Adhesive Sulfabetaine Polymer Hydrogels for the Sandwich Cell Culture.

Sandwich culture systems are techniques that cultivate cells by sandwiching them between the top and bottom substrates. Since the substrates can be separated, the system is expected to be applied to the construct layering of patterned cells and to the isolation of stacked cells. In this study, we prepared hydrogels composed of zwitterionic sulfabetaine polymers, poly[2-(2-(methacryloyloxyethyl)dimethylammonio)ethyl-1-sulfate] (PZBMA). The ZBMA homopolymers have been shown to form aggregates in aqueous solutions due to their intermolecular interactions. The water content of the PZBMA hydrogels in water was ∼70% regardless of N,N'-methylenebis(acrylamide), BIS, content as the cross-linker. The results indicated that the intermolecular interaction contributed more to the swelling behaviors than the chemical cross-linker. However, PZBMA hydrogels with 0.1 mol % BIS showed not only high elongation (∼850%) properties but also high adhesiveness and self-healing properties. When this PZBMA hydrogel was impregnated with collagen and subjected to sandwich culture using Madin-Darby canine kidney (MDCK) cells, a three-dimensional morphology of MDCK cell aggregates was constructed. Such a sulfabetaine hydrogel is expected to be developed for regenerative medicine.

Tim4, a macrophage receptor for apoptotic cells, binds polystyrene microplastics via aromatic-aromatic interactions.

Understanding the interface between microplastics and biological systems will provide new insights into the impacts of microplastics on living organisms. When microplastics enter the body, they are engulfed preferentially by phagocytes such as macrophages. However, it is not fully understood how phagocytes recognize microplastics and how microplastics impact phagocyte functions. In this study, we demonstrate that T cell immunoglobulin mucin 4 (Tim4), a macrophage receptor for phosphatidylserine (PtdSer) on apoptotic cells, binds polystyrene (PS) microparticles as well as multi-walled carbon nanotubes (MWCNTs) through the extracellular aromatic cluster, revealing a novel interface between microplastics and biological systems via aromatic-aromatic interactions. Genetic deletion of Tim4 demonstrated that Tim4 is involved in macrophage engulfment of PS microplastics as well as of MWCNTs. While Tim4-mediated engulfment of MWCNTs causes NLRP3-dependent IL-1ß secretion, that of PS microparticles does not. PS microparticles neither induce TNF-α, reactive oxygen species, nor nitric oxide production. These data indicate that PS microparticles are not inflammatory. The PtdSer-binding site of Tim4 contains an aromatic cluster that binds PS, and Tim4-mediated macrophage engulfment of apoptotic cells, a process called efferocytosis, was competitively blocked by PS microparticles. These data suggest that PS microplastics do not directly cause acute inflammation but perturb efferocytosis, raising concerns that chronic exposure to large amounts of PS microplastics may cause chronic inflammation leading to autoimmune diseases.

Van der Waals interactions regulating the hydration of 2-methacryloyloxyethyl phosphorylcholine, the constructing monomer of biocompatible polymers.

Van der Waals (VDW) interactions provide fantastic properties for biological systems that function at room temperature. The VDW interaction, which primarily contributes to weak hydrogen bonding, is expected to play a key role in regulating hydrophobic hydration to express the biologically inert biocompatible function of polymerized MPCs (2-methacryloyloxyethyl phosphorylcholine). This report explores at the molecular level the biologically inert function of polymerized MPCs through an array of vibrational spectroscopic and computational characterization of MPC monomers, as temperature-dependent change of intramolecular weak hydrogen bonding. Synchrotron Fourier transform infrared microspectroscopy and terahertz time-domain spectroscopy were used to investigate temperature-dependent spectral changes in the low frequency vibrations of the MPC over the temperature range from cryogenic to room temperature, and the results were analysed by highly reliable well-established density functional theory (DFT) calculations. Complicated spectral features in the low frequency energy region and the uncertain conformations of the MPC in the amorphous powder state are clearly resolved under a polarizable continuum model and dispersion correction to pure DFT calculations.

Sulfobetaine polymers for effective permeability into multicellular tumor spheroids (MCTSs).

Multicellular tumor spheroids (MCTSs) are attractive for drug screening before animal tests because they emulate an in vivo microenvironment. The permeability of the MCTSs and tumor tissues towards the candidate drugs is not sufficient even though the drugs can penetrate monolayer cultured cells; therefore, nanocarriers are required to enhance permeability and deliver drugs. In this study, we prepared zwitterionic polymers of sulfobetaine methacrylates and (meth)acrylamides with or without hydroxy groups between the zwitterions to serve as highly permeable nanocarriers. In the sulfobetaine polymers, poly(2-hydroxy-3-((3-methacrylamidopropyl)dimethylammonio)propane-1-sulfonate), P(OH-MAAmSB), the hydroxy group containing methacrylamide polymer exhibited little cytotoxicity and membrane translocation ability against monolayer cultured cells. Moreover, the excellent permeability of the hepatocyte MCTS enabled P(OH-MAAmSB) to permeate it and reach the center region (∼325 µm in diameter) at approximately 150 s, although poly(trimethyl-2-methacroyloxyethylammonium), a cationic polymer, penetrated just 1 to 2 layers from the periphery. The superior permeability of P(OH-MAAmSB) might be due to its good solubility and side chain conformation. P(OH-MAAmSB) is a promising nanocarrier with membrane translocation and permeability.

Heat-Induced Flower Nanogels of Both Cholesterol End-Capped Poly(N-isopropylacrylamide)s in Water.

Thermoresponsive self-assembled nanogels were conveniently prepared by cholesterol end-capped poly(N-isopropylacrylamide) (PNIPAM) in water. Both cholesterol end-capped PNIPAMs (telelchelic cholesterol PNIPAM, tCH-PNIPAM) formed flower-like nanogels by the self-assembling of four to five polymer chains with multiple domains of cholesterol in water at 20 °C. Meanwhile, one end-group cholesterol-capped PNIPAM (semitelechelic cholesterol PNIPAM, stCH-PNIPAM) was also formed as a nanogel by the self-assembling of 15-20 polymer chains with 3 to 4 cholesterol domains. The hydrophobic cholesterol domains of tCH-PNIPAM nanogels were maintained above the lower critical solution temperature (LCST) of PNIPAM (>32 °C). Differently, the hydrophobic domains of stCH-PNIPAM were disrupted by cholesterol-free PNIPAM chain ends and formed large mesoglobules above the LCST. These transition controls of hydrophilic end-capped smart polymers may open new methodologies to design thermoresponsive nanosystems.

Design of an LCST-UCST-Like Thermoresponsive Zwitterionic Copolymer.

Thermoresponsive polymers that possess both UCST- and LCST-like behaviors have generally been designed using diblock copolymers that are mostly composed of an LCST-like polymer and a UCST polymer. Herein, we prepared an LCST-UCST-type polymer composed of UCST-like thermoresponsive zwitterionic sulfabetaine methacrylate and nonthermoresponsive PEG methacrylate, ZB-PEG, by reversible addition-fragmentation chain transfer (RAFT) copolymerization. By adjusting the PEG composition, ZB-PEG formed a mesoglobule, a microglobule, and the dissociated states in phosphate-buffered saline (PBS). These states were found to be reversible via temperature control. Moreover, this behavior showed high reversibility and succeeded in stabilizing horseradish peroxidase (HRP) in the dilute condition. Such thermoresponsive ZB-PEG can be applied over a wide range of applications in biotechnology and other fields.

Effective Permeation of Anticancer Drugs into Glioblastoma Spheroids via Conjugation with a Sulfobetaine Copolymer.

Three-dimensional cell aggregates (spheroids) are becoming a research focus because their construction is similar to that in vivo microenvironments, enabling the acceleration of drug discovery and reducing the need for animal tests, and other advantages. However, the delivery of drugs to the inside of spheroids is time-consuming and has low efficiency. In this study, we selected a sulfobetaine copolymer that translocates to the cell membrane in monolayer cultured cells as a nanocarrier of anticancer drugs. Doxorubicin (Dox) and 17-demethoxy-17-allylamino geldanamycin (17AAG) were modified to the copolymer of sulfobetaine methacrylate and poly(ethylene glycol) methacrylate, P(SB-PEG), and added to glioblastoma A-172 cell spheroids. Dox-P(SB-PEG) showed fast permeation into A-172 spheroids, and the fluorescence in cells was observed in the center area of the spheroids within 1 h of polymer addition. Conversely, only the outer one to two cell layers of spheroids were observed when Dox was added to the spheroids. Dox-P(SB-PEG) in A-172 spheroids was localized in the mitochondria of each cell and exhibited comparable drug efficacy to that of Dox in growth inhibition assays of A-172 spheroids. Moreover, approximately 10-fold higher drug efficacy in growth inhibition and invasion of A-172 spheroids was found using 17AAG-P(SB-PEG). Conjugating anticancer drugs with P(SB-PEG) is a promising strategy to enhance drug permeation and efficacy against spheroid cells.

Control of Mitochondrial Localization Using Thermoresponsive Sulfobetaine Polymer.

Fast intracellular migration and controlled localization of molecules represent significant challenges for future applications of drug discovery and related fields. In this study, thermoresponsive sulfobetaine polymers with pyridinium cations are evaluated as biocompatible and mitochondria-localizing agents. Among the polymers, poly(3-(4-(2-methacrylamido)ethyl pyridinio-1-yl)propane-1-sulfonate), P(E-PySMAAm)14k (Mn  = 14 000 g mol-1 ) exhibit thermoresponsiveness with an upper critical solution temperature like behavior in cell culture medium containing serum with minimal cytotoxicity. Upon the addition of P(E-PySMAAm)14k to HeLa cells at temperatures above the clearing point at 37 °C, effective localization is observed in mitochondria. However, increased intensity but nonspecific localization is observed below the clearing point at 4 °C. Doxorubicin is conjugated to the P(E-PySMAAm) and achieves effective mitochondrial delivery while maintaining drug efficacy. Such sulfobetaine polymers represent promising tools for intracellular delivery of molecules.

Internalization Mechanisms of Pyridinium Sulfobetaine Polymers Evaluated by Induced Protic Perturbations on Cell Surfaces.

Understanding the interactions of soft nanomatters with cell membranes is particularly important for research into nanocarrier-based drug delivery systems, cell engineering, and subcellular imaging. Most nanoparticles, vesicles, micelles, and polymeric aggregates are internalized into endosomes and, eventually, lysosomes in the cytosol because of energy-dependent endocytic processes. Endocytic uptake substantially limits the access to the cytoplasm where a cargo agent acts. Bypassing the endocytic pathways by direct penetration into plasma membrane barriers would enhance the efficacy of nanomedicines. Some zwitterionic polymer nanoaggregates have been shown to permeate into the cell interior in an energy-independent manner. We have elucidated this phenomenon by observing changes in the biomembrane barrier functions against protons as the smallest indicator and have used these results to further design and develop poly(betaines). In this work, we investigated the translocation mechanisms for a series of zwitterionic poly(methacrylamide) and poly(methacrylate) species bearing a pyridinium propane sulfonate moiety in the monomers. Minor differences in the monomer structures and functional groups were observed to have dramatic effects on the interaction with plasma membranes during translocation. The ability to cross the plasma membrane involves a balance among the betaine dipole-dipole interaction, NH-π interaction, π-π interaction, cation-π interaction, and amide hydrogen bonding. We found that the cell-penetrating polysulfobetaines had limited or no detrimental effect on cell proliferation. Our findings enhance the opportunity to design and synthesize soft nanomatters for cell manipulations by passing across biomembrane partitions.

Assessment of the VDW interaction converting DMAPS from the thermal-motion form to the hydrogen-bonded form.

Assessment of van der Waals (VDW) interactions is fundamental to all of the central quest of structure that regulates the biological function. VDW interactions contributing to intramolecular weak hydrogen bonding are regarded as an important force to regulate the thermal stimuli-sensitive function of sulfobetaine methacrylate, DMAPS. We present here the conversion from the thermal-motion form at room temperature to the weak-hydrogen-bonded form against thermal motion as a terahertz spectral change with a definite isosbestic point from an absorption peak of one form to the other. Vibrational absorptions are used as a probe for assessing VDW interactions in conjunction with highly reliable and well-established density functional theory (DFT) calculations for analysis. Complicated spectral features and uncertain conformations of DMAPS in the amorphous state are clearly resolved under the polarizable continuum model and the dispersion correction for the pure DFT calculations.

Fast and effective mitochondrial delivery of ω-Rhodamine-B-polysulfobetaine-PEG copolymers.

Mitochondrial targeting and entry, two crucial steps in fighting severe diseases resulting from mitochondria dysfunction, pose important challenges in current nanomedicine. Cell-penetrating peptides or targeting groups, such as Rhodamine-B (Rho), are known to localize in mitochondria, but little is known on how to enhance their effectiveness through structural properties of polymeric carriers. To address this issue, we prepared 8 copolymers of 3-dimethyl(methacryloyloxyethyl)ammonium propane sulfonate and poly(ethyleneglycol) methacrylate, p(DMAPS-ran-PEGMA) (molecular weight, 18.0 < M n < 74.0 kg/mol) with two different endgroups. We labeled them with Rho groups attached along the chain or on one of the two endgroups (α or ω). From studies by flow cytometry and confocal fluorescence microscopy of the copolymers internalization in HeLa cells in the absence and presence of pharmacological inhibitors, we established that the polymers cross the cell membrane foremost by translocation and also by endocytosis, primarily clathrin-dependent endocytosis. The most effective mitochondrial entry was achieved by copolymers of M n < 30.0 kg/mol, lightly grafted with PEG chains (< 5 mol %) labeled with Rho in the ω-position. Our findings may be generalized to the uptake and mitochondrial targeting of prodrugs and imaging agents with a similar polymeric scaffold.

SiO2 and TiO2 nanoparticles synergistically trigger macrophage inflammatory responses.

Silicon dioxide (SiO2) nanoparticles (NPs) and titanium dioxide (TiO2) NPs are the most widely used inorganic nanomaterials. Although the individual toxicities of SiO2 and TiO2 NPs have been extensively studied, the combined toxicity of these NPs is much less understood. In this study, we observed unexpected and drastic activation of the caspase-1 inflammasome and production of IL-1ß in mouse bone marrow-derived macrophages stimulated simultaneously with SiO2 and TiO2 NPs at concentrations at which these NPs individually do not cause macrophage activation. Consistent with this, marked lung inflammation was observed in mice treated intratracheally with both SiO2 and TiO2 NPs. In macrophages, SiO2 NPs localized in lysosomes and TiO2 NPs did not; while only TiO2 NPs produced ROS, suggesting that these NPs induce distinct cellular damage leading to caspase-1 inflammasome activation. Intriguingly, dynamic light scattering measurements revealed that, although individual SiO2 and TiO2 NPs immediately aggregated to be micrometer size, the mixture of these NPs formed a stable and relatively monodisperse complex with a size of ~250 nm in the presence of divalent cations. Taken together, these results suggest that SiO2 and TiO2 NPs synergistically induce macrophage inflammatory responses and subsequent lung inflammation. Thus, we propose that it is important to assess the synergistic toxicity of various combinations of nanomaterials.

SR-B1 Is a Silica Receptor that Mediates Canonical Inflammasome Activation.

The inhalation of silica dust is associated with fibrosis and lung cancer, which are triggered by macrophage inflammatory responses; however, how macrophages recognize silica remains largely unknown. Here, we identify by functional expression cloning the class B scavenger receptor SR-B1 as a silica receptor. Through an extracellular α-helix, both mouse and human SR-B1 specifically recognized amorphous and crystalline silica, but not titanium dioxide nanoparticles, latex nanoparticles, or monosodium urate crystals, although all particles exhibited negative surface potentials. Genetic deletion of SR-B1 and masking of SR-B1 by monoclonal antibodies showed that SR-B1-mediated recognition of silica is associated with caspase-1-mediated inflammatory responses in mouse macrophages and human peripheral blood monocytes. Furthermore, SR-B1 was involved in silica-induced pulmonary inflammation in mice. These results indicate that SR-B1 is a silica receptor associated with canonical inflammasome activation.

Strong Dependence of Hydration State of F-Actin on the Bound Mg(2+)/Ca(2+) Ions.

Understanding of the hydration state is an important issue in the chemomechanical energetics of versatile biological functions of polymerized actin (F-actin). In this study, hydration-state differences of F-actin by the bound divalent cations are revealed through precision microwave dielectric relaxation (DR) spectroscopy. G- and F-actin in Ca- and Mg-containing buffer solutions exhibit dual hydration components comprising restrained water with DR frequency f2 (fw). The hydration state of F-actin is strongly dependent on the ionic composition. In every buffer tested, the HMW signal Dhyme (≡ (f1 - fw)δ1/(fwδw)) of F-actin is stronger than that of G-actin, where δw is DR-amplitude of bulk solvent and δ1 is that of HMW in a fixed-volume ellipsoid containing an F-actin and surrounding water in solution. Dhyme value of F-actin in Ca2.0-buffer (containing 2 mM Ca(2+)) is markedly higher than in Mg2.0-buffer (containing 2 mM Mg(2+)). Moreover, in the presence of 2 mM Mg(2+), the hydration state of F-actin is changed by adding a small fraction of Ca(2+) (∼0.1 mM) and becomes closer to that of the Ca-bound form in Ca2.0-buffer. This is consistent with the results of the partial specific volume and the Cotton effect around 290 nm in the CD spectra, indicating a change in the tertiary structure and less apparent change in the secondary structure of actin. The number of restrained water molecules per actin (N2) is estimated to be 1600-2100 for Ca2.0- and F-buffer and ∼2500 for Mg2.0-buffer at 10-15 °C. These numbers are comparable to those estimated from the available F-actin atomic structures as in the first water layer. The number of HMW molecules is roughly explained by the volume between the equipotential surface of -kT/2e and the first water layer of the actin surface by solving the Poisson-Boltzmann equation using UCSF Chimera.

Membrane Translocation and Organelle-Selective Delivery Steered by Polymeric Zwitterionic Nanospheres.

The majority of nanoparticles designed for cellular delivery of drugs and imaging agents enter the cell via endocytotic pathways leading to their entrapment in endosomes that present a robust barrier to further trafficking of the nanoparticles within the cells. A few materials, such as the cell penetrating peptides (CPPs), are known to enter cells not only via endocytosis, but also via translocation through the cell membrane into the cytoplasm, successfully bypassing the endosomes. We report here that random copolymers of 3-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate and poly(ethylene glycol) methacrylate, p(DMAPS-ran-PEGMA), are internalized in cells primarily via translocation through the cell membrane rather than endocytosis. The properties of the polymers and their modes of uptake were investigated systematically by dynamic light scattering, confocal fluorescence microscopy, and flow cytometry. Using specific inhibitors of the cellular uptake machinery in a human cervical carcinoma cell line (HeLa), we show that these nontoxic synthetic polyzwitterions exist in cell media as self-assembled nanospheres that unravel as they adsorb on the plasma membrane and translocate through it. Conjugates of p(DMAPS-ran-PEGMA) with rhodamine B were delivered selectively to the mitochondria, whereas doxorubicin (Dox)-p(DMAPS-ran-PEGMA) conjugates were accumulated in both the nucleus and the mitochondria, effectively inducing apoptosis in HeLa cells. These findings suggest that the noncytotoxic and readily synthesized p(DMAPS-ran-PEGMA) can find applications as bioimaging tools and drug nanocarriers.

Trading polymeric microspheres: exchanging DNA molecules via microsphere interaction.

A new class of artificial molecular transport system is constructed by polymeric microspheres. The microspheres are prepared by self-assembly of poly(ethylene glycol)-block-poly(3-dimethyl(methacryloyloxyethyl)ammonium propane sulfonate), PEG-b-PDMAPS, by intermolecular dipole-dipole interaction of sulfobetaine side chains in water. Below the upper critical solution temperature (UCST) of PEG-b-PDMAPS, the microspheres (∼1µm) interact with other microspheres by partial and transit fusion. In order to apply the interaction between microspheres, a 3'-TAMRA-labeled single-stranded DNA oligomer (ssDNA) is encapsulated into a PEG-b-PDMAPS microsphere by thermal treatment. The exchange of ssDNA between microspheres is confirmed by fluorescence resonance energy transfer (FRET) quenching derived from double-stranded formation with complementary 5'-BHQ-2-labeled ssDNA encapsulated in PEG-b-PDMAPS microspheres. The exchange rate of ssDNA is controllable by tuning the composition of the polymer. The contact-dependent transport of molecules can be applied in the areas of microreactors, sensor devices, etc.

Rotational motion of rhodamine 6G tethered to actin through oligo(ethylene glycol) linkers studied by frequency-domain fluorescence anisotropy.

Investigation of the rotational motion of a fluorescent probe tethered to a protein helps to elucidate the local properties of the solvent and protein near the conjugation site of the probe. In this study, we have developed an instrument for frequency-domain fluorescence (FDF) anisotropy measurements, and studied how the local properties around a protein, actin, can be elucidated from the rotational motion of a dye tethered to actin. Rhodamine 6G (R6G) was attached to Cys-374 using newly-synthesized R6G-maleimide with three different oligo(ethylene glycol) (OEG) linker lengths. The time-resolved anisotropy decay of R6G tethered to G-actin was revealed to be a combination of the two modes of the wobbling motion of R6G and the tumbling motion of G-actin. The rotational diffusion coefficient (RDC) of R6G wobbling was ~0.1 ns(-1) at 20°C and increased with OEG linker length. The use of the three R6G-actin conjugates of different linker lengths was useful to not only figure out the linker length dependence of the rotational motion of R6G but also validate the analyses. In the presence of a cosolvent of glycerol, although the tumbling motion of G-actin was retarded in response to the bulk viscosity, the wobbling motion of R6G tethered to actin exhibited an increase of RDC as glycerol concentration increased. This finding suggests an intricate relationship between the fluid properties of the bulk solvent and the local environment around actin.

Temperature-responsive telechelic dipalmitoylglyceryl poly(N-isopropylacrylamide) vesicles: real-time morphology observation in aqueous suspension and in the presence of giant liposomes.

Telechelic α,ω-di(twin-tailed poly(N-isopropylacrylamides)) form polymersomes in water that increase in size by fusion when the water temperature exceeds the polymers cloud point temperature. Hybrid vesicles form in mixed suspensions of giant phospholipid liposomes and polymersomes by adsorption/fusion, and undergo further transformations, such as fission.

Self-assembled microspheres driven by dipole-dipole interactions: UCST-type transition in water.

A double hydrophilic block copolymer, poly(ethylene glycol)-poly(3-dimethyl (methacryloyloxyethyl) ammonium propane sulfonate) (PEG-SB), is synthesized by reversible addition-fragmentation transfer (RAFT) polymerization using PEG methyl ether (4-cyano-4-pentanoate dodecyl trithiocarbonate) as a chain transfer agent. PEG-SB forms multi-layered microspheres with dipole-dipole interactions of the SB side chains as the driving force. The PEG-SB polymers show an upper critical solution temperature (UCST) and the UCST is controllable by the polymerization degree. The PEG-SB microspheres are dissociated above the UCST and then monodispersed microspheres (∼1 µm) are obtained when the solution temperature is decreased below the UCST again. The disassociation/association of the microspheres is also controllable using the concentration of NaCl. These multi-responsive microspheres could be a powerful tool in the field of nano-biotechnology.

1,3-Diethylurea-enhanced Mg-ATPase activity of skeletal muscle myosin with a converse effect on the sliding motility.

We investigate the effects of urea and its derivatives on the ATPase activity and on the in vitro motility of chicken skeletal muscle actomyosin. Mg-ATPase rate of myosin subfragment-1 (S1) is increased by 4-fold by 0.3M 1,3-diethylurea (DEU), but it is unaffected by urea, thiourea, and 1,3-dimethylurea at ≤1M concentration. Thus, we further examine the effects of DEU in comparison to those of urea as reference. In in vitro motility assay, we find that in the presence of 0.3M DEU, the sliding speeds of actin filaments driven by myosin and heavy meromyosin (HMM) are significantly decreased to 1/16 and 1/6.6, respectively, compared with the controls. However, the measurement of the actin-activated ATPase activity of HMM shows that the maximal rate, Vmax, is almost unchanged with DEU. Thus, the myosin-driven sliding motility of actin filaments is significantly impeded in the presence of 0.3M DEU, whereas the cyclic interaction of myosin with F-actin occurs during the ATP turnover, the rate of which is close to that without DEU. In contrast to DEU, 0.3M urea exhibits only modest effects on both actin-activated ATPase and sliding motility of actomyosin. Thus, DEU has the effect of uncoupling the sliding motility of actomyosin from its ATP turnover.