CN108578694A - The hollow gold nanometer ball of CpG oligodeoxynucleotides modification and preparation method, application - Google Patents
The hollow gold nanometer ball of CpG oligodeoxynucleotides modification and preparation method, application Download PDFInfo
- Publication number
- CN108578694A CN108578694A CN201810370104.XA CN201810370104A CN108578694A CN 108578694 A CN108578694 A CN 108578694A CN 201810370104 A CN201810370104 A CN 201810370104A CN 108578694 A CN108578694 A CN 108578694A
- Authority
- CN
- China
- Prior art keywords
- cpg
- nanometer ball
- hollow gold
- gold nanometer
- molecular weight
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Oncology (AREA)
- Inorganic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of preparation method of the hollow gold nanometer ball of CpG oligodeoxynucleotides modification, applications.Using sodium borohydride reduction cobalt chloride, cobalt nanometer particle is obtained, gold chloride is restored with cobalt nanometer particle, prepares hollow gold nanometer ball.CpG oligodeoxynucleotides, surfactant and buffer solution, shaken cultivation are added in hollow gold nanometer ball solution, obtains the hollow gold nanometer ball material of CpG oligodeoxynucleotides modification.The present invention is big using hollow gold nanometer ball load capacity, and by self assembly used load CpG, synthetic method is simple, is suitable for large-scale production application；The cellular uptake ability of CpG oligodeoxynucleotides can be enhanced, and biocompatibility is good；The hollow gold nanometer ball of CpG oligodeoxynucleotides modification has near-infrared surface plasma sink effect, primary tumo(u)r is eliminated using photo-thermal ablation, tumour antigen is generated in situ, under the promotion of CpG oligodeoxynucleotides, inducing systemic antineoplastic immune further treats the tumour of far-end transfer.
Description
Technical field
The present invention relates to cytosine-phosphate-guanine (CpG) oligodeoxynucleotide modification hollow gold nanometer ball material,
The invention further relates to the preparation method of the hollow gold nanometer ball material of this CpG oligodeoxynucleotides modification, this CpG widow's deoxidations
The hollow gold nanometer ball material of nucleotide modification can be applied to the photo-thermal therapy of cancer and the combination therapy of immunization therapy.
Background technology
In recent years, the preparation of nano material and the application in treatment of cancer attract wide attention.Gold nano-material
Since with good biocompatibility and hypotoxicity, surface is easily modified, and with diagnosis and photo-thermal property, is become cancer and controlled
Treat common nano material in research.Some applications of gold nano-material have been in clinical experimental stage.Gold nano-material
Including gold nanorods, gold nanometer cage, solid gold nanoparticle and hollow gold nanometer ball (HGNs) etc..HGNs is as a kind of novel
Gold nano-material, pattern is similar to sphere, and compare gold nanorods, has more regular form, more conducively transmits and through thin
After birth；And it is simpler than the preparation method of gold nanometer cage；With compared with solid gold nanoparticle with higher specific surface area, and
Surface plasmon absorption can be adjusted near infrared band, the Raman scattering (SERS) of surface enhanced be shown, in photo-thermal
It melts in (PTA) treatment, near infrared light can penetrate into tissue depth, have the side effect weakened for normal structure.
But the photo-thermal therapy of hollow gold nanometer ball mediation can only eliminate the local tumor of original site, cannot effectively control
Metastatic tumo(u)r processed.Ideal cancer therapy can not only eliminate primary tumo(u)r, but also can induce systemic anti-tumor and be immunized, treatment
The tumour of far-end transfer.Currently, most potential strategy is to combine photo-thermal therapy with immunization therapy.Tumour cell itself cannot
Effective induction immune response, for this purpose, we are presented carefully using the immunologic adjuvant that can be used for cancer immunotherapy come enhancement antigen
The intake and presentation of born of the same parents, to induce effective immune response.
The verified oligodeoxynucleotide (ODNs) containing non-methylated cytosine-phosphoric acid-guanine (CpG) motif is
The effective stimulus agent of innate immune system, flank are 5 ˊ purine and 3 ˊ pyrimidines, the strong cell immune response of potential initiation
And humoral immune reaction.These sequences combine Toll-like receptor 9 (TLR9) in antigen presenting cell (APC), to promote such as
Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), the expression of inflammatory cytokines such as IL-12 (IL-12) and
CD8+T cell response, and the inhibition cell (MDSC) of these sequence pair macrophages and derived from bone marrow plays an important roll.So
And the main channel of CpG ODNs stimulation cell effect is cellular uptake, but individually the water solubility and stability of ODNs compared with
Difference, it is difficult to through cell membrane, and easily removed by the nuclease in serum and cytoplasm, this greatly hinders the immune of ODNs
Using.It is reported that Thiolation CpG ODNs can carry out self assembly, the nanometer material of these functionalization on gold nanoparticle surface
Material is highly stable in physiological environment, nuclease-resistant degradation, can increase the delivery capability and enhancing CpG ODNs of CpG ODNs
Immunostimulation.
Therefore, we using HGNs as the carrier of CpG ODNs, Thiolation CpG ODNs pass through on the surface of HGNs
Au-S keys form self-assembled monolayer, and the hollow gold nanometer ball of this CpG modifications can play photo-thermal ablation and be immunized simultaneously
Stimulation realizes the combination therapy of photo-thermal therapy and immunization therapy.
Invention content
As in a first aspect, the first purpose of the invention is to provide the hollow gold nanometers of CpG oligodeoxynucleotides modification
Ball material.It can not only eliminate primary tumo(u)r, but also can induce systemic anti-tumor and be immunized, and treat the tumour of far-end transfer.It is empty
The surface plasmon absorption of heart gold nanosphere can be adjusted near infrared band, and near infrared light can penetrate into tissue depth
Place.The hollow gold nanometer ball of CpG oligodeoxynucleotides modification is detained (EPR) effect arrival tumor tissues by enhancing to permeate, and uses
The photo-thermal effect of near infrared light tumor locus, hollow gold nanometer ball melts primary tumo(u)r, and it is anti-to generate tumour correlation in situ
It is former.Under the double action of tumor associated antigen and CpG oligodeoxynucleotides, activation dendritic cells (DC) are to increase to tumour spy
Specific T cell stimulates to promote the recession of metastatic tumour.
Second object of the present invention is to provide the system of the hollow gold nanometer ball material of above-mentioned CpG oligodeoxynucleotides modification
Preparation Method.
The hollow gold nanometer ball material of CpG oligodeoxynucleotides of the present invention modification is that inventor passes through pair through a large number of experiments
It is summed up than screening.Sodium borohydride reduction cobalt chloride is used first, is obtained cobalt nanometer particle, is then restored with cobalt nanometer particle
Gold chloride prepares hollow gold nanometer ball.Then CpG oligodeoxynucleotides, surface-active are added in hollow gold nanometer ball solution
Agent and buffer solution, shaken cultivation obtain the hollow gold nanometer ball material of CpG oligodeoxynucleotides modification.
In the above-mentioned technical solutions, the raw material components is a concentration of：Hollow gold nanometer ball 5.0 × 10-9~0.1mol/L,
CpG oligodeoxynucleotides 1.0 × 10-8~1.0mol/L, surfactant 1.0 × 10-5~1.0mol/L, buffer solution (pH 6.0
~8.5) 5.0 × 10-3~1.0mol/L.
As the second aspect, in the above-mentioned technical solutions, the specific preparation method of the hollow gold nanometer ball solution
For：
Step 101, by 20~1000mL water and 1.0 × 10-2~2.0mol/L citrate solutions and 5.0 × 10-2~
10.0mol/L cobalt chloride solutions are mixed with mixed liquor；Above-mentioned mixed liquor is subjected to deoxidation, at least 1 minute in argon gas, and to
The 1.0 × 10 of preparation are rapidly joined in above-mentioned mixed liquor-2~6.0mol/L sodium borohydrides, while persistently being vibrated；
Step 102, oscillating reactions 1~injection 1.0 × 10 after sixty minutes is waited for-3~2.0mol/L citric acid solutions, continue into
Row oscillation；
Step 103, after oscillating reactions at least carries out 1 minute, cobalt liquor is moved to and carries out the 1.0 × 10 of deoxidation operation-4~
In 1.0mol/L chlorauric acid solutions, continuation is vibrated at least 1 minute in argon gas stream；
Step 104, stop deoxidation, hollow gold nanometer ball material is obtained at least 1 minute at least centrifugal force of 1000g
Material, and the hollow gold nanometer ball solution is made.
As third aspect, in the above-mentioned technical solutions, the hollow gold nanometer of CpG oligodeoxynucleotides modification
The specific preparation method of ball, includes the following steps：
Step 201, by 1.0 × 10-8~1.0mol/L CpG oligodeoxynucleotides are added to 5.0 × 10-9~0.1mol/L
In hollow gold nanometer ball solution, mixture is continued into slight oscillatory 1 hour or more in certain temperature (0~30 DEG C)；
Step 202,1.0 × 10 will be added in mixture-5~1.0mol/L surfactants and 5.0 × 10-3~
1.0mol/L buffer solutions (pH 6.0~8.5), further constant temperature (0~30 DEG C) shaken cultivation 1 hour or more；
Step 203, mixture is at least centrifugal force of 1000g, supernatant decantation and with 5.0 × 10-3~1.0mol/
L buffer solutions (pH 6.0~8.5) are rinsed, and repeatedly remove unbonded CpG oligodeoxynucleotides, and it is de- to obtain CpG widow
The hollow gold nanometer ball of oxygen nucleotide modification.
In above-mentioned technical proposal, the cobalt chloride solution for one or more of following chlorination cobalt compounds prepare and
At：1. cobalt chloride hexahydrate (molecular weight 237.93)；2. waterless cobaltous chloride (molecular weight 129.84).
In above-mentioned technical proposal, the chlorauric acid solution for one or more of following gold chloride compounds prepare and
At：1. gold chloride tetrahydrate (molecular weight 411.85)；2. gold chloride trihydrate (molecular weight 393.85)；3. gold chloride
Sodium dihydrate (molecular weight 397.8).
In above-mentioned technical proposal, the citric acid solution for one or more of following citrate compounds prepare and
At：1. citric acid monohydrate closes object (molecular weight 210.14)；2. anhydrous citric acid (molecular weight 192.13)；3. two water of citric acid
Close object (molecular weight 228.14).
In above-mentioned technical proposal, the citrate solution for one or more of following citrates prepare and
At：1. citrate trisodium dihydrate (molecular weight 294.1)；2. citric acid tri potassium monohydrate (molecular weight 324.41)；
3. Triammonium citrate (molecular weight 243.22)；4. calcium citrate tetrahydrate (molecular weight 570.49)；5. anhydrous citric acid sodium
(molecular weight 258.07)；6. ironic citrate (molecular weight 244.94)；7. ten tetrahydrate of magnesium citrate (molecular weight 703.4)；
8. magnesium citrate (molecular weight 451.11)；9. copper citrate (molecular weight 360.22)；10. zinc citrate (molecular weight 574.37).
In above-mentioned technical proposal, the CpG oligodeoxynucleotides are following Thiolation CpG oligodeoxynucleotide sequences
One or more of：
(1)CpG 1826(5′-TCCATGACGTTCCTGACGTT-SH-3′)；
(2)CpG-T20(5′-TCCATGACGTTCCTGACGT T-T20-SH-3′)；
(3)bi-CpG(5′-TCCATG ACGTTCC TGAC GTTTC CATGACGTTCCTGACGTT-SH-3′)；
(4)CpG T8(5′-TCGTCGTC GTCGTCGTCGTCGTCG-SH-3′)；
(5)CpG 1(5′-ACGCGTCGTCGTCGTCGTCGTCGT-SH-3′)；
(6)CpG 2(5′-TCGTCGATCGATCGACGAGGGG GG–SH-3′)；
(7)CpG 3(5′-TGTGCATCGATGCAGGGGGG-SH-3′)；
(8)CpG 4(5′-TC GACAACGTTAACGTCG TT-SH-3′)；
(9)CpG 5(5′-ACGACGACGACGACGACGTT-SH-3′)；
(10)CpG 6(5′-GACGTTCGAACGTAGA CGTT-SH-3′)；
(11)CpG 7(5′-ACGTCGACGTCGACGTCGACGTCG-SH-3′)；
(12)CpG 8(5′-TCGTCGTCGTCGACGACGACGACG-SH-3′)；
(13)CpG 9(5′-TCGCGATCGTCGTCGTCGACGCGT-SH-3′)；
(14)CpG 10(5′-TTTTCATCGATGCAGCGCGC-SH-3′)；
(15)CpG 11(5′-TCCTCCGTCGTTTTGTCGTT-SH-3′)；
(16)CpG 12(5′-GTCGTCGTCGTCGTCGTCCGCGCG-SH-3′)；
(17)CpG 13(5′-TCGGTCGTTTTGTCGTTGCGCGC-SH-3′)；
(18)CpG 14(5′-TCGGTCGTTGTCGTCGCGCGC-SH-3′)；
(19)CpG 15(5′-GGTGCATCGATGCAGGGGGG-SH-3′)；
(20)CpG 16(5′-TCGTCGTTTTGTCGTTTTGTCGTT-SH-3′)；
(21)CpG 17(5′-TGCTGCTTTTGTGCTTTTGTGCTT-SH-3′)；
(22)CpG 1911(5′-TCCAGGACTTTCCTCAGGT-SH-3′)；
(23)CpG 2216(5′-GGGGGACGATCGT CGGGGGG-SH-3′)；
(24)CpG M362(5′-TCGTCGTCGTTCGAACGACGTTGAT-SH-3′)；
(25)CpGM383(5′-TGCTGC TGCTTGCAAG CAGCTTGAT-SH-3′)；
(26)CpG 2006(5′-tcgtcgttttgtcgttttgtcgtt-SH-3′)；
(27)CpG 1585(5′-ggGGTCAACGTTGAgggggG-SH-3′)；
(28)CpG 2118(5′-ggGGTCAAGCTT GAg ggggG-SH-3′)；
(29)CpG 2197(5′-gmGGTCAACGTTGAgggmggG-SH-3′)；
(30)CpG 2198(5′-ggGGAGTTCGTTGAgggggG-SH-3′)；
(31)CpG 2204(5′-ggGGTCATCGATGAgggggG-SH-3′)；
(32)CpG 2216(5′-ggGGGACGATCGTCgggggG-SH-3′)；
(33)CpG 2243(5′-ggGGGAGCAT GCTCgggggG-SH-3′)；
(34)CpG 2217(5′-ggGGGTCGTACGACgggggG-SH-3′)。
In above-mentioned technical proposal, the surfactant is one or more of following surfactant compounds system
It is standby to form：1. lauryl sodium sulfate (molecular weight 288.38)；2. alkyl polyglycoside (molecular weight 320.22).
In the above-mentioned technical solutions, the buffer solution (pH 6.0~8.5) is sodium chloride, potassium chloride, citric acid, phosphoric acid hydrogen
One or more at room temperature uniform mixed in disodium, potassium dihydrogen phosphate, trishydroxymethylaminomethane, hydrochloric acid, sodium hydroxide
Close object.
The preparation method of the hollow gold nanometer ball material of CpG oligodeoxynucleotides modification, further includes following steps：With boron hydrogen
Change sodium reduction cobalt ions, obtain cobalt nanometer particle, gold chloride is then restored with cobalt nanometer particle, prepares hollow gold nanometer ball.So
CpG oligodeoxynucleotides, surfactant and buffer solution, shaken cultivation are added in hollow gold nanometer ball solution afterwards to obtain
The hollow gold nanometer ball material of CpG oligodeoxynucleotides modification.
Advantageous effect：
The hollow gold nanometer ball material of CpG oligodeoxynucleotides modification of the present invention has following features：1. this CpG functionalization
Nano material have high load amount oligodeoxynucleotide, and with hypotoxicity, good biocompatibility；2. CpG widow is de-
The hollow gold nanometer ball material of oxygen nucleotide modification has near-infrared plasmon absorption effect, can play photo-thermal therapy
Effect；3. the hollow gold nanometer ball material of CpG oligodeoxynucleotides modification can be swollen by enhancing infiltration delay (EPR) effect arrival
There is tumor tissue enhancing CpG oligodeoxynucleotides to absorb ability；4. the hollow gold nanometer ball material of CpG oligodeoxynucleotides modification
The near infrared light fuel factor of material can melt primary tumo(u)r, generate tumor associated antigen in situ, in CpG oligodeoxynucleotides
Under promotion, activation dendritic cells (DC) promote the recession of metastatic tumour to increase the stimulation to tumor specific T cells.
Description of the drawings
Fig. 1 is the hollow gold nanometer ball of cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODNs) modification
(HGNs) synthesis flow schematic diagram.
Fig. 2 is the transmission electron microscope picture of the hollow gold nanometer ball (CpG-HGNs) of CpG oligodeoxynucleotides modification.
Fig. 3 be CpG ODNs and CpG oligodeoxynucleotides modification hollow gold nanometer ball (CpG-HGNs) respectively with
Raw264.7 cells co-culture 24 hours after cytotoxicity experiment result.
The temperature variation that Fig. 4 is the solution containing HGNs in 808nm laser illuminations 10 minutes.
Fig. 5 is after the CpG ODNs of CpG-HGNs and the Cy5 label of Cy5 labels are co-cultured with Raw264.7 cells respectively
Confocal laser scanning microscope, CLSM figure；Wherein (a) is the Raw264.7 nuclear targeting fluorograms cultivated with CpG-HGNs, (b)
It is (c) CpG- of Raw264.7 nuclear targetings fluorogram and Cy5 labels for the CpG-HGNs fluorescent staining figures of Cy5 labels
The overlapping figure of HGNs fluorescent staining figures is (d) the Raw264.7 nuclear targeting fluorograms of CpG ODNs cultures, is (b) that Cy5 is marked
The CpG ODNs fluorescent staining figures of note (c) dye for Raw264.7 nuclear targetings fluorogram with the CpG ODNs that Cy5 is marked glimmering
The overlapping figure of light figure.
Fig. 6 is the sky that myeloid dendritic cell (mDCs) is modified with 4T1 breast tumor cells, HGNs, CpG oligodeoxynucleotide
The residue that heart gold nanosphere (CpG-HGNs) near-infrared photo-thermal (Laser) melts 4T1 breast tumor cells co-cultures 12 hours
Mature condition figure afterwards.
Specific implementation mode
Below with reference to the embodiments and with reference to the accompanying drawing the technical solutions of the present invention will be further described.Obviously,
Described embodiment is only the section Example of the present invention.
In order to further appreciate that the present invention, embodiment of the present invention is described with reference to embodiment, but is answered
Work as understanding, these descriptions are only the feature and advantage further illustrated the present invention rather than to the limit of patent requirements of the present invention
System.
TEM is tested：
All raw materials of this experiment, are not particularly limited its source, buying on the market or according to people in the art
It is prepared by conventional method known to member.In order to intuitively observe the hollow gold nanometer ball of CpG oligodeoxynucleotides modification
Pattern and size, we select the functionalized nano material that an example obtains as observation object.CpG oligodeoxynucleotides are modified
Hollow gold nanometer ball sample distilled water 1% phosphotungstic acid pretreatment, then sample is placed on grid, is done at room temperature
It is dry, and be imaged by TEM (JEM-2100 (HR), 200KV).
The cytotoxicity experiment of the nano material of functionalization
The hollow gold nanometer ball (CpG-HGNs) that the modification of CpG oligodeoxynucleotides is measured by mtt assay is thin to RAW 264.7
The vitro cytotoxicity of born of the same parents.By the cell dilution in 100 μ L DMEM culture mediums, then directly it is seeded in the hole of 96 orifice plates, 37
DEG C be incubated 24 hours.Then waste liquid is sucked out, contains the fresh culture of particular agent with 100 μ L, wherein containing various concentration
The hollow gold nanometer ball (2.5 × 10 of CpG oligodeoxynucleotides modification-6μmol/mL,5.0×10-6μm ol/mL) and 5.0 ×
10-6The free CpG of μm ol/mL.Cell and specific reagent are incubated 24 hours altogether at 37 DEG C.Then culture medium is discarded to be used in combination
100 μ L fresh cultures replace, and 20 μ L MTT are added in every hole, are then incubated 4 hours at 37 DEG C.Hereafter, it carefully removes
Supernatant is removed, and 200 μ L DMSO are added in every hole.Solution is measured at 570nm using microplate reader (Bio-Rad 550)
Absorbance is to determine OD values.Cell activity calculates as follows：
Cell activity=ODAdd material/ODControl× 100%
Wherein ODAdd materialIndicate the OD values obtained by the processed cell of particular agent, ODControlIt indicates without passing through any place
The OD values that the cell of reason obtains.
The photo-thermal of hollow gold nanometer ball is tested
Detect the photo-thermal effect of hollow gold nanometer ball.By 808nm laser with 3Wcm-2Power density irradiate hollow Jenner
The suspension of rice ball.Laser microprobe is placed on to induce release behavior on the test tube containing HGNs solution, with the temperature of suspension
Spend the function as irradiation time.With the preparation of laser irradiation predetermined time interval, irradiation suspension 2,4,6,8,10 minutes, note
Record variation of the sample temperature with irradiation time.Wherein as a control group with water sample.
The cellular uptake of the nano material of functionalization is tested：
The hollow gold nanometer of the CpG oligodeoxynucleotides modification of Cy5 labels is observed by confocal laser scanning microscope, CLSM
The cellular uptake situation of ball (CpG-HGNs).264.7 cell inoculations of RAW in the 1mL culture mediums containing 10%FBS are in glass
In the culture dish of glass bottom, 37 DEG C are incubated 24 hours.Then culture medium is removed, PBS buffer solutions washing cell is used in combination 3 times, is added
1mL contains particular agent (the hollow gold nanometer ball of the CpG oligodeoxynucleotides modification of CpG ODNs and the Cy5 labels of Cy5 labels
(Cy5-CpG-HGNs) fresh culture (10%FBS).After being co-cultured 4 hours at 37 DEG C, carefully washed with PBS buffer solutions
Cell is washed repeatedly to remove not by the reagent of cellular uptake, is incubated 12 minutes.Then cell is washed three times with PBS buffer solutions,
It is fixed, is incubated 13 minutes with 3% paraformaldehyde again.Nucleus uses 3 μ g/mL Hoechst, 33342 (Molecular later
Probes it) dyes, is finally washed with PBS buffer solutions and remove extra dyestuff three times.Pass through confocal laser scanning microscope, CLSM
(Nikon Ni-E C2+) observes cell under amplifying at 400 times.
External dendritic cells stimulation test：
The hollow gold nanometer ball that will be modified through HGNs or CpG oligodeoxynucleotides using transwell co-culture systems
(CpG-HGNs) residue after photo-thermal ablation 4T1 breast tumor cells, which is added in mDCs, cultivates 12 hours.MDCs anti-
CD11c FITC, anti-CD86 PE and anti-CD80 APC dyeing, then pass through flow cytometer (BD FACSCalibur)
Sorting.
Experimental result:
1) TEM is tested
Pass through the pattern and size of the hollow gold nanometer ball that the CpG oligodeoxynucleotides of transmission electron microscope observation are modified
As a result see Fig. 2.
It can be clearly seen that the hollow and thin-wall construction for the hollow gold nanometer ball that CpG oligodeoxynucleotides are modified from Fig. 2,
Core size is smaller, and golden shell completely exists, this illustrates that the CpG of self assembly does not influence the pattern of hollow gold nanometer ball, and functionalization
Nano material can individually be stabilized.And it is observed from fig. 1 that hollow gold nanometer ball periphery combines one layer of few deoxidation
Nucleotide further proves that CpG oligodeoxynucleotides are successfully incorporated in hollow gold nanometer ball surface.
The cytotoxicity analysis of the nano material of functionalization
Toxicity data of the hollow gold nanometer ball (CpG-HGNs) of CpG oligodeoxynucleotides modification to 264.7 cells of Raw
See Fig. 3.
From figure 3, it can be seen that 264.7 cells of Raw pass through the hollow of the CpG oligodeoxynucleotides modification of various concentration
Cell survival rate is 80% or more after gold nanosphere processing.This illustrates the hollow gold nanometer ball of CpG oligodeoxynucleotides modification
There are lower toxicity, good biocompatibility.
2) photo-thermal effect of hollow gold nanometer ball
The photo-thermal effect result of hollow gold nanometer ball is shown in Fig. 4.
As can be seen from Figure 4 near-infrared laser irradiation causes the raising of HGNs bulk solution temperature, the temperature at 10 minutes
Degree has increased 18 DEG C, is observed according to Fig. 4, higher in preceding 8 minutes temperature increase rates, and subsequent temperature increases slower.
And water sample temperature under near-infrared laser irradiation is almost unchanged.These are statistics indicate that hollow gold nanometer ball is a kind of effective photo-thermal
Mediator can utilize the photo-thermal effect of hollow gold nanometer ball to kill swollen when tumor locus carries out local near infrared light
Oncocyte.
3) cellular uptake
After the hollow gold nanometer ball and CpG ODNs of CpG oligodeoxynucleotides modification are co-cultured with Raw264.7 cells respectively
Cellular uptake result see Fig. 5, wherein (a) be Cy5 label CpG-HGNs culture Raw264.7 cells nucleus warp
Hoechst is dyed, and actual effect is rendered as blue；(b) it is the CpG-HGNs of Cy5 labels, actual effect is rendered as red；(c)
For the superposition of (a) and (b) the two figure layers；(d) it is the nucleus warp of the Raw264.7 cells of the CpG ODNs cultures of Cy5 labels
Hoechst is dyed, and actual effect is rendered as blue；(e) it is the CpG ODNs of Cy5 labels, actual effect is rendered as red；(f)
For the superposition of (d) and (e) the two figure layers.
(a), (b), (c) three figures in the cell of the CpG-HGNs cultures of Cy5 labels it is found that have accumulated red in Figure 5
Fluorescence, these red fluorescence dispersion ratios are more uniform, illustrate that the CpG-HGNs of Cy5 labels has been entered into the cell.And combine (d),
(e), (f) three figures in the Raw264.7 cells of the CpG ODNs cultures of Cy5 labels it is found that do not detect apparent red glimmering
Light, only seldom red fluorescence are difficult penetration cell film this demonstrates individual CpG ODNs in cell periphery.This shows
HGNs is a kind of effective carrier, and CpG ODNs can effectively be transported to RAW 264.7 into the cell, it is de- to enhance CpG widow
The cellular uptake ability of oxygen nucleotide, and then improve the immunostimulation of CpG ODNs.
4) external dendritic cells stimulation test：
The result of external dendritic cells stimulation test is shown in Fig. 6.
From fig. 6 it can be seen that the hollow gold nanometer ball (CpG-HGNs) that the right is modified with CpG oligodeoxynucleotides carries out
After the photo-thermal ablation of near infrared light (Laser) induction, the residue of 4T1 breast tumor cells can significantly increase mDCs maturity,
Carrying out the residue of the photo-thermal ablation 4T1 cells of near infrared light induction by HGNs far above centre enhances the water of mDCs maturity
It is flat.
Embodiment 1:
The hollow gold nanometer ball (surface plasmon absorption wavelength is 746nm) of CpG oligodeoxynucleotides modification
Preparation method：
1.0 × 10 are added in the neck round bottom flask containing 20~1000mL water-2~2.0mol/L anhydrous citric acid sodium
Solution and 5.0 × 10-2~10.0mol/L waterless cobaltous chloride solution.By this mixed liquor in argon gas deoxidation 20~30 minutes.To this
Freshly prepared 1.0 × 10 are rapidly joined in solution-2~6.0mol/L sodium borohydride solutions are vibrated when being added.Wait for reaction 3~
1.0 × 10 are injected after five minutes-3~2.0mol/L citric acid monohydrate solution, persistent oscillation allow sodium borohydride to be fully hydrolyzed.10
After~45 minutes, takes 20~40mL cobalt liquors to move to rapidly in the graduated cylinder of argon gas purging, be then quickly poured into just in deoxidation
1.0 × 10-4In three aqueous solution of~1.0mol/L gold chlorides, kept for 50~60 minutes after persistent oscillation and in argon gas stream.So
After stop deoxidation, expose the solution in air, aoxidize 45~60 minutes.Finally with 30~50 points of the centrifugal force of 4000g
Clock removes additional substance, and then deposit is resuspended in ultra-pure water, you can obtains hollow gold goal solution.
Chlorauric acid solution is added in the present invention, following chemical reaction is induced, referring to formula (I)
3Co+2AuCl4-→2Au+3Co2++8Cl-(I)。
HGNs is concentrated into 5.0 × 10 by centrifuging-9~0.1mol/L.Then, 1.0 × 10-8~1.0mol/L is Thiolation
CpG oligodeoxynucleotides are added in HGNs.Mixture is incubated to simultaneously slight oscillatory 8~10 under certain temperature (26~29 DEG C)
Hour, and it is adjusted to 1.0 × 10-5~1.0mol/L lauryl sodium sulfate and 5.0 × 10-3~1.0mol/L PBS bufferings are molten
Liquid (pH 6.0~8.5).Then by the way that NaCl is added dropwise, NaCl concentration in solution is made to be slowly increased to 5.0 in 3~5 hours
×10-2~1.0mol/L.By the further constant temperature of final mixture (26~29 DEG C) shaken cultivation 25~30 hours, with 3000~
The centrifugal force of 5000g, is used in combination 5.0 × 10-3~1.0mol/L PBS buffer solution (pH 6.0~8.5) is rinsed, repeatedly
Centrifugation-rinse cycle finally obtains Thiolation CpG widow's deoxyribonucleoside to remove unbonded Thiolation CpG oligodeoxynucleotides
The hollow gold nanometer ball of acid modification.
It is measured by laser particle potentiometer and ultraviolet-visible spectrometer, the hollow gold of this CpG oligodeoxynucleotide modification
The average grain diameter of nanosphere is about 82nm, and average surface potential is -19.2 ± 0.5mV, surface plasmon resonance absorption wavelength
For 746nm.
Embodiment 2:
The hollow gold nanometer ball (surface plasmon absorption wavelength is 961nm) of CpG oligodeoxynucleotides modification
Preparation method：
1.0 × 10 are added in the neck round bottom flask containing 20~1000mL ultra-pure waters-2~2.0mol/L citric acids
Sodium two aqueous solution and 5.0 × 10-2Six aqueous solution of~10.0mol/L cobalt chlorides.By this mixed liquor in argon gas deoxidation 40~
50 minutes.Freshly prepared 1.0 × 10 are rapidly joined into the solution-2~6.0mol/L sodium borohydride solutions shake when being added
It swings.1.0 × 10 are injected after reaction 5~7 minutes-3~2.0mol/L citric acid two aqueous solutions, persistent oscillation allow sodium borohydride
It is fully hydrolyzed.5~after ten minutes, it takes 10~30mL cobalt liquors to move to rapidly in the graduated cylinder of argon gas purging, then quickly falls it
Enter just the 1.0 × 10 of deoxidation-4~1.0 × 10-2In three aqueous solution of mol/L gold chlorides, protected after persistent oscillation and in argon gas stream
It holds 60~80 minutes.Then stop deoxidation, expose the solution in air, aoxidize 30~50 minutes.Finally with the centrifugation of 7000g
Power centrifuges 20~40 minutes and removes additional substance, then deposit is resuspended in ultra-pure water, you can it is molten to obtain hollow gold goal
Liquid.
HGNs is concentrated into 5.0 × 10 by centrifuging-8~0.1mol/L.Then, 1.0 × 10-8The mercaptan of~1.0mol/L
Change CpG oligodeoxynucleotides to be added in HGNs.Mixture is incubated to simultaneously slight oscillatory 10 under certain temperature (26~29 DEG C)
~20 hours, and it is adjusted to 1.0 × 10-5~1.0mol/L lauryl sodium sulfate and 5.0 × 10-3~1.0mol/L PBS are slow
Rush solution (pH 6.0~8.5).Then by the way that NaCl is added dropwise, NaCl concentration in solution is made to be slowly increased in 1~3 hour
5.0×10-2~1.0mol/L.By the further constant temperature of final mixture (26~29 DEG C) shaken cultivation 25~30 hours, with 7000
The centrifugal force of~9000g and with 5.0 × 10-3~1.0mol/L PBS buffer solution (pH 6.0~8.5) is rinsed, repeatedly
Centrifugation-rinse cycle finally obtains the hollow of CpG oligodeoxynucleotides modification to remove unbonded CpG oligodeoxynucleotides
Gold nanosphere.
It is measured by laser particle potentiometer and ultraviolet-visible spectrometer, the hollow gold of this CpG oligodeoxynucleotide modification
The average grain diameter of nanosphere is about 54nm, and average surface potential is -14.6 ± 0.8mV, surface plasmon resonance absorption wavelength
For 961nm.
Embodiment 3:
The hollow gold nanometer ball (surface plasmon absorption wavelength is 806nm) of CpG oligodeoxynucleotides modification
Preparation method：
1.0 × 10 are added in the neck round bottom flask containing 20~1000mL ultra-pure waters-3~1.0mol/L citric acids
Sodium two aqueous solution and 5.0 × 10-1~5.0mol/L waterless cobaltous chlorides.By this mixed liquor in argon gas deoxidation 50~80 minutes.
Freshly prepared 1.0 × 10 are rapidly joined into the solution-2~6.0mol/L sodium borohydride solutions are vibrated when being added.It waits for anti-
Answer 5~injection 1.0 × 10 after ten minutes-3~2.0mol/L citric acid monohydrate solution, persistent oscillation allow the abundant water of sodium borohydride
Solution.30~after sixty minutes, it takes 20~40mL cobalt liquors to move to rapidly in the graduated cylinder of argon gas purging, is then quickly poured into
The 1.0 × 10 of deoxidation-4In four aqueous solution of~0.1mol/L gold chlorides, after persistent oscillation and 50~60 points of the holding in argon gas stream
Clock.Then stop deoxidation, expose the solution in air, aoxidize 45~80 minutes.Finally with the centrifugal force of 6000g 30~
It removes additional substance within 50 minutes, then deposit is resuspended in ultra-pure water, you can obtain hollow gold goal solution.
HGNs is concentrated into 5.0 × 10 by centrifuging-9~0.1mol/L.Then, 1.0 × 10-8~1.0mol/L is Thiolation
CpG oligodeoxynucleotides are added in HGNs.Mixture is incubated to simultaneously slight oscillatory 5~20 under certain temperature (26~29 DEG C)
Hour, and it is adjusted to 1.0 × 10-5~1.0mol/L lauryl sodium sulfate and 5.0 × 10-3~1.0mol/L PBS bufferings are molten
Liquid (pH 6.0~8.5).Then by the way that NaCl is added dropwise, NaCl concentration in solution is made to be slowly increased to 5.0 in 1~3 hour
×10-2~1.0mol/L.By the further constant temperature of final mixture (26~29 DEG C) shaken cultivation 25~30 hours, with 3000~
The centrifugal force of 5000g, is used in combination 5.0 × 10-3~1.0mol/L PBS buffer solution (pH 6.0~8.5) is rinsed, repeatedly
Centrifugation-rinse cycle finally obtains the hollow of CpG oligodeoxynucleotides modification to remove unbonded CpG oligodeoxynucleotides
Gold nanosphere.
It is measured by laser particle potentiometer and ultraviolet-visible spectrometer, the hollow gold of this CpG oligodeoxynucleotide modification
The average grain diameter of nanosphere is about 65nm, and average surface potential is -15.4 ± 0.6mV, surface plasmon resonance absorption wavelength
For 806nm.
The present invention also provides the hollow Jenners of the CpG oligodeoxynucleotides modification described in above-mentioned technical proposal any one
The hollow gold nanometer ball material of CpG oligodeoxynucleotides modification prepared by rice ball material or above-mentioned technical proposal any one exists
The application of oncotherapy.
The specific aspect of the application is not particularly limited in the present invention, similar with routine well known to those skilled in the art
Using those skilled in the art can be adjusted and select, this hair according to actual conditions, product requirement and function and usage
The bright application includes oncotherapy, is specifically as follows the immunostimulation for enhancing CpG oligodeoxynucleotides, sends out simultaneously
Wave the photo-thermal effect of hollow gold nanometer ball.The present invention passes through self assembly used load CpG, synthetic method using hollow gold nanometer ball
Simply, it is suitable for large-scale production application；Hollow gold nanometer ball load capacity is big, can enhance the cell of CpG oligodeoxynucleotides
Intake ability, and biocompatibility is good；CpG oligodeoxynucleotides modification hollow gold nanometer ball have near-infrared surface etc. from
Daughter sink effect can utilize photo-thermal ablation to eliminate primary tumo(u)r；Meanwhile the modification of CpG oligodeoxynucleotides is hollow
Gold nanosphere can enhance the immunostimulatory activity of CpG oligodeoxynucleotides, and Immunotherapy is played while photo-thermal therapy,
Inducing systemic antineoplastic immune further treats the tumour of far-end transfer.
Above to the preparation of the hollow gold nanometer ball of CpG oligodeoxynucleotides provided by the invention modification, using progress
Detailed introduction, principle and implementation of the present invention are described for specific case used herein, above example
Explanation be merely used to help understand the method and its core concept of the present invention, including best mode, and but also this field
Any technical staff can put into practice the present invention, including manufacture and use any device or system, and implement any combination
Method.It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, also
Can be with several improvements and modifications are made to the present invention, these improvement and modification also fall into the protection domain of the claims in the present invention
It is interior.The range of patent protection of the present invention is defined by the claims, and may include those skilled in the art it is conceivable that its
His embodiment.If these other embodiments, which have, is not different from the structural element of claim character express, or if
They include with equivalent structural elements of the character express of claim without essence difference, then these other embodiments should also wrap
Containing within the scope of the claims.
Claims (12)
1. a kind of hollow gold nanometer ball of CpG oligodeoxynucleotides modification, which is characterized in that the CpG oligodeoxynucleotides are repaiied
The hollow gold nanometer ball of decorations is by including but not limited to hollow gold nanometer ball solution, CpG oligodeoxynucleotides, surfactant, delaying
Obtained by mixed liquor shaken cultivation prepared by fliud flushing.
2. a kind of hollow gold nanometer ball of CpG oligodeoxynucleotides modification according to claim 1, it is characterised in that the sky
Heart gold nanosphere solution concentration is 5.0 × 10-9~0.1mol/L, CpG oligodeoxynucleotide a concentration of 1.0 × 10-8~
1.0mol/L, surfactant concentration are 1.0 × 10-5~1.0mol/L, buffer concentration are 5.0 × 10-3~1.0mol/L；
The pH value range of the wherein described buffer solution is 6.0~8.5.
3. a kind of hollow gold nanometer ball of CpG oligodeoxynucleotides modification according to claim 1 or claim 2, it is characterised in that institute
The preparation method step for the hollow gold nanometer ball solution stated is：
Step 101, by 20~1000mL water and 1.0 × 10-2~2.0mol/L citrate solutions and 0.05~10.0mol/L chlorine
Change cobalt liquor and is mixed with mixed liquor；Above-mentioned mixed liquor carries out in argon gas to no less than 1 minute deoxygenation, and to above-mentioned
A concentration of 1.0 × 10 are added in mixed liquor-2The sodium borohydride of~6.0mol/L, while persistently being vibrated；
Step 102, wait for that the implantation concentration of oscillating reactions 1~after sixty minutes is 1.0 × 10-3The citric acid solution of~2.0mol/L, holds
It is continuous to be vibrated；
Step 103, after oscillating reactions at least carries out 1 minute, the cobalt chloride solution is moved to and carries out a concentration of of deoxidation operation
1.0×10-4In the chlorauric acid solution of~1.0mol/L, continue to carry out oscillation in no less than 1 minute in argon gas stream；
Step 104, stop deoxidation, the centrifugal force to be no less than 1000g carries out no less than 1 minute centrifugally operated, obtains hollow
The hollow gold nanometer ball solution is made in gold nanosphere material.
4. a kind of hollow gold nanometer ball of CpG oligodeoxynucleotides modification according to claim 3, it is characterised in that described
The preparation method step of hollow gold nanometer ball of CpG oligodeoxynucleotides modification is：
Step 201, by a concentration of 1.0 × 10-8The oligodeoxynucleotide of~1.0mol/L CpG is added to a concentration of 5.0 × 10-9
It is mixed in the hollow gold nanometer ball solution of~0.1mol/L, the mixture is continued slightly to shake in 0~30 DEG C of temperature range
It swings 1 hour or more；
Step 202, a concentration of 1.0 × 10 are added in the mixture-5The surfactant of~1.0mol/L and a concentration of 5.0
×10-3The pH range of the buffer solution of~1.0mol/L, the buffer solution is 6.0~8.5, permanent in 0~30 DEG C of temperature range
Warm shaken cultivation 1 hour or more；
Step 203, mixture carries out centrifugally operated to be no less than 1000g centrifugal force, with a concentration of 5.0 × 10-3~1.0mol/L
And the buffer solution that pH range is 6.0~8.5 repeatedly rinses the sediment of centrifugally operated, removes unbonded CpG widow's deoxidation core
Thuja acid obtains the hollow gold nanometer ball of CpG oligodeoxynucleotides modification.
5. a kind of hollow gold nanometer ball of CpG oligodeoxynucleotides modification according to claim 3, it is characterised in that described
Cobalt chloride solution is cobalt chloride hexahydrate, the one or more of waterless cobaltous chloride are prepared；Six water of the cobalt chloride
The molecular weight for closing object is 237.93；The molecular weight of the waterless cobaltous chloride is 129.84.
6. a kind of hollow gold nanometer ball of CpG oligodeoxynucleotides modification according to claim 3, it is characterised in that described
Chlorauric acid solution is gold chloride tetrahydrate, gold chloride trihydrate, one kind in sodium chloraurate dihydrate or several
Kind is prepared；The molecular weight of the gold chloride tetrahydrate is 411.85；The molecular weight of the gold chloride trihydrate
It is 393.85；The molecular weight of the sodium chloraurate dihydrate is 397.8.
7. a kind of hollow gold nanometer ball of CpG oligodeoxynucleotides modification according to claim 3, it is characterised in that described
Citric acid solution for citric acid monohydrate close object, anhydrous citric acid, one or more of citric acid dihydrate prepare and
At；The molecular weight that the citric acid monohydrate closes object is 210.14；The molecular weight of the anhydrous citric acid is 192.13；The lemon
The molecular weight of lemon acid dihydrate is 228.14.
8. a kind of hollow gold nanometer ball of CpG oligodeoxynucleotides modification according to claim 3, it is characterised in that described
Citrate solution is citrate trisodium dihydrate, citric acid tri potassium monohydrate, Triammonium citrate, calcium citrate
Tetrahydrate, anhydrous citric acid sodium, ironic citrate, ten tetrahydrate of magnesium citrate, magnesium citrate, copper citrate, citric acid
One or more of zinc is prepared；The molecular weight of the citrate trisodium dihydrate is 294.1；The citric acid three
The molecular weight of potassium monohydrate is 324.41；The molecular weight of the Triammonium citrate is 243.22；The calcium citrate four
The molecular weight of hydrate is 570.49；The molecular weight of the anhydrous citric acid sodium is 258.07；The molecular weight of the ironic citrate
It is 244.94；The molecular weight of ten tetrahydrate of the magnesium citrate is 703.4；The magnesium citrate volume molecular weight is
451.11；The copper citrate volume molecular weight is 360.22；The zinc citrate volume molecular weight is 574.37.
9. according to a kind of hollow gold nanometer ball of CpG oligodeoxynucleotides modification of claim 2 or 4, it is characterised in that institute
The CpG oligodeoxynucleotides stated are following Thiolation one or more of CpG oligodeoxynucleotide sequences：
(1)CpG1826(5′-TCCATGACGTTCCTGACGTT-SH-3′)；
(2)CpG-T20(5′-TCCATGACGTTCCTGACGT T-T20-SH-3′)；
(3)bi-CpG(5′-TCCATG ACGTTCC TGAC GTTTC CATGACGTTCCTGACGTT-SH-3′)；
(4)CpG T8(5′-TCGTCGTC GTCGTCGTCGTCGTCG-SH-3′)；
(5)CpG 1(5′-ACGCGTCGTCGTCGTCGTCGTCGT-SH-3′)；
(6)CpG 2(5′-TCGTCGATCGATCGACGAGGGG GG–SH-3′)；
(7)CpG 3(5′-TGTGCATCGATGCAGGGGGG-SH-3′)；
(8)CpG 4(5′-TC GACAACGTTAACGTCG TT-SH-3′)；
(9)CpG5(5′-ACGACGACGACGACGACGTT-SH-3′)；
(10)CpG 6(5′-GACGTTCGAACGTAGA CGTT-SH-3′)；
(11)CpG 7(5′-ACGTCGACGTCGACGTCGACGTCG-SH-3′)；
(12)CpG 8(5′-TCGTCGTCGTCGACGACGACGACG-SH-3′)；
(13)CpG 9(5′-TCGCGATCGTCGTCGTCGACGCGT-SH-3′)；
(14)CpG10(5′-TTTTCATCGATGCAGCGCGC-SH-3′)；
(15)CpG 11(5′-TCCTCCGTCGTTTTGTCGTT-SH-3′)；
(16)CpG12(5′-GTCGTCGTCGTCGTCGTCCGCGCG-SH-3′)；
(17)CpG 13(5′-TCGGTCGTTTTGTCGTTGCGCGC-SH-3′)；
(18)CpG 14(5′-TCGGTCGTTGTCGTCGCGCGC-SH-3′)；
(19)CpG15(5′-GGTGCATCGATGCAGGGGGG-SH-3′)；
(20)CpG 16(5′-TCGTCGTTTTGTCGTTTTGTCGTT-SH-3′)；
(21)CpG17(5′-TGCTGCTTTTGTGCTTTTGTGCTT-SH-3′)；
(22)CpG 1911(5′-TCCAGGACTTTCCTCAGGT-SH-3′)；
(23)CpG 2216(5′-GGGGGACGATCGT CGGGGGG-SH-3′)；
(24)CpG M362(5′-TCGTCGTCGTTCGAACGACGTTGAT-SH-3′)；
(25)CpGM383(5′-TGCTGC TGCTTGCAAG CAGCTTGAT-SH-3′)；
(26)CpG 2006(5′-tcgtcgttttgtcgttttgtcgtt-SH-3′)；
(27)CpG 1585(5′-ggGGTCAACGTTGAgggggG-SH-3′)；
(28)CpG 2118(5′-ggGGTCAAGCTT GAg ggggG-SH-3′)；
(29)CpG 2197(5′-gmGGTCAACGTTGAgggmggG-SH-3′)；
(30)CpG 2198(5′-ggGGAGTTCGTTGAgggggG-SH-3′)；
(31)CpG 2204(5′-ggGGTCATCGATGAgggggG-SH-3′)；
(32)CpG 2216(5′-ggGGGACGATCGTCgggggG-SH-3′)；
(33)CpG 2243(5′-ggGGGAGCAT GCTCgggggG-SH-3′)；
(34)CpG 2217(5′-ggGGGTCGTACGACgggggG-SH-3′)。
10. according to a kind of hollow gold nanometer ball of CpG oligodeoxynucleotides modification of claim 2 or 4, it is characterised in that institute
The surfactant stated is that one or more of lauryl sodium sulfate, alkyl polyglycoside are prepared；The dodecyl sulphur
The molecular weight of sour sodium is 288.38；The molecular weight of Quito glycosides is 320.22.
11. according to a kind of hollow gold nanometer ball of CpG oligodeoxynucleotides modification of claim 2 or 4, it is characterised in that institute
The buffer solution stated be sodium chloride, potassium chloride, citric acid, disodium hydrogen phosphate, potassium dihydrogen phosphate, trishydroxymethylaminomethane, hydrochloric acid,
One or more in sodium hydroxide are uniformly mixed at room temperature.
12. a kind of hollow gold nanometer ball of CpG oligodeoxynucleotides modification according to claim 3, it is characterised in that institute
The preparation method for stating hollow gold nanometer ball solution further includes following steps：With sodium borohydride reduction cobalt ions, cobalt nanoparticle is obtained
Then son restores gold chloride with cobalt nanometer particle, prepares hollow gold nanometer ball solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810370104.XA CN108578694A (en) | 2018-04-24 | 2018-04-24 | The hollow gold nanometer ball of CpG oligodeoxynucleotides modification and preparation method, application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810370104.XA CN108578694A (en) | 2018-04-24 | 2018-04-24 | The hollow gold nanometer ball of CpG oligodeoxynucleotides modification and preparation method, application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108578694A true CN108578694A (en) | 2018-09-28 |
Family
ID=63614256
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810370104.XA Pending CN108578694A (en) | 2018-04-24 | 2018-04-24 | The hollow gold nanometer ball of CpG oligodeoxynucleotides modification and preparation method, application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108578694A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110090203A (en) * | 2019-04-29 | 2019-08-06 | 南京锐利施生物技术有限公司 | A kind of degradable microsphere preparation of the load Pramipexole of near-infrared response type |
| CN113144211A (en) * | 2021-03-25 | 2021-07-23 | 长沙理工大学 | RNA packaging method |
| CN113332311A (en) * | 2021-06-01 | 2021-09-03 | 山东第一医科大学(山东省医学科学院) | Preparation method and application of manganese ion-CpG oligonucleotide nanoparticles |
| CN115531540A (en) * | 2022-09-21 | 2022-12-30 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of hollow gold nanosphere loaded in extracellular vesicles |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103284951A (en) * | 2013-04-27 | 2013-09-11 | 浙江大学 | Photosensitive liposome with encapsulated water-soluble medicament |
| CN104551008A (en) * | 2015-01-16 | 2015-04-29 | 吉林大学 | Adjustable spectrum gold nanoshell preparation method |
| CN105079802A (en) * | 2015-08-27 | 2015-11-25 | 中国人民解放军军事医学科学院野战输血研究所 | Accelerator capable of improving functionalization process of adoptive DCs (Dendritic Cells), as well as preparation method and application of accelerator |
| CN106581689A (en) * | 2016-12-08 | 2017-04-26 | 扬州大学 | SiRap2b-GNs coupling compound for targeted therapy of malignant tumor and preparation method of siRap2b-GNs coupling compound |
| CN107670053A (en) * | 2017-08-29 | 2018-02-09 | 杨蕾 | A kind of preparation method of the immunostimulation system based on MNPs@Au load C pG nucleic acid |
-
2018
- 2018-04-24 CN CN201810370104.XA patent/CN108578694A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103284951A (en) * | 2013-04-27 | 2013-09-11 | 浙江大学 | Photosensitive liposome with encapsulated water-soluble medicament |
| CN104551008A (en) * | 2015-01-16 | 2015-04-29 | 吉林大学 | Adjustable spectrum gold nanoshell preparation method |
| CN105079802A (en) * | 2015-08-27 | 2015-11-25 | 中国人民解放军军事医学科学院野战输血研究所 | Accelerator capable of improving functionalization process of adoptive DCs (Dendritic Cells), as well as preparation method and application of accelerator |
| CN106581689A (en) * | 2016-12-08 | 2017-04-26 | 扬州大学 | SiRap2b-GNs coupling compound for targeted therapy of malignant tumor and preparation method of siRap2b-GNs coupling compound |
| CN107670053A (en) * | 2017-08-29 | 2018-02-09 | 杨蕾 | A kind of preparation method of the immunostimulation system based on MNPs@Au load C pG nucleic acid |
Non-Patent Citations (3)
| Title |
|---|
| ADAM M. SCHWARTZBERG ET AL: "Improving Nanoprobes Using Surface-Enhanced Raman Scattering from 30-nm Hollow Gold Particles", 《ANALYTICAL CHEMISTRY》 * |
| MIN WEI ET AL: "Polyvalent Immunostimulatory Nanoagents with Self-Assembled CpG Oligonucleotide-Conjugated Gold Nanoparticles", 《ANGEW. CHEM. INT. ED.》 * |
| STACI ADAMS ET AL: "Key Factors Affecting the Reproducibility of Synthesis and Growth Mechanism of Near-Infrared Absorbing Hollow Gold Nanospheres", 《CHEMISTRY OF MATERIALS》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110090203A (en) * | 2019-04-29 | 2019-08-06 | 南京锐利施生物技术有限公司 | A kind of degradable microsphere preparation of the load Pramipexole of near-infrared response type |
| CN113144211A (en) * | 2021-03-25 | 2021-07-23 | 长沙理工大学 | RNA packaging method |
| CN113332311A (en) * | 2021-06-01 | 2021-09-03 | 山东第一医科大学(山东省医学科学院) | Preparation method and application of manganese ion-CpG oligonucleotide nanoparticles |
| CN115531540A (en) * | 2022-09-21 | 2022-12-30 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of hollow gold nanosphere loaded in extracellular vesicles |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108578694A (en) | The hollow gold nanometer ball of CpG oligodeoxynucleotides modification and preparation method, application | |
| CN108543074B (en) | Exosome-encapsulated nano drug delivery system for tumor treatment and preparation thereof | |
| He et al. | A smart ZnO@ polydopamine-nucleic acid nanosystem for ultrasensitive live cell mRNA imaging by the target-triggered intracellular self-assembly of active DNAzyme nanostructures | |
| Montalti et al. | Nanodiamonds and silicon quantum dots: ultrastable and biocompatible luminescent nanoprobes for long-term bioimaging | |
| Panyala et al. | Gold and nano-gold in medicine: overview, toxicology and perspectives. | |
| Song et al. | Cuprous oxide nanoparticles inhibit angiogenesis via down regulation of VEGFR2 expression | |
| CN107753464A (en) | It is encapsulated hollow silicon dioxide nano-particle, its preparation method and the application of bioactive ingredients | |
| CN107802840A (en) | A kind of tumor microenvironment response nano grain based on the dendrimer-modified fluorescent carbon point of peptides and preparation method thereof | |
| CN106620725B (en) | Optical and photoacoustic integrated bimodal molecular imaging probe and preparation method and application thereof | |
| CN104548109B (en) | biomedical compositions | |
| CN110237264A (en) | A kind of PLGA nano particle and preparation method thereof for TA-Fe (III) modification containing doxorubicin hydrochloride | |
| Zhang et al. | Research progress in the synthesis and biological application of quantum dots | |
| US20230109863A1 (en) | Compositions and methods for targeted delivery of therapeutic and/or diagnostic species | |
| WO2017097165A1 (en) | Preparation method for exogenous molecule-loaded cells, substrate for preparation of exogenous molecule-loaded cells, and exogenous molecule-loaded cells | |
| CN107737336A (en) | A kind of method for the gold nano star pharmaceutical carrier for preparing mesoporous silicon cladding | |
| Gang et al. | Synthesis and biological evaluation of fluorescent hyaluronic acid modified amorphous calcium phosphate drug carriers for tumor-targeting | |
| CN111303314B (en) | Hypoxia-sensitive nitrobenzylated chitosan, preparation and application thereof | |
| CN110393807B (en) | Silicon dioxide nano gene delivery system and preparation method and application thereof | |
| CN107837400B (en) | Novel nano gene vector and preparation method thereof | |
| CN105079802B (en) | A kind of accelerating agent and the preparation method and application thereof that adoptive dendritic cell function process can be improved | |
| CN111840305A (en) | Nano particles and preparation method and application thereof | |
| CN107243000B (en) | Drug-loaded hybrid nanoparticles and preparation method thereof | |
| KR102632530B1 (en) | Stomach cancer specific targeting exosome composition as a drug anticancer delivery and use thereof | |
| CN113769111B (en) | Dual miRNA (micro ribonucleic acid) -triggered universal key-drug delivery system as well as construction method and application thereof | |
| Cheng et al. | 3D-bioprintable endothelial cell-laden sacrificial ink for fabrication of microvessel networks |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180928 |