Endoplasmic reticulum targeted polypeptide nano-drug, preparation method and characterization method

Abstract

The invention relates to an endoplasmic reticulum targeting polypeptide nano-drug with glutathione responding to morphology transformation, a preparation method and a related performance characterization method. The endoplasmic reticulum targeting polypeptide nano-drug with glutathione responding to morphology transformation comprises a targeting unit, an assembling unit and a functional unit, wherein the targeting unit comprises one or more of targeting peptide sequences RVMLIGK, RVMLIEK, KDEL and sulfonyl groups with an endoplasmic reticulum targeting function; the functional unit comprises a glutathione response group disulfide bond, and the assembly primitive sequence and the assembly regulation sequence are covalently connected through the disulfide bond. The assembly structure of the polypeptide nano-drug has sensitive glutathione responsiveness, so that the polypeptide nano-drug can generate assembly morphology transformation in glutathione overexpressed tumor cells, and the polypeptide nano-drug is simple and convenient to synthesize and prepare and has wide application prospects in the aspects of drug delivery, tumor treatment and the like.

Description

Technical Field

[0001] The present application belongs to the technical field of polypeptide nanomedicines, and in particular relates to an endoplasmic reticulum-targeted polypeptide nanomedicine that responds to morphological changes in response to glutathione, a preparation method of an endoplasmic reticulum-targeted polypeptide nanomedicine that responds to morphological changes in response to glutathione, and a performance characterization method of an endoplasmic reticulum-targeted polypeptide nanomedicine that responds to morphological changes in response to glutathione. Background Art

[0002] The physiological environment of tumor tissues and lesions of complex diseases is complex and diverse, and is heterogeneous compared to normal tissues. Therefore, traditional drugs that cannot adapt to the complex conditions of the lesion site often show low drug utilization and drug resistance. The development of adaptive responsive nanomedicines shows great potential in the diagnosis and treatment of diseases.

[0003] The adaptability of nanomedicines usually depends on their own stimulus responsiveness. It is feasible to develop glutathione-responsive peptide nanomedicines for the special condition of high glutathione concentration in tumor tissue. Although some glutathione-responsive groups have been introduced into nanomedicines, it is still challenging to precisely control the nanoassembly structure of peptides, which limits the advantages of peptide nanomedicines such as natural biocompatibility.

[0004] The endoplasmic reticulum plays an important role in the life activities of cells and is directly involved in the synthesis and processing of intracellular proteins. When the protein is disturbed during the processing and folding process, the cell will initiate the unfolded protein response to maintain the intracellular protein homeostasis. However, when the stimulus persists, it will lead to the generation of endoplasmic reticulum stress, which will eventually lead to cell dysfunction and even cell apoptosis. Therefore, endoplasmic reticulum stress has become an effective strategy for disease treatment and has been used in many diseases such as tumor treatment. However, since the unfolded protein response of the cell itself can regulate endoplasmic reticulum stress to alleviate endoplasmic reticulum stress, the therapeutic effect of targeting endoplasmic reticulum stress is limited, which limits its further application. Summary of the invention

[0005] In view of this, the development of polypeptide nanomedicines with endoplasmic reticulum targeting and endoplasmic reticulum stress stimulating properties is of great significance and broad application prospects. The present application aims to provide an endoplasmic reticulum-targeted polypeptide nanomedicine with glutathione-responsive morphological transformation.

[0006] The first aspect of the present application provides an endoplasmic reticulum-targeted polypeptide nanomedicine that responds to morphological changes in response to glutathione, wherein the endoplasmic reticulum-targeted polypeptide nanomedicine includes a targeting unit, an assembly unit and a functional unit, and the targeting unit includes one or more of the targeting peptide sequences RVMLIGK, RVMLIEK, KDEL, and sulfonyl groups with endoplasmic reticulum targeting function.

[0007] In some embodiments of the present application, the assembly unit includes an assembly motif sequence and an assembly regulatory sequence, and the combination of the assembly motif sequence and the assembly regulatory sequence includes at least one of the following: 1) the assembly motif sequence EEEK (C 16 )GGC 12 VVAA and assembly regulatory sequence RRRRRR; 2) assembly motif sequence EEEK (C 16 )EEC 12 VVAA and assembly regulatory sequence RRRRRR.

[0008] In some embodiments of the present application, the functional unit includes a glutathione-responsive group disulfide bond, and the assembly motif sequence and the assembly regulatory sequence are covalently linked via the disulfide bond.

[0009] The second aspect of the present application provides a method for preparing an endoplasmic reticulum-targeted polypeptide nanodrug with glutathione-responsive morphological transformation, and the preparation method specifically comprises: S1, synthesizing an assembly primitive sequence, an assembly regulatory sequence and a glutathione-responsive group disulfide bond by a standard Fmoc solid phase peptide synthesis (SPPS) method, using piperidine as a deprotecting agent and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) as a condensing agent, and designing and synthesizing a polypeptide assembly sequence containing glutathione responsiveness; S2, mixing the targeting peptide sequence with endoplasmic reticulum targeting function with the polypeptide assembly sequence obtained in step S1 in proportion to obtain a mixed solution for standby use; S3, annealing the mixed solution obtained in step S2 to obtain a polypeptide co-assembly system solution, which contains an endoplasmic reticulum-targeted polypeptide nanodrug with glutathione-responsive morphological transformation.

[0010] In some embodiments of the present application, the solvent of the mixed solution in step S2 and the polypeptide co-assembly system solution in step S3 is one of buffer, ultrapure water, deionized water, and Milli-Q water.

[0011] In some embodiments of the present application, in step S2, the targeting peptide sequence accounts for 0.1%-50% of the total molar number of the mixed solution, and the molar concentration of the mixed solution is 0.1 μmol / L-10 mmol / L.

[0012] In some embodiments of the present application, the annealing temperature of the annealing operation in step S3 is 10° C.-100° C., and the annealing time is 0.1 h-100 h.

[0013] The third aspect of the present application provides a method for characterizing the performance of an endoplasmic reticulum-targeted polypeptide nanodrug that responds to morphological changes in response to glutathione, wherein the redox method of the endoplasmic reticulum-targeted polypeptide nanodrug obtained according to the above-mentioned preparation method: the reducing agent that responds to the morphological changes of the endoplasmic reticulum-targeted polypeptide nanodrug includes a glutathione reducing agent, the concentration of the glutathione reducing agent is 0.1μmol / L-1mol / L, the redox temperature is 0℃-100℃, and the oxidation reaction time is 0.1h-100h.

[0014] In some embodiments of the present application, cell experiments are included, in which the normal cells used are mouse embryonic fibroblasts (3T3), and the cancer cells used are mouse breast cancer cells (4T1).

[0015] Beneficial effects of this application:

[0016] (1) This application relates to An endoplasmic reticulum-targeted peptide nanodrug with glutathione-responsive morphological transformation :The glutathione-responsive morphological transformation polypeptide nanodrug molecule contains a disulfide bond, which can be broken and reduced in the presence of glutathione, resulting in a change in the assembly structure of the polypeptide molecule, inducing the polypeptide assembly structure to change from nanoparticles to nanofibers; the endoplasmic reticulum-targeted polypeptide nanodrug has good biocompatibility, biological activity and biodegradability. As an important biologically active substance in organisms, polypeptides have good biocompatibility, and the molecular structure in the assembly sequence can be reasonably designed and regulated to achieve precise regulation of the polypeptide assembly morphology.

[0017] (2) This application relates to an endoplasmic reticulum-targeted polypeptide nanomedicine that responds to morphological changes of glutathione Preparation Law :This method rationally designs a polypeptide assembly sequence containing disulfide bonds, and transforms the assembly structure from nanoparticles to nanofibers in the presence of glutathione; and co-assembles a polypeptide nanodrug with endoplasmic reticulum targeting properties by connecting a polypeptide sequence with endoplasmic reticulum targeting function to the polypeptide assembly sequence through a covalent bond. The preparation method of the endoplasmic reticulum targeting polypeptide nanodrug with glutathione-responsive morphological transformation is simple, with mild reaction conditions, simple operation, and easy industrialization, and has great application potential in anti-inflammatory, anti-tumor, and antibacterial aspects.

[0018] (3) This application relates to an endoplasmic reticulum-targeted polypeptide nanomedicine that responds to morphological changes of glutathione Performance Table Collection MethodThe endoplasmic reticulum-targeted peptide nanodrug can be reduced under the condition of high glutathione expression in tumor cells, so that its assembly morphology is transformed into nanofibers. Under the action of the endoplasmic reticulum targeting group, it is targeted to the endoplasmic reticulum. A large number of nanofibers are aggregated in the endoplasmic reticulum, causing endoplasmic reticulum stress and inducing cell apoptosis. The drug is transformed from nanoparticles to nanofibers, so that the peptide nanodrug is aggregated in the tumor in the form of nanoparticles, which is conducive to the uptake of tumor cells. It is reduced to nanofibers by glutathione in tumor cells, which is conducive to improving intracellular retention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings.

[0020] Figure 1 The following are some of the polypeptide sequences and molecular structure diagrams of functionalized polypeptides in the examples of this application;

[0021] Figure 2 The HPLC chromatogram of some polypeptide sequences in the examples of this application;

[0022] Figure 3 The atomic force microscope image and transmission electron microscope image of the shape-changing polypeptide in the examples of the present application before glutathione reduction;

[0023] Figure 4 The atomic force microscope image and transmission electron microscope image of the shape-changing polypeptide in the examples of the present application after glutathione reduction;

[0024] Figure 5 : is a circular dichroism spectrum of the shape-changing polypeptide in the examples of the present application before and after glutathione reduction;

[0025] Figure 6 IR spectra of the shape-changing polypeptide in the examples of the present application before and after glutathione reduction;

[0026] Figure 7 The atomic force microscope image of the endoplasmic reticulum-targeted polypeptide nanomedicine with glutathione-responsive morphological transformation before and after glutathione reduction in the examples of the present application;

[0027] Figure 8 The confocal laser microscope images of the endoplasmic reticulum targeting polypeptide nanomedicine with glutathione responsive morphological transformation in the examples of the present application, imaged by the endoplasmic reticulum probe and the endoplasmic reticulum targeting group, after 1 hour and 6 hours of cell uptake;

[0028] Fig. 9The confocal laser microscope images of the endoplasmic reticulum targeting polypeptide nanomedicine with glutathione responsive morphological transformation in the examples of the present application, imaged by the endoplasmic reticulum probe and the endoplasmic reticulum targeting group, after 12 hours and 24 hours of cell uptake;

[0029] Fig.10 This is a cytotoxicity analysis of the glutathione-responsive morphological transformation endoplasmic reticulum-targeted polypeptide nanomedicine and a polypeptide without an endoplasmic reticulum-targeting group in the examples of this application. DETAILED DESCRIPTION

[0030] In order to make the application purpose, technical solution and beneficial technical effect of the present application clearer, the present application is further described in detail in conjunction with the embodiments below. It should be understood that the embodiments described in this specification are only for explaining the present application, not for limiting the present application.

[0031] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unclearly recorded range; and any lower limit can be combined with other lower limits to form an unclearly recorded range, and any upper limit can be combined with any other upper limit to form an unclearly recorded range. In addition, although not clearly recorded, each point or single value between the range endpoints is included in the range. Thus, each point or single value can be combined with any other point or single value as its own lower limit or upper limit or with other lower limits or upper limits to form an unclearly recorded range.

[0032] The first aspect of the present application provides an endoplasmic reticulum-targeted polypeptide nanomedicine that responds to morphological changes of glutathione, wherein the endoplasmic reticulum-targeted polypeptide nanomedicine includes a targeting unit, an assembly unit and a functional unit, and the targeting unit includes a targeting peptide sequence RVMLIGK having an endoplasmic reticulum targeting function. In other specific embodiments, the targeting peptide sequence can be selected from one or more of polypeptides or functional groups such as RVMLIEK, KDEL, and sulfonyl groups.

[0033] In some embodiments, the assembly unit includes an assembly motif sequence and an assembly regulatory sequence. The combination of the assembly motif sequence and the assembly regulatory sequence may be: the assembly motif sequence EEEK(C 16 )GGC 12 VVAA, assembly regulatory sequence RRRRRR. In other embodiments, the assembly motif sequence can be selected from EEEK (C 16 )EEC 12 VVAA、RRRK(C 16 )EEC 12 VVAA、RRRK(C 16 )EEC 12 VVAA, EEEK(C 16 )EEC12 In some other embodiments, the assembly regulatory sequence can also be selected from the EEEEEE sequence.

[0034] In some embodiments, the functional unit comprises a glutathione-responsive group disulfide bond, and the assembly motif sequence and the assembly regulatory sequence are covalently linked via the disulfide bond.

[0035] The second aspect of the present application provides a method for preparing an endoplasmic reticulum-targeted polypeptide nanomedicine that responds to morphological changes of glutathione, which specifically comprises:

[0036] S1. Design a polypeptide assembly sequence with a β-folded secondary structure and a nanofiber assembly structure, namely, an assembly primitive sequence and an assembly regulatory sequence, and select a disulfide bond with glutathione responsiveness as a linker, use a hydrophilic polypeptide sequence to regulate the assembly structure of the polypeptide assembly, synthesize a polypeptide molecule with hydrophilic ends and hydrophobic middle, so that it is more inclined to assemble into an assembly morphology of nanoparticles, thereby constructing a polypeptide assembly with glutathione responsiveness that can be transformed from nanoparticles to nanofibers. The synthesis of the polypeptide sequence is synthesized by the standard Fmoc solid phase polypeptide synthesis (SPPS) method, using piperidine as a deprotecting agent and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) as a condensing agent, and designing and synthesizing a polypeptide assembly sequence containing glutathione responsiveness; the polypeptide assembly sequence containing glutathione responsiveness designed and synthesized includes but is not limited to EEEK (C 16 )GGC 12 VVAA-SS-RRRRRR, EEEK(C 16 )EEC 12 VVAA-SS-RRRRRR, RRRK(C 16 )EEC 12 VVAA-SS-RRRRRR, RRRK(C 16 )EEC 12 VVAA-SS-EEEEEE, and EEEK(C 16 )EEC 12 VVAA-SS-EEEEEE and other polypeptide sequences;

[0037] The peptide assembly sequence EEEK (C 16 )GGC 12Take VVAA-SS-RRRRRR as an example. The two ends of the sequence are hydrophilic amino acids glutamic acid and arginine, and the middle is a hydrophobic amino acid. This dumbbell-shaped structure promotes the assembly of polypeptides to tend to the nanoparticle assembly morphology. When the disulfide bond in the molecule is cut by glutathione response, RRRRRR is removed, which promotes the transformation of the assembly structure. The new assembly unit is more inclined to assemble into the assembly morphology of nanofibers, thereby constructing a polypeptide assembly that transforms from nanoparticles to nanofibers with glutathione responsiveness.

[0038] S2. On the basis of the polypeptide assembly sequence containing glutathione responsiveness synthesized in step S1, a targeting peptide sequence with endoplasmic reticulum targeting function is covalently linked to the polypeptide nitrogen end of the polypeptide assembly sequence by an amide condensation method, and a mixed solution is obtained by co-assembly, and the polypeptide assembly sequence is endoplasmic reticulum targeting performance is given to obtain a polypeptide assembly with endoplasmic reticulum targeting function; in some specific embodiments, the solvent of the mixed solution is PBS buffer or other buffer, ultrapure water, deionized water, and Milli-Q water; in some specific embodiments, the targeting peptide sequence accounts for 0.1%-99% of the total molar number of the mixed solution, preferably 0.1%-50%; in other specific embodiments, the substance concentration of the mixed solution is 0.1μmol / L-10mmol / L;

[0039] S3. Annealing the mixed solution obtained in step S2 to obtain a polypeptide co-assembly system solution, which contains an endoplasmic reticulum-targeted polypeptide nanodrug with glutathione-responsive morphological transformation; in some specific embodiments, the annealing temperature is 10°C-100°C, and the annealing time is 0.1h-100h; in some specific embodiments, the solvent of the polypeptide co-assembly system solution is one of PBS buffer or other buffer, ultrapure water, deionized water, and Milli-Q water.

[0040] The third aspect of the present application provides a method for characterizing the performance of an endoplasmic reticulum-targeted polypeptide nanodrug that responds to morphological changes in response to glutathione, and a redox method for the endoplasmic reticulum-targeted polypeptide nanodrug obtained according to the above preparation method: the endoplasmic reticulum-targeted polypeptide nanodrug obtained in step S1 is placed under reducing conditions, and a certain concentration of reducing agent solution is added to change the assembly structure of the polypeptide from nanoparticles to nanofibers. The reducing agent solution for the response morphological change of the endoplasmic reticulum-targeted polypeptide nanodrug includes but is not limited to a glutathione reducing agent, the concentration of the glutathione reducing agent is 0.1 μmol / L-1 mol / L, the redox temperature is 0°C-100°C, and the oxidation reaction time is 0.1h-100h.

[0041] In some embodiments of the present application, the performance characterization method also includes further cell experiments, in which the normal cells used are mouse embryonic fibroblasts (3T3) and the cancer cells used are mouse breast cancer cells (4T1), which are used to prove that the polypeptide nanomedicine has endoplasmic reticulum targeting and inducing cell apoptosis performance.

[0042] When the environment changes and contains the reducing agent glutathione, the disulfide bonds in the polypeptide molecules are reduced and broken, causing changes in the polypeptide molecular structure, which in turn causes the polypeptide assembly structure to transform into nanofibers, thereby realizing the transformation of the assembly structure from nanoparticles to nanofibers, which are enriched in the endoplasmic reticulum under the action of the endoplasmic reticulum targeting peptide, leading to endoplasmic reticulum stress and cell apoptosis.

[0043] The present application will be described in further detail below in conjunction with specific exemplary embodiments. It should be understood that the specific embodiments described herein are intended only to explain the present application, rather than to limit the present application. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are all set to three replicates, and the data are the mean values ​​or mean ± standard deviations of the three replicates.

[0044] Example 1

[0045] This embodiment provides an endoplasmic reticulum-targeted polypeptide nanomedicine containing a disulfide bond and glutathione that responds to morphological changes, and the preparation process includes:

[0046] S1. Design and synthesize polypeptide sequences such as assembly motif sequences and assembly regulatory sequences by solid phase peptide synthesis, specifically including the following sequences:

[0047] Assembly motif sequence: Glutamic acid-Glutamic acid-Glutamic acid-Lysine-Palmitane-Glutamic acid-Glutamic acid-Dodecane-Valine-Valine-Alanine-Alanine;

[0048] Assemble the regulatory sequence: Arginine-Arginine-Arginine-Arginine-Arginine-Arginine;

[0049] Glutathione responsive group: disulfide bond;

[0050] Targeting peptide sequences: arginine-valine-methionine-leucine-isoleucine-glutamic acid-lysine, lysine-aspartic acid-glutamic acid-leucine;

[0051] The solid phase resin feed was 0.5 mmol. The peptide sequence was synthesized by the standard Fmoc solid phase peptide synthesis (SPPS) method, with piperidine as a deprotecting agent and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) as a condensing agent. The yield was 90%. The product was separated and purified by high performance liquid chromatography, and the purity of the product was ≥99%. The high performance liquid chromatogram of the purified product is shown in the figure Figure 2 shown.

[0052] S2, the two polypeptides designed and synthesized in step S1 are assembled into peptides such as glutamic acid-glutamic acid-glutamic acid-lysine-palmitane-glutamic acid-glutamic acid-dodecane-valine-valine-alanine-alanine- Arginine-Arginine-Arginine- Arginine-Arginine-Arginine ( Figure 1-1 ) , endoplasmic reticulum targeted assembly peptides arginine-valine-methionine-leucine-isoleucine-glutamate-lysine, lysine-aspartic acid-glutamate-leucine-glutamate-glutamate-glutamate-lysine-palmitane-glutamate-glutamate-dodecane-valine-valine-alanine-alanine- Arginine-Arginine-Arginine Acid-Arg-Arg-Arg ( Figure 1-2 ) Add it into the aqueous solution at a molar ratio of 80:20 to obtain a mixed solution for standby use;

[0053] S3, annealing and assembling the mixed solution, the annealing temperature is 80°C, the annealing time is 48 hours, and a polypeptide co-assembly system solution is obtained, wherein the endoplasmic reticulum-targeted polypeptide nanomedicine with glutathione-responsive morphological transformation is contained, and the obtained assembly structure is observed under an atomic force microscope and a field emission transmission electron microscope, as shown in FIG. Figure 3 shown.

[0054] The obvious nanoparticle structure of the endoplasmic reticulum-targeted peptide nanodrug can be observed from the atomic force microscopy images and field emission transmission electron microscopy images, indicating that the endoplasmic reticulum-targeted peptide nanodrug can be assembled into nanoparticles with a particle size range of 30-60nm.

[0055] Example 2

[0056] The endoplasmic reticulum targeting polypeptide nanomedicine prepared in Example 1 of the present application was reduced in vitro by simulating tumor cell glutathione, with a glutathione concentration of 10 mmol, a reduction temperature of 37°C, and a reduction time of 12 hours. After the reduction was completed, annealing and assembly were performed in an aqueous solution, with an annealing temperature of 80°C and an assembly time of 48 hours. The obtained assembly structure was observed under an atomic force microscope and a field emission transmission electron microscope. The results are as follows: Figure 4 shown.

[0057] It can be observed from the atomic force microscopy images and field emission transmission electron microscopy images that the nanoparticle assembly structure of the polypeptide assembly after glutathione reduction disappears and transforms into a nanoparticle assembly morphology, indicating that the polypeptide assembly has the property of glutathione-responsive morphological transformation.

[0058] Test Example 1

[0059] The endoplasmic reticulum targeting polypeptide nanomedicine prepared in Example 1 of the present application and the endoplasmic reticulum targeting polypeptide nanomedicine after glutathione reduction in Example 2 were characterized by circular dichroism and infrared spectroscopy. The results are as follows: Figure 5 and Figure 6 shown.

[0060] From the circular dichroism spectrum, it can be seen that before glutathione reduction, the peptide nanomedicine has a negative peak at 200nm and no other absorption peaks, which is a typical random coil secondary structure; after glutathione reduction, the circular dichroism spectrum changes, the peak at 200nm disappears, a positive peak appears at 192nm, and a negative peak appears at 205nm, which is a typical β-fold secondary structure. This shows that the peptide nanomedicine can be reduced by glutathione, transforming from random coil to β-fold, realizing the transformation of the secondary structure responsive to glutathione.

[0061] In the infrared spectrum, the peptide nanomedicine was at 1652 cm before reduction. -1 There is an absorption peak at 1632 cm, which is a random curl absorption peak. After glutathione reduction, the absorption peak blue-shifts to a low wave number of 1632 cm -1 The secondary structure is transformed into β-folding. In addition, at 1650 cm -1 There is a shoulder peak at the middle, which may be the peak of the random coil polyarginine sequence removed after the disulfide bond is broken.

[0062] Test Example 2

[0063] The endoplasmic reticulum-targeted polypeptide nanomedicine prepared in Example 1 of the present application was compared and observed under an atomic force microscope before and after glutathione reduction. Figure 7 shown.

[0064] Experimental results: In the atomic force microscope images, it was observed that before glutathione reduction, the assembled morphology was nanoparticles. After glutathione reduction, the assembled morphology of the peptide nanomedicine changed from nanoparticles to nanofibers. This shows that after the modification of the endoplasmic reticulum targeting group, the endoplasmic reticulum targeting peptide nanomedicine formed after co-assembly with the assembly peptide also has the characteristics of glutathione response morphology change.

[0065] Test Example 3

[0066] The endoplasmic reticulum targeting polypeptide nanodrug prepared in Example 1 of the present application was incubated in 4T1 cells for 1 hour, 6 hours, 12 hours, and 24 hours, and the endoplasmic reticulum was labeled with an endoplasmic reticulum probe. The endoplasmic reticulum targeting performance of the polypeptide nanodrug was observed by confocal fluorescence microscopy. The results are as follows: Figure 8 and Fig. 9 shown.

[0067] Experimental results: The confocal fluorescence image shows that the peptide nanodrug is taken up by 4T1 cells, and when the incubation time is extended to 6 hours, the co-localization coefficient of the peptide nanodrug and the endoplasmic reticulum reaches 0.8, indicating that the peptide nanodrug has good co-localization with the endoplasmic reticulum. When the incubation time is extended to 12 hours, the co-localization coefficient of the peptide nanodrug and the endoplasmic reticulum is maintained at about 0.78. At 24 hours, the co-localization coefficient decreases, but still maintains at about 0.66. This shows that the peptide nanodrug has good endoplasmic reticulum targeting performance and can target and locate to the endoplasmic reticulum.

[0068] Test Example 4

[0069] The glutathione-responsive endoplasmic reticulum-targeted polypeptide nanomedicine prepared in Example 1 of the present application was subjected to a cytotoxicity experiment to evaluate the biocompatibility and tumor cell killing ability of the nanomedicine.

[0070] The cytotoxicity of peptide nanomedicine was detected by MTT method: 4T1 cells were cultured at 6×10 3 The cells were seeded at a density of 100 μg / well in a 96-well plate and cultured in a CO2 incubator for 24 hours. Fresh culture medium containing different concentrations of peptide nanomedicine was added to the 96-well plate and cultured for another 24 hours. Subsequently, 10 μl of MTT solution was added to each well and incubated in the incubator for another 4 hours. After 4 hours, the culture medium was discarded, 100 μl of dimethyl sulfoxide was added to each well, and the absorbance at 490 nm was measured using an enzyme marker.

[0071] Experimental results: Fig. 9 As shown, when there is no endoplasmic reticulum targeting group, the cell viability of the polypeptide assembly peptide after co-incubation with 4T1 cells is maintained at more than 80%, indicating that the polypeptide assembly has good biocompatibility. When it contains an endoplasmic reticulum targeting group, the cell viability is significantly reduced after incubation with cells, and the cell viability is less than 50% when the polypeptide concentration is 50μM, indicating that the polypeptide nanomedicine is lethal to tumor cells. This is because the polypeptide nanomedicine with an endoplasmic reticulum targeting group can target the endoplasmic reticulum, resulting in endoplasmic reticulum stress-induced cell apoptosis.

[0072] It should be noted that, in addition to the cases listed in Examples 1 to 6 above, it is also feasible to select other raw material ratios and preparation method parameters.

[0073] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed in the present application, and these modifications or replacements should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A glutathione-responsive endoplasmic reticulum-targeted polypeptide nanomedicine, wherein: The endoplasmic reticulum targeting polypeptide nanomedicine comprises a targeting unit, an assembly unit and a functional unit, wherein the targeting unit comprises one or more of the targeting peptide sequences RVMLIGK, RVMLIEK, KDEL and a sulfonyl group having an endoplasmic reticulum targeting function.

2. The endoplasmic reticulum-targeted polypeptide nanodrug according to claim 1, wherein: The assembly unit includes an assembly motif sequence and an assembly regulatory sequence, and the combination of the assembly motif sequence and the assembly regulatory sequence includes at least one of the following: 1) Assembling the motif sequence EEEK(C 16 )GGC 12 VVAA and assembly regulatory sequence RRRRRR; 2) Assembling the motif sequence EEEK(C 16 )EEC 12 VVAA and assembly regulatory sequence RRRRRR.

3. The endoplasmic reticulum-targeted polypeptide nanodrug according to claim 2, wherein: The functional unit includes a glutathione responsive group disulfide bond, and the assembly motif sequence and the assembly regulatory sequence are covalently linked via the disulfide bond.

4. The method for preparing the endoplasmic reticulum-targeted polypeptide nanomedicine according to claim 3, wherein: The preparation method comprises: S1. The assembly motif sequence, assembly regulatory sequence and glutathione responsive group disulfide bond were synthesized by standard Fmoc solid phase peptide synthesis (SPPS) method, piperidine was used as deprotection agent, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) was used as condensation agent, and a peptide assembly sequence containing glutathione responsiveness was designed and synthesized; S2, mixing the targeting peptide sequence with endoplasmic reticulum targeting function with the polypeptide assembly sequence obtained in step S1 in proportion to obtain a mixed solution for standby use; S3. Annealing the mixed solution obtained in step S2 to obtain a polypeptide co-assembly system solution containing an endoplasmic reticulum-targeted polypeptide nanomedicine with glutathione-responsive morphology transformation.

5. The preparation method according to claim 4, wherein: The solvent of the mixed solution in step S2 and the polypeptide co-assembly system solution in step S3 is one of buffer, ultrapure water, deionized water and Milli-Q water.

6. The preparation method according to claim 4 or 5, wherein: In step S2, the targeting peptide sequence accounts for 0.1%-50% of the total molar number of the mixed solution, and the molar concentration of the mixed solution is 0.1 μmol / L-10 mmol / L.

7. The preparation method according to claim 6, wherein: The annealing temperature of the annealing operation in step S3 is 10° C.-100° C., and the annealing time is 0.1 h-100 h.

8. A method for characterizing the performance of endoplasmic reticulum-targeted polypeptide nanomedicine, wherein: The redox method of the endoplasmic reticulum-targeted polypeptide nanodrug obtained by the preparation method according to any one of claims 4 to 7: the reducing agent of the endoplasmic reticulum-targeted polypeptide nanodrug in response to the morphological change includes a glutathione reducing agent, the concentration of the glutathione reducing agent is 0.1 μmol / L-1 mol / L, the redox temperature is 0°C-100°C, and the oxidation reaction time is 0.1h-100h.

9. The performance characterization method according to claim 8, wherein: The invention comprises a cell experiment, wherein the normal cells used in the cell experiment are mouse embryonic fibroblasts (3T3), and the cancer cells used are mouse breast cancer cells (4T1).

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