CN109602702A - A kind of resveratrol nanoparticle and its preparation process with Brain targeting effect - Google Patents
A kind of resveratrol nanoparticle and its preparation process with Brain targeting effect Download PDFInfo
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Abstract
A kind of resveratrol nano particle preparations with Brain targeting function, including component: resveratrol, BCA, lactoferrin solution, OH-PEG-MAL and water for injection.Include the following steps: the preparation of one, resveratrol nanoparticle；Two, the preparation of eluent A；Three, the preparation of lactoferrin modification resveratrol nanoparticle.After resveratrol nano particle preparations provided by the invention with lactoferrin by being modified, the resveratrol nanoparticle of lactoferrin modification is made, the nanoparticle pharmaceutical dosage form with Brain targeting enhances Brain targeting and the anti-AD effect of resveratrol.
Description
Technical field
The present invention relates to a kind of resveratrol nanoparticles and its preparation process with Brain targeting effect, belong to pharmaceutical preparation
Technical field.
Background technique
Alzheimer disease (Alzheimer ' s disease, AD) is a kind of common Neuro-degenerative of the elderly
Disease, clinical manifestation be memory fade, recognize and learning ability decline, apathetic and behaviouristics obstacle.Its characteristic
It includes: neurofibrillary tangles caused by brain neuron missing, Protein tau Hyperphosphorylationof that pathology, which mainly change,
(Neurofibrillary tangles, NFT) and amyloid beta (β eta amyloid peptides, A β) deposit institute
Senile plaque outside caused nerve cell forms (Senile plaque, SP).Although a century has the study of incident mechanism of AD
It is remaining, but so far still without final conclusion, there are numerous hypothesis, including cholinergic nerve unitarian hypothesis, amyloid beta toxicity hypothesis, tau
Abnormal protein phosphorylation hypothesis and radical damage hypothesis etc..It is investigated and analysed within 2016 and is reported by Alzheimer disease association, the U.S.
In point out: in the U.S. man-year age >=65 year long-term care of senile dementia and the estimation of hospice care be 236,000,000,000 beauty
Member.As it can be seen that AD patient causes strong influence to family and society.Although countries in the world put into substantial contribution for Ah
The research of Alzheimer's disease, but clinically there is no the drug of radical cure AD.Currently, the drug of clinical main prevention and treatment AD has: cholinolytic
The first-line drugs such as energy drug donepezil, excitatory amino acid receptor antagonists Memantine and antioxidant vitamin E.In addition,
There are also for A β vaccine, Chinese medicine and preventive health care's drug etc..The first-line treatment drug and A β vaccine of AD is secondary because of curative effect or poison
The reasons such as effect do not make a breakthrough always in clinical application, so people begin to focus on Chinese medicine and preventive health care's drug
Effect and mechanism to AD is delayed.Traditional Chinese medicine causes vast because its distinctive advantage has broad prospects in terms for the treatment of AD
The interest of scholar.It has now been discovered that ginkgo leaf and its extract, Radix Angelicae Sinensis, resveratrol (Resveratrol, Res) etc. have to change
The cognitive function of kind AD rat model.The good neuro-protective effect of especially Res has attracted a large amount of researchers to go
It is further to explore.
Entitled 3,5,4- trihydroxy -1, the 2- talan of Res chemistry, is to be widely present in the plants such as grape, polygonum cuspidate, peanut
Polyphenol compound in property food or drug.The study found that Res is a kind of phytoalexin, about 70 various plants can
To generate Res to resist the following pathogen challenges such as fungi and bacterium.Zoopery confirms that Res has extensive pharmacological action, mainly
Have following several respects: anticancer, anti-cardiovascular disease, anti-cell membrane lipid peroxidatio, anti-inflammatory, neuroprotection and oestrogen-like hormone are made
With etc..Wherein, cone is thin in the effect of Res directed against amyloid-beta protein toxic, the generation of reduction free radical and improvement AD hippocampal tissue
The neuroprotection of cellular damage has become the hot spot of research.The discovery of this laboratory early-stage study, Res can improve AD mouse model
Study and cognitive ability effectively inhibit the expression of AD hippocampus of mice A β, and are in certain dosage correlation.But because Res is liposoluble
Property compound, ordinary preparation is low by the bioavilability of oral or drug administration by injection in vivo, and how to guarantee drug
It can smoothly enter into brain tissue and effective treatment concentration is kept not yet to solve.Therefore, it improves Res bioavilability and passes through blood
The concentration that brain barrier (blood brain barrier, BBB) reaches brain tissue is the necessity for being conducive to Res and playing therapeutic effect
Condition.
Currently, in drug brain delivery systematic research, Non-Invasive administration route have become domestic and international intracerebral target to
The research emphasis of medicine.Non-Invasive administration route mainly includes via intranasal application drug delivery system, monoclonal antibody guiding drug delivery system, receives
Grain of rice delivery system, carrier or receptor-mediated drug delivery system etc..The barrier that these brain targeting drug delivery systems " can challenge " BBB is made
With, and obtain certain therapeutic effect.Wherein, especially brain delivery systematic research is administered the most by receptor-mediated particle
Success.Its modified by the function inhibitio to reticuloendothelial system or to the surface of particulate delivery system with extend particle to
Medicine system makes drug have the sufficient time to penetrate BBB and enters intracerebral and play treatment and make in the retention time of blood circulation system
With.In such research, generally select nanoparticle (Nanoparticles, NPs) as drug administration carrier, and connect on NPs
It connects ligand and drug delivery is entered into brain in the form of ligand-receptor.
NPs is used to deliver drug and has caused people greatly to pay close attention in recent years through the method for BBB.NPs is art of pharmacy
The general designation of the more active small drug delivery system of a series of new ultra micro in research, partial size is in 10-1000nm.Study earliest with
NPs is that be transmitted into the drug of brain be hexapeptide compounds dalargin to carrier, with Central Analgesic Effect, but peripherally administered no treatment
Effect, pharmacodynamic experiment proves dalargin-PBCA-NPs, and through Tween-80, Bolos intravenous administration can be by dalargin after surface modification
Smooth delivery of power enters brain and plays analgesic activity.Then, the research of dipeptide compound kytorpH in and tubocurarine etc. is also obtained
Obtained similar result.In addition, the Superparamagnetic Iron Oxide nanoparticle using optimization can also be produced as pharmaceutical carrier through BBB
Raw biological effect.There are also studies have shown that monoclonal antibody target treats nano-carrier systemic drug ACNPs-DOX-DSPE-
PEG2000-anti-CD20 is effective preparation of a monoclonal antibody and chemotherapeutics, and it is thin to can be applied to targeted chemotherapy specificity
Born of the same parents.Koziara etc., which demonstrates taxol-NPs using In situ perfusion method model, can significantly improve brain capture amount, and albumin combines purple
The NPs injection suspension (Abraxane) of China fir alcohol obtains the approval of FDA at the beginning of 2005.In the research of NPs, with paracyanogen base
Alkyl acrylate (Polyalkylcyanoacrylate, ACA) is the carrier material of representative, is a kind of relatively conventional biology drop
The artificial synthesized high molecular material of solution type.ACA includes that Methyl 2-cyanoacrylate, ethyl ester, N-butyl, isobutyl ester and dissident's ester etc. spread out
Biology, degradation in vivo mode mainly have two approach: first is that generating formaldehyde；Second is that it is logical to be degraded to isobutanol under esterase effect
Urine after renal metabolism is crossed to be discharged, ACA is mainly degraded by esterase under alkaline environment, degradation speed with the growth of alkyl chain and
It reduces.Because n-butyl cyanoacrylate (Butylcyanoacrylate, BCA) alkyl chain is longer, degradation is slow, internal tolerance
It is good, the deep favor by numerous scholars.Due to containing two electron-withdrawing groups of cyano and carboxyl on ACA monomer structure, so that β-C is former
Son shows very strong electropositive.Therefore, as long as with the presence of anion, even if under the effect of minimal amount of water, alcohol and weak base
It can make monomer that anionic polymerisation occur rapidly.This peculiar chemical activity of exactly ACA, thus can by control polymerization speed come
It prepares nanoparticle with a certain size and shape, adjusts under polymerization environment liquid phase PH valve to cause or stop polymerization reaction.
This preparation process is relatively simple, is suitable for the research of nanoparticle pharmaceutical delivery system.
Studies have found that the lactoferrin under the pathological states such as AD and Parkinson's disease on cerebral microvascular and neuron
(lactoferrin, Lf) expression of receptor will increase.Lf is a kind of cationic glycoproteins being present in the mammalian body, is belonged to
In transferrins family.It is made of about 690 amino acid, these amino acid form polypeptide chains round into two spherical leaves
Piece respectively includes an iron binding site.In addition to participating in iron metabolism, Lf also has antibacterial, a variety of lifes such as antiviral and immunological regulation
Manage function.Oneself has research to confirm, Lf can be transported into brain by receptor-mediated transcytosis, and the transport process is uniport,
No apparent across cell degradation [27].In addition, studies have found that, LDH receptor related protein antagonist is being added
Afterwards, which can be suppressed 70% or more, illustrate Lf be also possible to transport by LDH receptor related protein into
Brain.The brain drug delivery research of biodegradable NPs through Lf modification also demonstrates that Brain targeting index of the Lf-NPs than blank NPs increases
Add 2.49 times, and cell viability is held in 80 or more when Lf-NPs concentration is 3mg/mL, illustrates the good peace of Lf-NPs
Quan Xing.
In conclusion this experimental selection BCA is that carrier material prepares Res-NPs, by hydroxyl polyethylene glycol maleimide
Amine (Hydroxyl-PEG-Maleimide, OH-PEG-MAL) links Res-NPs and Lf as intermediate " bridge ", obtains Lf and repairs
The Res-NPs (Lactoferrin modified Resveratrol NanoParticles, Lf-Res-NPs) of decorations, it is expected that
It is transported by Lf with the intracerebral that receptor mediated endocytosis transporting mode increases Res, and therapeutic effect of the Res to AD is improved.
Summary of the invention
It is an object of the invention to overcome resveratrol vivo biodistribution availability low and brain distribution it is less, one kind is provided
Bioavilability is higher and has the resveratrol nano particle preparations of Brain targeting function.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:
A kind of resveratrol nano particle preparations with Brain targeting function, including component: resveratrol, BCA, lactoferrin are molten
Liquid, OH-PEG-MAL and water for injection.
A kind of resveratrol nano particle preparations preparation process with Brain targeting function, includes the following steps:
Step 1: the preparation of resveratrol nanoparticle: 50.0 mg of precision weighing macrodex is dissolved in 8 mL waters for injection
In, pH to 2.0 is adjusted, as water phase.48 mg resveratrol of precision weighing again, and accurate measurement 52 μ L of BCA, are dissolved in 4
In mL acetone, as oily phase.Under stirring conditions, oil is mutually slowly added dropwise in water phase, mixing speed is 800 rpm, is stirred
After mixing 3.7 h, pH to 7.0 is adjusted, is further continued for 0.5 h of stirring, this suspension is finally removed into acetone in 40 DEG C of rotary evaporations,
Resveratrol nanoparticle suspension is made；
Step 2: the preparation of eluent A: precision weighing N-2- hydroxyethyl piperazine-N ˊ -2-ethanesulfonic acid 2.3840g, NaCl
8.7652g is dissolved with appropriate distilled water, finally adjusts pH to 7.0 with 1 mol/L NaOH, distilled water is settled to 1000mL, standby
With；
Step 3: the preparation of lactoferrin modification resveratrol nanoparticle: being operated according to above-mentioned steps one, wherein when preparing water phase
10.0 mg of OH-PEG-MAL that precision weighing is added also is needed, PEG-Res-NPs suspension is thus prepared；Take PEG-Res-
NPs suspension is centrifuged 10 min in 3500 rpm, discards supernatant liquid, and deposit is dispersed again with 0.5 mL eluent A, then plus
The concentration for entering activated thiol groups is 1g/L lactoferrin solution 1mL, and the white Chenopodiaceae that 9 h of reaction modify to get lactoferrin is stirred at room temperature
Reed alcohol nanoparticle suspension.
After resveratrol nano particle preparations provided by the invention with lactoferrin by being modified, lactoferrin modification is made
Resveratrol nanoparticle, the nanoparticle pharmaceutical dosage form with Brain targeting enhance Brain targeting and the anti-AD effect of resveratrol.
Detailed description of the invention
Fig. 1 is the transmission electron microscope picture of resveratrol nanoparticle.
Fig. 2 is the transmission electron microscope picture that the lactoferrin modification resveratrol nanoparticle of primary antibody is not added.
Fig. 3 is the transmission electron microscope picture that lactoferrin modifies resveratrol nanoparticle.
Fig. 4 is that lactoferrin modifies resveratrol nanoparticle grain size distribution.
Fig. 5 is each group mice plasma pharmaceutical concentration-time curve (n=10) figure.
Specific embodiment
The foundation of 1 Res analysis method of embodiment:
Precision weighing Res 0.4mg, is dissolved with methanol and is settled to 10mL and obtain Res solution.Respectively within the scope of 200-400nm
Scan Res solution and blank NPs suspension.
Precision weighing Res standard items 4.0mg, methanol dissolve and are settled to 100mL, obtain the Res that concentration is 40 μ g/mL and store up
Standby liquid.It measures 1mL, 1.5mL, 2mL, 2.5mL, 3mL respectively from stock solution, is separately added into methanol dilution and is settled to 10mL, obtain
The Res standard solution for being 4,6,8,10,12 μ g/mL to concentration.Ultraviolet specrophotometer measures its absorbance under 309nm, with
Absorbance A carries out linear regression to concentration C.
Precision weighing Res standard items 4.0mg, methanol dissolve and are settled to 100mL, obtain the Res that concentration is 40 μ g/mL and store up
Standby liquid.Measure 5mL, 10mL, 15mL respectively from stock solution, be separately added into methanol dilution and be settled to 50mL, obtain concentration be 4,
8, the basic, normal, high standard solution of Res of 12 μ g/mL.Ultraviolet specrophotometer surveys its absorbance under 309nm, substitutes into standard curve
Equation calculation concentration.Measuring method is that withinday precision requires to measure 5 times in one day；Day to day precision requires to survey 1 time daily, even
Continuous measurement 5 days.
The measurement of 2 Res-NPs encapsulation rate of embodiment and drugloading rate:
Res recovery test: precision measures 0.1mL concentration as 4,8,12 μ g/mL Res standard solution to 2mL ultra-filtration centrifuge tube
In, 0.5mL ethyl alcohol is added and mixes, 4 DEG C, 15000rpm ultracentrifugation 2h.Precision measures filtrate 0.3mL, and it is molten that 5mL DMSO is added
Solution, finally with methanol constant volume to 10mL.Ultraviolet specrophotometer surveys its absorbance under 309nm, substitutes into calibration curve equation meter
Calculate concentration.Calibration curve equation A=0.1439C-0.0002, R2=0.9993.Show Res concentration in 4 ~ 12 μ g/mL in good
Linear relationship.
The assay of Res in Res-NPs: precision measures blank NPs suspension 1mL, and 5mL DMSO ultrasonic emulsion breaking is added
Afterwards, with methanol constant volume to 10mL, it is centrifuged 10min in 3500rpm, takes supernatant as blank control.It is mixed that precision measures Res-NPs
5mL DMSO ultrasonic emulsion breaking is added in suspension 1mL, with methanol constant volume to 10mL, is centrifuged 10min, uv-spectrophotometric in 3500rpm
It counts and surveys its absorbance under 309nm, and calculate medicament contg.
The measurement of Res-NPs encapsulation rate and drugloading rate: precision measures 0.1mL Res-NPs suspension and is centrifuged to 2mL ultrafiltration
Guan Zhong is added 0.5mL ethyl alcohol and mixes, and 4 DEG C, 15000rpm ultracentrifugation 2h.Precision measures filtrate 0.3mL, and 5mL DMSO is added
After ultrasonic emulsion breaking, with methanol constant volume to 10mL.Ultraviolet specrophotometer measures absorbance under 309nm wavelength, and it is bent to substitute into standard
Its concentration of line equation calculation simultaneously calculates free drug quality, by formula EE%=[(dosage-dissociate dose) ÷ dosage] × 100%
Calculate the encapsulation rate (Encapsulation efficiency, EE%) of Res-NPs.
The 1.5mL EP of constant weight is taken to manage, number A weighs its weight and is denoted as W0.Precision measures 1mL Res-NPs and is suspended
In A pipe, 80 DEG C of dryings to constant weight weigh its weight and are denoted as W liquidAlways, 1mL Res-NPs suspension quality W=WAlways-W0.By formula
DL%=[(dosage-dissociate dose) ÷ W] × 100% calculates the drugloading rate of Res-NPs suspension.
The research of 3 Res-NPs formulation and technology of embodiment:
The preparation of Res-NPs: dextran-70 and PLURONICS F87 are dissolved in purified water, and adjusting pH to acidity forms water phase,
It is spare.Res and BCA are dissolved in acetone, oily phase is formed.Oil is mutually slowly added dropwise in water phase under stirring condition, stirring is certain
PH to 7.0 is adjusted with 0.1mol/L NaOH after time, continues to stir certain time, this suspension is removed in 40 DEG C of rotary evaporations
Acetone is removed, Res-NPs suspension is made.
The preparation of blank NPs: it is identical as the preparation method of above-mentioned Res-NPs, Res is not added to get blank NPs.
Single factor exploration: fixed other preparation conditions are constant, change single factor, only with partial size, encapsulation rate and drugloading rate
For index, to investigate the prescription and technique of Res-NPs.
(1) the oil mutually investigation of example compared with water:
Select grease volume ratio for 8mL:8mL, 4mL:8mL, 2.7mL:8mL, 2mL:8mL respectively.40 μ of Res 32mg, BCA dosage
L, dextran-70 and each 50mg of PLURONICS F87, water phase pH 2.0, mixing speed 800rpm, mixing time 3h continue to stir
Time 0.5h is mixed, Res-NPs is prepared.
(2) investigation of aqueous pH values:
Aqueous pH values are adjusted to 2.0,3.0,4.0 respectively.Grease volume ratio is 4mL:8mL, 40 μ L of Res 32mg, BCA dosage, dextrorotation
Sugared acid anhydride -70 and each 50mg of PLURONICS F87, mixing speed 800rpm, mixing time 3h, continue mixing time 0.5h, preparation
Res-NPs。
(3) investigation of BCA dosage:
Select BCA monomer input amount for 20 μ L, 40 μ L, 64 μ L, 96 μ L respectively.Grease volume ratio be 4mL:8mL, Res 32mg,
Dextran-70 and each 50mg of PLURONICS F87, mixing speed 800rpm, mixing time 3h continue mixing time 0.5h, system
Standby Res-NPs.
(4) investigation of mixing time
After oily mutually instillation water phase, mixing time is set as 2h, 3h, 4h, 5h, 6h.Grease volume ratio be 4mL:8mL, Res 32mg,
40 μ L of BCA dosage, dextran-70 and each 50mg of PLURONICS F87, mixing speed 800rpm continue mixing time 0.5h, system
Standby Res-NPs.
(5) continue the investigation of mixing time
Continue mixing time after adjusting pH to 7.0 and is set as 0.5h, 1h, 2h.Grease volume ratio be 4mL:8mL, Res 32mg,
40 μ L of BCA dosage, dextran-70 and each 50mg of PLURONICS F87, mixing speed 800rpm, mixing time 3h prepare Res-
NPs。
(6) investigation of mixing speed
Mixing speed is respectively set to 400,600,800 and 1000rpm.Grease volume ratio is 4mL:8mL, Res 32mg, BCA use
40 μ L, dextran-70 and each 50mg of PLURONICS F87, mixing time 3h are measured, mixing time 0.5h is continued, prepares Res-
NPs。
(7) investigation of kinds of surfactants and dosage
Dextran-70 50mg, PLURONICS F87 50mg, dextran-70 and each 40mg of PLURONICS F87, dextrose
Acid anhydride -70 and each 80mg of PLURONICS F87, dextran-70 and each 120mg of PLURONICS F87.Grease volume ratio is 4mL:
8mL, Res 32mg, BCA dosage 40 μ L, mixing speed 800rpm, mixing time 3h continue mixing time 0.5h, prepare Res-
NPs。
(8) investigation of dosage
The quality for putting into Res respectively is 6,16,32,48,64,80mg.Grease volume ratio is 4mL:8mL, 40 μ L of BCA dosage, the right side
Sugared acid anhydride -70 50mg, mixing speed 800rpm, mixing time 3h are revolved, mixing time 0.5h is continued, prepares Res-NPs.
The characterization of Res-NPs: Res-NPs suspension is observed into nanoparticle under transmission electron microscope after 0.5% phosphotungstic acid negative staining
Form and size.It measures Res-NPs 0.5mL and is diluted to 1.8mL with distilled water, detect its partial size with laser particle size analyzer.
(9) characterization of Res-NPs
The form of transmission electron microscope observing Res-NPs, the result is shown in Figure 1.Particle size determination result is shown in Fig. 4.
The preparation of 4 Lf-Res-NPs of embodiment
Related solution is prepared: (1) preparation of secondary antibody buffer: precision weighing polysorbas20 0.050g, NaCL 2.922g, cow's serum
Albumin 0.100g, precision measure calf serum 5mL, and distilled water dissolves and is settled to 100mL.
(2) preparation of 0.15mol/L dobell's solution: precision weighing 5.720g sodium tetraborate adds 90mL distilled water to dissolve,
With 1mol/L NaOH tune pH to 8.5, distilled water is added to be settled to 100mL.
(3) 2- iminothiolane hydrochloride (2-Iminothiolane hydrochloride, Traut's) solution is matched
System: precision weighing Traut's reagent 0.010g adds 0.15 mol/L dobell's solution to dissolve and be settled to 10mL, with 200 μ L bodies
It is saved after integral dress in -20 DEG C.
(4) 8.0 pH, the preparation of the 0.1 mol/L phosphate buffer (PBS) of pH 7.0: the preparation of solution A: precision claims
Amount NaH2PO42 H2O 15.601g is dissolved in 500mL distilled water and obtains 0.2mol/L NaH2PO4 solution.The preparation of solution B:
Precision weighing Na2HPO4 12H2O 35.524g is dissolved in 500mL distilled water and obtains 0.2mol/L Na2HPO4 solution.pH 8.0
0.1mol/L PBS preparation: 5.3mL solution A and 94.7mL solution B are mixed, add distilled water to 200mL to obtain the final product.pH 7.0
0.1mol/L PBS preparation: 39mL solution A and 61mL solution B are mixed, add distilled water to 200mL to obtain the final product.
(5) preparation of eluent A: precision weighing N-2- hydroxyethyl piperazine-N ˊ -2-ethanesulfonic acid 2.383g, NaCl
8.7750g, distilled water dissolution, with 1 mol/L NaOH tune pH to 7.0, adds distilled water to be settled to 1000mL.
(6) preparation of Ellman ' s reagent: 5,5 ˊ-two thiobis of precision weighing (2- nitrobenzoic acid) [5,5'-
Dithio bis- (2-nitrobenzoic acid), DTNB] 0.0396g, add pH7.0 0.1mol/L PBS to dissolve and determine
Hold to 10mL to obtain the final product, 4 DEG C of refrigerators are kept in dark place.
The preparation of PEG-Res-NPs: precision weighing OH-PEG-MAL 0.01g is pure in 8mL with dextran-70 0.050g
Change and dissolved in water, adjust pH to 2.0 with 0.1mol/L HCL, at room temperature 800rpm magnetic agitation, forms water phase, it is spare.It is accurate
It weighs appropriate Res to be dissolved in acetone, BCA is added and forms oily phase.This oil is mutually slowly added dropwise in water phase again, persistently stirs 3.7h
PH to 7.0 is adjusted with 0.1mol/L NaOH afterwards, continues to stir that this suspension is removed into acetone in 40 DEG C of rotary evaporations after 30min,
PEG-Res-NPs suspension is made.
The preparation of Lf-Res-NPs: PEG-Res-NPs is centrifuged 10min in 3500rpm, precipitating is collected, is eluted with 0.5mL
The Lf solution of activated thiol groups is added after liquid A dispersion, after reaction 9h is stirred at room temperature, obtained nanoparticle suspension is with 0.01mol/L
PBS is stripped Sepharose CL-4B gel column (1.0 × 60cm, 0.2mL/min flow velocity), remove not connected albumen to get
Lf-Res-NPs。
The characterization of Lf-Res-NPs: Res-NPs 0.5mL is measured with distilled water and is diluted to 1.8mL, detects its partial size.Transmission
The Lf of the Electronic Speculum identification surface Lf-Res-NPs connection: taking appropriate Lf-Res-NPs, be dispersed in 500 μ L 0.01mol/L PBS,
The anti-20 μ L of Lf antibody (primary antibody) of the diluted goat of l:1000 is added, 37 DEG C of shaking tables (100rpm) are incubated for lh, use Sepharose
CL-4B gel column is separated off unbonded primary antibody, is eluted with PBS, collect the PBS containing nanoparticle, in 10000g, 4 DEG C from
Heart lh abandons supernatant.The dispersion of 1mL secondary antibody buffer is added in precipitating, and 100 μ L colloid gold label rabbit-anti anti-goat IgG antibodies (two are added
It is anti-), continue 37 DEG C of shaking tables (100rpm) and be incubated for l.5h, in 10000g, 4 DEG C of centrifugation lh, abandons supernatant.Precipitating plus 300 μ L PBS
Dispersion, unbonded colloid gold label secondary antibody is separated off with Sepharose CL-4B gel column, and collection contains nanoparticle
PBS carries out transmission electron microscope observing after phosphotungstic acid dyeing, as a result sees Fig. 3.It separately takes Lf-Res-NPs to be added without primary antibody and repeats above-mentioned examination
Step is tested, whether investigate has false positive phenomenon.It takes Res-NPs to repeat above-mentioned test as negative control, as a result sees Fig. 2.
5 lactoferrin of embodiment modifies resveratrol nanoparticle pharmacokinetics and Tissue distribution research in Mice Body
Dosage regimen: feeding environment temperature maintains (24 ~ 28) DEG C, relative humidity 50% ~ 60%, special room sub-cage rearing, the next day
Padding is replaced, water is freely taken the photograph, ingests (standard feed nursing), intraperitoneal injection after raising 1 week, mouse fasting 12h before being administered,
It is administered according to mouse weight, point 3 groups: Res group, Res-NPs group and Lf-Res-NPs group (preparing before use), Res dosage
For 44mg/kg；3% chloraldurate (0.1mL/10g) anesthetized mice, after administration in 0.17,0.5,0.75,1,2,6,10,12, for 24 hours
Blood sampling, each time point 10.After blood sampling immediately with physiological saline cardiac perfusion to it is each tissue it is white, take out the heart, liver, spleen,
Lung, kidney, brain, normal saline flushing clean surface bloodstain, filter paper suck dry moisture, -80 DEG C of preservations.
HPLC method analyzes the drug concentration in blood plasma
(1) preparation of Res stock solution and standard solution
Precision weighing Res 10.0mg, methanol constant volume to 10mL obtain the stock solution that Res concentration is 1mg/mL.Precision measures Res
Stock solution 0.0l, 0.25,0.5,1,2,3,4mL obtain 1,2.5,5,10,20,30,40 μ g/ of concentration with methanol constant volume to 10mL
The Res standard solution of mL.
(2) HPLC chromatogram testing conditions
Mobile phase: methanol: water=5:5；Flow velocity: 1.0 mL/min；
Detection wavelength: 306nm；Retention time: 14min；Sample volume: 60 μ L.
(3) processing of plasma sample
It plucks eyeball and takes 3500rpm centrifugation 10min after blood, separated plasma is saved in 4 DEG C.Precision measures 0.2mL blood plasma, is added
The ethyl acetate of 0.5mL, vortex oscillation 1min, sufficiently extraction drug.Stood after vortex it is visible have two layers of liquid of grease, by upper layer
Oil phase liquid take out, be added 1.5mL EP pipe in, 40 DEG C of water-baths are dried with nitrogen, be added 1mL methanol, vortex 1min, 4000rpm from
Heart 10min takes supernatant by method " 2.2.2 " sample detection.
(4) specificity is investigated
By method " 2.2.2 " sample detection as control after taking mouse blank plasma samples to handle by " 2.2.3 " method；Blank blood
Res stock solution is added in slurry by method " 2.2.2 " sample detection is pressed after the processing of " 2.2.3 " method, compares its result and investigates detection
The specificity of method.
(5) in blood plasma Res standard curve foundation
Precision measures mouse blank plasma 0.2mL, and Res series standard solution 1mL is added, and methanol constant volume to 10mL obtains concentration and is
0.1, the Res plasma standard solution of 0.25,0.5,1,2,3,4 μ g/mL.Precision draws Res plasma standard solution 1mL, and HPLC is surveyed
Determine Res concentration.
(6) precision and recovery test
Precision measures the basic, normal, high Res plasma standard solution 1mL prepared, and sample measures Res concentration after processing, with HPLC.
Withinday precision requires to measure 5 times in 1 day；Day to day precision requires to survey 1 time, METHOD FOR CONTINUOUS DETERMINATION 5 days daily.Precision measures preparation
Basic, normal, high Res plasma standard solution 1mL, sample measure drug concentration after processing, with HPLC, calculate the Res rate of recovery.
(7) Res group, Res-NPs group, pharmacokinetic results in Lf-Res-NPs group Mice Body, are as a result shown in Fig. 5.Mainly
Pharmacokinetic parameters be shown in Table 1.
1 each group pharmacokinetics of table statistics away from parameter (n=10)
Remarks:* P< 0.05 vs Res group# P< 0.05 vs Res-NPs group
6 Res, Res-NPs and Lf-Res-NPs mouse of embodiment targets Journal of Sex Research
Each tissue drug content is calculated according to each tissue Res concentration and each tissue organ weights, passes through formula DTI (%)=target
The drug targeting for calculating each time point in each tissue to the non-targeted Drug content in tissues of Drug content in tissues ÷ × 100% refers to
Number (Drug targeting index, DTI), the tissue-targeting of evaluation Res group, Res-NPs group and Lf-Res-NPs group.Knot
Fruit is shown in Table 2 ~ table 7.
2 Res solution group of table Res in mouse is respectively organized content (n=10)
"-": represents below the minimum detection line
Targeting index of the 3 Res group of table in mouse is respectively organized
4 Res-NPs group of table Res in mouse is respectively organized content (n=10)
Targeting index of the 5 Res-NPs group of table in mouse is respectively organized
6 Lf-Res-NPs group of table Res in mouse is respectively organized content (n=10)
Targeting index of the 7 Lf-Res-NPs group of table in mouse is respectively organized
Claims (2)
1. a kind of resveratrol nano particle preparations with Brain targeting function, it is characterised in that: it includes component: resveratrol,
BCA, lactoferrin solution, OH-PEG-MAL and water for injection.
2. a kind of resveratrol nano particle preparations preparation process with Brain targeting function, it is characterised in that: it includes following step
It is rapid:
Step 1: the preparation of resveratrol nanoparticle: 50.0 mg of precision weighing macrodex is dissolved in 8 mL waters for injection
In, pH to 2.0 is adjusted, as water phase；
48 mg resveratrol of precision weighing again, and accurate measurement 52 μ L of BCA, are dissolved in 4 mL acetone, as oily phase；
Under stirring conditions, oil is mutually slowly added dropwise in water phase, mixing speed is 800 rpm, after stirring 3.7 h, is adjusted
PH to 7.0 is further continued for 0.5 h of stirring, this suspension is finally removed acetone in 40 DEG C of rotary evaporations, obtained resveratrol is received
Grain of rice suspension；
Step 2: the preparation of eluent A: precision weighing N-2- hydroxyethyl piperazine-N ˊ -2-ethanesulfonic acid 2.3840g, NaCl
8.7652g is dissolved with appropriate distilled water, finally adjusts pH to 7.0 with 1 mol/L NaOH, distilled water is settled to 1000mL, standby
With；
Step 3: the preparation of lactoferrin modification resveratrol nanoparticle: being operated according to above-mentioned steps one, wherein when preparing water phase
10.0 mg of OH-PEG-MAL that precision weighing is added also is needed, PEG-Res-NPs suspension is thus prepared；Take PEG-Res-
NPs suspension is centrifuged 10 min in 3500 rpm, discards supernatant liquid, and deposit is dispersed again with 0.5 mL eluent A, then plus
The concentration for entering activated thiol groups is 1g/L lactoferrin solution 1mL, and the white Chenopodiaceae that 9 h of reaction modify to get lactoferrin is stirred at room temperature
Reed alcohol nanoparticle suspension.
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Cited By (1)
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|---|---|---|---|---|
| CN111166765A (en) * | 2020-01-16 | 2020-05-19 | 华南理工大学 | Iron lactoprotein modified gold-bismuth selenide quantum dot material and preparation method and application thereof |
Citations (2)
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|---|---|---|---|---|
| EP1239849A2 (en) * | 1999-10-29 | 2002-09-18 | Pharmascience Inc. | Pharmaceutical formulations comprising resveratrol and use thereof |
| CN104382888A (en) * | 2014-11-06 | 2015-03-04 | 江苏隆力奇生物科技股份有限公司 | Preparation method of resveratrol phospholipid complex self-microemulsion particles |
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2019
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1239849A2 (en) * | 1999-10-29 | 2002-09-18 | Pharmascience Inc. | Pharmaceutical formulations comprising resveratrol and use thereof |
| CN104382888A (en) * | 2014-11-06 | 2015-03-04 | 江苏隆力奇生物科技股份有限公司 | Preparation method of resveratrol phospholipid complex self-microemulsion particles |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111166765A (en) * | 2020-01-16 | 2020-05-19 | 华南理工大学 | Iron lactoprotein modified gold-bismuth selenide quantum dot material and preparation method and application thereof |
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