Harnessing biomaterials for lymphatic system modulation

Abstract

The lymphatic system plays an integral part in regulating immune cell trafficking and the transport of macromolecules. However, its influence on disease progression and drug uptake is understood less than that of the vascular system. To bridge this knowledge gap, biomaterials can be used to investigate the lymphatic system and to provide novel understanding into complex disease processes, including cancer metastasis and inflammation. Insight gained from these mechanistic studies can be further used to design innovative biomaterials to modulate the immune system, improve drug delivery, and promote tissue regeneration. This review article focuses on recent advances in (i) biomaterials used for lymphatic vessel formation, (ii) models for studying lymphatic-immune cells interactions, (iii) pharmaceuticals and their interactions with the lymphatic system, (iv) and strategies for drug delivery via the lymphatic system. Finally, several challenges regarding adopting biomaterials for immunomodulation and future perspectives are discussed. STATEMENT OF SIGNIFICANCE: The lymphatic system plays an integral part in regulating immune cell trafficking and the transport of macromolecules. However, its influence on disease progression and drug uptake is understood less than that of the vascular system. This review article focuses on recent progresses in biomaterials to investigate the lymphatic system and to provide novel understanding into complex disease states. Insight gained from these mechanistic studies can be further used to design innovative biomaterials to modulate the immune system, improve drug delivery, and promote tissue regeneration. Finally, a number of challenges in adopting biomaterials for immunomodulation and future perspectives are discussed.

Keywords: Biomaterials; Drug delivery; Immune system; Lymphatic vessels; Tissue engineering.

Figure 1.. Multiple types of models can be used to better understand the lymphatic system and its influence on disease progression, immune response, and drug uptake.

(A) These models include co-cultures, microfluidics, and animal models to develop and study lymphatic vessels. Biomaterials, specifically collagen, hyaluronic acid (HA), poly(ethylene glycol) (PEG), and fibrin, are utilized to develop biomimetic environments conducive to lymphatic vessels. Lymphatic vessels are composed of LECs that form lymphatic capillaries, which are characterized by discontinuous, button-like endothelial cell junctions, and pre-collecting and collecting vessels, which exhibit continuous, zipper-like cell-cell junctions and are surrounded by a contractile layer of non-striated muscle cells, referred to as lymphatic muscle cells that propel the flow of lymph to the LNs and contain valves to prevent backflow. Immune cells can enter through the capillaries and flow with lymph into the LNs where they are stored until an immune response triggers them to egress and reenter circulation. (B) A zoomed-in view of the structures within the LN. The parenchyma encompasses all of the internal functional tissue, which is comprised of reticular fibers, and is demarcated with the red outline. The sectional view (i) of the LN focuses on the structures that are utilized for immune cell entry, storage, and egress.

Figure 2.. Biomaterials to modulate the lymphatic system.

(A) Matrix stiffness of hyaluronic acid (HA)-hydrogels prime lymphatic tube formation directed by VEGF-C, as demonstrated by fluorescent microscopy of F-actin (green) and nuclei (blue). Scale bars are 50 μm. (B) Confocal images of lymphatic tubes formed on soft HA-hydrogels showing junctional markers for CD31 and VE-Cad. Enlarged rendering of confocal image stacks indicate cellular junctions (arrowheads) with discontinuous (arrows) and overlapping (asterisks) junctions. Scale bars are 50 μm and 25 μm (inset). (C) TEM analyses of lymphatic tubes formed after 12 hours showed LECs degrading the HA-hydrogels (H) to generate intracellular vacuoles (V), some of which were observed in the process of merging (asterisk) into coalescent vacuoles (CV). Scale bar is 20 μm. Illustration was adapted with permission from [26]. (D) FACS-sorted LECs mixed with 40% fibroblasts developed lymphatic capillaries (CD31, red) within Collagen type-1. (E) Lymphatic capillaries expressed the lymphatic marker Prox-1 (green). Scale bars are 40 μm. Illustration was adapted with permission from [34].

Figure 3.. Co-culture and Microfluidic systems to model the lymphatic system.

(A) Co-culture system with a 3D matrix and flow to investigate crosstalk between LECs and tumor cells. The cross-section shows the interface for the tumor suspension, porous membrane, and LECs, which are indicated with the black arrowheads. The black arrows indicate tumor cells migrating through the membrane’s pores. (B) Confocal microscopy of the underside of a transwell membrane. The LEC monolayer is stained with CD31 in red and the tumor cells were stained with PHAKT-GFP, a fluorescent protein that binds to AKT protein kinase and selectively migrates to the membrane when exposed to a chemoattractant. One of these tumor cells is adhering (the white arrowhead) and one is transmigrating through a pore (white arrow). The nuclei are stained blue, and the scale bar is 20 μm. Illustration was adapted with permission from [44]. (C) Microfluidics system that contain low-density (LD) or high-density (HD) collagen gels, LECs, and MDA-MB-231 cells, a metastatic breast cancer cell line. (D) Top-view and cross-section view of immunofluorescent images of lymphatic vessels co-cultured with MDA-MB-231 in LD (left) and HD (right) matrices. F-actin was stained with purple, CD31 with red, MDA-MB-231-GFP with green, and the nuclei with blue. The dashed lines indicate where LEC detachment was present in the vessel walls. The scale bar is 140 μm. Illustration was adapted with permission from [65].

Figure 4.. Multiple factors must be considered when designing therapeutics for immunomodulation via the lymphatic systems.

(A) Different types and sizes of therapeutics, such as monoclonal antibodies (mAb, 10 nm), solid lipid nanoparticles (200 nm), and nanostructured lipid carries (500 nm) that can be used to target the lymphatic system. As a reference, chylomicrons up to 1,000nm in diameter can be transported across the lacteals [92]. The specific structure of the lymphatic vessels being targeted must be considered, as their structure is organ-specific and vary in permeability depending on the types of junctions (button-type and zipper-type junctions), which will influence the possible biomaterials used for therapeutic delivery. (B) Schematic diagram of the different mechanisms of transport pathways following oral drug or pro-drug administration. The intestinal transport of lipid-based formulations (nanostructured lipid carries) through blood (major) and lymphatic circulation (minor). Pro-drug formulation as a drug-triglyceride undergoes hydrolysis into drug-monoglyceride, which will be further assembled into lipoprotein to enter the mesenteric lymph. Therefore, the drug can enter the systemic circulation by avoiding the first pass metabolism in the liver. Illustration in panel B was adapted with permission from [79].