A photochemical synthesis method of whole peptide nanoparticles

Abstract

The present invention relates to a photochemical synthesis method of a full-peptide nano-colloid. In this method, a double-terminal dityrosine sequence peptide containing aspartic acid D and aminocaproic acid X is used as a cross-linkable precursor, and a mixed solution of a precursor, a photosensitizer, and an oxidant is photochemically synthesized in water. The precursor can be designed from scratch with the help of a peptide database, and standardized solid-phase synthesis can be achieved. The primary structure of the polypeptide chain segment can be transformed into a higher-level structure of a functional colloid within a few minutes by irradiation with a high-intensity photoreactor. The morphology and particle size of the colloid can be regulated by the concentration of the precursor and the pH environment, the degree of cross-linking and the speed of the synthetic reaction can be regulated by the intensity of illumination and the ratio of photosensitizer / oxidant, and the space occupancy, amphiphilicity, water solubility, charge distribution, and encapsulation characteristics of the colloid can be regulated by designing a hydrophilic / hydrophobic / ionic repeating peptide sequence. The nano-colloid is based on a complete peptide unit structure, has excellent biocompatibility, can encapsulate molecules and ions to construct nano-probes, and realizes biomedical applications such as fluorescence imaging and magnetic resonance imaging.

Description

Technical Field

[0001] The present invention relates to a photochemical synthesis method of full-peptide nano-colloids. The method uses a polypeptide (YY polypeptide monomer) with a double-terminal dityrosine sequence containing aspartic acid D and aminocaproic acid X as a cross-linkable precursor, and utilizes a high-intensity photoreactor for illumination to achieve the transformation of the primary structure of the polypeptide chain segment into the higher-level structure of the functional colloid within a few minutes. Background Art

[0002] At present, the materials used to construct nanosystems are mainly divided into organic materials, inorganic materials and composite materials. Inorganic materials include mesoporous silicon, quantum dots, gold nanoparticles, carbon nanotubes, iron oxide, manganese oxide nanoparticles, etc. Due to their weak ability to bind to organisms, they are easily deposited in organs and difficult to metabolize. They have hidden safety issues such as poor compatibility and poor degradability. They are often coated or modified with organic materials to achieve better biological effects. Organic materials mainly include proteins, peptides, polysaccharides, nucleic acids, high molecular polymers, etc. Because they are homologous to the composition of organisms, have good compatibility, are rich in active sites in the body, and exhibit excellent targeting and responsiveness, this type of material has received extensive attention and research in the development of nanotechnology.

[0003] Supramolecular assembly utilizes discrete building blocks to produce biocompatible functional materials, which have been widely developed and applied in imaging, drug delivery, and tissue engineering. Peptides and amino acids, as one of the most important natural building blocks, have attracted much attention due to their many advantages such as routable synthesis, functional sequence encoding, biocompatibility and degradation, and bottom-up high-level structural architecture. The assembly of peptides is controlled by non-covalent or covalent interactions, including hydrogen bonds, π-π stacking, hydrophobic interactions, disulfide SS bonds, phenylalanine polymerization, and dityrosine cross-linking, which drive the assembly process of peptides.

[0004] Aromatic dipeptides such as diphenylalanine (FF) are often used as self-folding motifs to drive the construction of amphiphilic nanofibers. Under this condition, π-π stacking is the core driving force for the self-assembly of tubular structures, and the desired functions can be given to them through synthetic peptide encoding. For example, Ulijn et al. (ACS Nano. 2020, 14, 15056) from the City University of New York introduced positively charged lysine (K) residues at the peptide end, and the resulting nanofibers can be electrostatically labeled with anionic sulfonates of Alexa-488 dye and applied to in situ super-resolution imaging. Theoretically, the discrete distribution of aromatic residues such as phenyl (F) and tyrosine (Y) on peptide amphiphiles is related to the corresponding three-dimensional double-junction network. In addition, the cross-stacking of aromatic peptides, accompanied by alternating spacing, hydrogen bonds or hydrophobic interactions, can even drive the formation of nanospheres, nanorods, nanovesicles, nanotubes, and liquid-liquid phase separation (LLPS) in cells.

[0005] As an aromatic amino acid, tyrosine, as one of the building blocks of polypeptides, plays an important role in the formation of elastin, connective tissue, and biofilm in animals and plants. It can undergo cross-linking processes such as Fenton reaction or enzyme catalysis to form a dityrosine structure, which exhibits important physiological functions. For example, the dityrosine structure in the elastin of dragonfly wings has excellent mechanical properties, which can greatly enhance the structural strength of the wings and is sufficient to withstand high-frequency shear vibrations during flight. Another example is that there is a special biofilm composed of dityrosine in the intestines of African Aedes mosquitoes. Due to its dense structure, it can completely isolate the possibility of Aedes mosquitoes being infected with parasites when sucking host blood. In particular, this dityrosine structure can also be cross-linked through photochemical reactions. Under the excitation of light, a tyrosine free radical intermediate is formed, which isomerizes with nearby tyrosine. The isomer removes hydrogen atoms through proton-coupled electron transfer to form dityrosine.

[0006] In the traditional examples of amino acid dyads as driving sequences, nanosystems are mostly constructed in a non-covalent manner, such as the π-π stacking and hydrophobic interaction formed between diphenylalanine. This interaction has strict requirements on the reaction conditions of synthesis and is still limited by many factors such as solvent, temperature, pH environment, ionic strength, pre-assembly method, etc., and its morphology is difficult to control and size is difficult to adjust. It is crucial to find a construction method that can be easily controlled and less affected by the environment, so as to achieve controllable size adjustment and improve its biological utilization value. Summary of the invention

[0007] The purpose of the present invention is to provide a photochemical synthesis method for constructing all-peptide nanoparticles with adjustable particle size and probe encapsulation under light, which is driven by double-terminal dityrosine sequence cross-linking. The method has excellent biocompatibility and can realize a variety of biomedical applications, such as bioimaging, biosensing, gene carriers, etc.

[0008] The whole peptide nano-gel spheres of the present invention are characterized in that the synthesis reaction is carried out in an aqueous environment, and the obtained product is highly hydrophilic and well dispersed in an aqueous environment.

[0009] The whole peptide nano-gel sphere of the present invention is characterized by having a spherical nanostructure, and the size of the spherical particles is between 50 and 400 nm.

[0010] The whole peptide nanoparticles of the present invention are characterized in that the synthesis speed of the whole peptide nanoparticles can be controlled by the light intensity, and the typical light intensity used in the light reaction is 1-500 mW / cm 2 The light source used is a single-wavelength LED high-power photoreactor, and the light intensity is controlled by adjusting its current to regulate the reaction time.

[0011] The full-peptide nano-gel spheres of the present invention are characterized in that a photosensitizer and an oxidant are added during the synthesis to promote the photochemical synthesis of the gel spheres. According to the type of light source, the photoinitiator used is tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate Ru(bpy)3Cl2·6H2O, phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide C 26 H 27 O3P, 2-Hydroxy-2-methyl-1-phenylpropanone C 10 H 12 O2, the oxidant is ammonium persulfate (NH4)2S2O8, potassium persulfate K2S2O8, sodium persulfate Na2S2O8, and the reaction time is controlled within a few minutes.

[0012] The full-peptide nano-gel spheres of the present invention are characterized by designing a cross-linkable polypeptide precursor, that is, designing a sequence peptide containing double-terminal dityrosine to be a hydrophilic, hydrophobic, and ionic peptide repeating sequence, so as to regulate the spatial occupancy, amphiphilicity, water solubility, charge distribution, and encapsulation characteristics of the polypeptide nano-gel spheres.

[0013] The whole peptide nanoparticles of the present invention are characterized by encapsulating functional molecules or ions, and the encapsulation amount can be adjusted according to the feed ratio. The encapsulation mass percentage is usually 0-35%, which is called encapsulated whole peptide nanoparticles.

[0014] The encapsulated whole-peptide nano-gel spheres of the present invention are characterized by a co-assembly photo-crosslinking synthesis method, that is, when the whole-peptide nano-gel spheres are synthesized according to a ratio, further adding functional molecules or ions into the system can directly obtain the whole-peptide nano-gel spheres encapsulating functional molecules or ions, thereby realizing further functionalization of the whole-peptide nano-gel spheres.

[0015] The entrapped all-peptide nanoparticles of the present invention are characterized in that the entrapped functional molecules are selected from any one or more of near-infrared dyes such as rhodamine, methylene blue, IR780, or anticancer drugs such as adriamycin, paclitaxel, and camptothecin. The entrapped functional ions are selected from any one or more of Gd, Mn, Pt, Cu, Fe, Zn, and Au.

[0016] The encapsulated nano-colloid spheres described in the present invention are characterized in that they can adapt to a variety of functional molecules by adjusting the type of peptide repeating sequence in the YY polypeptide monomer. For example, increasing the number of repeats of hydrophobic amino acids such as phenylalanine can increase the encapsulation ratio of hydrophobic functional molecules; increasing the number of repeats of anionic amino acids such as aspartic acid can increase the encapsulation ratio of positively charged functional molecules or ions.

[0017] The core design of the present invention is to introduce 6-aminocaproic acid and aspartic acid to adjust the spatial occupancy, amphiphilicity, water solubility, charge distribution and encapsulation characteristics of the molecule, and to alternately form six double-terminal dityrosine polypeptide monomers 0 to 5DYY in different quantities. Under the action of a photosensitizer and an oxidant, the monomers are placed in an aqueous phase system and cross-linked to form a cross-linked nanostructure.

[0018] Preferably, the whole peptide nano-gel spheres of the present invention are characterized in that the YY polypeptide monomer is a 3DYY polypeptide monomer, the final concentration of which is 0.5 to 5 mM, and with the increase of feed concentration, the size range of the obtained nano-gel spheres is 50 to 400 nm.

[0019] Preferably, the whole peptide nanoparticles of the present invention are characterized in that the light source is a 405nm LED high-power photoreactor with a power range of 1 to 500mW / cm 2 , the reaction time ranges from 5 to 20 minutes

[0020] Preferably, the whole peptide nano-colloid spheres of the present invention are characterized in that the photosensitizer is tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate, namely Ru(bpy)3Cl2·6H2O, and its concentration is 1-25% of the 3DYY polypeptide monomer. The oxidant is ammonium persulfate (NH4)2S2O8, and its concentration is 1-10 times of the 3DYY polypeptide monomer.

[0021] Preferably, the whole peptide nano-gel spheres of the present invention are characterized in that the pH of the aqueous phase is 5 to 9, and the yield obtained varies according to the pH.

[0022] The present invention has the following beneficial effects

[0023] The nanoparticles are based on a complete peptide unit structure and have excellent biocompatibility.

[0024] The YY polypeptide precursor was designed from scratch with the help of a polypeptide database and synthesized in a standardized manner using conventional polypeptide solid-phase synthesis technology.

[0025] Compared with traditional particle synthesis methods, this method can easily control the morphology and yield of nano-colloid spheres through the solution pH environment.

[0026] Compared with non-covalently driven synthesis methods, the cross-linked network obtained by this method has better stability and structural strength.

[0027] Compared with the double-phenylalanine driven synthesis method, this method can easily control the particle size of polypeptide nanospheres by adjusting the concentration of polypeptide precursors.

[0028] The synthesis method is carried out under light and water environment, the reaction conditions are mild, there are no extreme high temperature, high pressure and other environmental requirements, and the reaction is safe, green and pollution-free.

[0029] The photoreactor used in the synthesis method is strong light with a constant wavelength. The rate and yield of the reaction process can be controlled by adjusting the current, voltage, power, illumination time, etc.

[0030] The synthesis method can also regulate the space occupancy, amphiphilicity, water solubility, charge distribution and encapsulation characteristics of the nano-colloid spheres by designing hydrophilic / hydrophobic / ionic peptide repeating sequences.

[0031] The whole peptide nanoparticles synthesized by this method can encapsulate stable functional fluorescent molecules or metal cations, giving them the application potential for in vivo tumor imaging. DETAILED DESCRIPTION

[0032] In order to further illustrate the present invention, a series of examples are given below. These examples are completely illustrative, and they are only used to specifically describe the present invention and should not be understood as limiting the present invention.

[0033] The present invention first designs six double-terminal dityrosine-containing polypeptide monomers 0-5DYY, and their structures are shown in Example 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the 0DYY structure diagram of Example 1.

[0035] Figure 2 It is the 0DYY hydrogen nuclear magnetic resonance spectrum of Example 1.

[0036] Figure 3This is the 1DYY structure diagram of Example 1.

[0037] Figure 4 This is the 1DYY hydrogen nuclear magnetic resonance spectrum of Example 1.

[0038] Figure 5 This is the 2DYY structure diagram of Example 1.

[0039] Figure 6 This is the 2DYY hydrogen nuclear magnetic resonance spectrum of Example 1.

[0040] Figure 7 This is the 3DYY structure diagram of Example 1.

[0041] Figure 8 This is the 3DYY hydrogen nuclear magnetic resonance spectrum of Example 1.

[0042] Fig. 9 This is the 4DYY structure diagram of Example 1.

[0043] Fig.10 This is the 4DYY hydrogen nuclear magnetic resonance spectrum of Example 1.

[0044] Fig.11 This is the 5DYY structure diagram of Example 1.

[0045] Fig.12 This is the 5DYY hydrogen nuclear magnetic resonance spectrum of Example 1.

[0046] Fig.13 This is the TEM image of Example 2.

[0047] Fig.14 This is the DLS graph of Example 2.

[0048] Fig.15 This is the TEM image of Example 3.

[0049] Fig.16 This is the DLS graph of Example 3.

[0050] Fig.17 This is the TEM image of Example 4.

[0051] Fig.18 This is the DLS graph of Example 4.

[0052] Fig.19 This is the TEM image of Example 5.

[0053] Fig. 20 This is the UV absorption spectrum of Example 5.

[0054] Fig.21 This is the FL emission spectrum of Example 5.

[0055] Fig. 22 These are the XRD and SAED spectra of Example 6.

[0056] Fig.23 This is the EDS spectrum of Example 6.

[0057] Fig.24 This is the XPS spectrum of Example 6.

[0058] Fig.25 These are in vivo and in vitro imaging images of nanogel balls in mice.

[0059] Example 1

[0060] According to the design, a 0-5DYY polypeptide monomer with double-terminal dityrosine was obtained. The steps of solid phase synthesis were as follows: in a polypeptide synthesis tube, 2-chlorotrityl chloride resin was used as a carrier, and side chain protected amino acid synthons were gradually introduced, and then recrystallized with ice ether, washed, and washed to obtain an undeprotected polypeptide. It was dissolved in a deprotecting agent, reacted for 5 minutes, recrystallized with ice ether, washed, and filtered to obtain an off-white powder, which was weighed and marked and stored in a refrigerator at 4°C. The type and amount of amino acid synthons added were adjusted, and 6 polypeptide monomers consisting of different amounts of aspartic acid and 6-aminocaproic acid alternating were gradually synthesized.

[0061] NH2-YYXYY-COOH(0DYY) was prepared by using 1.1mmol of 2-chlorotrityl chloride resin as carrier, piperidine as activator, 1-hydroxybenzotriazole as catalyst, and gradually introducing 3mmol of side chain protected tyrosine and 6-aminocaproic acid to obtain peptide reaction solution alternately. The peptide was recrystallized with ice ether and washed by grinding to obtain undeprotected 0DYY peptide ( Figure 1 ). It was dissolved in a deprotection agent trifluoroacetic acid: water: triisopropylsilane = 95%: 2.5%: 2.5% (v / v), reacted for 5 minutes, recrystallized with ice ether, washed, filtered, and obtained an off-white powder, weighed to obtain about 300 mg, with a yield of about 36.3%. It was marked and stored in a refrigerator at 4°C. Its mass spectrum and magnetic resonance hydrogen spectrum were measured to obtain the following data. ( Figure 2 )HR-ESIMS:m / z=Cacld.784.3558,found 784.3560(err.+0.25ppm)for[M+H] + ,C 42 H 50 N5O 10 + ;Calcd.806.3377,found 806.3375(err.-0.25ppm)for[M+Na] + ,C 42 H 49 N5NaO 10+ ;Calcd.828.3197,found 828.3194(err.-0.36ppm)for[M-H+2Na] + ,C 42 H 48 N5Na2O 10 + . 1 H NMR(600MHz,DMSO-d6,ppm)δ:12.71(br,1H),9.25(m,4H),8.69(br,1H),7.98(m,5H),7.01(br,8H) ,6.65(br,8H),4.42(m,3H),3.91(m,1H),2.81(m,10H),1.98(m,2H),1.32(m,6H).Anal.Calcd.for C 42 H 49 N5O 10 :C,64.35;H,6.30;N,8.93.Found:C,64.59;H,6.01;N,8.57.

[0062] The synthesis method of NH2-YYXDXYY-COOH (1DYY) is similar to the above 0DYY. Using 1.1 mmol of 2-chlorotrityl chloride resin as a carrier, piperidine as an activator, and 1-hydroxybenzotriazole as a catalyst, 3 mmol of side chain protected tyrosine, aspartic acid, and 6-aminocaproic acid are gradually introduced to alternately obtain a polypeptide reaction solution, which is recrystallized with ice ether and washed to obtain an undeprotected 1DYY polypeptide ( Figure 3 ). It was dissolved in a deprotecting agent trifluoroacetic acid: water: triisopropylsilane = 95%: 2.5%: 2.5% (v / v), reacted for 5 minutes, recrystallized with icy ether, washed, and filtered to obtain an off-white powder, weighed to obtain about 350 mg, with a yield of about 34.6%, marked and stored in a 4°C refrigerator, and its mass spectrum and magnetic resonance hydrogen spectrum were measured to obtain the following data ( Figure 4 ). HR-ESIMS:m / z=Cacld.1012.4668,found1012.4674(err.+0.59ppm)for[M+H] + ,C 52 H 66 N7O 14 + ;Calcd.1034.4487,found 1034.4490(err.+0.29ppm)for[M+Na] + ,C 52 H 65 N7NaO 14+ ;Calcd.1056.4307,found 1056.4315(err.+0.76ppm)for[M-H+2Na] + ,C 52 H 64 N7Na2O 14 + ;Calcd.1078.4126,found 1078.4134(err.+0.74ppm)for[M-2H+3Na] + ,C 52 H 63 N7Na3O 14 + . 1 HNMR(600MHz,DMSO-d6,ppm)δ:12.45(br,2H),9.22(m,4H),8.69(br,1H),7.97(m,7H),7.00(br,8H),6.65 (br,8H),4.42(m,4H),3.91(m,1H),2.96(m,14H),2.08(m,2H),1.97(m,2H),1.31(m,12H).Anal.Calcd.for C 52 H 65 N7O 14 :C,61.71;H,6.47;N,9.69. Found:C,62.08;H,6.21;N,9.93.

[0063] The synthesis method of NH2-YYXDXDXYY-COOH (2DYY) is similar to the above 0DYY. Using 1.1 mmol of 2-chlorotrityl chloride resin as a carrier, piperidine as an activator, and 1-hydroxybenzotriazole as a catalyst, 3 mmol of side chain protected tyrosine, aspartic acid, and 6-aminocaproic acid are gradually introduced to alternately obtain a polypeptide reaction solution, which is recrystallized with ice ether and washed to obtain an undeprotected 2DYY polypeptide ( Figure 5 ). It was dissolved in a deprotecting agent trifluoroacetic acid: water: triisopropylsilane = 95%: 2.5%: 2.5% (v / v), reacted for 5 minutes, recrystallized with ice ether, washed, filtered, and obtained an off-white powder, weighed to obtain about 380 mg, with a yield of about 30.7%, marked and stored in a 4°C refrigerator, and its mass spectrum and magnetic resonance hydrogen spectrum were measured to obtain the following data ( Figure 6 ). HR-ESIMS:m / z=Cacld.1238.5621,found1238.5615(err.-0.48ppm)for[MH] - ,C 62 H80 N9O 18 - ;Calcd.1260.5441,found 1260.5430(err.-0.87ppm)for[M-2H+Na]-,C 72 H 95 N 11 NaO 22 - ;Calcd.1282.5260,found 1282.5248(err.-0.94ppm)for[M-3H+2Na] - ,C 72 H 94 N 11 Na2O 22 - . 1 H NMR(600MHz,DMSO-d6,ppm)δ:12.38(br,3H),9.22(m,4H),8.69(br,1H),8.00(m,9H),7.01(br,8H),6.65( br,8H),4.43(m,5H),3.92(m,1H),2.97(m,18H),2.08(m,4H),1.98(m,2H),1.33(m,18H).Anal.Calcd.for C 62 H 81 N9O 18 :C,60.04;H,6.58;N,10.16.Found:C,60.58;H,6.41;N,9.97.

[0064] The synthesis method of NH2-YYXDXDXDXYY-COOH (3DYY) is similar to the above 0DYY. Using 1.1 mmol of 2-chlorotrityl chloride resin as a carrier, piperidine as an activator, and 1-hydroxybenzotriazole as a catalyst, 3 mmol of side chain protected tyrosine, aspartic acid, and 6-aminocaproic acid are gradually introduced to alternately obtain a polypeptide reaction solution, which is recrystallized with ice ether and washed to obtain an undeprotected 3DYY polypeptide ( Figure 7 ). It was dissolved in a deprotecting agent trifluoroacetic acid: water: triisopropylsilane = 95%: 2.5%: 2.5% (v / v), reacted for 5 minutes, recrystallized with ice ether, washed, filtered, and obtained an off-white powder, weighed to obtain about 450 mg, with a yield of about 30.2%, marked and stored in a 4°C refrigerator, and its mass spectrum and magnetic resonance hydrogen spectrum were measured to obtain the following data ( Figure 8). HR-ESIMS:m / z=Cacld.1466.6731,found 1466.6741(err.+0.68ppm)for[MH] - ,C 72 H 96 N 11 O 22 - ;Calcd.1488.6551,found 1488.6558(err.+0.47ppm)for[M-2H+Na] - ,C 72 H 95 N 11 NaO 22 - ;Calcd.1510.6370,found 1510.6375(err.+0.33ppm)for[M-3H+2Na] - ,C 72 H 94 N 11 Na2O 22 - ;Calcd.1532.6190,found 1532.6193(err.+0.20ppm)for[M-4H+3Na] - ,C 72 H 93 N 11 Na3O 22 - . 1 H NMR(600MHz,DMSO-d6,ppm)δ:12.37(br,4H),9.22(m,4H),8.69(br,2H),8.00(m,10H),7.01(br,8H),6. 66(br,8H),4.52(m,7H),2.99(m,8H),2.83(m,4H),2.60(m,4H),2.51(m,6H),2.09(m,8H),1.34(m,24H).

[0065] The synthesis method of NH2-YYXDXDXDXDXYY-COOH (4DYY) is similar to the above 0DYY. Using 1.1 mmol of 2-chlorotrityl chloride resin as a carrier, piperidine as an activator, and 1-hydroxybenzotriazole as a catalyst, 3 mmol of side chain protected tyrosine, aspartic acid, and 6-aminocaproic acid are gradually introduced to alternately obtain a polypeptide reaction solution, which is recrystallized with ice ether and washed to obtain an undeprotected 4DYY polypeptide ( Fig. 9). It was dissolved in a deprotecting agent trifluoroacetic acid: water: triisopropylsilane = 95%: 2.5%: 2.5% (v / v), reacted for 5 minutes, recrystallized with ice ether, washed, filtered, and obtained an off-white powder, weighed to obtain about 550 mg, with a yield of about 32.4%, marked and stored in a 4°C refrigerator, and its mass spectrum and magnetic resonance hydrogen spectrum were measured to obtain the following data ( Fig.10 ). HR-ESIMS:m / z=Cacld.1696.7998,found 1696.7992(err.-0.35ppm)for[M+H] + ,C 82 H 114 N 13 O 26 + ;Calcd.1718.7817,found 1718.7805(err.-0.70ppm)for[M+Na] + ,C 82 H 113 N 13 NaO 26 + . 1 H NMR(600MHz,DMSO-d6,ppm)δ:12.30(br,5H),9.22(m,4H),8.69(br,1H),8.01(m,13H),7.01(br,8H),6.65 (br,8H),4.52(m,7H),3.92(m,1H),2.99(m,26H),2.08(m,8H),1.98(m,2H),1.35(m,30H).Anal.Calcd.for C 82 H 113 N 13 O 26 :C,58.04;H,6.71;N,10.73.Found:C,58.59;H,6.55;N,10.36.

[0066] The synthesis method of NH2-YYXDXDXDXDXDXYY-COOH (5DYY) is similar to the above 0DYY. Using 1.1 mmol of 2-chlorotrityl chloride resin as a carrier, piperidine as an activator, and 1-hydroxybenzotriazole as a catalyst, 3 mmol of side chain protected tyrosine, aspartic acid, and 6-aminocaproic acid are gradually introduced to alternately obtain a polypeptide reaction solution, which is recrystallized with ice ether and washed to obtain an undeprotected 5DYY polypeptide ( Fig.11). It was dissolved in a deprotecting agent trifluoroacetic acid: water: triisopropylsilane = 95%: 2.5%: 2.5% (v / v), reacted for 5 minutes, recrystallized with ice ether, washed, filtered, and obtained an off-white powder, weighed to obtain about 600 mg, with a yield of about 31.2%, marked and stored in a 4°C refrigerator, and its mass spectrum and magnetic resonance hydrogen spectrum were measured to obtain the following data ( Fig.12 ). HR-ESIMS:m / z=Cacld.1924.9108,found 1924.9080(err.-1.45ppm)for[M+H] + ,C 92 H 130 N 15 O 30 + ;Calcd.1946.8927,found 1946.8918(err.-0.46ppm)for[M+Na] + ,C 92 H 129 N 15 NaO 30 + . 1 H NMR(600MHz,DMSO-d6,ppm)δ:12.26(br,6H),9.22(m,4H),8.69(br,1H),8.01(m,15H),7.01(br,8H),6.65( br,8H),4.52(m,8H),3.92(m,1H),2.99(m,30H),2.08(m,10H),1.98(m,2H),1.35(m,36H).Anal.Calcd.for C 92 H 129 N 15 O 30 :C,57.40;H,6.75;N,10.91.Found:C,57.90;H,6.92;N,10.63.

[0067] After high-resolution mass spectrometry and nuclear magnetic resonance hydrogen spectrum analysis, the actual measured values ​​of the above six polypeptides were consistent with the theoretical values, indicating that they were successfully synthesized. Therefore, the above six polypeptide sequences were respectively configured with 1mM and 1% tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate, namely Ru(bpy)3Cl2·6H2O, and 4 times the concentration of ammonium persulfate (NH4)2S2O8, and placed together under a photoreactor, reacted at a current of 0.1A for 10 minutes, and the reaction liquid was in a colloidal state. It was then collected by centrifugation at 8000rpm for 3 minutes and washed with water to obtain the product.

[0068] Example 2

[0069] Preferably, the 3DYY sequence is used as an example. A method for preparing small-sized nano-colloid balls comprises the following steps: preparing a 1.5mM polypeptide 3DYY stock solution, a 0.1mM Ru(bpy)3Cl2·6H2O stock solution, and a 6.67mM (NH4)2S2O8 stock solution, mixing the above stock solutions in a ratio of 1:1:3 (v / v), adjusting the pH value of the mixed solution to 7, placing it in a photoreactor, reacting with a current of 0.1A for 10 minutes, the reaction solution is in a colloidal state, and then centrifuged at 8000rpm for 3 minutes to collect, and washing with water to obtain a colloid ball of about 100nm. The characterization results of TEM, DLS, etc. show that the particle size distribution of the nano-colloid ball is relatively uniform and the morphology is relatively regular. Fig.13 This is the TEM image of small-sized nano-colloid balls. It can be seen that the nano-colloid balls have regular morphology. Through data statistical analysis, it is found that their particle size is normally distributed with a mean of 109nm. The size is uniform and the dispersion is good. Fig.14 This is the DLS image of small-sized nano-colloid balls. It can be seen that the hydrated particle size of the nano-colloid balls is about 130nm, and the peak is narrow and sharp, indicating that it has good dispersion in deionized water.

[0070] Example 3

[0071] Preferably, the 3DYY sequence is used as an example. A method for preparing medium-sized nano-colloid balls comprises the following steps: preparing a 5mM polypeptide 3DYY stock solution, a 0.1mM Ru(bpy)3Cl2·6H2O stock solution, and a 6.67mM (NH4)2S2O8 stock solution, mixing the above stock solutions in a ratio of 1:1:3 (v / v), adjusting the pH value of the mixed solution to 7, placing it in a photoreactor, reacting with a current of 0.1A for 10 minutes, the reaction solution is in a colloidal state, and then centrifuged at 8000rpm for 3 minutes to collect, and washing with water to obtain a colloid ball of about 200nm. The characterization results of TEM, DLS, etc. show that the particle size distribution of the nano-colloid ball is relatively uniform and the morphology is relatively regular. Fig.15 This is the TEM image of medium-sized nano-colloid balls. It can be seen that the nano-colloid balls have regular morphology. Through data statistical analysis, it is found that their particle size is normally distributed with a mean of 223nm. The size is uniform and the dispersion is good. Fig.16 This is the DLS image of medium-sized nano-colloid spheres. It can be seen that the hydrated particle size of the nano-colloid spheres is about 250nm, and the peak is narrow and sharp, indicating that they have good dispersion in deionized water.

[0072] Example 4

[0073] Preferably, the 3DYY sequence is used as an example. A method for preparing large-sized nano-colloid balls comprises the following steps: preparing a 10mM polypeptide 3DYY stock solution, a 0.1mM Ru(bpy)3Cl2·6H2O stock solution, and a 6.67mM (NH4)2S2O8 stock solution, mixing the above stock solutions in a ratio of 1:1:3 (v / v), adjusting the pH value of the mixed solution to 7, placing it in a photoreactor, reacting with a current of 0.1A for 10 minutes, the reaction solution is in a colloidal state, and then centrifuged at 8000rpm for 3 minutes to collect, and washing with water to obtain a colloid ball of about 300nm. The characterization results of TEM, DLS, etc. show that the particle size distribution of the nano-colloid ball is relatively uniform and the morphology is relatively regular. Fig.17 This is the TEM image of large-sized nano-colloid balls. It can be seen that the nano-colloid balls have regular morphology. Through data statistical analysis, it is found that their particle size is normally distributed with a mean of 309nm. The size is uniform and the dispersion is good. Fig.18 This is the DLS image of large-sized nano-colloid balls. It can be seen that the hydrated particle size of the nano-colloid balls is about 320nm, and the peak is narrow and sharp, indicating that it has good dispersion in deionized water.

[0074] Example 5

[0075] Preferably, the 3DYY sequence is used as an example. A method for preparing a nano-colloid ball encapsulating the fluorescent molecule rhodamine 800 comprises the following steps: preparing a 5 mM polypeptide 3DYY stock solution, a 0.1 mM Ru(bpy)3Cl2·6H2O stock solution, and a 6.67 mM (NH4)2S2O8 stock solution, mixing the above stock solutions at a ratio of 1:1:3 (v / v), adding 10 μL of a 5 mg / mL rhodamine 800 stock solution (dissolved with DMSO), adjusting the pH value of the mixed solution to 7, placing it in a photoreactor, reacting with a current of 0.1 A for 10 minutes, the reaction solution is in a colloidal state, and then centrifuged at 8000 rpm for 3 minutes to collect, and washing with water to obtain a nano-colloid ball encapsulating rhodamine 800. UV, FL and other spectra characterized the successful encapsulation of rhodamine 800. Fig.19 This is the TEM image of the nanospheres after encapsulating Rhodamine 800. It was observed that the nanospheres after encapsulation still maintained their original good morphology and size. Fig. 20 This is the UV absorption spectrum of the nanospheres after encapsulation of Rhodamine 800. The nanospheres after encapsulation show obvious Rhodamine 800 absorption peaks at around 626 and 686 nm, indicating that Rhodamine 800 is successfully encapsulated in the nanospheres. Fig.21This is the FL emission spectrum of the nano-colloidal balls after loading Rhodamine 800. With 450nm as the excitation light, the nano-colloidal balls loaded with Rhodamine 800 show an obvious emission peak at around 710nm, and the emission intensity is stronger than that of single-molecule Rhodamine 800 at the same concentration, indicating that after Rhodamine 800 is loaded into the colloidal balls, a fluorescence enhancement amplification effect occurs.

[0076] Example 6

[0077] Preferably, the 3DYY sequence is used as an example. 3+ ) preparation method of nano-colloid spheres, comprising the following steps: preparing a polypeptide 3DYY stock solution with a concentration of 5mM, a Ru(bpy)3Cl2·6H2O stock solution with a concentration of 0.1mM, and a (NH4)2S2O8 stock solution with a concentration of 6.67mM, mixing the above stock solutions at a ratio of 1:1:3 (v / v), adding 10μL of a GdCl3·6H2O stock solution with a concentration of 10mg / mL, adjusting the pH value of the mixed solution to 7, placing it in a photoreactor, reacting with a current of 0.1A for 10 minutes, the reaction solution is in a colloidal state, and then centrifuging at 8000rpm for 3 minutes to collect, and washing with water to obtain nano-colloid spheres encapsulating gadolinium ions. EDS, XPS, XRD, SAED, etc. characterized the successful encapsulation of gadolinium ions and the encapsulation method. Fig. 22 The XRD and SAED spectra of the nano-colloid spheres together characterize that the nano-colloid spheres are amorphous. Fig.23 This is the EDS spectrum of the nano-colloid balls loaded with gadolinium ions. It can be observed that the gadolinium element is evenly distributed in the nano-colloid balls. Fig.24 This is the XPS spectrum of the nanocolloid ball loaded with gadolinium ions. In the whole spectrum, the characteristic binding energies of Gd 3d and 4d are observed, indicating the successful loading of gadolinium. In the high-resolution spectrum of Gd 4d, 148.6eV and 143.3eV are the characteristic binding energies of 4d, respectively. 3 / 2 and 4d 5 / 2 The characteristic binding energy of gadolinium indicates that gadolinium exists in a trivalent form. In the high-resolution spectrum of O, the characteristic binding energy of metal oxides was observed at 534.1 eV. The ratio of polypeptide monomer to gadolinium atom was calculated by using the peak area to be 1:1, which is consistent with the theoretical calculation value, indicating that the gadolinium encapsulation behavior is related to aspartic acid on the polypeptide chain.

[0078] Example 7

[0079] Preferably, the 3DYY sequence is used as an example. A method for preparing a whole peptide nanosphere comprises the following steps: preparing a 5 mM peptide 3DYY stock solution, a 0.1 mM phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide C 26 H 27O3P stock solution and K2S2O8 stock solution with a concentration of 6.67mM were mixed in a ratio of 1:1:3 (v / v), and the pH value of the mixed solution was adjusted to 7. The mixed solution was placed in a photoreactor and reacted at a current of 0.1A for 10 minutes. The reaction solution was in a colloidal state, and then collected by centrifugation at 8000rpm for 3 minutes, and washed with water to obtain nano-colloid balls. The particle size distribution of the nano-colloid balls was relatively uniform and the morphology was relatively regular.

[0080] Example 8

[0081] Preferably, the 3DYY sequence is used as an example. A method for preparing nano-gel spheres comprises the following steps: preparing a 5 mM polypeptide 3DYY stock solution, a 0.1 mM 2-hydroxy-2-methyl-1-phenylacetone C 10 H 12 O2 stock solution and Na2S2O8 stock solution with a concentration of 6.67mM were mixed in a ratio of 1:1:3 (v / v), and the pH value of the mixed solution was adjusted to 7. The mixed solution was placed in a photoreactor and reacted at a current of 0.1A for 10 minutes. The reaction solution was in a colloid state, and then collected by centrifugation at 8000rpm for 3 minutes. After washing with water, nano-colloid balls were obtained. The particle size distribution of the nano-colloid balls was relatively uniform and the morphology was relatively regular.

[0082] Example 9

[0083] Preferably, the 3DYY sequence is used as an example. A method for preparing nano-colloid spheres encapsulating the anticancer drug doxorubicin comprises the following steps: preparing a 5 mM polypeptide 3DYY stock solution, a 0.1 mM Ru(bpy)3Cl2·6H2O stock solution, and a 6.67 mM (NH4)2S2O8 stock solution, mixing the above stock solutions at a ratio of 1:1:3 (v / v), adding 10 μL of a 5 mg / mL doxorubicin hydrochloride stock solution, adjusting the pH value of the mixed solution to 7, placing it in a photoreactor, reacting with a current of 0.1 A for 10 minutes, the reaction solution is in a colloidal state, and then centrifuged at 8000 rpm for 3 minutes to collect, and washing with water to obtain nano-colloid spheres encapsulating doxorubicin. The encapsulation ratio is measured to be about 18% using the standard curve.

[0084] Example 10

[0085] Preferably, the 3DYY sequence is used as an example. A metal cation manganese (Mn 2+) nano-gel spheres, comprising the following steps: preparing a 5mM polypeptide 3DYY stock solution, a 0.1mM Ru(bpy)3Cl2·6H2O stock solution, and a 6.67mM (NH4)2S2O8 stock solution, mixing the above stock solutions at a ratio of 1:1:3 (v / v), adding 10μL of a 10mg / mL MnCl2·4H2O stock solution, adjusting the pH value of the mixed solution to 7, placing the mixed solution in a photoreactor, reacting at a current of 0.1A for 10 minutes, the reaction solution being in a colloidal state, then centrifuging at 8000rpm for 3 minutes to collect, washing with water to obtain nano-gel spheres encapsulating doxorubicin. The encapsulation ratio is measured to be about 13% using a standard curve.

[0086] application

[0087] Tumors develop rapidly, and the immaturity of the vascular system leads to discontinuity of angiogenic endothelial cells, which allows particles with a size of less than 200nm to pass through the blood vessel wall and enter the tissue space, i.e., the high permeability and retention effect (EPR effect) of solid tumors. Based on this, after establishing a CT26 tumor-bearing mouse model, nanoparticles of about 100nm loaded with probes were injected through the tail vein to observe the fluorescence imaging and magnetic resonance imaging of the nanoparticles in tumor-bearing mice. Fig.25 , obvious near-infrared fluorescence appeared at the tumor site of the mouse, indicating that the probe-loaded full-peptide nanospheres exhibited good tumor imaging performance, as did magnetic resonance imaging. In vitro tissue fluorescence imaging also showed that the full-peptide nanospheres had good tumor enrichment performance. The above results together proved that the full-peptide nanospheres can achieve biomedical applications such as tumor imaging.

[0088] The above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A whole peptide nanoparticle, characterized in that: A double-terminal dityrosine (YY) sequence peptide NH2-YYXDXDXDXYY-COOH containing aspartic acid (D) and 6-aminohexanoic acid is used as a cross-linking precursor, wherein X is 6-aminohexanoic acid. The sequence peptide is abbreviated as 3DYY. The structure is based on a complete peptide unit and is synthesized in an aqueous environment under light conditions.

2. The whole peptide nanoparticles according to claim 1, characterized in that: It has a spherical nanostructure and the particle size is between 50 and 400 nm.

3. The whole peptide nanoparticles according to claim 1, characterized in that: The synthesis speed of the peptide nanospheres can be controlled by the light intensity. The typical light intensity used in the light reaction is 1-500 mW / cm 2 .

4. The whole peptide nanoparticles according to claim 1, characterized in that: During the synthesis, photosensitizers and oxidants were added to promote the photochemical synthesis of the colloids. The photoinitiators used were tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate Ru(bpy)3Cl2·6H2O, phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide C 26 H 27 O3P or 2-hydroxy-2-methyl-1-phenylpropanone C 10 H 12 O2, the oxidant is ammonium persulfate (NH4)2S2O8, potassium persulfate K2S2O8 or sodium persulfate Na2S2O8, and the reaction time is controlled at 5 to 20 minutes.

5. A type of encapsulated nano-gel ball, characterized in that: In the process of synthesizing the full-peptide nano-gel spheres according to claim 1 according to the ratio, functional molecules or ions are added at the same time, and the synthesis is carried out by co-assembly photo-crosslinking to directly form nano-gel spheres encapsulating functional molecules or ions; the encapsulated functional molecules are selected from any one or more of the near-infrared dyes rhodamine, methylene blue, IR780, or the anti-cancer drugs doxorubicin, paclitaxel, and camptothecin; the encapsulated functional ions are selected from any one or more of Gd, Mn, Pt, Cu, Fe, Zn, and Au.

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