CN110354281B - Double-targeting multi-modal molecular imaging probe and preparation method and application thereof - Google Patents
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- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0058—Antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/14—Peptides, e.g. proteins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/14—Peptides, e.g. proteins
- A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
Abstract
The invention discloses a double-targeting multi-modal molecular imaging probe and a preparation method and application thereof. The probe provided by the invention is a three-mode double-targeting probe with nuclear magnetism, optoacoustic and near infrared fluorescence signals, and a unique EGFR and SEC61G double-targeting strategy is applied, so that the precision of preoperative tumor diagnosis and potential intraoperative navigation application can be obviously improved, and the probe has a wide application prospect and a wide conversion value.
Description
Technical Field
The invention belongs to the technical field of molecular imaging probes, and relates to a double-targeting multi-modal molecular imaging probe, and a preparation method and application thereof.
Background
Gliomas are the most common malignant primary brain tumors of the central nervous system. Statistical reports of primary brain tumors and other central nervous system tumor diagnosis cases collected from 2010 to 2014 as published by the american brain tumor registry (CBTRUS) show: approximately half (47.1%) of the cases with brain tumors were glioma patients; and among patients diagnosed with gliomas, there are near sexually mature (56.1%) Glioblastomas (GBMs) that are diagnosed with the highest degree of malignancy. Because glioma tumor cells are infiltrated and grown along white matter fiber bundles and can invade adjacent normal brain tissues, tiny tumor residues are very difficult to identify in the operation process and cannot be completely eliminated, so that inevitable tumor recurrence after the operation is caused, and the prognosis is often poor. Surgical resection is the major means and cornerstone treatment for GBM currently used clinically, which not only can rapidly alleviate some of the clinical symptoms of a patient, but the completeness of surgery for tumor resection is closely related to the prognosis of the patient. A large phase iii clinical trial study confirmed that: the higher the extent of surgical resection of the tumor, the better the patient prognosis, and its progression-free and overall survival are significantly prolonged. However, due to the nature of brain glioma infiltration and growth, adjacent normal tissues and structures are invaded, so that it is difficult to accurately define the true tumor boundary and perform empirical resection, no matter the diagnostic image before the operation of the imaging surgeon or the real-time observation or "perception" during the operation of the surgeon. The consequences of this are: on one hand, incomplete resection causes tumor residues which are easy to cause tumor recurrence; on the other hand, excessive resection has an increased chance of causing damage to vital blood vessels and functional nuclei, resulting in impairment of neurological function and affecting the quality and level of life of the patient. Therefore, the method for 'finding' or 'seeing' the real boundary of the tumor infiltration of the GBM patient improves the precision of surgical excision, and has important value and significance for saving the important functions of the patient and prolonging the prognosis of the patient.
Molecular imaging is a labeled imaging technology based on cell or tissue specific targets, and complex physiological and pathological phenomena needing to be observed can be visually or indirectly reflected on a cell or molecular level through the medium effect of a molecular probe. Compared with the traditional anatomical imaging, the molecular imaging has the greatest characteristic that the change of the molecular level in a certain physiological or pathological state can be reflected quantitatively in real time in a non-invasive mode, and the change often occurs before the pathological change of the tissue structure, so that the molecular imaging has wide application in the aspects of early diagnosis imaging of tumors, clinical treatment decision, drug research and development, even precise medical treatment and the like. Imaging modalities that are currently widely used and studied in clinical practice and at the pre-clinical level are CT, MRI, PET, SPECT, US, optical molecular imaging, and photoacoustic imaging, among others. Different imaging modalities are based on different imaging principles, and have advantages and disadvantages in the aspects of diagnostic sensitivity, resolution, imaging depth, cost, safety and the like, and the provided diagnostic information is emphasized and tends to be single. Therefore, when the information and value provided by an imaging modality is limited, our knowledge of physiological or pathological phenomena is limited or even biased. By integrating the advantages of multiple imaging modalities, a fusion imaging means capable of acquiring multi-dimensional imaging modalities including molecular, functional and anatomical structure information and the like is developed, so that the system can help people to know and understand diseases from different layers, realize accurate diagnosis and achieve individualized diagnosis and treatment.
With the deep basic research and the development of high-throughput sequencing technology, a series of important breakthroughs are made for the research on the mechanism of the brain glioma in the process of forming tumor and malignant progression, a plurality of key molecular markers (biomarkers) related to the brain glioma are also determined, and corresponding biological agents are developed aiming at the targets, but the treatment effect of the biological agents is not ideal. More and more research and opinion reveals: brain glioma is a highly heterogeneous solid tumor, possessing multiple distinct oncogenic pathways; at different stages of spatiotemporal evolution, the expression molecular markers and the dependent oncogenic pathways are changed. Therefore, there is an urgent need to develop a molecular imaging probe and method for accurately diagnosing glioma and accurately defining tumor boundary, which will have great significance for the diagnosis and treatment of glioma.
Disclosure of Invention
The invention aims to provide a three-mode double-targeting probe with nuclear magnetism, photoacoustic and near infrared fluorescence signals, a preparation method and application thereof, and a unique EGFR and SEC61G double-targeting strategy is applied, so that the precision of preoperative tumor diagnosis and potential intraoperative navigation application can be obviously improved, and the probe has wide application prospect and conversion value.
The invention relates to a double-targeting multi-modal molecular imaging probe, which is a multi-modal molecular imaging probe targeting tumor cells by taking epidermal growth factor EGF polypeptide and anti-SEC 61G antibody as targeting groups.
Further, EGF polypeptide and anti-SEC 61G antibody are conjugated to a multi-modal signaling molecule group that enables imaging of a variety of molecular images.
Further, the group of the multi-modal signal molecules capable of realizing imaging of various molecular images comprises: the fluorescent probe has three-mode signal molecular groups of nuclear magnetism, optoacoustic and near infrared fluorescence signals.
Further, the trimodal signal molecule group with nuclear magnetic, photoacoustic and near-infrared fluorescence signals comprises superparamagnetic nano iron oxide particles with MRIT2 enhanced contrast effect of a core part, and indocyanine green with near-infrared fluorescence and photoacoustic imaging signals.
The invention relates to a preparation method of a double-targeting multi-modal molecular imaging probe, which comprises the following steps: firstly, synthesizing a super-paramagnetic iron oxide nano inner core wrapped by a lipophilic group; wrapping with DSPE-PEG phospholipid (including DSPE-PEG5000-COOH, DPSE-PEG5000-NHS, DSPE-PEG2000-COOH or DSPE-PEG2000-NHS available from Aiwei Tuo (Shanghai) pharmaceutical science and technology Co., Ltd.) to make it water soluble; then introducing indocyanine green into a hydrophobic area of the product; and finally activating carboxyl through the reaction of NHS/EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide), coupling corresponding antibody and polypeptide and purifying to prepare the double-targeting probe.
Further, the preparation method of the double-target multi-modal molecular imaging probe comprises the following steps:
(1) synthesizing a superparamagnetic iron oxide nano inner core wrapped by lipophilic oleylamine/oleic acid chains, namely a hydrophobic SPIO nano inner core, by a high-temperature thermal decomposition method; then, the hydrophobic SPIO nanometer inner core is wrapped by the DSPE-PEG phospholipid by using a rotary evaporation method so as to be converted into water solubility;
(2) by means of the amphiphilic property of indocyanine green (ICG), under the action of hydrophobic acting force, the indocyanine green (ICG) autonomously enters a hydrophobic area at the joint of a hydrophobic SPIO nanometer inner core and DSPE-PEGylated phospholipid;
(3) and finally activating carboxyl through the reaction of NHS/EDC, coupling corresponding antibody or polypeptide and purifying to prepare the double-targeting probe.
Furthermore, the preparation method of the double-targeting multi-modal molecular imaging probe specifically comprises the following steps:
1) synthesis of hydrophobic SPIO nanometer inner core: 0.7g of ferric acetylacetonate, 4.45g of oleic acid and 4.07g of oleylamine are taken and placed in a flask to be shaken and uniformly mixed, and argon is introduced to expel air in the system; heating to 120 ℃ within half an hour to react for 2 hours; then, heating to 220 ℃ within half an hour, and reacting for 30 min; finally, the temperature is adjusted to 300 ℃ within 1 hour, and the reaction is continued for 30 min; after the mixture is naturally cooled, adding 100ml of ethanol, mixing, centrifuging to precipitate a product, removing a supernatant, re-suspending with 50ml of hexane, repeating the four periods of precipitation and re-suspending, finally centrifugally collecting the precipitate, dissolving the precipitate in toluene, adjusting the iron content to 10mg/ml, and storing at 4 ℃;
2) synthesis of DSPE-PEG5000-COOH coated Water soluble SPIO: the method comprises the following steps of mixing the iron content in the hydrophobic SPIO nanometer inner core and DSPE-PEG5000-COOH according to the mass ratio of 1: 5, placing the mixture into a flask, adding 20ml of toluene, and fully shaking and dissolving; turning on a rotary evaporator, switching on a condensing device (condensation has the effect of enabling toluene vapor evaporated by heating to recover liquid state, and the toluene vapor enters a waste liquid bottle for recycling, so as to prevent danger caused by air entering), adjusting the rotating speed to 40 r/min, maintaining the temperature at 50 ℃, adding 20ml of deionized water after toluene in the system is completely evaporated, and oscillating for 1 hour under ultrasound; filtering the system solution with 0.22 μm needle filter for 3 times to remove large polymer; then 100000g of ultra-high speed centrifugation is carried out, and the precipitate is collected and resuspended; (100000 g of idle micelle which is not coated with the SPIO in the system is removed by ultra-high speed centrifugation, because the DSPE-PEG5000-COOH can spontaneously form empty carriers which are not coated with the SPIO in the system, the filtration can only remove large particles, but the centrifugation can separate the empty carriers which are not coated with the SPIO); the pellet was collected and resuspended.
3) Self-loading of indocyanine green: according to the mass ratio of iron: the indocyanine green is 20: 1, adding indocyanine green into water-soluble SPIO, oscillating on a shaking table for 4 hours, dialyzing by using a dialysis bag with the molecular weight of 3500Da, changing water for the first 4 hours respectively at 10 minutes, 30 minutes, 60 minutes, 2 hours and 4 hours, and collecting after staying overnight;
4) coupling of anti-SEC 61G antibody (purchased from Abcam, ab209867) and EGF polypeptide (purchased from Abcam, ab9697) to nanocarriers: in the nano carrier system obtained in the step 3), taking 1ml of solution with the SPIO iron concentration of 1mg/ml, reacting 5mgEDC and 10mgNHS at room temperature for 15 minutes, activating carboxyl, and then removing free EDC/NHS which does not react with the carboxyl by using an ultrafiltration tube with the molecular cut-off of 100 Kd; after the precipitate is resuspended, the concentration of iron is adjusted to be 1mg/ml, 1ml of solution is taken, 10ul of EGF polypeptide solution of 1mg/ml and 10ul of anti-SEC 61G antibody solution of 1mg/ml are added, after the reaction is continued for 4 hours at room temperature, the precipitate is centrifugally collected, protein molecules which are not coupled with the carrier are removed, and the precipitate is stored in a refrigerator at 4 ℃ for standby.
The invention also relates to application of the double-targeting multi-modal molecular imaging probe in preparation of a preparation for tumor diagnosis, in particular to a preparation for brain glioma diagnosis.
The physical and chemical properties of the double-targeting multi-modal molecular imaging probe prepared by the invention are measured by Dynamic Light Scattering (DLS), a Transmission Electron Microscope (TEM), a Vibrating Sample Magnetometer (VSM), an ultraviolet-visible spectrophotometer (Uv-Vis) and the like.
The in vitro toxicity detection and tumor cell targeting performance research of the prepared double-targeting multi-modal molecular imaging probe of the invention comprises the following steps: detecting the toxic effect of the nanoprobes under different concentration gradients on the cells by adopting a CellCountingkit-8(CCK-8) method; in order to verify the in vitro probe uptake and target binding capacity of tumor cells, the cells and probes marked by different targeting groups are incubated together, and the targeting effects of the cells are qualitatively and quantitatively compared by utilizing laser confocal technology and flow cytometry.
The nuclear magnetism-optoacoustic-fluorescence multi-mode imaging research of the double-targeting multi-mode molecular imaging probe prepared by the invention comprises the following steps: the targeted and non-targeted nano probes are independently compared with the imaging performance of the targeted group and the non-targeted group in each mode in a tail vein injection mode, and finally the advantage information of the three modes is comprehensively analyzed.
The invention has the advantages that:
firstly, the synthetic raw materials or components adopted by the invention are both ICG and SPIO which are FDA approved materials which can be used in clinic;
secondly, the greatest innovation of the present invention is the finding that there is a tendency for co-overexpression of the Epidermal Growth Factor Receptor (EGFR) and the gamma subunit of the protein transporter Sec61 (Sec61G) in GBM patients. By targeting two molecular targets simultaneously, the sensitivity and specificity of diagnosis can be significantly improved.
Thirdly, the combination of the imaging characteristics of different imaging modalities can help the clinician to obtain more accurate diagnostic information. Meanwhile, the long-term enhanced fluorescence, photoacoustic and nuclear magnetic imaging effects of the molecular probe can provide richer information for preoperative diagnosis, surgical planning and intraoperative navigation, make more reasonable decisions, and have the prospect and the conversion value of clinical application.
Drawings
FIG. 1: FIG. 1A is a schematic diagram of a flow of synthesis of a nanoprobe, FIG. 1B is a transmission electron micrograph of SPIO, FIG. 1C is a hysteresis loop of SPIO at room temperature, and FIG. 1D is a distribution diagram of hydrated particle size of the probe; fig. 1E is a different Iron: the mass ratio of ICG to the absorbance A of the mixed probe in the wavelength range of 400-900 nm.
FIG. 2: TCGA (cancer genome map) data analysis results of co-expression trend of EGFR and SEC61G in GBM;
a is oncopoint and heatmap of EGFR and SEC61G in GBM patients,
b: correlation of EGFR and SEC61G at the genomic copy number level;
c: correlation of EGFR and SEC61G at mRNA expression level;
d: results of EGFR and SEC61G co-existence trend prediction analysis;
e: the effect of a change in the copy number of SEC61G on its mRNA expression;
f: the prognosis is worse in cases of SEC61G expansion.
FIG. 3: western Blot to verify the coexpression trend of EGFR and SEC61G in glioma cell line;
a: protein quantification of EGFR and SEC61G in U87 and T98 cells;
b and C: the co-expression trend of EGFR and SEC61G in U87 and T98 was analyzed as gray scale values.
FIG. 4: and (4) detecting the toxicity of the nano probe.
FIG. 5: qualitative and quantitative detection results of the in vitro affinity of the nanoprobe and the glioma cell line;
a: qualitative analysis of laser confocal experiments shows that the affinity of the double-targeting group with tumor cells is strongest compared with that of the single-targeting group, and the enhanced binding capacity can be blocked by corresponding excessive free antibodies;
b: flow cytometry experimental quantitation showed that the dual-targeting group had the strongest affinity for tumor cells than the single-targeting group, and that this enhanced binding capacity could be blocked by a corresponding excess of free antibody.
FIG. 6: fluorescence imaging conditions at different time points in the body of the probe;
a: in vivo metabolic status of the probe and fluorescence imaging of the tumor site 0,4, 6, 12, 24, 48h after tail vein injection of the dual targeting probe;
b: co-localization results of biological self-luminescence imaging and fluorescence imaging of the tumor at 24 h;
c: change of fluorescence intensity of tumor region and contralateral background region at different time points;
d: the magnitude of the imaging signal-to-noise ratio of the tumor region at different points in time.
FIG. 7: t2 at different points in the body enhances the contrast effect;
a and B: the dual-target probe set in situ tumor nuclear magnetic T2 enhanced images (original and pseudocolor) changed from the T2 values of the tumor area and contralateral normal brain tissue;
c and D: the non-target probe set in situ tumor nuclear magnetic T2 enhanced images (original and false color) varied from the T2 values of the tumor area and contralateral normal brain tissue.
FIG. 8: photoacoustic imaging and histology verification of the probe in vivo;
a: imaging contrast and corresponding H & E staining of probes in the tumor region and the boundary of the double-targeting group and the non-targeting group;
b: comparing the signal intensity of the probes in the tumor region and the boundary of the dual-targeting group and the non-targeting group;
c: immunohistochemical staining results of tumor tissue EGFR and SEC61G as a whole;
d: enlarged partial images of EGFR and SEC61G immunohistochemical staining of tumor tissues.
Detailed Description
The present invention is further described below with reference to specific examples, and the features of the present invention will be more apparent as the examples are described. These examples are merely illustrative and do not set any limit to the scope of the invention.
The first embodiment is as follows: probe preparation process
1. Synthesis of hydrophobic SPIO nanometer inner core: 0.7g of ferric acetylacetonate, 4.45g of oleic acid and 4.07g of oleylamine are put into a 100ml double-neck flask to be uniformly stirred by shaking, fixed on a constant-temperature magnetic stirrer, and connected with a guide pipe to be introduced with argon for 5min so as to expel air in the system. Switching on a power supply to heat the system, heating the system to 120 ℃ within half an hour, and maintaining the temperature at 120 ℃ for reaction for 2 hours; then, gradually raising the temperature to 220 ℃ within half an hour, and controlling the system to react for 30min at 220 ℃; finally, after adjusting the temperature to 300 ℃ within 1 hour, the reaction was continued for 30min under these conditions. After the mixture is naturally cooled, ethanol and hexane which are subjected to four periods of precipitation and heavy suspension are respectively carried out, finally, the precipitate is centrifugally collected, dissolved in toluene, the iron content of the precipitate is adjusted to be 10mg/ml, and the precipitate is placed at 4 ℃ for long-term storage.
2. Synthesis of DSPE-PEG-COOH-5000 coated Water soluble SPIO: mixing the content of iron in SPIO and DSPE-PEG-COOH-5000 according to a mass ratio of 1: 5, placing the mixture into a flask, adding 20ml of toluene, and fully shaking and dissolving; starting a condensing device and a vacuum pump, adjusting the rotating speed to 40 revolutions per minute, maintaining the temperature at 50 ℃, adding 20ml of deionized water after toluene in the system is completely evaporated, and oscillating for 1 hour under ultrasonic waves; filtering the system solution with 0.22um needle filter for 3 times to remove large polymer; then 100000g of ultra-high speed centrifugation is used for removing the unloaded micelle which is not coated with the SPIO in the system; the pellet was collected and resuspended.
Self-loading of ICG: iron in the SPIO according to the mass ratio: ICG is 5: 1,10: 1,20: 1 adding ICG into water-soluble SPIO, shaking on a shaking table for 4h, dialyzing by using a dialysis bag with the molecular weight of 3500Da, changing water for the first 4h at 10min, 30min, 60 min, 2h and 4h respectively, and collecting after overnight.
4. In the nano carrier system obtained in the step 3, taking 1ml of solution with the SPIO iron concentration of 1mg/ml, reacting 5mgEDC and 10mgNHS for 15 minutes at room temperature, activating carboxyl, and then removing free EDC/NHS which does not react with the carboxyl by using an ultrafiltration tube with the molecular cut-off of 100 Kd; after the precipitate is resuspended, the concentration of iron is adjusted to be 1mg/ml, 1ml of solution is taken, 10ul of EGF polypeptide solution of 1mg/ml and 10ul of anti-SEC 61G antibody solution of 1mg/ml are added, after the reaction is continued for 4 hours at room temperature, the precipitate is centrifugally collected, protein molecules which are not coupled with the carrier are removed, and the precipitate is stored in a refrigerator at 4 ℃ for standby.
5. In vitro characterization
And (4) scanning by a transmission electron microscope, namely dripping a proper amount of the solution into a copper mesh, naturally airing the copper mesh, and scanning by the transmission electron microscope. The operation of the electron microscope is completed by the help of the instructor of the physical and chemical research institute of Chinese academy of sciences; after the scanning is finished, the particle size is measured, and the overall appearance is observed, which is shown in figure 1B. It can be seen that: the shape of the SPIO nano particle is relatively regular and is approximately spherical or elliptical; the dispersion is uniform, no obvious aggregation is caused, and the stability is better. Through measurement, the following results can be obtained: the diameter of the SPIO core is around 12.26 ± 0.35 nm.
And (3) measuring the saturation magnetic strength, namely weighing 1mg of dried sample at room temperature by using a magnetic Vibration Sample Magnetometer (VSM), wrapping the dried sample by nonmagnetic plastic, putting the wrapped sample into a sample cup, compacting, starting measurement, recording and storing experimental data, drawing a magnetic hysteresis loop, and judging the superparamagnetism and relaxation performance of the magnetic hysteresis loop. As can be seen from the hysteresis loop results (fig. 1C): the saturation magnetic strength is about 65emu/g, and the magnetic field has superparamagnetism (the curve passes through the origin), and can be used as a contrast agent for enhancing the scanning by using MRIT 2.
Determination of hydrated particle size: the solution to be detected is subjected to ultrasonic oscillation for 10min before detection, the cuvette is rinsed by alcohol after being cleaned by deionized water, and the cuvette is quickly dried by a blower; then adding 1ul of solution to be detected into a cuvette filled with 1ml of deionized water, fully and uniformly mixing, then placing the cuvette into a Malvern particle size analyzer for detection, and repeating the previous cleaning step after changing the sample each time. After the measurements were completed, the experimental data were saved and exported for mapping, see fig. 1D. It can be seen that: the average particle size of the nanoprobe coupled with the EGF and the anti-SEC 61G antibody is about 43.80 nm.
Measuring absorbance, namely, before measuring the absorbance by using an ultraviolet-visible spectrophotometer, washing two quartz cuvettes by using deionized water, rinsing the quartz cuvettes by using alcohol, and quickly drying the quartz cuvettes; initially, baseline calibration was performed: 3ml of deionized water was added to both cuvettes for calibration, setting the wavelength range between 400 and 900 nm. After the measurement is finished, the deionized water in one cuvette is replaced by the liquid to be measured with the same volume, and the absorbance of the liquid in the wavelength range is measured in times respectively. Record and save experimental data, see fig. 1E: as the specific gravity of ICG increases, a remarkable absorption peak can be obtained at 780nm, and the value of absorbance also increases, which shows that the loading amount of ICG also increases. In contrast, only SPIO was observed, and no significant absorption peak was observed in this wavelength range.
Example two: dual targeting probes detect EGFR and SEC61G in GBM
1. As can be seen from the results of fig. 2: EGFR and SEC61G have a tendency to "symbiotic" at both the genomic copy number level and the mRNA expression pattern; in the study cohort we selected, 33% of GBM patients had amplification of the SEC61G gene copy number at the genomic level; interestingly, these GBM patients with the SEC61G amplification event also all possessed amplification of the EGFR gene; moreover, such amplification events can have a direct effect on the transcriptional formation of their mRNA: cases with amplification events, whose levels of corresponding mRNA were also significantly increased (fig. 2A); at the same time, the changes in copy number of EGFR and SEC61G at the genomic level tended to be synchronized (fig. 2B); furthermore, the expression levels of both proteins on the mRNA water were 0.58 for the spearman correlation coefficient, 0.65 for the pearson correlation coefficient, and less than 0.05 for the P value, with statistical significance (fig. 2C). It can thus be determined that this trend of coexistence of EGFR and SEC61G is not a natural event. Furthermore, as the copy number of SEC61G increased on the genome, the transcription of mRNA increased, and the difference was statistically significant (fig. 2E). Finally, like EGFR, SEC61G also serves as an indicator of prognosis in patients with GBM, i.e., when SEC61G is highly expressed, the prognosis is worse (P < 0.05, FIG. 2F).
2. Western Blot, namely removing a culture medium after cell climbing in a culture dish to about 80 percent, washing with PBS for 3 times, adding 200ul of cell lysate, and placing on ice for 2 minutes; adherent portions were then scraped off with a cell scraper. Collecting lysate, centrifuging, taking supernatant, and quantifying protein by using a BCA method; boiling the sample with the same volume, and then carrying out sample loading, electrophoresis, membrane conversion and sealing; primary anti-EGFR and SEC61G dilution ratios were 1: 5000, selecting GAPDH as an internal reference protein, incubating at 4 ℃ in a refrigerator, and then incubating with a secondary antibody for 1 hour for developing; and finally, carrying out gray value recording and analysis on the image strip values. The results are shown in fig. 3, where it can be seen that: in U87 cells (human glioblastoma cell line U87), EGFR expression was relatively low, and SEC61G expression was also relatively low; whereas in the T98 cell line (human brain glioma cell line), increased expression of EGFR increased the expression level of SEC 61G.
3. Performing toxicity detection on the probe, namely digesting and collecting adherent culture cells, and adjusting the number of the cells to be 106One/ml and then 104Cell concentrations per well were plated in 96-well plates and grown overnight in an incubator. 5 differently labeled molecular probes, such as SPIO, SPIO-ICG, SPIO-ICG-EGF, SPIO-ICG-SEC61G, SPIO-ICG-Dual (EGF and SEC61G), were incubated with the cells at a gradient of 0,5,10,20,40,60,80 ug/ml iron for 48 hours. Cleaning and replacing the probe solution 2 hours before detection on a machine, adding 10ul (100ul of mixed solution) of CCK-8 into each hole, culturing at room temperature, measuring the light absorption value (OD value) of the probe solution at 450nm within 4 hours after adding the reagent solution by using an enzyme-labeling instrument every half hour, and calculating the cell activity according to the following formula to evaluate the toxicity of the probe:
cell viability (%) ═ a medicated-a blank/[ a0 medicated-a blank ] X100%
Wherein:
blank A: absorbance of wells with medium, CCK-8, without cells
A0 dosing: absorbance of wells with media, cells and CCK-8 without nanoprobes
A, adding medicine: absorbance of each well with media, cells, nanoprobe and CCK-8.
The results are shown in FIG. 4: whether the nanoprobe is coupled with a targeting group or not, the nanoprobe is incubated with cells for 48 hours under different concentrations, the effect of inhibiting the cell growth is more obvious along with the increase of the concentration of the probe, but even when the concentration (Iron) is up to 80ug/ml, the activity of the whole cell is still maintained to be more than 85 percent, which shows that the effect of the nanoprobe on the toxicity and the growth inhibition of the cell in vitro is not obvious. Therefore, it is suitable as a contrast agent for in vivo examination.
4. Qualitative and quantitative detection of in-vitro affinity of double-target probe by laser confocal experiment and flow cytometry
Laser confocal experiments: taking the vigorously growing cells, digesting, centrifuging, collecting and blending to 106Cell suspension per ml. Get 105Adding the cells into a laser confocal dish, culturing for 24h, washing with PBS for 3 times, adding a probe, incubating for 2h, fixing with paraformaldehyde for 30min, performing triton and membrane rupture for 5min, adding DAPI (Dacron-based fluorescent dye) for 3min, sealing, and performing on-machine detection.
Flow cytometry: the cell count concentration was 106Cell suspension at 10/ml5Adding 6-pore plates at the concentration per pore for overnight incubation, washing with PBS for 3 times, adding a probe for incubation for 2 hours, digesting, centrifuging, collecting cell-shaped precipitates, and carrying out on-machine detection after the PBS is resuspended.
Note:
a. probes used in confocal laser and flow cytometry have been fluorescently labeled with NHS-FITC for antibody or polypeptide molecules. Fluorescence acquisition of the FITC channel can be performed directly.
b. Blocking experiments were performed with 20-fold excess free antibody or polypeptide pre-treatment followed by incubation with probe.
From the results of laser confocal we can see (fig. 5A): the EGFR and SEC61G dual-target nanoprobes were more taken up or bound by tumor cells than the EGFR single-target and SEC61G single-target groups; furthermore, such an enhancement of binding ability can be partially blocked by previously using a corresponding excess amount of free antibody or polypeptide. It can be seen from this that: under the premise of taking a targeting group (EGF polypeptide or anti-SEC 61G antibody) as a guide, the double targets with EGFR and SEC61G can more remarkably improve the uptake and binding capacity of tumor cells to the nanoprobe.
Meanwhile, post-flow detection findings were performed (fig. 5B): the single EGF targeting group probe bound cells at a rate of 37.5% and the single SEC61G targeting group probe bound cells at a rate of 48.2%, whereas the EGFR and SEC61G group probes achieved the highest binding rate of 63.8%; to verify that this enhanced binding capacity has been attributed to the targeting groups (EGF and SEC 61G). Similarly, we performed pre-incubation with corresponding excess, free antibody or polypeptide before co-incubation of each set of probes with cells, respectively, and showed that there could be different degrees of blocking: the blocking group against EGFR and the blocking group of SEC61G had effects of blocking binding of 10% and 23.6%, respectively; moreover, in concert with the confocal laser results, the blocking effect on the EGFR and SEC61G dual targeting group was also most significant, up to 32.6%.
Example three: experiment of in-vivo fluorescence, nuclear magnetism and photoacoustic imaging effects of double-target imaging probe
1. Establishment of animal model
Constructing a subcutaneous tumor model: cells with strong cell viability in exponential growth phase were selected for digestion, centrifugation, collection, washed 3 times with PBS and then treated with PBS: uniformly mixing matrigel according to the volume ratio of 1:1, and adjusting the cell concentration to 107Per ml; sucking 100ul of cell mixed suspension by an insulin needle, injecting the cell mixed suspension into the back of a nude mouse for subcutaneous injection, inserting the naked mouse in parallel for 2-3mm when the needle point has a breakthrough feeling, taking care not to puncture the skin, injecting the cell mixed suspension slowly, indicating that the injection is successful when the skin is raised and whitened, putting the nude mouse back to continue breeding, observing the mouse at regular intervals, and performing the experiment when the diameter of the mouse is about 0.5 mm.
Constructing an in-situ model of the brain tumor of the allograft: selecting cells which grow vigorously (in a logarithmic growth period) and have good activity, washing with PBS, digesting with pancreatin, centrifuging, collecting, and sucking out excessive liquid, wherein the weight ratio of PBS: matrigel 1:1 after equal volume mixing, resuspending the cells, adjusting the concentration to 107And (4) putting the rice in an ice box or on ice for standby. After anaesthetizing, the mouse is fixed on a platform deck of a stereotaxic apparatus, and the head of the mouse is disinfected for 3 times by using iodophors; the scalp is carefully dissected along the midline using sterile scissors, preferably with a clear exposure of the crown, sagittal sutures, and midline. The needle insertion point was selected in the right brain, 1.5mm from the sagittal suture, 2mm posterior to the coronal suture. First, the skull is penetrated by the osteoclasts without entering the brain parenchyma; before each injection, the cell suspension is beaten by fingertips again and uniformly mixed, and then 6ul of the cell suspension is slowly sucked by a micro liquid inlet device (avoiding generating bubbles) to be injectedFixing and finding the previous needle inserting point of the osteoclast needle, vertically and slowly inserting the osteoclast needle into the brain parenchyma, firstly inserting the osteoclast needle by 3mm, and then retreating by 0.5mm, thus injecting cell suspension, and observing whether the cell suspension overflows at the needle inserting position (if so, wiping and removing the osteoclast needle in time and adjusting the needle inserting depth) in the injection process; and repeating the steps at intervals of 30s every time 1ul of cell suspension is pushed in until all liquid injection is finished, and slowly pulling out the needle after the needle stays for 2 minutes after the last injection is finished, wherein the breathing and other conditions of the mouse are observed. Then the scalp is sutured and disinfected. And putting the mixture back into the cage to be revived. The growth detection of intracranial tumors is monitored by IVIS or MRI until the tumor grows to a suitable size for subsequent experiments.
2. In vivo multimodal imaging
Fluorescence and biological spontaneous light detection of a small animal living body: the probe is administrated by tail vein injection, after gas anesthesia, a small animal living body imager is used for detecting the fluorescent signal of the probe at 0h, 1h, 2h, 4h, 6h, 12h,24h and 48h after administration, and 780nm and 810nm are respectively selected as excitation and emission wavelengths for signal acquisition according to the wave band close to the excitation emission of ICG; for biological self-luminescence collection, 100ul of D-fluorescein 15mg/ml is extracted from each mouse and injected into the abdominal cavity, a signal collection module is switched to be a BLI collection module, and images are collected 10-15min after substrate injection. The results are shown in FIG. 6: from the point 6 hours before the tail vein injection probe, we have difficulty in directly distinguishing the tumor and the surrounding tissue region on the living body, and the fluorescence signal is mainly concentrated in the liver region; at this 12 hour time point, the tumor and surrounding tissue contrast becomes increasingly apparent, and when 24 hours are reached, the signal accumulation in the tumor area increases to a maximum, where the tumor: the background signal-to-noise ratio was highest (1.974 ± 0.061) and this apparent contrast remained at a higher level (1.830 ± 0.027) up to 48h (panels a, C, D). In order to directly verify that the contrast-enhanced area under fluorescence imaging is indeed the tumor, 15mg/kg of luciferase substrate D-luciferin is intraperitoneally injected at the point of 24h, and as the tumor cells express luciferase, biological self-luminescence with higher specificity is formed through the action of the D-luciferin. From (FIG. 6B), it can be seen that the fluorescence excitation region and the bioluminescent region are fused at the tumor site, so that it can be confirmed that the region with high fluorescence is the tumor site in the living body.
Collecting nuclear magnetic data of small animals: grouping experimental mice, and administering through tail vein to respectively obtain nuclear magnetic images of tumor regions at different time points of 0h,4h,12h,24h and 48h, wherein basic parameters are set; FOV 40mm, scan layer thickness 0.8mm, TR 6000ms, TE 60 ms. The comparison was then made by determining the T2 value for the tumor area. For better visualization of this trend (since SPIO is a negative contrast effect), we draw a corresponding pseudo-color image for display, and see fig. 7: the contrast enhancement of the tumor region of the EGFR and SEC61G double-targeting probe imaging group at 24 hours by T2 can reach 34.3% (the average value of T2 is reduced from 18968.4ms to 12458.8ms), the change is more obvious than that of the normal brain tissue of the contralateral side, and the reduction of the signal value is more obvious (fig. 7A and B); in contrast, the tumor area of the mice imaged with the non-targeted probe had only 10.9% reduction in the T2-enhancing effect (from 18020.8ms to 16060.5ms on average). This trend can be more intuitively displayed from the magnitude of the color change of the corresponding pseudo-color map (fig. 7C and D).
And (3) collecting photoacoustic data of the small animal: after the experimental mouse is administrated by a tail vein way, the experimental mouse is fixed on a carrying platform under an anesthesia state, and in order to reduce artifacts, an ultrasonic coupling liquid is smeared on an imaging area. Before measurement, signal to be Imaged (ICG) absorption spectrum is selected, scanning wavelengths are set to be 720nm, 740nm, 780nm, 810nm and 830nm, and Hb and HbO are selected simultaneously2According to the time point information of the optical imaging of the living body, considering that the load of the photoacoustic imaging on the small animals is large, several special time points (0h, 24h and 48h) are selected for signal acquisition, and the result is shown in fig. 8. It can be seen that: the EGFR and SEC61G double-targeting labeled nanoprobes can more clearly display the peripheral area of the tumor, and the enhancement of the central part of the tumor is not obvious; for the non-targeted group, it was not only difficult to determine the tumor area and contour as a whole, but also did not have the sharp edge enhancement of the dual-targeted group (fig. 8A and B). We are right toAfter tumor tissue was sectioned, immunohistochemical staining was performed on EGFR and SEC61G, respectively, to find: EGFR and SEC61G were more strongly expressed in the periphery of the tumor, while closer to the central region, the expression was weaker (fig. 8C and D).
Claims (5)
1. A preparation method of a double-targeting multi-modal molecular imaging probe is characterized by comprising the following steps: firstly, synthesizing a super-paramagnetic iron oxide nano inner core wrapped by a lipophilic group; wrapping with DSPE-PEG to make it water-soluble; then introducing indocyanine green into a hydrophobic area of the product; finally activating carboxyl through EDC/NHS reaction, coupling corresponding antibody and polypeptide, and preparing the double-targeting probe; the antibody is an anti-SEC 61G antibody, and the polypeptide is an epidermal growth factor EGF polypeptide.
2. The method for preparing a dual-targeting multi-modal molecular imaging probe according to claim 1, wherein:
(1) synthesizing a superparamagnetic iron oxide nano inner core wrapped by lipophilic oleylamine/oleic acid chains, namely a hydrophobic SPIO nano inner core, by a high-temperature thermal decomposition method; then, the hydrophobic SPIO nanometer inner core is wrapped by DSPE-PEG by a rotary evaporation method to be converted into water solubility;
(2) by means of the amphiphilic property of indocyanine green, the indocyanine green autonomously enters a hydrophobic area at the joint of a hydrophobic SPIO nanometer inner core and DSPE-PEG under the action of hydrophobic acting force;
(3) and finally activating carboxyl through the reaction of NHS/EDC, coupling corresponding antibody or polypeptide and purifying to prepare the double-targeting probe.
3. The method for preparing a dual-targeting multi-modal molecular imaging probe according to claim 1, wherein:
the method specifically comprises the following steps:
1) synthesis of hydrophobic SPIO nanometer inner core: 0.7g of ferric acetylacetonate, 4.45g of oleic acid and 4.07g of oleylamine are taken and placed in a flask to be shaken and uniformly mixed, and argon is introduced to exhaust air in the system; heating to 120 ℃ within half an hour to react for 2 hours; then, heating to 220 ℃ within half an hour, and reacting for 30 min; finally, the temperature is adjusted to 300 ℃ within 1 hour, and the reaction is continued for 30 min; after the precipitate is naturally cooled, adding 100ml of ethanol, mixing, centrifuging to precipitate the product, removing supernatant, re-suspending with 50ml of hexane, repeating the four precipitation re-suspending periods, finally centrifugally collecting the precipitate, dissolving the precipitate in toluene, adjusting the iron content to 10mg/ml, and storing at 4 ℃;
2) synthesis of DSPE-PEG5000-COOH coated Water soluble SPIO: the method comprises the following steps of mixing the iron content in the hydrophobic SPIO nanometer inner core and DSPE-PEG5000-COOH according to the mass ratio of 1: 5, placing the mixture into a flask, adding 20ml of toluene, and fully shaking and dissolving; starting a rotary evaporator, switching on a condensing device, adjusting the rotating speed to 40 revolutions per minute, setting the reaction temperature at 50 ℃, adding 20ml of deionized water after toluene in the system is completely evaporated, and oscillating for 1 hour under ultrasonic; filtering the system solution with 0.22 μm needle filter for 3 times to remove large polymer; then 100000g of ultra-high speed centrifugation is carried out, and the precipitate is collected and resuspended;
3) self-loading of indocyanine green: according to the mass ratio of iron: the indocyanine green is 20: 1, adding indocyanine green into water-soluble SPIO, oscillating on a shaking table for 4 hours, dialyzing by using a dialysis bag with the molecular weight of 3500Da, changing water for the first 4 hours respectively at 10 minutes, 30 minutes, 60 minutes, 2 hours and 4 hours, and collecting after staying overnight;
4) coupling of anti-SEC 61G antibody and EGF polypeptide to nanocarriers: in the nano carrier system obtained in the step 3), taking 1ml of solution with the SPIO iron concentration of 1mg/ml, reacting 5mgEDC and 10mgNHS at room temperature for 15 minutes, activating carboxyl, and then removing free EDC/NHS which does not react with the carboxyl by using an ultrafiltration tube with the molecular cut-off of 100 Kd; adjusting the iron concentration to 1mg/ml after the precipitate is resuspended, taking 1ml of solution, adding 10 mu l of 1mg/ml EGF polypeptide solution and 10 mu l of 1mg/ml anti-SEC 61G antibody solution, continuing the reaction for 4 hours at room temperature, centrifuging to collect the precipitate, removing protein molecules which are not coupled with the carrier, and storing the precipitate in a refrigerator at 4 ℃ for later use.
4. Use of the bi-targeted multi-modal molecular imaging probe obtained by the preparation method according to any one of claims 1 to 3 for preparing a preparation for tumor diagnosis.
5. The use of the dual-targeting multi-modal molecular imaging probe according to claim 4, for preparing a preparation for diagnosing brain glioma.
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