Lymphatic Transport Research Articles

Multi-physics approach to model the lymph transport in the murine immune system

Abstract We formulate a compartmental model of the murine lymphatic system with the transfer rate parameters derived from the data on the geometric characteristics of the lymphatic system (LS) graph structure and the Hagen–Poiseuille-based values of the lymph flows through the system components, i.e., vertices and edges. It is supplemented by the physics-based model of lymph node draining-related function which considers a paradigmatic view of its geometry with one- and three-afferent lymphatic vessels and one efferent vessel, and the lymph flow described by the Darcy–Starling equations. We discuss further modelling work needed to gain a predictive understanding of the LS function in response to various perturbations including infections and therapeutic treatments.

Enabling oral novel Taxanes-based Chemotherapy with Lipophilic prodrug Self-nanoemulsifying drug delivery system

Larotaxel (LTX) and SB-T-1214 (SBT), two new synthetic experimental toxoids, have shown broad-spectrum antitumor activity, especially against tumors that are resistant to other drugs. However, their poor solubility, membrane permeability, and first-pass effect limits their use in oral administration. We designed and synthesized two long-chain triglyceride-mimic prodrugs of LTX (LTXSSTG) and SBT (SBTSSTG), which are bridged by disulfide bonds and efficiently incorporated them into Self-nanoemulsifying drug delivery system (SNEDDS). These prodrugs can bypass hepatic metabolism by entering the blood through intestinal lymphatic transport, following a similar oral absorption pathway to dietary lipids. It was found that LTXSSTG and SBTSSTG significantly improved oral bioavailability (about 4.5-fold for LTX and 3.4-fold for SBT) compared to their solution forms. Moreover, with LTXSSTG and SBTSSTG incorporating reduction stimulus-responsive spacer were much more effective in suppressing tumor growth in vivo with eliminated adverse effects than solution form. To sum up, this strategy provides a new avenue to enhance oral delivery of new toxoids.

Delivery of imiquimod to intestinal lymph nodes following oral administration

Intestinal lymph nodes are involved in the progression of colorectal cancer (CRC). Tumours suppress the activation of dendritic cells (DCs) in draining lymph nodes, diminishing anti-cancer immune response. Imiquimod (IMQ) facilitates DCs activation via toll-like receptor 7, suggesting that targeted delivery of IMQ to intestinal lymph nodes can improve the treatment of CRC. This study aims to enhance the delivery of IMQ to intestinal lymph nodes by a highly lipophilic prodrug approach. Amide prodrugs were synthesised by conjugating IMQ with saturated and unsaturated medium- to long-chain fatty acids. Their potential for intestinal lymphatic transport was assessed by their affinity to chylomicrons and solubility in long-chain triglycerides. Further selection of prodrug candidates was determined by resistance to enzymatic hydrolysis in intestinal lumen and release of IMQ in the lymphatics using fasting state simulated intestinal fluid supplemented with esterases, brush border enzyme vesicles and plasma. Key pharmacokinetic parameters and biodistribution in rats were assessed for the most promising compounds, prodrugs 5 and 8. The plasma concentration–time profile of IMQ following oral administration of the prodrugs was less erratic in comparison to the administration of unmodified IMQ. The lymph-to-plasma ratios of IMQ concentration increased 1.9- and 1.7-fold using prodrugs 5 and 8 in comparison to administration of unmodified IMQ, respectively. Importantly, the average concentration of IMQ in mesenteric lymph nodes (MLN) was 11.2- and 7.6-fold higher than in plasma following the administration of prodrugs 5 and 8, respectively. Additionally, the non-specific wide distribution of IMQ into various organs and tissues was reduced with prodrugs. This work suggests that the highly lipophilic prodrug approach can efficiently deliver IMQ to intestinal lymphatics. In addition, this study demonstrates the feasibility of an amide prodrug approach for intestinal lymphatic targeting.

In Vitro Cytotoxicity and Pharmacokinetic Study for Bosutinib Solid Lipid Nanoparticles.

Bosutinib (BST) is a Biopharmaceutics Classification System Class II drug having very low solubility and high permeability. Low aqueous solubility and poor dissolution of BST lead to poor bioavailability, Thus, limited aqueous solubility is the bottleneck for the therapeutic outcome of BST. Animal data suggest that the absolute bioavailability of BST is about 14-34% due to an extensive first-pass effect. To overcome hepatic first-pass metabolism and to enhance oral bioavailability, lipid-based drug delivery systems such as solid lipid nanoparticles (SLNs) can be used. SLNs are submicron colloidal carriers having a size range of 50-1000 nm. These are prepared with physiological lipid and dispersed in water or aqueous surfactant solution. BST can be conveniently loaded into SLNs to improve the oral bioavailability by exploiting the intestinal lymphatic transport. An optimal system was evaluated for bioavailability study in rats compared with that of BST suspension (SUS). An in vitro cytotoxicity study was done by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay method through ATCC cell lines; the percent inhibition was more in SLN when compared with SUS. The pharmacokinetics of BST-SLNs after oral administration in male Wistar rats was studied. The bioavailability of BST was increased by 2.28 fold when compared with that of a BST SUS. The results are indicative of SLNs as suitable lipid-based carrier system for improving the oral bioavailability of BST.

Multiscale computational analysis of the steady fluid flow through a lymph node.

Lymph Nodes (LNs) are crucial to the immune and lymphatic systems, filtering harmful substances and regulating lymph transport. LNs consist of a lymphoid compartment (LC) that forms a porous bulk region, and a subcapsular sinus (SCS), which is a free-fluid region. Mathematical and mechanical challenges arise in understanding lymph flow dynamics. The highly vascularized lymph node connects the lymphatic and blood systems, emphasizing its essential role in maintaining the fluid balance in the body. In this work, we describe a mathematical model in a steady setting to describe the lymph transport in a lymph node. We couple the fluid flow in the SCS governed by an incompressible Stokes equation with the fluid flow in LC, described by a model obtained by means of asymptotic homogenisation technique, taking into account the multiscale nature of the node and the fluid exchange with the blood vessels inside it. We solve this model using numerical simulations and we analyze the lymph transport inside the node to elucidate its regulatory mechanisms and significance. Our results highlight the crucial role of the microstructure of the lymph node in regularising its fluid balance. These results can pave the way to a better understanding of the mechanisms underlying the lymph node's multiscale functionalities which can be significantly affected by specific physiological and pathological conditions, such as those characterising malignant tissues.

Acute Neuroinflammation Alters the Transport of a Model Therapeutic Protein from the Brain into Lymph and Blood.

The drainage of fluid and solutes along lymphatic pathways from the brain has been found to be impaired in mouse models of multiple sclerosis, Alzheimer's disease, and Parkinson's disease where neuroinflammation is present. We recently demonstrated that 3H-albumin, a model therapeutic protein (∼65 kDa), undergoes preferential lymphatic transport from the brain using a cervical lymph cannulation model in healthy rats. We thus hypothesized that neuroinflammation would impede the lymphatic transport of 3H-albumin from the brain. Our aim was to quantify the impact of acute neuroinflammation on drainage of the model therapeutic protein (3H-albumin) from the rat brain into blood and deep cervical lymph. To establish the required neuroinflammation model, male Sprague-Dawley rats were administered an intraperitoneal (IP) dose of 0.5-2 mg/kg lipopolysaccharide (LPS, Escherichia coli) or a saline control. After 12 or 24 h, brain samples were collected and analyzed for concentrations of interferon gamma (IFN-γ) using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The impact of neuroinflammation on the drainage of 3H-albumin from the brain was determined via IP administration of 2 mg/kg LPS or saline followed by cannulation of the carotid artery for blood collection 24 h later with/without cannulation or ligation at the efferent deep cervical lymph trunk. Rats were then administered 3H-albumin via direct injection into the brain striatum or via intravenous (IV) injection (lymph-intact group only). Blood ± lymph samples were collected for up to 8 h following dosing. At the end of the study, brain and lymph node samples were harvested for biodistribution analysis, with samples analyzed for radioactivity levels via scintillation counting. Brain concentrations of the pro-inflammatory cytokine IFN-γ were only significantly elevated 24 h after IP administration of 2 mg/kg LPS compared to saline control. Therefore, this induction regimen was utilized for subsequent studies. The plasma concentrations of 3H-albumin over time were elevated in LPS-induced rats compared to saline-injected rats in the lymph-intact and lymph-ligated groups but not in the lymph-cannulated group. In the deep cervical lymph-cannulated animals, the lymph transport of 3H-albumin was not increased and appeared to be slower in the LPS-administered rats. Acute LPS-induced neuroinflammation therefore led to an enhanced overall transport of 3H-albumin from the brain into the systemic circulation. This appeared to be primarily due to increased transport of 3H-albumin from the brain directly into the blood circulation as 3H-albumin transport from the brain via the lymphatics was not increased in the LPS-induced neuroinflammation model. Such changes in the clearance of therapeutic proteins from the brain in the setting of neuroinflammation may impact the therapeutic efficacy and safety.

Intestinal Lymphatic Biology, Drug Delivery and Therapeutics: Current status and Future directions.

Historically, the intestinal lymphatics were considered passive conduits for fluids, immune cells, dietary lipids, lipid soluble vitamins and lipophilic drugs. Studies of intestinal lymphatic drug delivery in the late 20th century focussed primarily on the drug physicochemical properties, especially high lipophilicity, that resulted in intestinal lymphatic transport. More recent discoveries have changed our traditional view by demonstrating that the lymphatics are active, plastic and tissue-specific players in a range of biological and pathological processes, including within the intestine. These findings have, in turn, inspired exploration of lymph-specific therapies for a range of diseases, as well as the development of more sophisticated strategies to actively deliver drugs or vaccines to the intestinal lymph, including a range of nanotechnologies, lipid prodrugs and lipid-conjugated materials that 'hitchhike' on lymphatic transport pathways. With the increasing development of novel biological therapeutics there has been interest in whether these novel therapeutics are absorbed and transported through intestinal lymph after oral administration. Here we review the current state of understanding of the anatomy and physiology of the gastrointestinal lymphatic system in health and disease, with a focus on aspects relevant to drug delivery. We summarise the current state-of-the-art approaches to deliver drugs and quantify their uptake into the intestinal lymphatic system. Finally, and excitingly, we discuss recent examples of significant pharmacokinetic and therapeutic benefits achieved via intestinal lymphatic drug delivery. We also propose approaches to advance the development and clinical application of intestinal lymphatic delivery strategies into the future. Significance Statement This comprehensive review details understanding of the anatomy and physiology of the intestinal lymphatic system in health and disease, with a focus on aspects relevant to drug delivery. It highlights current state-of-the-art approaches to deliver drugs to the intestinal lymphatics and the shift toward the use of these strategies to achieve pharmacokinetic and therapeutic benefits for patients.

Three-Dimensional Lymphatics-on-a-Chip Reveals Distinct, Size-Dependent Nanoparticle Transport Mechanisms in Lymphatic Drug Delivery.

Although nanoparticle-based lymphatic drug delivery systems promise better treatment of cancer, infectious disease, and immune disease, their clinical translations are limited by low delivery efficiencies and unclear transport mechanisms. Here, we employed a three-dimensional (3D) lymphatics-on-a-chip featuring an engineered lymphatic vessel (LV) capable of draining interstitial fluids including nanoparticles. We tested lymphatic drainage of different sizes (30, 50, and 70 nm) of PLGA-b-PEG nanoparticles (NPs) using the lymphatics-on-a-chip device. In this study, we discovered that smaller NPs (30 and 50 nm) transported faster than larger NPs (70 nm) through the interstitial space, as expected, but the smaller NPs were captured by lymphatic endothelial cells (LECs) and accumulated within their cytosol, delaying NP transport into the lymphatic lumen, which was not observed in larger NPs. To examine the mechanisms of size-dependent NP transports, we employed four inhibitors, dynasore, nystatin, amiloride, and adrenomedullin, to selectively block dynamin-, caveolin-, macropinocytosis-mediated endocytosis-, and cell junction-mediated paracellular transport. Inhibiting dynamin using dynasore enhanced the transport of smaller NPs (30 and 50 nm) into the lymphatic lumen, minimizing cytosolic accumulation, but showed no effect on larger NP transport. Interestingly, the inhibition of caveolin by nystatin decreased the lymphatic transport of larger NPs without affecting the smaller NP transport, indicating distinct endocytosis mechanisms used by different sizes of NPs. Macropinocytosis inhibition by amiloride did not change the drainage of all sizes of NPs; however, paracellular transport inhibition by adrenomedullin blocked the lymphatic transport of NPs of all sizes. We further revealed that smaller NPs were captured in the Rab7-positive late-stage lymphatic endosomes to delay their lymphatic drainage, which was reversed by dynamin inhibition, suggesting that Rab7 is a potential target to enhance the lymphatic delivery of smaller NPs. Together, our 3D lymphatics-on-a-chip model unveils size-dependent NP transport mechanisms in lymphatic drug delivery.

Bile salt integrated cerasomes: A potential nanocarrier for enhancement of the oral bioavailability of idarubicin hydrochloride

Cerasomes are a modified form of liposomes containing both inorganic and organic parts and due to their strong polyorganosiloxane surface have remarkably high morphological stability and provide easier functionalization compared with conventional liposomes. To investigate the potential of these nanocarriers for oral delivery, bile salt integrated cerasomes (named bilocerasomes) encapsulating idarubicin hydrochloride (IDA) were prepared and characterized. The optimum formulation showed excellent stability in the simulated gastrointestinal fluids as well as under storage conditions. The oral pharmacokinetics of the IDA solution, empty nanocarrier + drug solution, and IDA-loaded bilocerasome were evaluated. The nanoformulation significantly increased the area under the drug concentration–time curve and the mean residence time (∼14.3- and 9-fold, respectively). The results obtained from cell uptake and chylomicron flow blocking approach revealed that bilocerasomes are absorbed into the intestinal cells via a clathrin/caveolin-independent endocytosis pathway and transported to the systemic circulation extensively via the intestinal lymphatic vessels. Considering the high stability of the prepared bilocerasome, noticeable participation of lymphatic transport in its systemic absorption and marked enhancement in the oral absorption of IDA, bilocerasomes can be introduced as a capable carrier for improving the oral bioavailability of drugs, particularly those that hepatic first-pass metabolism seriously limits their oral absorption.

In vivo turnover and biodistribution of soluble AXL: implications for biomarker development

Soluble biomarkers are paramount to personalized medicine. However, the in vivo turnover and biodistribution of soluble proteins is seldom characterized. The cleaved extracellular domain of the AXL receptor (sAXL) is a prognostic biomarker in several diseases and a predictive marker of AXL targeting agents. Plasma sAXL reflects a balance between production in tissues with lymphatic transport into the circulation and removal from blood by degradation or excretion. It is unclear how this transport cycle affects plasma sAXL levels that are the metric for biomarker development. Radiolabeled mouse sAxl was monitored after intravenous injection to measure degradation and urinary excretion of sAxl, and after intradermal injection to mimic tissue or tumor production. sAxl was rapidly taken-up and degraded by the liver and kidney cortex. Surprisingly, intact sAxl was detectable in urine, indicating passage through the glomerular filter and a unique sampling opportunity. The structure of sAxl showed an elongated, flexible molecule with a length of 18 nm and a thickness of only 3 nm, allowing passage through the glomerulus and excretion into the urine. Intradermally injected sAxl passed through local and distant lymph nodes, followed by uptake in liver and kidney cortex. Low levels of sAxl were seen in the plasma, consistent with an extended transit time from local tissue to circulation. The rapid plasma clearance of sAxl suggests that steady-state levels in blood will sensitively and dynamically reflect the rate of production of sAxl in the tissues but will be influenced by perturbations of liver and kidney function.

Hyaluronic acid nanoparticles for targeted oral delivery of doxorubicin: Lymphatic transport and CD44 engagement

The oral delivery of doxorubicin (DOX), an anti-cancer drug, encounters multiple hurdles such as limited gastrointestinal permeability, P-glycoprotein-mediated efflux, brief intestinal residence, and rapid degradation. This study introduced a novel approach utilizing hyaluronic acid (HA)-grafted fatty acid monoglycerides (HGD) to encapsulate DOX, forming HGD-DOX nanoparticles, aimed at enhancing its oral bioavailability. Drug encapsulated by HGD provided several advantages, including extended drug retention in the gastrointestinal tract, controlled release kinetics, and promotion of lymphatic absorption in the intestine. Additionally, HGD-DOX nanoparticles could specifically target CD44 receptors, potentially increasing therapeutic efficacy. The uptake mechanism of HGD-DOX nanoparticles primarily involved clathrin-mediated, caveolin-mediated and macropinocytosis endocytosis. Pharmacokinetic analysis further revealed that HGD significantly prolonged the in vivo residence time of DOX. In vivo imaging and pharmacodynamic studies indicated that HGD possessed tumor-targeting capabilities and exhibited a significant inhibitory effect on tumor growth, while maintaining an acceptable safety profile. Collectively, these findings position HGD-DOX nanoparticles as a promising strategy to boost the oral bioavailability of DOX, offering a potential avenue for improved cancer treatment.

Lymphatic vessel transit seeds cytotoxic resident memory T cells in skin draining lymph nodes.

Lymphatic transport shapes the homeostatic immune repertoire of lymph nodes (LNs). LN-resident memory T cells (TRMs) play an important role in site-specific immune memory, yet how LN TRMs form de novo after viral infection remains unclear. Here, we tracked the anatomical distribution of antiviral CD8+ T cells as they seeded skin and LN TRMs using a model of vaccinia virus-induced skin infection. LN TRMs localized to the draining LNs (dLNs) of infected skin, and their formation depended on the lymphatic egress of effector CD8+ T cells from the skin, already poised for residence. Effector CD8+ T cell transit through skin was required to populate LN TRMs in dLNs, a process reinforced by antigen encounter in skin. Furthermore, LN TRMs were protective against viral rechallenge in the absence of circulating memory T cells. These data suggest that a subset of tissue-infiltrating CD8+ T cells egress from tissues during viral clearance and establish a layer of regional protection in the dLN basin.

2-Monoacylglycerol Mimetic Liposomes to Promote Intestinal Lymphatic Transport for Improving Oral Bioavailability of Dihydroartemisinin.

Reducing the first-pass hepatic effect via intestinal lymphatic transport is an effective way to increase the oral absorption of drugs. 2-Monoacylglycerol (2-MAG) as a primary digestive product of dietary lipids triglyceride, can be assembled in chylomicrons and then transported from the intestine into the lymphatic system. Herein, we propose a biomimetic strategy and report a 2-MAG mimetic nanocarrier to target the intestinal lymphatic system via the lipid absorption pathway and improve oral bioavailability. The 2-MAG mimetic liposomes were designed by covalently bonding serinol (SER) on the surface of liposomes named SER-LPs to simulate the structure of 2-MAG. Dihydroartemisinin (DHA) was chosen as the model drug because of its disadvantages such as poor solubility and high first-pass effect. The endocytosis and exocytosis mechanisms were investigated in Caco-2 cells and Caco-2 cell monolayers. The capacity of intestinal lymphatic transport was evaluated by ex vivo biodistribution and in vivo pharmacokinetic experiments. DHA loaded SER-LPs (SER-LPs-DHA) had a particle size of 70 nm and a desirable entrapment efficiency of 93%. SER-LPs showed sustained release for DHA in the simulated gastrointestinal environment. In vitro cell studies demonstrated that the cellular uptake of SER-LPs primarily relied on the caveolae- rather than clathrin-mediated endocytosis pathway and preferred to integrate into the chylomicron assembly process through the endoplasmic reticulum/Golgi apparatus route. After oral administration, SER-LPs efficiently promoted drug accumulation in mesenteric lymphatic nodes. The oral bioavailability of DHA from SER-LPs was 10.40-fold and 1.17-fold larger than that of free DHA and unmodified liposomes at the same dose, respectively. SER-LPs improved oral bioavailability through efficient intestinal lymphatic transport. These findings of the current study provide a good alternative strategy for oral delivery of drugs with high first-pass hepatic metabolism.

Near-Infrared-II Imaging Revealed Hypothermia Regulates Neuroinflammation Following Brain Injury by Increasing the Glymphatic Influx.

Advanced in vivo imaging techniques have facilitated the comprehensive visual exploration of animal biological processes, leading to groundbreaking discoveries such as the glymphatic system. However, current limitations of macroscopic imaging techniques impede the precise investigation of physiological parameters regulating this specialized lymphatic transport system. While NIR-II fluorescence imaging has demonstrated advantages in peripheral lymphatic imaging, there are few reports regarding its utilization in the glymphatic system. To address this, a noninvasive transcranial macroscopic NIR-II fluorescence imaging model is developed using a cyanine dye-protein coupled nanoprobe. NIR-II imaging with high temporal and spatial resolution reveals that hypothermia can increase the glymphatic influx by reducing the flow rate of cerebrospinal fluid. In addition, respiratory rate, respiratory amplitude, and heart rate all play a role in regulating the glymphatic influx. Thus, targeting the glymphatic influx may alter the trajectory of immune inflammation following brain injury, providing therapeutic prospects for treating brain injury with mild hypothermia.

Intestinal lymphatic transport of Smilax china L. pectic polysaccharide via Peyer's patches and its uptake and transport mechanisms in mononuclear phagocytes

Recently, the intestinal lymphatic transport based on Peyer's patches (PPs) is emerging as a promising absorption pathway for natural polysaccharides. Herein, the aim of this study is to investigate the PP-based oral absorption of a pectic polysaccharide from Smilax china L. (SCLP), as well as its uptake and transport mechanisms in related immune cells. Taking advantages of the traceability of fluorescently labeled SCLP, we confirmed that SCLP could be absorbed into PPs and captured by their mononuclear phagocytes (dendritic cells and macrophages) following oral administration. Subsequently, the systematic in vitro study suggested that the endocytic mechanisms of SCLP by model mononuclear phagocytes (BMDCs and RAW264.7 cells) mainly involved caveolae-mediated endocytosis, macropinocytosis and phagocytosis. More importantly, SCLP directly binds and interacts with toll-like receptor 2 (TLR2) and galectin 3 (Gal-3) receptor, and was taken up by mononuclear phagocytes in receptor-mediated manner. After internalization, SCLP was intracellularly transported primarily through endolysosomal pathway and ultimately localized in lysosomes. In summary, this work reveals novel information and perspectives about the in vivo fate of SCLP, which will contribute to further research and utilization of SCLP and other pectic polysaccharides.

A Comprehensive Guide to the Development, Evaluation, and Future Prospects of Self-nanoemulsifying Drug Delivery Systems for Poorly Water-soluble Drugs.

Self-nano Emulsifying Drug Delivery Systems (SNEDDS) are novel formulations that can enhance the solubility and bioavailability of poorly water-soluble drugs. SNEDDS are composed of lipids, surfactants, co-solvents, and drugs and can spontaneously form nanoemulsions when mixed with water under mild agitation. SNEDDS can be formulated as liquid or solid dosage forms and can improve drug absorption by increasing the interfacial area, protecting the drug from degradation, and facilitating lymphatic transport. SNEDDS is characterized by various parameters such as particle size, zeta potential, droplet morphology, emulsification efficiency, drug solubility, and stability. SNEDDS offers several advantages over conventional dosage forms, such as dose reduction, faster onset of action, reduced variability, versatility, and ease of formulation. However, SNEDDS also face some limitations and challenges, such as drug precipitation, cost-effectiveness, compatibility with capsule shells, and lack of predictive in vitro models. SNEDDS has a promising future in the field of pharmaceuticals, especially for personalized medicine and targeted drug delivery.

Lymphatic vessels in the age of cancer immunotherapy.

Lymphatic transport maintains homeostatic health and is necessary for immune surveillance, and yet lymphatic growth is often associated with solid tumour development and dissemination. Although tumour-associated lymphatic remodelling and growth were initially presumed tosimply expand a passive route for regional metastasis, emerging research puts lymphatic vessels and their active transport at the interface of metastasis, tumour-associated inflammation and systemic immune surveillance. Here, we discuss active mechanisms through which lymphatic vessels shape their transport function to influence peripheral tissue immunity and the current understanding of how tumour-associated lymphatic vessels may both augment and disrupt antitumour immune surveillance. We end by looking forward to emerging areas of interest in the field of cancer immunotherapy in which lymphatic vessels and their transport function are likely key players: the formation of tertiary lymphoid structures, immune surveillance in the central nervous system, the microbiome, obesity and ageing. The lessons learnt support a working framework that defines the lymphatic system as a key determinant of both local and systemic inflammatory networks and thereby a crucial player in the response to cancer immunotherapy.

Quantifying the Lymphatic Transport of Model Therapeutics from the Brain in Rats.

In recent years, the drainage of fluids, immune cells, antigens, fluorescent tracers, and other solutes from the brain has been demonstrated to occur along lymphatic outflow pathways to the deep cervical lymph nodes in the neck. To the best of our knowledge, no studies have evaluated the lymphatic transport of therapeutics from the brain. The objective of this study was to determine the lymphatic transport of model therapeutics of different molecular weights and lipophilicity from the brain using cervical lymph cannulation and ligation models in rats. To do this, anesthetized Sprague-Dawley rats were cannulated at the carotid artery and cannulated, ligated, or left intact at the cervical lymph duct. Rats were administered 14C-ibuprofen (206.29 g/mol, logP 3.84), 3H-halofantrine HCl (536.89 g/mol, logP 8.06), or 3H-albumin (∼65,000 g/mol) via direct injection into the brain striatum at a rate of 0.5 μL/min over 16 min. Plasma or cervical lymph samples were collected for up to 6-8 h following dosing, and brain and lymph nodes were collected at 6 or 8 h. Samples were subsequently analyzed for radioactivity levels via scintillation counting. For 14C-ibuprofen, plasma concentrations over time (plasma AUC0-6h) were >2 fold higher in lymph-ligated rats than in lymph-intact rats, suggesting that ibuprofen is cleared from the brain primarily via nonlymphatic routes (e.g., across the blood-brain barrier) but that this clearance is influenced by changes in lymphatic flow. For 3H-halofantrine, >73% of the dose was retained at the brain dosing site in lymph-intact and lymph-ligated groups, and plasma AUC0-8h values were low in both groups (<0.3% dose.h/mL), consistent with the high retention in the brain. It was therefore not possible to determine whether halofantrine undergoes lymphatic transport from the brain within the duration of the study. For 3H-albumin, plasma AUC0-8h values were not significantly different between lymph-intact, lymph-ligated, and lymph-cannulated rats. However, >4% of the dose was recovered in cervical lymph over 8 h. Lymph/plasma concentration ratios of 3H-albumin were also very high (up to 53:1). Together, these results indicate that 3H-albumin is transported from the brain not only via lymphatic routes but also via the blood. Similar to other tissues, the lymphatics may thus play a significant role in the transport of macromolecules, including therapeutic proteins, from the brain but are unlikely to be a major transport pathway from the brain for small molecule drugs that are not lipophilic. Our rat cervical lymph cannulation model can be used to quantify the lymphatic drainage of different molecules and factors from the brain.

Impact of conjugation to different lipids on the lymphatic uptake and biodistribution of brush PEG polymers

Delivery to peripheral lymphatics can be achieved following interstitial administration of nano-sized delivery systems (nanoparticles, liposomes, dendrimers etc) or molecules that hitchhike on endogenous nano-sized carriers (such as albumin). The published work concerning the hitchhiking approach has mostly focussed on the lymphatic uptake of vaccines conjugated directly to albumin binding moieties (ABMs such as lipids, Evans blue dye derivatives or peptides) and their subsequent trafficking into draining lymph nodes. The mechanisms underpinning access and transport of these constructs into lymph fluid, including potential interaction with other endogenous nanocarriers such as lipoproteins, have largely been ignored. Recently, we described a series of brush polyethylene glycol (PEG) polymers containing end terminal short-chain or medium-chain hydrocarbon tails (1C2 or 1C12, respectively), cholesterol moiety (Cho), or medium-chain or long-chain diacylglycerols (2C12 or 2C18, respectively). We evaluated the association of these materials with albumin and lipoprotein in rat plasma, and their intravenous (IV) and subcutaneous (SC) pharmacokinetic profiles. Here we fully detail the association of this suite of polymers with albumin and lipoproteins in rat lymph, which is expected to facilitate lymph transport of the materials from the SC injection site. Additionally, we characterise the thoracic lymph uptake, tissue and lymph node biodistribution of the lipidated brush PEG polymers following SC administration to thoracic lymph cannulated rats. All polymers had moderate lymphatic uptake in rats following SC dosing with the lymph uptake higher for 1C2-PEG, 2C12-PEG and 2C18-PEG (5.8%, 5.9% and 6.7% dose in lymph, respectively) compared with 1C12-PEG and Cho-PEG (both 1.5% dose in lymph). The enhanced lymph uptake of 1C2-PEG, 2C12-PEG and 2C18-PEG appeared related to their association profile with different lipoproteins. The five polymers displayed different biodistribution patterns in major organs and tissues in mice. All polymers reached immune cells deep within the inguinal lymph nodes of mice following SC dosing. The ability to access these immune cells suggests the potential of the polymers as platforms for the delivery of vaccines and immunotherapies. Future studies will focus on evaluating the lymphatic targeting and therapeutic potential of drug or vaccine-loaded polymers in pre-clinical disease models.

Piezo1 regulates meningeal lymphatic vessel drainage and alleviates excessive CSF accumulation

Piezo1 regulates multiple aspects of the vascular system by converting mechanical signals generated by fluid flow into biological processes. Here, we find that Piezo1 is necessary for the proper development and function of meningeal lymphatic vessels and that activating Piezo1 through transgenic overexpression or treatment with the chemical agonist Yoda1 is sufficient to increase cerebrospinal fluid (CSF) outflow by improving lymphatic absorption and transport. The abnormal accumulation of CSF, which often leads to hydrocephalus and ventriculomegaly, currently lacks effective treatments. We discovered that meningeal lymphatics in mouse models of Down syndrome were incompletely developed and abnormally formed. Selective overexpression of Piezo1 in lymphatics or systemic administration of Yoda1 in mice with hydrocephalus or Down syndrome resulted in a notable decrease in pathological CSF accumulation, ventricular enlargement and other associated disease symptoms. Together, our study highlights the importance of Piezo1-mediated lymphatic mechanotransduction in maintaining brain fluid drainage and identifies Piezo1 as a promising therapeutic target for treating excessive CSF accumulation and ventricular enlargement.