CN108721649B - Design, synthesis and application of tumor-targeted near-infrared fluorescence imaging agent - Google Patents
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- 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
- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
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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
- A61K49/0056—Peptides, proteins, polyamino acids
Abstract
The invention relates to design, synthesis and application of a tumor-targeted near-infrared fluorescence imaging agent, wherein the structural formula is ICG-OSu- (PEG) n-G (CGNVVRQGC), the ICG is near-infrared fluorescence imaging agent indocyanine green, the ICG-OSu is sulfonic group indocyanine green activated grease which is an amino-reactive derivative of the ICG, the G (CGNVVRQGC) is tumor-targeted cyclic polypeptide TMTP1 which is carboxyl-reactive, the two are bridged by polyethylene glycol (PEG), and n is an integer from 2 to 20. The novel tumor-targeted molecular developer provided by the invention greatly improves the specificity of ICG imaging of cervical cancer, breast cancer focus and lymph node metastasis thereof, and provides good indication for clinical diagnosis of cervical cancer and breast cancer and tumor lymph node removal by using a fluorescence endoscope.
Description
Technical Field
The invention relates to the technical field of medical imaging, in particular to an accurate malignant tumor excision operation under a fluorescence endoscope.
Background
Malignant tumor has become the first disease threatening human health, 1 of 5 people in every death of Chinese residents in 2010, namely 1 person is dead of cancer, 2000 ten thousand new cancer cases are expected to occur every year in 2020, and the death number of cancer patients breaks through 1000 ten thousand. At present, the treatment mode of solid tumor patients is mainly surgical resection, and is assisted by chemotherapy, radiotherapy and molecular targeted therapy. The key to the treatment of cancer patients is early discovery and early intervention. The imaging examination CT/MR/PET/B ultrasonic examination and the laboratory examination have become the basis for the early tumor diagnosis of the clinician, but the specificity and the sensitivity of the existing diagnosis method are still insufficient, and researchers still strive to develop a specific diagnosis method to guide the clinical diagnosis and treatment decision. For example, standard surgical treatment of cervical cancer patients requires pelvic lymph node cleaning, but if a specific diagnostic method is developed to determine whether a cervical cancer patient has lymph node metastasis or which lymph nodes have tumor metastasis, unnecessary pelvic lymph node cleaning is reduced.
In patent application No. 2007101878437, we invented a targeting polypeptide specifically targeting tumors and their metastases, named TMTP 1. TMTP1 is prepared by performing four-wheel positive and negative screening on a high-transfer prostate cancer cell line PC-3M-1E8 and a non-transfer prostate cancer cell line PC-3M-2B4 by using a bacterial flagellar peptide library display technology, verifying bacterial clones specifically compatible with PC-3M-1E8 cells in vitro by an immunofluorescence method to obtain a repetitive sequence (NVVRQ), modifying a polypeptide sequence into G (CGNVVRQGC) disulfide bond ring formation to increase the stability of the polypeptide in vivo. In vitro experiments verified that TMTP1 has high affinity for high metastatic tumor cells and low affinity for low metastatic tumor cells. In vivo experiments prove that TMTP1 can target primary focuses and micrometastases of high-metastatic tumors. In the applications of patent numbers 2012103403857, 2012103658343, 2013103540685, we linked TMTP1 to antimicrobial peptide (TMTP1-KLA), diphtheria toxin (DT390-tritMTP1) and radionuclide (R) ((R) ())(99m)TC-HYNIC/EDDA-TMTP1), and the finding shows that TMTP1 can carry corresponding preparations to reach the tumor site in a targeted manner, so as to achieve the effect of treating tumors or imaging the tumors.
Molecular diagnosis has become the main index for target treatment and prognosis judgment of many malignant tumors, for example, patients with positive breast cancer HER2, ER and PgR2 can select herceptin Trastuzumab and estrogen receptor antagonist. Tumor vasculature VEGF receptor can be selected for bevacizumab inhibition of angiogenesis if it is positive for expression. Octreotide and RGD polypeptides are currently the most classical polypeptides for molecular diagnosis and treatment in clinical applications.(99m)TC-EDDA/HYNIC-TOC can be used for diagnosing nerve secretory tumor such as pancreatic cancer, brain glioma, etc. [18F ]]Galacto-RGD can diagnose the tumor with strong neovascular capacity.
Indocyanine Green (ICG), a highly sensitive near-infrared fluorescein, has been approved for clinical use by the FDA in the united states. The safety is high, and the fluorescence laparoscope developed based on ICG is applied to clinic. After ICG is injected into human body intravenously, 98% -99% of ICG is combined with plasma high molecular protein (such as albumin), the excitation wavelength of ICG-protein complex is 750-810nm, and the transmissivity of biological tissue in the near infrared region reaches the maximum. In europe and the united states, it is mainly used for ocular angiography, cardiac output measurement, and liver function evaluation, and in recent years, a large number of studies have been made on the diagnosis and treatment of ICG. ICG is used for sentinel lymph node imaging of cervical cancer and endometrial cancer under a fluorescence laparoscope, and the positive rate of sentinel lymph node biopsy is increased. ICG is therefore a good sentinel lymph node indicator, but it has obvious limitations on the visualization of lymph nodes, lacks specificity, and cannot distinguish between tumor metastasizing lymph nodes and inflamed hyperplastic lymph nodes.
PEG modification is a mature technology in drug development of proteins and polypeptides to increase water solubility, biocompatibility, and drug action time. PEG (polyethylene glycol) is a hydrophilic polymer with neutral pH, no toxicity and high water solubility, and has repeating units of ethylene oxide groups and end groups of two hydroxyl groups, and is in a linear or branched chain structure. PEG polymers are the polymers known to date to have the lowest levels of protein and cellular uptake, and PEG has been approved by the FDA as a polymer for in vivo injection as a result of its non-toxicity and good biocompatibility. When the PEG is coupled to the drug molecules or the surface of the drug, the PEG can endow the modified drug molecules with excellent properties, change the biological distribution behavior and the solubility of the modified drug molecules in an aqueous solution, generate a spatial barrier around the modified drug, reduce the enzymolysis of the drug, and avoid the rapid elimination in the metabolism of the kidney, thereby effectively prolonging the half-life period of the drug and enhancing the stability of the drug; the water solubility of the PEG modified drug is generally increased, and the drug improves the properties of pharmacokinetics, pharmacodynamics and the like by changing the molecular structure, and improves the blood concentration of an action part. However, the introduction of PEG also brings new problems, such as slow clearance of the drug system, possible poisoning of the drug after long-term retention in the body, and limited delivery rate of the drug from blood to the target tissue. Therefore, how to design the PEG bridging mode and the length of the PEG chain are crucial to the design of the drug.
Clinically, no matter whether the cervical cancer patients with the stage IA2 or above have lymph node metastasis, the lymph node cleaning is carried out conventionally, so that the postoperative lymphatic return disorder and the lower limb edema of some patients are caused, and the postoperative life quality of the patients is influenced. Therefore, there is an urgent need to develop a technique for discriminating the presence or absence of lymph node metastasis and then deciding whether to perform lymph node dissection. Colposcopy is primarily used to assess women with abnormal cervical cytological findings by identifying the source of abnormal cells found in cytological examinations, diagnosing with direct biopsy under colposcopy, the type and grade of pathology, and determining the appropriate treatment by determining the extent of the lesion in the cervix. Colposcopy has limitations in that it is impossible to see lesions in the cervical canal, and it is difficult to identify the presence or absence of interstitial infiltrates, and it is limited in diagnosing cervical lesions, especially in postmenopausal women. Therefore, there is a need for reliable indicators to guide sampling in colposcopic cervical biopsy sampling. The ICG connection TMTP1 offers a possibility to solve such problems. Conventional ICG was not capable of direct attachment of the polypeptide, and we attempted to bridge TMTP1 with the derivative ICG-OSu of ICG via (PEG) n, where ICG-OSu is amino reactive and TMTP1 is carboxy reactive, both attached to the two ends of (PEG) n, in such a way that the ICG attachment of the polypeptide was initiated. At present, the fluorescence laparoscope is clinically applied, and the ICG-OSu- (PEG) n-TMTP1 is a tumor molecule targeted fluorescence imaging agent with good application prospect.
Disclosure of Invention
In order to overcome the defect of low tumor specificity of the traditional ICG imaging, the invention provides a tumor targeted near-infrared fluorescent imaging agent ICG-OSu- (PEG4) n-TMTP1, which is designed and modified on the basis of the original tumor targeted polypeptide TMTP1(NVVRQ) to obtain the fluorescent imaging agent with the structural formula of ICG-OSu- (PEG) n-G (CGNVVRQGC).
The technical scheme of the invention is as follows:
TMTP1 can specifically target highly invasive metastatic tumor cells, is designed into cyclic (CGNVVRQGC) G with a ring structure to increase the stability, and is covalently linked with ICG-OSu through (PEG) n to construct a novel imaging agent ICG-OSu- (PEG) n-TMTP1 shown in the invention. On the one hand, the imaging agent still maintains the targeting characteristic of the tumor and can be specifically combined with an in-situ focus and a metastasis focus of the malignant tumor. On the other hand, the fluorescence characteristics of the ICG are kept unchanged, and a fluorescence image can be clearly displayed under the imaging device with the fluorescence exciter. Active hydroxyl groups at two ends of PEG can modify protein and polypeptide, can improve biocompatibility and drug action time, but introduction and modification of PEG bring new technical problems, the slower system clearance is, the poisoning phenomenon can occur in vivo after long-term retention of the drug, and the delivery speed of the drug from blood to target tissues is also limited. Here, we passed a number of cytotoxicity experiments, targeting experiments in vitro, on one hand by designing a PEG bridging structure and a PEG chain, one end of which is linked to the amino group of ICG-OSu and the other end is linked to the carboxyl group of TMTP 1. The imaging agent designed by the method has enhanced water solubility and prolonged circulation time in vivo, liver and kidney are gathered most in vivo within 1-2h, and the imaging agent is mainly retained on a targeted tumor part after 6h, so that the imaging agent is safe and effective, and the fluorescence background caused by nonspecific binding is effectively removed by utilizing the time difference. The imaging agent provided by the invention is a stable compound, has no cytotoxicity and less side effects, can be imaged under any instrument (such as a fluorescence laparoscope and a fluorescence colposcope) provided with a fluorescence detection system after local administration or intravenous administration in early clinical experimental research, and provides a new thought for detecting and accurately excising a tiny focus clinically.
The structural formula of the tumor-targeted near-infrared fluorescence imaging agent is ICG-OSu- (PEG) n-G (CGNVVRQGC), wherein ICG is near-infrared fluorescence imaging agent indocyanine green, ICG-OSu is sulfonic group indocyanine green activated lipid, G (CGNVVRQGC) is tumor-targeted cyclic polypeptide TMTP1, and polyethylene glycol (PEG) is bridged, wherein n is an integer from 2 to 20. Preferably n is an integer from 3 to 10, more preferably n is 4, 5,6, 7, most preferably n is 4.
Wherein ICG-OSu is an amino-reactive derivative of ICG and the cyclic polypeptide TMTP1 is carboxy-reactive.
The tumor targeted near-infrared fluorescence imaging agent ICG-OSu- (PEG) n-TMTP1 is synthesized by the following specific steps of 1) synthesizing polypeptide GCGNVVRQGC by utilizing a solid phase carrier according to a designed amino acid sequence, and covalently connecting the polypeptide GCGNVVRQGC to (PEG) n; 2) cleaving the polypeptide from the resin with MEOH/I2Oxidizing, bridging the polypeptide sequence by disulfide bonds to form a ring, namely (PEG) n-G (CGNVVRQGC), and then separating, purifying and freeze-drying; 3) mixing the product obtained in the step 2) with ICG-OSu according to a ratio of 1:1, dissolving in DMF, adding 2 times of DIEA by volume, reacting for 10 minutes, carrying out LCMS (liquid crystal display system) identification reaction, separating, purifying and freeze-drying to obtain the imaging agent ICG-OSu- (PEG) n-TMTP 1.
The tumor-targeted near-infrared fluorescence imaging agent can be further applied to the preparation of tumor diagnostic reagents, wherein the tumors are selected from cervical cancer, breast cancer, lung cancer, prostatic cancer and kidney cancer, and preferably cervical cancer and breast cancer.
The tumor targeted near-infrared fluorescent imaging agent is applied to a lymph node diagnostic reagent for distinguishing tumor metastasis lymph nodes from inflammatory hyperplasia. The tumor targeted near-infrared fluorescence imaging agent can also be applied to the preparation of an excision indicating reagent for guiding tumor focuses and lymph node metastasis focuses in fluorescence endoscopic surgery, and the tumors are cervical cancer and breast cancer.
The tumor targeted near-infrared fluorescence imaging agent can be applied to preparation of a sampling indicating reagent of a fluorescence colposcope, and is characterized in that the tumor is cervical cancer.
The physical and chemical properties of the imaging agent ICG-OSu- (PEG) n-TMTP1 are tested, and the safety and the tumor targeting property of the ICG-OSu-PEG4-TMTP1 are tested through cell experiments and animal experiments according to the in vitro cell binding efficiency of the imaging agent, most preferably n is 4. The synthesized developer has high purity and the fluorescence property is consistent with ICG. The CCK-8 test proves that the compound has no toxicity to a tumor cell line and a normal cell line; acute toxicity experiments of mice prove that the pharmaceutical composition has no toxic or side effect on visceral organs of the mice and high safety; cell combination experiments prove that the compound can be specifically combined with tumor cells, and the effect is obviously superior to ICG; the tumor-bearing mouse animal experiment inoculated with the cervical cancer cells and the breast cancer cells proves that the tumor-bearing mouse animal experiment can be specifically combined with tumor tissues, and the effect is obviously superior to ICG; the mouse animal experiment of the breast cancer lymphatic metastasis model proves that the compound can specifically image tumor metastasis lymph nodes. The imaging agent is currently in phase II clinical trial (registration number: ChiCTR-INR-17013087), is used for accurate cleaning of guide lymph nodes in cervical carcinoma operation, and initially shows a specific indication effect superior to ICG.
Compared with the prior art, the invention has the beneficial effects that: compared with the current near-infrared fluorescent imaging agent ICG which is only approved by FDA to be used for human bodies, the ICG-Osu-PEG4-TMTP1 has better specificity for imaging tumor focuses and lymphatic metastasis focuses on the premise of stability and safety, and has wider application prospect.
Drawings
FIG. 1 is a scheme showing the synthesis of ICG-OSu-PEG4-TMTP 1.
FIG. 2 is a quality measurement chart of ICG-OSu-PEG4-TMTP 1.
FIG. 3 shows the results of cytotoxicity experiments with ICG-OSu-PEG4-TMTP 1.
FIG. 4 is a graph showing the binding efficiency of ICG-OSu- (PEG) n-TMTP1 to tumor cells.
FIG. 5 shows the results of the cell binding experiments with ICG-OSu-PEG4-TMTP 1.
FIG. 6 shows the results of in vivo distribution and pharmacokinetic measurements of ICG-OSu-PEG4-TMTP 1.
FIG. 7 shows the comparison of the images of ICG-OSu-PEG4-TMTP1 and ICG in a HeLa-luc tumor-bearing mouse.
FIG. 8 shows the comparison between ICG and ICG in 4T1-Luc tumor-bearing mouse ICG-OSu-PEG4-TMTP 1.
FIG. 9 shows the comparison of the imaging of ICG and ICG in 4T1 lymph node metastasis tumor-bearing mouse ICG-OSu-PEG4-TMTP 1.
FIG. 10 shows the results of one example of the clinical trial of ICG-OSu-PEG4-TMTP 1.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
EXAMPLE 1 preparation of ICG-OSu-PEG4-TMTP1
Referring to FIG. 1, the preparation scheme of ICG-OSu-PEG4-TMTP1 is: 1) synthesizing the polypeptide GCGNVVRQGC according to the designed amino acid sequence by using a solid phase carrier, and covalently connecting the polypeptide GCGNVVRQGC to PEG 4; 2) cleaving the polypeptide from the resin with MEOH/I2Oxidizing, bridging the polypeptide sequence by disulfide bonds to form a ring, namely PEG4-G (CGNVVRQGC), and then separating, purifying and freeze-drying; 3) mixing the product obtained in the step 2) with ICG-Osu according to a ratio of 1:1, dissolving in DMF, adding 2 times of DIEA by volume, reacting for 10 minutes, carrying out LCMS (liquid Crystal display System) identification reaction, separating, purifying and freeze-drying to obtain the imaging agent ICG-Osu-PEG4-TMTP 1. The synthesis process is completed by Shanghai Mingkude new drug development Co.
Referring to fig. 2, fig. 2a and 2b demonstrate the correct synthesis of the imaging agent of the present invention as measured by a combination of liquid mass spectrometry and chromatography (LC-MS, Agilent 1200HPLC &6410Triple Quad, usa), fig. 2c shows the purity (> 95%) of the imaging agent as measured by a high performance liquid chromatograph (HPLC, Agilent 1200, usa), and fig. 2d shows the absorbance spectrum of the imaging agent as measured by a uv spectrophotometer (JH754PC, shanghai, china), with peaks consistent with ICG.
Example 2 cytotoxicity assay:
1. cell culture
Mouse breast cancer cell line 4T1 (purchased from ATCC cell Bank, USA) was cultured in RPMI-1640 medium (Gibco, ThermoFish, USA) containing 10% by volume fetal bovine serum, cervical cancer cell lines HeLa, SiHa (purchased from ATCC cell Bank, USA) and normal skin keratinized epithelial cells (purchased from CCTCC cell Bank, China) were cultured in DMEM medium (Gibco, ThermoFish, USA) containing 10% by volume fetal bovine serum, and cervical intraepithelial neoplasia cell line S12 (proffered by Kenneth Raj, UK) was cultured in DMEM medium containing 5% by volume fetal bovine serum, 5. mu.g/ml insulin (Sigma-Aldrich, USA), 8.4ng/ml cholera toxin (Sigma-A-Raj)ldrich, usa), 24.3 μ g/ml adenine (Sigma-Aldrich, usa), 0.5 μ g/ml hydrocortisone (Sigma-Aldrich, usa) and 10ng/ml endoglin (EGF, Peprotech) in F12 and DF12 medium (Gibco, ThermoFish, usa) as 1:1 proportion of the culture medium. The cells all contained 5% C0 at 37 deg.C2The incubator of (2) for cultivation.
CCK-8 test
4T1, SiHa, HeLa, S12 and HaCaT cells were mixed at 1 × 104Density of 96-well plates per well, 5% CO at 37 ℃2After 24h of incubation in the incubator, fresh media containing 0,3.125,6.5,12.5,25,50umol/L ICG-OSu-PEG4-TMTP1 were replaced, each set having 3 secondary wells. After 24h incubation, the medium was aspirated and replaced with CCK-8 reagent (Dojindo Molecular Technologies, Japan), and after 2.5h incubation, OD was measured at 450nm wavelength using a microplate reader (Biorad, USA). The calculation formula of the cell activity degree is as follows: (OD) is a value of viable rate ═ ODtreated-ODblank)/(ODcontrol-ODblank)*100％。
The experimental results are shown in fig. 3, and no matter cancer cells or normal cells, no matter the concentration of the drug is high or low, no obvious influence is caused on the proliferation of the cells.
Example 3 tumor cell binding efficiency experiments at different PEG numbers
The imaging agents were synthesized in different PEG numbers as shown in example 1, with n being 2,4,6,8,10, 20, respectively. 4T1 cells and HeLa cells were cultured as described in example 2, and 4T1 and HeLa cells were cultured in the same manner as in 1 × 105After overnight incubation in 24-well plates at a density of one well, 500ul of 1 μ M ICG, ICG-OSu- (PEG) n-TMTP1 was added to each well, and the wells with PBS were incubated on ice for 20 minutes, washed 3 times with cold PBS, and placed under a small animal Living body imager to collect fluorescence images from the wells. The fluorescence values of each well were then quantitatively analyzed by the ROI tool of IVIS visualization software.
Experimental results referring to fig. 4, fig. 4a is a fluorescence image of 4T1 cells after incubation with the present imaging agent at different PEG numbers, and fig. 4b is a quantitative analysis of fluorescence intensity of each well. The imaging agent has the best binding efficiency with tumor cells when taking n-4. Consistent results were also obtained in the HeLa cell experiments. This experiment shows that PEG chains and their number will significantly affect the binding efficiency of imaging agents to tumor cells.
Example 4 in vitro cell binding assay
Cell culture 4T1, SiHa, HeLa, S12, HaCaT cells were cultured as described in example 2 according to 1 × 105The cells are cultured in 6-well plates at a density of one well, 1ml of 1 mu M ICG-OSu-PEG4-TMTP1 or ICG is added when the cell density reaches 80 percent after 48 hours of culture, PBS is used as a control group, the cells are digested and collected by pancreatin solution (containing pancreatin with the mass fraction of 0.25 percent and EDTA with the mass fraction of 0.1 percent) after 1 hour of ice incubation, and the fluorescence positive rate of the ICG channel cells is analyzed by a flow cytometer (BD FACSCalibur, USA) after 2 times of PBS washing.
Results of the experiments see FIG. 5, the binding was significantly higher for the tumor cell line 4T1, SiHa, HeLa, ICG-OSu-PEG4-TMTP1 than for ICG, and less for the precancerous lesion cell line S12 and the normal cell line HaCaT.
Example 5 in vivo distribution and pharmacokinetic experiments
Normal BALB/c mice (purchased from Beijing Huafukang Biotechnology GmbH) were imaged (ex/em 745/840nm) at 100uL and 25umol/L ICG-OSu-PEG4-TMTP1 in the tail vein at different time points (30min, 1h, 2h, 12h, 24h, 48h) with a small animal imager (PerkinElmer, USA), the mice were dissected at each time point, the main organs (heart, liver, spleen, lung, kidney, colon) were extracted for imaging, the fluorescence organ values at each time point were measured by the ROI tool of IVIS imaging software, and 3 mice per group were obtained.
The experimental results are shown in fig. 6, fig. 6a and 6b are typical images of mice and organs at various time points, fig. 6c is a fluorescence quantification statistical chart of organs, and table 1 is a corresponding statistical table. It is shown that ICG-OSu-PEG4-TMTP1 is metabolized by liver and kidney, and is accumulated most in liver after 1h administration, and accumulated most in kidney after 2h administration, and then is cleared rapidly. After 24h of administration, liver clearance reaches 95.9%, and kidney clearance reaches 88.4%. After 48 hours, the liver clearance reaches 96.1 percent, and the kidney clearance reaches 94.4 percent.
Table 1: in vivo distribution and pharmacokinetic experiment results
| heart | liver | spleen | lung | kidney | colon | |
| 30min | 9.11E+08 | 2.55E+10 | 6.57E+08 | 4.16E+09 | 3.61E+09 | 4.71E+09 |
| 1h | 7.73E+08 | 2.66E+10 | 4.37E+08 | 3.24E+09 | 3.47E+09 | 6.17E+09 |
| 2h | 6.86E+08 | 1.86E+10 | 5.16E+08 | 2.68E+09 | 3.72E+09 | 5.82E+09 |
| 12h | 8.20E+07 | 5.56E+09 | 1.12E+08 | 5.24E+08 | 1.03E+09 | 5.40E+08 |
| 24h | 3.17E+07 | 1.11E+09 | 5.32E+07 | 3.44E+08 | 4.33E+08 | 2.98E+08 |
| 48h | 3.39E+07 | 1.03E+09 | 4.81E+07 | 2.34E+08 | 2.09E+08 | 1.18E+08 |
Example 6 in vivo tumor targeting experiments
Luciferase (l) transfected by human cervical cancer cell line HeLa and mouse breast cancer cell line 4T1uciferase) lentivirus, enabling it to monitor the size of tumors in real time by detecting biotin light. Culturing and amplifying HeLa-Luc cells, 1 × 107One mouse/mouse was inoculated to the left back of a BALB/c-nude mouse (purchased from Beijing Huafukang Biotechnology GmbH) for 4 weeks, the tumor grew to about 1cm in diameter after 3 weeks, 100uL and 25umol/L of ICG-OSu-PEG4-TMTP1 or ICG were applied to the tail vein, and then visualized under a small animal imager (1h, 2h, 6h, 12h, 24h and 48h), tumor tissues and organs were dissected and extracted for visualization, and 3 mice per group were obtained. Culturing and amplifying 4T1-Luc cells, 1X 105One/4-week-old BALB/c mouse (purchased from Beijing Huafukang Biotech, Inc.) was inoculated into the right breast pad, and 3 weeks later, the tumor grew to about 1cm in diameter, and the in vivo targeting experiment was performed as described above.
The experimental results are shown in fig. 7 and 8, fig. 7a and 8a are visualizations in the biological light mode, which illustrate successful establishment of a subcutaneous tumor model, fig. 7b and 8b are typical visualizations at various time points in the ICG fluorescence mode, and fig. 7c and 8c are typical organ fluorescence visualizations at different time points. FIGS. 7d and 8d are graphs showing the T/N ratio at each time point in ICG-OSu-PEG4-TMTP1 and ICG groups, which were obtained by measuring the fluorescence of tumor tissues and mouse ears (as controls) using the ROI tool, using GraphPad Prism 5.0 software. The result shows that the tumor targeting capability of the ICG-OSu-PEG4-TMTP1 is obviously superior to that of the ICG, and the ICG can be specifically combined with tumor tissues.
Example 7 mouse metastatic lymph node specific imaging experiment
Amplification of murine mammary carcinoma cell line 4T1 at 4X 105A density of 25 uL/ml/mouse is locally inoculated to BALB/c mice at 4 weeks, lymph node obvious swelling is observed after 3 weeks, after 20uL, 25umol/L ICG-OSu-PEG4-TMTP1 or ICG is locally administrated to foot pad tumors, the tumors are imaged under a small animal imager (2h, 12h and 24h), the lymph nodes are dissected after imaging is finished, and HE staining is carried out after paraffin embedding sections, so that establishment of a tumor lymph node metastasis model is further determined.
Experimental results referring to FIG. 9, FIG. 9a is a typical visualization of each time point in ICG fluorescence mode, and FIG. 9b is a statistical graph of fluorescence values measured at metastatic lymph nodes at different time points using ROI tool, obtained by using GraphPad Prism 5.0 software for ICG-OSu-PEG4-TMTP1 and ICG group at each time point. The ICG-OSu-PEG4-TMTP1 has obviously better capability of targeting the tumor metastasis lymph nodes than ICG and can be specifically combined with the tumor metastasis lymph nodes.
Example 8 Exception test
15 BALB/c mice (purchased from Beijing Huafukang biological Co., Ltd.) with the age of 4 weeks are selected and randomly divided into 3 groups (tail vein injection group, callus local injection group and control group), each group comprises 5 mice, the mice are raised according to normal raising conditions before the test and during the whole observation period of the test, and the weight of the mice is weighed before the test. The group of tail vein injections was injected with 100 uL/250 umol/L ICG-OSu-PEG4-TMTP1 per mouse, the group of local injections was injected with 25 uL/250 umol/L ICG-OSu-PEG4-TMTP1 per mouse footpad, and the blank control was not treated. The mice in each experimental group and control group were observed for diet, respiration, activity, reflex, defecation, pain sensation, and skin for 7 days. After 7 days, the weight of each mouse is weighed, blood is taken from the orbit to measure the liver and kidney functions, the mice are killed and dissected, and the color and the shape of each organ of the mice in the experimental group are measured.
The experimental results are as follows: the mice in the drug-treated group did not die throughout the experiment and were found to have no diet, respiration, activity, reflexes, defecation, pain sensation, or skin abnormalities. See table 2 that the weight change of the drug-treated mice and the untreated mice is not obviously different (P is more than 0.05), the organs are not obviously diseased, and the liver and kidney functions are not obviously different.
Table 2: results of abnormal toxicity test
Example 9 Security detection
1. And (3) detecting bacterial endotoxin: according to the content of Chinese pharmacopoeia (2010 version), the endotoxin detection level lambda of the test sample is determined to be 0.125 EU/ml. A sensitivity check experiment is carried out firstly, and the sensitivity of the limulus reagent is determined to be 0.125 EU/m. And then carrying out an interference experiment, wherein the test sample and the detection reagent do not have interference reaction.
The test results are shown in Table 3, and the endotoxin level of the sample is detected by the gel test, so that the endotoxin level of the sample is proved to be in accordance with the requirements.
Table 3:
2. and (3) sterility detection: 1mL of the product was cultured in nutrient medium of microorganism clinical test group of clinical laboratory of Wuhan Tongji Hospital for 3 days, and no bacterial growth was observed.
Example 10 clinical application of ICG-OSu-PEG4-TMTP1 in cervical cancer surgery
The reagent is used for guiding lymph node cleaning under a fluorescence laparoscope in cervical carcinoma operation, and the specific use method comprises the following steps: the preparation is administered by local (3 degree and 9 degree) cervical injection 30min before operation, and the preparation is prepared immediately, sterile water for injection is used to dissolve the powder, 1mg/mL, and 200ul of developing agent is injected at each point. For patients with stage IIB cervical cancer, 200ul of developer is respectively added to the left and right sides of the upper middle part of the vagina. The same dose of ICG injection served as control. And (3) respectively observing the lymph node imaging condition under the fluorescence laparoscope in the operation, performing wide hysterectomy and pelvic lymph node cleaning operation in a conventional way, and comparing the sensitivity and the specificity of the lymph node imaging condition on the metastatic lymph node imaging condition and the metastatic lymph node imaging condition according to the lymph biopsy result reported by the pathology department after the operation.
Test results referring to fig. 10, the reagent of the present invention can accurately identify lymph nodes positive for cervical cancer metastasis.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
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Claims (7)
1. A tumor-targeted near-infrared fluorescence imaging agent has a structural formula of ICG-OSu-PEG4-g (cgnvvrqgc), wherein ICG is near-infrared fluorescent imaging agent indocyanine green, ICG-OSu is sulfonic indocyanine green activated ester, core NVVRQ of g (cgnvvrqgc) is tumor targeting cyclic polypeptide TMTP1, carboxyl reactive, polyethylene glycol (PEG) bridging, the structural formula is specifically:
2. the tumor-targeted near-infrared fluorescence imaging agent according to claim 1, characterized in that the preparation method comprises the following steps:
1) synthesis of polypeptide GCGNVVRQGC Using solid phase support, attached to PEG4；
2) Cleaving the polypeptide from the resin with MEOH/I2Oxidizing to form a ring by bridging the polypeptide sequence via disulfide bonds, i.e. PEG4-g (cgnvvrqgc), followed by isolation, purification, lyophilization;
3) mixing the product obtained in the step 2) with ICG-Osu according to a ratio of 1:1, dissolving in DMF, adding DIEA for reaction, carrying out LCMS (liquid crystal display system) identification reaction, separating, purifying and freeze-drying to obtain the developer.
3. The use of the tumor-targeted near-infrared fluorescence imaging agent according to any one of claims 1 or 2 in the preparation of a tumor diagnostic reagent.
4. Use according to claim 3, characterized in that said tumors are selected from the group consisting of cervical, breast, lung, prostate and renal cancers.
5. Use of the tumor-targeted near-infrared fluorescent imaging agent according to any one of claims 1 or 2 for preparing a lymph node diagnostic agent for differentiating tumor metastasis from inflammatory hyperplasia.
6. The use of the tumor-targeted near-infrared fluorescent imaging agent according to any one of claims 1 or 2 in the preparation of an excision indicator reagent for guiding tumor lesions and lymph node metastasis in fluorescence endoscopic surgery, wherein the tumors are cervical cancer and breast cancer.
7. The use of the tumor-targeted near-infrared fluorescence imaging agent according to any one of claims 1 or 2 in the preparation of a fluorescence colposcope sampling indicator reagent, wherein the tumor is cervical cancer.
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