CN112546246A - Chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells and preparation method and application thereof - Google Patents
Chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells, and a preparation method and application thereof. The SP94 short peptide can be specifically combined with GRP78 protein, the GRP78 protein is specifically expressed on the surface of tumor cells, and the targeting material SP94 short peptide-polyethylene glycol 2000-chitosan prepared by the invention can be used as a raw material for preparing an ultrasonic contrast agent, can play the role of targeting tumor cells and can prepare the targeted ultrasonic contrast agent. The chitosan derivative nano-scale ultrasonic contrast agent for targeting tumor cells prepared by the invention has better drug loading capacity, is in a nano-scale range, has targeting and high efficiency for tumor treatment, can promote the drug to enter the tumor cells by virtue of a sound hole effect generated by ultrasonic irradiation, specifically kills the tumor cells and enhances the treatment effect of the tumor cells, and the chitosan derivative nano-scale ultrasonic contrast agent carrying adriamycin for targeting tumor cells is prepared.
Description
The technical field is as follows:
the invention relates to a chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells, and a preparation method and application thereof, and belongs to the technical field of ultrasonic molecular imaging.
Background art:
the tumor seriously threatens the life health of human beings, and chemotherapy is one of the commonly used treatment methods, but the chemotherapy has the problems of poor targeting property, large systemic toxic and side effects and non-ideal treatment effect. Therefore, how to safely and efficiently deliver the chemotherapeutic drugs to the tumor focus is an urgent problem to be solved for tumor treatment.
Glucose regulated protein 78(GRP78) is present in the endoplasmic reticulum and aids in proper protein folding and assembly in normal cells, as well as being involved in regulating the degradation of misfolded proteins, the binding of calcium ions in the endoplasmic reticulum, and the activation of endoplasmic reticulum stress signals. Solid tumors are usually in a hypoxic, low glucose and pH acidic microenvironment, and these tumor microenvironment factors can promote the transfer of GRP78 protein to the cell surface through endoplasmic reticulum stress, whereas such transfer is not observed on the membrane surface of normal cells. Therefore, the unique GRP78 protein on the surface of the tumor cell membrane can become a specific molecular target for treating tumor cells.
Some drug delivery systems targeting GRP78 are currently being developed, but no relevant research has been seen in the field of ultrasound contrast agents. The targeted system of ultrasound-targeted microbubble destruction (UTMD) has unique treatment advantages, is more accurate in positioning and real-time visible compared with other imaging modes, and can promote anticancer drugs to enter tumor cells to enhance treatment effects during targeted delivery. Many studies show that compared with the traditional ultrasound contrast microbubbles, the perfluoropentane (PFP) nano droplet has smaller particle size, is easier to penetrate through the wall of a tumor neoformation capillary, and can more stably deliver the drug to the targeted tumor tissue, but the current common wrapping material for preparing the PFP nano droplet needs to be dissolved by an organic solvent, and the residual organic solvent is easy to accumulate in the body to cause toxic and side effects. The chitosan is a natural polymer material, is easy to dissolve in water, safe and nontoxic, has good biocompatibility, contains rich free amino, carboxyl and other active groups, has good modification property and is an ideal shell membrane material of the ultrasonic contrast agent.
The invention content is as follows:
aiming at the problem that no ultrasonic contrast agent related research targeting GRP78 protein exists at present, the invention provides a chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells, and a preparation method and application thereof, wherein SP94 short peptide-polyethylene glycol 2000-chitosan is used as a shell membrane material, perfluoropentane is used as an inner core liquid, and the nano-scale ultrasonic contrast agent is prepared. The invention also verifies the drug-loading (adriamycin) capability of the nano-scale ultrasonic contrast agent, and proves the excellent effect of the chitosan derivative nano-scale ultrasonic contrast agent carrying adriamycin on the aspect of targeted killing of tumor cells.
Description of terms:
glucose regulatory protein 78: glucose regulated protein 78(GRP78), is a molecular chaperone. In normal cells, help the protein fold and assemble correctly, present in the endoplasmic reticulum; can be transferred to the surface of tumor cells through endoplasmic reticulum stress reaction in the tumor microenvironment.
SP94 short peptide: can be combined with GRP78 protein, and has an amino acid sequence of SFSIIHTPILPLGGC.
MAL-polyethylene glycol 2000-NHS: maleimide polyethylene glycol active ester.
Perfluoropentane: perfluoropentane (PFP), a liquid fluorocarbon with a boiling point of 29 ℃ may be used as the ultrasound contrast agent core.
Adriamycin: doxorubicin (DOX) can inhibit the synthesis of RNA and DNA, has wide anti-tumor spectrum, has effect on various tumors, belongs to a periodic nonspecific medicine, and has killing effect on tumor cells in various growth periods.
Room temperature: having a meaning well known in the art, typically 25. + -. 2 ℃.
The technical scheme of the invention is as follows:
a targeting material SP94 short peptide-polyethylene glycol 2000-chitosan has the following structure:
the preparation method of the targeting material SP94 short peptide-polyethylene glycol 2000-chitosan is prepared by a sulfydryl-maleimide reaction, and comprises the following steps:
dissolving chitosan in deionized water, adding MAL-polyethylene glycol 2000-NHS to dissolve completely, wherein the molar equivalent ratio of the chitosan to the MAL-polyethylene glycol 2000-NHS is 1:1, and stirring for reaction at room temperature; slowly dropwise adding DMF solution of SP94 short peptide into the reaction solution, wherein the molar equivalent ratio of chitosan to SP94 short peptide is 1:1, and continuously reacting at room temperature; and (4) after dialysis and purification, freeze-drying to obtain the targeting material SP94 short peptide-polyethylene glycol 2000-chitosan.
The reaction route is as follows:
preferably, according to the invention, the weight average molecular weight of the chitosan is 3kDa to 160 kDa; further preferably 5 kDa.
According to the invention, the reaction time after adding the MAL-polyethylene glycol 2000-NHS is preferably 12-36 h; further preferably 24 hours.
According to the invention, the mass-to-volume ratio of the chitosan to the DMF solution of the SP94 short peptide is (0.01-0.03): 1 in g/mL, more preferably 0.025:1 in g/mL.
According to the invention, the reaction time after the SP94 short peptide is dripped is preferably 12-36h, and more preferably 24 h.
According to the invention, the cut-off molecular weight of the dialysis is 3000-10000 Da, and is further preferably 3500 Da.
The targeting material SP94 short peptide-polyethylene glycol 2000-chitosan is applied to the preparation of the chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells.
A chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells takes SP94 short peptide-polyethylene glycol 2000-chitosan as a shell membrane material, wherein SP94 short peptide is a targeting ligand, and perfluoropentane is wrapped inside the shell membrane; the chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells has the particle size of 250-300 nm.
The preparation method of the chitosan derivative nano-scale ultrasonic contrast agent targeting the tumor cells comprises the following steps:
(1) dissolving SP94 short peptide-polyethylene glycol 2000-chitosan in deionized water to obtain SP94 short peptide-polyethylene glycol 2000-chitosan solution;
(2) dispersing perfluoropentane, Tween 20 and lecithin in deionized water, and homogenizing to obtain a suspension;
(3) dropwise adding the SP94 short peptide-polyethylene glycol 2000-chitosan solution prepared in the step (1) into the suspension prepared in the step (2), and homogenizing to prepare a suspension emulsion;
(4) and (4) standing the suspension emulsion prepared in the step (3) at a temperature, centrifuging at a low speed, and taking the upper layer liquid to obtain the chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells.
Preferably, the mass volume percentage of the SP94 short peptide-polyethylene glycol 2000-chitosan in the SP94 short peptide-polyethylene glycol 2000-chitosan solution in the step (1) is 1-3%, and the unit is g/mL; further preferably 2% in g/mL.
Preferably, the mass volume percentage of the lecithin in the suspension in the step (2) is 0.01-0.2% in g/mL; the mass-volume ratio of the lecithin to the perfluoropentane is (0.01-0.05):1, and the unit is g/mL; the mass-volume ratio of the lecithin to the Tween 20 is (0.2-0.8):1, and the unit is g/mL; further preferably, the mass volume percentage of the lecithin in the suspension is 0.13%, and the unit is g/mL; the mass-volume ratio of the lecithin to the perfluoropentane is 0.03:1, and the unit is g/mL; the mass-volume ratio of the lecithin to the Tween 20 is 0.6:1, and the unit is g/mL.
Preferably, according to the present invention, the homogenizing speed in step (2) is 12000-20000rpm for 1-3 min; further preferably, the homogenization speed is 20000rpm and the time is 1 min.
According to the invention, the volume ratio of the suspension in the step (3) to the SP94 short peptide-polyethylene glycol 2000-chitosan solution is (10-15): 1; further preferably, the volume ratio of the suspension to the SP94 short peptide-polyethylene glycol 2000-chitosan solution is 13: 1.
Preferably according to the present invention, the homogenization in step (3) is carried out at 12000-20000rpm for 1-3 min; further preferably, the homogenization speed is 20000rpm and the time is 2 min.
Preferably, the standing time at room temperature in the step (4) is 10-30 min; further preferably, the standing time at room temperature is 20 min.
According to the invention, the low-speed centrifugation in the step (4) is performed for 2-8min at 800rpm and 100-; more preferably, the centrifugation is carried out at 300rpm for 5 min.
A chitosan derivative nano-scale ultrasonic contrast agent carrying adriamycin and targeting tumor cells takes SP94 short peptide-polyethylene glycol 2000-chitosan as a shell membrane material, wherein SP94 short peptide is a targeting ligand; the inside of the shell membrane is wrapped with adriamycin and perfluoropentane; the mass ratio of SP94 short peptide-polyethylene glycol 2000-chitosan to adriamycin is 1 (1-2); the particle size of the nano-scale ultrasonic contrast agent is 300-400 nm.
The preparation method of the doxorubicin-carrying chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells comprises the following steps:
(1) dissolving SP94 short peptide-polyethylene glycol 2000-chitosan in deionized water to obtain SP94 short peptide-polyethylene glycol 2000-chitosan solution;
(2) dispersing perfluoropentane, Tween 20, lecithin and adriamycin in deionized water, and homogenizing to obtain a suspension;
(3) dropwise adding the SP94 short peptide-polyethylene glycol 2000-chitosan solution prepared in the step (1) into the suspension prepared in the step (2), and homogenizing to prepare a suspension emulsion;
(4) and (4) standing the suspended emulsion prepared in the step (3) at a warm state, centrifuging at a low speed, and taking the upper layer liquid to obtain the doxorubicin-carried chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells.
Preferably, the mass volume percentage of the SP94 short peptide-polyethylene glycol 2000-chitosan in the SP94 short peptide-polyethylene glycol 2000-chitosan solution in the step (1) is 1-3%, and the unit is g/mL; further preferably 2% in g/mL.
Preferably, the mass volume percentage of the lecithin in the suspension in the step (2) is 0.01-0.2% in g/mL; the mass-volume ratio of the lecithin to the perfluoropentane is (0.01-0.05):1, and the unit is g/mL; the mass-volume ratio of the lecithin to the Tween 20 is (0.2-0.8):1, and the unit is g/mL; the concentration of the adriamycin in the suspension is 1.5-3 mg/mL; further preferably, the mass volume percentage of the lecithin in the suspension is 0.13%, and the unit is g/mL; the mass-volume ratio of the lecithin to the perfluoropentane is 0.03:1, and the unit is g/mL; the mass-volume ratio of the lecithin to the Tween 20 is 0.6:1, and the unit is g/mL; the concentration of the adriamycin in the suspension is 3 mg/mL.
Preferably, according to the present invention, the homogenizing speed in step (2) is 12000-20000rpm for 1-3 min; further preferably, the homogenization speed is 20000rpm and the time is 1 min.
According to the invention, the volume ratio of the suspension in the step (3) to the SP94 short peptide-polyethylene glycol 2000-chitosan solution is (10-15): 1; further preferably, the volume ratio of the suspension to the SP94 short peptide-polyethylene glycol 2000-chitosan solution is 13: 1.
Preferably according to the present invention, the homogenization in step (3) is carried out at 12000-20000rpm for 1-3 min; further preferably, the homogenization speed is 20000rpm and the time is 2 min.
Preferably, the standing time at room temperature in the step (4) is 10-30 min; further preferably, the standing time at room temperature is 20 min.
According to the invention, the low-speed centrifugation in the step (4) is performed for 2-8min at 800rpm and 100-; more preferably, the centrifugation is carried out at 300rpm for 5 min.
The application of the doxorubicin-carrying chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells in preparing antitumor drugs.
The invention has the technical characteristics that:
according to the invention, firstly, chitosan reacts with MAL-polyethylene glycol 2000-NHS to synthesize MAL-polyethylene glycol 2000-chitosan, and then the MAL-polyethylene glycol 2000-chitosan reacts with SP94 short peptide through sulfydryl-maleimide to synthesize a targeting material SP94 short peptide-polyethylene glycol 2000-chitosan, wherein the material can be used as a shell membrane material of an ultrasonic contrast agent and can be combined with tumor cells in a targeted manner.
The method also comprises the steps of homogenizing perfluoropentane, Tween 20, lecithin and adriamycin to prepare a suspension (the concentration of adriamycin in the suspension is 1.5-3mg/mL), dropwise adding SP 94-polyethylene glycol 2000-chitosan solution, homogenizing to obtain a suspension emulsion with SP94 oligopeptide-polyethylene glycol 2000-chitosan as a shell and adriamycin and perfluoropentane as an inner core, standing at room temperature, centrifuging at low speed, and taking the upper layer liquid to obtain the targeted nanoscale ultrasonic contrast agent.
Has the advantages that:
1. the SP94 short peptide is a polypeptide with an amino acid sequence of SFSIIHTPILPLGGC, can be specifically combined with GRP78 protein, and the GRP78 protein is specifically expressed on the surface of a tumor cell, so the SP94 short peptide can be used as a targeting ligand of the tumor cell. The targeting material SP94 short peptide-polyethylene glycol 2000-chitosan prepared by the invention can be used as a raw material for preparing an ultrasonic contrast agent, and can play a role of targeting tumor cells to prepare the targeted ultrasonic contrast agent.
2. The chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells, which is prepared by the invention, takes SP94 short peptide-polyethylene glycol 2000-chitosan as a shell and perfluoropentane as an inner core, has the particle size of 250-300 nm and the average particle size of about 280nm, and can penetrate through gaps of blood vessel walls of tumor tissues in a nano-scale range; the biological safety is high, the capability of targeting and combining tumor cells is strong, and the targeting and combining capability is positively correlated with the expression level of GRP78 protein on a tumor cell membrane; the perfluoropentane polymer composite material also has excellent enhanced imaging capability because the core material perfluoropentane can generate liquid-gas phase change under the ultrasonic action to enhance imaging.
3. The chitosan derivative nano-scale ultrasonic contrast agent carrying the adriamycin and targeting the tumor cells, prepared by the invention, has better drug (adriamycin) carrying capacity, takes SP94 short peptide-polyethylene glycol 2000-chitosan as a shell and takes the adriamycin and perfluoropentane as an inner core, the particle size is 300-400 nm, the average particle size is about 367nm, the chitosan derivative nano-scale ultrasonic contrast agent can penetrate through gaps of blood vessel walls of tumor tissues in a nano-scale range, has targeting and high efficiency for tumor treatment, can promote more drugs (adriamycin) to enter the tumor cells by virtue of a sound hole effect generated by ultrasonic irradiation, specifically kills the tumor cells, and enhances the treatment effect of the tumor cells.
Description of the drawings:
FIG. 1 is a synthetic scheme of targeting material SP94 short peptide-polyethylene glycol 2000-chitosan;
FIG. 2 is a hydrogen spectrum (1HNMR) of a targeting material SP94 short peptide-polyethylene glycol 2000-chitosan;
FIG. 3 is an optical microscope photograph of a chitosan derivative nanoscale ultrasound contrast agent targeting tumor cells;
FIG. 4 is a particle size distribution diagram of a chitosan derivative nanoscale ultrasound contrast agent targeting tumor cells;
FIG. 5 is zeta potential diagram of chitosan derivative nanoscale ultrasound contrast agent targeting tumor cells;
FIG. 6 is a bar graph of cell viability under different concentrations of tumor cell-targeting chitosan derivative nanoscale ultrasound contrast agent; in the figure, the abscissa is the contrast agent concentration and the ordinate is the cell viability;
FIG. 7 is a bar graph of hemolysis rate under different concentrations of tumor cell-targeting chitosan derivative nanoscale ultrasound contrast agents; in the figure, the abscissa is the concentration of the contrast agent, and the ordinate is the hemolysis rate;
FIG. 8 is a dynamic imaging diagram of a chitosan derivative nanoscale ultrasound contrast agent targeting tumor cells;
FIG. 9 is a time intensity curve for imaging of a chitosan derivative nanoscale ultrasound contrast agent targeting tumor cells;
FIG. 10 shows the distribution of GRP78 protein in WPMY-1 cells and 22RV1 cells; wherein; nuclei were stained blue with DAPI and GRP78 protein green with Dylight 488;
FIG. 11 is a targeted fluorescence picture and corresponding flow cytometric analysis of chitosan derivative nanoscale ultrasound contrast agent targeting tumor cells with WPMY-1 cells and 22RV1 cells;
FIG. 12 is a targeted fluorescence image of a chitosan derivative nanoscale ultrasound contrast agent targeted to tumor cells and 22RV1 cells after 4-PBA treatment or not;
FIG. 13 is a fluorescence image of a chitosan derivative nanoscale ultrasound contrast agent carrying doxorubicin targeting tumor cells under a 100-fold oil-scope;
FIG. 14 particle size distribution plot of doxorubicin-loaded chitosan derivative nanoscale ultrasound contrast agents targeting tumor cells;
FIG. 15 is a drug loading and encapsulation efficiency curve for a tumor cell targeted doxorubicin-loaded chitosan derivative nanoscale ultrasound contrast agent;
FIG. 16 is a fluorescence image of tumor cell targeted chitosan derivative nanoscale ultrasound contrast agent carrying doxorubicin incubated with 22RV1 cells for 1h, 2h, 4 h;
FIG. 17 is a bar graph of fluorescence intensity after 1h, 2h, 4h incubation of doxorubicin-loaded chitosan derivative nanoscale ultrasound contrast agent targeted to tumor cells with 22RV1 cells;
figure 18 is a bar graph of 22RV1 cell viability under different treatment conditions.
The specific implementation mode is as follows:
the technical solution of the present invention is further illustrated by the following examples, but the scope of the present invention is not limited thereto.
The raw materials and reagents mentioned in the examples are, unless otherwise specified, all common commercial products.
SP94 short peptide was purchased from Sairanxi, chitosan (weight average molecular weight 5kDa, degree of deacetylation 90%) from Sigma-Aldrich.
Example 1: synthesis and identification of targeting material SP94 short peptide-polyethylene glycol 2000-chitosan
The synthesis of targeting material SP94 short peptide-polyethylene glycol 2000-chitosan is prepared by a sulfhydryl-maleimide reaction, the synthetic route is shown in figure 1, and the method comprises the following steps:
weighing 50mg of chitosan, dissolving in 10mL of deionized water, adding MAL-polyethylene glycol 2000-NHS to dissolve completely, wherein the molar equivalent ratio of the chitosan to the MAL-polyethylene glycol 2000-NHS is 1:1, and stirring and reacting for 24 hours at room temperature; slowly dropwise adding 2mL of DMF solution of SP94 short peptide into the reaction solution, wherein the molar equivalent ratio of the chitosan to the SP94 short peptide is 1:1, and continuously reacting for 24h at room temperature; then transferring the reaction solution to a dialysis bag (with molecular weight cutoff of 3500Da) to dialyze and purify in deionized water for 48h, taking the dialysate to freeze-dry to obtain SP94 short peptide-polyethylene glycol 2000-chitosan.
Identification of targeting material SP94 short peptide-polyethylene glycol 2000-chitosan:
identifying the final product by hydrogen spectrum (1HNMR), with the identification result shown in FIG. 2; in the figure, the characteristic peak of SP94 short peptide is 7.15-7.26ppm, the characteristic peak of chitosan is 3.61-3.79ppm, and two characteristic peaks can be observed on SP94 short peptide-polyethylene glycol 2000-chitosan, thereby verifying the successful synthesis of the targeting material SP94 short peptide-polyethylene glycol 2000-chitosan.
Example 2: preparation of chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells
A preparation method of a chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells comprises the following steps:
(1) adding 0.02g of SP94 oligopeptide-polyethylene glycol 2000-chitosan prepared in example 1 into 1mL of deionized water, and dissolving to obtain 0.02g/mL of SP94 oligopeptide-polyethylene glycol 2000-chitosan solution;
(2) dispersing 0.15mL of perfluoropentane, 0.006mL of Tween 20 and 4mg of lecithin in 2.7mL of deionized water, and homogenizing at 20000rpm for 1min to obtain a suspension;
(3) dropwise adding 0.225mL of SP94 oligopeptide-polyethylene glycol 2000-chitosan solution prepared in the step (1) into the suspension prepared in the step (2), and homogenizing at 20000rpm for 2min to obtain a suspension emulsion;
(4) and (4) standing the suspension emulsion prepared in the step (3) at room temperature for 10min, centrifuging at 300rpm for 5min, and taking the upper layer liquid to obtain the chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells.
The nano-scale ultrasonic contrast agent is observed by an optical microscope, and the result is shown in figure 3, wherein the nano-scale ultrasonic contrast agent is round under the optical microscope of 1000 times, uniform in particle size, uniform in dispersion and free of aggregation.
After the nanoscale ultrasonic contrast agent is diluted by deionized water, the particle size and the Zeta potential of the nanoscale ultrasonic contrast agent are detected by a nanometer laser particle size and Zeta potential analyzer, and the results are shown in fig. 4 and 5, and it can be seen from the graphs that the particle size of the nanoscale ultrasonic contrast agent is 250-300 nm, the average particle size is about 280nm, the dispersion index is less than 0.3, and the Zeta potential is +14.94 mV.
Example 3: evaluation of biocompatibility of chitosan derivative nanoscale ultrasound contrast agent targeting tumor cells
(1) At 2X 104(density per well) prostate cancer 22RV1 cells are inoculated in a 96-well plate, fresh RMPI-1640 culture medium containing chitosan derivative nano-scale ultrasonic contrast agents prepared in example 2 with different concentrations (5-800. mu.g/mL) is added, after incubation for 24h, the culture medium is changed into fresh culture medium containing 10% CCK-8, incubation is continued for 2h, absorbance is detected by a microplate reader (450nm), and the cell survival rate is calculated. The results are shown in FIG. 6, and the cell viability was over 80% in all concentration groups.
(2) The hemolysis test was approved by the ethical committee of the study of the university of Shandong, Qilu Hospital. The donor signed an informed consent before the experiment began. Taking 4mL of anticoagulated blood, diluting with 0.9% sterile physiological saline, and centrifuging at 1500rpm for 10min to obtain a blood cell suspension; 5mL of the chitosan derivative nano-scale ultrasonic contrast agent prepared in the embodiment 2 with different concentrations (100-800 μ g/mL) is added into 100 μ L of the obtained blood cell suspension, the blood cell suspension is incubated at 37 ℃ for 1h and then centrifuged at 1200rpm for 5min, supernatant is taken, absorbance is measured by using an enzyme-labeling instrument (545nm), deionized water is used as a positive control, and physiological saline is used as a negative control. The hemolysis ratio of each group is shown in FIG. 7, and the hemolysis ratio of all concentration groups is 2% or less.
The above results all show that the chitosan derivative ultrasound contrast agent prepared in example 2 has high biosafety.
Example 4: ultrasonic imaging capability of chitosan derivative nano-scale ultrasonic contrast agent for targeting tumor cells
The ultrasonic imaging ability of the nanoscale ultrasonic contrast agent was examined using a 9L probe of a clinical diagnostic ultrasound apparatus (LOGIQ E9; GE, USA), the chitosan derivative nanoscale ultrasonic contrast agent prepared in example 2 or PBS (control) was placed in a pipette and clamped closed, main parameters: center frequency 9.0 MHz; the emission power is 70%; the dynamic range is 60dB, the two-dimensional and contrast modes synchronously observe the change in continuous time, the change is recorded as a dynamic imaging file to perform TIC analysis, the imaging result is shown in figure 8, and a time intensity curve is drawn, as shown in figure 9. As can be seen from fig. 8, the nanoscale ultrasound contrast agent achieves significant ultrasound enhancement compared to the control group (PBS), and the ultrasound contrast intensity gradually decreases with time; fig. 9 records the decay of the nanoscale ultrasound contrast agent over the first 10 minutes, which is seen to be relatively slow over 10 minutes.
Example 5: distribution of GRP78 protein in different cell lines
Many studies show that the GRP78 protein is highly expressed on the cell membrane of prostate cancer, however, no study is currently made to directly verify whether the protein is expressed on the castration-resistant prostate cancer 22RV1 cell line, so that the immunofluorescence experiment is used to verify that the human normal prostate stromal cell line WPMY-1 is used as a control group. At 4X 104Density per well two cells were seeded separately on a slide in a 24-well plate,after overnight adherence was fixed with 4% paraformaldehyde for 20min, washed three times with PBS and blocked, then anti-GRP 78 antibody was added and left at 4 ℃ overnight. The following day was incubated with Dylight488 (green) labeled fluorescent secondary antibody and observed by fluorescent microscope. As a result, as shown in FIG. 10, much green fluorescence was attached to the cell membrane of 22RV1 cell, whereas no significant green fluorescence was observed on the cell membrane of WPMY-1. This indicates that the GRP78 protein is highly expressed on the cell membrane of 22RV1 cells and is less expressed on the cell membrane of WPMY-1 cells.
Example 6: cell targeting capability of chitosan derivative nano-scale ultrasonic contrast agent for targeting tumor cells
(1) WPMY-1 cells and 22RV1 cells at 2X 105Inoculating the cells/well into a 6-well plate, respectively adding a DiI-labeled chitosan derivative nano-scale ultrasonic contrast agent (SP94-NDs) or chitosan nano-scale ultrasonic contrast agent (NDs) for targeting tumor cells prepared in example 2 after adhesion, performing co-incubation for 2h, washing away unbound contrast agent by PBS, staining cell nuclei by DAPI, and analyzing the cell targeting capability of the contrast agent by a fluorescence microscope and a flow cytometer, wherein the result is shown in FIG. 11, and SP94-NDs and 22RV1 cells are more strongly targeted to bind compared with the NDs; the targeted binding of SP94-NDs to 22RV1 cells was significantly higher than that of WPMY-1 cells.
To further confirm that the stronger cell targeting ability of the chitosan derivative nanoscale ultrasound contrast agent for targeting tumor cells prepared in example 2 is mediated by GRP78 protein, an inhibition test was performed: before adding a contrast agent, 22RV1 cells and an endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) are incubated for 2h, and then a fluorescence microscope is used for observation, so that the result is shown in figure 12, the specific binding of SP94-NDs and 22RV1 cells can be blocked by 4-PBA, the SP94-NDs can be targeted and bound to 22RV1 cells, and the targeted binding capacity is positively correlated with the expression level of GRP78 proteins of the cells.
The preparation method of the chitosan nano-scale ultrasonic contrast agent (NDs) refers to the preparation method of the chitosan derivative nano-scale ultrasonic contrast agent in example 2, and the difference is that: 0.02g/mL of chitosan solution (0.225 mL) was added dropwise in step (3).
Example 7: preparation and optimization of doxorubicin-carrying chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells
A preparation method of a chitosan derivative nano-scale ultrasonic contrast agent carrying adriamycin and targeting tumor cells comprises the following steps:
(1) adding 0.02g of SP94 oligopeptide-polyethylene glycol 2000-chitosan prepared in example 1 into 1mL of deionized water, and dissolving to obtain 0.02g/mL of SP94 oligopeptide-polyethylene glycol 2000-chitosan solution;
(2) dispersing 0.15mL of perfluoropentane, 0.006mL of Tween 20, 4mg of lecithin and adriamycin in 2.7mL of deionized water, and homogenizing at 20000rpm for 1min to obtain a suspension; preparing four groups of suspensions, wherein the concentrations of the adriamycin in the suspensions are 1.5, 2.0, 2.5 and 3.0mg/mL respectively;
(3) dropwise adding 0.225mL of SP94 oligopeptide-polyethylene glycol 2000-chitosan solution prepared in the step (1) into the suspension prepared in the step (2), and homogenizing at 20000rpm for 2min to obtain a suspension emulsion;
(4) and (4) standing the suspoemulsion prepared in the step (3) at room temperature for 10min, centrifuging at 300rpm for 5min, and taking the upper layer liquid to obtain the doxorubicin-carrying chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells.
The chitosan derivative nano-scale ultrasonic contrast agent carrying the adriamycin is diluted and then dripped on a glass slide, and the result of observation by a fluorescence microscope is shown in figure 13, which shows that the chitosan derivative nano-scale ultrasonic contrast agent is spherical and is uniformly dispersed without aggregation.
The particle size is detected by a nano laser particle size analyzer, the result is shown in figure 14, and the result shows that the particle size of the drug-loaded nano-scale ultrasonic contrast agent is 300-400 nm, the average particle size is about 367nm, and the dispersion index is less than 0.3.
The drug loading and encapsulation efficiency of contrast agents prepared with doxorubicin at different concentrations are shown in fig. 15, and are most desirable at a concentration of 3.0 mg/mL.
Example 8: cell uptake of doxorubicin-loaded chitosan derivative nanoscale ultrasound contrast agents targeting tumor cells
22RV1 cells at 4X 104One/well was inoculated in a 24-well plate and the system of example 7 was added thereto after overnight adherenceThe prepared doxorubicin-carrying chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells is incubated for 1, 2 and 4 hours, then fixed by 4% paraformaldehyde, washed three times by PBS and observed by a fluorescence microscope, and the result is shown in FIG. 16, and the fluorescence intensity is quantitatively analyzed by image J1.46 r, and the quantitative result is shown in FIG. 17. As can be seen from fig. 16 and 17, the doxorubicin-loaded nanoscale ultrasound contrast agent prepared in example 7 can aggregate around 22RV1 cells, and this aggregation is time-dependent, with the more doxorubicin-loaded contrast agent that aggregates around 22RV1 cells as the incubation time is extended.
Example 9: killing effect of targeted tumor cell chitosan derivative nano-scale ultrasonic contrast agent carrying adriamycin on tumor in combination with ultrasonic irradiation
The half lethal dose of doxorubicin (IC50) was first established by a dose-cell activity curve, followed by the CCK-8 experiment. 22RV1 cells at 4X 104One/well is inoculated on a 96-well plate, different treatments (PBS, DOX, DOX-NDs, SP94-DOX-NDs, DOX + US, DOX-NDs + US, SP94-DOX-NDs + US) are respectively given, and the ultrasonic irradiation parameters are as follows: sound intensity 0.5W/cm2Time 60 sec. CCK-8 measures cytotoxicity in each treatment group.
The results are shown in fig. 18, for each group of cytotoxicity:
SP94-DOX-NDs+US>DOX-NDs+US>DOX+US>DOX>SP94-DOX-NDs>DOX-NDs>PBS。
the results of the higher cytotoxicity of SP94-DOX-NDs + US group (30.67% ± 1.16%) and DOX-NDs + US group (39.33% ± 2.52%) compared to DOX + US group (47.33% ± 2.89%), the lower cytotoxicity of SP94-DOX-NDs group (68.67% ± 1.53%) and DOX-NDs group (71.33% ± 2.89%) compared to DOX group (53.00% ± 3.00%), indicate that ultrasound radiation plays a key role in tumor therapy mediated by nanoscale ultrasound contrast agents, and that when ultrasound destroys nanoscale ultrasound contrast agents, transient shock waves form larger pores in cell membranes, possibly leading to more drug entry into cells and enhancing therapeutic effect; meanwhile, the nanoscale ultrasonic contrast agent can protect DOX from being released, so that the systemic toxicity caused by DOX is reduced; in addition, the cell survival rate of the SP94-DOX-NDs + US group (30.67% + -1.16%) was lower than that of the DOX-NDs + US group (39.33% + -2.52%), which indicates that the modification of the SP94 short peptide further enhances the therapeutic ability of the drug-loaded nanoscale ultrasound contrast agent.
The preparation method of the chitosan nano-scale ultrasound contrast agent (DOX-NDs) carrying doxorubicin, which is different from the preparation method of the chitosan derivative nano-scale ultrasound contrast agent carrying doxorubicin in example 7, is as follows: 0.02g/mL of chitosan solution (0.225 mL) was added dropwise in step (3).
Claims (10)
1. A targeting material SP94 short peptide-polyethylene glycol 2000-chitosan is characterized by having the following structure:
2. the preparation method of the targeting material SP94 short peptide-polyethylene glycol 2000-chitosan as claimed in claim 1, which is prepared by thiol-maleimide reaction, comprising the following steps:
dissolving chitosan in deionized water, adding MAL-polyethylene glycol 2000-NHS to dissolve completely, wherein the molar equivalent ratio of the chitosan to the MAL-polyethylene glycol 2000-NHS is 1:1, and stirring for reaction at room temperature; slowly dropwise adding DMF solution of SP94 short peptide into the reaction solution, wherein the molar equivalent ratio of chitosan to SP94 short peptide is 1:1, and continuously reacting at room temperature; and (4) after dialysis and purification, freeze-drying to obtain the targeting material SP94 short peptide-polyethylene glycol 2000-chitosan.
3. The method of claim 2, wherein one or more of the following conditions are satisfied:
i. the weight average molecular weight of the chitosan is 3 kDa-160 kDa;
the reaction time after adding the MAL-polyethylene glycol 2000-NHS is 12-36 h;
the mass-to-volume ratio of the chitosan to the SP94 short peptide in DMF is (0.01-0.03): 1, unit is g/mL;
the reaction time after the SP94 short peptide is dripped is 12-36 h;
v. the cut-off molecular weight of the dialysis is 3000-10000 Da.
4. The use of the targeting material SP94 short peptide-polyethylene glycol 2000-chitosan of claim 1 in the preparation of chitosan derivative nano-scale ultrasound contrast agent for targeting tumor cells.
5. A chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells is characterized in that SP94 short peptide-polyethylene glycol 2000-chitosan is used as a shell membrane material, wherein SP94 short peptide is used as a targeting ligand, and perfluoropentane is wrapped inside the shell membrane; the chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells has the particle size of 250-300 nm.
6. The method for preparing the tumor cell-targeted chitosan derivative nanoscale ultrasound contrast agent as claimed in claim 5, comprising the steps of:
(1) dissolving SP94 short peptide-polyethylene glycol 2000-chitosan in deionized water to obtain SP94 short peptide-polyethylene glycol 2000-chitosan solution;
(2) dispersing perfluoropentane, Tween 20 and lecithin in deionized water, and homogenizing to obtain a suspension;
(3) dropwise adding the SP94 short peptide-polyethylene glycol 2000-chitosan solution prepared in the step (1) into the suspension prepared in the step (2), and homogenizing to prepare a suspension emulsion;
(4) and (4) standing the suspension emulsion prepared in the step (3) at a temperature, centrifuging at a low speed, and taking the upper layer liquid to obtain the chitosan derivative nano-scale ultrasonic contrast agent targeting tumor cells.
7. The method of claim 6, wherein one or more of the following conditions are satisfied:
i. the mass volume percentage of the SP94 short peptide-polyethylene glycol 2000-chitosan in the SP94 short peptide-polyethylene glycol 2000-chitosan solution in the step (1) is 1-3%, and the unit is g/mL;
the mass volume percentage of the lecithin in the suspension in the step (2) is 0.01-0.2%, and the unit is g/mL; the mass-volume ratio of the lecithin to the perfluoropentane is (0.01-0.05):1, and the unit is g/mL; the mass-volume ratio of the lecithin to the Tween 20 is (0.2-0.8):1, and the unit is g/mL;
the homogenizing speed in the step (2) is 12000-20000rpm, and the time is 1-3 min;
the volume ratio of the suspension to the SP94 short peptide-polyethylene glycol 2000-chitosan solution in the step (3) is (10-15): 1;
v. the homogenizing speed in step (3) is 12000-20000rpm for 1-3 min;
standing at room temperature in the step (4) for 10-30 min;
the low-speed centrifugation in the step (4) is performed at 800rpm for 2-8min and 100-.
8. A chitosan derivative nano-scale ultrasonic contrast agent carrying adriamycin and targeting tumor cells is characterized in that SP94 short peptide-polyethylene glycol 2000-chitosan is used as a shell membrane material, wherein SP94 short peptide is used as a targeting ligand; the inside of the shell membrane is wrapped with adriamycin and perfluoropentane; the mass ratio of SP94 short peptide-polyethylene glycol 2000-chitosan to adriamycin is 1 (1-2); the particle size of the nano-scale ultrasonic contrast agent is 300-400 nm.
9. The method for preparing the tumor cell targeted chitosan derivative nano-scale ultrasound contrast agent carrying doxorubicin of claim 8, comprising the steps of:
(1) dissolving SP94 short peptide-polyethylene glycol 2000-chitosan in deionized water to obtain SP94 short peptide-polyethylene glycol 2000-chitosan solution;
(2) dispersing perfluoropentane, Tween 20, lecithin and adriamycin in deionized water, and homogenizing to obtain a suspension;
(3) dropwise adding the SP94 short peptide-polyethylene glycol 2000-chitosan solution prepared in the step (1) into the suspension prepared in the step (2), and homogenizing to prepare a suspension emulsion;
(4) standing the suspended emulsion prepared in the step (3) at a temperature, centrifuging at a low speed, and taking the upper layer liquid to obtain the chitosan derivative nano-scale ultrasonic contrast agent carrying the adriamycin and targeting tumor cells;
preferably, the above preparation method satisfies one or more of the following conditions:
i. the mass volume percentage of the SP94 short peptide-polyethylene glycol 2000-chitosan in the SP94 short peptide-polyethylene glycol 2000-chitosan solution in the step (1) is 1-3%, and the unit is g/mL;
preferably according to the invention, the mass volume percentage of lecithin in the suspension in the step (2) is 0.01-0.2%, and the unit is g/mL; the mass-volume ratio of the lecithin to the perfluoropentane is (0.01-0.05):1, and the unit is g/mL; the mass-volume ratio of the lecithin to the Tween 20 is (0.2-0.8):1, and the unit is g/mL; the concentration of the adriamycin in the suspension is 1.5-3 mg/mL;
preferably according to the invention, the homogenization in step (2) is carried out at 12000-20000rpm for a period of 1-3 min;
preferably, the volume ratio of the suspension to the SP94 short peptide-polyethylene glycol 2000-chitosan solution in the step (3) is (10-15): 1;
v. preferably according to the invention, the homogenisation in step (3) is carried out at a speed of 12000-20000rpm for a period of 1-3 min;
preferably, the standing time at room temperature in the step (4) is 10-30 min;
preferably, the low-speed centrifugation in step (4) is performed at 100-800rpm for 2-8 min.
10. The use of the tumor cell targeted chitosan derivative nanoscale ultrasound contrast agent carrying doxorubicin according to claim 8 for the preparation of an antitumor drug.
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