Three-Dimensional Bacterial Motions near a Surface Investigated by Digital Holographic Microscopy: Effect of Surface Stiffness.

Surface stiffness plays a critical role in bacterial adhesion, but the mechanism is unclear since the bacterial motion before adhesion is overlooked. Herein, the three-dimensional (3D) motions of Escherichia coli and Pseudonomas sp. nov 776 onto poly(dimethylsiloxane) (PDMS) surfaces with varying stiffness before adhering were monitored by digital holographic microscopy (DHM). As Young's modulus (E) of the PDMS surface decreases from 278.1 to 3.4 MPa, the adhered E. coli and Pseudonomas sp. decrease in number by 40.4 and 34.9%, respectively. Atomic force microscopy (AFM) measurements show that the adhesion force of bacteria to the surface declines with the decreased surface stiffness. In contrast, a nontumbling mutant of adhered E. coli (HCB1414 with the adaptive function being partially deficient) decreases much less (by 18.4%). On the other hand, the tumble frequency (Ft) of E. coli HCB1 and flick frequency (Ff) of Pseudomonas sp. increase as the surface stiffness decreases, and the motion bias (Bθ) of Pseudomonas sp. also increases. These facts clearly indicate that the bacteria have adapted responses to the surface stiffness. RNA sequencing (RNA-seq) reveals that the downregulated Cph2 and CsrA as well as the upregulated GcvA of swimming E. coli HCB1 in bulk near the softer surface promote the bacterial motility.

A mobile precursor determines protein resistance on nanostructured surfaces.

Biomaterials are often engineered with nanostructured surfaces to control interactions with proteins and thus regulate their biofunctions. However, the mechanism of how nanostructured surfaces resist or attract proteins together with the underlying design rules remains poorly understood at a molecular level, greatly limiting attempts to develop high-performance biomaterials and devices through the rational design of nanostructures. Here, we study the dynamics of nonspecific protein adsorption on block copolymer nanostructures of varying adhesive domain areas in a resistant matrix. Using surface plasmon resonance and single molecule tracking techniques, we show that weakly adsorbed proteins with two-dimensional diffusivity are critical precursors to protein resistance on nanostructured surfaces. The adhesive domain areas must be more than tens or hundreds of times those of the protein footprints to slow down the 2D-mobility of the precursor proteins for their irreversible adsorption. This precursor model can be used to quantitatively analyze the kinetics of nonspecific protein adsorption on nanostructured surfaces. Our method is applicable to precisely manipulate protein adsorption and resistance on various nanostructured surfaces, e.g., amphiphilic, low-surface-energy, and charged nanostructures, for the design of protein-compatible materials.

Landing Dynamics of Swimming Bacteria on a Polymeric Surface: Effect of Surface Properties.

Landing of bacteria for adhesion on a surface is a common phenomenon in our life. However, how surface properties are involved in this process remains largely unclear. Using digital holographic microscopy, we investigated the three-dimensional motions of flagellate Escherichia coli swimming near polymeric surfaces with different properties in aqueous solution before adhesion. We monitored the bacteria landing dynamics, which shows that the density distribution, the probability, and the orientation for collisions of the bacteria are determined by their motility but are slightly affected by the surface properties. However, surface hydrophobicity reduces the near-wall velocity of the bacteria through collisions and slightly increases the collision duration. This promotes the landing and adhesion of bacteria. By contrast, most bacteria collide with the surface using their flagella, which resist adhesion.

Three-Dimensional Bacterial Behavior near Dynamic Surfaces Formed by Degradable Polymers.

Understanding the behavior of bacteria near biodegradable surfaces is critical for the development of biomedical and antibiofouling materials. By using digital holographic microscopy (DHM), we investigated the three-dimensional (3D) behavior of Escherichia coli and Pseudomonas sp. in lipase-containing aquatic environments near dynamic surfaces constructed by biodegradable poly(ε-caprolactone) (PCL)-based polymers in real time. As the enzymatic degradation rate increases, the percentage of near-surface subdiffusive bacteria and consequently, the irreversible adhesion decreases. Atomic force microscopy (AFM) measurements reveal that the adhesion force between bacteria and the surfaces decreases with an increasing degradation rate. In addition, the degradation products elicit a negative chemotactic response in E. coli, further driving them away from the dynamic surfaces through more frequent tumbling motion. Our study clearly demonstrates that bacterial adhesion can be reduced on dynamic surfaces formed by degradable polymers.

Measuring the Surface-Surface Interactions Induced by Serum Proteins in a Physiological Environment.

In this work, we applied total internal reflection microscopy (TIRM) to directly measure the interactions between three different kinds of macroscopic surfaces: namely bare polystyrene (PS) particle and bare silica surface (bare-PS/bare-silica), PS particle and silica surfaces both coated with bovine serum albumin (BSA) (BSA-PS/BSA-silica), and PS particle and silica surfaces both modified with polyethylene glycol (PEG) (PEG-PS/PEG-silica) polymers, in phosphate buffer solution (PBS) and fetal bovine serum (FBS). Our results showed that in PBS, all the bare-PS, BSA-PS, and PEG-PS particles were irreversibly deposited onto the bare silica surface or surfaces coated either with BSA or PEG. However, in FBS, the interaction potentials between the particle and surface exhibited both free-diffusing particle and stuck particle profiles. Dynamic light scattering (DLS) and elliposmeter measurements indicated that there was a layer of serum proteins adsorbed on the PS particle and silica surface. TIRM measurement revealed that such adsorbed serum proteins can mediate the surface-surface interactions by providing additional stabilization under certain conditions, but also promoting bridging effect between the two surfaces. The measured potential profile of the stuck particle in FBS thus was much wider than in PBS. These quantitative measurements provide insights that serum proteins adsorbed onto surfaces can regulate surface-surface interactions, thus leading to unique moving behavior and stability of colloidal particles in the serum environment.

Measurements of long-range interactions between protein-functionalized surfaces by total internal reflection microscopy.

Understanding the interaction between protein-functionalized surfaces is an important subject in a variety of protein-related processes, ranging from coatings for biomedical implants to targeted drug carriers and biosensors. In this work, utilizing a total internal reflection microscope (TIRM), we have directly measured the interactions between micron-sized particles decorated with three types of common proteins concanavalin A (ConA), bovine serum albumin (BSA), lysozyme (LYZ), and glass surface coated with soy proteins (SP). Our results show that the protein adsorption greatly affects the charge property of the surfaces, and the interactions between those protein-functionalized surfaces depend on solution pH values. At pH 7.5-10.0, all these three protein-functionalized particles are highly negatively charged, and they move freely above the negatively charged SP-functionalized surface. The net interaction between protein-functionalized surfaces captured by TIRM was found as a long-range, nonspecific double-layer repulsion. When pH was decreased to 5.0, both protein-functionalized surfaces became neutral and double-layer repulsion was greatly reduced, resulting in adhesion of all three protein-functionalized particles to the SP-functionalized surface due to the hydrophobic attraction. The situation is very different at pH = 4.0: BSA-decorated particles, which are highly charged, can move freely above the SP-functionalized surfaces, while ConA- and LYZ-decorated particles can only move restrictively in a limited range. Our results quantify these nonspecific kT-scale interactions between protein-functionalized surfaces, which will enable the design of surfaces for use in biomedical applications and study of biomolecular interactions.

3D monitoring of the microphase separations inside the intraocular lens.

Glistenings often occur after implanting the intraocular lens (IOL) due to the formation of numerous microvacuoles (MVs) and may lead to deterioration of vision quality. Previous studies showed the formation of MVs was associated with the hydrophobicity of IOL materials. Yet, the mechanism remains an open question due to the complexity of IOL polymer networks. In this study, two commercialized IOLs with similar hydrophobicity are found distinct in the formation of MVs. The 3D growth kinetics of MVs during cooling processes are captured for the first time by digital holographic microscopy (DHM) and the components of MVs are measured by DHM and Raman spectroscopy. The results reveal that the growth of MVs stems from the microphase separation of water and surrounding IOL polymers. A polymer swelling model is thus proposed to describe the microphase separation process which is found dependent on the elasticity of IOL polymer networks. The total volume of MVs is determined by the IOL hydrophobicity, while the elastic force of IOL polymer networks determines the number density and size of MVs. This study demonstrates an approach for characterizing the phase separation of crosslinked polymeric materials in biosystems and sheds lights on the refinement of IOL materials. STATEMENT OF SIGNIFICANCE: Glistenings due to the formation of numerous microvacuoles (MVs) in intraocular lens (IOL) can occur after IOL implantation, which may induce poor quality of vision. However, the underlying mechanism of MVs formation is still an open question. This study establishes an in-situ 3D imaging platform to monitor growth kinetics of the MVs in IOLs, which allows to uncover the mechanism of glistenings formation resulting from the microphase separation. The findings imply the material hydrophobicity influences the total volume of MVs, while the local elasticity of IOL polymer networks determines the number density and the size of MVs. This study offers a new approach for characterizing phase separation in crosslinking biosystems and sheds lights on the refinement of IOL materials.

Interactions between solid surfaces with preadsorbed poly(ethylenimine) (PEI) layers: effect of unadsorbed free PEI chains.

Poly(ethylenimine) (PEI) polyelectrolytes have been widely used to tune the stability, rheology, or adhesion properties of colloidal suspensions due to their strong tendency to adsorb to solid surfaces. They have also gained importance as gene carriers in biomedical applications, in which the anionic DNA chains are complexed and condensed to form PEI/DNA polyplexes. Some reported literatures have recently shown that the overdosed PEI chains, which are free in the solution mixture, also play a vital role in promoting the gene transfection, but the reason is unclear. In this work, we present the results of using total internal reflection microscopy (TIRM) to measure the interaction forces between a Brownian colloidal sphere and a flat glass plate in the presence of overdosed free PEI cationic chains, when both surfaces were saturated adsorbed with the PEI chains. The colloidal sphere preadsorbed with PEI chains was chosen to mimic the PEI/DNA polyplex. Results for the potential energy of interaction measured for model polyplex (e.g., PEI-coated sphere) interacting with a PEI-coated glass surface in the presence of overdosed free PEI chains at various pH values and salt concentrations were presented. As can be shown by direct force measurements, the interaction potentials in NaCl salt solution are dominated by repulsive forces originating from diffuse layer overlap and gravitational attraction. However, the presence of free PEI chains in the solution mixture produces a long-ranged (>60 nm) attractive force between two PEI-coated surfaces with the range and magnitude tunable by pH value, PEI, and salt concentrations. The possible mechanisms of this long-ranged attractive force are discussed. A better understanding of this free PEI-induced attractive force will be useful in the development of improved PEI/DNA polyplexes systems for biomedical applications.

pH Window for High Selectivity of Ionizable Antimicrobial Polymers toward Bacteria.

Antimicrobial polymers exhibit great potential for treating drug-resistant bacteria; however, designing antimicrobial polymers that can selectively kill bacteria and cause relatively low toxicity to normal tissues/cells remains a key challenge. Here, we report a pH window for ionizable polymers that exhibit high selectivity toward bacteria. Ionizable polymer PC6A showed the greatest selectivity (131.6) at pH 7.4, exhibiting low hemolytic activity and high antimicrobial activity against bacteria, whereas a very high or low protonation degree (PD) produced relatively low selectivity (≤35.6). Bactericidal mechanism of PC6A primarily comprised membrane lysis without inducing drug resistance even after consecutive incubation for 32 passages. Furthermore, PC6A demonstrated synergistic effects in combination with antibiotics at pH 7.4. Hence, this study provides a strategy for designing selective antimicrobial polymers.

Betulin-Constituted Multiblock Amphiphiles for Broad-Spectrum Protein Resistance.

Multiblock-like amphiphilic polyurethanes constituted by poly(ethylene oxide) and biosourced betulin are designed for antifouling and synthesized by a convenient organocatalytic route comprising tandem chain-growth and step-growth polymerizations. The doping density of betulin (DB) in the polymer chain structure is readily varied by a mixed-initiator strategy. The spin-coated polymer films exhibit unique nanophase separation and protein resistance behaviors. Higher DB leads to enhanced surface hydrophobicity and, unexpectedly, improved protein resistance. It is found that the surface holds molecular-level heterogeneity when DB is substantially high due to restricted phase separation; therefore, broad-spectrum protein resistance is achieved despite considerable surface hydrophobicity. As DB decreases, the distance between adjacent betulin units increases so that hydrophobic nanodomains are formed, which provide enough landing areas for relatively small-sized proteins to adsorb on the surface.

Novel differential refractometry study of the enzymatic degradation kinetics of poly(ethylene oxide)-b-poly(epsilon-caprolactone) particles dispersed in water.

A poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PEO-b-PCL) diblock copolymer was micronized into small micelle-like particles (approximately 80 nm) via dialysis-induced microphase inversion. The enzymatic biodegradation of the PCL portion of these particles in water was in situ investigated inside a recently developed novel differential refractometer. Using this refractometry method, we were able to monitor the real-time biodegradation via the refractive index change (Deltan) of the dispersion because Deltan is directly proportional to the particle mass concentration. We found that the degradation rate is proportional to either the polymer or enzyme concentration. Our results directly support previous speculation on the basis of the light-scattering data that the biodegradation follows the first-order kinetics for a given enzyme concentration. This study not only leads to a better understanding of the enzymatic biodegradation of PCL, but also demonstrates a novel, rapid, noninvasive, and convenient way to test the degradability of polymers.

[Characterization of the physical properties of biofilms].

It was known that bacteria adhere to surfaces and form sessile colonies called biofilms. Biofilms show potential applications for biodegradation and biocatalysis, whilst they also cause healthy and environmental problems. In particular, they lead to human infections and biofouling problems in industry. Physical properties of biofilms reflect the architecture and mechanical stability of biofilms that are highly related to their resistance to environmental challenges and their survival. In this article, we reviewed the physical properties involved in the development of biofilms and the related characterization techniques. The surface adhesion of bacteria plays a crucial role in the biofilm formation, which is determined by the motion of bacteria near a surface as well as the interaction between the bacteria and the surface. As far as the biofilms become mature, they behave like a polymer glassy material revealed by rheological measurements.

Long-range interactions between protein-coated particles and POEGMA brush layers in a serum environment.

Hydrophilic poly[oligo(ethylene glycol) methyl methacrylate] (POEGMA) brush layers with different thickness and graft densities were prepared by surface-initiated atom transfer radical polymerization (SI-ATRP) to construct a model surface to examine protein-surface interactions in a serum environment. The thickness of the POEGMA brush layers could be well controlled by the polymerization time and density of the immobilized initiators. The interactions between these brush-modified surfaces and the protein-coated polystyrene (PS) particles in newborn calf serum (NBCS) environment were then measured by total internal reflection microscopy (TIRM). In addition, protein adsorption properties onto the polymer brush surface layers were examined by atomic force microscopy (AFM). Relatively large amounts of protein adsorbed to short (4nm and 9nm-thick) POEGMA-coated surfaces or surfaces grafted with a low density of polymer chains. It was considered that shorter polymer chains or chains with low grafted density cannot fully cover the surfaces, proteins in serum could directly interact with the material surface and then deposited to form an adsorbed layer. The TIRM measurements showed that such adsorbed protein layer could mediate the interactions between the two surfaces by generating steric or bridging forces, resulting in different interaction potentials. Some particles were freely diffusing, some experienced intermittent diffusion and more than 50% of particles were irreversibly deposited to the surfaces covered by short polymer brushes. However, for longer (17 and 30nm-thick) POEGMA brush layer surfaces, material surface would be sufficiently covered by the dense coating and the first step of protein adsorption on surface was avoided. TIRM measurements showed that around 95% of the protein-coated particles could freely move in the serum and no attractive force between two surfaces was detected. The steric repulsion generated from the long POEGMA brush layer in the swollen state was long-range and strong so that the protein adsorption is very unlikely. These results concluded that the adsorbed protein layer on POEGMA surfaces plays an important role in regulating the interaction between protein-coated particles and POEGMA surfaces which are highly repellent toward protein adsorption.

Microparticle templating as a route to nanoscale polymer vesicles with controlled size distribution for anticancer drug delivery.

Polymer vesicles are self-assembled shells of amphiphilic block copolymers (BCPs) that have attracted tremendous interest due to their encapsulation ability and intracellular delivery of therapeutic agents. However, typical processes for the formation of polymer vesicles lead to ensembles of structures with a broad size distribution (from nanometer to micrometer scale) which result in a limitation for efficient cellular uptake. In this study, we present a simple and efficient approach for the fabrication of polymer vesicles with uniform nanoscale dimensions from template formation of electrosprayed particles in a high throughput manner. First, electrospraying was applied to produce micrometer-sized templates of a block copolymer before polymer vesicles were formed from the pre-prepared microparticles via rehydration. Four different biocompatible diblock and triblock copolymers were used to successfully fabricate polymer vesicles with uniform size around 150nm using this approach. Furthermore, we encapsulate anticancer drug doxorubicin (DOX) within the polymer vesicles via this method. The kinetics of cellular uptake (HeLa cell) and intracellular distribution of DOX-loaded polymer vesicles have been quntified and monitored by flow cytometry and confocal microscopy, respectively. The results show that our new method provides a promising way to fabricate drug-loaded polymer vesicles with controllable nanoscale size for intracellular anticancer drug delivery.

Investigation of cell behaviors on thermo-responsive PNIPAM microgel films.

The use of poly(N-isopropylacrylamide) (PNIPAM) as building blocks for engineering responsive coatings and their potential use as switchable substrates such as biosensors have attracted great attention in recent years. However, few studies have been conducted regarding the cell behaviors and the related mechanism on thermos-responsive surfaces consisting of PNIPAM microgel particles. In this work, monodisperse PNIPAM microgels were synthesized and used to prepare PINPAM microgel films. Uniform microgel surfaces can be fabricated by drop-coating with the precoating of a polyethylenimine (PEI) layer. Cell experiments indicate that unlike PNIPAM polymer brushes reported with controllable detachment ability, HepG2, which is a human liver carcinoma cell line, remains adherent on the microgel films upon cooling. Surface plasmon resonance (SPR) experiments show an irreversible adsorption of serum proteins on the microgel surface upon cooling, whose adsorption is a prerequisite of cell adhesion during cell culture. This fact may account for the irreversible adhesion of HepG2 cells.

Direct measurements of particle-surface interactions in aqueous solutions with total internal reflection microscopy.

Non-covalent intermolecular forces, such as van der Waals, electrostatic, steric, and hydrophobic interactions, have played essential roles in determining the association, aggregation, adhesion and sedimentation processes of colloidal particles, surfactant micelles, and macromolecules, in solutions and biological systems. These interaction forces, however, are normally weak (

An active one-particle microrheometer: incorporating magnetic tweezers to total internal reflection microscopy.

We present a novel microrheometer by incorporating magnetic tweezers in the total internal reflection microscopy (TIRM) that enables measuring of viscoelastic properties of materials near solid surface. An evanescent wave generated by a solid∕liquid interface in the TIRM is used as the incident light source in the microrheometer. When a probe particle (of a few micrometers diameter) moves near the interface, it can interact with the evanescent field and reflect its position with respect to the interface by the scattered light intensity. The exponential distance dependence of the evanescent field, on the one hand, makes this technique extremely sensitive to small changes from z-fluctuations of the probe (with a resolution of several nanometers), and on the other, it does not require imaging of the probe with high lateral resolution. Another distinct advantage is the high sensitivity in determining the z position of the probe in the absence of any labeling. The incorporated magnetic tweezers enable us to effectively manipulate the distance of the embedded particle from the interface either by a constant or an oscillatory force. The force ramp is easy to implement through a coil current ramp. In this way, the local viscous and elastic properties of a given system under different confinements can therefore be measured by resolving the near-surface particle motion. To test the feasibility of applying this microrheology to soft materials, we measured the viscoelastic properties of sucrose and poly(ethylene glycol) solutions and compared the results to bulk rheometry. In addition, we applied this technique in monitoring the structure and properties of deformable microgel particles near the flat surface.