CN115337409B - Albumin combined near infrared fluorescent probe-fatty acid conjugate, and preparation method and application thereof - Google Patents
Albumin combined near infrared fluorescent probe-fatty acid conjugate, and preparation method and application thereof Download PDFInfo
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Classifications
-
- 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
- A61K49/0052—Small organic molecules
-
- 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
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
- A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/56—Ring systems containing three or more rings
- C07D209/58—[b]- or [c]-condensed
- C07D209/60—Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
AlbuminThe albumin combined near infrared fluorescent probe-fatty acid conjugate is prepared by replacing chlorine atoms in a cyanine dye conjugated chain with amino-substituted fatty acids, and modifying the optical properties of the albumin combined near infrared fluorescent probe-fatty acid conjugate by intramolecular charge transfer effect, so that the albumin combined near infrared fluorescent probe-fatty acid conjugate has longer Stokes shift and can enhance resolution of in-vivo imaging. Secondly, the fatty acid groups in the conjugate can interact with albumin in plasma through non-covalent bonds, so that the conjugate has good lymphatic homing capacity and tumor targeting. The near infrared fluorescent probe modified by fatty acid overcomes the defects of unstable fluorescent dye and low imaging resolution in clinical tumor and sentinel lymph node diagnosis, so that the near infrared fluorescent probe can realize good tumor and lymph node positioning imaging function in vivo.
Description
Technical Field
The invention belongs to the field of new auxiliary materials and new dosage forms of pharmaceutical preparations, and particularly relates to a near infrared fluorescent probe-fatty acid conjugate which takes endogenous plasma albumin as a carrier, is designed and synthesized in vivo and combines with albumin in plasma through non-covalent bonds, and application thereof in the aspects of sentinel lymph node and tumor diagnosis of tumor metastasis.
Background
Cancer is severely threatening the life safety and health of humans. Surgical excision remains the primary means of treating cancer, particularly for solid tumors. More than 90% of cancer-related deaths are reported to be due to metastasis. Sentinel lymph nodes serve as the first site for tumor cell metastasis to distant tissues or organs. Thus, accurate identification of primary tumors and sentinel nodes is of great importance to guide the surgeon in formulating a treatment regimen for a clinical patient. Currently, some radiotracers or blue dyes (methylene blue) are commonly used for clinical diagnosis of primary tumors or sentinel lymph nodes. However, radioactive elements are harmful to human bodies, blue dye can be dispersed in tissues to cause inaccurate diagnosis of sentinel lymph nodes, and popularization of clinical application is limited. Near infrared fluorescence imaging technology is widely used for diagnosing clinical diseases due to the advantages of non-invasiveness, good stability, low tissue interference and the like, and has certain advantages in the field of disease diagnosis.
Currently, the near infrared fluorescent dye indocyanine green (ICG) and its derivatives are widely used for sentinel lymph node or tumor fluorescence imaging. However, ICG also has the disadvantages of low in vivo imaging resolution, poor photostability, quick in vivo elimination, etc., which reduces the accuracy of sentinel lymph node or tumor localization. The ideal near infrared fluorescent probe for in-vivo high-resolution imaging has the following characteristics: (1) has good light stability; (2) high fluorescence quantum yield; (3) a large Stokes shift. Therefore, the development of high-resolution fluorescent probes to improve the accurate diagnosis of sentinel lymph nodes and tumors is of great importance.
Albumin is the most abundant protein in blood plasma, and has the characteristics of good biocompatibility, low immunogenicity, long in vivo half-life (19 days), tumor targeting, lymph homing and the like. Currently, albumin is widely used as a drug delivery vehicle in the field of drug delivery. Based on this, the fluorescence imaging efficiency is improved by using albumin in the near infrared fluorescent probe carrier.
Disclosure of Invention
Aiming at the defects of the existing sentinel lymph node and imaging method in tumor diagnosis, the invention designs an albumin combined near infrared fluorescent probe-fatty acid conjugate with good biological safety, high tumor targeting, good lymph homing capability and high imaging resolution, which is used for the precise positioning of sentinel lymph node and in-situ tumor of tumor metastasis.
The invention realizes the aim through the following technical scheme:
the invention provides three albumin combined near infrared fluorescent probe-fatty acid conjugates, wherein the near infrared fluorescent probe-fatty acid conjugates are obtained by substitution reaction of near infrared fluorescent probes and amino substituted fatty acids; the near infrared fluorescence probeThe needle is indocyanine green derivative containing chlorbenzene ring, and is selected from new indocyanine green (IR 820), IR780 and IR808; the structure of the amino-substituted fatty acid selected by the near infrared fluorescent probe-fatty acid conjugate is NH 2 -(CH 2 ) n-COOH, wherein n=2-23, preferably 6-16.
Further, the present invention preferably provides near infrared fluorescent probe-fatty acid conjugates obtained by coupling new indocyanine green (IR 820) with 8-aminocaprylic acid, 12-aminocaprylic acid and 16-aminopalmitic acid, respectively, by substitution reaction.
In the new indocyanine green and amino substituted fatty acid conjugate of near infrared fluorescent probe-fatty acid obtained by substitution, new indocyanine green (IR 820) is selected as a model fluorescent probe and is connected with 8-aminocaprylic acid, 12-aminolauric acid and 16-aminopalmitic acid. The structural formula is as follows:
the synthetic route and the preparation method of the three albumin-binding near infrared fluorescent probe-fatty acid conjugates are as follows:
IR820, 8-aminocaprylic acid, 12-aminopropionic acid and 16-aminopalmitic acid are respectively dissolved in a solvent in a molar ratio of 1:1-1:5, and stirred for 5-10 minutes to be completely dissolved. Then Triethylamine (TEA) was added to the reaction system and nitrogen was charged, and the reaction system was heated to 70-90℃and stirred for 1-6 hours in the dark, and the color of the reactant was observed to gradually change from green to blue. After the reaction, the solvent was removed and purified to give the products IR-OA, IR-LA and IR-PA.
In the preparation method, the solvent is anhydrous DMF, ethyl Acetate (EA), acetone (CP) or tetrahydrofuran.
The invention relates to an application of albumin binding type near infrared fluorescent probe-fatty acid conjugate in preparation of albumin binding type conjugate.
The invention relates to an application of albumin-combined near infrared fluorescent probe-fatty acid conjugate in preparing a tumor or tumor metastasis lymph node diagnostic imaging agent.
The albumin-binding near infrared fluorescent probe-fatty acid conjugate is applied to injection or subcutaneous local administration as an imaging agent.
The invention has the following beneficial effects:
by modifying IR820 with amino-substituted fatty acids, the stokes shift of IR-OA, IR-LA and IR-PA increases due to the shortening of the conjugated structure of IR820 as the electron withdrawing group chlorine atom of the conjugated chain of IR820 is replaced by the co-electron group secondary amine. Although the chlorine atom of IR820 is substituted, its fluorescence properties are not affected and it still has near infrared fluorescence imaging capability, so IR-OA, IR-LA and IR-PA can be applied as fluorescence imaging probes. As fatty acid groups are modified on the IR820 molecules, the IR-OA, IR-LA and IR-PA can be combined with albumin through non-covalent bonds, so that the imaging device has the lymphatic homing and tumor targeting capabilities and improves the resolution and imaging efficiency of lymphatic and tumor imaging. The invention provides a new strategy and thought for developing safe, effective, stable and portable clinical diagnosis of sentinel lymph nodes and in-situ tumors, and meets the urgent clinical demands for sentinel lymph nodes and in-situ tumor fluorescence imaging agents for tumor metastasis.
Drawings
FIG. 1 shows a near infrared fluorescent probe-fatty acid conjugate (IR-OA) of example 1 of the invention 1 H NMR nuclear magnetic spectrum.
FIG. 2 is a high resolution mass spectrum of near infrared fluorescent probe-fatty acid conjugate (IR-OA) of example 1 of the present invention.
FIG. 3 shows a near infrared fluorescent probe-fatty acid conjugate (IR-LA) of example 1 of the present invention 1 H NMR nuclear magnetic spectrum.
FIG. 4 is a high resolution mass spectrum of near infrared fluorescent probe-fatty acid conjugate (IR-LA) of example 1 of the present invention.
FIG. 5 shows the near infrared fluorescent probe-fatty acid conjugate (IR-PA) of example 1 of the present invention 1 H NMR nuclear magnetic spectrum.
FIG. 6 is a high resolution mass spectrum of near infrared fluorescent probe-fatty acid conjugate (IR-PA) of example 1 of the present invention.
FIG. 7 is a graph showing albumin binding gel results for near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) of example 2 of the present invention.
FIG. 8 is a graph showing fluorescence emission spectra of the albumin complex (BSA-IR-OA) of the near infrared fluorescent probe-fatty acid conjugate IR-OA of example 3 according to the present invention after various long-term illumination.
FIG. 9 is a graph showing fluorescence emission spectra of albumin complex (BSA-IR-LA) of near infrared fluorescent probe-fatty acid conjugate IR-LA according to example 3 of the present invention after various long-term illumination.
FIG. 10 is a fluorescence emission spectrum of albumin complex (BSA-IR-PA) of near infrared fluorescent probe-fatty acid conjugate IR-PA of example 3 of the present invention after various long-time illumination.
FIG. 11 is a graph showing ultraviolet absorption spectra of near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) and albumin complexes (BSA-IR-OA, BSA-IR-LA and BSA-IR-PA) according to example 4 of the present invention.
FIG. 12 is a graph showing fluorescence emission spectra of near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) and albumin complexes (BSA-IR-OA, BSA-IR-LA and BSA-IR-PA) according to example 4 of the present invention.
FIG. 13 is a cytotoxicity pattern of near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) and IR820 against 4T1 cells, PC3 cells and L02 according to example 5 of the present invention.
FIG. 14 is a graph showing the uptake results of near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) of example 6 of the present invention in 4T1 cells for 1 hour.
FIG. 15 is a graph showing the uptake results of near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) of example 6 of the present invention in 4T1 cells for 4 h.
FIG. 16 is a fluorescence imaging diagram of near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) and IR820 in tumor metastasis lymph nodes according to example 7 of the present invention.
FIG. 17 is a fluorescence imaging diagram of near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) and IR820 of example 8 of the present invention in tumor.
FIG. 18 is an H & E staining chart of major organs of near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) of example 9 of the present invention after intravenous injection to mice to demonstrate the long-term safety of IR-OA, IR-LA and IR-PA to individual organs.
FIG. 19 is an H & E staining chart of major organs of near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) of example 9 of the present invention after subcutaneous injection into footpads of mice to demonstrate the long-term safety of IR-OA, IR-LA and IR-PA to individual organs.
FIG. 20 is a graph showing the results of liver and kidney function index of near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) of example 9 of the present invention after intravenous injection to mice, to demonstrate the long-term and short-term safety of IR-OA, IR-LA and IR-PA against liver and kidney functions.
FIG. 21 is a graph showing the results of liver and kidney function index of near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) of example 9 of the present invention after subcutaneous injection into footpads of mice, to demonstrate the long-term and short-term safety of IR-OA, IR-LA and IR-PA against liver and kidney functions.
Detailed Description
The foregoing of the invention is further illustrated by the following specific examples, which are not intended to limit the invention thereto.
Example 1: synthesis of albumin-bound near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA)
IR820 (200 mg,0.236 mmol) and 8-aminocaprylic acid (112.7 mg, 0.706 mmol) or 12-aminododecanoic acid (152.5 mg, 0.706 mmol) or 16-aminohexadecanoic acid (192.2 mg, 0.706 mmol) were dissolved in anhydrous DMF (6 mL) and stirred for 5 min. Then, triethylamine (TEA, 134. Mu.L, 0.946 mmol) was added to the reaction system and nitrogen gas was introduced. The reaction was carried out in the absence of light at a temperature of 85℃for 3 hours. The color of the solution was observed to change from green to blue during the reaction. And the reaction process was monitored by Thin Layer Chromatography (TLC). After the reaction, DMF was removed by rotary evaporation under reduced pressure. Finally, the product was purified by silica gel column chromatography using ethyl acetate/methanol (v/v=10:1 to 3:1) as eluent gradient elution to give blue solid IR-OA, IR-LA and IR-PA in yields of 82%, 79% and 77%, respectively.
The structures of IR-OA, IR-LA and IR-PA prepared in example 1 were confirmed using High Resolution Mass Spectrometry (HRMS) and hydrogen nuclear magnetic resonance spectroscopy. The results are shown in FIGS. 1-6. d-DMSO is the solvent of choice in nuclear magnetic resonance, and the Bopu analysis results are as follows:
IR-OA： 1 H NMR(600MHz,DMSO-d6)δ8.15(d,J＝8.5Hz,2H),7.97(dd,J＝8.6,5.6Hz,4H),7.79–7.70(m,2H),7.57(dd,J＝16.1,8.4Hz,4H),7.39(t,J＝7.5Hz,2H),5.86(d,J＝13.0Hz,2H),4.08(d,J＝7.5Hz,4H),3.75(t,J＝6.8Hz,2H),2.55(dd,J＝14.7,8.1Hz,4H),2.20(t,J＝7.4Hz,2H),1.90(s,12H),1.77(ddd,J＝27.7,14.7,7.5Hz,12H),1.52(d,J＝7.1Hz,2H),1.41(q,J＝6.5,6.0Hz,2H),1.38–1.20(m,6H),0.89–0.84(m,2H).ESI-MS:m/z＝948.432[M-Na] - 。
IR-LA： 1 H NMR(600MHz,DMSO-d6)δ8.14(d,J＝8.7Hz,2H),7.97(dd,J＝8.8,5.5Hz,4H),7.75(d,J＝12.9Hz,2H),7.59(d,J＝8.9Hz,2H),7.56(t,J＝7.7Hz,2H),7.39(t,J＝7.5Hz,2H),5.87(d,J＝12.7Hz,2H),4.10(s,4H),3.75(d,J＝7.0Hz,2H),2.56(t,J＝7.3Hz,8H),2.12(q,J＝7.3,6.8Hz,2H),1.91(s,12H),1.79(dq,J＝18.9,7.0Hz,10H),1.45(t,J＝7.1Hz,2H),1.42–1.38(m,2H),1.36–1.31(m,2H),1.29–1.22(m,8H),1.21(s,4H).ESI-MS:m/z＝1004.49384[M-Na] - 。
IR-PA： 1 H NMR(600MHz,DMSO-d6)δ8.13(d,J＝8.3Hz,2H),7.97(t,J＝7.4Hz,4H),7.74(d,J＝13.1Hz,2H),7.65–7.52(m,4H),7.39(t,J＝7.6Hz,2H),5.86(d,J＝12.8Hz,2H),4.08(s,4H),3.75(s,2H),2.13(t,J＝7.7Hz,4H),2.56(s,2H),1.90(s,11H),1.78(t,J＝16.8Hz,14H),1.41(s,4H),1.22(s,12H),1.14(s,10H).ESI-MS:m/z＝1050.51120[M-Na] - 。
example 2: binding experiments of albumin-binding near-infrared fluorescent probe-fatty acid conjugate to Bovine Serum Albumin (BSA)
BSA was dissolved in Phosphate Buffer (PBS) at pH7.4 to give a BSA solution at a concentration of 35. Mu.M. BSA solution was mixed with IR-OA, IR-LA and IR-PA to give BSA-IR-OA, BSA-IR-LA and BSA-IR-PA solutions and incubated in a shaking table at 37℃for 12 hours. All samples were then analysed for binding to BSA using 10% SDS-polyacrylamide gel (SDS-PAGE). After the electrophoresis, the peeled gel plate was stained with coomassie blue dye, washed, and placed in a fluorescence imager, and fluorescence of BSA-IR-OA, BSA-IR-LA and BSA-IR-PA was observed at an excitation wavelength of 660nm and an emission wavelength of 790 nm.
As a result, as shown in FIG. 7, in the Coomassie brilliant blue staining results, the group containing albumin appeared with a blue band in the range of 63-75 kDa; in the fluorescence imaging results, only the BSA-IR-LA and BSA-IR-PA groups appeared in the same positions where the protein bands appeared, and the BSA-IR-OA groups did not appear fluorescence at the positions of albumin, which indicated that the binding capacity of IR-LA and IR-PA to albumin was stronger than that of IR-OA. Since IR-OA, IR-LA and IR-PA are small molecule compounds, the movement speed in electrophoresis is high and occurs at the bottom of the gel plate.
Example 3: light stability study of albumin-binding near-infrared fluorescent Probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) in vitro binding to albumin
Illumination is an indispensable condition for sentinel lymph node and clinical diagnosis of tumor, and thus it is very necessary to examine the photostability of fluorescent probes. Equimolar amounts of the complex of BSA-IR-OA, BSA-IR-LA and BSA-IR-PA were dissolved in the same volume of phosphate buffer solution (pH 7.4), sampled at different times under continuous illumination of a fluorescent lamp (0 min, 30min, 1h, 2h, 3h, 5 h), and then the samples were placed in a black 96-well plate, and their fluorescence emission spectra (excitation wavelength 660 nm) were measured using a multifunctional microplate reader.
The results of the light stability of BSA-IR-OA, BSA-IR-LA and BSA-IR-PA are shown in FIGS. 8,9 and 10, and after continuous illumination for 3 hours, the fluorescence intensities of BSA-IR-LA and BSA-IR-PA were hardly changed, whereas the fluorescence intensity of BSA-IR-OA was reduced by 7%. In addition, the fluorescence intensities of BSA-IR-OA, BSA-IR-LA and BSA-IR-PA after 5 hours of illumination were reduced by 36%, 26% and 10%, respectively, indicating different degrees of degradation of the albumin conjugates. Among them, BSA-IR-PA has the best light stability.
Example 4: characterization of optical Properties of Albumin-bound near-infrared fluorescent Probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA)
IR-OA, IR-LA, IR-PA and BSA-IR-OA, BSA-IR-LA, BSA-IR-PA complexes were prepared in the same molar concentrations and the ultraviolet absorbance spectra of the samples were measured using an enzyme-labeled instrument. The fluorescence emission spectrum (excitation wavelength: 660 nm) of the sample was also measured.
As a result, as shown in FIG. 11, after incubation with BSA, the ultraviolet absorption peaks of IR-LA and IR-PA were observed to be significantly shifted toward the long wave (red shift). However, the absorption peak of BSA-IR-OA was hardly changed compared to IR-OA. The above results indicate that IR-LA and IR-PA are capable of binding to BSA by strong hydrophobic forces. The fluorescence intensities of IR-OA, IR-LA and IR-PA were observed to increase significantly after incubation with BSA by about 3, 36 and 149-fold, respectively, in the fluorescence emission spectra shown in FIG. 12.
Example 5: cytotoxicity of albumin-bound near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA)
Mouse breast cancer cells (4T 1), human prostate cancer cells (PC 3) and human normal hepatocytes (L02) were embedded in a sterile 96-well plate at a density of 2000 cells/well at 37℃and 5% CO 2 Is cultured in an incubator for 12 hours. The medium was then poured off and the cells incubated with various concentrations of IR-OA, IR-LA, IR-PA and IR820 for 48 hours. After the incubation, the cell viability of the three cells was determined by MTT method (absorption wavelength 570 nm).
Cytotoxicity results of IR-OA, IR-LA, IR-PA and IR820 on 4T1 cells, PC3 cells and L02 cells As shown in FIG. 13, the cell viability of the three cells after 48 hours incubation of IR-OA, IR-LA and IR-PA was nearly 100%, indicating that IR-OA, IR-LA and IR-PA were safe for the three cell lines in the concentration range of 30.0625-1250 nM. However, IR820 showed significant cytotoxicity to three cells at concentrations of 625nM and 1250 nM. These results indicate that the safety of IR-OA, IR-LA and IR-PA is higher than that of IR820.
Example 6: cellular uptake of albumin-bound near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA)
Uptake of IR-OA, IR-LA and IR-PA by 4T1 cells was determined using flow cytometry. 4T1 cells were plated at 3X 10 5 Cell/well density was seeded into 12-well cell culture plates and incubated overnight. Cells were then incubated with the same molar concentrations of IR-OA, IR-LA and IR-PA (5. Mu.M) for 1 or 4 hours. After the incubation was completed, the cells were washed three times with ice-cold PBS, digested with trypsin, centrifuged and collected. Finally, cellular uptake was determined using a flow cytometer.
The uptake results are shown in FIGS. 14 and 15, and the order of the fluorescence intensities of IR-OA, IR-LA and IR-PA in the cells was IR-PA > IR-LA > IR-OA, whether at 1 hour or 4 hours, indicating that IR-PA has the highest cell uptake efficiency.
Example 7: fluorescent imaging of albumin-bound near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) in tumor metastasis sentinel lymph nodes
4T1 cells were plated at 5X 10 in PBS per 20. Mu.L 5 Cell density was inoculated subcutaneously into the right hind footpad of female Balb/c mice to establish a sentinel lymph node animal model of tumor metastasis. After successful model establishment, mice were given subcutaneously injected with equal amounts of IR-OA, IR-LA, IR-PA and IR820 (2 mg/kg, 20. Mu.L) in the right hind paw pads. At predetermined time points (5, 15, 30, 45, 60, 80, 100 and 120 min), the fluorescent imaging conditions of IR-OA, IR-LA, IR-PA and IR820 at sentinel lymph nodes of tumor metastasis (excitation wavelength 660nm, emission wavelength 790 nm) were observed using a small animal biopsy imager.
As a result, as shown in FIG. 16, IR-OA, IR-LA and IR-PA reached the sentinel lymph node by lymphatic drainage within 5min, and the accumulation amount in the sentinel lymph node increased with time. The IR-OA, IR-LA and IR-PA show enhanced fluorescence after binding to albumin in vivo compared to IR820, and thus show stronger fluorescence signals and imaging resolution. Furthermore, IR820 was observed to be significantly distributed in the liver at 5-80min of subcutaneous injection, possibly due to rapid diffusion of small molecules in the blood. Notably, the higher fluorescence intensity of IR-PA than IR-OA and IR-LA may be due to its higher photostability. The above results indicate that IR-OA, IR-LA and IR-PA have a pronounced lymphatic targeting after binding to albumin in vivo.
Example 8: fluorescent imaging of albumin-bound near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA) at tumor sites
Will contain 5X 10 6 100 μl PBS of 4T1 cells was inoculated subcutaneously into the right back blank region of female Balb/c mice to establish a tumor-bearing mouse model. When the tumor volume reaches about 300mm 3 At this time, IR-OA, IR-LA, IR-PA and IR820 were injected into mice by tail vein administration (dose of 2 mg/kg). Fluorescence signals of IR-OA, IR-LA, IR-PA and IR820 at the tumor sites were then monitored using a small animal in vivo imager at various time points (1, 2, 4, 8, 12, 24 and 48 h) after dosing.
Fluorescent imaging of IR-OA, IR-LA, IR-PA and IR820 at tumor sites in 4T1 tumor-bearing mice As shown in FIG. 17, the fluorescent signals were stronger in the IR-OA, IR-LA and IR-PA groups compared to the IR 820-administered group. In addition, the IR-PA group has higher fluorescence intensity and imaging resolution than IR-OA, IR-LA and IR820. The above results indicate that IR-OA, IR-LA and IR-PA are able to target the albumin in the carrier effectively to the tumor.
Example 9: in vivo safety of albumin-bound near infrared fluorescent probe-fatty acid conjugates (IR-OA, IR-LA and IR-PA)
To investigate the short-term and long-term in vivo safety of IR-OA, IR-LA and IR-PA, healthy female Balb/c mice were randomly divided into 6 groups (n=3). Of these 3 groups, IR-OA, IR-LA and IR-PA (2 mg/kg) were subcutaneously injected into the mouse footpad, mice were sacrificed 2 hours or 3 weeks after administration, serum was collected, and heart, liver, spleen, lung and kidney of the mice were dissected, and liver and kidney function indexes and histological analysis of the mice were examined. In another 3 groups, IR-OA, IR-LA and IR-PA (2 mg/kg) were injected into mice by tail vein, mice were sacrificed 2 hours or 3 weeks after administration, serum was collected, and liver and kidney function index and histological analysis of the mice were examined by dissecting out the major organs. In addition, 2 control groups were set, and mice were sacrificed after 3 weeks by subcutaneous injection of physiological saline into the footpad or tail vein of each mouse, and plasma and major organs were collected for liver and kidney function examination and H & E staining analysis, respectively.
Acute and chronic toxicity of IR-OA, IR-LA and IR-PA to healthy Balb/c mice was evaluated by H & E staining and liver and kidney function index. The results are shown in figures 18, 19, with no significant damage to tissue sections of the major organs of the mice after 2 hours and 3 weeks following IR-OA, IR-LA and IR-PA intravenous injection and footpad subcutaneous injection. Similarly, the results of toxicology examination are shown in FIGS. 20 and 21, and the indices of liver and kidney functions of the IR-OA, IR-LA and IR-PA intravenous and subcutaneous administration groups were all in the normal range, and were not significantly different from those of the physiological saline group. The above experimental results show that IR-OA, IR-LA and IR-PA have good in vivo biosafety.
Claims (4)
1. An albumin-bound near infrared fluorescent probe-fatty acid conjugate, characterized in that the albumin-bound near infrared fluorescent probe-fatty acid conjugate is any one of near infrared fluorescent probe-fatty acid conjugates obtained by connecting IR820 with 12-amino lauric acid and 16-amino palmitic acid respectively through substitution reaction, and the structural formulas are as follows:
2. use of the albumin-bound near infrared fluorescent probe-fatty acid conjugate of claim 1 in the preparation of an albumin-bound conjugate.
3. Use of the albumin-bound near infrared fluorescent probe-fatty acid conjugate of claim 1 in the preparation of a diagnostic imaging agent for tumors or tumor metastasis lymph nodes.
4. Use of the albumin-bound near infrared fluorescent probe-fatty acid conjugate of claim 1 for the preparation of an imaging agent for injectable or subcutaneous topical administration.
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