CN109432439B - Metal-porphyrin nanoparticle with sonodynamic treatment effect, preparation method and application thereof - Google Patents
Metal-porphyrin nanoparticle with sonodynamic treatment effect, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a metal-porphyrin nanoparticle with an acoustic dynamic treatment effect, and a preparation method and application thereof. First, the sulfonate ion and iron ion (Fe) of porphyrin are utilized3+) Binding occurs and porphyrin-like metal organic nanoparticles are formed by self-assembly. The nano structure has more excellent acoustic dynamic treatment performance and molecular image monitoring and tracing performance. In the present invention, Zn (ii) -lutidine amine dimer (Bis (DPA-Zn) -RGD) with targeting peptide modification was prepared. Furthermore, Bis (DPA-Zn) -RGD with tumor targeting capability is modified on the surface by applying the capability of combining the sulfonate ions on the surface of the nano TPPS and Zn (II) -lutidine (DPA-Zn). The siRNA can be loaded through the combination of the nano structure after surface modification and phosphate ions of the siRNA.
Description
Technical Field
The invention relates to a targeting polypeptide modified metal-porphyrin nanoparticle, a preparation method and application thereof.
Background
Liver cancer is one of the most common clinical tumors, is the sixth incidence of malignant tumors and the third incidence of fatality, and has about 50 ten thousand new cases each year. The existing treatment methods for liver cancer mainly comprise surgical resection, radio frequency ablation, chemotherapy, Transcatheter Arterial Chemoembolization (TACE) and the like, but have great limitations. Surgical resection and radiofrequency ablation are less than 40% suitable for patients; chemotherapy has serious side effects and drug resistance, and has poor treatment effect; TACE has many contraindications and adverse reactions. Photodynamic therapy (PDT) uses non-toxic light and photosensitizers to generate Reactive Oxygen Species (ROS) to kill tumor cells, has attracted considerable attention by researchers, and has the advantage of effectively and selectively destroying tumor tissue without damaging adjacent healthy tissue. Although photodynamic therapy has its unique advantages over the currently available treatments, its clinical utility remains minimal. This is because current photodynamic therapy techniques have serious limitations such as shallow penetration depth of light, skin phototoxicity due to nonspecific distribution of drugs, and the like. Acoustic dynamic force (SDT) therapy utilizes low frequency ultrasound to activate sonosensitizer, generate ROS to kill tumor cells, has a very good penetration depth compared with PDT, and can be used for treating deep tumors. However, the targeting of the sonosensitizer is poor, the concentration of the drug at the tumor part is low, and the effect of resisting oxidation substances in cells is poor, so the treatment effect is not good enough.
Disclosure of Invention
The main purpose of the present invention is to provide porphyrin-based metal-organic nanoparticles that can be specifically concentrated in tumor regions and can reduce the amount of antioxidant SOD in tumor cells2The content of (A) achieves better acoustodynamic treatment effect.
The technical scheme of the invention is as follows:
a metal-porphyrin nanoparticle with sonodynamic therapeutic effect is prepared by reacting sulfonic acid group of porphyrin with metal ion to form nanoparticle, modifying targeting polypeptide on the surface of the nanoparticle, and loading functional siRNA to form multifunctional metal-porphyrin nanoparticle with tumor specific targeting effect; wherein the content of the first and second substances,
the porphyrin is a porphyrin molecule modified by sulfonate ions;
the metal ions include iron ions (Fe)3+) Manganese ionSeed (Mn)2+) Gadolinium (Gd)2+) Zinc ion (Zn)2+) Copper ion (Cu)2+) At least one of;
the targeting polypeptide is at least one of cRGDfC (cyclo (Arg-Gly-Asp-D-Phe-Cys)), cRGDyK (cyclo (Arg-Gly-Asp-D-Tyr-Lys)), cRGDyC (cyclo (Arg-Gly-Asp-D-Tyr-Cys)), cRGDfK (cyclo (Arg-Gly-Asp-D-Phe-Lys));
the siRNA can reduce SOD in cells2Expressed siRNA.
Preferably, the porphyrin-like molecule is meso-tetrakis (4-sodium sulfophenyl) porphyrin (TPPS).
The invention also aims to provide the application of the metal-porphyrin nanoparticle with the sonodynamic treatment effect in tumor treatment preparations.
Preferably, the tumor treatment preparation is a sonodynamic and gene combined treatment cancer preparation.
Another object of the present invention is to provide a method for preparing metal-porphyrin nanoparticles having sonodynamic therapeutic effect, comprising the following steps:
1) preparing a nano structure assembled by porphyrin and metal ions; the porphyrin is a porphyrin molecule modified by sulfonate ions;
the metal ions include iron ions (Fe)3+) Manganese ion (Mn)2+) Gadolinium (Gd)2+) Zinc ion (Zn)2+) Copper ion (Cu)2+) At least one of;
2) preparing Zn (II) -lutidine amine dimer (Bis (DPA-Zn) with targeting polypeptide coupling; the targeting polypeptide is one of cRGDfC, cRGDyK, cRGDyC and cRGDfK;
3) adding the organic molecules prepared in the step 2) into the nano-structure prepared in the step a) for surface modification.
Preferably, the preparation method of the metal-porphyrin nanoparticle with the sonodynamic therapeutic effect comprises the following steps:
a) dissolving ferric trichloride and porphyrin in deionized water, and stirring at 100-2000rpm at room temperature for 12-36 h;
b) transferring the solution into a 30KD ultrafiltration tube, and centrifuging at 3000-5000rpm for 10-20min at room temperature;
c) removing supernatant, collecting precipitate, blowing with 1-5mL deionized water, mixing, centrifuging in ultrafiltration tube at 3000-;
d) resuspending the precipitate with 1-5mL of deionized water to prepare primary nanoparticles;
e) firstly, preparing dimer of DPA, bis (DPA); then coupling by using prepared bis (DPA) and RGD;
first, 2-4mg of Bis (DPA) and 2-4mg of Sulfo-SMCC were added to 1-3mL of DMSO, shaken until uniformly dispersed, and reacted for 2h-10 h.
Secondly, dispersing 1-2mg of cRGDyK polypeptide into 1-3mL of PBS, and then adding 0.5-0.7mg of Traut reagent; reacting at 3-5 ℃ for 0.5-5h to prepare RGD-SH;
thirdly, dispersing bis (DPA) -SMCC into DMSO, dispersing RGD-SH into PBS, reacting at 3-5 ℃ for 12-36h according to the molar ratio of 1-2:1-2, and purifying the prepared bis (DPA) -RGD by HPLC;
finally, a mixed solution containing 1-3mM of bis (DPA) -RGD and 3-5mM of Zn dispersed in DMSO was prepared2+Stirring the solution for 1-3h to prepare Bis (DPA-Zn) -RGD;
f) taking 0.5-2mL of the primary nanoparticles prepared in the step d), adding 10-100ul of Bis (DPA-Zn) -RGD, stirring at room temperature (1000rpm) overnight, repeating the step (c), and resuspending the precipitate with 0.5-2mL of deionized water to prepare targeting nanoparticles;
g) the targeting nanoparticles and siRNA were mixed according to a ratio of 50-100 ug: mixing the materials according to the proportion of 10pM, standing for 30-60min to obtain the final porphyrin-metal nanoparticles.
Preferably, in the above method, step 1) is:
a) dissolving ferric trichloride and porphyrin in deionized water, and stirring at room temperature (1000rpm) for 24 h;
b) transferring the above solution into 30KD ultrafilter tube, and centrifuging at room temperature (4000rpm) for 15 min;
c) uniformly blowing the precipitate with 3mL of deionized water, placing the precipitate into an ultrafiltration tube, centrifuging (4000rpm) for 15-25min, and repeating the step for 3 times;
d) the above precipitate was resuspended in 3mL of deionized water to make the nanostructure.
Preferably, in the above method, step 2) is: firstly, preparing dimer of DPA, bis (DPA), and then coupling by using the prepared bis (DPA) and RGD;
firstly, 3.38mg of Bis (DPA) and 2.18mg of Sulfo-SMCC are added into 2mL of DMSO, shaken until the mixture is uniformly dispersed, and reacted for more than 2 hours;
second, 1.73mg of cRGDyK polypeptide was dispersed in 2mL of PBS (pH 7.4), followed by the addition of 0.6mg of Traut reagent. Reacting at 4 ℃ for 2h to prepare RGD-SH;
thirdly, dispersing bis (DPA) -SMCC into DMSO, dispersing RGD-SH into PBS (pH 7.4) according to a molar ratio of 1:1, reacting at 4 ℃ for 24h, and purifying the prepared bis (DPA) -RGD by HPLC;
finally, 2mL of Bis (DPA) -RGD solution (2mM, dispersed in DMSO) and 2mL of Zn were simply mixed2+The solution (4mM) was stirred for 2 hours to prepare Bis (DPA-Zn) -RGD.
And (3) taking 1mL of preliminarily prepared nanoparticles, adding 10-100ul of Bis (DPA-Zn) -RGD, stirring at room temperature (1000rpm) overnight, repeating the step (c), and resuspending the precipitate with 1mL of deionized water to prepare the targeting nanoparticles.
According to the invention, the nanoparticles have a good passive targeting effect on tumors through a high-permeability retention (EPR) effect of solid tumors, and can increase the accumulation of drugs in tumor sites and reduce nonspecific distribution. Besides passive targeting, the active targeting function of the nanoparticles can be realized by using ligands and receptors, and the tumor specificity distribution is further improved. Wherein, arginine-glycine-aspartic acid (RGD) can be specifically combined with alpha v beta 3 integrin highly expressed by tumor new vessels. The process of forming tumors of all solid tumor cells (breast cancer, prostatic cancer, liver cancer and the like) is necessarily accompanied with the generation of tumor neoangiogenesis integrin alphavbeta 3 which is used as a common substance in the process of generating and developing tumors and has certain specificity.
The antioxidant substances in the cells mainly comprise superoxide dismutase (SOD1, SOD2), Glutathione (GSH), vitamin E and the like, and the cells can resist the killing effect of ROS through the antioxidant substances, so that the killing effect of ROS can be enhanced by reducing the content of the antioxidant substances in the cells. The metal ions such as iron, copper, manganese and the like can oxidize glutathione and reduce the oxidation resistance of cells. The content of superoxide dismutase can be reduced by delivering siRNA with a specific sequence into cells, and the synergistic curative effect of two kinds of anti-tumor treatment is realized.
According to the technical scheme, the metal-porphyrin nanoparticle provided by the disclosure has the following beneficial effects:
porphyrin is made into nano particles by a chemical method, and targeting polypeptide is modified on the surface, so that the targeting enrichment of the drug at the tumor can be improved, and the acoustodynamic effect of the drug can be enhanced. The siRNA delivered can reduce the content of antioxidant enzyme SOD2 in tumor cells, and can further enhance the killing effect of sonodynamic force on tumors, thereby realizing more thorough treatment on deep liver cancer.
Drawings
Fig. 1 is a flow chart of a preparation prepared according to an embodiment of the present disclosure.
Fig. 2A is a TEM image of nanoparticles made according to an embodiment of the present disclosure.
Fig. 2B is a graph of particle size analysis of nanoparticles made according to embodiments of the present disclosure.
Fig. 2C is an absorption profile of nanoparticles prepared according to an embodiment of the disclosure with a common porphyrin.
Fig. 2D is a fluorescence spectrum of nanoparticles prepared according to an embodiment of the disclosure and a common porphyrin.
Fig. 3A, 3B are graphs of the sonodynamic ROS generation performance of nanoparticles made according to embodiments of the present disclosure.
Fig. 3C is a gel electrophoresis image of nanoparticles prepared according to embodiments of the present disclosure.
Fig. 3D is a graph of surface potential changes during nanoparticle synthesis prepared according to an embodiment of the present disclosure.
Fig. 4A is a western blot of nanoparticles prepared according to embodiments of the present disclosure down-regulating intracellular superoxide dismutase 2.
Fig. 4B is a graph of relative quantification of superoxide dismutase 2 down-regulated by nanoparticles made according to embodiments of the present disclosure.
Detailed Description
For purposes of promoting a better understanding of the objects, aspects and advantages of the disclosure, reference is made to the following detailed description taken in conjunction with the accompanying drawings.
The invention provides a preparation method of porphyrin-based metal-organic nanoparticles, which comprises the following steps:
a) dissolving ferric trichloride and porphyrin in deionized water, and stirring at room temperature (1000rpm) for 24 h;
b) transferring the above solution into 30KD ultrafilter tube, and centrifuging at room temperature (4000rpm) for 15 min;
c) uniformly blowing the precipitate with 3mL of deionized water, placing the precipitate into an ultrafiltration tube, centrifuging (4000rpm) for 15-25min, and repeating the step for 3 times;
d) resuspending the precipitate with 3mL of deionized water to prepare nanoparticles preliminarily;
e) the dimer of DPA, bis (DPA), was first prepared in conjunction with applicants previous work (g.liu, k.y.choi, a.bharde, m.swiercewska, x.chen, angelw.chem.int.ed.2012, 51, 445-. Coupling was then performed using the prepared lutidine amine dimer (bis (DPA)) and RGD. First, 3.38mg of Bis (DPA) and 2.18mg of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt (Sulfo-SMCC) were added to 2mL of dimethyl sulfoxide (DMSO), shaken to be uniformly dispersed, and reacted for 2 hours or more. Second, 1.73mg of cRGDyK polypeptide was dispersed in 2mL of PBS (pH 7.4), followed by the addition of 0.6mg of Traut reagent. Reacting for 2h at 4 ℃ to prepare RGD-SH. Thirdly, bis (DPA) -SMCC was dispersed in DMSO and RGD-SH was dispersed in PBS (pH 7.4) at a molar ratio of 1:1 and reacted at 4 ℃ for 24 hours, and prepared bis (DPA) -RGD was purified by HPLC. Finally, 2mL of Bis (DPA) -RGD solution (2mM, dispersed in DMSO) and Zn were simply mixed2+The solution (4mM) was stirred for 2 hours to prepare Bis (DPA-Zn) -RGD.
f) And (3) taking 1mL of preliminarily prepared nanoparticles, adding 10-100ul of Bis (DPA-Zn) -RGD, stirring at room temperature (1000rpm) overnight, repeating the step (c), and resuspending the precipitate with 1mL of deionized water to prepare the targeting nanoparticles.
g) The targeting nanoparticles and siRNA were mixed according to a ratio of 50-100 ug: mixing the materials according to the proportion of 10pM, standing for 30-60min to obtain the final porphyrin-metal nanoparticles.
The metal-porphyrin nanoparticle provided by the disclosure modifies targeting polypeptide through the combination of metal ions and porphyrin (TPPS), co-delivers siRNA, and is prepared into the metal-porphyrin nanoparticle (NanoTPPS), the particle size of the metal-porphyrin nanoparticle is about 100nm, the metal-porphyrin nanoparticle has better tumor targeting performance and ROS (reactive oxygen species) generation performance through acoustic power, and meanwhile, the metal-porphyrin nanoparticle is combined with gene therapy, and has a good effect of killing tumor cells.
Fig. 1 is a flowchart of a method for preparing nanoparticles according to an embodiment of the present disclosure, and as shown in fig. 1, the method for preparing porphyrin-metal nanoparticles according to the present disclosure includes:
step 1: adding porphyrin and metal ions into ultrapure water, stirring at room temperature, and ultrafiltering to obtain a nanoparticle precursor;
the porphyrin in this example was meso-tetra (4-sulfophenyl) porphyrin, the mass of which was 1mg, and the volume of ultrapure water was
5ml of the solution; the metal ion in the present embodiment is iron ion (Fe)3+) The mass is 20mg, in other embodiments,
for other scenarios, other ions are possible, such as manganese ion (Mn)2+) Gadolinium (Gd)2+) Zinc ion (Zn)2 +) Copper ion (Cu)2+) Etc.;
the stirring speed in this example is 1000rpm, and the stirring time is 12 hours;
in the embodiment, the molecular weight of an ultrafiltration tube used in the ultrafiltration process is 30KD, the centrifugal speed is 4000rpm, and the centrifugal time is 15 min;
step 2: adding bis (DPA) and Sulfo-SMCC into DMSO, shaking and uniformly mixing, and standing for reaction to obtain bis (DPA) -SMCC.
In this example, the mass of bis (DPA) was 3.38mg, the mass of Sulfo-SMCC was 2.18mg, the volume of DMSO was 2mL, and the reaction time was 2-4 h.
And step 3: adding a Traut reagent into the PBS solution of the cRGDyK polypeptide, and reacting to obtain RGD-SH.
In this example, the mass of the crgyk polypeptide is 1.73mg, the pH of the PBS solution is 7.4, and the volume is 2 mL;
in this example, the Traut reagent had a mass of 0.6 mg;
the reaction temperature in this example was 4 ℃ and the reaction time was 2 hours;
and 4, step 4: mixing a DMSO solution of Bis (DPA) -SMCC with a PBS solution of RGD-SH, and reacting and purifying to obtain Bis (DPA-Zn) -RGD;
in the embodiment, the molar ratio of bis (DPA) -SMCC to RGD-SH is 1: 1;
the reaction temperature in this example is 4 ℃ and the reaction time is 24 hours;
in this example, it was purified by HPLC;
and 5: adding zinc ions (Zn) into the Bis (DPA-Zn) -RGD solution2+) And preparing Bis (DPA-Zn) -RGD.
In this example, Bis (DPA-Zn) -RGD was dissolved in DMSO at a concentration of 2 mM;
in this example, Zn2+The concentration is 4mM, the reaction condition is stirring (1000 revolutions per minute) and the time is 2 hours;
step 6: adding Bis (DPA-Zn) -RGD into the nanoparticle precursor solution, reacting overnight, and performing ultrafiltration purification to obtain the target nanoparticles.
In this example, 1mg of nanoparticle precursor was added to 5mL of ultrapure water;
in this example, the concentration of Bis (DPA-Zn) -RGD was 1mM, the volume was 100uL,
in this example, the reaction conditions were room temperature stirring (1000 rpm);
in the embodiment, the molecular weight of the ultrafiltration tube is 30KD, the centrifugal speed is 4000rpm, and the centrifugal time is 30 min;
and 7: adding siRNA into the targeting nanoparticle solution, reacting to obtain the final porphyrin-metal nanoparticles, and storing the final porphyrin-metal nanoparticles at the temperature of 4 ℃ in a dark place.
In this example, the siRNA was selected to have the sequence 5'-CAACAGGCCUUAUUCCACU-3'.
In this example, the ratio of targeting nanoparticles to siRNA is 50-100 ug: 10 pM.
In this example, the reaction was carried out at 4 ℃ for 60 min.
FIG. 2A is a TEM image of nanoparticles prepared according to this example; FIG. 2B is a graph showing the distribution of the particle size of nanoparticles prepared according to this example
As can be seen from fig. 2A and 2B, the nanoparticles are in the form of fusiform, uniform in particle size, and stable in structure; the average diameter of the nanoparticles in water is 100nm, and the particle size provides guarantee for the in vivo application of the nanoparticles.
Based on the nanoparticles prepared in the above examples, basic performance tests were performed.
Fig. 2C and 2D are ultraviolet absorption curve and fluorescence spectrum of nanoparticles and TPPS. As shown in fig. 2C and 2D, after nanocrystallization, the drug absorption curve is red-shifted, and the drug is significantly absorbed in the infrared region and can excite the fluorescence in the infrared region, which is of great significance for tracing the drug in vivo.
Fig. 3A and 3B are sonodynamic ROS generation performance detection diagrams of porphyrin-metal nanoparticles prepared according to an embodiment of the present disclosure, and ROS generation performance thereof is detected by using a ROS detection reagent DMA, so that it can be seen that the prepared nanoparticles have a better sonodynamic ROS generation performance and have a concentration dependency within a certain range.
Fig. 3C is a siRNA gel block electrophoretic assay of porphyrin-metal nanoparticles prepared according to an embodiment of the disclosure, showing that when the ratio of nanoparticles to siRNA is 50 μ g: 10pM and 100. mu.g: at 10pM, the nanoparticles and siRNA can be fully bound.
FIG. 3D is a graph of surface potential change during the synthesis of porphyrin-metal nanoparticles prepared according to the examples of the present disclosure, with a pure nanoparticle surface potential of-65 + -4.08 mV, a nanoparticle surface potential of-20 + -1.63 mV after the surface modification of the targeting polypeptide, and a targeted nanoparticle surface potential of-34 + -1.69 mV after loading with siRNA.
Fig. 4A and 4B are a Western blot result graph and a relative quantitative graph thereof of the effect of porphyrin-metal nanoparticles on intracellular SOD2 prepared according to the embodiments of the present disclosure, respectively, in which it can be seen that after the nanoparticles are loaded with siRNA that down-regulates SOD2 expression, the nanoparticles are incubated with cells for 24 hours, the content of antioxidant enzyme SOD2 in the cells can be significantly down-regulated, and the down-regulation effect is more significant after the nanoparticles are nanocrystallized compared to simple siRNA, because the targeted nanoparticles can deliver more siRNA into the cells to produce the effect.
Claims (9)
1. A metal-porphyrin nanoparticle with sonodynamic therapeutic effect is prepared by reacting sulfonic acid group of porphyrin with metal ion to form nanoparticle, modifying targeting polypeptide on the surface of the nanoparticle, and loading functional siRNA to form multifunctional metal-porphyrin nanoparticle with tumor specific targeting effect; wherein the content of the first and second substances,
the porphyrin is a porphyrin molecule modified by sulfonate ions;
the metal ions comprise at least one of iron ions, manganese ions, gadolinium ions, zinc ions and copper ions;
the target polypeptide is at least one of cRGDfC, cRGDyK, cRGDyC and cRGDfK;
the siRNA can reduce SOD in cells2Expressed siRNA.
2. The metal-porphyrin nanoparticle for sonodynamic therapy according to claim 1, wherein said porphyrin molecule is meso-tetrakis (4-sodium sulfophenyl) porphyrin.
3. Use of the metal-porphyrin nanoparticle with sonodynamic therapeutic effect according to claim 1 or 2 for the preparation of a formulation for the treatment of tumors.
4. The use of claim 3, wherein the tumor therapy preparation is a sonodynamic and gene combination therapy cancer preparation.
5. A preparation method of metal-porphyrin nanoparticles with sonodynamic therapeutic effect comprises the following steps:
1) preparing a nano structure assembled by porphyrin and metal ions; the porphyrin is a porphyrin molecule modified by sulfonate ions;
the metal ions comprise at least one of iron ions, manganese ions, gadolinium ions, zinc ions and copper ions;
2) preparing Zn (II) -lutidine dimer coupled with targeting polypeptide, wherein the targeting polypeptide refers to one of cRGDfC, cRGDyK, cRGDyC and cRGDfK;
3) adding Zn (II) -lutidine amine dimer with targeting polypeptide coupling prepared in the step 2) into the nano structure prepared in the step 1) for surface modification.
6. The method of claim 5, wherein the metal-porphyrin nanoparticle having an effect on sonodynamic therapy comprises the following steps:
a) dissolving ferric trichloride and porphyrin in deionized water, and stirring at 100-2000rpm at room temperature for 12-36 h;
b) transferring the solution into a 30KD ultrafiltration tube, and centrifuging at 3000-5000rpm for 10-20min at room temperature;
c) removing supernatant, collecting precipitate, blowing with 1-5mL deionized water, mixing, centrifuging in ultrafiltration tube at 3000-;
d) resuspending the precipitate with 1-5mL of deionized water to prepare primary nanoparticles;
e) firstly, preparing dimer of DPA, bis (DPA); then coupling by using prepared bis (DPA) and RGD;
firstly, adding 2-4mg of Bis (DPA) and 2-4mg of Sulfo-SMCC into 1-3mL of DMSO, shaking until the mixture is uniformly dispersed, and reacting for 2-10 h;
secondly, dispersing 1-2mg of cRGDyK polypeptide into 1-3mL of PBS, and then adding 0.5-0.7mg of Traut reagent; reacting at 3-5 ℃ for 0.5-5h to prepare RGD-SH;
thirdly, dispersing bis (DPA) -SMCC into DMSO, dispersing RGD-SH into PBS, reacting at 3-5 ℃ for 12-36h according to the molar ratio of 1-2:1-2, and purifying the prepared bis (DPA) -RGD by HPLC;
finally, a mixed solution containing 1-3mM of bis (DPA) -RGD and 3-5mM of Zn dispersed in DMSO was prepared2+Stirring the solution for 1-3h to prepare Bis(DPA-Zn)-RGD；
f) Taking 0.5-2mL of the primary nanoparticles prepared in the step d), adding 10-100ul of Bis (DPA-Zn) -RGD, stirring at room temperature overnight, repeating the step (c), and resuspending the precipitate with 0.5-2mL of deionized water to prepare targeted nanoparticles;
g) the targeting nanoparticles and siRNA were mixed according to a ratio of 50-100 ug: mixing the materials according to the proportion of 10pM, and standing for 30-60min to obtain the final metal-porphyrin nanoparticle.
7. The method for preparing metal-porphyrin nanoparticles having sonodynamic therapeutic effect of claim 6, wherein the step 1) is:
a) dissolving ferric trichloride and porphyrin in deionized water, and stirring at room temperature for 24 h;
b) transferring the above solution into 30KD ultrafilter tube, and centrifuging at room temperature for 15 min;
c) blowing the precipitate with 3mL of deionized water, mixing, placing in an ultrafiltration tube, centrifuging for 15-25min, and repeating the step for 3 times;
d) the above precipitate was resuspended in 3mL of deionized water to make the nanostructure.
8. The method for preparing metal-porphyrin nanoparticles having sonodynamic therapeutic effect of claim 6, wherein the step 2) is: firstly, preparing dimer of DPA, bis (DPA), and then coupling by using the prepared bis (DPA) and RGD;
firstly, 3.38mg of Bis (DPA) and 2.18mg of Sulfo-SMCC are added into 2mL of DMSO, shaken until the mixture is uniformly dispersed, and reacted for more than 2 hours;
second, 1.73mg of cRGDyK polypeptide was dispersed in 2mL PBS followed by the addition of 0.6mg of Traut reagent; reacting at 4 ℃ for 2h to prepare RGD-SH;
thirdly, bis (DPA) -SMCC is dispersed into DMSO, RGD-SH is dispersed into PBS to react for 24 hours at 4 ℃ according to the molar ratio of 1:1, and the prepared-RGD is purified by HPLC;
finally, 2mL of a 2mM solution of bis (DPA) -RGD dispersed in DMSO and 4mM of Zn were simply mixed2+Stirring the solution for 2h to prepare Bis (DPA-Zn)-RGD；
And (3) taking 1mL of preliminarily prepared nanoparticles, adding 10-100ul of Bis (DPA-Zn) -RGD, stirring overnight at room temperature, repeating the step (c), and resuspending the precipitate with 1mL of deionized water to prepare the targeting nanoparticles.
9. A metal-porphyrin nanoparticle having sonodynamic therapeutic effect, prepared by the method of any one of claims 6-8.
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