A novel method for targeting lymphatic vessel imaging: methylene blue nanoparticle integrated with dissolvable microneedles

Abstract

The lymphatic system serves many more functions than simply maintaining tissue fluid homeostasis, and its structural and functional changes indicate the occurrence of disease. Current clinical methods for the assessment of the lymphatic system, however, are severely limited because of their nontargeting ability, invasiveness, high cost, and radiation risk. Herein, we propose a simple and painless method for visualizing and quantifying the lymphatic system. This method is based on the noninvasive administration of a novel lymphatic tracer via dissolvable microneedles, followed by the application of a portable detection device for near-infrared (NIR) imaging. The tracer is prepared by incorporating the clinically approved NIR fluorescent dye methylene blue (ME) into the nanomaterial monomethoxyl poly(ethylene glycol)-b-poly(ε-caprolactone) (MPEG-PCL@ME). This novel tracer displays superior fluorescence properties, stability, biocompatibility, and targeting features in comparison with ME solution alone. Lymphography with MPEG-PCL@ME in vivo clearly revealed the lymphatic vessel morphology. Notably, compared with ME and indocyanine green, MPEG-PCL@ME can easily identify the dominant lymphatic vessels and nodes in rats with higher imaging quality. Furthermore, a series of segmental contracting sections are detected with MPEG-PCL@ME, allowing straightforward identification of the lymphatic pump, which provides direct evidence for exquisitely evaluating lymphatic functions.

Keywords

Methylene blue / Nanoparticle / Dissolving microneedles / Lymphatic vessel / Imaging tracer

References

[1] Janardhan HP, Jung R, Trivedi CM. Lymphatic system in organ development, function, and regeneration. Circ Res 2023; 132:1181-4. https://doi.org/10.1161/CIRCRESAHA.123.322867.

[2] Swartz MA. The physiology of the lymphatic system. Adv Drug Deliv Rev 2001; 50:3-20. https://doi.org/10.1016/s0169-409x(01)00150-8.

[3] Janardhan HP, Wachter BT, Trivedi CM. Lymphatic system development and function. Curr Cardiol Rep 2024; 26:1209-19. https://doi.org/10.1007/s11886-024-02120-8.

[4] Tso P, Bernier-Latmani J, Petrova TV, Liu M. Transport functions of intestinal lymphatic vessels. Nat Rev Gastroenterol Hepatol 2025; 22:127-45. https://doi.org/10.1038/s41575-024-00996-z.

[5] Igarashi A, Hayashi T, Shimizu T, Yuda K, Yamagami S. Inhibiting corneal transplantation rejection via lymphatic vessel ligation in a novel murine model. Sci Rep 2024; 14:25692. https://doi.org/10.1038/s41598-024-77160-9.

[6] Maclellan RA, Greene AK. Lymphedema. Semin Pediatr Surg 2014; 23:191-7. https://doi.org/10.1053/j.sempedsurg.2014.07.004.

[7] Warren AG, Brorson H, Borud LJ, Slavin SA. Lymphedema: a comprehensive review. Ann Plast Surg 2007; 59:464-72. https://doi.org/10.1097/01.sap.0000257149.42922.7e.

[8] Sudduth CL, Greene AK. Primary lymphedema: update on genetic basis and management. Adv Wound Care 2022; 11:374-81. https://doi.org/10.1089/wound.2020.1338.

[9] Leong SP, Pissas A, Scarato M, Gallon F, Pissas MH, Amore M, et al. The lymphatic system and sentinel lymph nodes: conduit for cancer metastasis. Clin Exp Metastasis 2022; 39:139-57. https://doi.org/10.1007/s10585-021-10123-w.

[10] Hu D, Li L, Li S, Wu M, Ge N, Cui Y, et al. Lymphatic system identification, pathophysiology and therapy in the cardiovascular diseases. J Mol Cell Cardiol 2019; 133:99-111. https://doi.org/10.1016/j.yjmcc.2019.06.002.

[11] Tsukiji N, Suzuki-Inoue K. Impact of hemostasis on the lymphatic system in development and disease. Arterioscler Thromb Vasc Biol 2023; 43:1747-54. https://doi.org/10.1161/ATVBAHA.123.318824.

[12] Pamarthi V, Pabon-Ramos WM, Marnell V, Hurwitz LM. MRI of the central lymphatic system: indications, imaging technique, and pre-procedural planning. Top Magn Reson Imaging 2017; 26:175-80. https://doi.org/10.1097/RMR.0000000000000130.

[13] Dori Y. Novel lymphatic imaging techniques. Tech Vasc Interv Radiol 2016; 19:255-61. https://doi.org/10.1053/j.tvir.2016.10.002.

[14] Clément O, Luciani A. Imaging the lymphatic system: possibilities and clinical applications. Eur Radiol 2004; 14:1498-507. https://doi.org/10.1007/s00330-004-2265-9.

[15] Barrett T, Choyke PL, Kobayashi H. Imaging of the lymphatic system: new horizons. Contrast Media Mol Imaging 2006; 1:230-45. https://doi.org/10.1002/cmmi.116.

[16] Albayram MS, Smith G, Tufan F, Tuna IS, Bostancıklıoğlu M, Zile M, et al. Non-invasive MR imaging of human brain lymphatic networks with connections to cervical lymph nodes. Nat Commun 2022; 13:203. https://doi.org/10.1038/s41467-021-27887-0.

[17] Sevick-Muraca EM. Translation of near-infrared fluorescence imaging technologies: emerging clinical applications. Annu Rev Med 2012; 63:217-31. https://doi.org/10.1146/annurev-med-070910-083323.

[18] Chao AH, Schulz SA, Povoski SP. The application of indocyanine green (ICG) and near-infrared (NIR) fluorescence imaging for assessment of the lymphatic system in reconstructive lymphaticovenular anastomosis surgery. Expert Rev Med Devices 2021; 18:367-74. https://doi.org/10.1080/17434440.2021.1900725.

[19] Rasmussen JC, Tan IC, Marshall MV, Fife CE, Sevick-Muraca EM. Lymphatic imaging in humans with near-infrared fluorescence. Curr Opin Biotechnol 2009; 20:74-82. https://doi.org/10.1016/j.copbio.2009.01.009.

[20] Nakajima Y, Asano K, Mukai K, Urai T, Okuwa M, Sugama J, et al. Near-infrared fluorescence imaging directly visualizes lymphatic drainage pathways and connections between superficial and deep lymphatic systems in the mouse hindlimb. Sci Rep 2018; 8:7078. https://doi.org/10.1038/s41598-018-25383-y.

[21] Li K, Nicoli F, Cui C, Wo Y, Liu NF, Feng S, et al. Vascularized lymph node flaps can survive on venous blood without an arterial inflow: an experimental model describing the dynamics of venous flow using indocyanine green angiography (with video). Burns Trauma 2023; 11:tkad019. https://doi.org/10.1093/burnst/tkad019.

[22] Chen QY, Xie JW, Zhong Q, Wang JB, Lin JX, Lu J, et al. Safety and efficacy of indocyanine green tracer-guided lymph node dissection during laparoscopic radical gastrectomy in patients with gastric cancer: a randomized clinical trial. JAMA Surg 2020; 155:300-11. https://doi.org/10.1001/jamasurg.2019.6033.

[23] Russell PS, Velivolu R, Maldonado Zimbrón VE, Hong J, Kavianinia I, Hickey AJR, et al. Fluorescent tracers for in vivo imaging of lymphatic targets. Front Pharmacol 2022; 13:952581. https://doi.org/10.3389/fphar.2022.952581.

[24] Guo W, Zhang L, Ji J, Gao W, Liu J, Tong M. Evaluation of the benefit of using blue dye in addition to indocyanine green fluorescence for sentinel lymph node biopsy in patients with breast cancer. World J Surg Oncol 2014; 12:290. https://doi.org/10.1186/1477-7819-12-290.

[25] Dai T, Zhou S, Yin C, Li S, Cao W, Liu W, et al. Dextran-based fluorescent nanoprobes for sentinel lymph node mapping. Biomaterials 2014; 35:8227-35. https://doi.org/10.1016/j.biomaterials.2014.06.012.

[26] Cwalinski T, Polom W, Marano L, Roviello G, D’Angelo A, Cwalina N, et al. Methylene blue-current knowledge, fluorescent properties, and its future use. J Clin Med 2020; 9:3538. https://doi.org/10.3390/jcm9113538.

[27] Clifton J 2nd, Leikin JB . Methylene blue. Am J Ther 2003; 10:289-91. https://doi.org/10.1097/00045391-200307000-00009.

[28] Gureev AP, Sadovnikova IS, Popov VN. Molecular mechanisms of the neuroprotective effect of methylene blue. Biochemistry (Mosc) 2022; 87:940-56. https://doi.org/10.1134/S0006297922090073.

[29] Fathi A, Asgari E, Danafar H, Salehabadi H, Fazli MM. A comprehensive study on methylene blue removal via polymer and protein nanoparticle adsorbents. Sci Rep 2024; 14:29434. https://doi.org/10.1038/s41598-024-80384-4.

[30] Al-Taher M, van den Bos J, Schols RM, Bouvy ND, Stassen LP. Fluorescence ureteral visualization in human laparoscopic colorectal surgery using methylene blue. J Laparoendosc Adv Surg Tech A 2016; 26:870-5. https://doi.org/10.1089/lap.2016.0264.

[31] Guo X, Ma Z, Yuan Y, Kang Y, Xu H, Mao Z, et al. Photoinduced absorption spectroscopy of photoelectrocatalytic methylene blue oxidation on titania and hematite: the thermodynamic and kinetic impacts on reaction pathways. Adv Sci (Weinh) 2023; 10:e2206685. https://doi.org/10.1002/advs.202206685.

[32] Barnes TG, Hompes R, Birks J, Mortensen NJ, Jones O, Lindsey I, et al. Methylene blue fluorescence of the ureter during colorectal surgery. Surg Endosc 2018; 32:4036-43. https://doi.org/10.1007/s00464-018-6219-8.

[33] Dean JC, Oblinsky DG, Rather SR, Scholes GD. Methylene blue exciton states steer nonradiative relaxation: ultrafast spectroscopy of methylene blue dimer. J Phys Chem B 2016; 120:440-54. https://doi.org/10.1021/acs.jpcb.5b11847.

[34] Pivetta TP, Ferreira Q, Vieira T, Silva JC, Simões S, Ribeiro PA, et al. Liposomes encapsulating methylene blue and acridine orange: an approach for phototherapy of skin cancer. Colloids Surf B Biointerfaces 2022; 220:112901. https://doi.org/10.1016/j.colsurfb.2022.112901.

[35] Maisel K, McClain CA, Bogseth A, Thomas SN. Nanotechnologies for physiology-informed drug delivery to the lymphatic system. Annu Rev Biomed Eng 2023; 25:233-56. https://doi.org/10.1146/annurev-bioeng-092222-034906.

[36] McCright J, Naiknavare R, Yarmovsky J, Maisel K. Targeting lymphatics for nanoparticle drug delivery. Front Pharmacol 2022; 13:887402. https://doi.org/10.3389/fphar.2022.887402.

[37] Correa S, Meany EL, Gale EC, Klich JH, Saouaf OM, Mayer AT, et al. Injectable nanoparticle-based hydrogels enable the safe and effective deployment of immunostimulatory CD40 agonist antibodies. Adv Sci (Weinh) 2022; 9:e2103677. https://doi.org/10.1002/advs.202103677.

[38] Kretsos K, Kasting GB. Dermal capillary clearance: physiology and modeling. Skin Pharmacol Physiol 2005; 18:55-74. https://doi.org/10.1159/000083706.

[39] Petkova M, Kraft M, Stritt S, Martinez-Corral I, Ortsäter H, Vanlandewijck M, et al. Immune-interacting lymphatic endothelial subtype at capillary terminals drives lymphatic malformation. J Exp Med 2023; 220:e20220741. https://doi.org/10.1084/jem.20220741.

[40] Polomska AK, Proulx ST, Brambilla D, Fehr D, Bonmarin M, Brändli S, et al. Minimally invasive method for the point-of-care quantification of lymphatic vessel function. JCI Insight 2019; 4:e126515. https://doi.org/10.1172/jci.insight.126515.

[41] Brambilla D, Proulx ST, Marschalkova P, Detmar M, Leroux JC. Microneedles for the noninvasive structural and functional assessment of dermal lymphatic vessels. Small 2016; 12:1053-61. https://doi.org/10.1002/smll.201503093.

[42] Yang S, Xu Y, Zhu M, Yu Y, Hu W, Zhang T, et al. Engineering the functional expansion of microneedles. Adv Mater 2025; 37:e2411112. https://doi.org/10.1002/adma.202411112.

[43] Xu J, Xu D, Xuan X, He H. Advances of microneedles in biomedical applications. Molecules 2021; 26:5912. https://doi.org/10.3390/molecules26195912.

[44] Xie Y, He J, He W, Iftikhar T, Zhang C, Su L, et al. Enhanced interstitial fluid extraction and rapid analysis via vacuum tube-integrated microneedle array device. Adv Sci (Weinh) 2024; 11:e2308716. https://doi.org/10.1002/advs.202308716.

[45] Sartawi Z, Blackshields C, Faisal W. Dissolving microneedles: applications and growing therapeutic potential. J Control Release 2022; 348:186-205. https://doi.org/10.1016/j.jconrel.2022.05.045.

[46] Ryan TJ . The blood vessels of the skin. J Invest Dermatol 1976; 67:110-8. https://doi.org/10.1111/1523-1747.ep12512516.

[47] Zhao Y, Liu J, Chen Z, Zhu X, Möller M. Hybrid nanostructured particles via surfactant-free double miniemulsion polymerization. Nat Commun 2018; 9:1918. https://doi.org/10.1038/s41467-018-04320-7.

[48] Zhang Y, Fang J, Ye S, Zhao Y, Wang A, Mao Q, et al. A hydrogen sulphide-responsive and depleting nanoplatform for cancer photodynamic therapy. Nat Commun 2022; 13:1685. https://doi.org/10.1038/s41467-022-29284-7.

[49] Weiler M, Dixon JB. Differential transport function of lymphatic vessels in the rat tail model and the long-term effects of indocyanine green as assessed with near-infrared imaging. Front Physiol 2013; 4:215. https://doi.org/10.3389/fphys.2013.00215.

[50] Meng X, Li H, Chen Y, Sai L, Feng S, Li K, et al. In vivo precision evaluation of lymphatic function by SWIR luminescence imaging with PbS quantum dots. Adv Sci (Weinh) 2023; 10:e2206579. https://doi.org/10.1002/advs.202206579.

[51] Deka G, Kalyani JN, Jahangir FB, Sabharwal P, Savithri HS, Murthy MRN. Structural and functional studies on salmonella typhimurium pyridoxal kinase: the first structural evidence for the formation of Schiff base with the substrate. FEBS J 2019; 286:3684-700.

[52] Li J, Xu X. Catalytic degradation of organic dyes induced by Tribo-electrification between insulating films. Materials (Basel) 2025; 18:2327.

[53] Münter R, Stavnsbjerg C, Christensen E, et al. Unravelling heterogeneities in complement and antibody opsonization of individual liposomes as a function of surface architecture. Small. 2022; 18:e2106529.

[54] Dethe MR, Prabakaran A, Ahmed H, Agrawal M, Roy U, Alexander A. PCL-PEG copolymer based injectable thermosensitive hydrogels. J Control Release 2022; 343:217-36.

[55] Sheffey VV, Siew EB, Tanner EEL, Eniola-Adefeso O. PLGA’s plight and the role of stealth surface modification strategies in its use for intravenous particulate drug delivery. Adv Healthc Mater 2022; 11:e2101536.

[56] Huang J, Zhou X, Shen Y, et al. Asiaticoside loading into polylactic-co-glycolic acid electrospun nanofibers attenuates host inflammatory response and promotes M2 macrophage polarization. J Biomed Mater Res A 2020; 108:69-80.

[57] Yang F, Niu X, Gu X, Xu C, Wang W, Fan Y. Biodegradable magnesium-incorporated poly(l-lactic acid) microspheres for manipulation of drug release and alleviation of inflammatory response. ACS Appl Mater Interfaces 2019; 11:23546-57.

[58] Paramshetti S, Angolkar M, Talath S, et al. Unravelling the in vivo dynamics of liposomes: insights into biodistribution and cellular membrane interactions. Life Sci 2024; 346:122616.

[59] Frigaard J, Jensen JL, Galtung HK, Hiorth M. The potential of chitosan in Nanomedicine: an overview of the cytotoxicity of chitosan based nanoparticles. Front Pharmacol 2022; 13:880377.

[60] Gao Y, Wu Y. Recent advances of chitosan-based nanoparticles for biomedical and biotechnological applications. Int J Biol Macromol 2022; 203:379-88.

[61] Shen J, Yuan W, Badv M, Moshaverinia A, Weiss PS. Modified poly(ε-caprolactone) with Tunable degradability and improved biofunctionality for regenerative medicine. ACS Mater Au 2023; 3:540-7.

[62] Ikhtiarini N, Kamil MZ, Bukit BF, et al. Biocompatible composites based on alginate, polycaprolactone, and nanocellulose-a review. Int J Biol Macromol 2025; 311:143423.

[63] Zhao P, Zhou Z, Wolter T, et al. Engineering microneedles for biosensing and drug delivery. Bioact Mater 2025; 52:36-59.

[64] Zhang H, Wang X, Zhang Y, et al. Hyaluronic acid modified indocyanine green nanoparticles: a novel targeted strategy for NIR-II fluorescence lymphatic imaging. Front Chem 2024; 12:1435627.

[65] Le QV, Kang S, Lee J, et al. Size-dependent effect of Indocyanine green Nanoimaging agent for metastatic lymph node detection. Biomater Res 2024; 28:0022.