CN105664183A - Preparation method of gamma-PGA (polyglutamic acid) hydrogel loaded with Au nanoparticles - Google Patents

Preparation method of gamma-PGA (polyglutamic acid) hydrogel loaded with Au nanoparticles Download PDF

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CN105664183A
CN105664183A CN201610068698.XA CN201610068698A CN105664183A CN 105664183 A CN105664183 A CN 105664183A CN 201610068698 A CN201610068698 A CN 201610068698A CN 105664183 A CN105664183 A CN 105664183A
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pga
nano
mpeg
pei
hydrogel
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史向阳
朱建志
孙文杰
周本青
周一伟
彭琛
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Donghua University
Shanghai Tenth Peoples Hospital
National Dong Hwa University
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Donghua University
Shanghai Tenth Peoples Hospital
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations

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Abstract

The invention relates to a preparation method of gamma-PGA (polyglutamic acid) hydrogel loaded with Au nanoparticles. The preparation method comprises the steps as follows: a PEI (polyetherimide) solution is added to an activated mPEG-COOH solution, PEI*NH2-mPEG is obtained and dissolved in ultrapure water, chloroauric acid is added and stirred, a sodium borohydride aqueous solution is added, and (Au<0>)200-PEI*NH2-mPEG is obtained; sodium bicarbonate is added to an activated gamma-PGA solution, an obtained solution is added to a DCM solution containing AOT, a W/O emulsion is obtained and added to a PVA solution, and a W/O/W polymer emulsion is obtained; an ultrapure water solution containing (Au<0>)200-PEI*NH2-mPEG is added dropwise to the polymer emulsion and stirred, and a product is obtained. The process is simple, and the cost is low; the prepared hydrogel has better X-ray attenuation performance, remarkable contrast effect as well as good water solubility, colloidal stability, cell compatibility and biocompatibility.

Description

A kind of preparation method in the γ-PGA hydrogel of load Au nano-particle
Technical field
The invention belongs to the preparation field of hydrogel, particularly to the preparation method in the γ-PGA hydrogel of a kind of load Au nano-particle.
Background technology
Computed tomography (CT) imaging, due to features such as its high spatial resolution, deep layer penetration capacity, easy Image Post-processing Techniques and lower costs, has had become as one of most widely used diagnostic detection technology clinically. In order to obtain the CT image of higher resolution, CT image-forming contrast medium arises at the historic moment. CT image-forming contrast medium conventional clinically at present is mainly containing iodine (I) contrast agent, such as iohexol (iohexol), iopromide (iopromide), iopamidol (iopamidol) etc. But this type of contrast agent imaging efficiency is not as high, therefore to obtain better imaging effect to generally require large bolus injection, so renal adverse effects is also more apparent. Therefore to obtain better CT imaging effect, it is necessary to development of new CT contrast agent. Gold (Au) nano-particle, as a kind of novel CT image-forming contrast medium, has the unrivaled advantage of traditional contrast agent at CT imaging side mask. The atomic number of Au is higher, and x-ray attenuation coefficient is bigger, so that can reduce Au nanoparticle contrast agent makes consumption to obtain identical imaging effect. Additionally, the chemical inertness of Au will not discharge poisonous ion in human body, Au nanoparticle contrast agent is made to have unique advantage in CT image-forming contrast medium.
The appearance of nanotechnology in recent years has greatly promoted the development in CT image-forming contrast medium of the Au nano-particle, and the Au nano-particle prepared has the features such as controlled, the surface easily modification of appearance and size. Early-stage Study adopts sodium borohydride (NaBH4) reducing process prepares the result of the stable Au nano-particle of polymine (PEI) and show, the Au nanoparticle size prepared by reducing process is less, particle diameter is comparatively uniform, and show good X-ray attenuation performance, and there is substantial amounts of amino active group in its surface, it is possible to carry out the further modification (preparation method of the multi-functional hyperbranched polyethyleneimine trapping gold nano-particle of a kind of folate-targeted as decorating site. Chinese invention patent, publication number: CN104288788A, publication date: 2015.01.21).
In the method for modifying of many nano-particle, nano-hydrogel has the advantage of uniqueness. Compared to single nanoparticle, nanogel has a good colloidal stability, biocompatibility, high load capability, be readily synthesized, size is controlled, be prone to the features such as multifunction. Gamma-polyglutamic acid-(γ-PGA) is a kind of natural polyamines, has excellent water solublity and biocompatibility, is a kind of excellent environment-friendly type macromolecule material, is widely used to the preparation of nano-hydrogel at present. According to the nano-hydrogel reported before, there is the better flexible feature easily swallowed by tumor cell with mobility, and nano-particle forms the advantage that Cluster Structures may produce the X-ray attenuation ability of cooperative effect reinforcing material.
Retrieval domestic and foreign literature finds, still not about with NaBH4The Au nano-particle stable for PEI of reducing process synthesis is the relevant report that cross-linking agent prepares that γ-PGA hydrogel is studied as CT image-forming contrast medium.
Summary of the invention
The technical problem to be solved is to provide the preparation method in the γ-PGA hydrogel of a kind of load Au nano-particle, and the method technique is simple, it is easy to operation separates, and raw material sources are extensive; γ-PGA the nano-hydrogel of preparation can stably be scattered in aqueous solution, even particle size distribution, and X-ray attenuation performance is higher, contrasting effects notable, has good medical prospect.
A kind of preparation method in the γ-PGA hydrogel of the load Au nano-particle of the present invention, including:
(1) the DMSO solution of PEI is joined in the dimethyl sulfoxide DMSO solution of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and the N-hydroxy-succinamide NHS carboxy methylation methoxy poly (ethylene glycol) mPEG-COOH activated, stir 2-5 days, dialysis, lyophilizing, obtains PEI NH2-mPEG powder; Wherein, the mass ratio of mPEG-COOH and PEI is 240:100;
(2) by gold chloride HAuCl4Solution joins the PEI NH in step (1)2In the ultra-pure water solution of-mPEG, stir 0.5h, be subsequently adding the NaBH that ice bath processes4Aqueous solution, continues stirring 1-2h, dialysis, lyophilizing, obtains (Au0)200-PEI·NH2-mPEG powder of nanometric particles; Wherein, PEI NH2-mPEG、HAuCl4And NaBH4Quality be 100:127:58.2;
(3) in the γ-PGA ultra-pure water solution activated through EDC and NHS, NaHCO is added3Reaction 0.5h, to reduce the electrostatic interaction of PEI surface amino groups and γ-PGA surface carboxyl groups; Solution after reaction being terminated joins dissolved with, in the dichloromethane DCM solution of Sodium docusate AOT, stirring 10min, forms W/O emulsion; Then W/O emulsion is added dropwise in the ultra-pure water solution of PVA, stirs 15min, form the polymer emulsion of W/O/W; Wherein, γ-PGA and NaHCO3Mass ratio be 1:1.3; γ-PGA ultra-pure water solution, the DCM solution of AOT and the volume ratio of PVA ultra-pure water solution are 1:2:15;
(4) by (Au in step (2)0)200-PEI·NH2The ultra-pure water dropwise of-mPEG joins in the polymer emulsion of the W/O/W of step (3), stirring, removes organic solvent, centrifuge washing, obtains γ-PGA/ [(Au0)200-PEI·NH2-mPEG], i.e. γ-PGA the hydrogel of load Au nano-particle; Wherein, γ-PGA and (Au0)200-PEI·NH2The mass ratio of-mPEG is 1:1.67.
In described step (1), EDC and NHS activation is: adds EDC and stirs 1-2h; It is subsequently adding NHS and continues stirring 1-2h; MPEG-COOH, EDC and NHS mass ratio be 240:230:140.
The bag filter distilled water that described step (1) and the middle dialysis of step (2) are molecular cut off 8000-14000 is dialysed 3 days.
Described step (2) HAuCl4Solution joins PEI NH2After the ultra-pure water solution of-mPEG, the time of stirring is 0.5h.
PEI NH in described step (2)2The concentration of the ultra-pure water solution of-mPEG is 1mg/mL.
In described step (3), EDC and NHS activation is: adds EDC and stirs 1-2h; It is subsequently adding NHS and continues stirring 1-2h; γ-PGA, EDC and NHS mass ratio be 20mg:30mg:34mg.
In described step (3), the concentration of γ-PGA ultra-pure water solution is 1mg/mL; The concentration of the DCM solution of AOT is 33.5mg/mL; The concentration of the ultra-pure water solution of PVA is 20mg/mL.
In described step (4), the time of stirring is 12h.
Described step (4) removes organic solvent and adopts the mode of evaporation.
In described step (4), washing is: with ultra-pure water centrifuge washing three times (6000rpm, 10min).
In described step (4), the γ-PGA hydrogel of load Au nano-particle is applied to CT image-forming contrast medium.
γ-PGA the nano-hydrogel of the load Au nano-particle prepared by the present invention is by γ-PGA and (Au0)200-PEI·NH2The electrostatic interaction of-mPEG and chemical action crosslinking form. The present invention is first with NaBH4The Au nano-particle that reducing process synthesis PEI is stable, is then added in the γ-PGA solution of EDC activation and double; two emulsifying, occurs cross-linking reaction to form the γ-PGA nano-hydrogel of load Au nano-particle. The present invention is simple to operate, it is easy to separating, raw material sources are extensive. γ-PGA nano-hydrogel the even particle size distribution of preparation, X-ray attenuation better performances, contrasting effects are notable, have good water solublity, colloidal stability and cell compatibility, it is easy to swallowed by tumor cell. Tumor imaging result shows, the γ-PGA nano-hydrogel of load Au nano-particle prepared by the present invention has significant contrasting effects, has potential using value in CT image-forming contrast medium field.
The present invention is with NaBH4The Au nano-particle stable for PEI of reducing process synthesis is cross-linking agent synthesis γ-PGA nano-hydrogel, γ-PGA the hydrogel constructing load Au nano-particle is used as CT image-forming contrast medium, the abundant carboxyl in its surface can rhetorical function reagent further, develop for the further investigation further of the present invention and provide very big space.
The present invention uses the means such as Zeta electric potential and dynamic scattering analysis (DLS), ultraviolet absorption spectroscopy (UV-Vis), X-ray diffraction analysis (XRD), fourier transform infrared spectroscopy analysis (FTIR), thermogravimetric analysis (TGA), transmission electron microscope (TEM), ICP-AES (ICP-OES) and X-ray attenuation performance to characterize the γ-PGA nano-hydrogel of the load Au nano-particle prepared. Then utilize MTT cell viability to evaluate the cytotoxicity of nano-hydrogel, and obtain the pattern of the cell after co-culturing with material with phase contrast microscope; Have rated the tumor cell phagocytic activity to this nano-hydrogel subsequently. Finally carry out the CT imaging experiment of cell in vitro, nude mice vivo tumor model, investigate γ-PGA/ [(Au0)200-PEI·NH2-mPEG] the inside and outside CT imaging effect of nano-hydrogel.
Beneficial effect
(1) present invention adopts the (Au that reducing process synthesizes0)200-PEI·NH2-mPEG nano-particle prepares γ-PGA/ [(Au as cross-linking agent0)200-PEI·NH2-mPEG] nano-hydrogel is for CT image-forming contrast medium, and the method technique is simple, it is easy to operation separates, and raw material sources are extensively inexpensive, biodegradable, have good development prospect;
(2) γ-the PGA/ [(Au that prepared by the present invention0)200-PEI·NH2-mPEG] nano-hydrogel even particle size distribution, there is good water solublity, colloidal stability and cell compatibility, organism is had no adverse effects;X-ray attenuation performance is good, and contrasting effects is notable, and this nano-hydrogel surface has substantial amounts of active group and can be used for further modifying and deeply developing, and has potential using value at CT imaging diagnostic field.
Accompanying drawing explanation
Fig. 1 is γ-PGA/ [(Au in embodiment 20)200-PEI·NH2-mPEG] nano-hydrogel aqueous solution difference store the time hydrodynamics diameter change;
Fig. 2 is γ-PGA/ [(Au in embodiment 20)200-PEI·NH2-mPEG] nano-hydrogel and (Au0)200-PEI·NH2The UV-Vis collection of illustrative plates of-mPEG nano-particle;
Fig. 3 is γ-PGA/ [(Au in embodiment 20)200-PEI·NH2-mPEG] nano-hydrogel and (Au0)200-PEI·NH2The XRD figure spectrum of-mPEG nano-particle;
Fig. 4 is γ-PGA/ [(Au in embodiment 20)200-PEI·NH2-mPEG] nano-hydrogel, (Au0)200-PEI·NH2The FTIR collection of illustrative plates of-mPEG nano-particle and γ-PGA;
Fig. 5 is γ-PGA/ [(Au in embodiment 20)200-PEI·NH2-mPEG] nano-hydrogel and (Au0)200-PEI·NH2The TGA of-mPEG nano-particle analyzes curve;
Fig. 6 is γ-PGA/ [(Au in embodiment 20)200-PEI·NH2-mPEG] the TEM picture of nano-hydrogel;
Fig. 7 is γ-PGA/ [(Au in embodiment 30)200-PEI·NH2-mPEG] nano-hydrogel CT imaging picture (a) under different Au concentration and γ-PGA/ [(Au0)200-PEI·NH2-mPEG] linear relationship chart (b) that changes with Au/I concentration of element of the CT value of nano-hydrogel (1) and iohexol (2);
Fig. 8 is γ-the PGA/ [(Au that in embodiment 4, HeLa cell is prepared through the embodiment of the present invention 10)200-PEI·NH2-mPEG] nano-hydrogel processes the MTT cell viability after 24h and analyzes result (Au concentration be 0.025,0.05,0.1,0.2 and 0.4mM);
Fig. 9 is γ-PGA/ [(Au in embodiment 40)200-PEI·NH2-mPEG] cell of (Au concentration 0 (a), 0.025 (b), 0.05 (c), 0.1 (d), 0.2 (e) and 0.4 (f) mM) after 24h that HeLa cell is processed of nano-hydrogel differs pattern picture;
Figure 10 is γ-PGA/ [(Au in embodiment 50)200-PEI·NH2-mPEG] nano-hydrogel and (Au0) the HeLa cytophagy amount of-γ-PGA nano-particle is with the variation relation (Au concentration be 0.025,0.05,0.1,0.2 and 0.4mM) of Au concentration;
Figure 11 is γ-PGA/ [(Au in embodiment 60)200-PEI·NH2-mPEG] nano-hydrogel cell is processed after cell CT imaging picture (a) and cell in vitro CT value with the graph of a relation (b) of Au concentration change;
Figure 12 is γ-PGA/ [(Au in embodiment 70)200-PEI·NH2-mPEG] after nano-hydrogel PBS solution ([Au]=0.5M, 0.1mL) under different time points CT imaging picture (a) of internal HeLa tumor model and signal value picture (b) thereof.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further. Should be understood that these embodiments are merely to illustrate the present invention rather than restriction the scope of the present invention. In addition, it is to be understood that after having read the content that the present invention lectures, the present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values fall within the application appended claims limited range equally.
Embodiment 1
(1) 240mgmPEG-COOH (Mw=2kDa) is dissolved in 10mLDMSO, adds 230mgEDC (being dissolved in 5mLDMSO) and stir 2h to activate COOH; Add 140mgNHS (being dissolved in 5mLDMSO) and continue stirring 1h. Afterwards 100mgPEI (Mw=25kDa) it is dissolved in 20mLDMSO and joins above-mentioned solution continuation stirring 3 days, finally dialysing 3 days with the bag filter of molecular cut off 8000-14000 distilled water, after lyophilizing, obtain PEI NH2-mPEG powder; (2) the 100mgPEI NH of above-mentioned preparation is taken2-mPEG is dissolved in 100mL ultra-pure water, adds 12.7mLHAuCl4Solution (10mg/mL), stirs 0.5h.It is added thereto to the 58.2mgNaBH that ice bath processes afterwards4(being dissolved in 5ml ultra-pure water) solution, continues stirring 2h, finally dialyses 3 days with the bag filter of molecular cut off 8000-14000 distilled water, (the Au obtained after lyophilizing0)200-PEI·NH2-mPEG powder of nanometric particles; (3) take 20mg γ-PGA and be dissolved in 2mL ultra-pure water, add 30mgEDC and activate 2h, add 34mgNHS and continue activation 1h; Add 26mgNaHCO3Continue reaction 0.5h, to reduce the electrostatic interaction of PEI surface amino groups and γ-PGA surface carboxyl groups; Then above-mentioned dropwise is joined dissolved with (4mL) in the DCM solution of 134mgAOT, stir 10min, form W/O emulsion; Then this W/O emulsion is added dropwise in the 30mL ultra-pure water solution containing 600mgPVA (alcoholysis degree 88%, Mw=20-30kDa), stirs 15min, form the polymer emulsion of W/O/W; (4) by the 33.4mg (Au of preparation0)200-PEI·NH2-mPEG nano-particle is dissolved in 5mL ultra-pure water, and it is added dropwise in the polymer emulsion of W/O/W prepared by above-mentioned steps, stirring 12h, evaporating organic solvent, then with three (6000rpm of ultra-pure water centrifuge washing, 10min), γ-the PGA/ [(Au of load Au nano-particle is namely obtained0)200-PEI·NH2-mPEG] nano-hydrogel.
Embodiment 2
γ-the PGA/ [(Au of preparation in Example 10)200-PEI·NH2-mPEG] nano-hydrogel, after it is diluted to debita spissitudo with ultra-pure water, it is used for surveying surface potential and hydrodynamic diameter. Zeta electric potential measurement result shows γ-PGA/ [(Au0)200-PEI·NH2-mPEG] surface potential of nano-hydrogel is-13.1 ± 0.32mV, thus effectively prevent (Au0)200-PEI·NH2The cytotoxicity that-mPEG nano grain surface amino produces, also demonstrates γ-PGA and (Au0)200-PEI·NH2The successful crosslinking of-mPEG. γ-PGA/ [(Au0)200-PEI·NH2-mPEG] nano-hydrogel hydrodynamics diameter is 210.8 ± 6.35nm, particle size distribution is homogeneous, and hydrodynamic diameter can keep almost constant (referring to Fig. 1) for a long time, thus γ-PGA/ [(Au prepared by embodiment 1 is described0)200-PEI·NH2-mPEG] nano-hydrogel has good colloidal stability. By measuring γ-the PGA/ [(Au of embodiment 1 preparation0)200-PEI·NH2-mPEG] nano-hydrogel and (Au0)200-PEI·NH2The UV-Vis collection of illustrative plates (referring to Fig. 2) of-mPEG nano-particle, result shows: obvious absworption peak occurs at 520nm place in prepared hydrogel, it was shown that after forming nano-hydrogel, the uv absorption peak position of Au nano-particle is not subjected to displacement. Then pass through γ-the PGA/ [(Au measuring embodiment 1 preparation0)200-PEI·NH2-mPEG] nano-hydrogel XRD figure spectrum (referring to Fig. 3), result shows: the diffraction maximum of prepared hydrogel is at site 111,200,220,311 and 222 place, very identical with the standard diffraction peak position point of Au crystalline texture. Diffraction peak-to-peak type is sharp-pointed, illustrates that reaction has prepared the Au nano-particle that crystal formation is good. Then γ-the PGA/ [(Au of embodiment 1 preparation is determined0)200-PEI·NH2-mPEG] the FTIR collection of illustrative plates (referring to Fig. 4) of nano-hydrogel shows: at 1377cm-1And 1737cm-1Place's absworption peak significant change. This illustrates (Au0)200-PEI·NH2-mPEG nano-particle has well cross-linked γ-PGA, forms nano-hydrogel. Afterwards to γ-the PGA/ [(Au of preparation in embodiment 10)200-PEI·NH2-mPEG] nano-hydrogel carried out TGA analysis (referring to Fig. 5), and result shows that in nano-hydrogel, the content of γ-PGA is 8.24%. Finally by tem observation embodiment 1 is prepared γ-PGA/ [(Au0)200-PEI·NH2-mPEG] pattern (referring to Fig. 6) of nano-hydrogel, result shows γ-the PGA/ [(Au formed0)200-PEI·NH2-mPEG] pattern of nano-hydrogel is spherical in shape, and size is more uniform, and gel diameter is about 108.6 ± 19.14nm, it does not have significantly agglomeration.
Embodiment 3
γ-the PGA/ [(Au of embodiment 1 preparation is measured by ICP-OES method of testing0)200-PEI·NH2-mPEG] content of Au element in nano-hydrogel. Preparation Au concentration is γ-the PGA/ [(Au of 0.005,0.01,0.02 and 0.04mM respectively0)200-PEI·NH2-mPEG] nano-hydrogel aqueous solution 2mL, measure material CT imaging picture under different Au concentration by CT imaging analysis instrument. By preparing the Medical iodine prevention mykol solution of identical I concentration as a comparison, prove γ-PGA/ [(Au0)200-PEI·NH2-mPEG] nano-hydrogel is for the superiority of CT imaging.
By γ-PGA/ [(Au prepared by embodiment 10)200-PEI·NH2-mPEG] CT imaging picture (referring to Fig. 7 a) of nano-hydrogel can be seen that prepared γ-PGA/ [(Au0)200-PEI·NH2-mPEG] the CT image of nano-hydrogel brightens gradually along with the increase of Au concentration. The CT imaging of variable concentrations sample can be seen that γ-PGA/ [(Au0)200-PEI·NH2-mPEG] nano-hydrogel has good external imaging effect. γ-PGA/ [(Au by relatively identical Au/I concentration of element0)200-PEI·NH2-mPEG] nano-hydrogel and iohexol CT imaging signal values (referring to Fig. 7 b) be it can be seen that γ-PGA/ [(Au0)200-PEI·NH2-mPEG] the X-ray attenuation performance of nano-hydrogel is better than Medical iodine prevention mykol reagent. Therefore, γ-the PGA/ [(Au prepared by the present invention0)200-PEI·NH2-mPEG] nano-hydrogel can as the excellent contrast agent in the diagnosis of CT molecular imaging.
Embodiment 4
γ-the PGA/ [(Au of Evaluation operation example 1 preparation is carried out by the vigor of MTT cell viability measuring HeLa cell0)200-PEI·NH2-mPEG] cell compatibility of nano-hydrogel. γ-the PGA/ [(Au of embodiment 1 preparation of the different Au concentration of preparation0)200-PEI·NH2-mPEG] PBS solution of nano-hydrogel. Collecting exponential phase HeLa cell, be seeded on 96 porocyte culture plates according to the density of 10000 cell per well, add 200 μ LDMEM culture medium, Tissue Culture Plate is placed in CO2Concentration is cultivate 24h in 5% and environment that temperature is 37 DEG C. After discarding culture medium, 180 μ LDMEM culture medium are changed in every hole, and add the γ-PGA/PEI-Fe of 20 μ L variable concentrations respectively3O4Nano-hydrogel (Au concentration be 0.025,0.05,0.1,0.2 and 0.4mM) and pure PBS (matched group), every kind of concentration sets 5 Duplicate Samples. Continue to be placed on 5%CO by Tissue Culture Plate2, 37 DEG C are continued to hatch 24h. Outwelling original culture medium and with aseptic PBS 3 times, every hole adds 20 μ LMTT solution, the lower 37 DEG C of constant temperature culture 4h of light protected environment. Culture supernatants 100 μ L is drawn in 96 orifice plates in every hole in order, detects each hole absorption value at excitation wavelength 490nm place in MK3 type microplate reader, and the size of absorption value can reflect the quantity (referring to Fig. 8) of living cells. Result shows, compared with PBS control group, and γ-PGA/ [(Au0)200-PEI·NH2-mPEG] HeLa cell do not have obvious cytotoxicity by nano-hydrogel within the scope of experimental concentration, and cell survival rate, all more than 90%, illustrates γ-PGA/ [(Au0)200-PEI·NH2-mPEG] nano-hydrogel has good cell compatibility, it is possible to it is applied in organism CT imaging safely. Meanwhile, the present invention demonstrates γ-PGA/ [(Au further by phase contrast microscope observational method0)200-PEI·NH2-mPEG] nano-hydrogel impact (referring to Fig. 9) on cell morphology. Phase contrast microscope picture shows, different Au concentration γ-PGA/ [(Au0)200-PEI·NH2-mPEG] nano-hydrogel (Au concentration 0,0.025,0.05,0.1,0.2 and 0.4mM) at 37 DEG C with co-culture of cells 24h after, the cell that cell morphology processes with PBS does not significantly change, and further illustrates γ-PGA/ [(Au0)200-PEI·NH2-mPEG] nano-hydrogel has good cell compatibility.
Embodiment 5
A kind of desirably contrast agent nano material should be easy to be swallowed by tumor cell, could better be applied to the CT imaging of tumor. With Au the nano-particle ((Au that γ-PGA is stable in the present invention0)-γ-PGA) and as a comparison material verify that nano-hydrogel is more easy to the feature swallowed by tumor cell relative to single nanoparticle.
γ-the PGA/ [(Au of embodiment 1 preparation of preparation variable concentrations0)200-PEI·NH2-mPEG] nano-hydrogel and (Au0) PBS solution of-γ-PGA nano-particle, HeLa cell being seeded on 6 porocyte culture plates (inoculum density is every hole 300,000), add 2mLDMEM culture medium, Tissue Culture Plate is placed in CO2Concentration is cultivate 12h in 5% and environment that temperature is 37 DEG C. 1800 μ LDMEM culture medium are changed in every hole afterwards, and add γ-the PGA/ [(Au of 200 μ L variable concentrations respectively0)200-PEI·NH2-mPEG] nano-hydrogel and (Au0) PBS solution (Au concentration be 0.025,0.05,0.1,0.2 and 0.4mM) of-γ-PGA nano-particle, at 5%CO2, co-culture 6h under 37 DEG C of conditions, cultivate as blank group using PBS. After PBS cell 3 times, with chloroazotic acid (hydrochloric acid/nitric acid; Volume ratio 3:1) digestion, then utilize ICP-OES to measure cytophagic Au concentration. Figure 10 result shows, relative to single (Au0)-γ-PGA nano-particle, through γ-PGA/ [(Au0)200-PEI·NH2-mPEG] the Au phagocytosis amount of cell that processes of nano-hydrogel presents notable difference. The result shows γ-PGA/ [(Au0)200-PEI·NH2-mPEG] nano-hydrogel is prone to be swallowed by tumor cell, thus obtaining desirable contrasting effects.
Embodiment 6
In vivo before experiment, have rated γ-the PGA/ [(Au of embodiment 1 preparation0)200-PEI·NH2-mPEG] the cell CT imaging effect of nano-hydrogel, γ-the PGA/ [(Au of embodiment 1 preparation of preparation variable concentrations0)200-PEI·NH2-mPEG] nano-hydrogel aqueous solution, HeLa cell is seeded on 6 porocyte culture plates (inoculum density is every hole 300,000), adds 2mLDMEM culture medium, Tissue Culture Plate is placed in CO2Concentration is cultivate 12h in 5% and environment that temperature is 37 DEG C. 1800 μ LDMEM culture medium are changed in every hole afterwards, and add γ-the PGA/ [(Au of 200 μ L variable concentrations respectively0)200-PEI·NH2-mPEG] PBS solution (Au concentration be 0.025,0.05,0.1,0.2 and 0.4mM) of nano-hydrogel, and with the PBS cell processed as a control group, cultivating cell PBS 5 times after 6h terminates, trypsinization again, centrifugal, finally it is dispersed in 0.2mL glutaraldehyde (2.5%) solution, surveys CT imaging picture (referring to Figure 11) of each cell sample with CT imager. In fig. 11 a, γ-PGA/ [(Au0)200-PEI·NH2-mPEG] nano-hydrogel process after HeLa cell along with the increase of Au concentration, its picture luminance substantially increases. Figure 11 b is cell γ-the PGA/ [(Au through variable concentrations0)200-PEI·NH2-mPEG] nano-hydrogel process after CT imaging value, it is evident that along with the increase of Au concentration, the CT imaging value of cell gradually rises from figure. These results illustrate γ-the PGA/ [(Au of preparation0)200-PEI·NH2-mPEG] nano-hydrogel has good cell CT imaging effect.
Embodiment 7
At nude mice construct in vitro HeLa subcutaneous tumors model, by γ-the PGA/ [(Au of tail vein injection embodiment 1 preparation0)200-PEI·NH2-mPEG] PBS solution ([Au]=0.5M, 0.1mL) of nano-hydrogel) evaluate tumor locus CT imaging effect (referring to Figure 12 a). Compared with the control mice before injection, after injection nano-hydrogel, nude mouse tumor brightens gradually, and after injection during 2h, the tumor locus of nude mice becomes the brightest, and γ-PGA/ [(Au is described0)200-PEI·NH2-mPEG] nano-hydrogel is easily successfully enriched in tumor locus, has obvious CT diagnosing tumor effect.4h after injection, nude mouse tumor position bright-dark degree recovers gradually. Illustrate that now nano-hydrogel is gone out from tumor locus metabolism gradually along with blood circulation. Figure 12 b is the tumor CT signal value change of corresponding injection time, and 2h after injection, nude mouse tumor position signal value reaches the highest, starts afterwards to recover gradually, and this is consistent with the result of Figure 12 a, illustrates that nano-hydrogel is gone out from tumor locus metabolism gradually. Tumor CT imaging results illustrates γ-PGA/ [(Au prepared by the present invention0)200-PEI·NH2-mPEG] nano-hydrogel can apply to in-vivo tumour CT imaging diagnosis contrast agent.
Comparative example 1
Take 10mg γ-PGA and be dissolved in 50mL ultra-pure water, add 5.1mLHAuCl4Solution (10mg/mL), stirs 0.5h; It is added thereto to the 14mgNaBH that ice bath processes afterwards4(being dissolved in 5ml ultra-pure water) solution, continues stirring 2h, finally dialyses 3 days with the bag filter of molecular cut off 8000-14000 distilled water, (the Au obtained after lyophilizing0)-γ-PGA nanoparticle powder. (the method that the Au nano-particle utilizing gamma-polyglutamic acid-stable detects trivalent chromic ion. Chinese invention patent, publication number: CN103471889A; Publication date: 2013.12.25).

Claims (10)

1. the preparation method in the γ-PGA hydrogel of load Au nano-particle, including:
(1) the DMSO solution of PEI is joined in the DMSO solution of the mPEG-COOH of EDC and NHS activation, stir 2-5 days, dialysis, lyophilizing, obtain PEI NH2-mPEG;
(2) by HAuCl4Solution joins the PEI NH in step (1)2In the ultra-pure water solution of-mPEG, stirring, it is subsequently adding the NaBH that ice bath processes4Aqueous solution, continues stirring 1-2h, dialysis, lyophilizing, obtains (Au0)200-PEI·NH2-mPEG nano-particle;
(3) in the γ-PGA ultra-pure water solution activated through EDC and NHS, NaHCO is added3Reaction, the solution after reaction being terminated joins dissolved with, in the dichloromethane DCM solution of Sodium docusate AOT, stirring, and forms W/O emulsion; Then W/O emulsion is joined in the ultra-pure water solution of PVA, stirring, form the polymer emulsion of W/O/W;
(4) by (Au in step (2)0)200-PEI·NH2The ultra-pure water solution of-mPEG joins in the polymer emulsion of the W/O/W of step (3), stirring, removes organic solvent, centrifuge washing, obtains γ-PGA/ [(Au0)200-PEI·NH2-mPEG], i.e. γ-PGA the hydrogel of load Au nano-particle.
2. the preparation method in the γ-PGA hydrogel of a kind of load Au nano-particle according to claim 1, it is characterised in that in described step (1), the mass ratio of mPEG-COOH and PEI is 240:100; EDC and NHS activation is: adds EDC and stirs 1-2h; It is subsequently adding NHS and continues stirring 1-2h; MPEG-COOH, EDC and NHS mass ratio be 240:230:140.
3. the preparation method in the γ-PGA hydrogel of a kind of load Au nano-particle according to claim 1, it is characterized in that, the bag filter distilled water that described step (1) and the middle dialysis of step (2) are molecular cut off 8000-14000 is dialysed 3 days.
4. the preparation method in the γ-PGA hydrogel of a kind of load Au nano-particle according to claim 1, it is characterised in that described step (2) HAuCl4Solution joins PEI NH2After the ultra-pure water solution of-mPEG, the time of stirring is 0.5h.
5. the preparation method in the γ-PGA hydrogel of a kind of load Au nano-particle according to claim 1, it is characterised in that PEI NH in described step (2)2-mPEG、HAuCl4And NaBH4Quality be 100:127:58.2;PEI NH2The concentration of the ultra-pure water solution of-mPEG is 1mg/mL.
6. the preparation method in the γ-PGA hydrogel of a kind of load Au nano-particle according to claim 1, it is characterised in that γ-PGA and NaHCO in described step (3)3Mass ratio be 1:1.3; γ-PGA ultra-pure water solution, the DCM solution of AOT and the volume ratio of PVA ultra-pure water solution are 1:2:15; EDC and NHS activation is: adds EDC and stirs 1-2h; It is subsequently adding NHS and continues stirring 1-2h; γ-PGA, EDC and NHS mass ratio be 20mg:30mg:34mg.
7. the preparation method in the γ-PGA hydrogel of a kind of load Au nano-particle according to claim 1, it is characterised in that in described step (3), the concentration of γ-PGA ultra-pure water solution is 1mg/mL; The concentration of the DCM solution of AOT is 33.5mg/mL; The concentration of the ultra-pure water solution of PVA is 20mg/mL.
8. the preparation method in the γ-PGA hydrogel of a kind of load Au nano-particle according to claim 1, it is characterised in that γ-PGA and (Au in described step (4)0)200-PEI·NH2The mass ratio of-mPEG is 1:1.67.
9. the preparation method in the γ-PGA hydrogel of a kind of load Au nano-particle according to claim 1, it is characterised in that in described step (4), the time of stirring is 12h.
10. the preparation method in the γ-PGA hydrogel of a kind of load Au nano-particle according to claim 1, it is characterised in that in described step (4), the γ-PGA hydrogel of load Au nano-particle is applied to CT image-forming contrast medium.
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CN112548110A (en) * 2020-10-28 2021-03-26 北京化工大学 Star-shaped gold nanoparticles synthesized in one step based on polyglutamic acid and preparation method thereof
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