Polypeptide nanoring material as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide nanoring material and a preparation method thereof, and belongs to the technical field of high polymer materials. The preparation method comprises the following steps: co-assembling poly (gamma-benzyl ester-L-glutamic acid) (PBLG) and achiral triphenylamine derivatives {N, N-bis (2-methylphenyl) aniline (MTPA), 4-(5-aldehyde-2, 2 ': 5', 2 ''-terthienyl)-N, N-bis (2-methylphenyl) aniline (MTTH) or 4-[5-(2, 2-dicyanovinyl) thiophene-2-yl]-N, N-bis (2-methylphenyl) aniline (MTTV)} to obtain the poly (gamma-benzyl ester-L-glutamic acid)-N, N-bis (2-methylphenyl) aniline derivative. According to the present invention, the emission of multi-color circularly polarized light (CPL) (near ultraviolet to deep red light, 370-660 nm) is achieved, and the maximum luminescence asymmetry factor (glum) is up to 1.1 * 10 <-2 >, and is improved by more than 30% compared with the prior art. Meanwhile, the generation inhibition rate of reactive oxygen species (ROS) is increased to 98% or above through a compact nanostructure of the material, the problem of phototoxicity of traditional triphenylamine derivatives is solved, and the material is suitable for high-resolution biological imaging and targeted diagnosis and treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of high polymer materials, and more particularly to a kind of poly-peptide nanoring material and its preparation method and application. BACKGROUND

[0002] In recent years, circularly polarized luminescent materials based on biomolecules have attracted much attention in the field of biomedical science. However, the existing technology has the following limitations: ① Difficulty in multi-color luminescence regulation - by covalently modifying fluorescent groups at the ends of poly-peptides, only single-wavelength circularly polarized luminescence can be achieved, and the luminescence asymmetry factor (g lum ) is less than 1x10 -2 , which cannot meet the needs of multi-color imaging; ② Low luminescence asymmetry factor (g lum ) - the g lum of existing water-dispersible poly-peptide-based circularly polarized luminescent materials is generally less than 1x10 -2 , which limits the signal-to-noise ratio of imaging; ③ Phototoxicity caused by reactive oxygen species (ROS) generation - traditional triphenylamine derivatives generate a large amount of ROS (free MTTV ROS generation is 47 times the fluorescence enhancement) under light, which seriously hinders their biological applications.

[0003] Poly-peptides have natural chirality and programmable self-assembly properties, and by co-assembling with functional molecules, materials with special optical properties can be obtained. Based on this, the preparation of water-dispersible poly-peptide nanoring materials with adjustable circularly polarized luminescence performance is of great significance for promoting the application of circularly polarized luminescence technology in the field of biomedical imaging. SUMMARY

[0004] The present application provides a kind of poly-peptide nanoring material and its preparation method and application, to solve the problem that circularly polarized luminescent materials in the prior art cannot have excellent multi-color luminescence performance, high imaging signal-to-noise ratio and low phototoxicity.

[0005] In a first aspect, the present application provides a method for preparing a poly-peptide nanoring material, comprising the following steps: mixing poly(γ-benzyl-L-glutamate) and achiral triphenylamine derivatives in an organic solvent, physically mixing to obtain an organic phase poly-peptide / photoreagent mixed solution; using solvent exchange method, under stirring conditions, adding deionized water to the organic phase poly-peptide / photoreagent mixed solution dropwise to form a nanoring structure; dialysis to remove the organic solvent to obtain the poly-peptide nanoring material.

[0006] As a possible implementation manner, the mass ratio of the poly(gamma-benzyl ester-L-glutamic acid) to the non-chiral triphenylamine derivative is 5-100:1; and / or, after being mixed in the organic solvent, the concentration of the non-chiral triphenylamine derivative is 0.1-0.4 mM; and / or, the ratio of the deionized water to the organic solvent is 50:1 in terms of volume ratio.

[0007] As a possible implementation manner, the dropping speed of the deionized water is 0.2-0.5 mL / min; and / or, the stirring speed of the stirring condition is 50-100 rpm; and / or, the dialysis condition is that the molecular weight cut-off of the dialysis bag is 14000, the dialysis medium is deionized water, and the dialysis time is 2-5 days, and the water is changed 3-5 times per day.

[0008] As a possible implementation manner, the organic solvent is tetrahydrofuran; and / or, the non-chiral triphenylamine derivative is any one of MTPA, MTTH and MTTV, and the structural formulae of the MTPA, the MTTH and the MTTV are as follows:

[0009]

[0010] In the second aspect, the application provides a poly-polypeptide nanoring material prepared by the preparation method in any possible implementation manner of the first aspect.

[0011] As a possible implementation manner, the diameter is 400±50 nm; and the inhibition rate of the active oxygen is greater than or equal to 80%.

[0012] As a possible implementation manner, the wavelength of the circularly polarized luminescence can be adjusted in the range of 370-660 nm; and the luminescence asymmetry factor can reach 1.1*10 -2 .

[0013] In the third aspect, the application provides an application of the poly-polypeptide nanoring material prepared by the preparation method in any possible implementation manner of the first aspect or the poly-polypeptide nanoring material in any possible implementation manner of the second aspect in medical imaging.

[0014] As a possible implementation manner, the application is realized by a circularly polarized luminescence technology.

[0015] The application provides a water-phase-dispersed homopolymer nanoring circularly polarized luminescence material and a preparation method thereof, constructs a luminescent chiral poly-polypeptide, and then successfully realizes the transfer of chirality by using a macromolecular self-assembly method, and prepares a water-phase-dispersed circularly polarized luminescence nanoparticle.

[0016] The preparation method provided by the present invention is simple and convenient, and provides a new idea for the preparation of aqueous circularly polarized luminescent materials; the circularly polarized luminescent signal of the system can be controlled by the chirality of the polypeptide and the co-assembly strategy; the circularly polarized light signal can be reversed after converting the chirality of the polypeptide; in the co-assembly system, the circularly polarized light signal can be controllably amplified by changing the mass ratio of the polypeptide to the small molecule; circularly polarized light can improve the signal-to-noise ratio in biological imaging. At present, the nanoparticle delivery diagnosis and treatment system in the biomedicine field is relatively mature. The aqueous phase dispersed homopolypeptide nanoring circularly polarized luminescent material in the present invention has good application prospects in the field of biomedical imaging.

[0017] Compared with the existing technology, the present invention achieves the following breakthroughs through the co-assembly strategy of achiral triphenylamine derivatives (MTPA, MTTH, MTTV) and polypeptides:

[0018] Tunable Multicolor Circularly Polarized Luminescence: By selecting triphenylamine derivatives with different molecular configurations (such as MTTH and MTTV), the emission wavelength of the nanoring material can cover the range from near-ultraviolet (370nm), yellow light (525nm), to deep red light (660nm). Furthermore, by adjusting the MTTV doping concentration (0.1-0.4mM), the emission wavelength can be further red-shifted by 30nm (630-660nm), breaking through the monochromatic emission limitations of traditional polypeptide materials.

[0019] Luminescence asymmetry factor (g lum ) significantly improved: the co-assembly system of the present invention achieves g under deep red circularly polarized emission (660nm) lum Up to 1.1×10 -2 , compared with the previously reported poly(peptide)-based materials (g lum <10 -2 ) increased by more than 30%.

[0020] Excellent reactive oxygen species (ROS) inhibition performance: The tightly packed nanoring structure of the polypeptide effectively blocks oxygen diffusion and inhibits exciton energy transfer, reducing the ROS generation of PBLG@MTTV to the same level as the blank control group. Compared with the ROS activity of free MTTV (47-fold fluorescence enhancement), the inhibition rate exceeds 98%, solving the phototoxicity problem of traditional triphenylamine derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Absorption spectra, emission spectra, fluorescence quantum yields and water fraction relationships of the achiral triphenylamine derivatives (MTTH, MTTV and MTPA) provided by the embodiments of the present application, wherein a is the ultraviolet spectrum of MTTH, MTTV and MTPA; b is the fluorescence spectrum of MTTH, MTTV and MTPA; c is the aggregation-induced emission characteristic curve of MTTH, MTTV and MTPA, (good solvent is selected as tetrahydrofuran, f w The ratio of water (H2O) is represented, and I / I0 refers to the relative fluorescence intensity of f w The ratio of water (H2O) is represented, and I / I0 refers to the relative fluorescence intensity of f w The ratio of water (H2O) is represented, and I / I0 refers to the relative fluorescence intensity of f

[0023] Figure 2 Circular polarization characterization results of the co-assembly products (PBLG@MTPA, PBLG@MTTH and PBLG@MTTV) provided by the embodiments of the present application, wherein a is the dynamic light scattering result of the MTTH, MTTV and MTPA assembly, the concentration of poly (γ-benzyl-L-glutamate) solution is 1.00 mg / mL, the concentration of photosensitizer small molecule solution is 0.200 mM, the following conditions are the same, b is the scanning electron microscope and transmission electron microscope image of the MTTH, MTTV and MTPA assembly, the scale is 200 nm, c is the fluorescence spectrum of the MTTH, MTTV and MTPA assembly, d is the circular dichroism spectrum of the MTTH, MTTV and MTPA assembly, e is the circularly polarized luminescence spectrum of the MTTH, MTTV and MTPA assembly, and f is the luminescence asymmetry factor value of the MTTH, MTTV and MTPA assembly.

[0024] Figure 3 Fluorescence intensity characterization and active oxygen inhibition performance test results of the co-assembly products (PBLG@MTPA, PBLG@MTTH and PBLG@MTTV) provided by the embodiments of the present application, wherein a is the dynamic light scattering result of the MTTV assembly, wherein 5:1, 10:1 and 20:1 represent the volume ratio of PBLG solution to MTTV solution used in the assembly process, the concentration of PBLG solution is 1 mg / mL, the concentration of photosensitizer small molecule solution is 1.00 mM, the following conditions are the same, b is the scanning electron microscope and transmission electron microscope image of the MTTH, MTTV and MTPA assembly, the scale is 200 nm, c is the fluorescence spectrum of the MTTH, MTTV and MTPA assembly, d is the circular dichroism spectrum of the MTTH, MTTV and MTPA assembly, e is the circularly polarized luminescence spectrum of the MTTH, MTTV and MTPA assembly, and f is the luminescence asymmetry factor value of the MTTH, MTTV and MTPA assembly.

[0025] Figure 4The active oxygen generation characterization results of the co-assembly products (PBLG@MTPA, PBLG@MTTH and PBLG@MTTV) provided in the embodiments of the present application are shown in the following table, wherein a is the change of the active oxygen generation intensity of DCFH as an indicator with time, b is the change of the active oxygen generation intensity of MTTV with time, c is the change of the active oxygen generation intensity of MTTV assembly (the concentration of MTTV is 0.2 mM) with time, and d is the change of the maximum active oxygen generation intensity at 525 nm in each group with irradiation time. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be clearly and completely described in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0027] To solve the problem that the circularly polarized luminescent material in the prior art cannot have excellent multi-color luminescent performance, high imaging signal-to-noise ratio and low phototoxicity, the embodiments of the present application provide a preparation method, application and performance detection experiment of a poly-polypeptide nanoring material.

[0028] The present application realizes the triple breakthrough of multi-color CPL emission, high g value and ROS inhibition through molecular design and co-assembly process, provides a new type of nano probe with high signal-to-noise ratio and low toxicity for biomedical imaging, and promotes the development of precise medical treatment and diagnosis. By adjusting the doping ratio of triphenylamine derivatives, we successfully realize the accurate regulation of circularly polarized luminescent performance; in the co-assembly system, by changing the mixing ratio of homopolypeptide and small molecules, we can significantly enhance the circularly polarized light signal. In view of the fact that circularly polarized light can improve the imaging quality and signal-to-noise ratio in biological imaging, and the mature application of nano particle delivery drug system in the current biological medicine field, the water-dispersible homopolypeptide-based circularly polarized luminescent nano material developed in the present application shows great application potential in biomedical imaging and treatment monitoring. In addition, due to the excellent biocompatibility and tunable luminescent properties of these nano materials, they may play a key role in precise medical treatment and personalized treatment, and provide a new direction for future biomedical imaging research and clinical application.

[0029] The technical solutions of the present application will be further described in combination with specific embodiments.

[0030] Embodiment 1

[0031] The present embodiment provides a preparation experiment of achiral triphenylamine derivatives.

[0032] A method for synthesizing a non-chiral triphenylamine derivative (MTTH) (a synthetic route thereof is shown in Formula I): 5-bromothiophene-2-carboxaldehyde (159 mg, 0.5 mmol), 4-(diphenylamino)phenylboronic acid (190 mg, 1 mmol), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4, 10 mol%), and potassium carbonate (K2CO3, 346 mg, 2.5 mmol) were added to a mixed solvent (dioxane:water = 10:1, by volume). The reaction mixture was heated to 95°C for 16 hours. After 16 hours, the reaction mixture was filtered and the solvent was removed. The residue was dissolved in dichloromethane (DCM) and washed with water. The combined organic layers were dried over anhydrous magnesium sulfate (MgSO4) and then filtered. After the solvent was removed, the residue was purified by silica gel column chromatography to obtain a yellow solid product (77 mg) with a yield of 40%.

[0033]

[0034] A method for synthesizing a non-chiral triphenylamine derivative (MTTH) (a synthetic route thereof is shown in Formula I): 5-bromothiophene-2-carboxaldehyde (159 mg, 0.5 mmol), 4-(diphenylamino)phenylboronic acid (190 mg, 1 mmol), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4, 10 mol%), and potassium carbonate (K2CO3, 346 mg, 2.5 mmol) were added to a mixed solvent (dioxane:water = 10:1, by volume). The reaction mixture was heated to 95°C for 16 hours. After 16 hours, the reaction mixture was filtered and the solvent was removed. The residue was dissolved in dichloromethane (DCM) and washed with water. The combined organic layers were dried over anhydrous magnesium sulfate (MgSO4) and then filtered. After the solvent was removed, the residue was purified by silica gel column chromatography to obtain a yellow solid product (77 mg) with a yield of 40%.

[0035]

[0036] The absorption spectrum, emission spectrum, and fluorescence quantum yield of MTPA and the prepared MTTH and MTTV in relation to water fraction are shown in Figure 1

[0037] Example 2

[0038] This example provides a circularly polarized luminescence performance modulation experiment.

[0039] Poly(γ-benzyl-L-glutamate) (PBLG) was co-assembled with MTTH, MTTV, and MTPA in Example 1, respectively. Taking MTPA as an example, the co-assembly process was as follows:

[0040] ​PBLG and MTPA were dissolved in THF to obtain a PBLG solution with a concentration of 1 mM and a MTPA solution with a concentration of 1 mM. 1 mL of the PBLG solution was taken into an assembled sample bottle, then 670 μL of the MTPA solution was added and stirred to prepare a mixed solution [c(MTPA) = 0.4 mM]. 2 mL of deionized water was added dropwise to the mixed solution using a peristaltic pump (2.1 rpm) while stirring (IKA 50 rpm). After assembly was completed, the reaction system was transferred to a dialysis bag for dialysis, the molecular weight cutoff value of the dialysis bag was 14000, water was changed four times a day, and stirring was continued for 2 days to obtain PBLG@MTPA. Samples with c(MTPA) = 0.2 mM and c(MTPA) = 0.1 mM were also co-assembled according to the above method.

[0041] The co-assembly steps of PBLG@MTTH and PBLG@MTTV were the same as described above.

[0042] The co-assembly products (PBLG@MTPA, PBLG@MTTH and PBLG@MTTV) were respectively characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), fluorescence spectroscopy (FL), circular dichroism spectroscopy (CD), and circularly polarized luminescence spectroscopy (CPL), and the results are shown in Figure 2 By changing the doping concentration of the achiral triphenylamine derivative (0.1, 0.2 and 0.4 mM), the size and morphology of the nanorings were characterized, and the results are shown in Figure 3 Figure 3 It can be seen that the diameter of the nanorings is about 400 nm; the effect of the nanorings on circularly polarized luminescence performance was studied, and MTTV was taken as an example for testing. With the increase of the doping concentration of MTTV, the emission wavelength of the nanorings red-shifted from 630 nm to 660 nm; the luminescence asymmetry factor increased from 0.6 x 10 -2 to 1.1 x 10 -2 .

[0043] By adjusting the type and doping concentration of the triphenylamine derivative, multi-color emission and signal enhancement of CPL were achieved:

[0044] Multi-color emission: from c in Figure 1 , PBLG@MTPA (370 nm, near ultraviolet), PBLG@MTTH (525 nm, yellow light), PBLG@MTTV (660 nm, deep red light);

[0045] g value improvement: from f in Figure 2 , g lum increased from 0.8 x 10 -2 to 1.1 x 10 -2 ​, which is more than 30% higher than the existing technology;

[0046] Wavelength control: Figure 3 As shown in Figure c, when the MTTV doping concentration increases from 0.1 mM to 0.4 mM, the emission wavelength red-shifts by 30 nm (630 → 660 nm).

[0047] Example 3

[0048] This example provides a test experiment for the active oxygen inhibition performance.

[0049] Using 2,7-dichlorofluorescein diacetate (DCFH) as a fluorescent probe, the blank group was tested, with only the photosensitizer small molecule (MTTV in this case) and the active oxygen generation ability of the PBLG@MTTV nanoring material. After DCFH was mixed with PBLG@MTTV, the active oxygen generation ability of the PBLG@MTTV nanoring material was tested under white light irradiation (4mW / cm 2 , tested every 30s, for a total of 5 minutes), and the changes in fluorescence intensity were detected. Figure 4 The results shown in a to c show that DCF fluorescence increased 47 times under free MTTV white light irradiation, indicating high ROS activity; and the ratio of the maximum value to the initial value at each time node was plotted in Figure d. The results showed that the fluorescence intensity of the PBLG@MTTV nanoring material under light conditions was equivalent to that of the blank group, and the ROS inhibition rate reached 98%, indicating that it has good reactive oxygen species inhibition performance and greatly eliminated the effects of phototoxicity.

[0050] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0051] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing a polypolypeptide nanoring material, characterized in that: The following steps are involved: Mixing poly(γ-benzyl ester-L-glutamic acid) and an achiral triphenylamine derivative in an organic solvent and physically mixing them to obtain an organic phase poly(polypeptide) / photosensitizer mixed solution; Using a solvent exchange method, deionized water is added dropwise to the organic phase polypolypeptide / photosensitizer mixed solution under stirring to form a nanoring structure; The organic solvent is removed by dialysis to obtain the polypolypeptide nanoring material.

2. The preparation method according to claim 1, characterized in that The molar ratio of the poly(γ-benzyl ester-L-glutamic acid) to the achiral triphenylamine derivative is 5 to 100:1; and / or, after said mixing in an organic solvent, the concentration of said achiral triphenylamine derivative is 0.1 to 0.4 mM; And / or, in terms of volume ratio, the ratio of the added amount of the deionized water to the added amount of the organic solvent is 50:

1.

3. The preparation method according to claim 1, characterized in that The dropwise addition rate of the deionized water is 0.2 to 0.5 mL / min; and / or, the stirring rate of the stirring condition is 50 to 100 rpm; And / or, the dialysis conditions are: the molecular weight cut-off of the dialysis bag is 14,000, the dialysis medium is deionized water, the dialysis time is 2 to 5 days, and the water is changed 3 to 5 times a day.

4. The preparation method according to claim 1, characterized in that The organic solvent is tetrahydrofuran; And / or, the achiral triphenylamine derivative is any one of MTPA, MTTH and MTTV, and the structural formulas of MTPA, MTTH and MTTV are respectively as shown below:

5. The polypolypeptide nanoring material prepared by the preparation method according to any one of claims 1 to 4.

6. The polypolypeptide nanoring material according to claim 5, characterized in that: Its diameter is 400 ± 50 nm; Its inhibition rate on active oxygen is ≥80%.

7. The polypolypeptide nanoring material according to claim 5, characterized in that: The wavelength of its circularly polarized light emission can be adjusted from 370 to 660 nm; Its luminescence asymmetry factor can reach 1.1×10 -2 .

8. Use of the polypolypeptide nanoring material prepared by the preparation method according to any one of claims 1 to 4 or the polypolypeptide nanoring material according to any one of claims 5 to 7 in medical imaging.

9. The use according to claim 8, characterized in that The application is achieved through circularly polarized luminescence technology.

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