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.

Corrigendum: Triglyceride-mimetic prodrugs of buprenorphine enhance oral bioavailability via promotion of lymphatic transport.

[This corrects the article DOI: 10.3389/fphar.2022.879660.].

Triglyceride-Mimetic Prodrugs of Buprenorphine Enhance Oral Bioavailability via Promotion of Lymphatic Transport.

Buprenorphine (BUP) is a potent opioid analgesic that is widely used for severe pain management and opioid replacement therapy. The oral bioavailability of BUP, however, is significantly limited by first-pass metabolism. Previous studies have shown that triglyceride (TG) mimetic prodrugs of the steroid hormone testosterone circumvent first-pass metabolism by directing drug transport through the intestinal lymphatics, bypassing the liver. The current study expanded this prodrug strategy to BUP. Here different self-immolative (SI) linkers were evaluated to conjugate BUP to the 2 position of the TG backbone via the phenol group on BUP. The SI linkers were designed to promote drug release in plasma. Lipolysis of the prodrug in the intestinal tract was examined via incubation with simulated intestinal fluid (SIF), and potential for parent drug liberation in the systemic circulation was evaluated via incubation in rat plasma. Lymphatic transport and bioavailability studies were subsequently conducted in mesenteric lymph duct or carotid artery-cannulated rats, respectively. TG prodrug derivatives were efficiently transported into the lymphatics (up to 45% of the dose in anaesthetised rats, vs. less than 0.1% for BUP). Incorporation of the SI linkers facilitated BUP release from the prodrugs in the plasma and in concert with high lymphatic transport led to a marked enhancement in oral bioavailability (up to 22-fold) compared to BUP alone. These data suggest the potential to develop an orally bioavailable BUP product which may have advantages with respect to patient preference when compared to current sublingual, transdermal patch or parenteral formulations.

Mesenteric lymphatic dysfunction promotes insulin resistance and represents a potential treatment target in obesity.

Visceral adipose tissue (VAT) encases mesenteric lymphatic vessels and lymph nodes through which lymph is transported from the intestine and mesentery. Whether mesenteric lymphatics contribute to adipose tissue inflammation and metabolism and insulin resistance is unclear. Here we show that obesity is associated with profound and progressive dysfunction of the mesenteric lymphatic system in mice and humans. We find that lymph from mice and humans consuming a high-fat diet (HFD) stimulates lymphatic vessel growth, leading to the formation of highly branched mesenteric lymphatic vessels that 'leak' HFD-lymph into VAT and, thereby, promote insulin resistance. Mesenteric lymphatic dysfunction is regulated by cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF)-C-VEGF receptor (R)3 signalling. Lymph-targeted inhibition of COX-2 using a glyceride prodrug approach reverses mesenteric lymphatic dysfunction, visceral obesity and inflammation and restores glycaemic control in mice. Targeting obesity-associated mesenteric lymphatic dysfunction thus represents a potential therapeutic option to treat metabolic disease.

Targeted delivery of mycophenolic acid to the mesenteric lymph node using a triglyceride mimetic prodrug approach enhances gut-specific immunomodulation in mice.

The mesenteric lymph nodes (MLN) are a key site for the generation of adaptive immune responses to gut-derived antigenic material and immune cells within the MLN contribute to the pathophysiology of a range of conditions including inflammatory and autoimmune diseases, viral infections, graft versus host disease and cancer. Targeting immunomodulating drugs to the MLN may thus be beneficial in a range of conditions. This paper investigates the potential benefit of targeting a model immunosuppressant drug, mycophenolic acid (MPA), to T cells in the MLN, using a triglyceride (TG) mimetic prodrug approach. We confirmed that administration of MPA in the TG prodrug form (MPA-TG), increased lymphatic transport of MPA-related species 83-fold and increased MLN concentrations of MPA >20 fold, when compared to MPA alone, for up to 4 h in mice. At the same time, the plasma exposure of MPA and MPA-TG was similar, limiting the opportunity for systemic side effects. Confocal microscopy and flow cytometry studies with a fluorescent model prodrug (Bodipy-TG) revealed that the prodrug accumulated in the MLN cortex and paracortex at 5 and 10 h following administration and was highly associated with B cells and T cells that are found in these regions of the MLN. Finally, we demonstrated that MPA-TG was significantly more effective than MPA at inhibiting CD4+ and CD8+ T cell proliferation in the MLN of mice in response to an oral ovalbumin antigen challenge. In contrast, MPA-TG was no more effective than MPA at inhibiting T cell proliferation in peripheral LN when mice were challenged via SC administration of ovalbumin. This paper provides the first evidence of an in vivo pharmacodynamic benefit of targeting the MLN using a TG mimetic prodrug approach. The TG mimetic prodrug technology has the potential to benefit the treatment of a range of conditions where aberrant immune responses are initiated in gut-associated lymphoid tissues.

The Impact of Conjugation Position and Linker Chemistry on the Lymphatic Transport of a Series of Glyceride and Phospholipid Mimetic Prodrugs.

Drug delivery to the lymphatic system is gaining increasing attention, particularly in fields such as immunotherapy where drug access to lymphocytes is central to activity. We have previously described a prodrug strategy that facilitates the lymphatic delivery of a model immunomodulator, mycophenolic acid (MPA) via incorporation into intestinal triglyceride transport pathways. The current study explored a series of structurally related glyceride and phospholipid mimetic prodrugs of MPA in an attempt to enhance lymph targeting and to better elucidate the design criteria for lipid mimetic prodrugs. MPA was conjugated to a glyceride or phospholipid backbone at various positions using different spacers employing ester, ether, carbonate and amide bonds. Patterns of prodrug hydrolysis were evaluated in rat digestive fluid, and lymphatic transport and plasma pharmacokinetics were assessed in lymph duct cannulated rats. Prodrugs with different spacers between MPA and the glyceride backbone resulted in up to 70-fold differences in gastrointestinal stability. MPA conjugation at the 2 position of the glyceride backbone and via an ester bond were most effective in promoting lymphatic transport. Phospholipid prodrug derivatives, or glyceride derivatives with MPA attached at the 1 position or when linked via ether, carbonate or amide bonds were poorly incorporated into lymphatic transport pathways.

Promoting intestinal lymphatic transport targets a liver-X receptor (LXR) agonist (WAY-252,623) to lymphocytes and enhances immunomodulation.

Lymphocytes play a central role in the pathology of a range of chronic conditions such as autoimmune disease, transplant rejection, leukemia, lymphoma HIV/AIDs and cardiometabolic diseases such as atherosclerosis. Current treatments for lymphocyte-associated conditions are incompletely effective and/or complicated by a range of off-target toxicities. One major challenge is poor drug access to lymphocytes via the systemic blood and this may be attributed, at least in part, to the fact that lymphocytes are concentrated within lymph fluid and lymphoid tissues, particularly in gut-associated lymphatics. Here we demonstrate that promoting drug uptake into the intestinal lymphatics with a long chain fatty acid, thereby increasing lymphocyte access, enhances the pharmacodynamic effect of a highly lipophilic liver X receptor (LXR) agonist, WAY-252623, that has been suggested as a potential treatment for atherosclerosis. This has been exemplified by: (1) increased mRNA expression of key markers of LXR activation (ABCA1) and regulatory T cells (Foxp3) in local lymphatic lymphocytes and (2) enhanced numbers of CD4+CD25+Foxp3+ regulatory T cells in the systemic circulation, after administration of a 5-fold lower dose with a lymph directing lipid formulation when compared with a non-lipid containing formulation. These data suggest that combining lipophilic, lymphotropic drug candidates such as WAY-252,623, with lymph-directing long chain lipid based formulations can enhance drug targeting to, and activity on, lymphocytes in lymph and that this effect persists through to the systemic circulation. This presents a promising approach to achieve more selective and effective therapeutic outcomes for the treatment of lymphocyte associated diseases.

Lymphatic Transport and Lymphocyte Targeting of a Triglyceride Mimetic Prodrug Is Enhanced in a Large Animal Model: Studies in Greyhound Dogs.

In previous studies, a triglyceride (TG) mimetic prodrug of the model immunomodulator mycophenolic acid (MPA) was shown to significantly enhance lymphatic transport of MPA-related species in the rat. The rat gastrointestinal tract, however, is somewhat different from that in higher order species such as dogs and humans and may underestimate lymphatic transport. Here the effectiveness of the prodrug strategy has been examined in conscious greyhound dogs, the GI physiology of which is more representative of that in humans. The bioavailability and lymphatic transport of free MPA and total MPA related materials were examined following oral administration of the parent drug (MPA) and the prodrug (2-MPA-TG) to both thoracic lymph duct cannulated and intact (noncannulated) greyhound dogs. The enrichment of free MPA in lymph nodes and lymph-derived lymphocytes was also determined to examine the efficiency of drug targeting to potential sites of action within the lymph. Via biochemical integration into a series of site-specific metabolic processes, the prodrug markedly increased (288-fold) lymphatic transport of total MPA related material (present as re-esterified 2-MPA-TG) when compared to the parent MPA and the extent of lymphatic transport was significantly greater in the dog (36.4% of the dose recovered in lymph) when compared to the previous data in the rat (13.4% of the dose). Conversion from 2-MPA-TG derivatives to parent MPA occurred in vivo, resulting in a marked increase in MPA concentrations in lymph nodes (5-6-fold) and lymph lymphocytes (21-fold), when compared to animals administered the parent drug. In conclusion, the data demonstrate that the TG prodrug of MPA facilitates efficient delivery of MPA to the lymphatic system in dogs and suggest that the TG prodrug strategy may more effectively facilitate targeted delivery in large animals than in rats.

Glyceride-Mimetic Prodrugs Incorporating Self-Immolative Spacers Promote Lymphatic Transport, Avoid First-Pass Metabolism, and Enhance Oral Bioavailability.

First-pass hepatic metabolism can significantly limit oral drug bioavailability. Drug transport from the intestine through the lymphatic system, rather than the portal vein, circumvents first-pass metabolism. However, the majority of drugs do not have the requisite physicochemical properties to facilitate lymphatic access. Herein, we describe a prodrug strategy that promotes selective transport through the intestinal lymph vessels and subsequent release of drug in the systemic circulation, thereby enhancing oral bioavailability. Using testosterone (TST) as a model high first-pass drug, glyceride-mimetic prodrugs incorporating self-immolative (SI) spacers, resulted in remarkable increases (up to 90-fold) in TST plasma exposure when compared to the current commercial product testosterone undecanoate (TU). This approach opens new opportunities for the effective development of drugs where oral delivery is limited by first-pass metabolism and provides a new avenue to enhance drug targeting to intestinal lymphoid tissue.

Constitutive Triglyceride Turnover into the Mesenteric Lymph Is Unable to Support Efficient Lymphatic Transport of a Biomimetic Triglyceride Prodrug.

The triglyceride (TG) mimetic prodrug (1,3-dipalmitoyl-2-mycophenoloyl glycerol, 2-MPA-TG) biochemically integrates into intestinal lipid transport and lipoprotein assembly pathways and thereby promotes the delivery of mycophenolic acid (MPA) into the lymphatic system. As lipoprotein (LP) formation occurs constitutively, even in the fasted state, the current study aimed to determine whether lymphatic transport of 2-MPA-TG was dependent on coadministered exogenous lipid. In vitro incubation of the prodrug with rat digestive fluid and in situ intestinal perfusion experiments revealed that hydrolysis and absorption of the prodrug were relatively unaffected by the quantity of lipid in formulations. In vivo studies in rats, however, showed that the lymphatic transport of TG and 2-MPA-TG was significantly higher following administration with higher quantities of lipid and that oleic acid (C18:1) was more effective in promoting prodrug transport than lipids with higher degrees of unsaturation. The recovery of 2-MPA-TG and TG in lymph correlated strongly (R(2) = 0.99) and more than 97% of the prodrug was associated with chylomicrons. Inhibition of LP assembly by Pluronic L81 simultaneously inhibited the lymphatic transport of 2-MPA-TG and TG. In conclusion, although the TG mimetic prodrug effectively incorporates into TG resynthetic pathways, lipid coadministration is still required to support efficient lymphatic transport.

The mesenteric lymph duct cannulated rat model: application to the assessment of intestinal lymphatic drug transport.

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Profiling the role of deacylation-reacylation in the lymphatic transport of a triglyceride-mimetic prodrug.

PURPOSE: Recent studies have demonstrated the potential for a triglyceride (TG) mimetic prodrug to promote the delivery of mycophenolic acid (MPA) to the lymphatic system. Here, the metabolic pathways that facilitate the lymphatic transport of the TG prodrug (1,3-dipalmitoyl-2-mycophenoloyl glycerol, 2-MPA-TG) were examined to better inform the design of next generation prodrugs. METHODS: In vitro hydrolysis experiments in simulated intestinal conditions and in vivo rat lymphatic transport experiments were conducted in the presence and absence of orlistat and A922500 (inhibitors of lipolysis and TG re-esterification, respectively), to evaluate the importance of 2-MPA-TG digestion and re-esterification of 2-MPA-MG (the 2-monoglyceride derivative) in promoting lymphatic transport. RESULTS: 2-MPA-TG was rapidly hydrolysed to 2-MPA-MG on incubation with fresh bile and pancreatic fluid (BPF), but not in simulated gastric fluid, heat-inactivated BPF or BPF + orlistat. Orlistat markedly decreased lymphatic transport and systemic exposure of 2-MPA-TG derivatives suggesting that inhibition of pancreatic lipase hindered luminal digestion and absorption of the prodrug. A922500 also significantly decreased lymphatic transport of 2-MPA-TG but redirected MPA to the portal blood, suggesting that hindered re-acylation of 2-MPA-MG resulted in intracellular degradation. CONCLUSION: Incorporation into TG deacylation-reacylation pathways is a critical determinant of the utility of lymph directed TG-mimetic prodrugs.

Targeted delivery of a model immunomodulator to the lymphatic system: comparison of alkyl ester versus triglyceride mimetic lipid prodrug strategies.

A lipophilic prodrug approach has been used to promote the delivery of a model immunomodulator, mycophenolic acid (MPA), to the lymphatic system after oral administration. Lymphatic transport was employed to facilitate enhanced drug uptake into lymphocytes, as recent studies demonstrate that targeted drug delivery to lymph resident lymphocytes may enhance immunomodulatory effects. Two classes of lymph-directing prodrugs were synthesised. Alkyl chain derivatives (octyl mycophenolate, MPA-C8E; octadecyl mycophenolate, MPA-C18E; and octadecyl mycophenolamide, MPA-C18AM), to promote passive partitioning into lipids in lymphatic transport pathways, and a triglyceride mimetic prodrug (1,3-dipalmitoyl-2-mycophenoloyl glycerol, 2-MPA-TG) to facilitate metabolic integration into triglyceride deacylation-reacylation pathways. Lymphatic transport, lymphocyte uptake and plasma pharmacokinetics were assessed in mesenteric lymph and carotid artery cannulated rats following intraduodenal infusion of lipid-based formulations containing MPA or MPA prodrugs. Patterns of prodrug hydrolysis in rat digestive fluid, and cellular re-esterification in vivo, were evaluated to examine the mechanisms responsible for lymphatic transport. Poor enzyme stability and low absorption appeared to limit lymphatic transport of the alkyl derivatives, although two of the three alkyl chain prodrugs - MPA-C18AM (6-fold) and MPA-C18E (13-fold) still increased lymphatic drug transport when compared to MPA. In contrast, 2-MPA-TG markedly increased lymphatic drug transport (80-fold) and drug concentrations in lymphocytes (103-fold), and this was achieved via biochemical incorporation into triglyceride deacylation-reacylation pathways. The prodrug was hydrolysed rapidly to 2-mycophenoloyl glycerol (2-MPA-MG) in the presence of rat digestive fluid, and 2-MPA-MG was subsequently re-esterified in the enterocyte with oleic acid (most likely originating from the co-administered formulation) prior to accessing the lymphatics and lymphocytes. Importantly, after administration of 2-MPA-TG, the concentrations of free MPA in the mesenteric lymph nodes were significantly enhanced (up to 28 fold) when compared to animals administered equimolar quantities of MPA, suggesting the efficient conversion of the esterified prodrug back to the pharmacologically active parent drug. The data suggest that triglyceride mimetic prodrugs have potential as a means of enhancing immunotherapy via drug targeting to lymphocytes and lymph nodes.

The impact of lymphatic transport on the systemic disposition of lipophilic drugs.

This work investigates the influence of drug absorption route (intestinal lymphatics vs. blood supply) on drug pharmacokinetics and tissue distribution. To achieve this aim, the pharmacokinetics and tissue distribution of model compounds [1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane, DDT; halofantrine] and lipids were assessed following intravenous delivery in lymph lipoproteins or plasma, and were found to differ significantly. For DDT, the clearance (CL) and volume of distribution (Vd ) were higher, whereas for halofantrine, CL and V(d) were lower, after entry in lymph versus plasma due, in particular, to differences in adipose tissue and liver uptake. In a recent study, halofantrine CL and V(d) were similar following entry in lymph or entry in plasma into the systemic circulation of animals predosed with lymph, whereas in the current study, predosing lymph did not influence DDT CL and V(d). For compounds such as DDT, changes to the route of absorption may thus directly impact on pharmacokinetics and tissue distribution, whereas for halofantrine factors that influence lymphatic transport may, by altering systemic lipoprotein concentrations, indirectly impact pharmacokinetics and tissue distribution. Ultimately, careful control of dosing conditions (formulation, prandial state), and thus the extent of lymphatic transport, may be important in assuring reproducible efficacy and toxicity for lymphatically transported drugs.

Transferrin-modified c[RGDfK]-paclitaxel loaded hybrid micelle for sequential blood-brain barrier penetration and glioma targeting therapy.

The effective chemotherapy for glioblastoma multiform (GBM) requires a nanomedicine that can both penetrate the blood-brain barrier (BBB) and target the glioma cells subsequently. In this study, Transferrin (Tf) modified cyclo-[Arg-Gly-Asp-d-Phe-Lys] (c[RGDfK])-paclitaxel conjugate (RP) loaded micelle (TRPM) was prepared and evaluated for its targeting efficiency, antiglioma activity, and toxicity in vitro and in vivo. Tf modification significantly enhanced the cellular uptake of TRPM by primary brain microvascular endothelial cells (BMEC) to 2.4-fold of RP loaded micelle (RPM) through Tf receptor mediated endocytosis, resulting in a high drug accumulation in the brain after intravenous injection.The c[RGDfK] modified paclitaxel (PTX) was released from micelle subsequently and targeted to integrin overexpressed glioma cells in vitro, and showed significantly prolonged retention in glioma tumor and peritumoral tissue. Most importantly, TRPM exhibited the strongest antiglioma activity, as the mean survival time of mice bearing intracranial U-87 MG glioma treated with TRPM (42.8 days) was significantly longer than those treated with Tf modified PTX loaded micelle (TPM) (39.5 days), PTX loaded micelle (PM) (34.8 days), Taxol (33.6 days), and saline (34.5 days). Noteworthy, TRPM did not lead to body weight loss compared with saline and was less toxic than TPM. These results indicated that TRPM could be a promising nanomedicine for glioma chemotherapy.

Transferrin-conjugated polyphosphoester hybrid micelle loading paclitaxel for brain-targeting delivery: synthesis, preparation and in vivo evaluation.

The successful treatment of central nervous system (CNS) disorders is hampered by inefficient drug delivery across the blood-brain barrier (BBB). Transferrin (Tf) could facilitate the transcytosis of coupled nanocarriers through Tf receptor (TfR) mediated pathway. In this study, Tf-modified paclitaxel-loaded polyphosphoester hybrid micells (TPM) was prepared and evaluated for its in vitro and in vivo brain-targeting efficiency. The polyphosphoester hybrid micelle formed a core-shell structure in aqueous solution, and demonstrated high drug entrapping efficiency (89.9±3.4%). In addition, the micelle showed negligible hemolysis even at 2.0 mg/mL. The TPM was 87.85±2.32 nm with ζ potentials -12.33±1.46 mV, and PTX showed sustained release from TPM. TPM demonstrated enhanced cellular uptake and brain accumulation, which were 2 and 1.8-fold of PM, respectively. TPM exhibited strongest anti-glioma activity, and the mean survival time of mice bearing intracranial U-87 MG glioma treated with TPM (39.5 days) was significantly longer than those treated with Taxol® (33.6 days). These results indicated that Tf conjugated micelle could be a promising carrier for brain-targeting drug delivery.

Poly(ε-caprolactone)-block-poly(ethyl ethylene phosphate) micelles for brain-targeting drug delivery: in vitro and in vivo valuation.

PURPOSE: The purpose of this work was to investigate the potential of poly(ε-caprolactone)-block-poly(ethyl ethylene phosphate) (PCL-PEEP) micelles for brain-targeting drug delivery. METHOD: The coumarin-6-loaded PCL-PEEP micelles (CMs) were prepared and characterized. The cellular uptake of CMs was evaluated on in vitro model of brain-blood barrier (BBB), and the brain biodistribution of CMs in ICR mice was investigated. RESULTS: PCL-PEEP could self-assemble into 20 nm micelles in water with the critical micelle concentration (CMC) 0.51 µg/ml and high coumarin-6 encapsulation efficiency (92.5 ± 0.7%), and the micelles were stable in 10% FBS with less than 25% leakage of incorporated coumarin-6 during 24 h incubation at 37°C. The cellular uptake of CMs by BBB model was significantly higher and more efficient than coumarin-6 solution (CS) at 50 ng/ml. Compared with CS, 2.6-fold of coumarin-6 was found in the brains of CM-treated mice, and C(max) of CMs was 4.74% of injected dose/g brain. The qualitative investigation on the brain distribution of CMs indicated that CMs were prone to accumulate in hippocampus and striatum. CONCLUSION: These results suggest that PCL-PEEP micelles could be a promising brain-targeting drug delivery system with low toxicity.