Intra-articular Injection of a B Cell Depletion Antibody Enhances Local Exposure to the Joint-Draining Lymph Node in Mice with Collagen-Induced Arthritis.

Changes to the number, type, and function of immune cells within the joint-draining lymphatics is a major contributor to the progression of inflammatory arthritis. In particular, there is a significant expansion in pathogenic B cells in the joint-draining lymph node (jdLN). These B cells appear to clog the lymphatic sinuses in the lymph node, inhibit lymph flow, and therefore, reduce the clearance of inflammatory fluid and cells from the joint. Taken together, there is potential to treat inflammatory arthritis more effectively, as well as reduce off-target side effects, with localized delivery of B-cell depleting therapies to the jdLNs. We recently reported that joint-draining lymphatic exposure of biologic disease-modifying anti-rheumatic drugs (DMARDs), including the B cell depletion antibody rituximab, is increased in healthy rats following intra-articular (IA) compared to subcutaneous (SC) or intravenous (IV) administration. This suggests that IA administration of B cell depleting antibodies may increase delivery to target cells in the jdLN and increase the effectiveness of B cell depletion compared to standard SC or IV administration. However, whether enhanced local delivery of DMARDs to the jdLN is also achieved after IA injection in the setting of inflammatory arthritis, where there is inflammation in the joint and jdLN B cell expansion is unknown. We, therefore, assessed the lymph node distribution, absorption and plasma pharmacokinetics, and B cell depletion at different sites after IA, SC, or IV administration of a fluorescently labeled mouse anti-CD20 B cell depleting antibody (Cy5-αCD20) in healthy mice compared to mice with collagen-induced arthritis (CIA). The absorption and plasma pharmacokinetics of Cy5-αCD20 appeared unaltered in mice with CIA whereas distribution of Cy5-αCD20 to the jdLNs was generally increased in mice with CIA, regardless of the route of administration. However, IA administration led to greater and more specific exposure to the jdLNs. Consistent with increased Cy5-αCD20 in the jdLNs of CIA compared to healthy mice, there was a greater reduction in jdLN weight and a trend toward greater jdLN B cell depletion at 24 h compared to 4 h after IA compared to SC and IV administration. Taken together, this data supports the potential to improve local efficacy of B cell depletion therapies through a jdLN-directed approach which will enable a reduction in dose and systemic toxicities.

Increasing Linker Chain Length and Intestinal Stability Enhances Lymphatic Transport and Lymph Node Exposure of Triglyceride Mimetic Prodrugs of a Model Immunomodulator Mycophenolic Acid.

Targeted delivery of immunomodulators to the lymphatic system has the potential to enhance therapeutic efficacy by increasing colocalization of drugs with immune targets such as lymphocytes. A triglyceride (TG)-mimetic prodrug strategy has been recently shown to enhance the lymphatic delivery of a model immunomodulator, mycophenolic acid (MPA), via incorporation into the intestinal TG deacylation-reacylation and lymph lipoprotein transport pathways. In the current study, a series of structurally related TG prodrugs of MPA were examined to optimize structure-lymphatic transport relationships for lymph-directing lipid-mimetic prodrugs. MPA was conjugated to the sn-2 position of the glyceride backbone of the prodrugs using linkers of different chain length (5-21 carbons) and the effect of methyl substitutions at the alpha and/or beta carbons to the glyceride end of the linker was examined. Lymphatic transport was assessed in mesenteric lymph duct cannulated rats, and drug exposure in lymph nodes was examined following oral administration to mice. Prodrug stability in simulated intestinal digestive fluid was also evaluated. Prodrugs with straight chain linkers were relatively unstable in simulated intestinal fluid; however, co-administration of lipase inhibitors (JZL184 and orlistat) was able to reduce instability and increase lymphatic transport (2-fold for a prodrug with a 6-carbon spacer, i.e., MPA-C6-TG). Methyl substitutions to the chain resulted in similar trends in improving intestinal stability and lymphatic transport. Medium- to long-chain spacers (C12, C15) between MPA and the glyceride backbone were most effective in promoting lymphatic transport, consistent with increases in lipophilicity. In contrast, short-chain (C6-C10) linkers appeared to be too unstable in the intestine and insufficiently lipophilic to associate with lymph lipid transport pathways, while very long-chain (C18, C21) linkers were also not preferred, likely as a result of increases in molecular weight reducing solubility or permeability. In addition to more effectively promoting drug transport into mesenteric lymph, TG-mimetic prodrugs based on a C12 linker resulted in marked increases (>40 fold) in the exposure of MPA in the mesenteric lymph nodes in mice when compared to administration of MPA alone, suggesting that optimizing prodrug design has the potential to provide benefit in targeting and modulating immune cells.

A lipid-anchored neurokinin 1 receptor antagonist prolongs pain relief by a three-pronged mechanism of action targeting the receptor at the plasma membrane and in endosomes.

G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NK1R) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NK1R antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NK1R signaling, and causes prolonged antinociception. However, the extent to which the Chol-anchor influences long-term location and activity is poorly understood. Herein, we used fluorescent correlation spectroscopy and targeted biosensors to characterize Span-Chol over time. The Chol-anchor increased local concentration of probe at the plasma membrane. Over time we observed an increase in NK1R-binding affinity and more potent inhibition of NK1R-mediated calcium signaling. Span-Chol, but not Span, caused a persistent decrease in NK1R recruitment of ß-arrestin and receptor internalization to early endosomes. Using targeted biosensors, we mapped the relative inhibition of NK1R signaling as the receptor moved into the cell. Span selectively inhibited cell surface signaling, whereas Span-Chol partitioned into endosomal membranes and blocked endosomal signaling. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a three-pronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NK1R endocytosis, and persistent inhibition of signaling from endosomes. Identifying the mechanisms that contribute to the increased preclinical efficacy of lipid-anchored NK1R antagonists is an important step toward understanding how we can effectively target intracellular GPCRs in disease.

Polymeric Nanotubes as Drug Delivery Vectors─Comparison of Covalently and Supramolecularly Assembled Constructs.

Rod-shaped nanoparticles have been identified as promising drug delivery candidates. In this report, the in vitro cell uptake and in vivo pharmacokinetic/bio-distribution behavior of molecular bottle-brush (BB) and cyclic peptide self-assembled nanotubes were studied in the size range of 36-41 nm in length. It was found that BB possessed the longest plasma circulation time (t1\2 > 35 h), with the cyclic peptide system displaying an intermediate half-life (14.6 h), although still substantially elevated over a non-assembling linear control (2.7 h). The covalently bound BB underwent substantial distribution into the liver, whereas the cyclic peptide nanotube was able to mostly circumvent organ accumulation, highlighting the advantage of the inherent degradability of the cyclic peptide systems through their reversible aggregation of hydrogen bonding core units.

Biocompatible Cationic Lipoamino Acids as Counterions for Oral Administration of API-Ionic Liquids.

PURPOSE: The use of ionic liquids (ILs) in drug delivery has focused attention on non-toxic IL counterions. Cationic lipids can be used to form ILs with weakly acidic drugs to enhance drug loading in lipid-based formulations (LBFs). However, cationic lipids are typically toxic. Here we explore the use of lipoaminoacids (LAAs) as cationic IL counterions that degrade or digest in vivo to non-toxic components. METHODS: LAAs were synthesised via esterification of amino acids with fatty alcohols to produce potentially digestible cationic LAAs. The LAAs were employed to form ILs with tolfenamic acid (Tol) and the Tol ILs loaded into LBF and examined in vitro and in vivo. RESULTS: Cationic LAAs complexed with Tol to generate lipophilic Tol ILs with high drug loading in LBFs. Assessment of the LAA under simulated digestion conditions revealed that they were susceptible to enzymatic degradation under intestinal conditions, forming biocompatible FAs and amino acids. In vitro dispersion and digestion studies of Tol ILs revealed that formulations containing digestible Tol ILs were able to maintain drug dispersion and solubilisation whilst the LAA were breaking down under digesting conditions. Finally, in vivo oral bioavailability studies demonstrated that oral delivery of a LBF containing a Tol IL comprising a digestible cationic lipid counterion was able to successfully support effective oral delivery of Tol. CONCLUSIONS: Digestible LAA cationic lipids are potential IL counterions for weakly acidic drug molecules and digest in situ to form non-toxic breakdown products.

Digestion of Lipid-Based Formulations Not Only Mediates Changes to Absorption of Poorly Soluble Drugs Due to Differences in Solubilization But Also Reflects Changes to Thermodynamic Activity and Permeability.

The aim of this study was to evaluate the effect of lipid digestion on the permeability and absorption of orally administered saquinavir (SQV), a biopharmaceutics classification system (BCS) class IV drug, in different lipid-based formulations. Three LBFs were prepared: a mixed short- and medium-chain lipid-based formulation (SMCF), a medium-chain lipid-based formulation (MCF), and a long-chain lipid-based formulation (LCF). SQV was loaded into these LBFs at 26.7 mg/g. To evaluate the pharmacokinetics of SQV in vivo, drug-loaded formulations were predispersed in purified water at 3% w/w and orally administered to rats. A low dose (0.8 mg/rat) was employed to limit confounding effects on drug solubilization, and consistent with this design, presolubilization of SQV in the LBFs did not increase in vivo exposure compared to a control suspension formulation. The areas under the plasma concentration-time curve were, however, significantly lower after administration of SQV as MCF and LCF compared to SMCF. To evaluate the key mechanisms underpinning absorption, each LBF containing SQV was digested, and the flux of SQV from the digests across a dialysis membrane was evaluated in in vitro permeation experiments. This study revealed that the absorption profiles were driven by the free concentration of SQV and that this varied due to differences in SQV solubilization in the digestion products generated by LBF digestion. The apparent first-order permeation rate constants of SQV (kapp,total) were estimated by dividing the flux of SQV in the dialysis membrane experiments by the concentration of total SQV on the donor side. kapp,total values strongly correlated with in vivo AUC. The data provide one of the first studies of the effect of digestion products on the free concentration of a drug in the GI fluid and oral absorption. This simple permeation model may be a useful tool for the evaluation of the impact of lipid digestion on apparent drug permeability from lipid-based formulations. These effects should be assessed alongside, and in addition to, the more well-known effects of lipids on enhancing intestinal solubilization of poorly water-soluble drugs.

Lipophilic Salts and Lipid-Based Formulations: Enhancing the Oral Delivery of Octreotide.

PURPOSE: Successful oral peptide delivery faces two major hurdles: low enzymatic stability in the gastro-intestinal lumen and poor intestinal membrane permeability. While lipid-based formulations (LBF) have the potential to overcome these barriers, effective formulation of peptides remains challenging. Lipophilic salt (LS) technology can increase the apparent lipophilicity of peptides, making them more suitable for LBF. METHODS: As a model therapeutic peptide, octreotide (OCT) was converted to the docusate LS (OCT.DoS2), and compared to the commercial acetate salt (OCT.OAc2) in oral absorption studies and related in vitro studies, including parallel artificial membrane permeability assay (PAMPA), Caco-2, in situ intestine perfusion, and simulated digestion in vitro models. The in vivo oral absorption of OCT.DoS2 and OCT.OAc2 formulated in self-emulsifying drug delivery systems (SEDDS) was studied in rats. RESULTS: LS formulation improved the solubility and loading of OCT in LBF excipients and OCT.DoS2 in combination with SEDDS showed higher OCT absorption than the acetate comparator in the in vivo studies in rats. The Caco-2 and in situ intestine perfusion models indicated no increases in permeability for OCT.DoS2. However, the in vitro digestion studies showed reduced enzymatic degradation of OCT.DoS2 when formulated in the SEDDS formulations. Further in vitro dissociation and release studies suggest that the enhanced bioavailability of OCT from SEDDS-incorporating OCT.DoS2 is likely a result of higher partitioning into and prolonged retention within lipid colloid structures. CONCLUSION: The combination of LS and LBF enhanced the in vivo oral absorption of OCT primarily via the protective effect of LBF sheltering the peptide from gastrointestinal degradation.

Molecular Dynamics Simulations and Experimental Results Provide Insight into Clinical Performance Differences between Sandimmune® and Neoral® Lipid-Based Formulations.

OBJECTIVE: Molecular dynamics (MD) simulations provide an in silico method to study the structure of lipid-based formulations (LBFs) and the incorporation of poorly water-soluble drugs within such formulations. In order to validate the ability of MD to effectively model the properties of LBFs, this work investigates the well-known cyclosporine A formulations, Sandimmune® and Neoral®. Sandimmune® exhibits poor dispersibility and its absorption from the gastrointestinal tract is enhanced when administered after food, whereas Neoral® disperses comparatively well and shows no food effect. METHODS: MD simulations were performed of both LBFs to investigate the differences observed in fasted and fed conditions. These conditions were also tested using an in vitro experimental model of dispersion and digestion. RESULTS: These MD simulations were able to show that the food effect observed for Sandimmune® can be explained by large changes in drug solubilization on addition of bile. In contrast, Neoral® is well dispersed in water or in simulated fasted conditions, and this dispersion is relatively unchanged on moving to fed conditions. These differences were confirmed using dispersion and digestion in vitro experimental model. CONCLUSIONS: The current data suggests that MD simulations are a potential method to model the fate of LBFs in the gastrointestinal tract, predict their dispersion and digestion, investigate behaviour of APIs within the formulations, and provide insights into the clinical performance of LBFs.

Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome.

Once activated at the surface of cells, G protein-coupled receptors (GPCRs) redistribute to endosomes, where they can continue to signal. Whether GPCRs in endosomes generate signals that contribute to human disease is unknown. We evaluated endosomal signaling of protease-activated receptor-2 (PAR2), which has been proposed to mediate pain in patients with irritable bowel syndrome (IBS). Trypsin, elastase, and cathepsin S, which are activated in the colonic mucosa of patients with IBS and in experimental animals with colitis, caused persistent PAR2-dependent hyperexcitability of nociceptors, sensitization of colonic afferent neurons to mechanical stimuli, and somatic mechanical allodynia. Inhibitors of clathrin- and dynamin-dependent endocytosis and of mitogen-activated protein kinase kinase-1 prevented trypsin-induced hyperexcitability, sensitization, and allodynia. However, they did not affect elastase- or cathepsin S-induced hyperexcitability, sensitization, or allodynia. Trypsin stimulated endocytosis of PAR2, which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR2, which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR2-dependent hyperexcitability of nociceptors, and PAR2 association with ß-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR2 A cholestanol-conjugated PAR2 antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR2 signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR2 antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases.

A ligand-induced structural change in fatty acid-binding protein 1 is associated with potentiation of peroxisome proliferator-activated receptor α agonists.

Peroxisome proliferator-activated receptor α (PPARα) is a transcriptional regulator of lipid metabolism. GW7647 is a potent PPARα agonist that must reach the nucleus to activate this receptor. In cells expressing human fatty acid-binding protein 1 (FABP1), GW7647 treatment increases FABP1's nuclear localization and potentiates GW7647-mediated PPARα activation; GW7647 is less effective in cells that do not express FABP1. To elucidate the underlying mechanism, here we substituted residues in FABP1 known to dictate lipid signaling by other intracellular lipid-binding proteins. Substitutions of Lys-20 and Lys-31 to Ala in the FABP1 helical cap affected neither its nuclear localization nor PPARα activation. In contrast, Ala substitution of Lys-57, Glu-77, and Lys-96, located in the loops adjacent to the ligand-binding portal region, abolished both FABP1 nuclear localization and GW7647-induced PPARα activation but had little effect on GW7647-FABP1 binding affinity. Using solution NMR spectroscopy, we determined the WT FABP1 structure and analyzed the dynamics in the apo and GW7647-bound structures of both the WT and the K57A/E77A/K96A triple mutant. We found that GW7647 binding causes little change in the FABP1 backbone, but solvent exposes several residues in the loops around the portal region, including Lys-57, Glu-77, and Lys-96. These residues also become more solvent-exposed upon binding of FABP1 with the endogenous PPARα agonist oleic acid. Together with previous observations, our findings suggest that GW7647 binding stabilizes a FABP1 conformation that promotes its interaction with PPARα. We conclude that full PPARα agonist activity of GW7647 requires FABP1-dependent transport and nuclear localization processes.

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.

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.

Quantifying In Vivo Luminal Drug Solubilization -Supersaturation-Precipitation Profiles to Explain the Performance of Lipid Based Formulations.

PURPOSE: To evaluate the role of supersaturation in the in vivo absorption of fenofibrate (FFB), after oral administration in a medium-chain lipid-based formulation (MCLBF). METHODS: FFB was loaded at 90% and 20% w/w of saturated solubility in MCLBF. The two formulations were pre-dispersed in purified water at 5% w/w (ME90% and 20%, respectively) and orally administered to rats to measure in vivo luminal drug concentrations. RESULTS: FFB precipitated in the stomach due to lipid digestion by gastric lipases and loss of solubilization capacity. This was most significant for ME90%. For ME90%, a high degree of supersaturation was also observed in the duodenum, however, precipitated FFB crystals rapidly re-dissolved. The combination of supersaturation and rapid re-dissolution appeared to drive effective absorption in the upper intestine. For ME20%, FFB precipitated in the stomach but not in the crystalline form and rapidly re-dissolved. Supersaturation in the duodenum again appeared to be the major driver of oral absorption. CONCLUSIONS: The data provide one of the first studies of in vivo luminal drug concentration, supersaturation and absorption from lipid based formulations and suggests that for FFB, whilst very high supersaturation may drive in vitro and in vivo precipitation, re-dissolution and drug absorption is rapid and efficient.

Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission.

G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8-37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8-37-cholestanol, but not unconjugated CGRP8-37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8-37-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP8-37 Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.

Pointing in the Right Direction: Controlling the Orientation of Proteins on Nanoparticles Improves Targeting Efficiency.

Protein-conjugated nanoparticles have the potential to precisely deliver therapeutics to target sites in the body by specifically binding to cell surface receptors. To maximize targeting efficiency, the three-dimensional presentation of ligands toward these receptors is crucial. Herein, we demonstrate significantly enhanced targeting of nanoparticles to cancer cells by controlling the protein orientation on the nanoparticle surface. To engineer the point of attachment, we used amber codon reassignment to incorporate a synthetic amino acid, p-azidophenylalanine (azPhe), at specific locations within a single domain antibody (sdAb or nanobody) that recognizes the human epidermal growth factor receptor (EGFR). The azPhe modified sdAb can be tethered to the nanoparticle in a specific orientation using a bioorthogonal click reaction with a strained cyclooctyne. The crystal structure of the sdAb bound to EGFR was used to rationally select sites likely to optimally display the sdAb upon conjugation to a fluorescent nanocrystal (Qdot). Qdots with sdAb attached at the azPhe13 position showed 6 times greater binding affinity to EGFR expressing A549 cells, compared to Qdots with conventionally (succinimidyl ester) conjugated sdAb. As ligand-targeted delivery systems move toward clinical application, this work shows that nanoparticle targeting can be optimized by engineering the site of protein conjugation.

Lymphatic Uptake of Liposomes after Intraperitoneal Administration Primarily Occurs via the Diaphragmatic Lymphatics and is Dependent on Liposome Surface Properties.

Drugs are commonly administered via the intraperitoneal (IP) route to treat localized infections and cancers in patients and to test drug efficacy and toxicity in preclinical studies. Despite this, there remain large gaps in our understanding of drug absorption routes (lymph vs blood) and pharmacokinetics following IP administration. This is particularly true when drugs are administered in complex delivery systems such as liposomes which are the main marketed formulation for several drugs that are administered intraperitoneally. This study investigated the impact of liposome surface properties (charge and PEGylation) on absorption into lymph and blood, and lymphatic disposition patterns, following IP administration. To achieve this, stable 3H-dipalmitoyl-phosphatidylcholine (DPPC) and 14C-sucrose-radiolabeled liposomes of 100-150 nm diameter with negative, neutral, or positive surface charge, or a PEGylated surface, were prepared and administered intraperitoneally to rats. Radiolabel concentrations were measured in lymph, blood, and lymph nodes (LNs). Lymph was collected from the thoracic lymph duct at either the abdomen (ABD) or the jugular-subclavian junction (JSJ). The lymphatic recovery of the radiolabels was substantially lower after administration in positively charged compared to the neutral, negative, or PEGylated liposomes. Radiolabel recovery was substantially greater (up to 18-fold) in the thoracic lymph collected at the JSJ when compared to that at the ABD, suggesting that liposomes entered the lymphatics at the diaphragm. Consistent with this, the concentration of the liposome labels was substantially higher (up to seven-fold) in mediastinal than in mesenteric LNs. Overall, this study shows how the peritoneal absorption and lymphatic disposition of drugs administered intraperitoneally can be manipulated through a careful selection of the drug delivery system and may thus be optimized to treat localized conditions such as cancers, infections, inflammatory diseases, and acute and critical illness.

Engineering Biocoatings To Prolong Drug Release from Supraparticles.

Supraparticles (SPs) assembled from smaller colloidal nanoparticles can serve as depots of therapeutic compounds and are of interest for long-term, sustained drug release in biomedical applications. However, a key challenge to achieving temporal control of drug release from SPs is the occurrence of an initial rapid release of the loaded drug (i.e., "burst" release) that limits sustained release and potentially causes burst release-associated drug toxicity. Herein, a biocoating strategy is presented for silica-SPs (Si-SPs) to reduce the extent of burst release of the loaded model protein lysozyme. Specifically, Si-SPs were coated with a fibrin film, formed by enzymatic conversion of fibrinogen into fibrin. The fibrin-coated Si-SPs, FSi-SPs, which could be loaded with 7.9 ± 0.9 µg of lysozyme per SP, released >60% of cargo protein over a considerably longer period of time of >20 days when compared with the uncoated Si-SPs that released the same amount of the cargo protein, however, within the first 3 days. Neurotrophins that support the survival and differentiation of neurons could also be loaded at ∼7.3 µg per SP, with fibrin coating also delaying neurotrophin release (only 10% of cargo released over 21 days compared with 60% from Si-SPs). In addition, the effects of incorporating a hydrogel-based system for surgical delivery and the opportunity to control drug release kinetics were investigated-an alginate-based hydrogel scaffold was used to encapsulate FSi-SPs. The introduction of the hydrogel further extended the initial release of the encapsulated lysozyme to ∼40 days (for the same amount of cargo released). The results demonstrate the increasing versatility of the SP drug delivery platform, combining large loading capacity with sustained drug release, that is tailorable using different modes of controlled delivery approaches.

Dietary docosahexaenoic acid supplementation enhances expression of fatty acid-binding protein 5 at the blood-brain barrier and brain docosahexaenoic acid levels.

The cytoplasmic trafficking of docosahexaenoic acid (DHA), a cognitively beneficial fatty acid, across the blood-brain barrier (BBB) is governed by fatty acid-binding protein 5 (FABP5). Lower levels of brain DHA have been observed in Alzheimer's disease (AD), which is associated with diminished BBB expression of FABP5. Therefore, up-regulating FABP5 expression at the BBB may be a novel approach for enhancing BBB transport of DHA in AD. DHA supplementation has been shown to be beneficial in various mouse models of AD, and therefore, the aim of this study was to determine whether DHA has the potential to up-regulate the BBB expression of FABP5, thereby enhancing its own uptake into the brain. Treating human brain microvascular brain endothelial (hCMEC/D3) cells with the maximum tolerable concentration of DHA (12.5 µM) for 72 h resulted in a 1.4-fold increase in FABP5 protein expression. Associated with this was increased expression of fatty acid transport proteins 1 and 4. To study the impact of dietary DHA supplementation, 6- to 8-week-old C57BL/6 mice were fed with a control diet or a DHA-enriched diet for 21 days. Brain microvascular FABP5 protein expression was up-regulated 1.7-fold in mice fed the DHA-enriched diet, and this was associated with increased brain DHA levels (1.3-fold). Despite an increase in brain DHA levels, reduced BBB transport of 14 C-DHA was observed over a 1 min perfusion, possibly as a result of competitive binding to FABP5 between dietary DHA and 14 C-DHA. This study has demonstrated that DHA can increase BBB expression of FABP5, as well as fatty acid transporters, overall increasing brain DHA levels.

Reduced blood-brain barrier expression of fatty acid-binding protein 5 is associated with increased vulnerability of APP/PS1 mice to cognitive deficits from low omega-3 fatty acid diets.

Lower levels of the cognitively beneficial docosahexaenoic acid (DHA) are often observed in Alzheimer's disease (AD) brains. Brain DHA levels are regulated by the blood-brain barrier (BBB) transport of plasma-derived DHA, a process facilitated by fatty acid-binding protein 5 (FABP5). This study reports a 42.1 ± 12.6% decrease in the BBB transport of 14 C-DHA in 8-month-old AD transgenic mice (APPswe,PSEN1∆E9) relative to wild-type mice, associated with a 34.5 ± 6.7% reduction in FABP5 expression in isolated brain capillaries of AD mice. Furthermore, short-term spatial and recognition memory deficits were observed in AD mice on a 6-month n-3 fatty acid-depleted diet, but not in AD mice on control diet. This intervention led to a dramatic reduction (41.5 ± 11.9%) of brain DHA levels in AD mice. This study demonstrates FABP5 deficiency and impaired DHA transport at the BBB are associated with increased vulnerability to cognitive deficits in mice fed an n-3 fatty acid-depleted diet, in line with our previous studies demonstrating a crucial role of FABP5 in BBB transport of DHA and cognitive function.