Lymphatic Targeting Imaging Agent Based On Indocyanine Green

BackgroundLymphatic circulatory system is one of the most important vascular systems in the body.It plays a vital role in lipid absorption,maintaining tissue fluid balance and immune monitoring.The pathological changes of lymphatic system are closely related to the occurrence and development of cardiovascular diseases,immune system diseases,nervous system diseases,inflammation,malignant tumors and other systemic diseases.However,compared with the rapid development of diagnosis and treatment technology of vascular system diseases,the development of lymphatic system diseases is still in its infancy due to the obstacles of visualization technology.Therefore,it is of great significance for the medical field to establish an efficient monitoring system that can image the lymphatic system in real time.Indocyanine green（ICG）is a near-infrared fluorescent dye,which has high binding rate with plasma protein and is not absorbed by extrahepatic tissues.More importantly,ICG,as the only near-infrared fluorescent dye approved for clinical use by the U.S.Food and Drug Administration（FDA）,has been in clinical use for more than 50 years and has good safety.In recent years,ICG near infrared fluorescence imaging has been widely used in the early diagnosis,clinical staging,intraoperative navigation and prognosis monitoring of lymphedema diseases,and has gradually become the best method to study the functional state of superficial lymphatic system.PurposeAlthough ICG has been widely used in the field of lymphatic imaging,ICG has the following shortcomings:（1）No lymphatic targeting.Due to the small molecular size of ICG,after subcutaneous injection,it enters the capillary vein（endothelial space<4 nm）and lymphatic capillaries（endothelial space 30～120 nm）at the same time.The imaging is mostly diffuse and star shaped,which interferes with the development of superficial lymphatic system;（2）Physical and chemical properties are not ideal,such as poor stability,self-quenching and low quantum yield;（3）The development depth of lymphoid tissue was limited（<2cm）.These defects limit the full potential of near-infrared lymphography.Therefore,the development of lymphatic system specific developers based on ICG with lymphatic targeting,enhanced fluorescence intensity,good stability and high biosafety will be of great significance to the clinical diagnosis and treatment of lymphatic system related diseases.MethodTo solve the above problems,we designed nanoparticle carriers with different structures and functions,loaded ICG molecules,and prepared two new nanoparticle optical imaging agents through a series of post modifications.In order to improve the imaging performance of ICG from two directions:improving the lymphatic targeting of ICG and enhancing the fluorescence intensity and stability of ICG.The specific research is as follows:1.The FDA-approved small-molecule dye,ICG,has been widely used in lymphatic imaging.However,this small molecule imaging agent does not show selective binding ability after entering the lymphatic system.It will enter the capillary blood vessels and lymphatic capillaries at the same time,which will interfere with the imaging results;at the same time,the half-life of small molecule ICG in the body is very short and ICG will be quickly cleared by human tissue.Therefore,in this paper,aminoated self-degradable organic mesoporous silicon with an average particle size of 90 nm was selected as the carrier to connect carboxylated ICG to it;then,the remaining amino groups on the surface of mesoporous silicon were used to further modify hyaluronic acid（HA）on the surface of mesoporous silicon.Using mesoporous silicon loaded ICG can increase the size of the imaging system;Hyaluronic acid modified on the surface of mesoporous silicon can interact with hyaluronic acid receptor-1（LYVE-1）of lymphatic endothelial cells distributed in lymphatic vessels,which improves the imaging specificity and residence time of the imaging system in lymphatic vessels;At the same time,the imaging system has good biocompatibility,degradability within seven days and high biosafety.2.Then the cytotoxicity of ICG@HMONs-HA was detected by MTT assay and compared with three other commercially available nano materials with the same size as ICG@HMONs-HA;Three representative cells were selected to investigate the selectivity of ICG@HMONs-HA to lymphatic endothelial cells;Finally,in order to further prove the targeting effect of ICG@HMONs-HA,sections of mouse leg tissues were prepared and studied by co-localization fluorescence staining.3.Due to the natural defects in structure,ICG still has some problems such as poor stability,self-quenching and low quantum yield.In view of the above defects,we designed and synthesized a porous organic polymer（POP）as a nano carrier to improve the imaging function of ICG.POP shows great application potential in the field of fluorescence imaging because of its open structure,excellent stability and adjustable structural composition.In this paper,amino grafted porous polymers were prepared according to the characteristic structure of indocyanine green molecule containing both sulfonic acid groups and conjugated fragments.ICG was subsequently loaded within POP to examine the improvement of stability and fluorescence luminescence properties.4.Then the cytotoxicity of ICG-POP-HA at different concentrations was detected by MTT assay;three representative cells were selected to investigate the selectivity of ICG-POP-HA to lymphatic endothelial cells;finally,in order to further prove the targeting effect of ICG-POP-HA,sections of mouse leg tissues were prepared and studied by co-localization fluorescence staining.Result1.The correct synthesis of ICG@HMONs-HA material was proved by TEM,DLS and zeta potential;UV-Vis spectra showed that the optical properties of ICG@HMONs-HA did not change significantly;The degradation experiment simulating physiological conditions showed that ICG@HMONs-HA could be biodegraded spontaneously under physiological conditions,which also ensured the safety of ICG@HMONs-HA in organisms and laid a foundation for its application in lymphatic imaging.2.Firstly,the cytotoxicity of ICG@HMONs-HA was detected by MTT assay.The experimental results showed that even when the concentration of ICG@HMONs-HA was as high as 2.0 mg/ml,no obvious cytotoxicity was observed.Compared with the other three commercially available nano materials with the same size as ICG@HMONs-HA,the biosafety of ICG@HMONs-HA was still the best,which also provided a prerequisite for the biological application of ICG@HMONs-HA.Next,in order to investigate the selectivity of ICG@HMONs-HA on lymphatic endothelial cells,we selected three kinds of cells for research:mouse normal lymphatic endothelial cells（MLEC）,mouse macrophage cells（RAW264.7）and mouse colon cancer cells（CT26）.The experimental results showed that when ICG@HMONs-HA was used to stain cells,both the selectivity of cells（specific binding to MLEC）and the firm binding to cells（not easy to be cleaned）could be observed.This showed that ICG@HMONs-HA produced a strong affinity between hyaluronic acid and LYVE-1 on the surface,which promoted the obvious specific recognition and residence ability of ICG@HMONs-HA to lymphatic endothelial cells,making it more suitable for lymphatic imaging.In order to further prove the targeting effect of ICG@HMONs-HA,we prepared sections of mouse leg tissues and performed co-localization fluorescence staining with ICG@HMONs-HA and LYVE-1antibodies.The experimental results showed that ICG@HMONs-HA could target and bind to the lymphatic region.3.The obtained NH₂-POP porous polymers had pore sizes centered at 1.2 and 3.5nm.The 3.5-nanometer mesoporous channel provided a large pore space for the rapid transport of indocyanine green molecules inside the particle,and the 1.2-nanometer micropore size incorperated the ICG through the synergistic effect of electrostatic bonding andπ-πinteractions.More importantly,ICG-POP complex could significantly improve the fluorescence intensity and stability of ICG.Then,the remaining amino groups on the surface of POP were used to further modify hyaluronic acid on the surface of POP to obtain the ICG-POP-HA composite imaging agent,in order to enhance the interaction between imaging agent and LYVE-1 in lymphatic vessels,and improve the imaging specificity and residence time of imaging system in lymphatic vessels.4.Firstly,the cytotoxicity of ICG-POP-HA was detected by MTT assay.The experimental results showed that even when the concentration of ICG-POP-HA was as high as 5.0 mg/ml,no obvious cytotoxicity was observed.Next,in order to investigate the selectivity of ICG-POP-HA on lymphatic endothelial cells,we selected three kinds of cells for research:mouse normal lymphatic endothelial cells（MLEC）,mouse macrophage cells（RAW264.7）and mouse colon cancer cells（CT26）.The experimental results showed that when ICG-POP-HA was used to stain cells,both cell selectivity and firm binding with cells could be observed.At the same time,the qualitative calculation using Adobe Photoshop CS3 software also showed that compared with ICG,ICG-POP-HA had higher green fluorescence intensity when imaging MLEC cells,which indicated that POP could not only stabilize ICG,but also improve the fluorescence intensity of ICG during imaging.Finally,we prepared sections of mouse leg tissues and performed co-localization fluorescence staining with ICG-POP-HA and LYVE-1 antibodies.The experimental results showed that ICG-POP-HA could bind to the lymphatic region.This work provides a practical method to enhance the fluorescence intensity and stability of ICG,and proves that POP based solid nano materials also have great application prospects in fluorescence imaging and biomedical applications.ConclusionIn conclusion,two novel ICG based lymphatic targeting nano imaging agents were designed and synthesized in this paper.Among them,ICG nano imaging agent with mesoporous silicon as carrier can improve the targeting and residence time of ICG imaging system in lymphatic vessels;ICG nano imaging agent with POP as carrier can further enhance the fluorescence intensity and stability of ICG on the basis of improving the targeting of imaging agent.The above work provides two practical methods to improve the imaging ability of ICG,and provides a new idea for the design of lymphatic imaging agent based on nano materials.