CN110343144A - A kind of therapeutic nano enzyme and preparation method thereof assembled by free yl induction - Google Patents
A kind of therapeutic nano enzyme and preparation method thereof assembled by free yl induction Download PDFInfo
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Abstract
The invention discloses a kind of therapeutic nano enzymes assembled by free yl induction, and by L-lysine and glutathione, dehydrating condensation is prepared under certain conditions.Specifically, L-lysine (Lys) and glutathione (GSH) are dissolved in suitable ultrapure water, it is uniformly mixed, wherein the molar ratio of L-lysine (Lys) and glutathione (GSH) is (1:1)~(3:1)；Then mixed solution is placed in microwave synthesizer, material is taken out after 60-90 DEG C of gradient-heated, cooling, separated, obtain small nano enzyme with effect of scavenging radical itself.Small nano enzyme size is in 1-2nm; it is easy to break through blood-brain barrier; it in addition can be by free yl induction; induction becomes larger, and reduces nano enzyme in the non-specificity accumulation of healthy organ, the big nano enzyme to become larger still has the catalytic activity of a variety of enzymes; when the region is invaded and harassed by free radical again; it can continue to play catalysis, accelerated decomposition free radical forms long-acting restoration and protection effect.
Description
Technical field
The present invention relates to artificial mimic enzyme preparation technical fields, and in particular to a kind of by the therapeutic of free yl induction aggregation
Nano enzyme and preparation method thereof.
Background technique
Biological enzyme is a kind of biocatalyst existing for life entity is rely, and can efficiently be urged under extremely mild physiological condition
Change series of chemical.Biological enzyme is mostly protein, also there is a small number of nucleic acid, has exquisite three-dimensional structure, to assign it
High catalytic activity and substrate selectivity.However, biological enzyme is difficult to extraction separation and purification, high production cost, stability is poor, easily
Inactivation, it is sensitive to environmental condition, limit the large-scale production and application of biological enzyme.
Nano enzyme is a kind of nano material with biological enzyme characteristic, is a kind of people for simulating biological enzyme on a molecular scale
Work catalyst had not only had the characteristic of nano material, but also the catalytic activity with analogue enztme.From 2007, ferromagnetic nano grain
Son be reported with peroxidase activity since, various metal nanoparticles, metal oxide, hydroxide and
Compound etc. is all used as classical artificial peroxidase.Especially as graphene quantum dot, carbon nanotube, fullerene and carbon
The c-based nanomaterials such as point, because of its high stability, low cost is easy to be mass produced and store, good biocompatibility and
Hypotoxicity is widely used in biologic medical, the fields such as bio-sensing and environmental project.But nano enzyme how is improved in physiological condition
Under catalytic activity, improve environmental selectivity and substrate selectivity, control nano enzyme in target cell high-efficient and lasting play treatment
And catalytic performance, it is still the hot spot of research.Such as, patent CN109046360A discloses a kind of Fe that catalytic activity improves3O4It receives
Rice enzyme, by Fe3O4Doped metallic elements improve routine Fe in nano enzyme preparation process3O4The catalytic activity of nano enzyme；Patent
CN108067309A discloses a kind of method for optimizing nano enzyme seed activity, and histidine, histamine, nitroimidazole etc. is used to contain
There is the compound-modified nanometer enzyme granulate of imidazole ring, by nanometer enzyme granulate to substrate H2O2Affinity improve 12 times, by nanometer
The catalytic efficiency of particle has been increased to 21 times；Patent CN109799313A discloses a kind of nitrogen based on nucleotide or derivatives thereof
Doped carbon material nano enzyme and preparation method thereof, wherein nitrogen-doped carbon material nano enzyme is set by nucleic acid base or derivatives thereof
The carbon material nano enzyme being carbonized at different temperatures in inert gas environment.
Currently, there are many research to nano enzyme, but still remain following problems:
(1) nano material size is larger, and organism is difficult to drain, some contain the substances such as metal, and there are long term toxicity products
Tired problem, nano enzyme size of the present invention can break through blood-brain barrier, and raw material in several nanometers, extra small size
For amino acid, biocompatibility is preferable, and toxicity is very low.
(2) what existing nano enzyme had has a single function, and it is limited to be applied to organism treatment function.
(3) existing nano enzyme is limited applied to organism by catalytic selectivity and environmental selectivity, is not easy in specific region
Efficient catalytic ability is played with physiological environment.
Summary of the invention:
In order to overcome the deficiencies in the prior art, the present invention provides a kind of by the therapeutic of free yl induction aggregation
Nano enzyme.
To achieve the goals above, the invention adopts the following technical scheme:
A kind of therapeutic nano enzyme assembled by free yl induction, by L-lysine (Lys) and glutathione (GSH)
Dehydrating condensation is prepared under certain conditions.
A kind of preparation method for the therapeutic nano enzyme assembled by free yl induction, specifically includes the following steps:
(1) L-lysine (Lys) and glutathione (GSH) are dissolved in suitable ultrapure water, are uniformly mixed, wherein L- relies
The molar ratio of propylhomoserin (Lys) and glutathione (GSH) is (1:1)~(3:1)；
(2) step (1) mixed solution is placed in microwave synthesizer, takes out material after 60-90 DEG C of gradient-heated, cooling
Material, separation, obtains small nano enzyme.
Gradient-heated in step (2) specifically: to 60 DEG C, 70 DEG C, 80 DEG C, 85 DEG C respectively keep 1min, gradient-heated purpose
Prevent bumping, then to 90 DEG C of holding 30min.
Above-mentioned nano enzyme is assembled under the induction of free radical and forms big nano enzyme.
The small nano enzyme size is in 1-2nm, and big nano enzyme size is in 200-300nm, wherein " big " and " small " is size
On in contrast, be not particularly limited.
Compared with prior art, the present invention having the advantage that (1) by adjusting raw material proportioning in experiment and temperature, system
It is standby a kind of with good catalytic activity and the antioxidative therapeutic nano enzyme assembled by free yl induction out, and preparation method
Step is simple, at low cost, is easy to be mass produced and save；(2) the small nano enzyme prepared not only has urging for conventional nano enzyme
Change activity, can speed up removing free radical, and there is inoxidizability, itself has the function of removing free radical, that is, have and control
Treatment effect, removing inflammation part, extra free radical achievees the purpose that treat disease；(3) small nano enzyme size is smaller, it is easy to
Blood-brain barrier is broken through, in addition can be cross-linked in situ by free yl induction and be gathered in inflammation part；(4) it participates in removing certainly at itself
During by base, small nano enzyme is become larger by free yl induction, is reduced nano enzyme in the non-specificity accumulation of healthy organ, is become
Big big nano enzyme still has the catalytic activity of a variety of enzymes, when the region is invaded and harassed by free radical again, can continue to play
Catalysis, accelerated decomposition free radical form long-acting restoration and protection effect；(5) using amino acid as raw material, nano enzyme is raw
Object compatibility is good, and toxicity is low.
Detailed description of the invention
Fig. 1 is the uv absorption spectra of mixed solution and small nanometer enzyme solutions in embodiment 1.
Fig. 2 is small nano enzyme in the Induction Process of hydrogen peroxide, different moments atomic force scanning probe microscopy photo.
Fig. 3 is small nano enzyme in the Induction Process of illumination, different moments atomic force scanning probe microscopy photo.
Fig. 4 is small nano enzyme in hydrogen peroxide and the Induction Process of illumination, and small nano enzyme partial size changes over time relational graph.
Fig. 5 is the mixed solution of the identical Lys+GSH of GSH concentration and the uv absorption spectra of small nano enzyme.
Fig. 6 is the GSH solution of various concentration and the absorbance figure of small nanometer enzyme solutions in total antioxidant capacity test process.
Fig. 7 is the GSH solution of various concentration and the clearance rate of small nanometer enzyme solutions in total antioxidant capacity test process
Figure.
Fig. 8 is the GSH solution of various concentration and the absorbance of small nanometer enzyme solutions in the reset procedure of active oxygen radical
Figure.
Fig. 9 is the removing speed of the GSH solution of various concentration and small nanometer enzyme solutions in the reset procedure of active oxygen radical
Rate figure.
Figure 10 is that (from left to right the concentration of small nano enzyme is followed successively by the hydrogen peroxide activated detection figure of small nanometer enzyme
3.4mg/ml, 2.6mg/ml, 1.7mg/ml, 0.85mg/ml and 0mg/ml).
Lys, the GSH and small enzyme solutions decomposing H of Figure 11 for comparable sodium2O2Figure
Figure 12 is the UV absorption spectrogram that hydrogen peroxide is catalytically decomposed in the small nano enzyme of various concentration, GSH and Lys.
Figure 13 is the small nano enzyme of various concentration in 5min to the clearance rate figure of hydrogen peroxide.
Figure 14 is the POD Activity determination figure of the small enzyme of various concentration
Figure 15 is the small nano enzyme of comparable sodium (SN) and nano enzyme (H two kinds big2O2Induce big enzyme OCN；The big enzyme of photoinduction
UCN free sulfhydryl group content measuring)
Figure 16 is that the big nanometer enzyme hydrogen peroxide of same concentration of the small nano enzyme and two methods preparation after consuming sulfydryl is living
Property detection figure.
During Figure 17 is the big nano enzyme catalyzing hydrogen peroxide of two methods preparation, hydrogen peroxide surplus and time
Relational graph.
Figure 18 is the big nano enzyme of two methods preparation and changes with time with the POD activity of the GSH and Lys of concentration
Figure.
Figure 19 is fluorescence excitation spectrum spectrogram of the small nanometer enzyme solutions under 365nm wavelength.
Figure 20 is fluorescence excitation spectrum spectrogram of the small nanometer enzyme solutions under 473nm wavelength.
Figure 21 is fluorescence excitation spectrum spectrogram of the small nanometer enzyme solutions under 532nm wavelength.
Figure 22 is H2O2Induce utilizing total internal reflection fluorescence microscope photo and the common location of nano enzyme aggregation real in living cells
It tests.
Specific embodiment
Below by specific embodiment and Figure of description, the present invention will be further described.
Embodiment 1
It is a kind of by free yl induction assemble therapeutic nano enzyme, specific preparation method the following steps are included:
(1) 1g L-lysine (Lys) and 0.701g glutathione (GSH) are dissolved in 5ml ultrapure water, are uniformly mixed, obtain
To mixed solution；
(2) step (1) mixed solution is placed in microwave synthesizer and is heated, until 60 DEG C, 70 DEG C, 80 DEG C, 85 DEG C of each holdings
1min, gradient-heated purpose prevent bumping, then to 90 DEG C of holding 30min, separate dialysis with the cellulose membrane of 500Da after cooling,
Obtaining small nanometer enzyme solutions, (mass concentration 340mg/ml, mass concentration are body of two kinds of amino acid precursor quality divided by solvent
Product)；
(3) in 1 × PBS solution of PH=7.4, the small nano enzyme of step (2) synthesis is added, H is then added2O2, mixing
After dilution, the mass concentration of small nano enzyme is 31mg/ml, H in solution2O2Concentration be 88mM, reaction a period of time received greatly
Rice enzyme solutions, survey its particle size in Particle Size Analyzer in different times；
Or (4) in 1 × PBS solution of PH=7.4, is added the small nano enzyme of step (2) synthesis, it is molten after mixed diluting
The mass concentration of small nano enzyme is 31mg/ml in liquid, then irradiates under xenon source, it is molten that big nano enzyme is obtained after a period of time
Liquid, different time survey its particle size in Particle Size Analyzer.
Small nano enzyme, big nano enzyme are detected, described in detail below:
1. big nano enzyme and small nanometer enzymatic property
1.1 uv-spectrophotometric
The small nanometer enzyme solutions for mixed solution and step (2) preparation that 1 step of embodiment (1) obtains are respectively adopted ultraviolet
Spectrophotometer is detected, as shown in Figure 1, there is new characteristic absorption peak at 367nm in nanometer enzyme solutions, and mixed solution
There is no absorption peak in 300nm or so, illustrates there is new substance to generate, in addition, above two solution is placed under 365nm ultraviolet lamp
The color of irradiation, mixed solution is light green color, and the color of nanometer enzyme solutions becomes navy blue.
1.2 particle diameter distribution
In the reaction process of 1 step of embodiment (3) and (4), the observation of atomic force scanning probe microscopy is respectively adopted not
The hydroxyl radical free radical (Fig. 3) that small nano enzyme is generated by hydrogen peroxide (Fig. 2) and illumination in the same time induces the process to become larger.With when
Between increase, small nanoparticle aggregation becomes big nanoparticle, in addition detects different moments small nanometer with nano-particle size analysis instrument
The process that enzyme solutions partial size becomes larger, as shown in figure 4, small nanoparticle aggregation becomes big nanoparticle with the increase in reaction time
Son finally reaches stabilization, it can be seen from the figure that small nano enzyme size is about in 1-2nm, big nano enzyme size is in 200-
300nm or so.
2, nano enzyme has the function of removing free radical
Free sulfhydryl group content in 2.1 small nano enzymes
The nano enzyme of lysine and glutathione synthesis has inoxidizability, can remove free radical, this is attributed to carbon source
The cysteine of middle composition glutathione contains an active sulfydryl, it can provide in electronics and free radical at single electron
It matches, and then the electronics of normal cell is protected to deprive it by free radical.It is surveyed using classical Ellman reagent method
Free sulfhydryl group content before and after fixed small nanometer enzymatic synthesis, detailed process is as follows:
It is dense that the small nanometer enzyme solutions for mixed solution and step (2) preparation that 1 step of embodiment (1) obtains are diluted to quality
Degree is 23mg/ml, then respectively takes 20 μ l, is dissolved separately in the not urea-containing Tris-Gly buffer of 3mL, 10 μ are then added
L Ellman reagent (DTNB, i.e. 5,5- dithiobis 2- nitrobenzoic acid are dissolved in Tris-Gly buffer, 4mg/mL), at 25 DEG C
Under the conditions of insulation reaction 60min, be centrifuged 15min under conditions of 5000g, to supernatant carry out uv-spectrophotometric detection (such as
Fig. 5), then measurement supernatant is in 412nm light absorption value, using the Tris-Gly buffer of the reagent containing Ellman as blank control, with
Ultrapure water is baseline.It is calculated, free sulfhydryl group content accounts for free mercapto before synthesis after synthesizing under comparable sodium (GSH concentration is identical)
The 92% of base content.
Removing of the 2.2 small nano enzymes to active nitrogen free radical
Total antioxidant capacity is measured using total antioxidant capacity test (ABTS method), is specially examined using total antioxidant capacity
Test agent box (the green skies Bioisystech Co., Ltd in Shanghai, number: S0121) measure respectively various concentration (0,3.5,14,35,
70 μ g/ml) GSH solution and existed using the GSH of comparable sodium according to small nanometer enzyme solutions prepared by 1 the method for embodiment
Absorbance under 414nm, and then characterize the total antioxidation energy of the GSH of above-mentioned concentration gradient and the small nano enzyme of step (2) preparation
Power, as a result as shown in fig. 6, being then based on absorbance data obtains the clearance rate of various concentration sample, as shown in Figure 7.As a result
Show: the small nano enzyme of synthesis and the glutathione of comparable sodium have comparable reproducibility, remain carbon source gluathione peptide former
Expect stronger oxidation resistance, under the low concentration of 70ug/ml, nearly 100% has been reached to the clearance rate of free radical.
Removing (ROS) of the 2.3 small nano enzymes to active oxygen radical
Removing using PTIO determining free radicals to active oxygen radical, specific steps are as follows:
12 parts of PTIO mother liquor of 214uM are taken, GSH is separately added into and use the GSH of comparable sodium according to side described in embodiment 1
The small nanometer enzyme solutions of method preparation, are settled to 3ml, the mass concentration of GSH is respectively 0,9.4,24,47,94 and 232 μ in solution
After g/ml, room temperature avoid light place 2h, the absorbance under spectrophotometric measurement 557nm is respectively adopted, as a result as shown in figure 8, then
The clearance rate of various concentration sample is obtained based on absorbance data, as shown in Figure 9.
3. effect of scavenging radical
Common nano enzyme can speed up removing free radical because of the catalytic activity with biological enzyme.And it is prepared by the present invention
Small nano enzyme, in addition to can speed up removing free radical, due to itself having the function of removing free radical, separately with inoxidizability
It forms big nano enzyme equally with catalytic activity during external removing free radical.
3.1 small nanometers of enzymatic activities
3.1.1 small nano enzyme CAT (catalase-like) activity
Catalase can be catalyzed H2O2It is decomposed into water and oxygen, the catalysis of hydrogen peroxide is reacted by small nano enzyme,
Verify whether small nano enzyme has catalase-like active.
The small nanometer enzyme solutions and H of the preparation of embodiment 1 are separately added into 1 × PBS system of 1ml PH=7.42O2, mix
After conjunction, H in solution2O2Concentration be 422mM, the mass concentration of small nano enzyme is respectively 3.4mg/ml, 2.6mg/ml, 1.7mg/
Ml, 0.85mg/ml and 0mg/ml.After half an hour, observation generates the amount of gas, with the life of the raising bubble of small nanometer enzyme concentration
It is increasing the GSH and Lys that comparable sodium compared with small nano enzyme (SN), is added (see Figure 10) at amount, then is generated without bubble
(see Figure 11) illustrates in certain H2O2Under concentration, small nano enzyme has CAT catalytic activity, can be by H2O2Catalytic decomposition be oxygen and
Water, and it is higher with small nanometer enzyme concentration to increase catalytic efficiency.
Add the H of 16mM in 1 × PBS system of 3ml PH=7.42O2, it is separately added into the small nano enzyme of the preparation of embodiment 1
Solution, GSH and Lys are configured to the small nanometer enzyme solutions, 2.2mg/ml that mass concentration is 0.6,1.1,1.7,2.2mg/ml
The Lys solution of GSH solution and 2.2mg/ml.It surveys absorbance of the above-mentioned solution at 240nm and changes over time situation, as a result such as
Shown in Figure 12, small nano enzyme is calculated to H according to ultraviolet absorptivity data2O2Clearance rate, as shown in figure 13.The result shows that: with small
The increase of nanometer enzyme concentration, decomposing H2O2Amount it is more, the small nano enzyme of 2.2mg/ml can remove 83% H in 5min2O2。
Small nano enzyme has catalytic action, can accelerate the decomposition of hydrogen peroxide, in addition, small nano enzyme has antioxidation, it can
With hydroperoxidation under the catalytic action of itself, the removal of hydrogen peroxide is common caused by both effects.And GSH
Because there is relatively low decomposing H without hydrogen peroxide enzymatic2O2Ability, Lys is then substantially without decomposing H2O2。
3.1.2 small nano enzyme POD (peroxidase) activity
Peroxidase being capable of catalyzing hydrogen peroxide and TMB generation chromogenic reaction.By small nano enzyme be added hydrogen peroxide with
In the reaction process of TMB, verify whether small nano enzyme has catalase-like active.
In the PBS system of the PH=7.4 of totally 500 μ l, TMB solution and H are sequentially added2O2, then it is separately added into small nano enzyme
Solution, after mixing, TMB concentration is 2.5mg/ml, H in solution2O2Concentration is 400mM, and the concentration of small nano enzyme is respectively
0mg/ml, 0.34mg/ml, 0.68mg/ml survey the absorption value that blue substrate is formed at 652nm respectively.Such as Figure 14: You Tuke
Know, small enzyme has peroxidase activity, can catalyzing hydrogen peroxide and TMB chromogenic reaction occurs, so the suction at 652nm
Receipts value can become larger；But contain free sulfhydryl group in small enzyme, can with hydrogen peroxide occur redox reaction, prevent hydrogen peroxide from after
It is continuous that chromogenic reaction occurs with TMB, so the characteristic absorption at 652nm has certain decline again.
3.2 big nanometers of enzymatic activities
Small nano enzyme forms big nano enzyme under free yl induction effect.But due to containing active mercapto in small nano enzyme
Base has reproducibility, can be with hydroperoxidation.Detecting big nano enzyme catalase-like and peroxidase activity
When, free sulfhydryl group can elder generation and hydroperoxidation.Therefore, in order to eliminate the influence of sulfydryl, to big nanometer enzymatic activity into
Before row detection, need to detect whether free sulfhydryl group in solution exhausts.Below by addition excess hydrogen peroxide and long-time light
According to two methods of hydroxyl radical free radical are formed to consume free sulfhydryl group, also induce small nano enzyme aggregation big while removing free sulfhydryl group
Nano enzyme.
Big nano enzyme preparation method, specifically uses following two:
The first: in 1 × PBS system of the PH=7.4 of total 3ml, sequentially adding H2O2With small nanometer enzyme solutions, mixing
After uniformly, H2O2Concentration is 16mM, and small nanometer enzyme concentration is 1.7mg/ml, and the small nano enzyme in solution is by hydrogen peroxide induced synthesis
Big nano enzyme, rear solution bubble-free generates for 24 hours, detects without free sulfhydryl group, obtains big nanometer enzyme solutions (OCN).
Second: in 1 × PBS of the PH=7.4 of total 3ml, small nanometer enzyme solutions are added, after mixing small nano enzyme
Concentration be 1.7mg/ml, irradiated under strong light, the small nano enzyme in the hydroxyl radical free radical inducing solution of generation forms big nanometer
Enzyme, free sulfhydryl group exhausts after 2h, obtains big nanometer enzyme solutions (UCN).
Wherein, the specific detection method of free sulfhydryl group content are as follows:
Not urea-containing Tris-Gly buffer will be separately added into big nano enzyme that both the above method obtains
Total 3ml (big nanometer enzyme concentration is 151 μ g/ml) in (0.086M Tris, 0.09M Gly and 0.04M EDTA, pH 8.0), and
After 10 μ L Ellman reagents are added (DTNB, i.e. 5,5- dithiobis 2- nitrobenzoic acid are dissolved in Tris-Gly buffer, 4mg/
ML), insulation reaction 60min under the conditions of 25 DEG C is centrifuged 15min under conditions of 5000g, measures supernatant at 412nm
Light absorption value is the control for not consuming sulfydryl to prepare the above two small nano enzyme (SN) for receiving nano enzyme greatly, using ultrapure water as baseline,
As shown in figure 15.It can be seen that do not consume the small nano enzyme of sulfydryl has higher absorption value at 412nm, illustrate free sulfhydryl group compared with
It is more；And the big nano enzyme (OCN) of hydrogen peroxide induced synthesis and illumination generate the big nano enzyme that hydroxyl radical free radical induced aggregation is formed
(UCN) there is no characteristic absorption peak in 412nm, illustrate to be free of free sulfydryl in big nanometer enzyme solutions.
3.2.1 big nano enzyme CAT (catalase-like) activity
Catalase can be catalyzed H2O2It is decomposed into water and oxygen, by big nano enzyme to H2O2Catalysis reaction, verifying
Whether big nano enzyme has catalase-like active.
In the big nanometer enzyme solutions (OCN) obtained to the first above-mentioned big nano enzyme preparation method, the H of 16mM is added2O2,
There are a large amount of bubbles to generate (see OCN in Figure 16) in half an hour, surveys in the big nanometer enzyme solutions 30min of 1.7mg/ml at 240nm
Absorbance, according to absorbance calculate residue H2O2The percentage of content, as shown in OCN in Figure 17.
In the big nanometer enzyme solutions obtained to above-mentioned second big nano enzyme preparation method, the H of 16mM is added2O2, half is small
When the interior suction for thering are a large amount of bubbles to generate (see UCN in Figure 16), surveying in the big nanometer enzyme solutions 30min of 1.7mg/ml at 240nm
Luminosity calculates residue H according to absorbance2O2The percentage of content, as shown in UCN in Figure 17.
3.2.2 big nano enzyme POD (peroxidase) activity
Peroxidase can be catalyzed H2O2Chromogenic reaction occurs with TMB.H is added in big nano enzyme2O2With reacting for TMB
In the process, it sees whether that H can be catalyzed2O2Chromogenic reaction occurs with TMB, being formed at 652nm has the product of characteristic absorption, tests
Demonstrate,prove whether big nano enzyme has catalase-like active.
In the PBS system of the PH=7.4 of totally 500 μ l, TMB solution and H are sequentially added2O2, after mixing, then distinguish
Nano enzyme solution O CN and UCN two kinds big is added, TMB concentration is 2.5mg/ml, H in solution2O2Concentration is 400mM, big nano enzyme
Solution O CN is 1.36mg/ml, and big nanometer enzyme solutions UCN is 1.36mg/ml, surveys the absorption that blue substrate is formed at its 652nm
Value is control with the GSH of comparable sodium and Lys, as a result as shown in figure 18.
4 fluorescence properties
4.1 fluorescent characteristic
The fluorescence of the small nanometer enzyme solutions of measurement 1 step of embodiment (1) obtains respectively mixed solution and step (2) preparation
Spectrogram.Figure 19 is fluorescence excitation spectrum spectrogram of the small nanometer enzyme solutions under 365nm wavelength, the results showed that, at 421nm
There is fluorescence emission peak.Figure 20 is fluorescence excitation spectrum spectrogram of the small nanometer enzyme solutions under 473nm wavelength, the results showed that,
There is fluorescence emission peak at 515nm.Figure 21 is fluorescence excitation spectrum spectrogram of the small nanometer enzyme solutions under 532nm wavelength, as a result table
It is bright, there is very weak fluorescence emission peak at 575nm.
4.2 H2O2Nano enzyme aggregation is induced in living cells
Can free radical induce nano enzyme to assemble in living cells, be that the nano enzyme is applied to organism and efficiently removes freedom
The key of base.The small nano enzyme of 1.7mg/ml and Hela cell (human cervical carcinoma cell) are not incubated for 12 hours by we jointly, and not
Add the blank control of small nano enzyme compared to (see No nanozyme in Figure 22), (405nm swashs under utilizing total internal reflection fluorescence microscope
Light, 490 long pass filters) observe that cell successfully takes in nano enzyme.In the H that physiological concentration is added2O2After (100 μM),
The process that small nano enzyme is significantly built up in the cell is observed in 10min (see 0-9min in Figure 22).We use MitoTracker
Red CMXRos red fluorescence dyestuff (Ex=579nm, Em=599nm) has carried out common location in distribution intracellular to nano enzyme
Experiment, the dyestuff only need simple incubation cell, can passively be transported through cell membrane and directly be gathered on active mitochondria.When
Cell is long to required abundance, absorbs culture solution, the MitoTracker Red CMXRos dyeing working fluid of 37 DEG C of preheatings is added
(50nM).20min is incubated under cell regular culture conditions.After dyeing, using in the PBS buffer solution replacement of PH=7.4
Dyeing liquor is stated, (532 laser, 590 long pass filters) is placed under utilizing total internal reflection fluorescence microscope and takes pictures (see b in Figure 22
MitoTracker Red), it is superimposed (see in Figure 22 with the nano enzyme fluorescence distribution photo (see figure Nanozyme) of cellular uptake
Overlap), common location coefficients R r=0.966 illustrates that nano enzyme is gathered in mitochondria position intracellular mostly.And mitochondria is
The intracellular main cell device for generating active oxygen promotees to confirm the nano enzyme induction of receptor 1 activity oxygen in the cell and assemble
Into the removing of free radical.
Claims (4)
1. a kind of therapeutic nano enzyme assembled by free yl induction, which is characterized in that it is existed by L-lysine and glutathione
Dehydrating condensation is prepared under conditions of certain.
2. the therapeutic nano enzyme according to claim 1 assembled by free yl induction, which is characterized in that small nano enzyme ruler
It is very little in 1-2nm, small nano enzyme can be assembled under the induction of free radical forms big nano enzyme, and big nano enzyme size is in 200-300nm.
3. a kind of preparation method of the therapeutic nano enzyme described in claim 1 assembled by free yl induction, which is characterized in that
The following steps are included:
(1) L-lysine (Lys) and glutathione (GSH) are dissolved in suitable ultrapure water, are uniformly mixed, wherein L-lysine
(Lys) and the molar ratio of glutathione (GSH) is (1:1)~(3:1)；
(2) step (1) mixed solution is placed in microwave synthesizer, takes out material after 60-90 DEG C of gradient-heated, cooling, point
From obtaining small nano enzyme.
4. the preparation method of Lys/GSH nano enzyme according to claim 2, which is characterized in that gradient-heated in step (2)
Specifically: to 60 DEG C, 70 DEG C, 80 DEG C, 85 DEG C each to keep 1min, and gradient-heated purpose prevents bumping, then keeps to 90 DEG C
30min。
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| CN116874556A (en) * | 2023-06-01 | 2023-10-13 | 四川农业大学 | Preparation method and application of selenium glutathione nano-enzyme with antioxidant activity |
| WO2024012071A1 (en) * | 2022-07-11 | 2024-01-18 | 深圳大学 | Preparation method for nano-enzyme inducing generation of reactive oxygen species under light regulation |
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| CN116874556A (en) * | 2023-06-01 | 2023-10-13 | 四川农业大学 | Preparation method and application of selenium glutathione nano-enzyme with antioxidant activity |
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