Biophysics of claudin proteins in tight junction architecture: Three decades of progress.

Tight junctions are cell-cell adhesion complexes that act as gatekeepers of the paracellular space. Formed by several transmembrane proteins, the claudin family performs the primary gate-keeping function. The claudin proteins form charge and size-selective diffusion barriers to maintain homeostasis across endothelial and epithelial tissue. Of the 27 known claudins in mammals, some are known to seal the paracellular space, while others provide selective permeability. The differences in permeability arise due to the varying expression levels of claudins in each tissue. The tight junctions are observed as strands in freeze-fracture electron monographs; however, at the molecular level, tight junction strands form when multiple claudin proteins assemble laterally (cis assembly) within a cell and head-on (trans assembly) with claudins of the adjacent cell in a zipper-like architecture, closing the gap between the neighboring cells. The disruption of tight junctions caused by changing claudin expression levels or mutations can lead to diseases. Therefore, knowledge of the molecular architecture of the tight junctions and how that is tied to tissue-specific function is critical for fighting diseases. Here, we review the current understanding of the tight junctions accrued over the last three decades from experimental and computational biophysics perspectives.

Supramolecular Peptoid Structure Strengthens Complexation with Polyacrylic Acid Microgels.

We have studied the complexation between cationic antimicrobials and polyanionic microgels to create self-defensive surfaces that responsively resist bacterial colonization. An essential property is the stable sequestration of the loaded (complexed) antimicrobial within the microgel under a physiological ionic strength. Here, we assess the complexation strength between poly(acrylic acid) [PAA] microgels and a series of cationic peptoids that display supramolecular structures ranging from an oligomeric monomer to a tetramer. We follow changes in loaded microgel diameter with increasing [Na+] as a measure of the counterion doping level. Consistent with prior findings on colistin/PAA complexation, we find that a monomeric peptoid is fully released at ionic strengths well below physiological conditions, despite its +5 charge. In contrast, progressively higher degrees of peptoid supramolecular structure display progressively greater resistance to salting out, which we attribute to the greater entropic stability associated with the complexation of multimeric peptoid bundles.

Lipidation alters the phase-separation of resilin-like polypeptides.

Biology exploits biomacromolecular phase separation to form condensates, known as membraneless organelles. Despite significant advancements in deciphering sequence determinants for phase separation, modulating these features in vivo remains challenging. A promising approach inspired by biology is to use post-translational modifications (PTMs)-to modulate the amino acid physicochemistry instead of altering protein sequences-to control the formation and characteristics of condensates. However, despite the identification of more than 300 types of PTMs, the detailed understanding of how they influence the formation and material properties of protein condensates remains incomplete. In this study, we investigated how modification with myristoyl lipid alters the formation and characteristics of the resilin-like polypeptide (RLP) condensates, a prototypical disordered protein with upper critical solution temperature (UCST) phase behaviour. Using turbidimetry, dynamic light scattering, confocal and electron microscopy, we demonstrated that lipidation-in synergy with the sequence of the lipidation site-significantly influences RLPs' thermodynamic propensity for phase separation and their condensate properties. Molecular simulations suggested these effects result from an expanded hydrophobic region created by the interaction between the lipid and lipidation site rather than changes in peptide rigidity. These findings emphasize the role of "sequence context" in modifying the properties of PTMs, suggesting that variations in lipidation sequences could be strategically used to fine-tune the effect of these motifs. Our study advances understanding of lipidation's impact on UCST phase behaviour, relevant to proteins critical in biological processes and diseases, and opens avenues for designing lipidated resilins for biomedical applications like heat-mediated drug elution.

The Role of ZO-2 in Modulating JAM-A and γ-Actin Junctional Recruitment, Apical Membrane and Tight Junction Tension, and Cell Response to Substrate Stiffness and Topography.

This work analyzes the role of the tight junction (TJ) protein ZO-2 on mechanosensation. We found that the lack of ZO-2 reduced apical membrane rigidity measured with atomic force microscopy, inhibited the association of γ-actin and JAM-A to the cell border, and instead facilitated p114RhoGEF and afadin accumulation at the junction, leading to an enhanced mechanical tension at the TJ measured by FRET, with a ZO-1 tension probe, and increased tricellular TJ tension. Simultaneously, adherens junction tension measured with an E-cadherin probe was unaltered. The stability of JAM-A and ZO-2 binding was assessed by a collaborative in silico study. The absence of ZO-2 also impacted the cell response to the substrate, as monolayers plated in 20 kPa hydrogels developed holes not seen in parental cultures and displayed a retarded elongation and formation of cell aggregates. The absence of ZO-2 was sufficient to induce YAP and Snail nuclear accumulation in cells cultured over glass, but when ZO-2 KD cells were plated in nanostructured ridge arrays, they displayed an increased abundance of nuclear Snail and conspicuous internalization of claudin-4. These results indicate that the absence of ZO-2 also impairs the response of cells to substrate stiffness and exacerbates transformation triggered by substrate topography.

Persister control by leveraging dormancy associated reduction of antibiotic efflux.

Persistent bacterial infections do not respond to current antibiotic treatments and thus present a great medical challenge. These conditions have been linked to the formation of dormant subpopulations of bacteria, known as persister cells, that are growth-arrested and highly tolerant to conventional antibiotics. Here, we report a new strategy of persister control and demonstrate that minocycline, an amphiphilic antibiotic that does not require active transport to penetrate bacterial membranes, is effective in killing Escherichia coli persister cells [by 70.8 ± 5.9% (0.53 log) at 100 µg/mL], while being ineffective in killing normal cells. Further mechanistic studies revealed that persister cells have reduced drug efflux and accumulate more minocycline than normal cells, leading to effective killing of this dormant subpopulation upon wake-up. Consistently, eravacycline, which also targets the ribosome but has a stronger binding affinity than minocycline, kills persister cells by 3 logs when treated at 100 µg/mL. In summary, the findings of this study reveal that while dormancy is a well-known cause of antibiotic tolerance, it also provides an Achilles' heel for controlling persister cells by leveraging dormancy associated reduction of drug efflux.

Lipidation Alters the Structure and Hydration of Myristoylated Intrinsically Disordered Proteins.

Lipidated proteins are an emerging class of hybrid biomaterials that can integrate the functional capabilities of proteins into precisely engineered nano-biomaterials with potential applications in biotechnology, nanoscience, and biomedical engineering. For instance, fatty-acid-modified elastin-like polypeptides (FAMEs) combine the hierarchical assembly of lipids with the thermoresponsive character of elastin-like polypeptides (ELPs) to form nanocarriers with emergent temperature-dependent structural (shape or size) characteristics. Here, we report the biophysical underpinnings of thermoresponsive behavior of FAMEs using computational nanoscopy, spectroscopy, scattering, and microscopy. This integrated approach revealed that temperature and molecular syntax alter the structure, contact, and hydration of lipid, lipidation site, and protein, aligning with the changes in the nanomorphology of FAMEs. These findings enable a better understanding of the biophysical consequence of lipidation in biology and the rational design of the biomaterials and therapeutics that rival the exquisite hierarchy and capabilities of biological systems.

Combined Computational-Biochemical Approach Offers an Accelerated Path to Membrane Protein Solubilization.

Membrane proteins are difficult to isolate and purify due to their dependence on the surrounding lipid membrane for structural stability. Detergents are often used to solubilize these proteins, with this approach requiring a careful balance between protein solubilization and denaturation. Determining which detergent is most appropriate for a given protein has largely been done empirically through screening, which requires large amounts of membrane protein and associated resources. Here, we describe an alternative to conventional detergent screening using a computational modeling approach to identify the most likely candidate detergents for solubilizing a protein of interest. We demonstrate our approach using ghrelin O-acyltransferase (GOAT), a member of the membrane-bound O-acyltransferase family of integral membrane enzymes that has not been solubilized or purified in active form. A computationally derived GOAT structural model provides the only structural information required for this approach. Using computational analysis of detergent ability to penetrate phospholipid bilayers and stabilize the GOAT structure, a panel of common detergents were rank-ordered for their proposed ability to solubilize GOAT. The simulations were performed at all-atom resolution for a combined simulation time of 24 µs. Independently, we biologically screened these detergents for their solubilization of fluorescently tagged GOAT constructs. We found computational prediction of protein structural stabilization was the better predictor of detergent solubilization ability, but neither approach was effective for predicting detergents that would support GOAT enzymatic function. The current rapid expansion of membrane protein computational models lacking experimental structural information and our computational detergent screening approach can greatly improve the efficiency of membrane protein detergent solubilization, supporting downstream functional and structural studies.

Adaptive Recombinant Nanoworms from Genetically Encodable Star Amphiphiles.

Recombinant nanoworms are promising candidates for materials and biomedical applications ranging from the templated synthesis of nanomaterials to multivalent display of bioactive peptides and targeted delivery of theranostic agents. However, molecular design principles to synthesize these assemblies (which are thermodynamically favorable only in a narrow region of the phase diagram) remain unclear. To advance the identification of design principles for the programmable assembly of proteins into well-defined nanoworms and to broaden their stability regimes, we were inspired by the ability of topologically engineered synthetic macromolecules to acess rare mesophases. To test this design principle in biomacromolecular assemblies, we used post-translational modifications (PTMs) to generate lipidated proteins with precise topological and compositional asymmetry. Using an integrated experimental and computational approach, we show that the material properties (thermoresponse and nanoscale assembly) of these hybrid amphiphiles are modulated by their amphiphilic architecture. Importantly, we demonstrate that the judicious choice of amphiphilic architecture can be used to program the assembly of proteins into adaptive nanoworms, which undergo a morphological transition (sphere-to-nanoworms) in response to temperature stimuli.

Development of the computational antibiotic screening platform (CLASP) to aid in the discovery of new antibiotics.

Bacterial colonization of biotic and abiotic surfaces and antibiotic resistance are grand challenges with paramount societal impacts. However, in the face of increasing bacterial resistance to all known antibiotics, efforts to discover new classes of antibiotics have languished, creating an urgent need to accelerate the antibiotic discovery pipeline. A major deterrent in the discovering of new antibiotics is the limited permeability of molecules across the bacterial envelope. Notably, the Gram-negative bacteria have nutrient specific protein channels (or porins) that restrict the permeability of non-essential molecules, including antibiotics. Here, we have developed the Computational Antibiotic Screening Platform (CLASP) for screening of potential drug molecules through the porins. The CLASP takes advantage of coarse grain (CG) resolution, advanced sampling techniques, and a parallel computing environment to maximize its performance. The CLASP yields comprehensive thermodynamic and kinetic output data of a potential drug molecule within a few hours of wall-clock time. Its output includes the potential of mean force profile, energy barrier, the rate constant, and contact analysis of the molecule with the pore-lining residues, and the orientational analysis of the molecule in the porin channel. In our first CLASP application, we report the transport properties of six carbapenem antibiotics-biapenem, doripenem, ertapenem, imipenem, meropenem, and panipenem-through OccD3, a major channel for carbapenem uptake in Pseudomonas aeruginosa. The CLASP is designed to screen small molecule libraries with a fast turnaround time to yield structure-property relationships to discover antibiotics with high permeability. The CLASP will be freely distributed to enable accelerated antibiotic drug discovery.

The ghrelin O-acyltransferase structure reveals a catalytic channel for transmembrane hormone acylation.

Integral membrane proteins represent a large and diverse portion of the proteome and are often recalcitrant to purification, impeding studies essential for understanding protein structure and function. By combining co-evolutionary constraints and computational modeling with biochemical validation through site-directed mutagenesis and enzyme activity assays, we demonstrate here a synergistic approach to structurally model purification-resistant topologically complex integral membrane proteins. We report the first structural model of a eukaryotic membrane-bound O-acyltransferase (MBOAT), ghrelin O-acyltransferase (GOAT), which modifies the metabolism-regulating hormone ghrelin. Our structure, generated in the absence of any experimental structural data, revealed an unanticipated strategy for transmembrane protein acylation with catalysis occurring in an internal channel connecting the endoplasmic reticulum lumen and cytoplasm. This finding validated the power of our approach to generate predictive structural models for other experimentally challenging integral membrane proteins. Our results illuminate novel aspects of membrane protein function and represent key steps for advancing structure-guided inhibitor design to target therapeutically important but experimentally intractable membrane proteins.

High-Level Antibiotic Tolerance of a Clinically Isolated Enterococcus faecalis Strain.

Bacteria can survive antibiotic treatment both by acquiring antibiotic resistance genes and through mechanisms of tolerance that are based on phenotypic changes and the formation of metabolically inactive cells. Here, we report an Enterococcus faecalis strain (E. faecalis UM001B) that was isolated from a cystic fibrosis patient and had no increase in resistance but extremely high-level tolerance to ampicillin, vancomycin, and tetracycline. Specifically, the percentages of cells that survived 3.5-h antibiotic treatment (at 100 µg · ml-1) were 25.4% ± 4.3% and 51.9% ± 4.0% for ampicillin and tetracycline, respectively; vancomycin did not exhibit any significant killing. Consistent with the changes in antibiotic susceptibility, UM001B was found to have reduced penetration of ampicillin and vancomycin and accumulation of tetracycline compared to the reference strain ATCC 29212. Based on whole-genome sequencing, four amino acid substitutions were identified in one of the tetracycline efflux pump repressors (TetRs), compared to ATCC 29212. Results of molecular simulations and experimental assays revealed that these mutations could lead to higher levels of tetracycline efflux activity. Consistently, replicating these mutations in Escherichia coli MG1655 increased its tolerance to tetracycline. Overall, these findings provide new insights into the development of multidrug tolerance in E. faecalis, which can facilitate future studies to better control enterococcal infections.IMPORTANCEEnterococcus faecalis represents a major group of pathogens causing nosocomial infections that are resistant to multiple classes of antibiotics. An important challenge associated with E. faecalis infection is the emergence of multidrug-tolerant strains, which have normal MICs but do not respond to antibiotic treatment. Here, we report a strain of E. faecalis that was isolated from a cystic fibrosis patient and demonstrated high-level tolerance to ampicillin, vancomycin, and tetracycline. Whole-genome sequencing revealed critical substitutions in one of the tetracycline efflux pump repressors that are consistent with the increased tolerance of E. faecalis UM001B to tetracycline. These findings provide new information about bacterial antibiotic tolerance and may help develop more effective therapeutics.

Paracellular Gatekeeping: What Does It Take for an Ion to Pass Through a Tight Junction Pore?

Tight junction pores are physiological gatekeepers of paracellular transport in epithelial tissues. Conventionally, tight junction permeability is determined via in vitro electrophysiology measurements; however, the macroscopic readout does not provide molecular-level understanding into the mechanism of ion permeation. Insight into the factors governing selectivity across the paracellular space is just emerging. In this study, we investigated tight junction pores comprising of claudin-2 and claudin-5 proteins that are structurally similar to subnanometer radii but have measurably different in vitro ion permeabilities. To evaluate the mechanistic differences in ion transport across the pores, we computed the free-energy profiles and relative rate constants for the transport of monovalent (Na+, K+, Cl-) and divalent (Mg2+ and Ca2+) ions through the pores using replica exchange metadynamics. In claudin-2, we demonstrate how a single residue dictates selective permeability of Na+ and K+ ions. In claudin-5, we found no clear preference for anion or cation selectivity; thus, pores formed by claudin-5 are indeed barriers to ion permeation. Mutations to claudin-5 that widen the pore's steric radius did not significantly impact pore selectivity, indicating that electrostatics dominate pore selectivity. The key takeaways from this work are as follows: (a) two pores that are similar in diameter and length can have dissimilar ion conductance, (b) existence of a physical pore does not guarantee ion permeability, and (c) the electrostatic environment created by the pore-lining residues dictates the ion conductivity. These mechanistic understandings of the tight junction pores are critical for the interpretation of tight junction physiology.

Interaction of amphiphilic coumarin with DPPC/DPPS lipid bilayer: effects of concentration and alkyl tail length.

In this work, interactions between amphiphilic amino methyl coumarin and dipalmitoyl-sn-glycero-3-phosphocholine/dipalmitoyl-sn-glycero-3-phosphoserine (DPPC/DPPS) lipid bilayer were investigated. A combination of experimental techniques (zeta potential, fluorescence spectroscopy, and differential scanning calorimetry) along with molecular dynamics simulations was employed to examine the influence of alkyl tail length and concentration of the amphiphilic coumarin on the lipid bilayer. Alkyl tails comprising 5(C5), 9(C9), and 12(C12) carbon atoms were conjugated to amino methyl coumarin via a single-step process. The binding and insertion mechanisms of the amphiphilic coumarins were studied in increasing concentrations for short-tailed (C5) and long-tailed (C12) coumarins. The simulation results show that C5 coumarin molecules penetrate the lipid bilayer, but owing to the short alkyl tail, they interact primarily with the lipid head groups resulting in lipid bilayer thinning; however, at high concentrations, the C5 coumarins undergo continuous insertion-ejection from the outer leaflet of the lipid bilayer. In contrast, C12 coumarins interact favorably with the hydrophobic lipid tails and lack the ejection-reinsertion behavior. Instead, the C12 coumarin molecules undergo flip-flops between the outer and inner leaflets of the lipid bilayer. At high concentrations, the high-frequency flip-flops lead to lipid destabilization, causing the lipid bilayer to rupture. The simulation results are in excellent agreement with the toxicity of amphiphilic coumarin activity in cancer cells. The efficacy of amphiphilic coumarins in liposomal lipid bilayers demonstrates the promise of these molecules as a tool in the treatment of cancer.

Computational Nanoscopy of Tight Junctions at the Blood-Brain Barrier Interface.

The selectivity of the blood-brain barrier (BBB) is primarily maintained by tight junctions (TJs), which act as gatekeepers of the paracellular space by blocking blood-borne toxins, drugs, and pathogens from entering the brain. The BBB presents a significant challenge in designing neurotherapeutics, so a comprehensive understanding of the TJ architecture can aid in the design of novel therapeutics. Unraveling the intricacies of TJs with conventional experimental techniques alone is challenging, but recently developed computational tools can provide a valuable molecular-level understanding of TJ architecture. We employed the computational methods toolkit to investigate claudin-5, a highly expressed TJ protein at the BBB interface. Our approach started with the prediction of claudin-5 structure, evaluation of stable dimer conformations and nanoscale assemblies, followed by the impact of lipid environments, and posttranslational modifications on these claudin-5 assemblies. These led to the study of TJ pores and barriers and finally understanding of ion and small molecule transport through the TJs. Some of these in silico, molecular-level findings, will need to be corroborated by future experiments. The resulting understanding can be advantageous towards the eventual goal of drug delivery across the BBB. This review provides key insights gleaned from a series of state-of-the-art nanoscale simulations (or computational nanoscopy studies) performed on the TJ architecture.

Dynamics of OmpF Trimer Formation in the Bacterial Outer Membrane of Escherichia coli.

The self-assembly of outer membrane protein F (OmpF) in the outer membrane of Escherichia coli Gram-negative bacteria was studied using multiscale molecular dynamics simulations. To accommodate the long time scale required for protein assembly, coarse-grained parametrization of E. coli outer membrane lipids was first developed. The OmpF monomers formed stable dimers at specific protein-protein interactions sites irrespective of the lipid membrane environment. The dimer intermediate was asymmetric but provided a template to form a symmetric trimer. Superposition analysis of the self-assembled trimer with the X-ray crystal structure of the trimer available in the protein data bank showed excellent agreement with global root-mean-square deviation of less than 2.2 Å. The free energy change associated with dimer formation was -26 ± 1 kcal mol-1, and for a dimer to bind to a monomer and to form a trimer yielded -56 ± 4 kcal mol-1. Based on thermodynamic data, an alternate path to trimer formation via interaction of two dimers is also presented.

Development of effective stochastic potential method using random matrix theory for efficient conformational sampling of semiconductor nanoparticles at non-zero temperatures.

The relationship between structure and property is central to chemistry and enables the understanding of chemical phenomena and processes. Need for an efficient conformational sampling of chemical systems arises from the presence of solvents and the existence of non-zero temperatures. However, conformational sampling of structures to compute molecular quantum mechanical properties is computationally expensive because a large number of electronic structure calculations are required. In this work, the development and implementation of the effective stochastic potential (ESP) method is presented to perform efficient conformational sampling of molecules. The overarching goal of this work is to alleviate the computational bottleneck associated with performing a large number of electronic structure calculations required for conformational sampling. We introduce the concept of a deformation potential and demonstrate its existence by the proof-by-construction approach. A statistical description of the fluctuations in the deformation potential due to non-zero temperature was obtained using infinite-order moment expansion of the distribution. The formal mathematical definition of the ESP was derived using the functional minimization approach to match the infinite-order moment expansion for the deformation potential. Practical implementation of the ESP was obtained using the random-matrix theory method. The developed method was applied to two proof-of-concept calculations of the distribution of HOMO-LUMO gaps in water molecules and solvated CdSe clusters at 300 K. The need for large sample size to obtain statistically meaningful results was demonstrated by performing 105 ESP calculations. The results from these prototype calculations demonstrated the efficacy of the ESP method for performing efficient conformational sampling. We envision that the fundamental nature of this work will not only extend our knowledge of chemical systems at non-zero temperatures but also generate new insights for innovative technological applications.

Combinatorial approaches to evaluate nanodiamond uptake and induced cellular fate.

Nanodiamonds (NDs) are an emerging class of engineered nanomaterials that hold great promise for the next generation of bionanotechnological products to be used for drug and gene delivery, or for bio-imaging and biosensing. Previous studies have shown that upon their cellular uptake, NDs exhibit high biocompatibility in various in vitro and in vivo set-ups. Herein we hypothesized that the increased NDs biocompatibility is a result of minimum membrane perturbations and their reduced ability to induce disruption or damage during cellular translocation. Using multi-scale combinatorial approaches that simulate ND-membrane interactions, we correlated NDs real-time cellular uptake and kinetics with the ND-induced membrane fluctuations to derive energy requirements for the uptake to occur. Our discrete and real-time analyses showed that the majority of NDs internalization occurs within 2 h of cellular exposure, however, with no effects on cellular viability, proliferation or cellular behavior. Furthermore, our simulation analyses using coarse-grained models identified key changes in the energy profile, membrane deformation and recovery time, all functions of the average ND or ND-based agglomerate size. Understanding the mechanisms responsible for ND-cell membrane interactions could possibly advance their implementation in various biomedical applications.

Multiscale approach to investigate self-assembly of telodendrimer based nanocarriers for anticancer drug delivery.

Delivery of poorly soluble anticancer drugs can be achieved by employing polymeric drug delivery systems, capable of forming stable self-assembled nanocarriers with drug encapsulated within their hydrophobic cores. Computational investigations can aid the design of efficient drug-delivery platforms; however, simulations of nanocarrier self-assembly process are challenging due to high computational cost associated with the large system sizes (millions of atoms) and long time scales required for equilibration. In this work, we overcome this challenge by employing a multiscale computational approach in conjunction with experiments to analyze the role of the individual building blocks in the self-assembly of a highly tunable linear poly(ethylene glycol)-b-dendritic oligo(cholic acid) block copolymer called telodendrimer. The multiscale approach involved developing a coarse grained description of the telodendrimer, performing simulations over several microseconds to capture the self-assembly process, followed by reverse mapping of the coarse grained system to atomistic representation for structural analysis. Overcoming the computational bottleneck allowed us to run multiple self-assembly simulations and determine average size, drug-telodendrimer micellar stoichiometry, optimal drug loading capacity, and atomistic details such hydrogen-bonding and solvent accessible area of the nanocarrier. Computed results are in agreement with the experimental data, highlighting the success of the multiscale approach applied here.

Signaling factor interactions with polysaccharide aggregates of bacterial biofilms.

Biofilms are surface-attached colonies of bacteria embedded in an extracellular polymeric substance (EPS). Inside the eukaryotic hosts, bacterial biofilms interact with the host cells through signaling factors (SFs). These signaling processes play important roles in the interaction between bacteria and host cells and the outcome of infections and symbiosis. However, how host immune factors diffuse through biofilms is not well understood. Here, we describe synergistic molecular dynamics and experimental approaches for studying the translocation of signaling factors through polysaccharide chain aggregates present in the extracellular matrix of bacterial biofilms. The effect of polysaccharide chain degradation on the energetics of SF-EPS interactions was examined by simulating an EPS consisting of various polysaccharide chain lengths. It is shown that the SF stabilization energy, defined as the average potential of mean force difference between the environments outside and within the matrix, increases linearly with decreasing chain length. This effect has been explained based on the changes in the polysaccharide configurations around the SF. Specifically, shorter chains are packed tightly around the SF, promoting favorable SF-EPS interactions, while longer chains are packed loosely resulting in screening of interactions with neighboring chains. We further investigated the translocation of SFs through the host cell membrane using molecular dynamics simulations. Further, simulations predict the existence of energy barriers greater than 1000 kJ mol(-1) associated with the translocation of the signaling factors necrosis factor-alpha (TNF-α) and granulocyte macrophage colony stimulating factor (GM-CSF) across the lipid bilayer. The agreement of computational and experimental findings motivates future computational studies using a more detailed description of the EPS aimed at understanding the role of the extracellular matrix on biofilm drug resistance.

A structure-property relationship study of the well-defined telodendrimers to improve hemocompatibility of nanocarriers for anticancer drug delivery.

A series of telodendrimer (a linear polyethyelene glycol-block-dendritic oligo-cholic acid) have been synthesized via a bottom-up approach to optimize the hemocompatibility of the nanocarrier. Numbers of hydrophilic glycerol groups were introduced onto the polar surface of cholic acid to reduce the plasma membrane lytic activity of telodendrimers. An interesting result was observed: only an optimum number of glycerol introduced could reduce the hemolytic properties of the nanocarrier; on the contrary, more glycerols or the amino-glycerol substitution onto cholic acid significantly increased the hemolytic properties of the nanocarriers. To further elucidate the structure-property relationship, the molecular dynamic approach was used to simulate the conformation of the subunits of telodendrimers with different glycerol substitution, and the binding energies and the polar surface areas of the hairpin conformations were calculated to explain the membrane activities of nanocarriers. In addition, these telodendrimer subunits were synthesized and their membrane activities were tested directly, which validated the computational prediction and correlated with the observed hemolytic activity of nanocarriers. The glycerol substitution sustained the facial amphiphilicity of cholic acid, maintaining the superior drug loading capacity (paclitaxel and doxorubicin), stability, cell uptake, and anticancer efficacy of payloads. The in vivo optical imaging study indicated that the optimized nanocarriers can specifically deliver drug molecules to the tumor sites more efficiently than free drug administration, which is essential for the enhanced cancer treatment.