CN111135314A - Nano-composite for early diagnosis and treatment of gastric cancer and preparation method thereof - Google Patents
Nano-composite for early diagnosis and treatment of gastric cancer and preparation method thereof Download PDFInfo
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- CN111135314A CN111135314A CN202010098157.8A CN202010098157A CN111135314A CN 111135314 A CN111135314 A CN 111135314A CN 202010098157 A CN202010098157 A CN 202010098157A CN 111135314 A CN111135314 A CN 111135314A
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Abstract
The invention discloses a nano-composite for early diagnosis and treatment of gastric cancer and a preparation method thereof. The nano composite has a core-shell structure formed by an amphiphilic copolymer, an inner core layer is formed by a distearoyl phosphatidyl ethanolamine fragment, an outer shell layer is formed by a polyethylene glycol fragment, near-indocyanine green and hydrophobic gastric cancer chemotherapy drugs are wrapped in the inner core layer, and the surface of the outer shell layer is connected with RGD short peptide. The nano-composite preparation integrates targeted gastric cancer treatment and fluorescence imaging tracing, can realize targeted aggregation on gastric cancer focus imaging, controls the release of drugs, increases the concentration of targeted site drugs and improves the action time of the drugs, and has wide application prospect in early gastric cancer targeted diagnosis and treatment and drug delivery.
Description
Technical Field
The invention relates to the technical field of high polymer materials and medical engineering, in particular to a nano-composite for early diagnosis and treatment of gastric cancer and a preparation method thereof.
Background
Gastric cancer is a common malignant tumor in China, the number of new cases is over 100 ten thousand per year, more than 80 percent of cases are diagnosed in middle and advanced stages, and the number of death cases is up to 70 ten thousand per year, so that diagnosis and treatment of gastric cancer and colorectal cancer become problems to be solved urgently. At present, the diagnosis of part of early lesions by an endoscope is difficult, the control of incisal margins under the endoscope is difficult, and the treatment of gastric cancer still generally adopts oral or injection anti-cancer chemotherapeutic drugs (chemotherapy) in clinic. However, the specific aggregation effect of the drug at the tumor site is poor, and an effective targeting technology is lacked, so that normal cells are attacked by the drug, and toxic and side effects are inevitably generated; and the treatment process and the imaging tracing or diagnosis cannot be carried out simultaneously, and the restraining effect on rapidly developing and metastatic tumors is limited. Therefore, there is an urgent need for developing a new technical means for gastric cancer diagnosis and treatment that integrates targeted therapy and imaging tracing.
Indocyanine green (ICG) is a tricarbocyanine dye that can be almost completely bound to plasma proteins in plasma and whole blood, and can be guaranteed to remain almost completely in blood vessels and not easily diffuse outward. Indocyanine green is a near-infrared imaging agent approved by the U.S. Food and Drug Administration (FDA) for clinical use at present, has an emission wavelength of 795-845 nm, has the characteristics of an amphiphilic structure, namely hydrophilicity and lipophilicity, and can be used as an excellent in-vivo tissue penetrating agent. The integrin is highly expressed on the surfaces of various tumor cells and tumor vascular endothelial cells, and is low expressed in normal cells and vascular cells, and the RGD molecule is a tripeptide sequence capable of being specifically combined with the integrin and is a hot molecule for tumor targeting research. A plurality of research results show that the RGD is connected with a fluorescent molecule or a radionuclide and has a certain tumor targeted imaging function, however, the RGD and the fluorescent molecule or radionuclide conjugate has small molecular weight, high metabolism speed in vivo and easy clearance, and poor targeting is caused. Therefore, the delivery of the fluorescent molecule and the RGD short peptide molecule to the tumor site using a carrier with good biocompatibility is an important issue facing the current gastric cancer diagnosis method.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a nano-composite for early diagnosis and treatment of gastric cancer and a preparation method thereof, the nano-composite integrates targeted gastric cancer treatment and fluorescence imaging tracing, can realize targeted aggregation on gastric cancer focus imaging, simultaneously controls the release of drugs, increases the concentration of targeted site drugs and improves the action time of the drugs, and has wide application prospect in the aspects of early gastric cancer targeted diagnosis and treatment and drug delivery.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a nano-composite for early diagnosis and treatment of gastric cancer has a core-shell structure formed by an amphiphilic copolymer, an inner core layer is formed by distearoyl phosphatidyl ethanolamine fragments, an outer shell layer is formed by polyethylene glycol fragments, indocyanine green and hydrophobic gastric cancer chemotherapeutic drugs are wrapped in the inner core layer, and the surface of the outer shell layer is connected with RGD short peptide.
According to the invention, fluorescent molecule indocyanine green is modified by using amphiphilic polymer, hydrophobic gastric cancer chemotherapy drugs are coated at the same time, RGD short peptide molecules are modified to synthesize a nano fluorescent probe, the purposes of integrating tumor treatment and fluorescence development are achieved by combining the passive targeting property of nano molecules, the active targeting property of RGD short peptide and the fluorescent molecule development, and a multifunctional targeting nano drug carrier system capable of effectively improving the diagnosis and treatment effects of gastric cancer is expected to be developed.
Preferably, the amphiphilic copolymer is distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide; the hydrophobic gastric cancer chemotherapeutic drug comprises at least one of adriamycin, 5-fluorouracil, paclitaxel and cisplatin.
Preferably, the amphiphilic copolymer is distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide, more preferably, the molecular weight of the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide is 2805.5, and an amphiphilic nano micelle can be formed.
The invention takes RGD short peptide (amino acid sequence is Arg-Gly-Asp) capable of targeting gastric cancer vascular endothelial cells as a target to prepare a polymer nano micelle carrier (DSPE-PEG-Mal), simultaneously assembles and compounds indocyanine green with the carrier through hydrophobic electrostatic interaction, finally adsorbs drug adriamycin in a hydrophobic inner core to synthesize drug-loaded micelle (DSPE-PEG/ICG/DOX-RGD), and constructs the nano compound for early diagnosis and treatment of gastric cancer.
The nano composite adopts a core-shell structure formed by amphiphilic copolymers, an inner core layer is formed by hydrophobic distearoyl phosphatidyl ethanolamine segments, an outer shell layer is formed by hydrophilic polyethylene glycol segments, hydrophobic gastric cancer chemotherapeutic drugs and ICG are wrapped in the inner core layer, and RGD polypeptide molecules are connected to the hydrophilic surface of the micelle through chemical bonds. The nano-composite can realize the imaging of target aggregation at the focus of the gastric cancer, and simultaneously control the release of the drug, and has wide application prospect in the aspects of target diagnosis and treatment of the gastric cancer and drug slow release.
Preferably, the nanocomposite comprises the following components in parts by weight: 5-20 parts of distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide, 1-5 parts of indocyanine green, 0.5-1 part of hydrophobic gastric cancer chemotherapy drug and one part per million to one hundred thousand parts of RGD oligopeptide. The components can form a nano composite drug-loaded micelle in which indocyanine green and hydrophobic gastric cancer chemotherapeutic drugs are coated on the nano composite hydrophobic layer, and the RGD short peptide is connected to the hydrophilic section.
Preferably, the particle size of the nano-composite is 50-200 nm.
Preferably, the amino acid sequence of the RGD short peptide is Arg-Gly-Asp.
The invention also provides a preparation method of the nano-composite for early diagnosis and treatment of gastric cancer, which comprises the following steps:
(1) adding indocyanine green into tetrahydrofuran, adding distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide and hydrophobic gastric cancer chemotherapeutic drugs, mixing, performing ultrasonic treatment at room temperature, and concentrating to remove tetrahydrofuran;
(2) cooling to room temperature, adding deionized water under ultrasonic condition, filtering, ultrafiltering, centrifuging, collecting upper layer transparent water phase solution, and freeze drying to obtain powdered solid;
(3) preparing the powdery solid obtained in the step (2) into a solution, adding RGD short peptide, and reacting;
(4) and (4) dialyzing the reaction solution obtained in the step (3) in distilled water, and freeze-drying the dialyzed solution to obtain the nano-composite.
Preferably, in the step (1), the ultrasonic treatment is performed for 5-10 min, so that the distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide, the gastric cancer chemotherapeutic drug and the indocyanine green are fully mixed and dispersed in tetrahydrofuran, and the mixture is concentrated to be subjected to vacuum pumping on a 70 ℃ water bath and then rotary evaporation.
Preferably, in the step (2), the ultrasonic time is 5-15 min, 20mL of deionized water is added, and the polymer is self-assembled in the ultrasonic process to coat the adriamycin and the indocyanine green to form the nano micelle particles; the aperture of the filter membrane used for filtering is 220nm, the freeze drying time is 10-20 h, and the obtained nano-composite has good dispersibility.
Preferably, in the step (3), the powdery solid obtained in the step (2) is prepared into a solution with a concentration of 2mg/mL, the reaction time of adding the RGD short peptide is 4-12 h, and the thiol of the RGD short peptide can fully react with the maleimide group on the surface of the nano-composite.
Preferably, in the step (4), the cut-off molecular weight of a dialysis bag used for dialysis is 5000D, and water is changed once every 6h during dialysis to remove free unreacted RGD short peptide.
Compared with the prior art, the invention has the beneficial effects that:
the nano-composite preparation integrates targeted gastric cancer treatment and fluorescence imaging tracing, can realize targeted aggregation on gastric cancer focus imaging, controls the release of drugs, increases the concentration of targeted site drugs and improves the action time of the drugs, and has wide application prospect in early gastric cancer targeted diagnosis and treatment and drug delivery.
Drawings
Fig. 1 is a photograph of a transmission electron microscope of the nanocomposite prepared in example 1.
Fig. 2 is a photograph of a transmission electron microscope of the nanocomposite prepared in example 2.
Fig. 3 is a photograph of a transmission electron microscope of the nanocomposite prepared in example 3.
Fig. 4 is a photograph of a transmission electron microscope of the nanocomposite prepared in example 4.
Fig. 5 is a transmission electron micrograph of the nanocomposite prepared in comparative example 1.
Fig. 6 is a transmission electron micrograph of the nanocomposite prepared in comparative example 2.
Fig. 7 shows ICG loading and encapsulation efficiencies in the nanocomposites of example 1, example 2, and comparative example 3.
Fig. 8 shows the drug loading and encapsulation efficiencies of doxorubicin in the nanocomposites of example 1, example 2 and comparative example 3.
Fig. 9 is a confocal laser image in effect example 1.
Fig. 10 is a graph showing the growth of tumor volume in effect example 2.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the examples, the experimental methods used were, unless otherwise specified, conventional methods, and materials, reagents, etc., used were, unless otherwise specified, commercially available, wherein the RGD short peptide was obtained from Shanghai Chu peptide Biotech, Inc., and the amino acid sequence thereof was Arg-Gly-Asp. The molecular weight of the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide selected below is 2805.5.
Example 1
One embodiment of the nanocomposite of the present invention, a method of making the same, comprises the steps of:
(1) weighing 1 part of indocyanine green by weight, adding 5mL of tetrahydrofuran, mixing the mixture with 5 parts of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide by weight and 0.5 part of adriamycin by weight, performing ultrasonic treatment at room temperature for 10min to fully disperse and dissolve the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide, the indocyanine green and the adriamycin, mixing, placing the round-bottomed flask on a rotary evaporator, performing water bath at 70 ℃, vacuumizing, and performing rotary evaporation; when the organic solvent is completely evaporated, the nanostructure is successfully transferred from the oil phase to the water phase;
(2) after the mixture is cooled to room temperature, the ultrasonic probe acts for 15min to disperse the mixture; filtering with a 220nm filter membrane, ultrafiltering and centrifuging, removing bottom precipitate, collecting upper layer transparent water phase nano-structured solution, and freeze drying for 20 hr to obtain powdery solid;
(3) preparing 2.5mL of micellar solution with the concentration of 2mg/mL from the powdery solid obtained in the step (2), adding one part per million of RGD short peptide in weight components, and reacting for 12 h;
(4) dialyzing in distilled water by using a 5000D dialysis bag to remove free RGD short peptide, and changing water once within 6 h; and (3) freeze-drying the micelle aqueous solution to obtain the nano-composite.
The particle size distribution of the nanocomposite of this example was measured by a dynamic laser light scattering apparatus, and fig. 1 is a transmission electron micrograph of the integrated preparation for diagnosis and treatment of this example. As is clear from FIG. 1, the average particle size in this example is 122nm, the particles are spherical, the polydispersity is 0.125, and the dispersion is relatively uniform.
Example 2
One embodiment of the nanocomposite of the present invention, a method of making the same, comprises the steps of:
(1) weighing 5 parts of indocyanine green by weight, adding 5mL of tetrahydrofuran, mixing the mixture with 20 parts of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide by weight and 1 part of adriamycin by weight, performing ultrasonic treatment for 10min at room temperature to fully disperse and dissolve the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide, the indocyanine green and the adriamycin, mixing, placing a round-bottomed flask on a rotary evaporator, performing water bath at 70 ℃, vacuumizing, and performing rotary evaporation; when the organic solvent is completely evaporated, the nanostructure is successfully transferred from the oil phase to the water phase;
(2) after the mixture is cooled to room temperature, the ultrasonic probe acts for 15min to disperse the mixture; filtering with a 220nm filter membrane, ultrafiltering and centrifuging, removing bottom precipitate, collecting upper layer transparent water phase nano-structured solution, and freeze drying for 20 hr to obtain powdery solid;
(3) preparing 2.5mL of micellar solution with the concentration of 2mg/mL from the powdery solid obtained in the step (2), adding one part of RGD short peptide which is one hundred ten thousand parts by weight, and reacting for 12 hours;
(4) dialyzing in distilled water by using a 5000D dialysis bag to remove free RGD short peptide, and changing water once within 6 h; and (3) freeze-drying the micelle aqueous solution to obtain the nano-composite.
The particle size distribution of the nanocomposite of this example was measured using a dynamic laser light scattering apparatus, and fig. 2 is a transmission electron micrograph of the integrated preparation for diagnosis and treatment of this example. As is clear from FIG. 2, the average particle diameter in this example was 134nm, the particles were spherical, the polydispersity was 0.104, and the dispersion was relatively uniform.
Example 3
One embodiment of the nanocomposite of the present invention, a method of making the same, comprises the steps of:
(1) weighing indocyanine green with the weight component of 2 parts, adding 5mL of tetrahydrofuran, mixing the mixture with distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide with the weight component of 10 parts and adriamycin with the weight component of 0.6 part, performing ultrasonic treatment for 10min at room temperature to fully disperse and dissolve the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide, the indocyanine green and the adriamycin, mixing, placing a round-bottomed flask on a rotary evaporator, performing water bath at 70 ℃, vacuumizing, and performing rotary evaporation; when the organic solvent is completely evaporated, the nanostructure is successfully transferred from the oil phase to the water phase;
(2) after the mixture is cooled to room temperature, the ultrasonic probe acts for 15min to disperse the mixture; filtering with a 220nm filter membrane, ultrafiltering and centrifuging, removing bottom precipitate, collecting upper layer transparent water phase nano-structured solution, and freeze drying for 20 hr to obtain powdery solid;
(3) preparing 2.5mL of micellar solution with the concentration of 2mg/mL from the powdery solid obtained in the step (2), adding one part of RGD short peptide of twenty-ten-thousandth of weight component, and reacting for 12 h;
(4) dialyzing in distilled water by using a 5000D dialysis bag to remove free RGD short peptide, and changing water once within 6 h; and (3) freeze-drying the micelle aqueous solution to obtain the nano-composite. As is clear from FIG. 3, the average particle size in this example was 128nm, the particles were spherical, the polydispersity was 0.124, and the dispersion was relatively uniform.
Example 4
One embodiment of the nanocomposite of the present invention, a method of making the same, comprises the steps of:
(1) weighing indocyanine green with the weight component of 2 parts, adding 5mL of tetrahydrofuran, mixing the mixture with distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide with the weight component of 10 parts and adriamycin with the weight component of 0.6 part, performing ultrasonic treatment for 5min at room temperature to fully disperse and dissolve the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide, the indocyanine green and the adriamycin, mixing, placing a round-bottomed flask on a rotary evaporator, performing water bath at 50 ℃, vacuumizing, and performing rotary evaporation; when the organic solvent is completely evaporated, the nanostructure is successfully transferred from the oil phase to the water phase;
(2) after the mixture is cooled to room temperature, the ultrasonic probe acts for 5min to disperse the mixture; filtering with a 220nm filter membrane, ultrafiltering and centrifuging, removing bottom precipitate, collecting upper layer transparent water phase nano-structured solution, and freeze drying for 10 hr to obtain powdery solid;
(3) preparing 2.5mL of micellar solution with the concentration of 2mg/mL from the powdery solid obtained in the step (2), adding one part of RGD short peptide of twenty-ten-thousandth of weight component, and reacting for 12 h;
(4) dialyzing in distilled water by using a 5000D dialysis bag to remove free RGD short peptide, and changing water once within 6 h; and (3) freeze-drying the micelle aqueous solution to obtain the nano-composite. As is clear from FIG. 4, the average particle diameter in this example is 143nm, the particles are spherical, the polydispersity is 0.247, and the dispersion is relatively uniform.
Comparative example 1
The preparation method of the nanocomposite of the present comparative example includes the steps of:
(1) weighing indocyanine green with the weight component of 1 part, adding 5mL of tetrahydrofuran, mixing the mixture with distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide with the weight component of 5 parts, performing ultrasonic treatment for 10min at room temperature to fully disperse and dissolve the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide and the indocyanine green, mixing, placing a round-bottomed flask on a rotary evaporator, performing water bath at 70 ℃, vacuumizing, and performing rotary evaporation; when the organic solvent is completely evaporated, the nanostructure is successfully transferred from the oil phase to the water phase;
(2) after the mixture is cooled to room temperature, the ultrasonic probe acts for 15min to disperse the mixture; filtering with a 220nm filter membrane, ultrafiltering, centrifuging, removing bottom precipitate, collecting upper layer transparent water phase nano-structured solution, and freeze drying for 20 hr to obtain powdered solid.
The particle size distribution of the nanocomposite of the comparative example was measured using a dynamic laser light scattering apparatus, and fig. 5 is a transmission electron micrograph of the formulation of the comparative example. As is clear from FIG. 5, the average particle diameter was 102nm, the particles were spherical, the polydispersity was 0.128, and the dispersion was relatively uniform.
Comparative example 2
The preparation method of the nanocomposite of the present comparative example includes the steps of:
(1) weighing indocyanine green of 1 part by weight and adriamycin of 0.5 part by weight, adding 5mL of tetrahydrofuran, mixing the mixture with distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide of 5 parts by weight, performing ultrasonic treatment for 10min at room temperature to fully disperse and dissolve the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide, the indocyanine green and the adriamycin, placing the round-bottomed flask on a rotary evaporator after mixing, performing water bath at 70 ℃, vacuumizing, and performing rotary evaporation; when the organic solvent is completely evaporated, the nanostructure is successfully transferred from the oil phase to the water phase;
(2) after the mixture is cooled to room temperature, the ultrasonic probe acts for 15min to disperse the mixture; filtering with a 220nm filter membrane, ultrafiltering, centrifuging, removing bottom precipitate, collecting upper layer transparent water phase nano-structured solution, and freeze drying for 20 hr to obtain powdered solid.
The particle size distribution of the nanocomposite of the comparative example was measured using a dynamic laser light scattering apparatus, and fig. 6 is a transmission electron micrograph of the formulation of the comparative example. As can be seen from FIG. 6, the average particle diameter was 134nm, the particles were spherical, the polydispersity was 0.135, and the dispersion was relatively uniform.
Comparative example 3
The preparation method of the nanocomposite of the present comparative example includes the steps of:
(1) weighing indocyanine green with the weight component of 0.5 part, adding 5mL of tetrahydrofuran, mixing the mixture with distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide with the weight component of 25 parts and adriamycin with the weight component of 1.5 parts, performing ultrasonic treatment at room temperature for 10min to fully disperse and dissolve the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide, the indocyanine green and the adriamycin, placing the round-bottomed flask on a rotary evaporator after mixing, performing water bath at 70 ℃, vacuumizing, and performing rotary evaporation; when the organic solvent is completely evaporated, the nanostructure is successfully transferred from the oil phase to the water phase;
(2) after the mixture is cooled to room temperature, the ultrasonic probe acts for 15min to disperse the mixture; filtering with a 220nm filter membrane, ultrafiltering and centrifuging, removing bottom precipitate, collecting upper layer transparent water phase nano-structured solution, and freeze drying for 20 hr to obtain powdery solid;
(3) preparing 2.5mL of micellar solution with the concentration of 2mg/mL from the powdery solid obtained in the step (2), adding two parts by weight of RGD short peptide into the micellar solution, and reacting for 12 hours;
(4) dialyzing in distilled water by using a 5000D dialysis bag to remove free RGD short peptide, and changing water once within 6 h; and (3) freeze-drying the micelle aqueous solution to obtain the nano-composite.
Effect example 1 comparison of ICG and Adriamycin entrapment Effect in nanocomposites of example 1, example 2 and comparative example 3
Scanning indocyanine green and adriamycin with different concentrations dissolved in DMSO by using a UV-Vis spectrophotometer, wherein the wavelength range is 550-950 nm, and taking the absorbance of the maximum absorption peak as a standard curve. The sample solutions of example 1, example 2 and comparative example 3, in which DMSO was dissolved, were subjected to UV spectrum scanning using a UV-Vis spectrophotometer to measure absorbance values at the maximum absorption peak thereof.
Calculating the concentration of the sample solution through a standard curve, and further calculating the drug loading rate and the encapsulation rate
Drug loading rate is the mass of drug contained in the carrier/total mass of drug carrier
The encapsulation rate is the mass of the drug contained in the carrier/the total mass of the fed drug
As shown in fig. 7 and 8, the drug loading rates of ICG in the nanocomposites of example 1, example 2 and comparative example 3 were 13.1%, 16.8% and 1.30%, respectively, and the encapsulation rates were 85.0%, 87.5% and 70.2%, respectively. Thus, the encapsulation effect of the nanocomposites of example 1 and example 2 on ICG is better than that of comparative example 3
Effect example 2 in vitro fluorescence imaging Effect of the nanocomposites of example 1 and comparative example 1
Will be 1 × 105SGC7901 cells are paved in a 30mm confocal special dish, after the cells are adhered to the wall at night, targeted and non-targeted nano drug-loaded materials are diluted to 30 mu g/mL (calculated by ICG content) by a DMEM medium, and are incubated with the cells for 12 hours at 37 ℃. After three washes with PBS, fixation was performed with 4% paraformaldehyde for 1 h. After 3 times of PBS washing, DAPI light-resistant staining is carried out for 2min, PBS is used for full washing for 3 times, quenching inhibitor is dripped into a bottom glass of the dish, and a cover glass is covered and mounted. Finally, the cells are placed under a laser confocal microscope, the fluorescence of cell nucleus and ICG is respectively observed by exciting light of 365nm and 633nm, and the recombination of the cells to the nano-particles is recordedThe cell uptake of the substance is shown in the confocal image in FIG. 9.
As can be seen from fig. 9, CLSM can not only qualitatively determine the uptake of the nanocomplex into the cell, but also clearly define the intracellular distribution of the nanoparticles that have entered the cell. Since DAPI localizes the nucleus (blue in the figure) and the whole cell is localized by Bright Field (Bright Field in the figure), the specific distribution of ICG in the cell is clarified by comparing ICG (red in the figure) with the coinciding region of DAPI in blue and Bright Field (large in the figure). The fluorescence intensity of the cells of the two experimental groups is observed by a confocal microscope, and the fluorescence intensity of the red ICG in the cytoplasm of the cells incubated by the targeting nano-composite (example 1) is obviously higher than that of the cells incubated by the non-targeting nano-composite (comparative example 1), so that the fluorescence developing function is good.
Effect example 3 therapeutic effects of the nanocomposites of example 2 and comparative example 2
BALB/c nude mice female (6 weeks old, 18-20g) all animal experimental processes were approved by the ethical committee of experimental animals, and were in line with the welfare requirements of experimental animals. Constructing a tumor model: subcutaneously implanting 3 × 10 SGC7901 cells into the upper right lower limb of each nude mouse; after 2 weeks of injection, nude mice subcutaneous transplantation tumor models with low permeability were obtained by screening. Tumor volume V ═ a × b2And/2, wherein a is the major diameter of the tumor and b is the minor diameter of the tumor. Nude mice bearing tumor were randomly divided into 3 groups of 5 mice each; groups 3 of nude mice were injected intravenously with PBS, no targeting nanocomplex (comparative example 2) and targeting nanocomplex (example 2), respectively; changes of tumor volume and body weight of nude mice in future 2 weeks are tracked and recorded
The curative effect of the targeting nano-composite on the tumor is further verified through a living tumor inhibition experiment. As can be seen from fig. 10, tumors receiving the targeted nanocomplex (example 2) showed significant tumor suppression effect compared to the non-targeted nanocomplex (comparative example 2) and PBS blank control group.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. The nano-composite for early diagnosis and treatment of gastric cancer is characterized by having a core-shell structure formed by an amphiphilic copolymer, wherein an inner core layer is formed by distearoyl phosphatidyl ethanolamine segments, an outer shell layer is formed by polyethylene glycol segments, near-indocyanine green and hydrophobic gastric cancer chemotherapy drugs are wrapped in the inner core layer, and the surface of the outer shell layer is connected with RGD short peptide.
2. The nanocomposite for the early diagnosis and treatment of gastric cancer according to claim 1, wherein the amphiphilic copolymer is distearoylphosphatidylethanolamine-polyethylene glycol-maleimide, preferably distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide; the hydrophobic gastric cancer chemotherapeutic drug comprises at least one of adriamycin, 5-fluorouracil, paclitaxel and cisplatin.
3. The nanocomposite for early diagnosis and treatment of gastric cancer according to claim 2, comprising the following components in parts by weight: 5-20 parts of distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide, 1-5 parts of indocyanine green, 0.5-1 part of hydrophobic gastric cancer chemotherapy drug and one part per million to one hundred thousand parts of RGD oligopeptide.
4. The nanocomposite for early diagnosis and treatment of gastric cancer according to any one of claims 1 to 3, wherein the particle size of the nanocomposite is 50 to 200 nm.
5. The nanocomposite for early diagnosis and treatment of gastric cancer according to claim 4, wherein the amino acid sequence of the RGD short peptide is Arg-Gly-Asp.
6. The method for preparing a nano-composite for the early diagnosis and treatment of gastric cancer according to any one of claims 1 to 5, comprising the steps of:
(1) adding indocyanine green into tetrahydrofuran, adding distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide and hydrophobic gastric cancer chemotherapeutic drugs, mixing, performing ultrasonic treatment at room temperature, and concentrating to remove tetrahydrofuran;
(2) cooling to room temperature, adding deionized water under ultrasonic condition, filtering, ultrafiltering, centrifuging, collecting upper layer transparent water phase solution, and freeze drying to obtain powdered solid;
(3) preparing the powdery solid obtained in the step (2) into a solution, adding RGD short peptide, and reacting;
(4) and (4) dialyzing the reaction solution obtained in the step (3) in distilled water, and freeze-drying the dialyzed solution to obtain the nano-composite.
7. The method for preparing a nano-composite for early diagnosis and treatment of gastric cancer according to claim 6, wherein in the step (1), the ultrasonic time is 5-10 min, and the nano-composite is concentrated to be subjected to rotary evaporation after water bath vacuum pumping at 50-70 ℃.
8. The method for preparing a nano-composite for early diagnosis and treatment of gastric cancer as claimed in claim 6, wherein in the step (2), the ultrasound time is 5-15 min, the pore diameter of the filter membrane used for filtering is 220nm, and the freeze-drying time is 10-20 h.
9. The method of claim 6, wherein in the step (3), the powdered solid obtained in the step (2) is prepared into a solution with a concentration of 2mg/mL, and the reaction time for adding the RGD short peptide is 4-12 h.
10. The method for preparing nano-composite for early diagnosis and treatment of gastric cancer according to claim 6, wherein in the step (4), the cut-off molecular weight of the dialysis bag used for dialysis is 5000D, and water is changed every 6h during dialysis.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113827742A (en) * | 2021-09-26 | 2021-12-24 | 厦门大学附属翔安医院 | Breast cancer sentinel lymph node tracing targeting molecular probe and preparation method thereof |
| CN114246955A (en) * | 2021-12-22 | 2022-03-29 | 中山大学附属第三医院（中山大学肝脏病医院） | Nanoparticle and preparation method and application thereof |
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