CN109833329A - A kind of hyaluronic acid-porous nano selenium compound and the preparation method and application thereof - Google Patents
A kind of hyaluronic acid-porous nano selenium compound and the preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to technical field of nano material, a kind of hyaluronic acid-porous nano selenium compound and the preparation method and application thereof is disclosed.It is activated after specially porous nano selenium aqueous solution is mixed with EDC and NHS, then by the porous nano selenium aqueous solution and HA hybrid reaction after activation, obtains hyaluronic acid-porous nano selenium compound aqueous solution.The nanometer selenium of gained extra specific surface area porous structure of the invention can elimination activity oxygen inhibition inflammation oxidative damage, compared to traditional nanometer selenium, with to the higher affinity of active oxygen and reactivity, enhance the electron affinity and conduction efficiency of active oxygen and nanometer selenium.And by the modification of HA, porous selenium is improved to the targeting of inflammatory macrophage, can increase the ingestion of medicines amount of cell, to guarantee that Intracellular drug maintains higher level.It enhances porous selenium to diminish inflammation intracellular and activity in vivo oxygen activity, the functional modification for selenium class antioxidant provides thinking.
Description
Technical field
The invention belongs to technical field of nano material, in particular to a kind of hyaluronic acid-porous nano selenium compound and its
Preparation method and application.
Background technique
Inflammation is present in many diseases as typical pathological characters, such as bacterium infection, and cancer and artery congee are hard
Change etc..Especially in septicemia, systemic inflammatorome can cause a variety of organ failures so as to cause the danger of acute death.Research is found
The macrophage largely activated is infiltrated in inflammatory microenvironment, to generate excessive active oxygen, causes extracellular toxicity.It is living
Property oxygen mainly includes superoxide anion (.O2), hydroxyl radical free radical (.OH) and hydrogen peroxide (H2O2), excessive active oxygen is often
The nucleic acid of normal cell, liposome and protein can be destroyed, serious damage is caused.Therefore efficient antioxidant is developed to inhibit
Inflammation damnification become septicemia therapeutic process in and clinical operation in there is an urgent need to.Selenium is natural antioxidant, and selenium is in body
It is interior to pass through the active oxidative damage to free radical resisting of raising reductase containing selenium.And recently as the hair of nanotechnology
Exhibition, the unique texture and physicochemical property of nano material promote nanometer selenium in the research and application of many diseases.Studies have found that
Selenium nano particles have the activity for eliminating hydroxyl radical free radical, but due to the low reaction activity of nanometer selenium, active oxygen eliminates effect
Rate only has 22%.Therefore, in order to further enhance the ability of nanometer selenium Scavenging active oxygen, it is high to need to develop a kind of New Target tropism
The nanometer selenium of reactivity.
In addition, being another major reason that nanometer selenium active oxygen eliminates low efficiency to the low targeting of macrophage.It is transparent
Matter acid is the molecule for specifically binding cell surface molecule CD44, and CD44 is a kind of highly expressed albumen point in macrophage
Son, therefore by being the available strategy for enhancing its targeting in nanometer selenium surface modification hyaluronan molecule.
Summary of the invention
In order to overcome the shortcomings and deficiencies of the prior art described above, the primary purpose of the present invention is that providing a kind of hyalomitome
Acid-porous nano selenium compound preparation method.
Another object of the present invention is to provide hyaluronic acid-porous nano selenium compound of above method preparation.
Still a further object of the present invention is that providing above-mentioned hyaluronic acid-porous nano selenium compound treats septicemia medicine in preparation
Application in object.
The purpose of the present invention is realized by following proposal:
A kind of preparation method of hyaluronic acid-porous nano selenium compound, comprising the following steps:
By porous nano selenium aqueous solution and 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and N-
It is activated after HOSu NHS (NHS) mixing, then by the porous nano selenium aqueous solution and hyaluronic acid after activation
(HA) hybrid reaction obtains hyaluronic acid-porous nano selenium compound (MSe-HA) aqueous solution.
The concentration of porous nano selenium is 0.1~10mg/mL in the porous nano selenium aqueous solution；Preferably 2mg/mL.
The molar ratio of the porous nano selenium, EDC and NHS is 1:0.1:0.1~1:20:20；Preferably 1:1:1~1:2:
2。
The time of the activation is 0.5~10h；Preferably 3~5h；More preferably 4h.
The mass ratio of the porous nano selenium and hyaluronic acid is 1:1~1:10；Preferably 1:3~1:5.
The molecular weight of the hyaluronic acid is 10KD~50KD.
The condition of the hybrid reaction is that 6~18h of reaction is stirred at room temperature；Mixed reaction time is preferably 8~14h；
More preferably 12h.
Preferably, further include purification process after hybrid reaction is complete, specially solution of the hybrid reaction after complete is collected by centrifugation
It is resuspended after precipitating and washing to get MSe-HA aqueous solution is arrived；The centrifugation is in 8000~10000rpm centrifugation, 10~15min；
The number of the washing is 3 times.
The preparation step of the porous nano selenium aqueous solution are as follows:
Meso-porous titanium dioxide Si powder is dissolved in after organic solvent and is mixed with 3- aminopropyl triethoxysilane (APTES), is returned
Stream reaction obtains amidized mesoporous silicon oxide solution, and sodium selenite aqueous solution is then added dropwise and is stirred to react, then reduction is added dropwise
Agent is persistently stirred to react, to obtain reaction solution after the reaction was completed, then be added hf etching fall mesoporous silicon oxide obtain it is more
Hole nanometer selenium.
The organic solvent is at least one of dehydrated alcohol and toluene.
The mass volume ratio of the mesoporous silicon oxide, organic solvent and APTES is 0.1g:5mL:10 μ L~20g:
100mL:100 μ L；Preferably 10g:10mL:50 μ L.
The time of the back flow reaction is 3~10h；Preferably 6h.
The concentration of sodium selenite is 0.1~1.0mol/L in the sodium selenite aqueous solution.
The volume ratio of the amidized mesoporous silicon oxide solution and sodium selenite aqueous solution is 1:2~1:10；It is preferred that
For 1:4.
The reducing agent is at least one of citric acid, aspartic acid, glutamic acid, vitamin A acid and L-AA.
The molar ratio of the sodium selenite and reducing agent is 1:1~1:5.
The time that the first time is stirred to react is 15~60min, and the time being stirred to react for the second time is 1~5h；
Preferably, the time that the first time is stirred to react is 30min, and the time being stirred to react for the second time is 2h.
The concentration of the hydrofluoric acid is 20~70wt%, preferably 40.0wt%；The reaction solution that dosage meets every 1mL is corresponding
The hydrofluoric acid of 5~200 μ L is added.
Mesoporous silicon oxide cetyl trimethylammonium bromide (CTAB) template hydrolyzing tetraethoxy orthosilane (TEOS)
Method be prepared, specific steps are as follows:
Cetyl trimethylammonium bromide is dissolved in after water, organic solvent A is added, then adjusting solution is to be mixed to alkalinity
Ethyl orthosilicate is added after dissolution, then increases temperature and is stirred to react to obtain reaction solution, then in acid solution and has reaction solution
Reflux obtains mesoporous silicon oxide in the mixed solution of solvent B.
The organic solvent A is at least one of ethyl alcohol and toluene.
The mass volume ratio of the cetyl trimethylammonium bromide, water and organic solvent A be 50mg:20mL:5mL~
200mg:100mL:50mL；Preferably 100mg:68mL:30mL.
The molar ratio of the ethyl orthosilicate and cetyl trimethylammonium bromide is 1:1~1:10.
PH value of solution is adjusted to 9~10 using ammonium hydroxide or NaOH aqueous solution to alkalinity by the adjusting solution.
The raising temperature continues to be stirred to react to be increased to 45~70 DEG C and then being stirred to react 12~36h；Preferably rise
Up to 60 DEG C are stirred to react for 24 hours.
It preferably, further include purification process after being stirred to react, the solution eccentric cleaning three after being specially stirred to react
It is secondary；More preferably cleaned using ethyl alcohol.
The acid solution is HCl, and organic solvent B is dehydrated alcohol；The mixing of the acid solution and organic solvent is molten
The pH value of liquid is 1~3.
The time of the reflux is 8~12h.
The present invention does not indicate that temperature is to carry out at room temperature, and the room temperature is 25~35 DEG C.
A kind of hyaluronic acid prepared by the above method-porous nano selenium compound.
Application of the hyaluronic acid-porous nano selenium compound in preparation treatment sepsis disease drug.
The present invention compared with the existing technology, have the following advantages and the utility model has the advantages that
(1) the present invention provides the nanometer seleniums of extra specific surface area porous structure can elimination activity oxygen inhibition inflammation oxidation damage
Wound, compared to traditional nanometer selenium, porous selenium has to the higher affinity of active oxygen and reactivity, enhances active oxygen and receives
The electron affinity and conduction efficiency of rice selenium, therefore there is porous nano selenium of the invention the active oxygen of high-efficiency broad spectrum to eliminate work
Property.
(2) the present invention provides targeting porous nano selenium elimination activity oxygen therapy septicemia application, by transparent
The modification of matter acid effectively increases porous selenium to the targeting of inflammatory macrophage, can increase the ingestion of medicines amount of cell, from
And guarantees Intracellular drug and maintain higher level.Further enhance porous selenium diminish inflammation it is intracellular and activity in vivo oxygen
Activity, the functional modification for selenium class antioxidant provide thinking.
(3) hyaluronic acid decorated porous nano selenium preparation process and product system provided by the invention are simple, and product can
It directly saves and uses, and preparation method is easy.
(4) hyaluronic acid decorated-porous nano selenium provided by the invention is with good stability in aqueous solution and divides
Property is dissipated, use can be saved in room temperature environment.In addition, hyaluronic acid and selenium are all necessary material and element in human body, have good
Good biocompatibility has lower irritation in vivo.With after reactive oxygen species it is degradable be non-toxic substance, have
Good clinical application potentiality.
Detailed description of the invention
The transmission electron microscope and specific surface detection figure that Fig. 1 is 1 gained MSe NPs of embodiment, wherein A is transmission electron microscope picture,
39000 times；B is transmission electron microscope picture, 65000 times；C is nitrogen adsorption De contamination curve graph；D is graph of pore diameter distribution.
Fig. 2 is the transmission electron microscope and composite diagram of 1 gained MSe-HA NPs of embodiment, and wherein A is transmission electron microscope picture, and B is red
External spectrum figure, C are ultraviolet spectrogram.
Fig. 3 is 1 gained Se NPs of embodiment, MSe NPs and MSe-HA NPs Scavenging active oxygen effect picture, and wherein A is hair
Fluorescence intensity figure when a length of 650nm of ejected wave；The fluorescence intensity figure that B is launch wavelength when being 510nm；C is that launch wavelength is
Fluorescence intensity figure when 425nm；D is that superoxide anion eliminates efficiency chart；E is hydroxyl radical free radical elimination factor figure；F is hydrogen peroxide
Elimination factor figure.
Fig. 4 is targeting figure of 1 gained MSe NPs and the MSe-HA NPs of embodiment in Macrophage Cell and mouse,
Middle A is that the cell of macrophage absorbs figure；B is fluorescence quantization figure during the cell of macrophage absorbs；C is in porous nano selenium body
Targeting figure；D is targeting fluorescence quantization figure in porous nano selenium body.
Fig. 5 is 1 gained Se NPs of embodiment, MSe NPs, MSe-HA NPs eliminate active oxygen lab diagram in macrophage,
Wherein A is laser copolymerization figure；B is fluidic cell figure.
Fig. 6 is 1 gained Se NPs of embodiment, MSe NPs, MSe-HA NPs eliminate active oxygen therapy septicemia in Mice Body
Lab diagram, wherein A is mouse ear damage figure；B is active oxygen probe DCFH-DA fluorogram；C is active oxygen probe DCFH-DA
Fluorescence quantization figure；D is the H&E slice map of Mouse Liver and lung.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited
In this.
Agents useful for same can routinely be bought unless otherwise specified from market in embodiment.
RAW264.7 cell (the American Type Culture Collection committee, Chinese Academy of Sciences cell bank)；C57BL/6 mouse (Guangdong Province
Animal experimental center)
The synthesis of 1 nanometer selenium of embodiment, porous nano selenium and hyaluronic acid-porous nano selenium compound
(1) synthetic method of porous nano selenium (MSe NPs) and nanometer selenium: by 100mg cetyl trimethylammonium bromide
(CTAB) it is dissolved in 68mL distilled water and stirring at normal temperature, 30mL ethyl alcohol is added, it is to be mixed then with ammonium hydroxide pH adjusting 9~10
80 μ L TEOS are added after dissolution, then heats to 60 DEG C, is persistently stirred to react for 24 hours, are centrifuged after reaction with dehydrated alcohol clear
It washes three times, and reflux 6h obtains mesoporous silicon oxide in HCl and the mixed solution (pH 1) of ethyl alcohol.Take 10g meso-porous titanium dioxide
Si powder is dissolved in 10mL dehydrated alcohol, is added 50 μ L APTES reflux 6h and is obtained amidized mesoporous silicon oxide.It utilizes
The positively charged property that can adsorb negatively charged sodium selenite of amination silica takes the mesoporous silicon oxide solution of 5mL, is added dropwise
The sodium selenite solution (5mg/mL) of 5mL stirs 30min, and 10mL reducing agent L-AA solution (10mg/mL) then is added dropwise
Persistently stir 2h.Three times to centrifuge washing after the reaction was completed, be added 100 μ L hf etching fall mesoporous silicon oxide obtain it is more
Hole nanometer selenium.
(2) synthetic method of MSe-HA NPs: 2mg/mL is configured to by porous nano selenium that step (1) obtains is soluble in water
Solution the HA (M=of 50mg is added after using EDC (30mg) and NHS (40mg) to activate 4h at room temperature acquired solution 10mL
105) 12h is stirred at room temperature, 8000rmp is collected by centrifugation precipitating and is resuspended after washing 3 times to get water-soluble to MSe-HA NPs
Liquid.
(3) synthetic method of nanometer selenium (Se NPs): the sodium selenite solution (5mg/mL) of 5mL is taken to stir 30min, then
10mL reducing agent L-AA solution (10mg/mL) is added dropwise and persistently stirs 2h.It is obtained three times to centrifuge washing after the reaction was completed
Nanometer selenium.
The transmission electron microscope and specific surface detection figure that Fig. 1 is 1 gained MSe NPs of embodiment, wherein A is transmission electron microscope picture,
39000 times；B is transmission electron microscope picture, 65000 times；C is nitrogen adsorption De contamination curve graph；D is graph of pore diameter distribution.From Figure 1A and
As can be seen that MSe NPs is about the finely dispersed nanoparticle of 150nm in Figure 1B, and porous structure is evenly distributed on MSe
The surface NPs is further detected display (Fig. 1 C and Fig. 1 D) by nitrogen adsorption detachment assays, and MSe NPs is apparent porous knot
Structure, aperture are concentrated mainly on 17.2nm, specific surface area 1160.196g/m2.Fig. 2 is 1 gained MSe-HA NPs of embodiment
Transmission electron microscope and composite diagram, wherein figure A is transmission electron microscope picture, figure B is infrared spectrogram, and figure C is ultraviolet spectrogram.From transmission electricity
Mirror figure can be seen that it is hyaluronic acid decorated after porous nano selenium nanometer particle size then increase to about 155nm, it is ultraviolet and infrared
Then show that hyaluronic acid is successfully modified on porous nano selenium.
Embodiment 2: Conventional nano selenium (Se NPs), porous selenium (MSe), hyaluronic acid-porous nano selenium (MSe-HA) disappear
Except active oxygen is tested in solution
MSe, MSe-HA and SeNPs are that embodiment 1 is prepared in the present embodiment.
(1) superoxide anion eliminates experiment: taking 2.28mg xanthine to be dissolved in the NaOH solution that 1mL concentration is 10mM and is made into
Xanthine concentration is that 15mM homogeneous solution is stand-by.Xanthine is taken to dilute 40 μ L of (0.6mM) liquid, 4.8 μ L of xanthine oxidase is together
It is added in 805.2 μ L PBS buffering, 37 DEG C of reaction 40min.Be then respectively adding 100 μ L concentration be 20 μ g/mL Se NPs,
MSe and MSe-HA solution is incubated for 40min jointly, while blank control group is arranged, and three repeating groups of every group of setting.Then distinguish
The dihydro second pyridine probe that 50 μ L are added is incubated for 40min, the fluorescence intensity of upper machine testing each component, excitation wave 470nm, transmitted wave
For 650nm, Fig. 3 is as a result seen.
(2) hydroxyl radical free radical eliminates experiment: taking 18mg FeSO4.7H2O is dissolved in 10mL deionized water (pH=3~4), together
When take 10 μ L hydrogen peroxide original solutions (30%) to be diluted to 5mL.Then 4mL hydrogen peroxide dilution liquid is taken to be added to 4mL FeSO4
10min is reacted in solution, to after the reaction was completed, solution is divided into four groups, being separately added into 100 μ L concentration is 200 μ g/mL Se
NPs, MSe and MSe-HA solution are incubated for 1h jointly, while blank control group is arranged, and three repeating groups of every group of setting.Then divide
Not Jia Ru 1mL salicylic acid solution (1.8mM) be incubated for 15min, then upper machine testing fluorescence intensity, excitation wave 385nm, transmitted wave
For 510nm, Fig. 3 is as a result seen.
(3) hydrogen peroxide eliminates experiment: taking terephthalic acid (TPA) to be dissolved in 10mL N,N-dimethylformamide (DMF) and is made
The solution for later use of 0.5mM.10 μ L hydrogen peroxide original solutions (30%) are taken to be diluted to 5mL.Then take 4mL hydrogen peroxide dilution liquid equal
It is divided into four groups, is separately added into 100 μ L concentration as 200 μ g/mL Se NPs, MSe and MSe-HA solution and is incubated for 6h jointly, set simultaneously
Set blank control group, and three repeating groups of every group of setting.Then 1mL terephthalic acid solution is added and is incubated for 30min, then upper machine
As a result fluorescence intensity, excitation wave 320nm, transmitted wave 425nm are shown in Fig. 3.
Fig. 3 is 1 gained Se NPs of embodiment, MSe NPs and MSe-HA NPs Scavenging active oxygen effect picture.Xanthine and Huang
Purine oxidase reaction can generate superoxide anion (.O2), it is raw that superoxide anion can aoxidize dihydro second pyridine fluorescence probe (HE)
At the oxidation second pyridine for having special absorption at 650nm, therefore the size of fluorescence intensity has directly reacted superoxide anion in solution
Concentration.As shown in Figure 3A, after by fluorescence detection and background correction, detection discovery blank control group, Se NPs group, MSe
NPs group and MSe-HA NPs group average fluorescent strength are respectively 292.1,127.6,69.3 and 64.9, as shown in Figure 3D, Se NPs
The elimination efficiency of group, MSe NPs group and MSe-HA NPs group superoxide anion is respectively 50.17%, 78.73% and 78.81%.
Therefore, it was demonstrated that effectively improve the efficiency that nanometer selenium eliminates ultra-oxygen anion free radical by increasing specific surface area.
Ferrous ion can generate active oxygen hydroxyl radical free radical (.OH) with reacting for hydrogen peroxide, and hydroxyl radical free radical can be with oxygen
Change salicylic acid (SA) and be created at 510nm 2, the 3- dihydroxy-benzoic acid for having special absorption, therefore the size of fluorescence intensity is direct
The concentration of hydroxyl radical free radical in solution is reacted.As shown in Figure 3B, after by fluorescence detection and background correction, detection discovery is empty
White control group, Se NPs group, MSe NPs group and MSe-HA NPs group mean fluorecence absorbance are respectively 0.58368,
0.29104,0.12391 and 0.1002.As shown in FIGURE 3 E, Se NPs group, MSe NPs group and MSe-HA NPs group hydroxyl radical free radical
Elimination efficiency be respectively 55.92%, 78.91% and 82.83%.Therefore, it was demonstrated that effectively promoted by increasing specific surface area
Nanometer selenium eliminates the efficiency of hydroxyl radical free radical.
Terephthalic acid (TPA) can result from the 2- hydroxyl terephthaldehyde for having special absorption at 425nm with reacting for hydrogen peroxide
Acid, therefore the size of fluorescence intensity has directly reacted the concentration of hydrogen peroxide in solution.As shown in Figure 3 C, simultaneously by fluorescence detection
After background correction, detection discovery blank control group, Se NPs group, MSe NPs group and MSe-HA NPs group mean fluorecence extinction
Degree is respectively 1746,1337,219.3 and 179.8.As illustrated in Figure 3 F, Se NPs group, MSe NPs group and MSe-HA NPs group
The elimination efficiency of hydroxyl radical free radical is respectively 32.12%, 87.32% and 89.81%.Therefore, it was demonstrated that by increasing specific surface
Product effectively improves the efficiency that nanometer selenium eliminates hydrogen peroxide.
Embodiment 3: porous selenium, hyaluronic acid-porous nano selenium targeting experiment
(1) RAW264.7 cell absorption experiment: in order to detect 1 gained porous nano selenium (MSe NPs) of embodiment and transparent
The targeting ability of matter acid-porous nano selenium (MSe-HA NPs), we have detected LPS activation using confocal laser microscope
Absorbability of the RAW264.7 cell to MSe NPs and MSe-HA NPs.In brief, the RAW264.7 cell of activation with 1 ×
105Density kind is incubated in the burnt capsule of copolymerization and is then separately added into 50 μ L for 24 hours containing MSe NPs (the 10 μ g/ marked with FITC
ML) and the culture medium of MSe-HA NPs (10 μ g/mL) is into cell, and cell and drug are incubated for 0.5h, 1h, 2h, 4h and 8h respectively
Afterwards, cell is washed three times with PBS, and dyes 15min with DAPI.Cell sample is washed one time again with PBS, and aobvious in confocal laser
Micro- microscopic observation, is as a result shown in Fig. 4.
(2) targeting is tested in inflammation Mice Body: porous in order to detect porous nano selenium (MSe NPs) and hyaluronic acid-
The targeting inflammation part of nanometer selenium (MSe-HA NPs) targets ability, using fluorescent vital imaging MSe NPs and MSe-
For HA NPs in the accumulation of inflammation part, the accumulation of fluorescence intensity more strong representation nanometer is more, and targeting ability is stronger.In short
10 μ L concentration are that 10ng/mL lipopolysaccharides (LPS) solution is injected into C57BL/6 auris dextra by it, and after 12h, ear occurs apparent
Redness indicates that auris dextra has apparent inflammatory reaction, then respectively will be containing with the FITC MSe NPs (10 μ g/mL) marked and MSe-HA
The PBS aqueous solution of NPs (10 μ g/mL) is by the way that in tail vein injection to Mice Body, injection volume is 200 μ g/kg (drugs/Mice Body
Weight), the distribution situation of drug fluorescence in body in 0.5h, 1h, 2h, 4h, 8h and 12h is then observed respectively, as a result sees Fig. 4.
Fig. 4 is targeting figure of 1 gained MSe NPs and the MSe-HA NPs of embodiment in Macrophage Cell and mouse,
Middle A is that the cell of macrophage absorbs figure；B is fluorescence quantization figure during the cell of macrophage absorbs；C is in porous nano selenium body
Targeting figure；D is targeting fluorescence quantization figure in porous nano selenium body.
Laser co-focusing detection shows that MSe and MSe-HA and RAW264.7 cell are incubated for fluorescence intensity in 2h jointly and present
Time dependence.And fluorescence intensity reaches saturation after 2h, does not enhance significantly.In addition, being can be found that by comparison
MSe-HA is significantly stronger than MSe in the fluorescence intensity of 2h, and fluorescence sxemiquantitative shows that the fluorescence intensity of MSe-HA is the 1.98 of MSe
Again (Fig. 4 B), therefore shown through the hyaluronic acid decorated porous nano selenium that can be enhanced by absorption experiment to the target of macrophage
Tropism.As shown in Figure 4 C, internal fluorescence experiments result, which further displays MSe-HA, can reach inflammation part in 1h, go forward side by side
The effective accumulation of row.And reaching maximum in 4h, fluorescence intensity is 4 times (Fig. 4 D) of MSe, is also demonstrated using hyaluronic acid
Modification-porous nano selenium has excellent targeting to inflammation.
Embodiment 4: Conventional nano selenium, porous selenium, hyaluronic acid-porous nano selenium eliminate active oxygen experiment in cell
Se NPs, MSe NPs and MSe-HA NPs are that embodiment 1 is prepared in the present embodiment.
In order to detect Conventional nano selenium (Se NPs), porous nano selenium (MSe NPs) and hyaluronic acid-porous nano selenium
(MSe-HA NPs) eliminates the ability of active oxygen in activated macrophage, we focus laser capture microdissection using flow cytometer together
Mirror has detected RAW264.7 cell by reactive oxygen species fluorescence intensity after various processing.In brief, activation
RAW264.7 cell is with 1 × 105Density kind is incubated for for 24 hours in the burnt capsule of copolymerization, and being then separately added into 50 μ L containing concentration is 10
Blank control group is arranged into cell in Se NPs, the MSe NPs of μ g/mL and the culture medium of MSe-HA NPs, cell and medicine
After object is incubated for 12h respectively, three times with PBS washing cell, and 30min is dyed with active oxygen probe (DCFH-DA).Cell sample with
PBS is washed one time again, and in confocal laser microscopically observation (excitation and launch wavelength are respectively 495nm and 525nm).Streaming
Cell instrument detection is then that will activate RAW264.7 cell with every hole 5 × 104Density kind is in six orifice plates, and in 37 DEG C, 5%CO2Training
It supports to cultivate in case and is separately added into culture medium of the 50 μ L containing Se NPs, MSe NPs and MSe-HA NPs that concentration is 10 μ g/mL for 24 hours
Into cell, while blank control group is set, after cell is incubated for 12h respectively with drug, three times with PBS washing cell, and with work
Property oxygen probe (DCFH-DA) dye 30min.Cell sample is washed one time again with PBS, and is detected in flow cytometer, is as a result seen
Fig. 5.
Fig. 5 is 1 gained Se NPs of embodiment, MSe NPs, MSe-HA NPs eliminate active oxygen lab diagram in macrophage.
Laser co-focusing detection shows as shown in Figure 5A, after the RAW 264.7 induced with LPS is incubated for 12h, blank control group (LPS)
Middle active oxygen fluorescence intensity is strongest, and reactive oxygen species fluorescence intensity in each experimental group of nanometer effect is added and then shows
Different degrees of decline out, and it can be found that the efficiency of MSe and MSe-HA Scavenging active oxygen is significantly stronger than Conventional nano selenium
(Se), it thus also demonstrates the nanometer selenium with porous structure and eliminates ability with stronger active oxygen.And MSe-HA because
With good targeting ability, the efficiency ratio MSe of Scavenging active oxygen is stronger, and reactive oxygen species are disappeared completely substantially after processing
It removes.Flow cytomery also shows identical trend, and as shown in Figure 5 B, as a result detection shows active in blank control group
Oxygen fluorescence intensity is that treated that reactive oxygen species fluorescence intensity is 1238 by 2315, Se NPs, and MSe and MSe-HA is processed
Fluorescence intensity afterwards then only has 900 and 804, therefore the MSe-HA of the super large specific surface with targeting has good macrophage thin
Intracellular reactive oxygen species generation eliminates ability.
Embodiment 5: Conventional nano selenium, porous selenium, hyaluronic acid-porous nano selenium eliminate activity in vivo oxygen therapy septicemia
Experiment
Se NPs, MSe NPs and MSe-HA NPs are the preparation gained of embodiment 1 in the present embodiment.
6-8 week old is taken, counterpoise is that the C57BL/6 mouse of 20.0g or so is equally divided into four groups, and every group of 5 progress modelings will
10 μ L are injected in mouse right ear dissolved with the normal saline solution (100ng/mL) of lipopolysaccharides, are occurred after 12h to mouse right ear
Redness is handled.Distinguish the PBS water of Se NPs, MSe NPs and MSe-HA NPs that tail vein injection concentration is 2mg/mL
Solution, injection volume is 200 μ g/Kg (drug/mouse weight), while blank control group is arranged.Hereafter right every observation mouse for 24 hours
Ear redness situation, and recorded with mobile phone photograph.Injected after treatment four days active oxygen probe (DCFH-DA) and with living body fluorescent at
As activity keto concentration in observation mouse right ear.Pass through observation liver lung organ damage sections observation septicemia treatment condition simultaneously.Knot
Fruit sees Fig. 6.
Fig. 6 is 1 gained Se NPs of embodiment, MSe NPs, MSe-HA NPs eliminate active oxygen therapy septicemia in Mice Body
Lab diagram.As shown in Figure 6A, the 4th day when, there is apparent degree of impairment in blank control group mouse auris dextra, this is control group inflammation
Oxidative damage caused by disease position activity keto concentration is high.And inject Se NPs, MSe NPs and MSe-HA NPs treatment group then shows
Different degrees of therapeutic effect out, wherein MSe NPs only has a little redness, and there is no degree of impairment, MSe-HA NPs is then complete
Damage is inhibited to be no different with normal rat entirely.As shown in Figure 6B, living imaging is detected and is then further demonstrated that, active oxygen in control group
Concentration is apparently higher than treatment group, active oxygen is then completely inhibited in MSe NPs and MSe-HA NPs treatment group, while cutting from liver lung
Piece is it is also seen that using the then damage (Fig. 6 D) of complete liver lung in MSe NPs and MSe-HA NPs treatment group.These result tables
Bright, also there is MSe NPs and MSe-HA NPs good active oxygen to eliminate ability in vivo restrained effectively inflammation oxidation damage
Hurt and treated septicemia.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of hyaluronic acid-porous nano selenium compound preparation method, which comprises the following steps:
It is activated after porous nano selenium aqueous solution is mixed with EDC and NHS, then by the porous nano selenium aqueous solution after activation
With hyaluronic acid hybrid reaction, hyaluronic acid-porous nano selenium compound aqueous solution is obtained.
2. hyaluronic acid according to claim 1-porous nano selenium compound preparation method, it is characterised in that:
The concentration of porous nano selenium is 0.1~10mg/mL in the porous nano selenium aqueous solution；
The molar ratio of the porous nano selenium, EDC and NHS is 1:0.1:0.1~1:20:20；
The molecular weight of the hyaluronic acid is 10KD~50KD；
The mass ratio of the porous nano selenium and hyaluronic acid is 1:1~1:10.
3. hyaluronic acid according to claim 1-porous nano selenium compound preparation method, it is characterised in that:
The time of the activation is 0.5~10h；
The condition of the hybrid reaction is that 6~18h of reaction is stirred at room temperature；
Further include purification process after the hybrid reaction is complete, specially solution of the hybrid reaction after complete is collected by centrifugation and precipitates and washes
It is resuspended after washing to get hyaluronic acid-porous nano selenium compound aqueous solution is arrived.
4. described in any item hyaluronic acids-porous nano selenium compound preparation method according to claim 1~3, feature
It is, the preparation step of the porous nano selenium aqueous solution are as follows:
Meso-porous titanium dioxide Si powder is dissolved in after organic solvent and is mixed with APTES, back flow reaction obtains amidized mesoporous dioxy
Then SiClx solution is added dropwise sodium selenite aqueous solution and is stirred to react, then reducing agent is added dropwise and is persistently stirred to react, to after the reaction was completed
Reaction solution is obtained, hf etching is then added and falls mesoporous silicon oxide and obtains porous nano selenium.
5. hyaluronic acid according to claim 4-porous nano selenium compound preparation method, it is characterised in that:
The mass volume ratio of the mesoporous silicon oxide, organic solvent and APTES is 0.1g:5mL:10 μ L~20g:100mL:
100μL；
The volume ratio of the amidized mesoporous silicon oxide solution and sodium selenite is 1:2~1:10；
The molar ratio of the sodium selenite and reducing agent is 1:1~1:5；
The time that the first time is stirred to react is 15~60min, and the time being stirred to react for the second time is 1~5h.
6. hyaluronic acid according to claim 4-porous nano selenium compound preparation method, it is characterised in that:
The concentration of sodium selenite is 0.1~1.0mol/L in the sodium selenite aqueous solution；
The reducing agent is at least one of citric acid, aspartic acid, glutamic acid, vitamin A acid and L-AA；
The concentration of the hydrofluoric acid is 20~70wt%.
7. hyaluronic acid according to claim 4-porous nano selenium compound preparation method, which is characterized in that described
The preparation step of mesoporous silicon oxide are as follows:
By cetyl trimethylammonium bromide rear addition organic solvent A soluble in water, then adjusting solution is to be mixed molten to alkalinity
Ethyl orthosilicate is added after solution, then increases temperature and is stirred to react to obtain reaction solution, then by reaction solution in acid solution and organic
Reflux obtains mesoporous silicon oxide in the mixed solution of solvent B.
8. hyaluronic acid according to claim 7-porous nano selenium compound preparation method, it is characterised in that:
The mass volume ratio of the cetyl trimethylammonium bromide, water and organic solvent A is 50mg:20mL:5mL~200mg:
100mL:50mL；
The molar ratio of the ethyl orthosilicate and cetyl trimethylammonium bromide is 1:1~1:10；
The raising temperature continues to be stirred to react to be increased to 45~70 DEG C and then being stirred to react 12~36h；
The time of the reflux is 8~12h.
9. a kind of hyaluronic acid-porous nano selenium compound of the method preparation of any one according to claim 1~8.
10. hyaluronic acid-porous nano selenium compound answering in preparation treatment sepsis disease drug according to claim 9
With.
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