State-of-the-art drug delivery system to target the lymphatics.

PRIMARY OBJECTIVE: The primary objective of the review is to assess the potential of lymphatic-targeted drug delivery systems, with a particular emphasis on their role in tumour therapy and vaccination efficacy. REASON FOR LYMPHATIC TARGETING: The lymphatic system's crucial functions in maintaining bodily equilibrium, regulating metabolism, and orchestrating immune responses make it an ideal target for drug delivery. Lymph nodes, being primary sites for tumour metastasis, underscore the importance of targeting the lymphatic system for effective treatment. OUTCOME: Nanotechnologies and innovative biomaterials have facilitated the development of lymphatic-targeted drug carriers, leveraging endogenous macromolecules to enhance drug delivery efficiency. Various systems such as liposomes, micelles, inorganic nanomaterials, hydrogels, and nano-capsules demonstrate significant potential for delivering drugs to the lymphatic system. CONCLUSION: Understanding the physiological functions of the lymphatic system and its involvement in diseases underscores the promise of targeted drug delivery in improving treatment outcomes. The strategic targeting of the lymphatic system presents opportunities to enhance patient prognosis and advance therapeutic interventions across various medical contexts, indicating the importance of ongoing research and development in this area.

Battle of the milky way: Lymphatic targeted drug delivery for pathogen eradication.

Many viruses, bacteria, and parasites rely on the lymphatic system for survival, replication, and dissemination. While conventional anti-infectives can combat infection-causing agents in the bloodstream, they do not reach the lymphatic system to eradicate the pathogens harboured there. This can result in ineffective drug exposure and reduce treatment effectiveness. By developing effective lymphatic delivery strategies for antiviral, antibacterial, and antiparasitic drugs, their systemic pharmacokinetics may be improved, as would their ability to reach their target pathogens within the lymphatics, thereby improving clinical outcomes in a variety of acute and chronic infections with lymphatic involvement (e.g., acquired immunodeficiency syndrome, tuberculosis, and filariasis). Here, we discuss approaches to targeting anti-infective drugs to the intestinal and dermal lymphatics, aiming to eliminate pathogen reservoirs and interfere with their survival and reproduction inside the lymphatic system. These include optimized lipophilic prodrugs and drug delivery systems that promote lymphatic transport after oral and dermal drug intake. For intestinal lymphatic delivery via the chylomicron pathway, molecules should have logP values >5 and long-chain triglyceride solubilities >50 mg/g, and for dermal lymphatic delivery via interstitial lymphatic drainage, nanoparticle formulations with particle size between 10 and 100 nm are generally preferred. Insight from this review may promote new and improved therapeutic solutions for pathogen eradication and combating infective diseases, as lymphatic system involvement in pathogen dissemination and drug resistance has been neglected compared to other pathways leading to treatment failure.

Low Efficacy of Genetic Tests for the Diagnosis of Primary Lymphedema Prompts Novel Insights into the Underlying Molecular Pathways.

Lymphedema is a chronic inflammatory disorder caused by ineffective fluid uptake by the lymphatic system, with effects mainly on the lower limbs. Lymphedema is either primary, when caused by genetic mutations, or secondary, when it follows injury, infection, or surgery. In this study, we aim to assess to what extent the current genetic tests detect genetic variants of lymphedema, and to identify the major molecular pathways that underlie this rather unknown disease. We recruited 147 individuals with a clinical diagnosis of primary lymphedema and used established genetic tests on their blood or saliva specimens. Only 11 of these were positive, while other probands were either negative (63) or inconclusive (73). The low efficacy of such tests calls for greater insight into the underlying mechanisms to increase accuracy. For this purpose, we built a molecular pathways diagram based on a literature analysis (OMIM, Kegg, PubMed, Scopus) of candidate and diagnostic genes. The PI3K/AKT and the RAS/MAPK pathways emerged as primary candidates responsible for lymphedema diagnosis, while the Rho/ROCK pathway appeared less critical. The results of this study suggest the most important pathways involved in the pathogenesis of lymphedema, and outline the most promising diagnostic and candidate genes to diagnose this disease.

Role of Angiopoietin-Tie axis in vascular and lymphatic systems and therapeutic interventions.

The Angiopoietin (Ang)-Tyrosine kinase with immunoglobulin-like and EGF-like domains (Tie) axis is an endothelial cell-specific ligand-receptor signaling pathway necessary for vascular and lymphatic development. The Ang-Tie axis is involved in regulating angiogenesis, vascular remodeling, vascular permeability, and inflammation to maintain vascular quiescence. Disruptions in the Ang-Tie axis are involved in many vascular and lymphatic diseases and play an important role in physiological and pathological vascular processes. Given recent advances in the Ang-Tie axis in the vascular and lymphatic systems, this review focuses on the multiple functions of the Ang-Tie axis in inflammation-induced vascular permeability, vascular remodeling, atherosclerosis, ocular angiogenesis, tumor angiogenesis, and metastasis. A summary of relevant therapeutic approaches to the Ang-Tie axis, including therapeutic antibodies, recombinant proteins and small molecule drugs are also discussed. The purpose of this review is to provide new hypotheses and identify potential therapeutic strategies based on the Ang-Tie signaling axis for the treatment of vascular and lymphatic-related diseases.

Lymphatic Dysfunction Exacerbates Cutaneous Tumorigenesis and Psoriasis-Like Skin Inflammation through Accumulation of Inflammatory Cytokines.

Lymphatic transport plays an important role in coordinating local immune responses. However, the biologic effects of impaired lymphatic flow in vivo are not fully understood. In this study, we investigated the roles of the lymphatic system in skin carcinogenesis and psoriasis-like inflammation using k-cyclin transgenic (kCYC+/-) mice, which demonstrate severe lymphatic dysfunction. kCYC+/- mice showed augmented tumor growth in the two-stage skin carcinogenesis model and severe clinical scores in imiquimod-induced psoriasis-like skin inflammation compared with wild-type mice. Although mRNA levels of inflammatory cytokines in skin after topical application of 12-O-tetradecanoylphorbol-13-acetate or imiquimod were comparable between kCYC+/- and wild-type mice, protein levels of inflammatory cytokines, such as IL-17A, IL-22, and IL-23, were significantly upregulated in kCYC+/- mice in both models. Consistently, signal transducer and activator of transcription 3 pathway and NF-κB signaling were augmented in epidermal keratinocytes in kCYC+/- mice. These results suggest that lymphatic dysfunction in kCYC+/- mice caused accumulation of inflammatory cytokines, leading to the exacerbation of two-stage skin carcinogenesis and imiquimod-induced psoriasis-like skin inflammation. These findings add insight into the clinical problems of secondary malignancies and inflammatory dermatoses that may occur with extremity lymphedema.

Smart design approaches for orally administered lipophilic prodrugs to promote lymphatic transport.

Challenges to effective delivery of drugs following oral administration has attracted growing interest over recent decades. Small molecule drugs (<1000 Da) are generally absorbed across the gastrointestinal tract into the portal blood and further transported to the systemic circulation via the liver. This can result in a significant reduction to the oral bioavailability of drugs that are metabolically labile and ultimately lead to ineffective exposure and treatment. Targeting drug delivery to the intestinal lymphatics is attracting increased attention as an alternative route of drug transportation providing multiple benefits. These include bypassing hepatic first-pass metabolism and selectively targeting disease reservoirs residing within the lymphatic system. The particular physicochemical requirements for drugs to be able to access the lymphatics after oral delivery include high lipophilicity (logP>5) and high long-chain triglyceride solubility (> 50 mg/g), properties required to enable drug association with the lipoprotein transport pathway. The majority of small molecule drugs, however, are not this lipophilic and therefore not substantially transported via the intestinal lymph. This has contributed to a growing body of investigation into prodrug approaches to deliver drugs to the lymphatic system by chemical manipulation. Optimised lipophilic prodrugs have the potential to increase lymphatic transport thereby improving oral pharmacokinetics via a reduction in first pass metabolism and may also target of disease-specific reservoirs within the lymphatics. This may provide advantages for current pharmacotherapy approaches for a wide array of pathological conditions, e.g. immune disease, cancer and metabolic disease, and also presents a promising approach for advanced vaccination strategies. In this review, specific emphasis is placed on medicinal chemistry strategies that have been successfully employed to design lipophilic prodrugs to deliberately enable lymphatic transport. Recent progress and opportunities in medicinal chemistry and drug delivery that enable new platforms for efficacious and safe delivery of drugs are critically evaluated.

[Advances of Angiopoietin-Tie axis in vascular and lymphatic system-related diseases].

Endothelial cells that form the inner layers of both blood and lymphatic vessels are important components of the vascular system and are involved in the pathogenesis of vascular and lymphatic diseases. Angiopoietin (Ang)-Tie axis in endothelial cells is the second endothelium-specific ligand-receptor signaling system necessary for embryonic cardiovascular and lymphatic development in addition to the vascular endothelial growth factor receptor pathway. The Ang-Tie axis also maintains vascular homeostasis by regulating postnatal angiogenesis, vessel remodeling, vascular permeability, and inflammation. Therefore, the dysfunction of this system leads to many vascular and lymphatic diseases. In light of the recent advances on the role of the Ang-Tie axis in vascular and lymphatic system-related diseases, this review summarizes the functions of the Ang-Tie axis in inflammation-induced vascular permeability, vascular remodeling, ocular angiogenesis, shear stress response, atherosclerosis, tumor angiogenesis, and metastasis. Moreover, this review summarizes the relevant therapeutic antibodies, recombinant proteins, and small molecular drugs associated with the Ang-Tie axis.

Hyaluronan and Its Receptors: Key Mediators of Immune Cell Entry and Trafficking in the Lymphatic System.

Entry to the afferent lymphatics marks the first committed step for immune cell migration from tissues to draining lymph nodes both for the generation of immune responses and for timely resolution of tissue inflammation. This critical process occurs primarily at specialised discontinuous junctions in initial lymphatic capillaries, directed by chemokines released from lymphatic endothelium and orchestrated by adhesion between lymphatic receptors and their immune cell ligands. Prominent amongst the latter is the large glycosaminoglycan hyaluronan (HA) that can form a bulky glycocalyx on the surface of certain tissue-migrating leucocytes and whose engagement with its key lymphatic receptor LYVE-1 mediates docking and entry of dendritic cells to afferent lymphatics. Here we outline the latest insights into the molecular mechanisms by which the HA glycocalyx together with LYVE-1 and the related leucocyte receptor CD44 co-operate in immune cell entry, and how the process is facilitated by the unusual character of LYVE-1 • HA-binding interactions. In addition, we describe how pro-inflammatory breakdown products of HA may also contribute to lymphatic entry by transducing signals through LYVE-1 for lymphangiogenesis and increased junctional permeability. Lastly, we outline some future perspectives and highlight the LYVE-1 • HA axis as a potential target for immunotherapy.

Inflammation rapidly recruits mammalian GMP and MDP from bone marrow into regional lymphatics.

Innate immune cellular effectors are actively consumed during systemic inflammation, but the systemic traffic and the mechanisms that support their replenishment remain unknown. Here, we demonstrate that acute systemic inflammation induces the emergent activation of a previously unrecognized system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors, but not other progenitors or stem cells, from bone marrow (BM) to regional lymphatic capillaries. The progenitor traffic to the systemic lymphatic circulation is mediated by Ccl19/Ccr7 and is NF-κB independent, Traf6/IκB-kinase/SNAP23 activation dependent, and is responsible for the secretion of pre-stored Ccl19 by a subpopulation of CD205+/CD172a+ conventional dendritic cells type 2 and upregulation of BM myeloid progenitor Ccr7 signaling. Mature myeloid Traf6 signaling is anti-inflammatory and necessary for lymph node myeloid cell development. This report unveils the existence and the mechanistic basis of a very early direct traffic of myeloid progenitors from BM to lymphatics during inflammation.

When the body becomes infected with disease-causing pathogens, such as bacteria, the immune system activates various mechanisms which help to fight off the infection. One of the immune system's first lines of defense is to launch an inflammatory response that helps remove the pathogen and recruit other immune cells. However, this response can become overactivated, leading to severe inflammatory conditions that damage healthy cells and tissues. A second group of cells counteract this over inflammation and are different to the ones involved in the early inflammatory response. Both types of cells ­ inflammatory and anti-inflammatory ­ develop from committed progenitors, which, unlike stem cells, are already destined to become a certain type of cell. These committed progenitors reside in the bone marrow and then rapidly travel to secondary lymphoid organs, such as the lymph nodes, where they mature into functioning immune cells. During this journey, committed progenitors pass from the bone marrow to the lymphatic vessels that connect up the different secondary lymphoid organs, and then spread to all tissues in the body. Yet, it is not fully understood what exact route these cells take and what guides them towards these lymphatic tissues during inflammation. To investigate this, Serrano-Lopez, Hegde et al. used a combination of techniques to examine the migration of progenitor cells in mice that had been treated with lethal doses of a bacterial product that triggers inflammation. This revealed that as early as one to three hours after the onset of infection, progenitor cells were already starting to travel from the bone marrow towards lymphatic vessels. Serrano-Lopez, Hegde et al. found that a chemical released by an "alarm" immune cell already residing in secondary lymphoid organs attracted these progenitor cells towards the lymphatic tissue. Further experiments showed that the progenitor cells travelling to secondary lymphoid organs were already activated by bacterial products. They then follow the chemical released by alarm immune cells ready to respond to the immune challenge and suppress inflammation. These committed progenitors were also found in the inflamed lymph nodes of patients. These findings suggest this rapid circulation of progenitors is a mechanism of defense that contributes to the fight against severe inflammation. Altering how these cells migrate from the bone marrow to secondary lymphoid organs could provide a more effective treatment for inflammatory conditions and severe infections. However, these approaches would need to be tested further in the laboratory and in clinical trials.

Lymphatic blood filling in CLEC-2-deficient mouse models.

C-type lectin-like receptor 2 (CLEC-2) is considered as a potential drug target in settings of wound healing, inflammation, and infection. A potential barrier to this is evidence that CLEC-2 and its ligand podoplanin play a critical role in preventing lymphatic vessel blood filling in mice throughout life. In this study, this aspect of CLEC-2/podoplanin function is investigated in more detail using new and established mouse models of CLEC-2 and podoplanin deficiency, and models of acute and chronic vascular remodeling. We report that CLEC-2 expression on platelets is not required to maintain a barrier between the blood and lymphatic systems in unchallenged mice, post-development. However, under certain conditions of chronic vascular remodeling, such as during tumorigenesis, deficiency in CLEC-2 can lead to lymphatic vessel blood filling. These data provide a new understanding of the function of CLEC-2 in adult mice and confirm the essential nature of CLEC-2-driven platelet activation in vascular developmental programs. This work expands our understanding of how lymphatic blood filling is prevented by CLEC-2-dependent platelet function and provides a context for the development of safe targeting strategies for CLEC-2 and podoplanin.

HMGB1 Promotes Myeloid Egress and Limits Lymphatic Clearance of Malignant Pleural Effusions.

Pleural effusions, when benign, are attributed to cardiac events and suffusion of fluid within the pleural space. When malignant, lymphatic obstruction by tumor and failure to absorb constitutively produced fluid is the predominant formulation. The prevailing view has been challenged recently, namely that the lymphatics are only passive vessels, carrying antigenic fluid to secondary lymphoid sites. Rather, lymphatic vessels can be a selective barrier, efficiently coordinating egress of immune cells and factors within tissues, limiting tumor spread and immune pathology. An alternative explanation, offered here, is that damage associated molecular pattern molecules, released in excess, maintain a local milieu associated with recruitment and retention of immune cells associated with failed lymphatic clearance and functional lymphatic obstruction. We found that levels of high mobility group box 1 (HMGB1) were equally elevated in both benign and malignant pleural effusions (MPEs) and that limited diversity of T cell receptor expressing gamma and delta chain were inversely associated with these levels in MPEs. Acellular fluid from MPEs enhanced γδ T cell proliferation in vitro, while inhibiting cytokine production from γδ T cells and monocytes as well as restricting monocyte chemotaxis. Novel therapeutic strategies, targeting HMGB1 and its neutralization in such effusions as well as direct delivery of immune cells into the pleural space to reconstitute normal physiology should be considered.

Lymph-directed immunotherapy - Harnessing endogenous lymphatic distribution pathways for enhanced therapeutic outcomes in cancer.

The advent of immunotherapy has revolutionised the treatment of some cancers. Harnessing the immune system to improve tumour cell killing is now standard clinical practice and immunotherapy is the first line of defence for many cancers that historically, were difficult to treat. A unifying concept in cancer immunotherapy is the activation of the immune system to mount an attack on malignant cells, allowing the body to recognise, and in some cases, eliminate cancer. However, in spite of a significant proportion of patients that respond well to treatment, there remains a subset who are non-responders and a number of cancers that cannot be treated with these therapies. These limitations highlight the need for targeted delivery of immunomodulators to both tumours and the effector cells of the immune system, the latter being highly concentrated in the lymphatic system. In this context, macromolecular therapies may provide a significant advantage. Macromolecules are too large to easily access blood capillaries and instead typically exhibit preferential uptake via the lymphatic system. In contexts where immune cells are the therapeutic target, particularly in cancer therapy, this may be advantageous. In this review, we examine in brief the current immunotherapy approaches in cancer and how macromolecular and nanomedicine strategies may improve the therapeutic profiles of these drugs. We subsequently discuss how therapeutics directed either by parenteral or mucosal administration, can be taken up by the lymphatics thereby accessing a larger proportion of the body's immune cells. Finally, we detail drug delivery strategies that have been successfully employed to target the lymphatics.

Cetuximab Exhibits Sex Differences in Lymphatic Exposure after Intravenous Administration in Rats in the Absence of Differences in Plasma Exposure.

PURPOSE: The aim of this work was to identify whether biochemical and physiological sources of mAb pharmacokinetic sex-effects could be identified in the rat model where target-mediated disposition is avoided. METHODS: Plasma and lymphatic pharmacokinetics of the humanised anti-EGFR antibody cetuximab, along with potential physiological and biochemical drivers of pharmacokinetic sex differences, were examined in male and female rats. Cetuximab was used as a model mAb since plasma clearance is slower in female patients. RESULTS: When plasma concentrations were normalised to dose, female rats displayed slower plasma clearance than males, but no significant differences were observed in liver and spleen biodistribution. Sex differences in apparent plasma clearance, however, were abolished after normalisation to body weight, surface area or fat-free mass. Significant sex differences were observed in plasma testosterone, endogenous IgG and fat free mass, but did not correlate with apparent clearance. Females did, however, show two-fold higher lymphatic exposure compared to males. CONCLUSIONS: These data suggested that mAbs more efficiently access lymph in females, but this does not affect plasma pharmacokinetics or biodistribution. Further, the data suggest that sex differences observed in humans could be a function of antigen density.

Decreased lymphatic HIF-2α accentuates lymphatic remodeling in lymphedema.

Pathologic lymphatic remodeling in lymphedema evolves during periods of tissue inflammation and hypoxia through poorly defined processes. In human and mouse lymphedema, there is a significant increase of hypoxia inducible factor 1 α (HIF-1α), but a reduction of HIF-2α protein expression in lymphatic endothelial cells (LECs). We questioned whether dysregulated expression of these transcription factors contributes to disease pathogenesis and found that LEC-specific deletion of Hif2α exacerbated lymphedema pathology. Even without lymphatic vascular injury, the loss of LEC-specific Hif2α caused anatomic pathology and a functional decline in fetal and adult mice. These findings suggest that HIF-2α is an important mediator of lymphatic health. HIF-2α promoted protective phosphorylated TIE2 (p-TIE2) signaling in LECs, a process also replicated by upregulating TIE2 signaling through adenovirus-mediated angiopoietin-1 (Angpt1) gene therapy. Our study suggests that HIF-2α normally promotes healthy lymphatic homeostasis and raises the exciting possibility that restoring HIF-2α pathways in lymphedema could mitigate long-term pathology and disability.

Oncogenic Herpesvirus Engages Endothelial Transcription Factors SOX18 and PROX1 to Increase Viral Genome Copies and Virus Production.

Kaposi sarcoma is a tumor caused by Kaposi sarcoma herpesvirus (KSHV) infection and is thought to originate from lymphatic endothelial cells (LEC). While KSHV establishes latency in virtually all susceptible cell types, LECs support spontaneous expression of oncogenic lytic genes, high viral genome copies, and release of infectious virus. It remains unknown the contribution of spontaneous virus production to the expansion of KSHV-infected tumor cells and the cellular factors that render the lymphatic environment unique to KSHV life cycle. We show here that expansion of the infected cell population, observed in LECs, but not in blood endothelial cells, is dependent on the spontaneous virus production from infected LECs. The drivers of lymphatic endothelium development, SOX18 and PROX1, regulated different steps of the KSHV life cycle. SOX18 enhanced the number of intracellular viral genome copies and bound to the viral origins of replication. Genetic depletion or chemical inhibition of SOX18 caused a decrease of KSHV genome copy numbers. PROX1 interacted with ORF50, the viral initiator of lytic replication, and bound to the KSHV genome in the promoter region of ORF50, increasing its transactivation activity and KSHV spontaneous lytic gene expression and infectious virus release. In Kaposi sarcoma tumors, SOX18 and PROX1 expression correlated with latent and lytic KSHV protein expression. These results demonstrate the importance of two key transcriptional drivers of LEC fate in the regulation of the tumorigenic KSHV life cycle. Moreover, they introduce molecular targeting of SOX18 as a potential novel therapeutic avenue in Kaposi sarcoma. SIGNIFICANCE: SOX18 and PROX1, central regulators of lymphatic development, are key factors for KSHV genome maintenance and lytic cycle in lymphatic endothelial cells, supporting Kaposi sarcoma tumorigenesis and representing attractive therapeutic targets.

Adrenomedullin - Current perspective on a peptide hormone with significant therapeutic potential.

The peptide hormone adrenomedullin (ADM) consists of 52 amino acids and plays a pivotal role in the regulation of many physiological processes, particularly those of the cardiovascular and lymphatic system. Like calcitonin (CT), calcitonin gene-related peptide (CGRP), intermedin (IMD) and amylin (AMY), it belongs to the CT/CGRP family of peptide hormones, which despite their low little sequence identity share certain characteristic structural features as well as a complex multicomponent receptor system. ADM, IMD and CGRP exert their biological effects by activation of the calcitonin receptor-like receptor (CLR) as a complex with one of three receptor activity-modifying proteins (RAMP), which alter the ligand affinity. Selectivity within the receptor system is largely mediated by the amidated C-terminus of the peptide hormones, which bind to the extracellular domains of the receptors. This enables their N-terminus consisting of a disulfide-bonded ring structure and a helical segment to bind within the transmembrane region and to induce an active receptor confirmation. ADM is expressed in a variety of tissues in the human body and is fundamentally involved in multitude biological processes. Thus, it is of interest as a diagnostic marker and a promising candidate for therapeutic interventions. In order to fully exploit the potential of ADM, it is necessary to improve its pharmacological profile by increasing the metabolic stability and, ideally, creating receptor subtype-selective analogs. While several successful attempts to prolong the half-life of ADM were recently reported, improving or even retaining receptor selectivity remains challenging.

Advanced drug delivery 2020 and beyond: Perspectives on the future.

Drug delivery systems are developed to maximize drug efficacy and minimize side effects. As drug delivery technologies improve, the drug becomes safer and more comfortable for patients to use. During the last seven decades, extraordinary progress has been made in drug delivery technologies, such as systems for long-term delivery for months and years, localized delivery, and targeted delivery. The advances, however, will face a next phase considering the future technologies we need to overcome many physicochemical barriers for new formulation development and biological unknowns for treating various diseases. For immediate and long-term progress into the future, the drug delivery field should use time and resources for more translatable research ideas. The drug delivery discipline has to continue working on basic, applied, translational, and clinical research in a concerted manner to produce drug delivery systems that work for patients. It is a time to focus our attention on things that matter. It is also a time to develop realistic research goals and outcomes, diversify drug delivery technologies, and take the collective responsibility for our actions.

Histopathologic Features of Lymphedema: A Molecular Review.

An estimated 5 million people in the United States are affected by secondary lymphedema, with most cases attributed to malignancies or malignancy-related treatments. The pathogenesis of secondary lymphedema has historically been attributed to lymphatic injury or dysfunction; however, recent studies illustrate the complexity of lymphedema as a disease process in which many of its clinical features such as inflammation, fibrosis, adipogenesis, and recurrent infections contribute to on-going lymphatic dysfunction in a vicious cycle. Investigations into the molecular underpinning of these features further our understanding of the pathophysiology of this disease and suggests new therapeutics.

Mechanisms of palmitic acid-conjugated antisense oligonucleotide distribution in mice.

Conjugation of antisense oligonucleotide (ASO) with a variety of distinct lipophilic moieties like fatty acids and cholesterol increases ASO accumulation and activity in multiple tissues. While lipid conjugation increases tissue exposure in mice and reduces excretion of ASO in urine, histological review of skeletal and cardiac muscle indicates that the increased tissue accumulation of lipid conjugated ASO is isolated to the interstitium. Administration of palmitic acid-conjugated ASO (Palm-ASO) in mice results in a rapid and substantial accumulation in the interstitium of muscle tissue followed by relatively rapid clearance and only slight increases in intracellular accumulation in myocytes. We propose a model whereby increased affinity for lipid particles, albumin, and other plasma proteins by lipid-conjugation facilitates ASO transport across endothelial barriers into tissue interstitium. However, this increased affinity for lipid particles and plasma proteins also facilitates the transport of ASO from the interstitium to the lymph and back into circulation. The cumulative effect is only a slight (∼2-fold) increase in tissue accumulation and similar increase in ASO activity. To support this proposal, we demonstrate that the activity of lipid conjugated ASO was reduced in two mouse models with defects in endothelial transport of macromolecules: caveolin-1 knockout (Cav1-/-) and FcRn knockout (FcRn-/-).