CN109045056A - Multiple medicine carries medicine targeted nano particle and its preparation method and application - Google Patents
Multiple medicine carries medicine targeted nano particle and its preparation method and application Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
- A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
- A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
Abstract
The invention belongs to biomedicine technical fields, and in particular to a kind of multiple medicine carries medicine targeted nano particle and its preparation method and application.The multiple medicine carries medicine targeted nano particle and is prepared by golden coated iron oxide core-shell nano (GNPs) load polyphenol compound, iRhom2 siRNA and TNF-α inhibitor, the gold coated iron oxide core-shell nano, polyphenol compound, iRhom2 siRNA and TNF-α inhibitor molar ratio be 1:1~3:1~3:1~3.Multiple medicine of the invention carries medicine targeted nano particle and improves polyphenol compound bioactivity and bioavilability, can efficiently prevent and treat related disease caused by septicemia caused by Listeria Monocytogenes.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of multiple medicine carries medicine targeted nano particle and its preparation side
Method and application.
Background technique
Innate immune system is the key that host and the first line of defence, by causing highly complex the acquired immune response
To resist foreign pathogen.When infectious organism is accredited, host's innate immune system identification invading micro-organism simultaneously causes
Defense reaction and inflammatory process.Dysfunctional inflammatory reaction is the important component of congenital immunity, and leads to the hair of many diseases
Raw, including lasting tissue damage, Balance disorders are physically different or even dead.Septicemia is that a kind of serious bacterium infection is drawn
The diseases associated with inflammation risen.Have proven to immunosupress and uncontrolled inflammatory reaction be septicemia main pathophysiological mechanism it
One.Epidemiological study shows that septicemia is the common and main cause of high mortality and disease incidence that China observes.Due to
The death rate caused by Chinese septicemia increased since 55 years old, and further increased since 75 years old, finally reached in one's mid-80s
To peak.
Lethal septicemia and inflammatory mediator (TNF-α, IL-6 and IL-1 β), chemotactic factor (CF) (MCP-1, Emr-1 and
Fractalkine the caused systemic inflammatory response syndrome (SIRS) and high oxidation stress level of high expression) are closely related.
The pathogenesis of multiple organ dysfunction syndrome (multiple organ dysfunction syndrome, MODS) is very
Complicated and unclear, MODS is a substantially uniform viewpoint as caused by SIRS.Cell factor is uncontrolled in macrophage
The release of system also plays a crucial role in the conversion process from apoplexy with SIRS to MODS.
It has recently been demonstrated that in various fruits and vegetables (including red onion, grape, blueberry, tea, broccoli and red Portugal
Grape wine) in find be known as fisetin (Fisetin, also known as fisetin) natural can be used as exempting from mouse disease
Epidemic disease promotor model includes anti-aging, anti-inflammatory and anticancer model.However, bioavilability is low due to fisetin poorly water-soluble,
Its medically application be restricted.
Application No. is a kind for the treatment of for septicemia of 201280055832.2 patent disclosure and/or improved medicines
Object, the drug contain thrombomodulin as effective component, for treating and/or improving severe septic patient organ dysfunction
Obstacle, the effect that thrombomodulin therein can selectively be combined with fibrin ferment, the work for promoting the PROTEIN C based on fibrin ferment
The effect of change, the effect for extending the clotting time based on fibrin ferment, the effect for inhibiting the platelet aggregation based on fibrin ferment and anti-
Inflammation effect.The visible drug improvement result of the effect of the albumen is greater than treatment, cannot effectively prevent and assist in the treatment of septicemia.
Application No. is a kind of Chinese medicine for treating septicemia of 201510310946.2 patent disclosure, the treatment septicemia
Chinese medicine, prescription include following raw material medicaments: 16 parts of sulphur, 32 parts of minium, 12 parts of alum, 12 parts of nitre, 28 parts of pepper.Above-mentioned medicine
It is spare that object is ground into fine powder, and medicinal powder vinegar is reconciled pelletization when use, be held in the hand in the minds of 4-5 hour, send out and have a good sweat to whole body, gently
Disease is with primary, and severe is with secondary.The drug is externally applied drug, cannot prevent and treat septicemia from internal.
Currently, Most patients still have initiation excessively and the mechanism of uncontrolled inflammatory reaction and death is unclear
Chu.Although treatment aspect achieves progress, the high mortality of septicemia is still significant challenge clinically so far.Lack chemistry
Prevention and adjuvant treatment are still the major obstacle of successful treatment septicemia.Therefore developing one kind can increase fisetin biological utilisation
Degree and the drug that can effectively prevent and assist in the treatment of bacterial septicemia related disease.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of multiple medicines to carry medicine targeted nano particle, which carries medicine targeting
Nano particle can prevent and treat acute liver failure caused by septicemia caused by Listeria Monocytogenes.
To achieve the above object, the technical solution of the present invention is as follows:
Multiple medicine carries medicine targeted nano particle, loads Polyphenols chemical combination by golden coated iron oxide core-shell nano (GNPs)
Object, iRhom2siRNA and TNF-α inhibitor are prepared, the gold coated iron oxide core-shell nano, Polyphenols chemical combination
The molar ratio of object, iRhom2siRNA and TNF-α inhibitor is 1:1~3:1~3:1~3.
Useful load of the iRhom2siRNA on nanoparticle and its being positively correlated property of molar concentration, therefore molar ratio mistake
Useful load up-regulation that is high then will lead to iRhom2siRNA, steric hindrance increase, and will lead to other polyphenols and TNF-α suppression
The useful load of preparation declines.It is very few to will lead to useful load of three substances on nano particle if molar ratio is too low, no
It is horizontal that expected drug effect can be reached.
Further, the polyphenol compound be include fisetin, Quercetin, juglanin, mulberrin (phellinus ketone)
It is a kind of.
As a preference, the polyphenol compound is fisetin.
As a preference, the gold coated iron oxide core-shell nano, polyphenol compound, iRhom2siRNA and
The molar ratio of TNF-α inhibitor is 1:1.5~2.5:1.5~2.5:1.5~2.5.
As a preference, the gold coated iron oxide core-shell nano, fisetin, iRhom2siRNA and TNF-α suppression
The molar ratio of preparation is 1:2.5:2.5:2.5.
The present invention loads to polyphenol compound on GNPs to enhance its bioactivity and bioavilability.
The effect of fisetin is natural, is improved immunity, anti-aging, anti-inflammatory and anticancer.But it is water-soluble
Difference, bioavilability is low, and application medically is restricted.It is natural to enhance this on GNPs by loading to fisetin
The bioactivity and bioavilability of product.
IRhom2 (also referred to as Rhbdf2) is sluggish member in protease family in diamond shape film, it be by with TNF-
α invertase (TACE/ADAM17) connects to control the important inflammation associated adjustment factor of TNF-α generation.The discovery of applicant team
IRhom2 missing may inhibit TNF-α correlation hepatic injury, and the inhibition of iRhom2/TACE/TNF- α activation shaft can reduce whole body inflammation
Disease restores metabolic balance and mitigates MODS (multiple organ dysfunction syndrome).
The present invention carries medicine targeted nano particle (FN nano particle) by building multiple medicine, by fisetin, iRhom2siRNA and
TNF-α inhibitor is supported on golden coated iron oxide core-shell nano, multiple groups strategy combination is realized, for iRhom2/
The efficient targeting of TNF-α signal path blocks caused by Listeria infection from Immune inflammatory reaction and oxidative stress angle
Acute liver failure.Wherein, fisetin is living with anti-oxidant and anti-inflammatory pharmacology as Polyphenols natural products monomer
Property.The necessary regulatory factor that iRhom2 is discharged as proinflammatory factor TNF-α caused by bacterium infection, the direct source iRhom2siRNA
Head inhibits the activity expression of iRhom2.In signal downstream, TNF-α inhibitor also can directly block the release of TNF-α.
Further, the TNF-α inhibitor is lenalidomide (CC-5013).Lenalidomide is the immunomodulator of representative
One critical function of object can exactly inhibit monocyte to generate TNF-α.Monocyte in our human bodies can produce one
Kind inflammatory mediator TNF-α, the mediated factor have several effects: local hemorrhage can be caused downright bad；Surrounding group can be damaged
It knits；Lymphocyte can be recruited to areas of inflammation；It can promote the expression of Some Adhesion Molecules on Endothelial Cells, and then accelerate newborn
The formation of blood vessel；It can promote the growth and transfer of certain tumour cells.And the generation of TNF-α is once inhibited, above-mentioned effect
It will weaken significantly.Lenalidomide can also stimulate the generation of T lymphocyte, can stimulate the T lymphocyte conversion of more naiveties
For the proliferation of T lymphocyte can be promoted with the mature T lymphocyte of tumor cell feature, at the same promote various kinds of cell because
The generation of son, and mature T lymphocyte, then have powerful lethal effect to tumour cell, enhance immunological regulation effect.
Further, the nucleotide sequence of the targeting iRhom2siRNA is as shown in SEQ ID NO.1.
Nonactive diamond shape albumen 2 (iRhom2) be by TNF-α invertase (TACE) act on so that TNF-α secretion it is required
Regulator inhibits the expression of iRhom2 to inhibit the secretion of TNF-α by iRhom2siRNA.
The second object of the present invention is to provide a kind of preparation method of above-mentioned nano particle, the polyphenol compound
For fisetin, comprising the following steps:
1) golden coated iron oxide core-shell nano (GNPs) is prepared；
2) Thiolation siRNA is dissolved in DEPC, is added to containing in mass percent 0.08%SDS GNPs, 4
10~20h is reacted at DEG C；
3) nano particle after isolating step 2) reaction, is scattered in DEPC water, removes not connected
IRhom2siRNA obtains the GNPs of iRhom2siRNA functionalization；
4) the HS-PEG fisetin and lenalidomide that are connected to are added to the iRhom2siRNA functionalization of step 3) preparation
GNPs mixed liquor in reaction 18~for 24 hours, after centrifugation and washing multiple medicine carry medicine targeted nano particle.
3 kinds of substance Mo-Co catalysts have a significant impact the pharmacological activity of GNPs in preparation method of the invention.Due to sky
Between steric effect, golden coated iron oxide core-shell nano (GNPs) mount first siRNA and control hybrid reaction time and
The concentration of siRNA, to reserve enough space mounting HS-PEG fisetins and lenalidomide.As first mounted HS-PEG paint in method
Flavine and lenalidomide, then the two the space occupied is excessive, and the amount for mounting siRNA is very few, cannot reach siRNA for target spot
Effective inhibiting effect.
Concentration of the Thiolation siRNA in DEPC is 10 μM in step 2)；Wherein contain mass percent 0.08%SDS
The concentration of GNPs is 10nM in GNPs.
Further, it is ultrasonically treated when reaction in step 2) and salinity is adjusted to 0.25M~0.35M NaCl, to increase
Coverage of the siRNA on nano grain surface.
Salinity is adjusted to 0.3M NaCl as a preference, being ultrasonically treated when reaction in step 2), to increase siRNA
Coverage on nano grain surface.
As a preference, reacting 15h at 4 DEG C in step 2).
As a preference, the temperature reacted in step 4) is 20~30 DEG C.
As a preference, the temperature reacted in step 4) is 25 DEG C, reaction is for 24 hours.
Further, in step 1) golden coated iron oxide core-shell nano the preparation method comprises the following steps:
1) the coated ferroferric oxide nano granules of citric acid are dispersed in water and are ultrasonically treated using ultrasonoscope, surpassed
It will be mixed with chlorogold solution after sonication, and obtain mixed liquor；
2) trisodium citrate dehydrating solution is added in the mixed liquor in step 1), is thoroughly mixed to obtain suspension；
3) washed, collect and dry suspension in golden coated iron oxide core-shell nano.
Further, the temperature being ultrasonically treated in step 1) is 70~85 DEG C, and the processing time is 15~30min；Ultrasonic treatment
It will be mixed afterwards with chlorogold solution under keeping warm mode, and obtain mixed liquor；
As a preference, the temperature being ultrasonically treated in step 1) is 80 DEG C, the processing time is 25min.
Further, wherein the mass volume ratio of ferroferric oxide nano granules and chlorogold solution is 1:0.5~3.
As a preference, wherein the mass volume ratio of ferroferric oxide nano granules and chlorogold solution is 1:1.
The third object of the present invention is that providing the above-mentioned multiple medicine of one kind carries medicine targeted nano particle in preparation prevention and control
Treat the application in the drug of related disease caused by septicemia caused by Listeria Monocytogenes, which is characterized in that
The related disease includes acute liver failure caused by septicemia caused by Listeria Monocytogenes.
The beneficial effects of the present invention are:
1) multiple medicine provided by the invention carries medicine targeted nano particle and can increase polyphenol compound bioavilability and can have
Effect prevention and adjuvant treatment bacterial septicemia related disease, can be effectively prevented and treated Listeria Monocytogenes and draw
Acute liver failure caused by the septicemia risen.
2) preparation method that multiple medicine provided by the invention carries medicine targeted nano particle increases the mounting of siRNA, directly presses down
The activity of the proinflammatory inflammation factor regulatory factor iRhom2 of macrophage processed and the activation level of macrophage.
3) multiple medicine provided by the invention carries the preparation method of medicine targeted nano particle by mounting TNF-α inhibitor come that degree
Amine, directly inhibits the release and expression of TNF-α, and then directly inhibits iRhom2/TNF- α whole signal path.
Detailed description of the invention
Fig. 1 is the structural schematic diagram that multiple medicine of the invention carries medicine targeted nano particle.
Fig. 2 is the reaction route figure for synthesizing the fisetin of HS-PEG-COOH connection.
Fig. 3 is the transmission electron microscope image of nano particle.
Fig. 4 is dynamic light scattering (DLS) image of FN nano particle.
Fig. 5 is the magnetometric analysis of FN nano particle.
Fig. 6 is the efficiency and concentration dependant sexual intercourse that iRhom2siRNA mounts FN nano particle.
Fig. 7 is the in-vitro release rate curve synoptic diagram of fisetin in FN nano particle.
Fig. 8 is the influence of Pathologic changes and metabolic syndrome caused by FN nano particle infects Listeria.
Wherein A curve for survival；B is respiratory rate curve；C is that mean blood pressure is horizontal；D is that Temperature changing is horizontal；E is real
Test the weight of animals variation；F is the variation of liver of laboratory animal weight in wet base；G is the variation of experimental animal spleen weight in wet base；H is that experimental animal is attached
Testis fat；I is experimental animal abdominal cavity fat；J is experimental animal brown fat.
Fig. 9 is the influence of peripheral inflammation reaction caused by FN nano particle infects Listeria.
Wherein A is the expression of TNF-α in serum；B is the expression of IL-10 in serum；C is IFN-γ in serum
Expression；D is the expression of IFN-β in serum；E is the expression of IL-6 in serum；F is IL-1 β in serum
Expression；G is the expression of PCT in serum；H is the expression of HMGB1 in serum；I is the expression water of MDC in serum
It is flat；J is the expression of MIP-1 β in serum；K is the expression of MIP-2 in serum；L is the expression water of MIP-3 β in serum
It is flat；M is the expression of MIP-1 α in serum；O is the expression of MCP-1 in serum；P is the expression water of MCP-3 in serum
It is flat；Q is the expression of GCP-2 in serum.
Specific embodiment
Hereinafter reference will be made to the drawings, and the preferred embodiment of the present invention is described in detail.Tool is not specified in preferred embodiment
The experimental method of concrete conditions in the establishment of a specific crime, usually according to normal condition, illustrated embodiment are to preferably say to the contents of the present invention
It is bright, but be not that the contents of the present invention are only limitted to illustrated embodiment.So those skilled in the art are according to foregoing invention
Content carries out nonessential modifications and adaptations to embodiment, still falls within protection scope of the present invention.
The reagent and material that following embodiment is used:
Fisetin(C15H10O6, Mw:286.24, HPLC >=98%, CAS:345909-34-4), ferric chloride hexahydrate
(III)(FeCl3·6H2O, >=99%), ferrous sulfate heptahydrate (II) (FeSO4·7H2O, >=98%), citric acid (C6H8O7,
99.5%), hydrochloric acid (HCl, 37%), sodium hydroxide (NaOH), sodium chloride (NaCl, >=99.5%) and tetra chlorauric acid salt
(HAuCl4·3H2O, 49-50%Au), HS-PEG (Cat:JKA5101, Mn:7,500) is purchased from Sigma-Aldrich (China).
Lenalidomide (CC-5013) is purchased from Selleckchem (China).Sodium citrate (Na3C6H5O7·2H2O, 99%) it is purchased from
KeyGENBioTECH Co., Ltd (Nanjing of China).IRhom2siRNA is by Sangon Biotech, and Co., Ltd. is (in China
Sea) preparation.The fisetin or lenalidomide of HS-PEG connection are by Sangon Biotech, Co., Ltd.(Chinese Shanghai) production.
The preparation of 1 multiple medicine of embodiment load medicine targeted nano particle
1. preparing golden coated iron oxide core-shell nano (GNPs)
By 0.1M FeSO4·7H2O aqueous solution and 0.2M FeCl36H O is placed in the 90ml solution containing 0.1M HCl
In.It is then careful that 80ml 2M sodium hydroxide is added dropwise, make to react under continual stirring conditions sufficiently distribution 70 minutes and protects nitrogen
Gas.Obtained black suspension is collected using high power neodymium alloy magnet (2500G) and with double steam water washings of nitrogen treatment
3-4 times.It undergoes 0.1g/mL citrate working solution under conditions of 6.8 pH and stablizes ferric oxide nanometer particle suspension,
It is stirred continuously mixture and is heated to 80 DEG C of 2h.Then the solution is kept to be cooled to room temperature with obtain citric acid it is stable four
Fe 3 O nano particle.The coated ferric oxide nanometer particle of 60mg citric acid is dispersed in 15mL distilled water and uses water-bath
Ultrasonoscope is ultrasonically treated 25 minutes.Sample is heated to 80 DEG C in ultrasonoscope under continual stirring conditions.Next, will
The freshly prepared 0.1M chlorogold solution mixing of 800 μ l.Finally, 4ml 0.1M trisodium citrate dehydrating solution is added to mixing
In object.At 80 DEG C by mixture solution slowly and continue stirring 60 minutes until sample formed in suspension it is stable dark red
Color.Ferric oxide nanometer particle using double steam carefully lightly by the gold coating of acquisition washs 4 times.Then, using neodymium alloy
Magnet (2500G) collects golden coated iron oxide core-shell nano (GNPs), and GNPs dries in vacuum drying oven and be maintained at 4
It is DEG C spare.
2. couple GNPs carries out functionalization
Reaction route figure is shown in Fig. 2, carries out function to nano particle with the siRNA that the iRhom2 gene targeted with DY647 is connect
Energyization.Thiolation siRNA is prepared in 1mL 0.1M DTT, is separated 3 times by ethyl acetate, and according to the manufacturer's instructions
It is further purified with desalination NAP-5 column (GE Healthcare, China).By the Thiolation siRNA of purifying with 10 μM of concentration
It maintains in DEPC water, it is then careful and be added in the GNPs containing 0.08%SDS (10nM) immediately, at ultrasonic in short-term
Salinity is adjusted to 0.3M NaCl by reason from 0.05M NaCl, to increase coverage of the siRNA on nano grain surface.4
After forming 15h at DEG C, at 4 DEG C, 20000 × g centrifugation 25min isolates nano particle, and is scattered in DEPC water again, the step
Suddenly in triplicate.By in microplate reader (Varioskan Flash Multimode Reader, Thermo Scientific)
The oligonucleotides being not connected in the supernatant harvested during being synthesized by DY647 fluorescent dye (excitation/emission, 645/663nm)
Determine the siRNA number in particle.It, will for the functionalization of fisetin and lenalidomide (TNF-α inhibitor (CC-5013))
The fisetin or lenalidomide of HS-PEG connection are added in the 10nM GNPs of final concentration of 5 μ g/mL and keep it anti-at 25 DEG C
It should for 24 hours DEG C.Later, it by nano particle at 4 DEG C, is centrifuged 25 minutes under 20000 × g, and obtains FN nanometers with Mili-Q water washing 3 times
Particle, structural schematic diagram are shown in Fig. 1, are resuspended in distilled water, then store for future use at 4 DEG C.
The characterization of 2 multiple medicine of embodiment load medicine targeted nano particle
The electricity of FN nano particle is checked using TEM (Tecnai G2 20200kV TEM (Fei, Electron Optics))
Sub- microphoto.A small amount of dropping liquid of the sample solution in distilled water is dried on the copper mesh that carbon coats to carry out tem analysis.This
Outside, it is examined using the dynamic light scattering (DLS) of LB-550DLS Particle Size Analyzer (Horiba Scientific, Edison, NJ)
Look into the average-size of our previously obtained FN nano particles.It is detected under room temperature (25 DEG C) using Lake Shore 735VSM
The magnetism of nano particle.
The tem analysis of nano particle shows that nano particle has spherical morphology (Fig. 3).Dynamic light scattering shows these nanometers
The average hydrodynamic diameter of particle is 15nm (Fig. 4).Due to the high electron density of gold, the gold coating in the region studied
Ferric oxide nano particles seem darker.In addition, density (Fig. 5) of the GNPs nanoparticle in the intensity of magnetization (Ms) is
34.742emu/g.Once a series of different materials including CC-5013 and fisetin are linked to GNPs nanometers including siRNA
On grain, Ms value is just down to 31.801.The reduction of Ms may be since the long range sequence magnetic exchange on nano grain surface is mutual
Caused by the turbulent flow for acting on coupling.In addition, the concentration in order to determine siRNA on nano particle, is increased with the siRNA of DY647 label
The emission spectrum of concentration is added to show that the Percentage bound between siRNA and GNPs increases (Fig. 6) with siRNA concentration.In order to analyze
External FN nano particle discharges fisetin, is accumulated in the PBS (0.1M, pH7.4) containing 0.1%Tween-80 (w/v)
Release detection.Fig. 7 display synthesis GNPs nano particle simultaneously loads fisetin to enhance the bioactivity of the natural products.About
30% fisetin is initially discharged in 1h implosive, this may be to be desorbed and discharged from nano grain surface due to drug.In 72h
Research in, nearly 99% fisetin is released from nano particle.
3 multiple medicine of embodiment carries the mouse metabolism exception and systemic inflammatorome that medicine targeted nano particle inhibits LM induction
Listeria monocytogenes (LM) lethal septicemia induced is accredited as metabolic system disorder and whole body
Then the reason of inflammatory reaction, assesses the intervention of FN nano particle to improvement abnormal physiology.In our study, with compare
Mouse is compared, and LM applies significant inducible metabolism illness.All mouse in model group are dead in 42h after injecting LM.FN nanometers
Grain intervention group mouse survival rate is respectively 0% (25/25；54h after injection；20mg/kg FN), 36% (9/25；40mg/kg FN)
With 60% (15/25；80mg/kg FN) (Fig. 8 A).In addition, LM infection changes mouse breathing frequency, blood pressure and body temperature.Fig. 8 B-
8D shows respiratory rate in the mouse of LM infection, the increase of hypothermia and dyskinesia.However, the treatment of FN nano particle inhibits
These abnormal symptoms, or even promote them and be restored to normal level, and action level is dose-dependent.With compare
Group is compared, and LM administration is significant to reduce weight, liver mass and spleen weight (Fig. 8 E-8G), but eWAT, iWAT and BAT weight
There is no notable difference (Fig. 8 H-8J).These metabolic disorders are significant inverse by being handled with FN nano particle with dosage-dependent manner
Turn, shows that these FN nano particles there may be the ability for improving the metabolic disorder of LM induction in mouse.
More and more evidences show that LM exposure can raise systemic inflammatorome, this may pass through the secretion of inhibition inflammatory factor
Release with chemotactic factor (CF) and be suppressed.Herein, we further detect whether LM infection increases in time course experiment
Add peripheral inflammation horizontal.Fig. 9 A-9F is shown in LM- pollution mouse 0-24, main inflammation index of correlation, including TNF-α,
IFN-γ, IL-6, IL-10, IFN-β and IL-1 β are significantly raised.Meanwhile the expression of inflammatory factor continues to increase after infection.It is important
, significant variation also has occurred in the expression of some important inflammation associated chemokines.Inspection when Fig. 9 G-9Q is shown in 6h
Measure including MIP-2, MCP-1, the chemotactic of MIP-3 β, MCP-3, PCT, HMGB1, MDC, MIP-1 β, MIP-1 α and GCP-2 because
Son after LM infection for 24 hours prompts the increase of chemotactic factor (CF) related to the generation of metabolic disorder and systemic inflammatorome, and applied by LM
Cause, the adjusting to the inhibiting effect of these chemokine expressions by FN concentrations of nanoparticles in mice serum.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the scope of the claims of invention.
<110>the second college of education of Chongqing
<120>multiple medicine carries medicine targeted nano particle and its preparation method and application
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<170> PatentIn version 3.3
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Claims (10)
1. multiple medicine carries medicine targeted nano particle, which is characterized in that loaded by golden coated iron oxide core-shell nano (GNPs) more
Phenolic compound, iRhom2 siRNA and TNF-α inhibitor are prepared, the gold coated iron oxide core-shell nano, more
The molar ratio of phenolic compound, iRhom2 siRNA and TNF-α inhibitor is 1:1~3:1~3:1~3.
2. multiple medicine according to claim 1 carries medicine targeted nano particle, which is characterized in that the polyphenol compound is packet
Include one kind of fisetin, Quercetin, juglanin, mulberrin (phellinus ketone).
3. multiple medicine according to claim 1 carries medicine targeted nano particle, which is characterized in that the TNF-α inhibitor is next
That degree amine.
4. multiple medicine according to claim 1 carries medicine targeted nano particle, which is characterized in that the targeting iRhom2 siRNA
Nucleotide sequence as shown in SEQ ID NO.1.
5. the preparation method that the described in any item multiple medicines of Claims 1 to 4 carry medicine targeted nano particle, which is characterized in that described
Polyphenol compound is fisetin, comprising the following steps:
1) golden coated iron oxide core-shell nano (GNPs) is prepared；
2) Thiolation siRNA is dissolved in DEPC, is added to containing in mass percent 0.08%SDSGNPs, it is anti-at 4 DEG C
Answer 12~18h；
3) nano particle after isolating step 2) reaction, is scattered in DEPC water, removes not connected iRhom2 siRNA, obtain
The GNPs of iRhom2 siRNA functionalization；
4) the HS-PEG fisetin and lenalidomide that are connected to are added to the iRhom2 siRNA functionalization of step 3) preparation
In GNPs mixed liquor reaction 18~for 24 hours, after centrifugation and washing multiple medicine carry medicine targeted nano particle.
6. preparation method according to claim 5, which is characterized in that be ultrasonically treated when reaction by salinity tune in step 2)
To 0.25M~0.35M NaCl, to increase coverage of the siRNA on nano grain surface.
7. preparation method according to claim 5, which is characterized in that golden coated iron oxide core-shell nano in step 1)
The preparation method comprises the following steps:
1) the coated ferroferric oxide nano granules of citric acid are dispersed in water and are ultrasonically treated using ultrasonoscope, at ultrasound
Reason will be mixed with chlorogold solution, obtain mixed liquor；
2) trisodium citrate dehydrating solution is added in the mixed liquor in step 1), is thoroughly mixed to obtain suspension；
3) washed, collect and dry suspension in golden coated iron oxide core-shell nano.
8. preparation method according to claim 7, which is characterized in that the temperature being ultrasonically treated in step 1) is 70~85
DEG C, the processing time is 15~30min；It will be mixed with chlorogold solution under keeping warm mode after ultrasonic treatment, and obtain mixed liquor.
9. preparation method according to claim 7, which is characterized in that wherein ferroferric oxide nano granules and chlorauride are molten
The mass volume ratio of liquid is 1:0.5~3.
10. the described in any item multiple medicines of Claims 1 to 4, which carry medicine targeted nano particle, prevents and treats monocyte increasing in preparation
Application in the drug of related disease caused by the raw microbial septicemia of Listeria, which is characterized in that the related disease
Including acute liver failure caused by septicemia caused by Listeria Monocytogenes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113577102A (en) * | 2021-08-09 | 2021-11-02 | 西北大学 | Application of iron nanocluster in preparation of anti-septicemia and myocardial damage drug induced by same |
| CN113855698A (en) * | 2021-09-29 | 2021-12-31 | 黄钟 | Application of metallothionein-1 in preparation of medicine for treating bacterial infection |
| CN114224869A (en) * | 2021-12-21 | 2022-03-25 | 湖南省人民医院(湖南师范大学附属第一医院) | Drug-loaded nanoparticle for efficiently delivering siRNA (small interfering ribonucleic acid), and preparation method and application thereof |
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| CN102307470A (en) * | 2009-01-08 | 2012-01-04 | 西北大学 | Inhibition of bacterial protein production by polyvalent oligonucleotide modified nanoparticle conjugates |
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| CN102307470A (en) * | 2009-01-08 | 2012-01-04 | 西北大学 | Inhibition of bacterial protein production by polyvalent oligonucleotide modified nanoparticle conjugates |
Non-Patent Citations (1)
| Title |
|---|
| MIN-XUAN XU等: "Multicombination Approach Suppresses Listeria monocytogenes-Induced Septicemia-Associated Acute Hepatic Failure: The Role of iRhom2 Signaling", 《ADVANCED HEALTHCARE MATERIALS》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113577102A (en) * | 2021-08-09 | 2021-11-02 | 西北大学 | Application of iron nanocluster in preparation of anti-septicemia and myocardial damage drug induced by same |
| CN113577102B (en) * | 2021-08-09 | 2022-09-16 | 西北大学 | Application of iron nanocluster in preparation of anti-septicemia and myocardial damage drug induced by same |
| CN113855698A (en) * | 2021-09-29 | 2021-12-31 | 黄钟 | Application of metallothionein-1 in preparation of medicine for treating bacterial infection |
| CN113855698B (en) * | 2021-09-29 | 2023-04-18 | 黄钟 | Application of MT-siRNA in preparation of medicine for killing bacteria and inhibiting bacterial infection |
| CN114224869A (en) * | 2021-12-21 | 2022-03-25 | 湖南省人民医院(湖南师范大学附属第一医院) | Drug-loaded nanoparticle for efficiently delivering siRNA (small interfering ribonucleic acid), and preparation method and application thereof |
| CN114224869B (en) * | 2021-12-21 | 2023-06-23 | 湖南省人民医院(湖南师范大学附属第一医院) | Drug-loaded nanoparticle for efficiently delivering siRNA as well as preparation method and application thereof |
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