Self-Assembly of Soft and Conformable Broadband Absorbing Nanocellulose-Gold Nanoparticle Composites.

Broadband light-absorbing materials are of large interest for numerous applications ranging from solar harvesting and photocatalysis to low reflection coatings. Fabrication of these materials is often complex and typically utilizes coating techniques optimized for flat and hard materials. Here, we show a self-assembly based strategy for generating robust but mechanically flexible broadband light-absorbing soft materials that can conform to curved surfaces and surface irregularities. The materials were fabricated by adsorbing large quantities of gold nanoparticles (AuNPs) on the nanofibrils of hydrated bacterial cellulose (BC) membranes by tailoring the interaction potential between the cellulose nanofibrils and the AuNPs. The highly efficient self-assembly process resulted in very dense multilayers of AuNPs on the nanofibrils, causing extensive broadening of the localized surface plasmon resonance band and a striking black appearance of the BC membranes. The nanocomposite materials showed an absorptance >96% in both the visible and the near-infrared wavelength range. The AuNP-functionalized BC membranes demonstrated excellent conformability to curved and structured surfaces and could adopt the shape of highly irregular surface structures without any obvious changes in their optical properties. The proposed self-assembly based strategy enables the fabrication of soft and conformable broadband light-absorbing nanocomposites with unique optical and mechanical properties using sustainable cellulose-based materials.

Enzymatically Triggered Peptide-Lipid Conjugation of Designed Membrane Active Peptides for Controlled Liposomal Release.

Possibilities for controlling the release of pharmaceuticals from liposomal drug delivery systems can enhance their efficacy and reduce their side effects. Membrane-active peptides (MAPs) can be tailored to promote liposomal release when conjugated to lipid head groups using thiol-maleimide chemistry. However, the rapid oxidation of thiols hampers the optimization of such conjugation-dependent release strategies. Here, we demonstrate a de novo designed MAP modified with an enzyme-labile Cys-protection group (phenylacetamidomethyl (Phacm)) that prevents oxidation and facilitates in situ peptide lipidation. Before deprotection, the peptide lacks a defined secondary structure and does not interact with maleimide-functionalized vesicles. After deprotection of Cys using penicillin G acylase (PGA), the peptide adopts an α-helical conformation and triggers rapid release of vesicle content. Both the peptide and PGA concentrations significantly influence the conjugation process and, consequently, the release kinetics. At a PGA concentration of 5 µM the conjugation and release kinetics closely mirror those of fully reduced, unprotected peptides. We anticipate that these findings will enable further refinement of MAP conjugation and release processes, facilitating the development of sophisticated bioresponsive MAP-based liposomal drug delivery systems.

Increased matrix stiffness enhances pro-tumorigenic traits in a physiologically relevant breast tissue- monocyte 3D model.

High mammographic density, associated with increased tissue stiffness, is a strong risk factor for breast cancer per se. In postmenopausal women there is no differences in the occurrence of ductal carcinoma in situ (DCIS) depending on breast density. Preliminary data suggest that dense breast tissue is associated with a pro-inflammatory microenvironment including infiltrating monocytes. However, the underlying mechanism(s) remains largely unknown. A major roadblock to understanding this risk factor is the lack of relevant in vitro models. A biologically relevant 3D model with tunable stiffness was developed by cross-linking hyaluronic acid. Breast cancer cells were cultured with and without freshly isolated human monocytes. In a unique clinical setting, extracellular proteins were sampled using microdialysis in situ from women with various breast densities. We show that tissue stiffness resembling high mammographic density increases the attachment of monocytes to the cancer cells, increase the expression of adhesion molecules and epithelia-mesenchymal-transition proteins in estrogen receptor (ER) positive breast cancer. Increased tissue stiffness results in increased secretion of similar pro-tumorigenic proteins as those found in human dense breast tissue including inflammatory cytokines, proteases, and growth factors. ER negative breast cancer cells were mostly unaffected suggesting that diverse cancer cell phenotypes may respond differently to tissue stiffness. We introduce a biological relevant model with tunable stiffness that resembles the densities found in normal breast tissue in women. The model will be key for further mechanistic studies. Additionally, our data revealed several pro-tumorigenic pathways that may be exploited for prevention and therapy against breast cancer. STATEMENT OF SIGNIFICANCE: Women with mammographic high-density breasts have a 4-6-fold higher risk of breast cancer than low-density breasts. Biological mechanisms behind this increase are not fully understood and no preventive therapeutics are available. One major reason being a lack of suitable experimental models. Having such models available would greatly enhance the discovery of relevant targets for breast cancer prevention. We present a biologically relevant 3D-model for studies of human dense breasts, providing a platform for investigating both biophysical and biochemical properties that may affect cancer progression. This model will have a major scientific impact on studies for identification of novel targets for breast cancer prevention.

Proteolytic remodeling of 3D bioprinted tumor microenvironments.

In native tissue, remodeling of the pericellular space is essential for cellular activities and is mediated by tightly regulated proteases. Protease activity is dysregulated in many diseases, including many forms of cancer. Increased proteolytic activity is directly linked to tumor invasion into stroma, metastasis, and angiogenesis as well as all other hallmarks of cancer. Here we show a strategy for 3D bioprinting of breast cancer models using well-defined protease degradable hydrogels that can facilitate exploration of the multifaceted roles of proteolytic extracellular matrix remodeling in tumor progression. We designed a set of bicyclo[6.1.0]nonyne functionalized hyaluronan (HA)-based bioinks cross-linked by azide-modified poly(ethylene glycol) (PEG) or matrix metalloproteinase (MMP) degradable azide-functionalized peptides. Bioprinted structures combining PEG and peptide-based hydrogels were proteolytically degraded with spatial selectivity, leaving non-degradable features intact. Bioprinting of tumor-mimicking microenvironments using bioinks comprising human breast cancer cells (MCF-7) and fibroblast in hydrogels with different susceptibilities to proteolytic degradation shows that MCF-7 proliferation and spheroid size were significantly increased in protease degradable hydrogel compartments, but only in the presence of fibroblasts. In the absence of fibroblasts in the stromal compartment, cancer cell proliferation was reduced and did not differ between degradable and nondegradable hydrogels. The interactions between spatially separated fibroblasts and MCF-7 cells consequently resulted in protease-mediated remodeling of the bioprinted structures and a significant increase in cancer cell spheroid size, highlighting the close interplay between cancer cells and stromal cells in the tumor microenvironment and the influence of proteases in tumor progression.

Nanocellulose Reinforced Hyaluronan-Based Bioinks.

Bioprinting of hydrogel-based bioinks can allow for the fabrication of elaborate, cell-laden 3D structures. In addition to providing an adequate extracellular matrix mimetic environment and high cell viability, the hydrogels must offer facile extrusion through the printing nozzle and retain the shape of the printed structure. We demonstrate a strategy to incorporate cellulose oxalate nanofibrils in hyaluronan-based hydrogels to generate shear thinning bioinks that allowed for printing of free-standing multilayer structures, covalently cross-linked after bioprinting, yielding long-term stability. The storage modulus of the hydrogels was tunable between 0.5 and 1.5 kPa. The nanocellulose containing hydrogels showed good biocompatibility, with viability of primary human dermal fibroblasts above 80% at day 7 after seeding. The cells were also shown to tolerate the printing process well, with viability above 80% 24 h after printing. We anticipate that this hydrogel system can find broad use as a bioink to produce complex geometries that can support cell growth.

Development of novel broad-spectrum antimicrobial lipopeptides derived from plantaricin NC8 ß.

Bacterial resistance towards antibiotics is a major global health issue. Very few novel antimicrobial agents and therapies have been made available for clinical use during the past decades, despite an increasing need. Antimicrobial peptides have been intensely studied, many of which have shown great promise in vitro. We have previously demonstrated that the bacteriocin Plantaricin NC8 αß (PLNC8 αß) from Lactobacillus plantarum effectively inhibits Staphylococcus spp., and shows little to no cytotoxicity towards human keratinocytes. However, due to its limitations in inhibiting gram-negative species, the aim of the present study was to identify novel antimicrobial peptidomimetic compounds with an enhanced spectrum of activity, derived from the ß peptide of PLNC8 αß. We have rationally designed and synthesized a small library of lipopeptides with significantly improved antimicrobial activity towards both gram-positive and gram-negative bacteria, including the ESKAPE pathogens. The lipopeptides consist of 16 amino acids with a terminal fatty acid chain and assemble into micelles that effectively inhibit and kill bacteria by permeabilizing their cell membranes. They demonstrate low hemolytic activity and liposome model systems further confirm selectivity for bacterial lipid membranes. The combination of lipopeptides with different antibiotics enhanced the effects in a synergistic or additive manner. Our data suggest that the novel lipopeptides are promising as future antimicrobial agents, however additional experiments using relevant animal models are necessary to further validate their in vivo efficacy.

Astrocyte 3D culture and bioprinting using peptide functionalized hyaluronan hydrogels.

Astrocytes play an important role in the central nervous system, contributing to the development of and maintenance of synapses, recycling of neurotransmitters, and the integrity and function of the blood-brain barrier. Astrocytes are also linked to the pathophysiology of various neurodegenerative diseases. Astrocyte function and organization are tightly regulated by interactions mediated by the extracellular matrix (ECM). Engineered hydrogels can mimic key aspects of the ECM and can allow for systematic studies of ECM-related factors that govern astrocyte behaviour. In this study, we explore the interactions between neuroblastoma (SH-SY5Y) and glioblastoma (U87) cell lines and human fetal primary astrocytes (FPA) with a modular hyaluronan-based hydrogel system. Morphological analysis reveals that FPA have a higher degree of interactions with the hyaluronan-based gels compared to the cell lines. This interaction is enhanced by conjugation of cell-adhesion peptides (cRGD and IKVAV) to the hyaluronan backbone. These effects are retained and pronounced in 3D bioprinted structures. Bioprinted FPA using cRGD functionalized hyaluronan show extensive and defined protrusions and multiple connections between neighboring cells. Possibilities to tailor and optimize astrocyte-compatible ECM-mimicking hydrogels that can be processed by means of additive biofabrication can facilitate the development of advanced tissue and disease models of the central nervous system.

Nanocellulose composite wound dressings for real-time pH wound monitoring.

The skin is the largest organ of the human body. Wounds disrupt the functions of the skin and can have catastrophic consequences for an individual resulting in significant morbidity and mortality. Wound infections are common and can substantially delay healing and can result in non-healing wounds and sepsis. Early diagnosis and treatment of infection reduce risk of complications and support wound healing. Methods for monitoring of wound pH can facilitate early detection of infection. Here we show a novel strategy for integrating pH sensing capabilities in state-of-the-art hydrogel-based wound dressings fabricated from bacterial nanocellulose (BC). A high surface area material was developed by self-assembly of mesoporous silica nanoparticles (MSNs) in BC. By encapsulating a pH-responsive dye in the MSNs, wound dressings for continuous pH sensing with spatiotemporal resolution were developed. The pH responsive BC-based nanocomposites demonstrated excellent wound dressing properties, with respect to conformability, mechanical properties, and water vapor transmission rate. In addition to facilitating rapid colorimetric assessment of wound pH, this strategy for generating functional BC-MSN nanocomposites can be further be adapted for encapsulation and release of bioactive compounds for treatment of hard-to-heal wounds, enabling development of novel wound care materials.

Self-Assembly of Chiro-Optical Materials from Nonchiral Oligothiophene-Porphyrin Derivatives and Random Coil Synthetic Peptides.

Biomimetic chiral optoelectronic materials can be utilized in electronic devices, biosensors and artificial enzymes. Herein, this work reports the chiro-optical properties and architectural arrangement of optoelectronic materials generated from self-assembly of initially nonchiral oligothiophene-porphyrin derivatives and random coil synthetic peptides. The photo-physical- and structural properties of the materials were assessed by absorption-, fluorescence- and circular dichroism spectroscopy, as well as dynamic light scattering, scanning electron microscopy and theoretical calculations. The materials display a three-dimensional ordered helical structure and optical activity that are observed due to an induced chirality of the optoelectronic element upon interaction with the peptide. Both these properties are influenced by the chemical composition of the oligothiophene-porphyrin derivative, as well as the peptide sequence. We foresee that our findings will aid in developing self-assembled optoelectronic materials with dynamic architectonical accuracies, as well as offer the possibility to generate the next generation of materials for a variety of bioelectronic applications.

Plantaricin NC8 αß rapidly and efficiently inhibits flaviviruses and SARS-CoV-2 by disrupting their envelopes.

Potent broad-spectrum antiviral agents are urgently needed to combat existing and emerging viral infections. This is particularly important considering that vaccine development is a costly and time consuming process and that viruses constantly mutate and render the vaccine ineffective. Antimicrobial peptides (AMP), such as bacteriocins, are attractive candidates as antiviral agents against enveloped viruses. One of these bacteriocins is PLNC8 αß, which consists of amphipathic peptides with positive net charges that display high affinity for negatively charged pathogen membrane structures, including phosphatidylserine rich lipid membranes of viral envelopes. Due to the morphological and physiological differences between viral envelopes and host cell plasma membranes, PLNC8 αß is thought to have high safety profile by specifically targeting viral envelopes without effecting host cell membranes. In this study, we have tested the antiviral effects of PLNC8 αß against the flaviviruses Langat and Kunjin, coronavirus SARS-CoV-2, influenza A virus (IAV), and human immunodeficiency virus-1 (HIV-1). The concentration of PLNC8 αß that is required to eliminate all the infective virus particles is in the range of nanomolar (nM) to micromolar (µM), which is surprisingly efficient considering the high content of cholesterol (8-35%) in their lipid envelopes. We found that viruses replicating in the endoplasmic reticulum (ER)/Golgi complex, e.g. SARS-CoV-2 and flaviviruses, are considerably more susceptible to PLNC8 αß, compared to viruses that acquire their lipid envelope from the plasma membrane, such as IAV and HIV-1. Development of novel broad-spectrum antiviral agents can significantly benefit human health by rapidly and efficiently eliminating infectious virions and thereby limit virus dissemination and spreading between individuals. PLNC8 αß can potentially be developed into an effective and safe antiviral agent that targets the lipid compartments of viral envelopes of extracellular virions, more or less independent of virus antigenic mutations, which faces many antiviral drugs and vaccines.

Intranasal Coronavirus SARS-CoV-2 Immunization with Lipid Adjuvants Provides Systemic and Mucosal Immune Response against SARS-CoV-2 S1 Spike and Nucleocapsid Protein.

In this preclinical two-dose mucosal immunization study, using a combination of S1 spike and nucleocapsid proteins with cationic (N3)/or anionic (L3) lipids were investigated using an intranasal delivery route. The study showed that nasal administration of low amounts of antigens/adjuvants induced a primary and secondary immune response in systemic IgG, mIL-5, and IFN-gamma secreting T lymphocytes, as well as humoral IgA in nasal and intestinal mucosal compartments. It is believed that recipients will benefit from receiving a combination of viral antigens in promoting a border immune response against present and evolving contagious viruses. Lipid adjuvants demonstrated an enhanced response in the vaccine effect. This was seen in the significant immunogenicity effect when using the cationic lipid N3. Unlike L3, which showed a recognizable effect when administrated at a slightly higher concentration. Moreover, the findings of the study proved the efficiency of an intranasally mucosal immunization strategy, which can be less painful and more effective in enhancing the respiratory tract immunity against respiratory infectious diseases.

Peptide-Folding Triggered Phase Separation and Lipid Membrane Destabilization in Cholesterol-Rich Lipid Vesicles.

Liposome-based drug delivery systems are widely used to improve drug pharmacokinetics but can suffer from slow and unspecific release of encapsulated drugs. Membrane-active peptides, based on sequences derived or inspired from antimicrobial peptides (AMPs), could offer means to trigger and control the release. Cholesterol is used in most liposomal drug delivery systems (DDS) to improve the stability of the formulation, but the activity of AMPs on cholesterol-rich membranes tends to be very low, complicating peptide-triggered release strategies. Here, we show a de novo designed AMP-mimetic peptide that efficiently triggers content release from cholesterol-containing lipid vesicles when covalently conjugated to headgroup-functionalized lipids. Binding to vesicles induces peptide folding and triggers a lipid phase separation, which in the presence of cholesterol results in high local peptide concentrations at the lipid bilayer surface and rapid content release. We anticipate that these results will facilitate the development of peptide-based strategies for controlling and triggering drug release from liposomal drug delivery systems.

Bioorthogonally Cross-Linked Hyaluronan-Laminin Hydrogels for 3D Neuronal Cell Culture and Biofabrication.

Laminins (LNs) are key components in the extracellular matrix of neuronal tissues in the developing brain and neural stem cell niches. LN-presenting hydrogels can provide a biologically relevant matrix for the 3D culture of neurons toward development of advanced tissue models and cell-based therapies for the treatment of neurological disorders. Biologically derived hydrogels are rich in fragmented LN and are poorly defined concerning composition, which hampers clinical translation. Engineered hydrogels require elaborate and often cytotoxic chemistries for cross-linking and LN conjugation and provide limited possibilities to tailor the properties of the materials. Here a modular hydrogel system for neural 3D cell cultures, based on hyaluronan and poly(ethylene glycol), that is cross-linked and functionalized with human recombinant LN-521 using bioorthogonal copper-free click chemistry, is shown. Encapsulated human neuroblastoma cells demonstrate high viability and grow into spheroids. Long-term neuroepithelial stem cells (lt-NES) cultured in the hydrogels can undergo spontaneous differentiation to neural fate and demonstrate significantly higher viability than cells cultured without LN. The hydrogels further support the structural integrity of 3D bioprinted structures and maintain high viability of bioprinted and syringe extruded lt-NES, which can facilitate biofabrication and development of cell-based therapies.

Plantaricin NC8 αß prevents Staphylococcus aureus-mediated cytotoxicity and inflammatory responses of human keratinocytes.

Multidrug resistance bacteria constitue an increasing global health problem and the development of novel therapeutic strategies to face this challenge is urgent. Antimicrobial peptides have been proven as potent agents against pathogenic bacteria shown by promising in vitro results. The aim of this study was to characterize the antimicrobial effects of PLNC8 αß on cell signaling pathways and inflammatory responses of human keratinocytes infected with S. aureus. PLNC8 αß did not affect the viability of human keratinocytes but upregulated several cytokines (IL-1ß, IL-6, CXCL8), MMPs (MMP1, MMP2, MMP9, MMP10) and growth factors (VEGF and PDGF-AA), which are essential in cell regeneration. S. aureus induced the expression of several inflammatory mediators at the gene and protein level and PLNC8 αß was able to significantly suppress these effects. Intracellular signaling events involved primarily c-Jun via JNK, c-Fos and NFκB, suggesting their essential role in the initiation of inflammatory responses in human keratinocytes. PLNC8 αß was shown to modulate early keratinocyte responses, without affecting their viability. The peptides have high selectivity towards S. aureus and were efficient at eliminating the bacteria and counteracting their inflammatory and cytotoxic effects, alone and in combination with low concentrations of gentamicin. We propose that PLNC8 αß may be developed to combat infections caused by Staphylococcus spp.

Coiled coil-based therapeutics and drug delivery systems.

Coiled coils are characterized by an arrangement of two or more α-helices into a superhelix and one of few protein motifs where the sequence-to-structure relationship to a large extent have been decoded and understood. The abundance of both natural and de novo designed coil coils provides a rich molecular toolbox for self-assembly of elaborate bespoke molecular architectures, nanostructures, and materials. Leveraging on the numerous possibilities to tune both affinities and preferences for polypeptide oligomerization, coiled coils offer unique possibilities to design modular and dynamic assemblies that can respond in a predictable manner to biomolecular interactions and subtle physicochemical cues. In this review, strategies to use coiled coils in design of novel therapeutics and advanced drug delivery systems are discussed. The applications of coiled coils for generating drug carriers and vaccines, and various aspects of using coiled coils for controlling and triggering drug release, and for improving drug targeting and drug uptake are described. The plethora of innovative coiled coil-based molecular systems provide new knowledge and techniques for improving efficacy of existing drugs and can facilitate development of novel therapeutic strategies.

Tyrosine Side-Chain Functionalities at Distinct Positions Determine the Chirooptical Properties and Supramolecular Structures of Pentameric Oligothiophenes.

Control over the photophysical properties and molecular organization of π-conjugated oligothiophenes is essential to their use in organic electronics. Herein we synthesized and characterized a variety of anionic pentameric oligothiophenes with different substitution patterns of L- or D-tyrosine at distinct positions along the thiophene backbone. Spectroscopic, microscopic, and theoretical studies of L- or D-tyrosine substituted pentameric oligothiophene conjugates revealed the formation of optically active π-stacked self-assembled aggregates under acid conditions. The distinct photophysical characteristics, as well as the supramolecular structures of the assemblies, were highly influenced by the positioning of the L- or D-tyrosine moieties along the thiophene backbone. Overall, the obtained results clearly demonstrate how fundamental changes in the position of the enantiomeric side-chain functionalities greatly affect the optical properties as well as the architecture of the self-assembled supramolecular structures.

Author Correction: Plantaricin NC8 αß exerts potent antimicrobial activity against Staphylococcus spp. and enhances the effects of antibiotics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dynamic peptide-folding mediated biofunctionalization and modulation of hydrogels for 4D bioprinting.

Hydrogels are used in a wide range of biomedical applications, including three-dimensional (3D) cell culture, cell therapy and bioprinting. To enable processing using advanced additive fabrication techniques and to mimic the dynamic nature of the extracellular matrix (ECM), the properties of the hydrogels must be possible to tailor and change over time with high precision. The design of hydrogels that are both structurally and functionally dynamic, while providing necessary mechanical support is challenging using conventional synthesis techniques. Here, we show a modular and 3D printable hydrogel system that combines a robust but tunable covalent bioorthogonal cross-linking strategy with specific peptide-folding mediated interactions for dynamic modulation of cross-linking and functionalization. The hyaluronan-based hydrogels were covalently cross-linked by strain-promoted alkyne-azide cycloaddition using multi-arm poly(ethylene glycol). In addition, a de novo designed helix-loop-helix peptide was conjugated to the hyaluronan backbone to enable specific peptide-folding modulation of cross-linking density and kinetics, and hydrogel functionality. An array of complementary peptides with different functionalities was developed and used as a toolbox for supramolecular tuning of cell-hydrogel interactions and for controlling enzyme-mediated biomineralization processes. The modular peptide system enabled dynamic modifications of the properties of 3D printed structures, demonstrating a novel route for design of more sophisticated bioinks for four-dimensional bioprinting.

Plantaricin NC8 αß exerts potent antimicrobial activity against Staphylococcus spp. and enhances the effects of antibiotics.

The use of conventional antibiotics has substantial clinical efficacy, however these vital antimicrobial agents are becoming less effective due to the dramatic increase in antibiotic-resistant bacteria. Novel approaches to combat bacterial infections are urgently needed and bacteriocins represent a promising alternative. In this study, the activities of the two-peptide bacteriocin PLNC8 αß were investigated against different Staphylococcus spp. The peptide sequences of PLNC8 α and ß were modified, either through truncation or replacement of all L-amino acids with D-amino acids. Both L- and D-PLNC8 αß caused rapid disruption of lipid membrane integrity and were effective against both susceptible and antibiotic resistant strains. The D-enantiomer was stable against proteolytic degradation by trypsin compared to the L-enantiomer. Of the truncated peptides, ß1-22, ß7-34 and ß1-20 retained an inhibitory activity. The peptides diffused rapidly (2 min) through the bacterial cell wall and permeabilized the cell membrane, causing swelling with a disorganized peptidoglycan layer. Interestingly, sub-MIC concentrations of PLNC8 αß substantially enhanced the effects of different antibiotics in an additive or synergistic manner. This study shows that PLNC8 αß is active against Staphylococcus spp. and may be developed as adjuvant in combination therapy to potentiate the effects of antibiotics and reduce their overall use.

Mechanism and Kinetics of Lipid Bilayer Formation in Solid-State Nanopores.

Solid-state nanopores provide a highly versatile platform for rapid electrical detection and analysis of single molecules. Lipid bilayer coating of the nanopores can reduce nonspecific analyte adsorption to the nanopore sidewalls and increase the sensing selectivity by providing possibilities for tethering specific ligands in a cell-membrane mimicking environment. However, the mechanism and kinetics of lipid bilayer formation from vesicles remain unclear in the presence of nanopores. In this work, we used a silicon-based, truncated pyramidal nanopore array as the support for lipid bilayer formation. Lipid bilayer formation in the nanopores was monitored in real time by the change in ionic current through the nanopores. Statistical analysis revealed that a lipid bilayer is formed from the instantaneous rupture of individual vesicle upon adsorption in the nanopores, differing from the generally agreed mechanism that lipid bilayer forms at a high vesicle surface coverage on a planar support. The dependence of the lipid bilayer formation process on the applied bias, vesicle size, and concentration was systematically studied. In addition, the nonfouling properties of the lipid bilayer coated nanopores were demonstrated during long single-stranded DNA translocation through the nanopore array. The findings indicate that the lipid bilayer formation process can be modulated by introducing nanocavities intentionally on the planar surface to create active sites or changing the vesicle size and concentration.