The beneficial effects of the present invention are:

[0016] The present invention provides a polypeptide, a polypeptide / siRNA complex and its application. The polypeptide is an amphiphilic self-assembling polypeptide that responds to an enzyme, and the polypeptide / siRNA complex is constructed by the electrostatic attraction between R and K and the base, and the hydrogen bond interaction between Gly and His and the base. The polypeptide / siRNA complex has uniform particle size and good stability, and can be used as a delivery carrier of nucleic acid drugs to achieve intelligent delivery of siRNA molecules: on the one hand, the hydrophobic end of the polypeptide / siRNA complex can respond to cathepsin B in the lysosome to assemble into nanofibers, destroy the membrane of the lysosome, and then promote the lysosomal escape of siRNA; on the other hand, after the siRNA / cationic peptide part is released into the cytoplasm, the cationic peptide responds to the change of cytoplasmic pH to reduce its positive charge to promote the release of siRNA, enhance the silencing effect of siRNA molecules, and thus achieve efficient cytoplasmic delivery of siRNA molecules. In addition, the polypeptide / siRNA complex has good in vivo biocompatibility. The embodiment shows that the Pep / siRNA complex of the present invention has a strong intratumoral retention ability in mice, and can effectively inhibit the growth of breast cancer tumors.

[0017] The polypeptide synthesis method of the present invention is simple and easy to implement, and is suitable for mass production. The present invention will help solve the problems of heterogeneity of siRNA delivery vector composition and complex synthesis, and provide a new method for developing safer, easier to prepare, and more efficient polypeptide vectors in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the solid phase synthesis of the polypeptides Pep1 (Figure A) and Pep2 (Figure B) of the present invention.

[0019] Figure 2 HPLC chromatograms of the polypeptides Pep1 (Figure A) and Pep2 (Figure B) of the present invention.

[0020] Figure 3 Mass spectra of the polypeptides Pep1 (Figure A) and Pep2 (Figure B) of the present invention.

[0021] Figure 4 Figure 2 shows the particle size (Figure A), PDI (Figure B) and Zeta potential (Figure C) of Pep1 / siRNA NPs and Pep2 / siRNA NPs prepared with different N / P ratios.

[0022] Figure 5 Gel electrophoresis images of Pep1 / siRNA NPs and Pep2 / siRNA NPs prepared with different N / P ratios.

[0023] Figure 6Gel electrophoresis images of Pep1 / siRNA NPs and Pep2 / siRNA NPs after incubation with 50% serum, respectively.

[0024] Figure 7 The computer simulation diagrams of the molecular docking between the polypeptide Pep1 of the present invention and the siRNA molecule (Figure A) and the computer simulation diagrams of the molecular docking between the polypeptide Pep2 and the siRNA molecule (Figure B).

[0025] Figure 8 The high performance liquid chromatograms of Pep1 and Pep1 / siRNA NPs after incubation with cathepsin B (Figure A) and the high performance liquid chromatograms of Pep2 and Pep2 / siRNA NPs after incubation with cathepsin B (Figure B).

[0026] Fig. 9 The MALDI-TOF mass spectra of Pep1 / siRNA NPs after incubation with cathepsin B (Figure A) and the MALDI-TOF mass spectra of Pep2 / siRNA NPs after incubation with cathepsin B (Figure B).

[0027] Fig.10 TEM electron micrographs of Pep1 / siRNA NPs and Pep2 / siRNA NPs, as well as TEM electron micrographs of Pep1 / siRNA NPs and Pep2 / siRNA NPs after incubation with CatB, respectively.

[0028] Fig.11 This is a graph showing the results of detecting the cell uptake after incubation of Pep / siRNA NPs of the present invention with 4T1 cells.

[0029] Fig.12 Lysosome co-localization images of Pep1 / siRNA NPs, Pep2 / siRNA NPs and Cy5-siRNA after incubation with 4T1 cells for 6h and 12h, respectively.

[0030] Fig.13 Figure 1 is a graph showing the cell viability detection result after incubation of 4T1 cells with Pep1 / siRNA NPs of the present invention (Figure A) and a graph showing the cell viability detection result after incubation of 4T1 cells with Pep2 / siRNA NPs (Figure B).

[0031] Fig.14 This is a graph showing the expression level of Bcl-2 mRNA after incubation of Pep / siRNA NPs of the present invention with 4T1 cells.

[0032] Fig.15These are the in vivo imaging evaluations of Pep1 / siRNA NPs, Pep2 / siRNA NPs and Cy5-siRNA at the tumor sites of 4T1 tumor-bearing Balb / c mice.

[0033] Fig.16 These are the semi-quantitative results of in vivo imaging of Pep1 / siRNA NPs, Pep2 / siRNA NPs and Cy5-siRNA at the tumor site of 4T1 tumor-bearing Balb / c mice.

[0034] Fig.17 The graph shows the tumor growth curves of mice in different drug administration groups.

[0035] Fig.18 The figures are the tumor weight test results of mice in different drug administration groups.

[0036] Fig.19 The expression levels of Bcl-2 mRNA in tumor tissues of mice in different drug administration groups.

[0037] Fig. 20 The hemolysis conditions of different drug-administered groups.

[0038] Fig.21 Bio-TEM images of tumor tissues of mice in different drug-dosing groups.

[0039] Fig. 22 HE images of heart, liver, spleen, lung and kidney tissues of mice in different drug-administered groups. DETAILED DESCRIPTION

[0040] The technical solutions and beneficial effects of the present invention are further illustrated below by way of examples, but are not intended to limit the scope of the present invention in any way.

[0041] Example 1

[0042] Solid Phase Synthesis of Peptides

[0043] The invention provides an enzyme-responsive amphipathic self-assembling polypeptide, wherein the amino acid sequence of the polypeptide is: Nap-FFKYGFRRGHHHRHHHR (Pep1) or Nap-FFKYGFRRGHHHKHHHK (Pep2).

[0044] The peptide was commissioned to Beijing Baipeptide Biotechnology Co., Ltd. to be synthesized using standard Fmoc-peptide solid phase synthesis technology. The specific synthetic routes of Pep1 and Pep2 are as follows: Figure 1 A and Figure 1 As shown in B, first, different amino acids are sequentially coupled to the resin using 2-CTC resin, and then the polypeptide (Pep) is cut off from the resin using a cutting solution and separated and purified by high performance liquid chromatography.

[0045] Pep1 and Pep2 were prepared into 1 mg / mL solutions, and the purity of Pep was investigated using HPLC. Figure 2 As shown, the purity of Pep1 (Figure A) and Pep2 (Figure B) were both >95%, indicating that they can be used for subsequent studies.

[0046] Among them, the HPLC detection conditions are as follows:

[0047] Chromatographic column: Agela Technologies 100-5C18 column; mobile phase composition: phase A: water; phase B: acetonitrile (both containing 0.05% TFA); mobile phase ratio: 0min: 30% B; 20min: 80% B; 20.1min: 30% B; 25min: Stop; column temperature: 40℃; injection volume: 10μL; flow rate: 1mL / min; detection wavelength: 220nm.

[0048] The molecular weights of Pep1 and Pep2 were determined using a 3200QTRAP mass spectrometer and analyzed in positive ion mode. Figure 3 As shown, the mass spectrometry results of Pep1 showed that its main ion peak was [M+4H] 4+ (m / z=621), [M+5H] 5+ (m / z=497.6), [M+6H] 6+ (m / z=414.3) (Figure A), which is consistent with the theoretical value (2480.7), confirming that Pep1 was successfully synthesized.

[0049] The mass spectrometry results of Pep2 showed that its main ion peak was [M+4H] 4+ (m / z=606.7), [M+5H] 5+ (m / z=485.3) (Figure B), which is consistent with the theoretical value (2424.7), confirming that Pep2 was successfully synthesized.

[0050] Example 2

[0051] Construction of peptide / siRNA complexes

[0052] After the Pep1 and Pep2 solutions were diluted with DEPC water respectively, an equal volume of siRNA solution (DEPC solution) was added dropwise under vortexing and allowed to stand at room temperature for 30 min to obtain complexes, namely Pep1 / siRNA NPs and Pep2 / siRNA NPs.

[0053] The particle size, polymer dispersity index (PDI) and Zeta potential of Pep / siRNA NPs with different N / P were detected by dynamic light scattering (DLS) using Nano ZS 90 particle size analyzer.

[0054] like Figure 4 As shown in Figure 1, the average particle size of Pep / siRNA NPs is 70-150nm (Figure A), and the PDI ranges from 0.1-0.3 (Figure B), confirming that Pep and siRNA electrostatically bind to form a complex. At the same time, its Zeta potential increases with the increase of N / P (Figure C).

[0055] The appearance of Pep / siRNA NPs was observed by TEM, and the nanoparticles were spherical.

[0056] Agarose gel electrophoresis (AGE) was used to examine the ability of Pep to bind to siRNA in order to screen the optimal molar ratio of N / phosphate P in siRNA (N / P ratio) in the Pep system.

[0057] Take 10 μL of sample solution from each group, mix with 2 μL of 6× RNA Loading buffer, and add 10 μL of mixed sample solution to each well. The voltage was set to 100 V and the time was set to 30 min. After the electrophoresis, the bands were observed and photographed using a gel imaging system.

[0058] like Figure 5 As shown, Pep can completely bind to siRNA when N / P ≥ 10; while a too high N / P ratio (30:1) will cause Pep to bind to siRNA too tightly, which may affect the release of siRNA; the optimal N / P ratio of peptide to siRNA is 20:1.

[0059] In the following studies, Pep / siRNA NPs were prepared with an N / P ratio of peptide to siRNA of 20:1.

[0060] Example 3

[0061] Detection of serum protection ability of Pep / siRNA complex against siRNA

[0062] AGE was used to investigate the serum protection ability of Pep / siRNA NPs against siRNA.

[0063] Pep1 / siRNA NPs and Pep2 / siRNA NPs (N / P 20:1) were incubated with 50% fetal bovine serum (FBS) in a 37°C water bath, respectively. At 0.5, 2, 4, 8, 24 and 48 h of incubation, 20 μL of sample solution was taken out, and 1 μL of 0.5 M Na2EDTA·2H2O solution was immediately added, followed by 4 μL of 1 mg / mL sodium heparin solution. The samples were incubated in a 37°C water bath for 1 h and stored at -80°C. After the experiment, agarose gel electrophoresis was performed.

[0064] like Figure 6 As shown, compared with free siRNA, clear electrophoretic bands can still be observed after Pep1 / siRNA NPs and Pep2 / siRNA NPs were incubated with 50% FBS solution for 48 hours, indicating that the Pep / siRNA complex (ie, Pep / siRNA NPs) of the present invention can improve the stability of siRNA in 50% FBS (serum).

[0065] Figure 7 The computer simulation diagrams of the molecular docking between the polypeptide Pep1 of the present invention and the siRNA molecule (Figure A) and the computer simulation diagram of the molecular docking between the polypeptide Pep2 and the siRNA molecule (Figure B) are respectively shown. The siRNA and polypeptide structures were obtained using AlphaFold3 prediction modeling. Among them, 2-naphthylacetic acid was manually modified to the modeled polypeptide and the structure was optimized using the MMFF94 force field. The siRNA and polypeptide were docked using the HDOCK program, and HDOCK used a grid step size of 1.2 and an angle step size of 15. After the docking was completed, the conformation with the best score in each group of docking results was selected for analysis.

[0066] It can be seen that the polypeptide of the present invention and the siRNA molecule form a complex mainly through the electrostatic attraction between R and K and the base, and the hydrogen bond interaction between Gly and His and the base. Specifically, Lys3, Arg 7, Arg 17 of Pep1 form salt bridges with the base due to electrostatic interaction and hydrogen bonding, Arg 8 forms an electrostatic interaction with the base, and Tyr4, Gly5, His10, His15 form hydrogen bonds with the base. Lys13 and Lys 17 of Pep2 form electrostatic interactions with the base, and His14 and His15 form hydrogen bonds with the base.

[0067] Example 4

[0068] Detection of enzymatic degradation of Pep / siRNA NPs after incubation with cathepsin B

[0069] Pep1 / siRNA NPs and Pep2 / siRNA NPs (N / P 20:1) were mixed with equal volumes of 5 μg / mL cathepsin B (CTSB) (pH 5, 25 mM MES buffer) solution, incubated in a 37°C water bath for 6 h, and 100 μL of the sample solution was taken for detection by HPLC.

[0070] like Figure 8 As shown, after Pep1 / siRNA NPs (Figure A) and Pep2 / siRNA NPs (Figure B) were incubated with CTSB, the original chromatographic peaks disappeared and new chromatographic peaks appeared, indicating that the Pep / siRNA NPs of the present invention can be enzymatically hydrolyzed by CTSB.

[0071] Pep1 / siRNA NPs and Pep2 / siRNA NPs (N / P 20:1) were mixed with 5 μg / mL cathepsin B (pH 5, 25 mM MES buffer) solution in equal volumes and incubated in a 37°C water bath for 24 h. 1 μL of sample solution was taken and air-dried, and then 1 μL of 15 mg / mL CHCA solution was added and mass spectrometry was performed by MALDI-TOF.

[0072] like Fig. 9 As shown, both Pep1 / siRNA NPs (Figure A) and Pep2 / siRNA NPs (Figure B) have ion peaks of m / z=1232.8 and 1288.9, indicating that the Pep / siRNA NPs of the present invention can be specifically cleaved by cathepsin B at the cleavage site (FRRG).

[0073] Pep1 / siRNA NPs and Pep2 / siRNA NPs (N / P 20:1) were mixed with equal volumes of 5 μg / mL cathepsin B (pH 5, 25 mM MES buffer) solution, incubated in a 37°C water bath for 24 h, and then the morphology was observed by TEM.

[0074] like Fig.10 As shown, it can be seen that the polypeptide of the present invention can form nanoparticles with uniform particle size with siRNA, and after Pep / siRNA NPs are incubated with CTSB, Pep / siRNA NPs are specifically cleaved by CTSB, and the hydrophobic part thereof self-assembles to form nanofibers.

[0075] Example 5

[0076] In vitro detection of the effect of Pep / siRNA NPs on mouse breast cancer (4T1 cells)

[0077] 1.4 Uptake of Pep / FAM-siRNA NPs by T1 cells (mouse breast cancer cell line)

[0078] The uptake of Pep / FAM-siRNA NPs by 4T1 cells (mouse breast cancer cell line) was quantitatively detected by flow cytometry.

[0079] 50 nM Pep1 / Cy5-siRNA NPs and Pep2 / Cy5-siRNA NPs were taken and incubated with 4T1 cells for 2 h and 6 h, respectively. The free Cy5-siRNA group was set as the negative control, and the cationic transfection reagent was set as the positive control. Flow cytometric analysis was performed using a BD FACSCalibur flow cytometer.

[0080] like Fig.11 As shown, the cellular uptake of Cy5-siRNA and Pep1 / Cy5-siRNA NPs and Pep2 / Cy5-siRNA NPs were all time-dependent, and Pep1 / Cy5-siRNA NPs and Pep2 / Cy5-siRNA NPs could increase the cellular uptake of siRNA, and the uptake was higher than that of the positive control group, indicating that the Pep / siRNA NPs of the present invention could significantly promote the uptake of Cy5-siRNA in 4T1 cells.

[0081] 2. Detection of lysosomal escape ability of Pep / siRNA NPs

[0082] CLSM was used to investigate the lysosomal escape ability of Pep / siRNA NPs.

[0083] Pep1 / Cy5-siRNA NPs (Cy5-siRNA final concentration was 200 nM) were incubated with Cy5-siRNA and 4T1 cells at 37°C, 5% CO2 for 6 h and 12 h, respectively. After washing three times with PBS, 500 μL Hoechst 33342 and 500 μL LysoTracker Green were added, respectively, incubated for 20 min, and observed under a laser scanning confocal microscope (Pep2 / Cy5-siRNA NPs were subjected to the same operation and observation).

[0084] like Fig.12As shown in the figure, 4T1 cells were incubated with Pep1 / Cy5-siRNA NPs and Pep2 / Cy5-siRNA NPs for 6 hours, and yellow fluorescence signals were observed, indicating that Cy5-siRNA (red) and lysosomes (green) were co-localized. This indicates that Pep1 / Cy5-siRNA NPs and Pep2 / Cy5-siRNA NPs entered the cells through the endocytosis pathway; when the incubation time was extended to 12 hours, more diffuse Cy5-siRNA red fluorescence signals were observed in the cytoplasm, indicating that Cy5-siRNA successfully escaped from the lysosome.

[0085] The above results indicate that the Pep / siRNA NPs of the present invention can effectively promote the escape of siRNA from lysosomes.

[0086] 3. Cytotoxicity assay of Pep / siBcl-2 NPs

[0087] First, Pep1 / siBcl-2 NPs and Pep2 / siBcl-2 NPs loaded with siBcl-2 molecules were constructed according to the method of Example 2, and the cytotoxicity of Pep / siBcl-2 NPs was investigated using the CCK-8 method.

[0088] Different concentrations of Pep1 / siBcl-2 NPs and Pep2 / siBcl-2 NPs (the final siRNA concentrations were 25, 50, 100, 150, 200, 250, 300 nM siBcl-2) were incubated with 4T1 cells for 48 hours, and the Control group (normal cell group), blank siRNA group (Pep / siN.C., 300 nM), and free siRNA group (siBcl-2, 300 nM) were set up at the same time. The drug solution was discarded, PBS was washed three times, 100 μL of 10% CCK-8 solution was added to each well, and the culture was continued for 2 hours. The absorbance of each well at 450 nm was measured using a multifunctional microplate reader to calculate the cell survival rate.

[0089] like Fig.13 As shown, the cytotoxicity of Pep1 / siBcl-2 NPs (Figure A) and Pep2 / siBcl-2 NPs (Figure B) to 4T1 cells was concentration-dependent. Under the same concentration conditions, compared with siBcl-2 and Pep / siBcl-2 NPs, the Pep / siRNA NPs of the present invention can significantly inhibit the viability of 4T1 cells.

[0090] 4. Silencing efficacy of Pep / siRNA NPs on 4T1 cells

[0091] qRT-PCR was used to detect the silencing efficacy of Pep / siRNA NPs on 4T1 cells.

[0092] Pep1 / siBcl-2 NPs and Pep2 / siBcl-2 NPs (siRNA final concentration was 200 nM) were incubated with 4T1 cells for 6 h, respectively. At the same time, the Control group (normal cell group), blank siRNA group (siN.C., 200 nM), and free siRNA group (siBcl-2, 200 nM) were set up. The drug solution was discarded, and each well was replaced with fresh culture medium containing serum. The cells were incubated at 37°C and 5% CO2 for 24 h, and the expression level of Bcl-2 mRNA was detected by qRT-PCR.

[0093] like Fig.14 As shown, compared with the free siBcl-2 group, Pep1 / siBcl-2 NPs and Pep2 / siBcl-2 NPs can significantly inhibit the expression of siBcl-2 mRNA. This result shows that the Pep / siRNA NPs of the present invention can inhibit the expression of Bcl-2 gene and have excellent cell silencing efficacy.

[0094] The siBcl-2 sequence is: sense: 5′-GGAGGAUUGUGGCCUUCUU-3′.

[0095] The primer sequences used for quantitative PCR analysis are as follows:

[0096]

[0097] Example 6

[0098] Detection of the retention ability of Pep / siRNA NPs in tumor sites of tumor-bearing Balb / c mice

[0099] Small animal in vivo imaging was used to investigate the retention ability of Pep / siRNA NPs in tumor sites of tumor-bearing Balb / c mice.

[0100] 5% glucose solution was used instead of DEPC water to dissolve Pep and siRNA, and Pep1 / siRNA NPs and Pep2 / siRNA NPs were prepared according to the above method. A subcutaneous 4T1 tumor model of Balb / c mice was established, and Pep1 / Cy5-siRNA NPs, Pep2 / Cy5-siRNA NPs and free Cy5-siRNA were injected into the tumor. At the specified time points (1, 2, 4, 8, 12, 24, 48h) after drug injection, the distribution of Pep / Cy5-siRNA NPs in the tumor site was observed using a small animal in vivo imaging device.

[0101] like Fig.15As shown, the in vivo stability of siRNA molecules is poor and they are easily degraded by various enzymes in the body, resulting in a faster degradation rate in the body. However, the fluorescence of Pep1 / siRNA NPs and Pep2 / siRNA NPs in the tumor site can be observed within 48 hours, indicating that the polypeptide of the present invention can effectively protect siRNA, improve its in vivo stability, and enhance its retention ability in the tumor site.

[0102] Semi-quantitative analysis of the fluorescence intensity of the tumor site at different time points was performed. Fig.16 As shown in the figure, as time goes by, the fluorescence intensity of Pep1 / siRNA NPs and Pep2 / siRNA NPs in the tumor site continues to increase, reaching a peak at around 4 hours, and the fluorescence intensity in the tumor site is the strongest. The fluorescence intensity of Cy5-siRNA is affected by both concentration and molecular aggregation. After the vector is injected into the tumor site, the aggregation between molecules is strong at the beginning, which may be affected by fluorescence quenching caused by aggregation. As time goes by, the vector penetrates into the tumor site, the aggregation between fluorescent molecules decreases, and the fluorescence intensity gradually increases. At the same time, Cy5-siRNA will also be gradually degraded by the body, reducing its fluorescence intensity.

[0103] The above results indicate that the Pep / siRNA NPs of the present invention can effectively protect siRNA molecules in Balb / c mice, improve their in vivo stability, enhance their retention capacity at tumor sites, and effectively inhibit tumor growth.

[0104] Example 7

[0105] In vivo antitumor efficacy testing of Pep / siRNA NPs

[0106] A subcutaneous 4T1 tumor model was established in Balb / c mice to evaluate the in vivo antitumor efficacy of Pep / siRNA NPs.

[0107] When the tumor volume of mice reached about 50 mm 3 Afterwards, Balb / c mice were randomly divided into six groups (n=5), and each group of mice was injected with Pep1 / siBcl-2 NPs, Pep2 / siBcl-2 NPs, Pep1, Pep2, siBcl-2, and 5% glucose (siBcl-2: 4μg / mouse, N / P=20) into the tumor, once every other day for three consecutive weeks. During the treatment, tumor growth was monitored. After the treatment, the mice were killed by cervical dislocation, and the tumors were dissected and weighed.

[0108] like Fig.17As shown, the tumor growth rate of the 5% glucose group (Glucose) was rapid, the free siBcl-2 and free Pep groups had weak ability to inhibit tumor growth, while the Pep1 / siBcl-2 NPs group and the Pep2 / siBcl-2 NPs group could effectively inhibit tumor growth.

[0109] Fig.18 The results showed that the in vitro tumor weights of the Pep1 / siBcl-2 NPs group and the Pep2 / siBcl-2 NPs group were the smallest, indicating that the Pep / siRNA NPs of the present invention have the strongest ability to inhibit tumor growth.

[0110] Mouse tumor tissues from different groups were collected, and qRT-PCR was used to analyze the expression of Bcl-2 gene in mouse tumor tissues. Mouse tumor tissues from different groups were washed with PBS, dried with filter paper, placed in sterile EP tubes, chopped with scissors, added with lysis buffer, mixed with a tissue homogenizer, and total mRNA was extracted according to the above method for subsequent qRT-PCR experiments to detect the inhibition of Bcl-2 gene in vivo by Pep1 / siBcl-2 NPs and Pep2 / siBcl-2 NPs.

[0111] like Fig.19 As shown in the figure, compared with the 5% glucose group, the ability of the free siBcl-2 group to silence Bcl-2 mRNA in tumor tissues was very weak. However, the Pep1 / siBcl-2 NPs and Pep2 / siBcl-2 NPs groups could significantly inhibit the expression level of Bcl-2 mRNA in tumor tissues, showing a strong silencing effect.

[0112] Prepare 2% erythrocyte suspension with saline. Prepare Pep1 / siRNA NPs and Pep2 / siRNA NPs with 5% glucose solution. Mix equal volumes of Pep1 solution and Pep1 / siRNA NPs with 2% erythrocyte suspension, and use 5% glucose solution and 1% Trition X-100 solution as negative control and positive control, respectively (Pep2 solution and Pep2 / siRNA NPs are operated in the same way). Incubate in a 37°C water bath for 1 hour. After incubation, centrifuge at 3500rpm and 4°C for 10 minutes. Remove the supernatant to a 96-well plate, measure the absorbance at λ=540nm, and calculate the hemolysis rate of each group.

[0113] like Fig. 20 As shown, the hemolysis rates of Pep1, Pep2, Pep1 / siRNA NPs and Pep2 / siRNA NPs were all less than 3%, indicating that the polypeptide and polypeptide / siRNA NPs of the present invention have good blood compatibility.

[0114] Tumor tissues from different groups were further collected, divided into small pieces, and fixed in glutaraldehyde. The fixed tumor tissues were made into specimens and observed using a transmission electron microscope.

[0115] like Fig.21 As shown, compared with the control group (5% glucose solution), obvious nanofibers can be observed in the Pep1 / siRNA NPs and Pep2 / siRNA NPs groups in tumor cells. The presence of ruptured vesicles in tumor cells can be inferred to be lysosomes with fiber destruction. The experimental results further show that the hydrophobic end of the polypeptide / siRNA complex of the present invention can respond to cathepsin B in the lysosome to assemble into nanofibers, destroy the lysosomal membrane, and promote the lysosomal escape of siRNA.

[0116] The H&E staining method was used to perform histological analysis of the tumor tissues and major organs of mice in the different drug injection groups. 24 hours after the last administration, the mice were killed by cervical dislocation, and the tumors and major organs such as the heart, liver, spleen, lungs and kidneys were dissected and collected. After fixation with 4% paraformaldehyde, slice specimens were made and H&E staining was performed. The tissue sections were observed using an optical microscope.

[0117] like Fig. 22 As shown, the H&E staining results of the main organs of tumor-bearing mice in each group showed that compared with the control group (5% glucose solution), no obvious lesions were found in the main organs of tumor-bearing mice in the Pep1 / siRNA NPs and Pep2 / siRNA NPs groups, indicating that the polypeptide / siRNA complex of the present invention (i.e., Pep / siRNA NPs) has good in vivo biocompatibility.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is: Nap-FFKYGFRRGHHHRHHHR or Nap-FFKYGFRRGHHHKHHHK.

2. A polypeptide / siRNA complex, characterized in that: The polypeptide / siRNA complex consists of the polypeptide according to claim 1 and siRNA.

3. The polypeptide / siRNA complex according to claim 2, characterized in that: The particle size of the polypeptide / siRNA complex is 70-150 nm.

4. The polypeptide / siRNA complex according to claim 2, characterized in that The N / P ratio of the polypeptide to the siRNA in the polypeptide / siRNA complex is (10-30):

1.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide / siRNA complex according to any one of claims 2 to 4.

6. Use of the polypeptide / siRNA complex according to any one of claims 2 to 4 in the preparation of a product for delivering siRNA to cells or tissues.

7. The use according to claim 6, characterized in that: The cells are breast cancer cells or the tissues are breast cancer tissues.

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