CN104865348A - Efficient detection method for nano-particle cardiotoxicity - Google Patents
Efficient detection method for nano-particle cardiotoxicity Download PDFInfo
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- CN104865348A CN104865348A CN201510214824.3A CN201510214824A CN104865348A CN 104865348 A CN104865348 A CN 104865348A CN 201510214824 A CN201510214824 A CN 201510214824A CN 104865348 A CN104865348 A CN 104865348A
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Abstract
The invention discloses an efficient detection method for nano-particle cardiotoxicity. The method comprises the following steps: 1) preparing a pattern fiber electrospinning receiving board, using photoetching technology and direct current magnetron sputtering technology for preparing a receiving board having pattern metallic silver layer; 2) preparing pattern electrospinning fiber, using the object prepared in the step 1) for executing electrospinning operation; 3) preparing PDMS cavity; 4) processing the objects obtained in the step 2) and step 3) via plasma and connecting; 5) inoculating the primary mouse myocardial cell to the pattern electrospinning fiber obtained in the step 4), using an injection pump for pumping the culture medium into the cavity obtained in the step 4), pumping the nano-particle, and recording cardiotoxicity index during the later 20 days after pumping the nano-particle. The efficient and high precision nano-particle cardiotoxicity detection can be executed by the method, and the detection effect is consistent to the in vivo animal experiment; the detection method is relatively fast compared with the in vivo animal experiment.
Description
Technical field
The invention belongs to nano particle toxicity detection field, be specifically related to a kind of method of efficient detection nano particle myocardium toxicity.
Technical background
At biomedicine field, nano particle is usually used as the carrier of drug delivery, when using nano particle, often need evaluate its toxicity.
Heart is the vitals of human body, and can a kind of nano particle carry out bio-medical, and it is important investigation object to cardiocellular toxicity.
In the prior art, the common method evaluating the myocardium toxicity of nano cell is carry out the zoopery of nano particle toxicity.But carry out zoopery, required sense cycle is long, with often causing metrical error due to the individual difference of animal used as test.For substituting zooperal experiment in vitro, complex steps in prior art, easily because misoperation causes error, degree of accuracy is difficult to ensure.
Summary of the invention
For the shortcoming of prior art, the object of the present invention is to provide a kind of method of efficient detection nano particle myocardium toxicity, it is characterized in that, described method comprises the steps:
1) patterned fibrous electrospinning dash receiver is prepared: first on insulating glass sheet, apply positive-working photoresist, then cover one deck photomask, utilize litho machine to etch; Again by direct magnetic control technology glass sheet after etching deposits layer of metal silver, the shape of the argent deposited comprises the one in square, rectangle, and the scope of the length of side is 20 μm-100 μm; Finally wash remaining positive-working photoresist;
2) prepare patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains as electrospinning dash receiver, prepared the patterning electrospinning fibre of square or rectangular composition by electrostatic spinning technique; The diameter controlling gained fiber is 100-300 nm;
Described medical high polymer comprises the one in PLA, polycaprolactone, polyurethane, polyacrylonitrile, and described organic solvent comprises at least one in acetone, dimethyl formamide;
3) negative epoxy resin type near ultraviolet ray photoetching glue SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain wide for 40-150 μm, high for the SU-8 rectangular parallelepiped of 70-100 μm is as mould; The PDMS of melting is placed on gained mould, after PDMS cooling, removes mould, obtain PDMS cavity;
4) by step 2) and step 3) gains at O
2or N
2atmosphere under, by Cement Composite Treated by Plasma 60 seconds, make step 2) and step 3) gains compact siro spinning technology;
5) by 5 × 10
6-1.0 × 10
7individual mouse primary cardiac muscle cell is inoculated on the patterning electrospinning fibre in step 4) gains, and utilize syringe pump nutrient culture media to be pumped into the cavity of step 4) gains, flow velocity is 0.2-0.3 ml/h; Again nanoparticle suspension is pumped into after PBS, after nano particle pumps into, the change of record mouse primary cardiac muscle cell jumping frequency rate, the enzyme work of superoxide dismutase, the activity of glutathione peroxidase, mda content, the damage of cell DNA, the change of apoptosis rate, draw the toxicity of nano particle;
Described nano particle comprises the one in nano ferriferrous oxide, nano titanium oxide, nano zine oxide, nano silicon, nano-Ag particles.
The present invention gropes to find by a large amount of experiments, be the electrospinning fibre of 100-300 nm when selecting diameter, and when being square or rectangular array by the pattern setting of tunica fibrosa, and arrange PDMS cavity be of a size of wide for 40-150 μm, high for 70-100 μm time, mouse primary cardiac muscle cell can be made well to grow in cavity, and almost identical with zoopery toxic reaction is produced to nano particle.
The present invention can make patterned fibrous and PDMS cavity and substrate of glass is seamless fits tightly, ensure that nano particle completely with cells contacting, ensure that the degree of accuracy of detection.Meanwhile, due to fitting with substrate and cavity of patterned fibrous film close, make the present invention can have good repetitive operation, save the cost of detection.
Importantly, any predictable change made according to the present invention, as fine setting pattern form and PDMS size, all belongs to protection scope of the present invention.
Special needs to be pointed out is, the present invention is applicable to the myocardium toxicity detecting nano particle.According to the general knowledge of this area, the myocardium toxicity detection that the present invention is applicable to any nano particle should be understood.Several nano particles cited by the present invention should being understood only for proving that the myocardium toxicity that the present invention is applicable to nano particle detects, should not be construed as limitation of the invention.
In the present invention, the argent of step 1) deposition can be prepared into any type of array, only needs the figure in array to be square or rectangular of the present invention.
Preferably, the shape of the argent deposited in described step 1) is square, and the scope of the length of side is 20 μm-100 μm.When the shape of deposited argent is quadrate array, primary cardiomyocytes has better growth conditions, is more conducive to the detection of the myocardium toxicity of nano particle.
Described step 2) in, when carrying out electrospinning, voltage is 15-25 KV, and flow velocity is 0.5-1.0 ml/h.
Preferably, described step 2) in, the diameter of fiber is 200 nm.When the diameter of fiber used is 200 nm, primary cardiomyocytes has better growth conditions, is more conducive to the detection of the myocardium toxicity of nano particle.
Described step 2) in medical high polymer be PLA, described organic solvent is the mixed solution of acetone and dimethyl formamide, and the volume ratio of acetone and dimethyl formamide is 9:1.When fiber is acid fiber by polylactic, primary cardiomyocytes has better growth conditions, is more conducive to the detection of the myocardium toxicity of nano particle.
Preferably, the wide of the mould in described step 3) is 100 μm, and height is 80 μm.
Preferred, the wide of the mould in described step 3) is 50 μm, and height is 70 μm.Detect cavity less, more integrated, a pick-up unit can be realized there is multiple cavity, realize the carrying out of more groups of parallel laboratory tests, more guarantee the degree of accuracy detected.In the prior art, not yet find to prepare so little, carry out myocardium toxicity detection based on electrospun fiber membrane cavity.
In described step 5), the number for the primary cardiomyocytes inoculated is 8 × 10
6individual.
In described step 5), the flow velocity of nutrient culture media is 0.25 ml/h.
Beneficial effect of the present invention:
1, the present invention can realize the detection of efficient, high-precision nano particle myocardium toxicity, and Detection results is consistent with interior animal experiment; More quick relative to interior animal experiment;
2, the present invention is raw materials used is easy to get, and cost is low, and technology maturation used is easily implemented, and has huge market application foreground.
Accompanying drawing explanation
Fig. 1 is gained patterned fibrous of the present invention.
Embodiment
Embodiment is by the following examples described in further detail foregoing of the present invention, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.
Embodiment 1
1) patterned fibrous electrospinning dash receiver is prepared: first on insulating glass sheet, apply positive-working photoresist, then cover one deck photomask, utilize litho machine to etch; Again by direct magnetic control technology glass sheet after etching deposits layer of metal silver, the shape of the argent deposited is quadrate array, and the scope of the length of side is 20 μm; Finally wash remaining positive-working photoresist;
2) prepare patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains as electrospinning dash receiver, prepared the patterning electrospinning fibre of quadrate array by electrostatic spinning technique; The diameter controlling gained fiber is 100 nm;
Described medical high polymer is polyacrylonitrile, and described organic solvent is acetone and dimethyl formamide mixed solution (9:1 v/v);
3) SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain wide be 100 μm, height is that the SU-8 rectangular parallelepiped of 80 μm is as mould; The PDMS of melting is placed on gained mould, after PDMS cooling, removes mould, obtain PDMS cavity;
4) pass through plasma treatment: open equipment vacuum room, glass sheet and the PDMS cavity of what loading prepared above deposit patterning electrospinning fibre, open RF driving source filament supply, open O
2:gas cylinder valve, Cement Composite Treated by Plasma is after 1 minute, by step 2) and step 3) gains fit tightly, make it connect;
5) by 8 × 10
6individual mouse primary cardiac muscle cell is inoculated on the patterning electrospinning fibre in step 4) gains, and utilize syringe pump nutrient culture media to be pumped into the cavity of step 4) gains, flow velocity is 0.25 ml/h; Again nanoparticle suspension is pumped into after PBS, after nano particle pumps into, the cell jumping frequency rate change of record mouse primary cardiac muscle cell, the enzyme work of superoxide dismutase, the activity of glutathione peroxidase, mda content, the damage of cell DNA, the change of apoptosis rate, draw the toxicity of nano particle;
Described nano particle comprises nano-Ag particles.
Embodiment 2
1) patterned fibrous electrospinning dash receiver is prepared: first on insulating glass sheet, apply positive-working photoresist, then cover one deck photomask, utilize litho machine to etch; Again by direct magnetic control technology glass sheet after etching deposits layer of metal silver, the shape of the argent deposited is rectangular array, and the length of side is 20 μm and 70 μm, finally washes remaining positive-working photoresist;
2) prepare patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains as electrospinning dash receiver, prepared the patterning electrospinning fibre of rectangular array by electrostatic spinning technique; The diameter controlling gained fiber is 150 nm;
Described medical high polymer comprises PLA, and described organic solvent is acetone;
3) SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain wide be 100 μm, height is that the SU-8 rectangular parallelepiped of 80 μm is as mould; The PDMS of melting is placed on gained mould, after PDMS cooling, removes mould, obtain PDMS cavity;
4) pass through plasma treatment: open equipment vacuum room, glass sheet and the PDMS cavity of what loading prepared above deposit patterning electrospinning fibre, open RF driving source filament supply, open N
2gas cylinder valve, Cement Composite Treated by Plasma is after 1 minute, by step 2) and step 3) gains fit tightly, make it connect;
5) by 8 × 10
6individual mouse primary cardiac muscle cell is inoculated on the patterning electrospinning fibre in step 4) gains, and utilize syringe pump nutrient culture media to be pumped into the cavity of step 4) gains, flow velocity is 0.25 ml/h; Again nanoparticle suspension is pumped into after PBS, after nano particle pumps into, the cell jumping frequency rate change of record mouse primary cardiac muscle cell, the enzyme work of superoxide dismutase, the activity of glutathione peroxidase, mda content, the damage of cell DNA, the change of apoptosis rate, draw the toxicity of nano particle;
Described nano particle comprises nano silicon.
Embodiment 3
1) patterned fibrous electrospinning dash receiver is prepared: first on insulating glass sheet, apply positive-working photoresist, then cover one deck photomask, utilize litho machine to etch; Again by direct magnetic control technology glass sheet after etching deposits layer of metal silver, the shape of the argent deposited is quadrate array, and the length of side is 50 μm; Finally wash remaining positive-working photoresist;
2) prepare patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains as electrospinning dash receiver, prepare square pattern electrospinning fibre by electrostatic spinning technique; The diameter controlling gained fiber is 200 nm;
Described medical high polymer comprises polyurethane, and described organic solvent is acetone;
3) SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain wide be 100 μm, height is that the SU-8 rectangular parallelepiped of 80 μm is as mould; The PDMS of melting is placed on gained mould, after PDMS cooling, removes mould, obtain PDMS cavity;
4) pass through plasma treatment: open equipment vacuum room, glass sheet and the PDMS cavity of what loading prepared above deposit patterning electrospinning fibre, open RF driving source filament supply, open N
2gas cylinder valve, Cement Composite Treated by Plasma is after 1 minute, by step 2) and step 3) gains fit tightly, make it connect;
5) by 8 × 10
6individual mouse primary cardiac muscle cell is inoculated on the patterning electrospinning fibre in step 4) gains, and utilize syringe pump nutrient culture media to be pumped into the cavity of step 4) gains, flow velocity is 0.2-0.3 ml/h; Again nanoparticle suspension is pumped into after PBS, after nano particle pumps into, the cell jumping frequency rate change of record mouse primary cardiac muscle cell, the enzyme work of superoxide dismutase, the activity of glutathione peroxidase, mda content, the damage of cell DNA, the change of apoptosis rate, draw the toxicity of nano particle;
Described nano particle is nano zine oxide.
Embodiment 4
1) patterned fibrous electrospinning dash receiver is prepared: first on insulating glass sheet, apply positive-working photoresist, then cover one deck photomask, utilize litho machine to etch; Again by direct magnetic control technology glass sheet after etching deposits layer of metal silver, the shape of the argent deposited is rectangular array, and the length of side is respectively 50 μm and 75 μm; Finally wash remaining positive-working photoresist;
2) prepare patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains as electrospinning dash receiver, prepare patterning electrospinning fibre by electrostatic spinning technique; The diameter controlling gained fiber is 250 nm;
Described medical high polymer is PLA, and described organic solvent is acetone;
3) SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain wide be 150 μm, height is that the SU-8 rectangular parallelepiped of 100 μm is as mould; The PDMS of melting is placed on gained mould, after PDMS cooling, removes mould, obtain PDMS cavity;
4) pass through plasma treatment: open equipment vacuum room, glass sheet and the PDMS cavity of what loading prepared above deposit patterning electrospinning fibre, open RF driving source filament supply, open O
2gas cylinder valve, Cement Composite Treated by Plasma is after 1 minute, by step 2) and step 3) gains fit tightly, make it connect;
5) by 1.0 × 10
7individual mouse primary cardiac muscle cell is inoculated on the patterning electrospinning fibre in step 4) gains, and utilize syringe pump nutrient culture media to be pumped into the cavity of step 4) gains, flow velocity is 0.3 ml/h; Again nanoparticle suspension is pumped into after PBS, after nano particle pumps into, the cell jumping frequency rate change of record mouse primary cardiac muscle cell, the enzyme work of superoxide dismutase, the activity of glutathione peroxidase, mda content, the damage of cell DNA, the change of apoptosis rate, draw the toxicity of nano particle;
Described nano particle is nano ferriferrous oxide.
Embodiment 5
1) patterned fibrous electrospinning dash receiver is prepared: first on insulating glass sheet, apply positive-working photoresist, then cover one deck photomask, utilize litho machine to etch; Again by direct magnetic control technology glass sheet after etching deposits layer of metal silver, the shape of the argent deposited is rectangular array, and the scope of the length of side is 20 μm and 100 μm; Finally wash remaining positive-working photoresist;
2) prepare patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains as electrospinning dash receiver, prepare patterning electrospinning fibre by electrostatic spinning technique; The diameter controlling gained fiber is 300 nm;
Described medical high polymer is polycaprolactone, and described organic solvent is acetone;
3) SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain wide be 40 μm, height is that the SU-8 rectangular parallelepiped of 70 μm is as mould; The PDMS of melting is placed on gained mould, after PDMS cooling, removes mould, obtain PDMS cavity;
4) pass through plasma treatment: open equipment vacuum room, glass sheet and the PDMS cavity of what loading prepared above deposit patterning electrospinning fibre, open RF driving source filament supply, open O
2gas cylinder valve, Cement Composite Treated by Plasma is after 1 minute, by step 2) and step 3) gains fit tightly, make it connect;
5) by 5 × 10
6individual mouse primary cardiac muscle cell is inoculated on the patterning electrospinning fibre in step 4) gains, and utilize syringe pump nutrient culture media to be pumped into the cavity of step 4) gains, flow velocity is 0.2 ml/h; Again nanoparticle suspension is pumped into after PBS, after nano particle pumps into, the cell jumping frequency rate change of record mouse primary cardiac muscle cell, the enzyme work of superoxide dismutase, the activity of glutathione peroxidase, mda content, the damage of cell DNA, the change of apoptosis rate, draw the toxicity of nano particle;
Described nano particle is nano titanium oxide.
Experimental example 1:
The method of the embodiment of the present invention 1 is utilized to carry out myocardium toxicity detection: when Primary mouse cardiac muscle cell in vitro culture after 20 days in system of the present invention, adding concentration is 80 μ g/ml, when particle diameter is about the nano-Ag particles of 80 nm, act on after 24 hours, the jumping frequency rate recording primary cardiomyocytes is 243 beats/min, the activity measuring its superoxide dismutase (SOD) is 10.44 U/mgprot, the activity of glutathione peroxidase (GSH-Px) is 47.14 μm of ol/gprot, the content of MDA (MDA) is 1.45 nmol/mgprot, the damage ratio of DNA is 8.32%, apoptosis rate is 6.74%.
Experimental example 2:
The method of the embodiment of the present invention 1 is utilized to carry out myocardium toxicity detection: when Primary mouse cardiac muscle cell in vitro culture after 15 days in system of the present invention, adding concentration is 60 μ g/ml, when particle diameter is about the nano silicon of 70 nm, act on after 24 hours, the jumping frequency rate recording primary cardiomyocytes is 174 beats/min, the activity measuring its superoxide dismutase (SOD) is 35.56 U/mgprot, the activity of glutathione peroxidase (GSH-Px) is 54.03 μm of ol/gprot, the content of MDA (MDA) is 10.61 nmol/mgprot, the damage ratio of DNA is 56.11%, apoptosis rate is 46.88%.
Experimental example 3:
The method of the embodiment of the present invention 1 is utilized to carry out myocardium toxicity detection: when Primary mouse cardiac muscle cell in vitro culture after 10 days in system of the present invention, adding concentration is 100 μ g/ml, when particle diameter is about the nano zine oxide of 50 nm, act on after 24 hours, the jumping frequency rate recording primary cardiomyocytes is 168 beats/min, the activity measuring its superoxide dismutase (SOD) is 54.32 U/mgprot, the activity of glutathione peroxidase (GSH-Px) is 67.35 μm of ol/gprot, the content of MDA (MDA) is 18.97 nmol/mgprot, the damage ratio of DNA is 68.15%, apoptosis rate is 60.04%.
Experimental example 4:
The method of the embodiment of the present invention 1 is utilized to carry out myocardium toxicity detection: when Primary mouse cardiac muscle cell in vitro culture after 10 days in system of the present invention, adding concentration is 400 μ g/ml, when particle diameter is about the tri-iron tetroxide of 30 nm, act on after 24 hours, the jumping frequency rate recording primary cardiomyocytes is 113 beats/min, the activity measuring its superoxide dismutase (SOD) is 87.46 U/mgprot, the activity of glutathione peroxidase (GSH-Px) is 79.57 U/mgprot, the content of MDA (MDA) is 45.33 nmol/mgprot, the damage ratio of DNA is 78.43%, apoptosis rate is 72.51%.
Experimental example 5:
The method of the embodiment of the present invention 1 is utilized to carry out myocardium toxicity detection: when Primary mouse cardiac muscle cell in vitro culture after 10 days in system of the present invention, adding concentration is 70 μ g/ml, when particle diameter is about the titania of 10 nm, act on after 24 hours, the jumping frequency rate recording primary cardiomyocytes is 207 beats/min, the activity measuring its superoxide dismutase (SOD) is 43.15 U/mgprot, the activity of glutathione peroxidase (GSH-Px) is 13.28 U/mgprot, the content of MDA (MDA) is 8.57 nmol/mgprot, the damage ratio of DNA is 28.67%, apoptosis rate is 22.74%.
Contrast experiment's example 1:
Tail vein injection experiment is carried out to mouse, implantation concentration is 80 μ g/ml, particle diameter is about Nano Silver about 5 ml of 80 nm, effect body is after 24 hours, recording mouse heart jumping frequency rate is 266 beats/min, take out mouse heart, carry out homogenate detection, the activity measuring the superoxide dismutase (SOD) in heart is 15.65 U/mgprot, the activity of glutathione peroxidase (GSH-Px) is 49.58 U/mgprot, the content of MDA (MDA) is the damage ratio of 2.37 nmol/mgprot, DNA is 3.58%, and apoptosis rate is 3.44%.
Contrast experiment's example 2:
Tail vein injection experiment is carried out to mouse, implantation concentration is 60 μ g/ml, particle diameter is about nano silicon about 5 ml of 70 nm, effect body is after 24 hours, recording mouse heart jumping frequency rate is 198 beats/min, take out mouse heart, carry out homogenate detection, the activity measuring the superoxide dismutase (SOD) in heart is 37.89 U/mgprot, the activity of glutathione peroxidase (GSH-Px) is 59.74 U/mgprot, the content of MDA (MDA) is the damage ratio of 12.54 nmol/mgprot, DNA is 50.33%, and apoptosis rate is 40.24%.
Contrast experiment's example 3:
Tail vein injection experiment is carried out to mouse, implantation concentration is 100 μ g/ml, particle diameter is about nano zine oxide about 5 ml of 50 nm, effect body is after 24 hours, recording mouse heart jumping frequency rate is 185 beats/min, take out mouse heart, carry out homogenate detection, the activity measuring the superoxide dismutase (SOD) in heart is 57.67 U/mgprot, the activity of glutathione peroxidase (GSH-Px) is 70.25 U/mgprot, the content of MDA (MDA) is the damage ratio of 21.07 nmol/mgprot, DNA is 59.18%, and apoptosis rate is 52.66%.
Contrast experiment's example 4:
Tail vein injection experiment is carried out to mouse, implantation concentration is 400 μ g/ml, particle diameter is about nano zine oxide about 5 ml of 30 nm, effect body is after 24 hours, recording mouse heart jumping frequency rate is 142 beats/min, take out mouse heart, carry out homogenate detection, the activity measuring the superoxide dismutase (SOD) in heart is 89.17 U/mgprot, the activity of glutathione peroxidase (GSH-Px) is 80.12 U/mgprot, the content of MDA (MDA) is the damage ratio of 47.33 nmol/mgprot, DNA is 69.54%, and apoptosis rate is 65.37%.
Contrast experiment's example 5:
Tail vein injection experiment is carried out to mouse, implantation concentration is 70 μ g/ml, particle diameter is about nano titanium oxide about 5 ml of 10 nm, effect body is after 24 hours, recording mouse heart jumping frequency rate is 232 beats/min, take out mouse heart, carry out homogenate detection, the activity measuring the superoxide dismutase (SOD) in heart is 47.54 U/mgprot, the activity of glutathione peroxidase (GSH-Px) is 16.35 U/mgprot, the content of MDA (MDA) is the damage ratio of 9.67 nmol/mgprot, DNA is 21.93%, and apoptosis rate is 20.07%.
From above-mentioned experimental example 1-5 and contrast experiment's example 1-5, in the scheming toxicity detection of nano particle, testing result of the present invention and interior animal experiment result are similar to, and can be used as the replacement scheme that interior animal experiment detects.Importantly, the present invention is more easy to control than experiment in vivo, and financial cost and time cost lower, there is very good market application foreground.
Claims (9)
1. a method for efficient detection nano particle myocardium toxicity, is characterized in that, described method comprises the steps:
1) patterned fibrous electrospinning dash receiver is prepared: first on insulating glass sheet, apply positive-working photoresist, then cover one deck photomask, utilize litho machine to etch; Again by direct magnetic control technology glass sheet after etching deposits layer of metal silver, the shape of the argent deposited comprises the one in square, rectangle, and the scope of the length of side is 20 μm-100 μm; Finally wash remaining positive-working photoresist;
2) prepare patterning electrospinning fibre: by medical high polymer organic solvent dissolution, utilize step 1) gains as electrospinning dash receiver, prepared the patterning electrospinning fibre of square or rectangular composition by electrostatic spinning technique; The diameter controlling gained fiber is 100-300 nm;
Described medical high polymer comprises the one in PLA, polycaprolactone, polyurethane, polyacrylonitrile, and described organic solvent comprises at least one in acetone, dimethyl formamide;
3) negative epoxy resin type near ultraviolet ray photoetching glue SU-8 is placed on silicon chip, utilizes optical etching technology, remove remaining SU-8, retain wide for 40-150 μm, high for the SU-8 rectangular parallelepiped of 70-100 μm is as mould; The PDMS of melting is placed on gained mould, after PDMS cooling, removes mould, obtain PDMS cavity;
4) by step 2) and step 3) gains at O
2or N
2atmosphere under, by Cement Composite Treated by Plasma 60 seconds, make step 2) and step 3) gains compact siro spinning technology;
5) by 5 × 10
6-1.0 × 10
7individual mouse primary cardiac muscle cell is inoculated on the patterning electrospinning fibre in step 4) gains, and utilize syringe pump nutrient culture media to be pumped into the cavity of step 4) gains, flow velocity is 0.2-0.3 ml/h; Again nanoparticle suspension is pumped into after PBS, after nano particle pumps into, the change of record mouse primary cardiac muscle cell jumping frequency rate, the enzyme work of superoxide dismutase, the activity of glutathione peroxidase, mda content, the damage of cell DNA, the change of apoptosis rate, draw the toxicity of nano particle;
Described nano particle comprises the one in nano ferriferrous oxide, nano titanium oxide, nano zine oxide, nano silicon, nano-Ag particles.
2. method according to claim 1, is characterized in that, the shape of the argent deposited in described step 1) is square, and the scope of the length of side is 20 μm-100 μm.
3. method according to claim 1, is characterized in that, described step 2) in, when carrying out electrospinning, voltage is 15-25 KV, and flow velocity is 0.5-1.0 ml/h.
4. method according to claim 1, is characterized in that, described step 2) in, the diameter of fiber is 200 nm.
5. method according to claim 1, is characterized in that, described step 2) in medical high polymer be PLA, described organic solvent is the mixed solution of acetone and dimethyl formamide, and the volume ratio of acetone and dimethyl formamide is 9:1.
6. method according to claim 1, is characterized in that, the wide of the mould in described step 3) is 100 μm, and height is 80 μm.
7. method according to claim 1, is characterized in that, the wide of the mould in described step 3) is 50 μm, and height is 70 μm.
8. method according to claim 1, is characterized in that, in described step 5), the number for the Primary mouse cardiac muscle cell inoculated is 8 × 10
6individual.
9. method according to claim 1, is characterized in that, in described step 5), the flow velocity of nutrient culture media is 0.3 ml/h.
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