Prediction of blood-brain barrier penetrating peptides based on data augmentation with Augur.

BACKGROUND: The blood-brain barrier serves as a critical interface between the bloodstream and brain tissue, mainly composed of pericytes, neurons, endothelial cells, and tightly connected basal membranes. It plays a pivotal role in safeguarding brain from harmful substances, thus protecting the integrity of the nervous system and preserving overall brain homeostasis. However, this remarkable selective transmission also poses a formidable challenge in the realm of central nervous system diseases treatment, hindering the delivery of large-molecule drugs into the brain. In response to this challenge, many researchers have devoted themselves to developing drug delivery systems capable of breaching the blood-brain barrier. Among these, blood-brain barrier penetrating peptides have emerged as promising candidates. These peptides had the advantages of high biosafety, ease of synthesis, and exceptional penetration efficiency, making them an effective drug delivery solution. While previous studies have developed a few prediction models for blood-brain barrier penetrating peptides, their performance has often been hampered by issue of limited positive data. RESULTS: In this study, we present Augur, a novel prediction model using borderline-SMOTE-based data augmentation and machine learning. we extract highly interpretable physicochemical properties of blood-brain barrier penetrating peptides while solving the issues of small sample size and imbalance of positive and negative samples. Experimental results demonstrate the superior prediction performance of Augur with an AUC value of 0.932 on the training set and 0.931 on the independent test set. CONCLUSIONS: This newly developed Augur model demonstrates superior performance in predicting blood-brain barrier penetrating peptides, offering valuable insights for drug development targeting neurological disorders. This breakthrough may enhance the efficiency of peptide-based drug discovery and pave the way for innovative treatment strategies for central nervous system diseases.

Penetration of Organophosphate Triesters and Diesters across the Blood-Cerebrospinal Fluid Barrier: Efficiencies, Impact Factors, and Mechanisms.

The penetration of organophosphate triesters (tri-OPEs) and diesters (di-OPEs) across the blood-brain barrier and their influencing factors remain unclear in humans. In this study, 21 tri-OPEs and 8 di-OPEs were measured in 288 paired serum and cerebrospinal fluid (CSF) samples collected in Jinan, China. Six tri-OPEs were frequently detected in both serum and CSF, with median concentrations ranging from 0.062 to 1.62 and 0.042-1.11 ng/mL, respectively. Their penetration efficiencies across the blood-CSF barrier (BCSFB) (RCSF/serum, CCSF/Cserum) were calculated at 0.667-2.80, and these efficiencies first increased and then decreased with their log Kow values. The reduced penetration efficiencies of triphenyl phosphate (TPHP) and 2-ethylhexyl diphenyl phosphate (EHDPP) may be attributed to their strong binding affinities for human serum albumin and p-glycoprotein due to their high hydrophobicity and aryl structure, as indicated by molecular docking. This suggests that active efflux transport may be involved in the penetration of TPHP and EHDPP in addition to passive diffusion similar to the other four tri-OPEs. Di-OPEs were found in few serum samples and even fewer CSF samples, indicating their limited BCSFB permeability. This may be due to their high polarity, low hydrophobicity, and ionic state in blood. This study has important implications for understanding the neurotoxicity of tri-OPEs and di-OPEs and the underlying mechanisms.

Modernistic and Emerging Developments of Nanotechnology in Glioblastoma-Targeted Theranostic Applications.

Brain tumors such as glioblastoma are typically associated with an unstoppable cell proliferation with aggressive infiltration behavior and a shortened life span. Though treatment options such as chemotherapy and radiotherapy are available in combating glioblastoma, satisfactory therapeutics are still not available due to the high impermeability of the blood-brain barrier. To address these concerns, recently, multifarious theranostics based on nanotechnology have been developed, which can deal with diagnosis and therapy together. The multifunctional nanomaterials find a strategic path against glioblastoma by adjoining novel thermal and magnetic therapy approaches. Their convenient combination of specific features such as real-time tracking, in-depth tissue penetration, drug-loading capacity, and contrasting performance is of great demand in the clinical investigation of glioblastoma. The potential benefits of nanomaterials including specificity, surface tunability, biodegradability, non-toxicity, ligand functionalization, and near-infrared (NIR) and photoacoustic (PA) imaging are sufficient in developing effective theranostics. This review discusses the recent developments in nanotechnology toward the diagnosis, drug delivery, and therapy regarding glioblastoma.

Central Nervous System Stimulants Limit Caffeine Transport at the Blood-Cerebrospinal Fluid Barrier.

Caffeine, a common ingredient in energy drinks, crosses the blood-brain barrier easily, but the kinetics of caffeine across the blood-cerebrospinal fluid barrier (BCSFB) has not been investigated. Therefore, 127 autopsy cases (Group A, 30 patients, stimulant-detected group; and Group B, 97 patients, no stimulant detected group) were examined. In addition, a BCSFB model was constructed using human vascular endothelial cells and human choroid plexus epithelial cells separated by a filter, and the kinetics of caffeine in the BCSFB and the effects of 4-aminopyridine (4-AP), a neuroexcitatory agent, were studied. Caffeine concentrations in right heart blood (Rs) and cerebrospinal fluid (CSF) were compared in the autopsy cases: caffeine concentrations were higher in Rs than CSF in Group A compared to Group B. In the BCSFB model, caffeine and 4-AP were added to the upper layer, and the concentration in the lower layer of choroid plexus epithelial cells was measured. The CSF caffeine concentration was suppressed, depending on the 4-AP concentration. Histomorphological examination suggested that choroid plexus epithelial cells were involved in inhibiting the efflux of caffeine to the CSF. Thus, the simultaneous presence of stimulants and caffeine inhibits caffeine transfer across the BCSFB.

Activated leukocyte cell adhesion molecule promotes leukocyte trafficking into the central nervous system.

Adhesion molecules of the immunoglobulin superfamily are crucial effectors of leukocyte trafficking into the central nervous system. Using a lipid raft-based proteomic approach, we identified ALCAM as an adhesion molecule involved in leukocyte migration across the blood-brain barrier (BBB). ALCAM expressed on BBB endothelium localized together with CD6 on leukocytes and with BBB endothelium transmigratory cups. ALCAM expression on BBB cells was upregulated in active multiple sclerosis and experimental autoimmune encephalomyelitis lesions. Moreover, ALCAM blockade restricted the transmigration of CD4+ lymphocytes and monocytes across BBB endothelium in vitro and in vivo and reduced the severity and delayed the time of onset of experimental autoimmune encephalomyelitis. Our findings indicate an important function for ALCAM in the recruitment of leukocytes into the brain and identify ALCAM as a potential target for the therapeutic dampening of neuroinflammation.

Fluorescently-labeled fremanezumab is distributed to sensory and autonomic ganglia and the dura but not to the brain of rats with uncompromised blood brain barrier.

BACKGROUND: The presence of calcitonin gene-related peptide and its receptors in multiple brain areas and peripheral tissues previously implicated in migraine initiation and its many associated symptoms raises the possibility that humanized monoclonal anti-calcitonin gene-related peptide antibodies (CGRP-mAbs) can prevent migraine by modulating neuronal behavior inside and outside the brain. Critical to our ability to conduct a fair discussion over the mechanisms of action of CGRP-mAbs in migraine prevention is data generation that determines which of the many possible peripheral and central sites are accessible to these antibodies - a question raised frequently due to their large size. MATERIAL AND METHODS: Rats with uncompromised and compromised blood-brain barrier (BBB) were injected with Alexa Fluor 594-conjugated fremanezumab (Frema594), sacrificed 4 h or 7 d later, and relevant tissues were examined for the presence of Frema594. RESULTS: In rats with uncompromised BBB, Frema594 was similarly observed at 4 h and 7 d in the dura, dural blood vessels, trigeminal ganglion, C2 dorsal root ganglion, the parasympathetic sphenopalatine ganglion and the sympathetic superior cervical ganglion but not in the spinal trigeminal nucleus, thalamus, hypothalamus or cortex. In rats with compromised BBB, Frema594 was detected in the cortex (100 µm surrounding the compromised BBB site) 4 h but not 7 d after injections. DISCUSSION: Our inability to detect fluorescent (CGRP-mAbs) in the brain supports the conclusion that CGRP-mAbs prevent the headache phase of migraine by acting mostly, if not exclusively, outside the brain as the amount of CGRP-mAbs that enters the brain (if any) is too small to be physiologically meaningful.

Predicting the Blood-Brain Barrier Permeability of New Drug-Like Compounds via HPLC with Various Stationary Phases.

The permeation of the blood-brain barrier is a very important consideration for new drug candidate molecules. In this research, the reversed-phase liquid chromatography with different columns (Purosphere RP-18e, IAM.PC.DD2 and Cosmosil Cholester) was used to predict the penetration of the blood-brain barrier by 65 newly-synthesized drug-like compounds. The linear free energy relationships (LFERs) model (log BB = c + eE + sS + aA + bB + vV) was established for a training set of 23 congeneric biologically active azole compounds with known experimental log BB (BB = Cblood/Cbrain) values (R2 = 0.9039). The reliability and predictive potency of the model were confirmed by leave-one-out cross validation as well as leave-50%-out cross validation. Multiple linear regression (MLR) was used to develop the quantitative structure-activity relationships (QSARs) to predict the log BB values of compounds that were tested, taking into account the chromatographic lipophilicity (log kw), polarizability and topological polar surface area. The excellent statistics of the developed MLR equations (R2 > 0.8 for all columns) showed that it is possible to use the HPLC technique and retention data to produce reliable blood-brain barrier permeability models and to predict the log BB values of our pharmaceutically important molecules.

A Near-Infrared-Controllable Artificial Metalloprotease Used for Degrading Amyloid-ß Monomers and Aggregates.

Proteolysis of amyloid-ß (Aß) is a promising approach against Alzheimer's disease. However, it is not feasible to employ natural hydrolases directly because of their cumbersome preparation and purification, poor stability, and hazardous immunogenicity. Therefore, artificial enzymes have been developed as potential alternatives to natural hydrolases. Since specific cleavage sites of Aß are usually embedded inside the ß-sheet structures that restrict access by artificial enzymes, this strongly hinders their efficiency for practical applications. Herein, we construct a NIR (near-IR) controllable artificial metalloprotease (MoS2 -Co) using a molybdenum disulfide nanosheet (MoS2 ) and a cobalt complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Codota). Evidenced by detailed experimental and theoretical studies, the NIR-enhanced MoS2 -Co can circumvent the restriction by simultaneously inhibition of ß-sheet formation and destroying ß-sheet structures of the preformed Aß aggregates in living cell. Furthermore, our designed MoS2 -Co is an easy to graft Aß-target agent that prevents misdirected or undesirable hydrolysis reactions, and has been demonstrated to cross the blood brain barrier. This method can be adapted for hydrolysis of other kinds of amyloids.

Expanding the MiniAp-4 BBB-shuttle family: Evaluation of proline cis-trans ratio as tool to fine-tune transport.

Venoms have recently emerged as a promising field in drug discovery due to their good selectivity and affinity for a wide range of biological targets. Among their multiple potential applications, venoms are a rich source of blood-brain barrier (BBB) peptide shuttles. We previously described a short nontoxic derivative of apamin, MiniAp-4, which can transport a wide range of cargoes across the BBB. Here, we have studied the conformation of the proline residue of a range of MiniAp-4 analogues by high-field NMR techniques, with the aim to identify whether there is a direct relation between the cis/trans population and a range of features, such as the capacity to transport molecules across a human-based cellular model and stability in various media. The most promising candidate showed improved transport properties for a relevant small fluorophore.

Efficiency of Penetration of Dopamine and Serotonin Peptide Derivatives through the Artificial Membranes.

A mass spectrometric method has been developed for determining the content of dopamine and serotonin derivatives, which allows evaluating the efficiency of their penetration through artificial membranes depending on the structure of their peptide fragment. In this case, the diffusion of dopamine and serotonin derivatives through the membrane occurred as a result of competitive interactions. It was shown which compounds in this mixture more easily penetrate through artificial membranes. It was found that the most promising in terms of overcoming the BBB are Boc-Pro-Srt and Boc-Pro-DOPA.

Seizure progression and inflammatory mediators promote pericytosis and pericyte-microglia clustering at the cerebrovasculature.

BACKGROUND: Cerebrovascular dysfunction and inflammation occur in epilepsy. Here we asked whether pericytes, a pivotal cellular component of brain capillaries, undergo pathological modifications during experimental epileptogenesis and in human epilepsy. We evaluated whether pro-inflammatory cytokines, present in the brain during seizures, contribute to pericyte morphological modifications. METHODS: In vivo, unilateral intra-hippocampal kainic acid (KA) injections were performed in NG2DsRed/C57BL6 mice to induce status epilepticus (SE), epileptogenesis, and spontaneous recurrent seizures (SRS). NG2DsRed mice were used to visualize pericytes during seizure progression. The effect triggered by recombinant IL-1ß, TNFα, or IL-6 on pericytes was evaluated in NG2DsRed hippocampal slices and in human-derived cell culture. Human brain specimens obtained from temporal lobe epilepsy (TLE) with or without sclerosis (HS) and focal cortical dysplasia (FCD-IIb) were evaluated for pericyte-microglial cerebrovascular assembly. RESULTS: A disarray of NG2DsRed+ pericyte soma and ramifications was found 72 h post-SE and 1 week post-SE (epileptogenesis) in the hippocampus. Pericyte modifications topographically overlapped with IBA1+ microglia clustering around the capillaries with cases of pericytes lodged within the microglial cells. Microglial clustering around the NG2DsRed pericytes lingered at SRS. Pericyte proliferation (Ki67+) occurred 72 h post-SE and during epileptogenesis and returned towards control levels at SRS. Human epileptic brain tissues showed pericyte-microglia assemblies with IBA1/HLA microglial cells outlining the capillary wall in TLE-HS and FCD-IIb specimens. Inflammatory mediators contributed to pericyte modifications, in particular IL-1ß elicited pericyte morphological changes and pericyte-microglia clustering in NG2DsRed hippocampal slices. Modifications also occurred when pro-inflammatory cytokines were added to an in vitro culture of pericytes. CONCLUSIONS: These results indicate the occurrence of pericytosis during seizures and introduce a pericyte-microglial mediated mechanism of blood-brain barrier dysfunction in epilepsy.

Potent 3-Hydroxy-2-Pyridine Aldoxime Reactivators of Organophosphate-Inhibited Cholinesterases with Predicted Blood-Brain Barrier Penetration.

A new series of 3-hydroxy-2-pyridine aldoxime compounds have been designed, synthesised and tested in vitro, in silico, and ex vivo as reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by organophosphates (OPs), for example, VX, sarin, cyclosarin, tabun, and paraoxon. The reactivation rates of three oximes (16-18) were determined to be greater than that of 2-PAM and comparable to that of HI-6, two pyridinium aldoximes currently used by the armies of several countries. The interactions important for a productive orientation of the oxime group within the OP-inhibited enzyme have been clarified by molecular-modelling studies, and by the resolution of the crystal structure of the complex of oxime 17 with Torpedo californica AChE. Blood-brain barrier penetration was predicted for oximes 15-18 based on their physicochemical properties and an in vitro brain membrane permeation assay. Among the evaluated compounds, two morpholine-3-hydroxypyridine aldoxime conjugates proved to be promising reactivators of OP-inhibited cholinesterases. Moreover, efficient ex vivo reactivation of phosphylated native cholinesterases by selected oximes enabled significant hydrolysis of VX, sarin, paraoxon, and cyclosarin in whole human blood, which indicates that the oximes have scavenging potential.

Strategies for Enhancing the Permeation of CNS-Active Drugs through the Blood-Brain Barrier: A Review.

Background: The blood brain barrier (BBB) is a dynamic and functional structure which poses a vast challenge in the development of drugs acting on the central nervous system (CNS). While most substances are denied BBB crossing, selective penetration of substances mainly occurs through diffusion, carrier mediated transport, or receptor mediated transcytosis. Methods: Strategies in enhancing BBB penetration have been reviewed and summarized in accordance with their type of formulation. Highlights in monoclonal antibodies, peptide-vectors, nanoparticles, and simple prodrugs were included. Conclusion: Nanoparticles and simple prodrugs, for example, can be used for efficient BBB penetration through inhibition of efflux mechanisms, however, monoclonal antibodies are the most promising strategy in BBB penetration. Close follow-up of future development in this area should confirm our expectation.

Use of Polyamidoamine Dendrimers in Brain Diseases.

Polyamidoamine (PAMAM) dendrimers are one of the smallest and most precise nanomolecules available today, which have promising applications for the treatment of brain diseases. Each aspect of the dendrimer (core, size or generation, size of cavities, and surface functional groups) can be precisely modulated to yield a variety of nanocarriers for delivery of drugs and genes to brain cells in vitro or in vivo. Two of the most important criteria to consider when using PAMAM dendrimers for neuroscience applications is their safety profile and their potential to be prepared in a reproducible manner. Based on these criteria, features of PAMAM dendrimers are described to help the neuroscience researcher to judiciously choose the right type of dendrimer and the appropriate method for loading the drug to form a safe and effective delivery system to the brain.

Design, Synthesis and Characterization of Novel Co-Polymers Decorated with Peptides for the Selective Nanoparticle Transport across the Cerebral Endothelium.

The development of new strategies for enhancing drug delivery to the brain represents a major challenge in treating cerebral diseases. In this paper, we report on the synthesis and structural characterization of a biocompatible nanoparticle (NP) made up of poly(lactic-co-glycolic acid) (PLGA)-polyethylene glycol (PEG) co-polymer (namely PELGA) functionalized with the membranotropic peptide gH625 (gH) and the iron-mimicking peptide CRTIGPSVC (CRT) for transport across the blood-brain barrier (BBB). gH possesses a high translocation potency of the cell membrane. Conversely, CRT selectively recognizes the brain endothelium, which interacts with transferrin (Tf) and its receptor (TfR) through a non-canonical ligand-directed mechanism. We hypothesize that the delivery across the BBB of PELGA NPs should be efficiently enhanced by the NP functionalization with both gH and CRT. Synthesis of peptides and their conjugation to the PLGA as well as NP physical-chemical characterization are performed. Moreover, NP uptake, co-localization, adhesion under dynamic conditions, and permeation across in vitro BBB model are evaluated as a function of gH/CRT functionalization ratio. Results establish that the cooperative effect of CRT and gH may change the intra-cellular distribution of NPs and strengthen NP delivery across the BBB at the functionalization ratio 33% gH⁻66% CRT.

Shiga toxin glycosphingolipid receptors and their lipid membrane ensemble in primary human blood-brain barrier endothelial cells.

Shiga toxin (Stx)-mediated injury to microvascular endothelial cells in the brain significantly contributes to the pathogenesis of the hemolytic-uremic syndrome caused by enterohemorrhagic Escherichia coli (EHEC). Stxs are AB5 toxins and the B-pentamers of the two major Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) expressed by human endothelial cells. Here we report on comprehensive structural analysis of the different lipoforms of Gb3Cer (Galα4Galß4Glcß1Cer) and globotetraosylceramide (Gb4Cer, GalNAcß3Galα4Galß4Glcß1Cer, the less effective Stx receptor) of primary human brain microvascular endothelial cells and their association with lipid rafts. Detergent-resistant membranes (DRMs), obtained by sucrose density gradient ultracentrifugation, were used as lipid raft-analogous microdomains of the liquid-ordered phase and nonDRM fractions were employed as equivalents for the liquid-disordered phase of cell membranes. Structures of the prevalent lipoforms of Gb3Cer and Gb4Cer were those with Cer (d18:1, C16:0), Cer (d18:1, C22:0) and Cer (d18:1, C24:1/C24:0) determined by electrospray ionization mass spectrometry that was combined with thin-layer chromatography immunodetection using anti-Gb3Cer and anti-Gb4Cer antibodies as well as Stx1a and Stx2a subtypes. Association of Stx receptor GSLs was determined by co-localization with lipid raft-specific membrane protein flotillin-2 and canonical lipid raft marker sphingomyelin with Cer (d18:1, C16:0) and Cer (d18:1, C24:1/C24:0) in the liquid-ordered phase, whereas lyso-phosphatidylcholine was detectable exclusively in the liquid-disordered phase. Defining the precise microdomain structures of primary endothelial cells may help to unravel the initial mechanisms by which Stxs interact with their target cells and will help to develop novel preventive and therapeutic measures for EHEC-mediated diseases.

The blood-brain barrier and protein-mediated fatty acid uptake: role of the blood-brain barrier as a metabolic barrier: An Editorial Comment for 'The blood-brain barrier fatty acid transport protein 1 (FATP1/SLC27A1) supplies docosahexaenoic acid to the brain, and insulin facilitates transport'.

Read the commented article 'The blood-brain barrier fatty acid transport protein 1 (FATP1/SLC27A1) supplies docosahexaenoic acid to the brain, and insulin facilitates transport' on page 400.

in vivo localization of the neuronal ceroid lipofuscinosis proteins, CLN3 and CLN7, at endogenous expression levels.

The neuronal ceroid lipofuscinoses are a group of recessively inherited, childhood-onset neurodegenerative conditions. Several forms are caused by mutations in genes encoding putative lysosomal membrane proteins. Studies of the cell biology underpinning these disorders are hampered by the poor antigenicity of the membrane proteins, which makes visualization of the endogenous proteins difficult. We have used Drosophila to generate knock-in YFP-fusions for two of the NCL membrane proteins: CLN7 and CLN3. The YFP-fusions are expressed at endogenous levels and the proteins can be visualized live without the need for overexpression. Unexpectedly, both CLN7 and CLN3 have restricted expression in the CNS of Drosophila larva and are predominantly expressed in the glia that form the insect blood-brain-barrier. CLN7 is also expressed in neurons in the developing visual system. Analogous with murine CLN3, Drosophila CLN3 is strongly expressed in the excretory and osmoregulatory Malpighian tubules, but the knock-in also reveals unexpected localization of the protein to the apical domain adjacent to the lumen. In addition, some CLN3 protein in the tubules is localized within mitochondria. Our in vivo imaging of CLN7 and CLN3 suggests new possibilities for function and promotes new ideas about the cell biology of the NCLs.

PEGylation of Reduced Graphene Oxide Induces Toxicity in Cells of the Blood-Brain Barrier: An in Vitro and in Vivo Study.

Polyethylene glycol (PEG) coating has been frequently used to improve the pharmacokinetic behavior of nanoparticles. Studies that contribute to better unravel the effects of PEGylation on the toxicity of nanoparticle formulation are therefore highly relevant. In the present study, reduced graphene oxide (rGO) was functionalized with PEG, and its effects on key components of the blood-brain barrier, such as astrocytes and endothelial cells, were analyzed in culture and in an in vivo rat model. The in vitro studies demonstrated concentration-dependent toxicity. The highest concentration (100 µg/mL) of non-PEGylated rGO had a lower toxic influence on cell viability in primary cultures of astrocytes and rat brain endothelial cells, while PEGylated rGO induced deleterious effects and cell death. We assessed hippocampal BBB integrity in vivo by evaluating astrocyte activation and the expression of the endothelial tight and adherens junctions proteins. From 1 h to 7 days post-rGO-PEG systemic injection, a notable and progressive down-regulation of protein markers of astrocytes (GFAP, connexin-43), the endothelial tight (occludin), and adherens (ß-catenin) junctions and basal lamina (laminin) were observed. The formation of intracellular reactive oxygen species demonstrated by increases in the enzymatic antioxidant system in the PEGylated rGO samples was indicative of oxidative stress-mediated damage. Under the experimental conditions and design of the present study the PEGylation of rGO did not improve interaction with components of the blood-brain barrier. In contrast, the attachment of PEG to rGO induced deleterious effects in comparison with the effects caused by non-PEGylated rGO.

Internal Structure of 15 nm 3-Helix Micelle Revealed by Small-Angle Neutron Scattering and Coarse-Grained MD Simulation.

3-Helix micelles (3HM) formed by self-assembly of peptide-polymer conjugate amphiphiles have shown promise as a nanocarrier platform due to their long-circulation, deep tumor penetration, selective accumulation in tumor, and ability to cross the blood-brain barrier (BBB) for glioblastoma therapy. There is a need to understand the structural contribution to the high in vivo stability and performance of 3HM. Using selective deuteration, the contrast variation technique in small-angle neutron scattering, and coarse-grained molecular dynamics simulation, we determined the spatial distribution of each component within 3HM. Our results show a slightly deformed polyethylene glycol (PEG) conformation within the micelle that is radially offset from its conjugation site toward the exterior of the micelle and a highly solvated shell. Surprisingly, ∼85 v/v % of 3HM is water, unusually higher than any micellar nanocarrier based on our knowledge. The result will provide important structural insights for future studies to uncover the molecular origin of 3HM's in vivo performance, and development of the nanocarriers.