CN110806402B - Neuromelanoidin-like nano material and preparation method and application thereof - Google Patents

Abstract

The invention discloses a neurotensin-like nano material, a method for preparing the neurotensin-like nano material and application of the neurotensin-like nano material. The chemical composition of the neurotensin-like nano material is amino acid and dopamine. The method comprises at least the following steps: 1) dissolving basic amino acid in an aqueous solution to obtain a solution I; 2) adding dopamine hydrochloride into the solution I, and centrifuging at a low speed to remove precipitates to obtain a solution II; 3) and (4) carrying out high-speed centrifugation on the solution II, and separating supernatant to obtain the neurotensin-like nano material. The method for preparing the neuromelanin-like nano material can effectively adjust the size of the neuromelanin-like nano particles, is simple and easy to operate, and the prepared neuromelanin-like nano material can be applied to Pb²⁺And (4) during detection.

Description

Neuromelanoidin-like nano material and preparation method and application thereof

Technical Field

The application belongs to the field of applied chemistry, and particularly relates to a method for preparing a melanin-like nano material, and a preparation method and application thereof.

Background

Neuromelanin (NM) is a pigment found in parts of the brain, such as the substantia nigra. It is produced by Dopamine (DA) in the Central Nervous System (CNS), and can protect the brain from oxidative stress, and its production is associated with neurodegenerative diseases such as parkinson's disease and alzheimer's disease. Unlike peripheral melanin found in integuments, NM does not have a well-defined particle shape and is more difficult to extract or purify. In addition, NM is a more complex, heterogeneous melanin and protein structure.

DA is one of the most important monoamine neurotransmitters in the mammalian central nervous system, which regulates a wide range of complex processes including motor control, attention span, and reinforcement of learning. In vitro, DA can self-polymerize under alkaline conditions to form fluorescent polydopamine (polydopamine)Melanogenesis) nanoparticles, the alkaline condition being by oxidation of H₂O₂Or reducing bases such as NH₃·H₂O, NaOH or Tris (hydroxymethyl) -aminomethane (Tris). The absence of these bases in biological tissues still requires researchers to use alternative methods of synthesizing melanin using biocompatible reagents for in vivo applications.

In the brain, L-arginine, L-lysine, L-histidine and DA are chemical messengers belonging to the overbalanced neurotransmitter complex to maintain normal brain function. Understanding that these basic amino acids catalyze the oxidation of DA in the human body, particularly in the brain, is very important for elucidating the new synthetic pathways for DA oxidation and NM provision in vivo.

Disclosure of Invention

The inventors of the present invention studied the reaction of dopamine with 20 amino acids and successfully demonstrated that basic amino acids promote the conversion of DA to neurotensin-like nanoparticles (NM-NPs). Specifically, the inventors selected L-lysine as a model basic amino acid to prepare NM-NP (5-160NM) with a controlled size. NM-NPs exhibit strong fluorescence when their size is around 5NM and their fluorescence can be selectively quenched by lead ions, and thus NM-NPs can be used as multifunctional probes to monitor their degradation and detect lead ions. The invention not only expands the understanding of people on DA self-polymerization, but also provides a new way for monitoring neurotoxic lead ions in the brain.

One aspect of the invention provides a neurotensin-like nanometer material, which contains basic amino acid and dopamine,

the particle size of the neurotensin-like nano material is 5 nm-160 nm;

preferably, the particle size of the neurotensin-like nanometer material is 5 nm-40 nm.

Preferably, the dopamine may be dopamine hydrochloride, and the other dopamine substances may be analogs such as epinephrine.

In a preferred embodiment, the neurotensin-like nanomaterial comprises at least the following peaks on an infrared spectrum:

a peak at a wavelength of 200 to 500 nm;

the wave number is 3300-3500 cm^-1A peak of (a);

wave number of 1600-1650 cm^-1A peak of (a);

wave number of 1050-1100 cm^-1Peak of (2).

It is to be understood that the characteristic peaks shown above are only characteristic peaks of neurotensin-like nanomaterial according to the preferred embodiment, and in other preferred embodiments, the characteristic peaks may be outside of this range.

In a preferred embodiment, when the particle size of the neurotensin-like nanomaterial is 5nm, the neurotensin-like nanomaterial has a fluorescence emission peak at 505nm to 515nm under the excitation of light with the wavelength of 430 nm.

Another aspect of the present invention provides a method for preparing a neurotensin-like nanomaterial, the method at least comprising the steps of:

1) Dissolving basic amino acid in an aqueous solution to obtain a solution I;

2) adding dopamine into the solution I, and centrifuging at a low speed to remove precipitates to obtain a solution II;

3) and (4) carrying out high-speed centrifugation on the solution II, and separating supernatant to obtain the neurotensin-like nano material.

In a preferred embodiment, the concentration of the basic amino acid is 0.01 to 10 mg/mL;

preferably, the concentration of the basic amino acid is 0.08 to 2.8 mg/mL.

In a preferred embodiment, the dopamine is dopamine hydrochloride, and the concentration of the dopamine hydrochloride is 1 to 10mg mL^-1；

Preferably, the ratio of the basic amino acid to dopamine hydrochloride is 1: 1 to 1: 10.

in a preferred embodiment, the reaction time of the dopamine hydrochloride with the solution I is 3 to 24 hours;

preferably, the speed of the low-speed centrifugation is 2500-5000 rpm, and the speed of the high-speed centrifugation is 8000-12000 rpm.

In a preferred embodiment, the molecular mass of the precipitate is above 8000 Da.

In a preferred embodiment, in the step 3), the step of washing with deionized water and freeze-drying the product is further included after separating the supernatant.

The invention further provides an application of the melanin-like nano material for detecting Pb ²⁺。

The beneficial effect that this application can produce includes:

1) the method for preparing the neuromelanin-like nano material can effectively adjust the size of the neuromelanin-like nano particles.

2) The method for preparing the neurotensin-like nano material is simple and easy to operate.

3) The neurothron-like nano material provided by the application can be applied to Pb²⁺And (4) during detection.

Drawings

FIG. 1A is a transmission electron micrograph showing that melanin formed by DA and HIS (HIS-NM) does not have well-controlled morphology; FIGS. 1B and 1C are transmission electron micrographs showing spherical melanin formation by ARG-NM and LYS-NM, respectively.

FIG. 2 shows the LYS-NM as a model system to represent the mechanism and properties of NM-NP synthesis.

FIG. 3A is a Fourier transform infrared spectrum of NM-NP of example 1 showing NP having a broad absorption of 200 to 500NM, and FIG. 3B shows NM-NP at 3300-^-1、1620cm^-1And 1089cm^-1Three major bands are shown.

FIG. 4 shows the reaction steps for DA generation of NM-NP by L-lysine protonation oxidation.

FIG. 5A shows that L-lysine concentration and reaction time are key to adjusting the size of NM-NPs. Increasing the L-lysine concentration from 0.15mg/mL to 2.6mg/mL, and decreasing NM-NP from 95NM to 5NM, the results are shown in FIG. 5A, and the yield of NM-NP remained unchanged. In addition, when the reaction time was increased from 3 hours to 24 hours, the size of NM-NP was increased from 95NM to 160NM, and the result is shown in FIG. 5B.

FIG. 6 shows the fluorescence characteristics of NM-NP 5NM in diameter.

FIG. 7A shows the emission peak of 5NM NM-NP at 510NM when using an excitation wavelength of 430 NM; FIG. 7B shows that the emission intensity depends on the excitation wavelength, which initially increases with increasing wavelength and then begins to decrease gradually; FIGS. 7C and 7D show that the fluorescence intensity of NM-NP increases as its concentration increases.

FIGS. 8A and 8B show, respectively, a cross-sectional view at H₂O₂After 12 hours of reaction, the fluorescence of NM-NP gradually increased.

FIG. 9 is a TEM image demonstrating degradation of NM-NPs.

FIG. 10A shows Pb²⁺Significant fluorescence quenching can be induced, and the fluorescence intensity of NM-NP is not obviously changed by other metal ions; FIG. 10B shows the evolution of Pb with Pb²⁺An increase in concentration, a decrease in NM-NP fluorescence; FIG. 10C shows the calibration curve display vs. Pb²⁺Concentration (0-50. mu.gL)^-1) Linear relationship of (2) with a detection limit of 2. mu.gL^-1。

FIG. 10D shows good biocompatibility of NM-NP.

Detailed Description

The present application will be described in detail with reference to examples, but the present application is not limited to these examples.

Unless otherwise specified, the raw materials used in the examples were all purchased commercially and used without treatment; the used instruments are all used according to the parameters recommended by manufacturers.

In the examples, DA & HCl, Fmoc-Lys (Me, Boc) -OH, and 20 amino acids (including lysine, arginine, histidine, glutamic acid, aspartic acid, alanine, valine, serine, asparagine, threonine, methionine, isoleucine, glutamine, proline, leucine, tryptophan, cysteine, tyrosine, phenylalanine, glycine) were purchased from Sigma Aldrich.

In the examples, the size of NM-NPs was measured by a Malvern Zetasizer Nano ZS instrument (Malvern, UK) using the Dynamic Light Scattering (DLS) method. The morphology of NM-NPs was characterized by Transmission Electron Microscopy (TEM) (FEI Tecnai Multipurpose TEM, ThermoFisher Scientific, USA). Fourier transform Infrared Spectroscopy (FTIR) (Thermo Fisher FTIR 6700) was used to characterize the chemical structure, and UV-Vis Nanodrop 2000C spectroscopy (Thermo Fisher Scientific, USA) was used to measure the absorption of the sample. Fluorescence spectra were obtained from a fluorescence spectrophotometer (Horiba Jobin Yvon, Fluorolog-3).

Example 1 Synthesis and modification of NM-NPs

Synthesis and characterization of NM-NP

In the specific experimental conditions, 8mg of L-lysine was completely dissolved in 95mL of an aqueous solution for 1 hour under stirring at 25 ℃. 5mL of DA & HCl (4mg mL) was added under stirring^-1) Slowly injecting into the solution. After 3 hours, all large precipitates were removed by low speed centrifugation (4,000rpm), and then the supernatant was separated by high speed centrifugation (10,000rpm) and washed three times with deionized water. The product was dried in a freeze-dryer for 12 hours. NM-NP was obtained after several washes with ethanol and Deionized (DI) water.

The remaining 19 amino acids were tested under the same conditions as for L-lysine, whereby it was investigated whether 20 amino acids were directly reacted with DA in an aqueous solution. The results show that only when basic amino acids (histidine, arginine and lysine) were added, the solution darkened and then turned black. Considering that this synthetic process is similar to the conversion of DA to NM in the brain, the product was named NM-NPs. Melanin formed by DA and HIS (HIS-NM) did not have well-controlled morphology, as shown in fig. 1A, and the other two types (ARG-NM and LYS-NM) showed spherical shapes, with the results shown in fig. 1B and 1C.

Due to the good morphology and relatively simple lysine chemical structure of LYS-NM, we used LYS-NM as a model system to express the synthetic mechanism and properties of NM-NP, as shown in FIG. 2.

NM-NP has a broad absorption of 200 to 500NM, as shown in FIG. 3A, compared to DA with a sharp absorption peak at 300 NM. NM-NP shows a broadband IR spectrum. NM-NPs are different from L-lysine and DA, indicating that NM-NPs are a new polymeric material. NM-NP 3500cm in 3300-^-1、1620cm^-1And 1089cm^-1Showing three main bands, e.g.As shown in fig. 3B. 3300-3500cm^-1Peak from NH (. about.3300 cm)^-1) And OH stretch (-3400 cm) in indole or pyrrole^-1) (ii) a The C ═ O extensional carbonate group is 1620cm^-1C ═ N and/or C ═ C aromatic ring vibration; 1089cm from C-O stretching vibration and/or C-H vibration^-1. NM-NPs are chemically different from synthetic melanin, which is 1089cm^-1There was no peak, probably due to the reaction of L-lysine with DA.

To understand the reaction of L-lysine and DA to form NM-NP, we replaced L-lysine with three chemical analogs of L-lysine (e.g., F-moc-Lys (Me, Boc) -OH, N-epsilon-acetyl-L-lysine, and N-alpha-acetyl-L-lysine). No color change was found in the reaction under the same conditions. DA is protonated by L-lysine (basic amino acid) and autoxidized under synthesis conditions to produce NM-NP, the reaction steps are shown in fig. 4. During the reaction, the pH of the solution decreased from 9.2 to 8.0. In the case of amino-blocked L-lysine analogs (i.e., N- ε -acetyl-L-lysine, N- α -acetyl-L-lysine and F-moc-Lys (Me, Boc) -OH), we observed neither color change nor nanoparticle formation in solution. This indicates that the two amino groups in L-lysine are critical for the preparation of NM-NP. The free amine of L-lysine acts as a nucleophile by "donating" the available lone pair of electrons. The reactive sites for DA are not primarily nitrogen, but are catechol moieties. Due to the electron donating effect from catechol, the aromatic ring is electron rich and is more easily oxidized to produce the o-quinone product. The oxidation reaction depends to some extent on the pH, but primarily on the presence of reactive oxygen species. After oxidation at the reactive site on the quinone moiety, it can act as a nucleophile and generate the indole unit (indoloquinone) by the michael addition reaction through attack of the indole product by the primary nitrogen. Indoloquinones react readily with NH of L-lysine by Schiff's base reaction (L-lysine-indolone) ₂Combined and further polymerized to NM-NP (higher oligomers of L-lysine-indoloquinone).

EXAMPLE 2 determination of the stability of NM-NPs

Three bottles of 2.0mL NM-NPs aqueous solution were prepared, all at a concentration of 0.1 wt%. In vial 1, 10. mu.LH was added₂O₂(30% by volume); in vial 2, at 50mM PBS (pH6.5) and 10. mu.LH₂O₂(30%) 10. mu.L 0.1mgmL of the additive was added^-1Horseradish peroxidase type VI (Sigma Aldrich). Sample vial 3 is a control without any additions. All three vials were capped and stirred by a magnetic stirrer at 250rpm at 25 ℃. After 24 hours of incubation, the samples were washed with deionized water and centrifuged at 10,000 rpm. TEM and DLS were used to study size variation of NM-NPs.

L-lysine concentration and reaction time are key to adjusting the size of NM-NPs. Increasing the L-lysine concentration from 0.15mg/mL to 2.6mg/mL, and decreasing the NM-NP from 95NM to 5NM, the results are shown in FIG. 5A, and the yield of NM-NP remained unchanged. In addition, when the reaction time was increased from 3 hours to 24 hours, the size of NM-NP was increased from 95NM to 160NM, and the result is shown in FIG. 5B.

To study the fluorescence properties of NM-NPs, different sizes of NM-NPs were prepared. NM-NPs with a diameter of 5NM were fluorescent as shown in fig. 6, but no fluorescence was observed for 40NM nanoparticles or any larger nanoparticles. For 5NM NM-NP, the emission peak was at 510NM when an excitation wavelength of 430NM was used, as shown in FIG. 7A. The emission intensity depends on the excitation wavelength, which initially increases with increasing wavelength and then starts to decrease gradually, as shown in fig. 7B. The fluorescence intensity of NM-NP increased from 0 to 0.24mg mL with its concentration, as shown in FIGS. 7C-7D ^-1But is increased.

Melanin is a powerful antioxidant both in vivo and in vitro. To facilitate our understanding of NM antioxidant behavior, we investigated the degradation properties of NM-NPs. In and H₂O₂After 12 hours of reaction, the fluorescence of NM-NP gradually increased, as shown in FIGS. 8A-8B, indicating that NM-NP decreased to less than 40 NM. After 48 hours, due to H₂O₂And the fluorescence disappears after the degradation. TEM also confirmed the degradation of NM-NP as shown in FIG. 9.

Example 3 Selective detection of Pb²⁺

Preparation of Pb in deionized Water²⁺、Cd²⁺、Hg²⁺、Cu²⁺、Mn²⁺、Mg²⁺、K¹⁺And Na¹⁺The stock solution of (1). Adding Pb²⁺(10μL，1-5000μgL^-1) And other metal ions (10. mu.L, 3000. mu.gL)^-1) And 990. mu.L of 0.1. mu.gmL^-1NM-NPs (N-N) were mixed separately at 25 ℃ over 1 minute. The fluorescence spectrum of the resulting solution was recorded at an excitation wavelength of 430nm (λ ex ═ 430nm) and an emission wavelength of 510nm (λ em ═ 510 nm).

We hypothesize that NM-NPs can bind to metal ions as melanin. To confirm the specificity of NM-NPs, we used 30. mu.g L^-17 kinds of metal ions (Cd)²⁺、Hg²⁺、Cu²⁺、Mn²⁺、Mg²⁺、K¹⁺And Na¹⁺) The fluorescence change is tracked.

Pb²⁺Significant fluorescence quenching can be induced without other metal ions significantly altering the fluorescence intensity of NM-NP, as shown in fig. 10A. This indicates a unique Pb²⁺Can effectively bind with NM-NP, so that NM-NP can be used for detecting Pb ²⁺. Further, with Pb²⁺The increase in concentration, the fluorescence of NM-NP decreased, as shown in FIG. 10B. Calibration curve shows the ratio of Pb to²⁺Concentration (0-50. mu.g L)^-1) Linear relationship of (2) with a detection limit of 2. mu.gL^-1As shown in fig. 10C. In humans, at low concentrations (10. mu.gL)^-1) Pb of²⁺It can cause behavioral problems, interfere with brain development, and slow down nerve conduction velocity. The use of NM-NP as a lead detector is practical and biocompatible, with a resolution comparable to that of the fiber optic probe method.

Example 4 cytotoxicity assays

Human epidermal keratinocytes (adult, HEKa cells) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco), 80U mL^-1Penicillin and 0.08mg mL^-1Streptomycin. To determine the cytotoxicity of NM-NP, HEKa cells were seeded at a density of 10⁴96-well plates per cell/well. NM-NP was dispersed in DMEM without FBS, added to a range of concentrations (0, 20, 40, 80 and 160 μ gmL)^-1) In the HEKa cell of (1). After 24 hours incubation, 100. mu.L/well was usedThiazolyl blue tetrazolium bromide (MTT) solution (KeyGEN BioTECH) replaced cell supernatant and was incubated for an additional 4 hours. The supernatant was discarded and DMSO was added. The optical density at 550nm was measured by a microtiter plate reader (Multiskan GO).

To further validate the potential use of NM-NP in biomedical science, we assessed NM-NP cytotoxicity by testing HEKa cell viability after 24 hours incubation with NM-NP. When the concentration is lower than 40 mu gmL^-1At this time, more than 95% of the cells survived, as shown in fig. 10D. When the concentration of NM-NP reached 80. mu.gmL^-1And above, slight inhibition of cellular activity was observed. However, even if the concentration reaches 160. mu.gmL^-1Cell viability remained above 80%, indicating relatively good biocompatibility.

Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.

Claims (8)

1. Application of a neurotensin-like nano material, which is characterized in that the neurotensin-like nano material is used for detecting Pb by a fluorescence quenching method²⁺；

The neuromelanin-like nano material is prepared from raw materials containing basic amino acid and dopamine hydrochloride,

The particle size of the neurotensin-like nano material is 5 nm-40 nm;

the infrared spectrogram of the neurotensin-like nanometer material at least comprises the following peaks:

a peak at a wavelength of 200 to 500 nm;

the wave number is 3300-3500 cm^-1A peak of (a);

wave number of 1600-1650 cm^-1A peak of (a);

wave number of 1050-1100 cm^-1A peak of (a);

when the particle size of the neurotensin-like nano material is 5nm, the neurotensin-like nano material has a fluorescence emission peak at 505 nm-515 nm under the excitation of light with the wavelength of 430 nm;

the method for preparing the neurotensin-like nano material at least comprises the following steps:

1) dissolving basic amino acid in an aqueous solution to obtain a solution I;

2) adding dopamine hydrochloride into the solution I, and centrifuging at a low speed to remove precipitates to obtain a solution II;

3) centrifuging the solution II at a high speed, and separating supernatant to obtain a neurotensin-like nano material;

the ratio of the basic amino acid to the dopamine hydrochloride is 1: 1 to 1: 10.

2. use according to claim 1, wherein the concentration of the basic amino acid is from 0.01 to 10 mg/mL.

3. Use according to claim 1, wherein the concentration of the basic amino acid is 0.08 to 2.8 mg/mL.

4. The use according to claim 1, wherein the concentration of dopamine hydrochloride is 1 to 10mg mL^-1。

5. The use according to claim 1, wherein the reaction time of dopamine hydrochloride with solution I is 3 to 24 hours.

6. The use according to claim 1, wherein the low speed centrifugation is at a speed of 2500 to 5000rpm and the high speed centrifugation is at a speed of 8000 to 12000 rpm.

7. Use according to claim 1, wherein the precipitate has a molecular mass above 8000 Da.

8. The use according to claim 1, wherein in step 3), the separation of the supernatant further comprises the steps of washing with deionized water and freeze-drying the product.

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