The therapeutic effect of GAS6 in remyelination is dependent upon Tyro3.

Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) characterized by demyelination, axonal damage and, for the majority of people, a decline in neurological function in the long-term. Remyelination could assist in the protection of axons and their functional recovery, but such therapies are not, as yet, available. The TAM (Tyro3, Axl, and MERTK) receptor ligand GAS6 potentiates myelination in vitro and promotes recovery in pre-clinical models of MS. However, it has remained unclear which TAM receptor is responsible for transducing this effect and whether post-translational modification of GAS6 is required. In this study, we show that the promotion of myelination requires post-translational modification of the GLA domain of GAS6 via vitamin K-dependent γ-carboxylation. We also confirmed that the intracerebroventricular provision of GAS6 for 2 weeks to demyelinated wild-type (WT) mice challenged with cuprizone increased the density of myelinated axons in the corpus callosum by over 2-fold compared with vehicle control. Conversely, the provision of GAS6 to Tyro3 KO mice did not significantly improve the density of myelinated axons. The improvement in remyelination following the provision of GAS6 to WT mice was also accompanied by an increased density of CC1+ve mature oligodendrocytes compared with vehicle control, whereas this improvement was not observed in the absence of Tyro3. This effect occurs independent of any influence on microglial activation. This work therefore establishes that the remyelinative activity of GAS6 is dependent on Tyro3 and includes potentiation of oligodendrocyte numbers.

Length-Dependent Cellular Internalization of Nanobody-Functionalized Poly(2-oxazoline) Nanorods.

The ability to target specific tissues and to be internalized by cells is critical for successful nanoparticle-based targeted drug delivery. Here, we combined "stealthy" rod-shaped poly(2-oxazoline) (POx) nanoparticles of different lengths with a cancer marker targeting nanobody and a fluorescent cell internalization sensor via a heat-induced living crystallization-driven self-assembly (CDSA) strategy. A significant increase in association and uptake driven by nanobody-receptor interactions was observed alongside nanorod-length-dependent kinetics. Importantly, the incorporation of the internalization sensor allowed for quantitative differentiation between cell surface association and internalization of the targeted nanorods, revealing unprecedented length-dependent cellular interactions of CDSA nanorods. This study highlights the modularity and versatility of the heat-induced CDSA process and further demonstrates the potential of POx nanorods as a modular nanomedicine platform.

Self-Immolative Polymer Nanoparticles with Precise and Controllable pH-Dependent Degradation.

Polymer nanoparticles have generated significant interest as delivery systems for therapeutic cargo. Self-immolative polymers (SIPs) are an interesting category of materials for delivery applications, as the characteristic property of end-to-end depolymerization allows for the disintegration of the delivery system, facilitating a more effective release of the cargo and clearance from the body after use. In this work, nanoparticles based on a pH-responsive polymer poly(ethylene glycol)-b-(2-diisopropyl)amino ethyl methacrylate) and a self-immolative polymer poly[N,N-(diisopropylamino)ethyl glyoxylamide-r-N,N-(dibutylamino)ethyl glyoxylamide] (P(DPAEGAm-r-DBAEGAm)) were developed. Four particles were synthesized based on P(DPAEGAm-r-DBAEGAm) polymers with varied diisopropylamino to dibutylamino ratios of 4:1, 2:1, 2:3, and 0:1, termed 4:1, 2:1, 2:3, and 0:1 PGAm particles. The pH of particle disassembly was tuned from pH 7.0 to pH 5.0 by adjusting the ratio of diisopropylamino to dibutylamino substituents on the pendant tertiary amine. The P(DPAEGAm-r-DBAEGAm) polymers were observed to depolymerize (60-80%) below the particle disassembly pH after ∼2 h, compared to <10% at pH 7.4 and maintained reasonable stability at pH 7.4 (20-50% depolymerization) after 1 week. While all particles exhibited the ability to load a peptide cargo, only the 4:1 PGAm particles had higher endosomal escape efficiency (∼4%) compared to the 2:3 or 0:1 PGAm particles (<1%). The 4:1 PGAm particle is a promising candidate for further optimization as an intracellular drug delivery system with rapid and precisely controlled degradation.

Nanoparticles for vaccine and gene therapy: Overcoming the barriers to nucleic acid delivery.

Nucleic acid therapeutics can be used to control virtually every aspect of cell behavior and therefore have significant potential to treat genetic disorders, infectious diseases, and cancer. However, while clinically approved to treat a small number of diseases, the full potential of nucleic acid therapeutics is hampered by inefficient delivery. Nucleic acids are large, highly charged biomolecules that are sensitive to degradation and so the approaches to deliver these molecules differ significantly from traditional small molecule drugs. Current studies suggest less than 1% of the injected nucleic acid dose is delivered to the target cell in an active form. This inefficient delivery increases costs and limits their use to applications where a small amount of nucleic acid is sufficient. In this review, we focus on two of the major barriers to efficient nucleic acid delivery: (1) delivery to the target cell and (2) transport to the subcellular compartment where the nucleic acids are therapeutically active. We explore how nanoparticles can be modified with targeting ligands to increase accumulation in specific cells, and how the composition of the nanoparticle can be engineered to manipulate or disrupt cellular membranes and facilitate delivery to the optimal subcellular compartments. Finally, we highlight how with intelligent material design, nanoparticle delivery systems have been developed to deliver nucleic acids that silence aberrant genes, correct genetic mutations, and act as both therapeutic and prophylactic vaccines. This article is categorized under: Nanotechnology Approaches to Biology > Cells at the Nanoscale Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Biology-Inspired Nanomaterials > Lipid-Based Structures.

Resolving subcellular pH with a quantitative fluorescent lifetime biosensor.

Changes in sub-cellular pH play a key role in metabolism, membrane transport, and triggering cargo release from therapeutic delivery systems. Most methods to measure pH rely on intensity changes of pH sensitive fluorophores, however, these measurements are hampered by high uncertainty in the inferred pH and the need for multiple fluorophores. To address this, here we combine pH dependant fluorescent lifetime imaging microscopy (pHLIM) with deep learning to accurately quantify sub-cellular pH in individual vesicles. We engineer the pH sensitive protein mApple to localise in the cytosol, endosomes, and lysosomes, and demonstrate that pHLIM can rapidly detect pH changes induced by drugs such as bafilomycin A1 and chloroquine. We also demonstrate that polyethylenimine (a common transfection reagent) does not exhibit a proton sponge effect and had no measurable impact on the pH of endocytic vesicles. pHLIM is a simple and quantitative method that will help to understand drug action and disease progression.

Identifying cell-to-cell variability in internalization using flow cytometry.

Biological heterogeneity is a primary contributor to the variation observed in experiments that probe dynamical processes, such as the internalization of material by cells. Given that internalization is a critical process by which many therapeutics and viruses reach their intracellular site of action, quantifying cell-to-cell variability in internalization is of high biological interest. Yet, it is common for studies of internalization to neglect cell-to-cell variability. We develop a simple mathematical model of internalization that captures the dynamical behaviour, cell-to-cell variation, and extrinsic noise introduced by flow cytometry. We calibrate our model through a novel distribution-matching approximate Bayesian computation algorithm to flow cytometry data of internalization of anti-transferrin receptor antibody in a human B-cell lymphoblastoid cell line. This approach provides information relating to the region of the parameter space, and consequentially the nature of cell-to-cell variability, that produces model realizations consistent with the experimental data. Given that our approach is agnostic to sample size and signal-to-noise ratio, our modelling framework is broadly applicable to identify biological variability in single-cell data from internalization assays and similar experiments that probe cellular dynamical processes.

Subcutaneous delivery of a dendrimer-BH3 mimetic improves lymphatic uptake and survival in lymphoma.

As a malignant tumour of lymphatic origin, B-cell lymphoma represents a significant challenge for drug delivery, where effective therapies must access malignant cells in the blood, organs and lymphatics while avoiding off-target toxicity. Subcutaneous (SC) administration of nanomedicines allows preferential access to both the lymphatic and blood systems and may therefore provide a route to enhanced drug exposure to lymphomas. Here we examine the impact of SC dosing on lymphatic exposure, pharmacokinetics (PK), and efficacy of AZD0466, a small molecule dual Bcl-2/Bcl-xL inhibitor conjugated to a 'DEP®' G5 poly-l-lysine dendrimer. PK studies reveal that the plasma half-life of the dendrimer-drug conjugate is 8-times longer than that of drug alone, providing evidence of slow release from the circulating dendrimer nanocarrier. The SC dosed construct also shows preferential lymphatic transport, with over 50% of the bioavailable dose recovered in thoracic lymph. Increases in dose (up to 400 mg/kg) are well tolerated after SC administration and studies in a model of disseminated lymphoma in mice show that high dose SC treatment outperforms IV administration using doses that lead to similar total plasma exposure (lower peak concentrations but extended exposure after SC). These data show that the DEP® dendrimer can act as a circulating drug depot accessing both the lymphatic and blood circulatory systems. SC administration improves lymphatic exposure and facilitates higher dose administration due to improved tolerability. Higher dose SC administration also results in improved efficacy, suggesting that drug delivery systems that access both plasma and lymph hold significant potential for the treatment of haematological cancers where lymphatic and extranodal dissemination are poor prognostic factors.

Understanding the Biological Interactions of pH-Swellable Nanoparticles.

pH-responsive nanoparticles have generated significant interest for use as drug delivery systems due to their potential for inducible release at low pH. The pH variation from the bloodstream (pH 7.4) to intracellular compartments of cells called endosomes/lysosomes (pH < 5.0) has been of particular interest. However, one of the limitations with nanoparticle delivery systems is the inability to migrate out of these compartments to the cytosol or other organelles, via a process termed endosomal escape. Previous studies have postulated that pH-responsive nanoparticles can facilitate endosomal escape through a range of mechanisms including membrane interaction, pH-induced swelling, and the proton-sponge effect. In this study, a series of pH-swellable nanoparticles (85-100 nm) are designed and their impact on biological interactions, particularly endosomal escape, are investigated. The particles exhibit tunable pH-induced swelling (from 120% to 200%) and have good buffering capacity. The cellular association is studied using flow cytometry and endosomal escape is determined using a calcein leakage assay. Interestingly, no endosomal escape with all nanoparticle formulations is found, which suggests there are limitations with both the proton-sponge effect and pH-induced swelling mechanism as the primary methods for inducing endosomal escape.

Association of a vaccine adjuvant with endogenous HDL increases lymph uptake and dendritic cell activation.

Vaccines are a powerful health intervention but there is still an unmet need for effective preventative and therapeutic vaccines for many diseases such as cancer and infections. Interstitial (e.g. subcutaneous (SC)) injection in nano-sized carriers such as high density lipoproteins (HDLs) can improve the access of vaccine subunit antigens or adjuvants to target immune cells in the lymphatics and potentiate vaccination responses such as cytotoxic T lymphocyte (CTL) responses (Kuai et al., 2016, 2018; Qian et al., 2016). Here we examined how cholesterol conjugation to the vaccine adjuvant CpG, and incorporation into HDL, changes lymphatic absorption and association with, and processing by, dendritic cells (DCs), ultimately influencing adjuvant efficacy. We investigated the lymphatic disposition of cholesterol conjugated CpG incorporated into HDL (HDL(Chol-CpG-Cy5)) relative to free cholesterol conjugated CpG (Chol-CpG-Cy5) and unconjugated CpG (free CpG-Cy5) after SC administration in rats and mice. HDL (Chol-CpG-Cy5) and Chol-CpG-Cy5 differentially altered CpG absorption into lymph vs. blood, but surprisingly resulted in similarly higher LN accumulation relative to free CpG. The mechanism of access of Chol-CpG-Cy5 into lymph might be partly due to association with endogenous HDL at the injection site followed by transport into lymph in association with the HDL. To measure CpG association with and processing by DCs and the strength of the immune response, mice were vaccinated with free ovalbumin (OVA) co-administered with the different CpG constructs. There were significant changes in DC activation that were reflective of the trend in LN accumulation at 24 h post-vaccination. However, T cell responses at 24 h and 7 days post-vaccination were not significantly different across the CpG groups although the response was less variable for Chol-CpG-Cy5 compared to free CpG Cy5 and also HDL(Chol-CpG-Cy5) - despite similar LN accumulation with the latter. Overall, our data indicate that cholesterol conjugation and incorporation into HDL increases adjuvant lymph disposition and DC activation.

Quantifying the Endosomal Escape of pH-Responsive Nanoparticles Using the Split Luciferase Endosomal Escape Quantification Assay.

All nanoparticles have the potential to revolutionize the delivery of therapeutic cargo such as peptides, proteins, and RNA. However, effective cytosolic delivery of cargo from nanoparticles represents a significant challenge in the design of more efficient drug delivery vehicles. Recently, research has centered on designing nanoparticles with the capacity to escape endosomes by responding to biological stimuli such as changes in pH, which occur when nanoparticles are internalized into the endo-/lysosomal pathway. Current endosomal escape assays rely on indirect measurements and yield little quantitative information, which hinders the design of more efficient drug delivery vehicles. Therefore, we adapted the highly sensitive split luciferase endosomal escape quantification (SLEEQ) assay to better understand nanoparticle-induced endosomal escape. We applied SLEEQ to evaluate the endosomal escape behavior of two pH-responsive nanoparticles: the first with a poly(2-diisopropylamino ethyl methacrylate) (PDPAEMA) core and the second with 1:1 ratio of poly(2-diethylamino ethyl methacrylate) (PDEAEMA) and PDPAEMA. SLEEQ directly measured the cytosolic delivery and showed that engineering the nanoparticle disassembly pH could improve the endosomal escape efficiency by fivefold. SLEEQ is a versatile assay that can be used for a wide range of nanomaterials and will improve the development of drug delivery vehicles in the future.

Opportunities for innovation: Building on the success of lipid nanoparticle vaccines.

Lipid nanoparticle (LNP) formulations of messenger RNA (mRNA) have demonstrated high efficacy as vaccines against SARS-CoV-2. The success of these nanoformulations underscores the potential of LNPs as a delivery system for next-generation biological therapies. In this article, we highlight the key considerations necessary for engineering LNPs as a vaccine delivery system and explore areas for further optimisation. There remain opportunities to improve the protection of mRNA, optimise cytosolic delivery, target specific cells, minimise adverse side-effects and control the release of RNA from the particle. The modular nature of LNP formulations and the flexibility of mRNA as a payload provide many pathways to implement these strategies. Innovation in LNP vaccines is likely to accelerate with increased enthusiasm following recent successes; however, any advances will have implications for a broad range of therapeutic applications beyond vaccination such as gene therapy.

Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay.

Cytosolic transport is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of cytosolic delivery mechanisms remains limited due to a significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we develop a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We apply SLEEQ to evaluate the cytosolic delivery of a range of widely studied cell-penetrating peptides (CPPs) fused to a model protein. We demonstrate that positively charged CPPs enhance cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how CPPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol.

In Vivo Quantification of Nanoparticle Association with Immune Cell Subsets in Blood.

Nanoparticles offer great promise for more effective drug delivery. However, their particulate nature typically results in rapid systemic clearance by immune cells in blood. Currently, to understand these interactions, nanoparticle association is probed ex vivo with whole blood. While ex vivo assays give important information about the relative cell association, they do not consider changes in immune cell homeostasis or the complex mixing behavior that occurs in vivo. To address this, a nanoparticle in vivo immune-cell association assay is developed to study the in vivo association of unmodified and poly(ethylene glycol) modified liposomes with immune cells, and compared this to the ex vivo association in static whole blood. In vivo, it is observed that neutrophils play a significantly greater role in nanoparticle binding than suggested by ex vivo assays. The increased influence of neutrophils in vivo is largely due to a significant increase in number of circulating neutrophils after intravenous injection. Conversely, the number of circulating monocytes significantly decreased after intravenous injection, leading to significantly less total association of liposomes to monocytes compared to ex vivo. This novel in vivo immune cell binding assay sheds new light on the fate of nanoparticles following intravenous delivery.

Key principles and methods for studying the endocytosis of biological and nanoparticle therapeutics.

Endocytosis is a critical step in the process by which many therapeutic nanomedicines reach their intracellular targets. Our understanding of cellular uptake mechanisms has developed substantially in the past five years. However, these advances in cell biology have not fully translated to the nanoscience and therapeutics literature. Misconceptions surrounding the role of different endocytic pathways and how to study these pathways are hindering progress in developing improved nanoparticle therapies. Here, we summarize the latest insights into cellular uptake mechanisms and pathways. We highlight limitations of current systems to study endocytosis, particularly problems with non-specific inhibitors. We also summarize alternative genetic approaches to robustly probe these pathways and discuss the need to understand how cells endocytose particles in vivo. We hope that this critical assessment of the current methods used in studying nanoparticle uptake will guide future studies at the interface of cell biology and nanomedicine.

Inducing immune tolerance with dendritic cell-targeting nanomedicines.

Induced tolerogenic dendritic cells are a powerful immunotherapy for autoimmune disease that have shown promise in laboratory models of disease and early clinical trials. In contrast to conventional immunosuppressive treatments, tolerogenic immunotherapy leverages the cells and function of the immune system to quell the autoreactive lymphocytes responsible for damage and disease. The principle techniques of isolating and reprogramming dendritic cells (DCs), central to this approach, are well characterized. However, the broader application of this technology is limited by its high cost and bespoke nature. Nanomedicine offers an alternative route by performing this reprogramming process in situ. Here, we review the challenges and opportunities in using nanoparticles as a delivery mechanism to target DCs and induce immunomodulation, emphasizing their versatility. We then highlight their potential to solve critical problems in organ transplantation and increasingly prevalent autoimmune disorders such as type 1 diabetes mellitus and multiple sclerosis, where new immunotherapy approaches have begun to show promise.

It's what's on the inside that counts: Techniques for investigating the uptake and recycling of nanoparticles and proteins in cells.

Many applications of nanomedicines depend on the therapeutic gaining access to the interior of cells. As most proteins and nanoparticles are taken up by endocytosis, determining the properties of nanoparticles that govern uptake is essential. In this review, we examine the current approaches for measuring the cellular uptake of nanoparticles and proteins. We explore the techniques distinguishing material internalized by the cell from material bound to the surface, with a particular focus on recent advances in sensor technology. We also highlight the requirements for quantifying internalization and the pitfalls that can limit data analysis. Finally, we explore the importance of understanding recycling of internalized material back to the cell surface, and the methods that can be used to quantify this. Delivering cargo to specific subcellular locations first requires uptake. Robust techniques that can quantify this event are the critical for developing the next generation of smart, targeted, therapeutic nanoparticles.

A molecular sensor to quantify the localization of proteins, DNA and nanoparticles in cells.

Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAPSwitch) that can be conjugated to biomolecules using click chemistry. SNAPSwitch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAPSwitch, we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAPSwitch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells.

High-Density Lipoprotein Composition Influences Lymphatic Transport after Subcutaneous Administration.

Interstitial administration (e.g., subcutaneous (SC) administration) of immunotherapies and vaccines within nanoparticles can improve access to lymph-resident immune cells, leading to enhanced efficacy and reduced off-target effects. Recently, endogenous high-density lipoproteins (HDLs) were found to return from peripheral tissue back to the systemic circulation via the lymphatic vessels and nodes. This suggests the potential utility of HDLs as biocompatible lymphatic-targeted therapeutic carriers. However, we have a limited understanding of the mechanisms that drive HDL uptake into peripheral lymphatics from the interstitium. This study investigated the influence of HDL physicochemical properties on lymphatic transport and lymph node (LN) retention of HDL after SC administration. A range of HDL particles was prepared and characterized. Sphere-shaped endogenous HDLs were isolated from biological fluids (rat lymph, rat plasma, and human plasma) and separated into two subclasses based on the density. Discoidal-shaped synthetic (reconstituted) HDLs (rHDLs) of similar sizes were assembled from lipids and apolipoprotein A-I. All HDLs had similar sizes of 10-20 nm and a slightly negative surface charge. All HDLs were radiolabeled with 3H-cholesteryl ester (3H-CE) and/or 14C-free cholesterol (14C FC) and administered SC into the hind leg of thoracic lymph-cannulated rats. The recovery of radiolabels in lymph, plasma, LN, and tissues was determined. From the interstitial injection site, all HDLs were preferentially transported into the lymph and not blood vessels as indicated by high lymph-to-plasma concentration ratios of the radiolabels (up to 100:1 during the absorption phase) and greater radiolabel recovery in LNs draining the injection site compared to the contralateral side. Several HDLs with unique composition demonstrated significantly higher lymphatic transport compared to other HDLs despite possessing similar physical properties, suggesting that HDL lymphatic transport is less influenced by physical properties. The LN retention of HDL was positively correlated to increasing the negative charge of HDL, which was related to surface composition. Overall, this study informs the optimal design of HDL-based nanoparticles to promote lymphatic targeting of immunotherapies and vaccines.

Silicon-Nanotube-Mediated Intracellular Delivery Enables Ex Vivo Gene Editing.

Engineered nano-bio cellular interfaces driven by vertical nanostructured materials are set to spur transformative progress in modulating cellular processes and interrogations. In particular, the intracellular delivery-a core concept in fundamental and translational biomedical research-holds great promise for developing novel cell therapies based on gene modification. This study demonstrates the development of a mechanotransfection platform comprising vertically aligned silicon nanotube (VA-SiNT) arrays for ex vivo gene editing. The internal hollow structure of SiNTs allows effective loading of various biomolecule cargoes; and SiNTs mediate delivery of those cargoes into GPE86 mouse embryonic fibroblasts without compromising their viability. Focused ion beam scanning electron microscopy (FIB-SEM) and confocal microscopy results demonstrate localized membrane invaginations and accumulation of caveolin-1 at the cell-NT interface, suggesting the presence of endocytic pits. Small-molecule inhibition of endocytosis suggests that active endocytic process plays a role in the intracellular delivery of cargo from SiNTs. SiNT-mediated siRNA intracellular delivery shows the capacity to reduce expression levels of F-actin binding protein (Triobp) and alter the cellular morphology of GPE86. Finally, the successful delivery of Cas9 ribonucleoprotein (RNP) to specifically target mouse Hprt gene is achieved. This NT-enhanced molecular delivery platform has strong potential to support gene editing technologies.

Organ-specific lymphatics play distinct roles in regulating HDL trafficking and composition.

Recently, peripheral lymphatic vessels were found to transport high-density lipoprotein (HDL) from interstitial tissues to the blood circulation during reverse cholesterol transport. This function is thought to be critical to the clearance of cholesterol from atherosclerotic plaques. The role of organ-specific lymphatics in modulating HDL transport and composition is, however, incompletely understood. This study aimed to 1) determine the contribution of the lymphatics draining the intestine and liver (which are major sites of HDL synthesis) to total (thoracic) lymph HDL transport and 2) verify whether the HDLs in lymph are derived from specific organs and are modified during trafficking in lymph. The mesenteric, hepatic, or thoracic lymph duct was cannulated in nonfasted Sprague-Dawley rats, and lymph was collected over 5 h under anesthesia. Whole lymph and specific lymph lipoproteins (isolated by ultracentrifugation) were analyzed for protein and lipid composition. The majority of thoracic lymph fluid, protein, and lipid mass was sourced from the mesenteric, and to a lesser extent, hepatic lymph. Mesenteric and thoracic lymph were both rich in chylomicrons and very low-density lipoprotein, whereas hepatic lymph and plasma were HDL-rich. The protein and lipid mass in thoracic lymph HDL was mostly sourced from mesenteric lymph, whereas the cholesterol mass was equally sourced from mesenteric and hepatic lymph. HDLs were compositionally distinct across the lymph sources and plasma. The composition of HDL also appeared to be modified during passage from the mesenteric and hepatic to the thoracic lymph duct. Overall, this study demonstrates that the lipoproteins in lymph are organ specific in composition, and the intestine and liver appear to be the main source of HDL in the lymph.NEW & NOTEWORTHY High-density lipoprotein in lymph are organ-specific in composition and derive mostly from the intestine and liver. High-density lipoprotein also appears to be remodeled during transport through the lymphatics. These findings have implications to cardiometabolic diseases that involve perturbations in lipoprotein distribution and metabolism.