Switching it Up: The Promise of Stimuli-Responsive Polymer Systems in Biomedical Science.

Responsive polymer systems have the ability to change properties or behavior in response to external stimuli. The properties of responsive polymer systems can be fine-tuned by adjusting the stimuli, enabling tailored responses for specific applications. These systems have applications in drug delivery, biosensors, tissue engineering, and more, as their ability to adapt and respond to dynamic environments leads to improved performance. However, challenges such as synthesis complexity, sensitivity limitations, and manufacturing issues need to be addressed for successful implementation. In our review, we provide a comprehensive summary on stimuli-responsive polymer systems, delving into the intricacies of their mechanisms and actions. Future developments should focus on precision medicine, multifunctionality, reversibility, bioinspired designs, and integration with advanced technologies, driving the dynamic growth of sensitive polymer systems in biomedical applications.

Temperature-Controlled Orientation of Proteins on Temperature-Responsive Grafted Polymer Brushes: Poly(butyl methacrylate) vs Poly(butyl acrylate): Morphology, Wetting, and Protein Adsorption.

Poly( n-butyl methacrylate) (PBMA) or poly( n-butyl acrylate) (PBA)-grafted brush coatings attached to glass were successfully prepared using atom-transfer radical polymerization "from the surface". The thicknesses and composition of the PBMA and PBA coatings were examined using ellipsometry and time-of-flight secondary ion mass spectrometry (ToF-SIMS), respectively. For PBMA, the glass-transition temperature constitutes a range close to the physiological limit, which is in contrast to PBA, where the glass-transition temperature is around -55 °C. Atomic force microscopy studies at different temperatures suggest a strong morphological transformation for PBMA coatings, in contrast to PBA, where such essential changes in the surface morphology are absent. Besides, for PBMA coatings, protein adsorption depicts a strong temperature dependence. The combination of bovine serum albumin and anti-IgG structure analysis with the principal component analysis of ToF-SIMS spectra revealed a different orientation of proteins adsorbed to PBMA coatings at different temperatures. In addition, the biological activity of anti-IgG molecules adsorbed at different temperatures was evaluated through tracing the specific binding with goat IgG.

Immobilization and detection of platelet-derived extracellular vesicles on functionalized silicon substrate: cytometric and spectrometric approach.

Among the various biomarkers that are used to diagnose or monitor disease, extracellular vesicles (EVs) represent one of the most promising targets in the development of new therapeutic strategies and the application of new diagnostic methods. The detection of circulating platelet-derived microvesicles (PMVs) is a considerable challenge for laboratory diagnostics, especially in the preliminary phase of a disease. In this study, we present a multistep approach to immobilizing and detecting PMVs in biological samples (microvesicles generated from activated platelets and human platelet-poor plasma) on functionalized silicon substrate. We describe the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and spectroscopic ellipsometry methods to the detection of immobilized PMVs in the context of a novel imaging flow cytometry (ISX) technique and atomic force microscopy (AFM). This novel approach allowed us to confirm the presence of the abundant microvesicle phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) on a surface with immobilized PMVs. Phosphatidylcholine groups (C5H12N+; C5H15PNO4+) were also detected. Moreover, we were able to show that ellipsometry permitted the immobilization of PMVs on a functionalized surface to be evaluated. The sensitivity of the ISX technique depends on the size and refractive index of the analyzed microvesicles. Graphical abstract Human platelets activated with thrombin (in concentration 1IU/mL) generate population of PMVs (platelet derived microvesicles), which can be detected and enumerated with fluorescent-label method (imaging cytometry). Alternatively, PMVs can be immobilized on the modified silicon substrate which is functionalized with a specific IgM murine monoclonal antibody against human glycoprotein IIb/IIIa complex (PAC-1). Immobilized PMVs can be subjected to label-free analyses by means ellipsometry, atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS).

Thermoresponsive Smart Copolymer Coatings Based on P(NIPAM-co-HEMA) and P(OEGMA-co-HEMA) Brushes for Regenerative Medicine.

The fabrication of multifunctional, thermoresponsive platforms for regenerative medicine based on polymers that can be easily functionalized is one of the most important challenges in modern biomaterials science. In this study, we utilized atom transfer radical polymerization (ATRP) to produce two series of novel smart copolymer brush coatings. These coatings were based on copolymerizing 2-hydroxyethyl methacrylate (HEMA) with either oligo(ethylene glycol) methyl ether methacrylate (OEGMA) or N-isopropylacrylamide (NIPAM). The chemical compositions of the resulting brush coatings, namely, poly(oligo(ethylene glycol) methyl ether methacrylate-co-2-hydroxyethyl methacrylate) (P(OEGMA-co-HEMA)) and poly(N-isopropylacrylamide-co-2-hydroxyethyl methacrylate) (P(NIPAM-co-HEMA)), were predicted using reactive ratios of the monomers. These predictions were then verified using time-of-flight-secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). The thermoresponsiveness of the coatings was examined through water contact angle (CA) measurements at different temperatures, revealing a transition driven by lower critical solution temperature (LCST) or upper critical solution temperature (UCST) or a vanishing transition. The type of transition observed depended on the chemical composition of the coatings. Furthermore, it was demonstrated that the transition temperature of the coatings could be easily adjusted by modifying their composition. The topography of the coatings was characterized using atomic force microscopy (AFM). To assess the biocompatibility of the coatings, dermal fibroblast cultures were employed, and the results indicated that none of the coatings exhibited cytotoxicity. However, the shape and arrangement of the cells were significantly influenced by the chemical structure of the coating. Additionally, the viability of the cells was correlated with the wettability and roughness of the coatings, which determined the initial adhesion of the cells. Lastly, the temperature-induced changes in the properties of the fabricated copolymer coatings effectively controlled cell morphology, adhesion, and spontaneous detachment in a noninvasive, enzyme-free manner that was confirmed using optical microscopy.

Dendrite Formation on the Poly(methyl methacrylate) Surface Reactively Sputtered with a Cesium Ion Beam.

The development of topography plays an important role when low-energy projectiles are used to modify the surface or analyze the properties of various materials. It can be a feature that allows one to create complex structures on the sputtered surface. It can also be a factor that limits depth resolution in ion-based depth profiling methods. In this work, we have studied the evolution of microdendrites on poly(methyl methacrylate) sputtered with a Cs 1 keV ion beam. Detailed analysis of the topography of the sputtered surface shows a sea of pillars with islands of densely packed pillars, which eventually evolve to fully formed dendrites. The development of the dendrites depends on the Cs fluence and temperature. Analysis of the sputtered surface by physicochemical methods shows that the mechanism responsible for the formation of the observed microstructures is reactive ion sputtering. It originates from the chemical reaction between the target material and primary projectile and is combined with mass transport induced by ion sputtering. The importance of chemical reaction for the formation of the described structures is shown directly by comparing the change in the surface morphology under the same dose of a nonreactive 1 keV xenon ion beam.

Grafted polymer brush coatings for growth of cow granulosa cells and oocyte-cumulus cell complexes.

In the present work, three types of grafted brush coatings [P4VP, POEGMA246, and P(4VP-co-POEGMA246)] were successfully fabricated using graft polymerization of monomers "from the surface." The composition, thickness, and morphology of the grafted brush coatings were analyzed by TOF-SIMS, ellipsometry, and AFM, respectively. The chemical nature of the polymer surface plays a crucial role in the growth and development of the cow granulosa cells and, therefore, also oocyte-cumulus complexes. In comparison with other coatings, the P(4VP-co-POEGMA246) copolymer coating enables the formation of dispersed and small but numerous cell conglomerates and high cumulus expansion in oocyte-cumulus complexes with highly homogeneous cumulus layers surrounding the oocytes. Moreover, the cellular oxygen uptake for this coating in the presence of NaF (inhibitor glycolysis) was stimulated. This new (4VP-co-POEGMA246) copolymer nanostructured coating is a promising material for granulosa cell and oocyte-cumulus complex cultivation and possibly will have great potential for applications in veterinary and reproductive medicine.

Selective protein adsorption on polymer patterns formed by self-organization and soft lithography.

Thin films, with both isotropic and ordered patterns of polymer domains, are used as substrates to study selective adsorption of two proteins (concanavalin A and lentil lectin) and to test reconstruction of polymer patterns by these proteins. Integral geometry approach is used to compare quantitatively fluorescence micrographs of protein patches with AFM images of original isotropic patterns, formed during blend casting of polystyrene/poly(methyl methacrylate) and PS/poly(ethylene oxide). Preferential adsorption of both lectins to PMMA phase domains, enhanced for PS/PMMA interfaces is concluded. In turn, protein binding to PS phase regions of PS/PEO blends is highly selective. Ordered protein grouping is obtained as a result of selective adsorption to alternating stripes of polystyrene (partly brominated to enable identification) and cross-linked PEO, prepared with solvent-assisted micromolding applied to PBrS/PEO bilayers. Biological activity test, performed with concanavalin A, confirms preserved functionality of a complementary protein, carboxypeptidase Y, adsorbed to polymer patterns.

Sequential binary protein patterning on surface domains of thermo-responsive polymer blends cast by horizontal-dipping.

We characterize an approach enabling dual protein positioning over broad polymer areas based on subsequent selective adsorption of two fluorescently labelled lectins, Concanavalin A (Con A) and Lentil Lectin (LcH), on self-assembled gradient patterns of thermoresponsive poly(N-isopropyl acrylamide) (PNIPAM) and polystyrene (PS) polymers blend, prepared by horizontal dipping technique. The film morphologies of gradient samples prior dual selective protein adsorption are mapped with scanning microscopy (AFM) and secondary ion mass spectrometry (ToF-SIMS), whereas adsorbed proteins are imaged with fluorescence microscope. ToF-SIMS analysis reveals surface composition consisting of PNIPAM-rich domains in PS-rich matrix. The two-step protein adsorption experiment results in selective adsorption of Con A and LcH to PNIPAM- and PS-rich phases, respectively. Integral geometry approach is used to compare quantitatively morphology of polymer patterns varied in domain size due to horizontal dipping casting. Minkowski measures are also used to compare quantitatively fluorescence micrographs of protein patches with SIMS images of original isotropic polymer patterns. It confirms that PNIPAM domains size increases with increasing speed. Further, Minkowski analysis unveiled that adsorbed proteins cover about 60-70% of polymer surface. What is more fluorescence micrographs acknowledge both no lectins contamination and no adsorption to interphase areas. Additionally, protein displacement effect is observed.

"Command" surfaces with thermo-switchable antibacterial activity.

In the presented work "smart" antibacterial surfaces based on silver nanoparticles (AgNPs) embedded in temperature-responsive poly(di(ethylene glycol)methyl ether methacrylate) - (POEGMA188) as well as poly(4-vinylpyridine) - (P4VP) coatings attached to a glass surface were successfully prepared. The composition, thickness, morphology and wettability of the resulting coatings were analyzed using ToF-SIMS, XPS, EDX, ellipsometry, AFM, SEM and CA measurements, respectively. Temperature-switched killing of the bacteria was tested against Escherichia coli ATCC 25922 (representative of Gram-negative bacteria) and Staphylococcus aureus ATCC 25923 (representative of Gram-positive bacteria) at 4 and 37 °C. In general at 4 °C no significant difference was observed between the amounts of bacteria accounted on the grafted brush coatings and within the control sample. In contrast, at 37 °C almost no bacteria were visible for temperature-responsive coating with AgNPs, whereas the growth of bacteria remains not disturbed for "pure" coating, indicating strong temperature-dependent antibacterial properties of AgNPs integrated into brushes.

Temperature-Controlled Three-Stage Switching of Wetting, Morphology, and Protein Adsorption.

The novel polymeric coatings of oligoperoxide-graft-poly(4-vinylpyridine-co-oligo(ethylene glycol)ethyl ether methacrylate246) [oligoperoxide-graft-P(4VP-co-OEGMA246)] attached to glass were successfully fabricated. The composition, thickness, morphology, and wettability of resulting coatings were analyzed using X-ray photoelectron spectroscopy, ellipsometry, atomic force microscopy, and contact angle measurements, respectively. In addition, adsorption of the bovine serum albumin was examined with fluorescence microscopy. The thermal response of wettability and morphology of the coatings followed by that of protein adsorption revealed two distinct transitions at 10 and 23 °C. For the first time, three stage switching was observed not only for surface wetting but also for morphology and protein adsorption. Moreover, the influence of the pH on thermo-sensitivity of modified surfaces was shown.

Orientation and biorecognition of immunoglobulin adsorbed on spin-cast poly(3-alkylthiophenes): Impact of polymer film crystallinity.

Many of bioelectronic and biosensor applications are based on poly(3-alkylthiophenes), conducting and solution-processable polymers. The most facile approach for the fabrication of such devices relies on biofunctionalization of P3AT surfaces with antibodies through adsorption. The success of this approach depends critically on antibody orientation that affects its biorecognition. As demonstrated here both these features are controlled by the surface structure of spin-cast P3ATs. In particular, a multi-technique and multivariate study that involved Atomic Force Microscopy, Grazing Incidence X-ray Diffraction, Angle-Resolved X-ray Photoelectron Spectroscopy, Enzyme-Linked ImmunoSorbent Assay, and Time-of-Flight Secondary Ion Mass Spectrometry combined with Principal Component Analysis is conducted in order to deduce the crystalline texture of three P3AT polymers as well as its effect on orientation of adsorbed rabbit immunoglobulin (IgG) molecules. An edge-on crystalline texture is concluded for regioregular poly(3-butylthiophene) (RP3BT) and poly(3-hexylthiophene) (RP3HT), while amorphous morphology is inferred for poly(3-butylthiophene) (P3BT). In addition, end-on and head-on orientations similar for all P3ATs were concluded, based on the amount of adsorbed rabbit IgG molecules. Examination of amino acids characteristic for F(ab')2 and Fc fragments, and dominant in the external regions of adsorbed immunoglobulin molecules, points to end-on IgG alignment on RP3BT and RP3HT, but not on P3BT. Moreover, the binding of an anti-rabbit IgG antibody on the absorbed rabbit IgG is higher (up to 71%) when the biorecognition reactions are performed on regioregular rather than regiorandom P3AT surfaces. In particular, the highest biorecognition efficiency and IgG orientational order is observed for the RP3BT surfaces with the more developed crystallinity.

PDMS substrate stiffness affects the morphology and growth profiles of cancerous prostate and melanoma cells.

A deep understanding of the interaction between cancerous cells and surfaces is particularly important for the design of lab-on-chip devices involving the use of polydimethylsiloxane (PDMS). In our studies, the effect of PDMS substrate stiffness on mechanical properties of cancerous cells was investigated in conditions where the PDMS substrate is not covered with any of extracellular matrix proteins. Two human prostate cancer (Du145 and PC-3) and two melanoma (WM115 and WM266-4) cell lines were cultured on two groups of PDMS substrates that were characterized by distinct stiffness, i.e. 0.75 ± 0.06 MPa and 2.92 ± 0.12 MPa. The results showed the strong effect on cellular behavior and morphology. The detailed analysis of chemical and physical properties of substrates revealed that cellular behavior occurs only due to substrate elasticity.

Temperature-responsive peptide-mimetic coating based on poly(N-methacryloyl-l-leucine): properties, protein adsorption and cell growth.

Poly(N-methacryloyl-l-leucine) (PNML) coatings were successfully fabricated via polymerization from peroxide initiator grafted to premodified glass substrate. Chemical composition and thickness of PNML coatings were determined using time of flight-secondary ion mass spectrometry (TOF- SIMS) and ellipsometry, respectively. PNML coatings exhibit thermal response of the wettability, between 4 and 28°C, which indicates a transition between hydrated loose coils and hydrophobic collapsed chains. Morphology of the PNML coating was observed with the AFM, transforming with increasing temperature from initially relatively smooth surface to rough and more structured surface. Protein adsorption observed by fluorescence microscopy for model proteins (bovine serum albumin and lentil lectin labeled with fluorescein isothiocyanate) at transition from 5 to 25°C, showed high affinity of PNML coating to proteins at all investigated temperatures and pH. Thus, PNML coating have significant potential for medical and biotechnological applications as protein capture agents or functional replacements of antibodies ("plastic antibodies"). The high proliferation growth of the human embryonic kidney cell (HEK 293) onto PNML coating was demonstrated, indicating its excellent cytocompatibility.

Temperature and pH dual-responsive POEGMA-based coatings for protein adsorption.

Poly(oligo(ethylene glycol)ethyl ether methacrylate (POEGMA246) coatings were successfully fabricated using novel approach via polymerization from oligoperoxide grafted to premodified glass substrate. Wettability, content and composition of coatings fabricated with different polymerization times were determined using contact angle measurements, ellipsometry and Time of Flight-Secondary Ion Mass Spectrometry (TOF-SIMS). Thermo- and pH-responsive properties of POEGMA246 coatings were found to depend significantly on concentration of the grafted POEGMA246. Coatings fabricated with polymerization time 30 h exhibit not only temperature- but also pH-dependence of wettability. Thermal response of wettability, measured between 20 and 32°C, was prominent at pH 9 and 7 and diminished or was absent at pH 5 and 3, indicating a transition between hydrated loose coils and hydrophobic collapsed chains, blocked at low pH. Protein adsorption, observed by fluorescence microscopy and analyzed semi-quantitatively using integral geometry approach, decreased dramatically for model protein (lentil lectin labeled with fluorescein isothiocyanate) at transition from pH 5 to pH 9, showing only very weak thermal-dependence. Strong protein adsorption response to pH and very weak one to temperature was confirmed by TOF-SIMS and Principal Component Analysis.

Reverse contrast and substructures in protein micro-patterns on 3D polymer surfaces.

We characterize an approach enabling protein patterning over broad polymer areas based on selective protein adsorption on surfaces of spin-cast amino-terminated polystyrene structured topographically with elastomer molds (capillary force lithography) and passivated locally against adsorption with poly(ethylene oxide)-silanes printed with flat elastomer stamps (inverted micro-contact printing). Atomic force microscopy reveals uniformity of PS-NH(2) films with stripes of grooves and elevations alternating with periodicity 4<λ<200 µm. Film morphologies, prior and after selective adsorption of model protein, are mapped with optical and fluorescence microscopes, respectively. The examination with the Fourier analysis shows that elevated regions of polymer relief are replicated as dark or bright stripes on fluorescent micrographs for elevations forming plateaus (>3 µm) or narrow ridges, respectively. Reverse contrast in protein micro-patterns is induced by modified relief geometry, which affects surface flux of silanes from stamp to polymer surface both within and away from contact zones of micro-contact printing. In addition, protein substructures with a fraction λ/n of relief periodicity are observed on surfaces with elevated ridges (n=2) and plateaus (n=2 and 4). This is due to the locally modified protein adsorption with silane concentration and surface topography, respectively.

Temperature and pH dual-responsive coatings of oligoperoxide-graft-poly(N-isopropylacrylamide): wettability, morphology, and protein adsorption.

Poly(N-isopropylacrylamide) (PNIPAM) coatings attached to glass with novel approach involving polymerization from oligoperoxide grafted to surface with (3-aminopropyl)triethoxysilane exhibit not only temperature- but also pH-dependence of wettability and protein adsorption. Wettability and composition of coatings, fabricated with different polymerization times, were determined using contact angle measurements and Time Of Flight-Secondary Ion Mass Spectrometry (TOF-SIMS), respectively. Thermal response of wettability, measured between 20 and 40°C, was prominent at pH 9 and 7 and diminished or absent at pH 5 and 3. This indicates a transition between hydrated loose coils and hydrophobic collapsed chains that is blocked at low pH. Higher surface roughness and dramatically increased adsorption of model protein (lentil lectin labeled with fluorescein isothiocyanate) were observed with AFM and fluorescence microscopy to occur in hydrophobic phases (at pH 3, for pH varied at constant temperature of 22°C and at ∼33°C, for temperature varied at constant pH 9). Protein adsorption response to pH was confirmed by TOF-SIMS and Principal Component Analysis.

Integral geometry analysis of fluorescence micrographs for quantitative relative comparison of protein adsorption onto polymer surfaces.

Most methods developed to study protein binding to distinct surfaces can only determine the average amount of adsorbed protein or merely provide (qualitative) information on its spatial distribution. Both these features can be characterized rigorously by integral geometry analysis of fluorescence micrographs. This approach is introduced here to compare the relative protein adsorption onto various polymer surfaces: polystyrene (PS), poly(methyl methacrylate) (PMMA), poly( n-butyl methacrylate) (PnBMA), poly( tert-butyl methacrylate) (PtBMA), and PS(PETA) and cross-linked poly(ethylene oxide) (PEO*(PETA)), admixed with pentaerythritol triacrylate (PETA). The polymeric surfaces were incubated for 15 min in phosphate-buffered saline (pH 7.4) containing 125 mug/mL fluorescently labeled lectins, either lentil lectin (LcH) or concanavalin A (ConA). Fluorescence images were recorded at identical conditions (physiological buffer, same exposure time, magnification, gain). For each image, taken a few times for each polymer, the distribution and average value of the normalized intensity were determined. The results show that the binding of LcH to PS(PETA), PtBMA, PS, PnBMA, PMMA, and PEO*(PETA) can be expressed by the ratio of the following values (mean +/- 95% confidence interval): 0.356 +/- 0.022, 0.298 +/- 0.030, 0.241 +/- 0.014, 0.083 +/- 0.008, 0.039 +/- 0.008, and 0.010 +/- 0.006, respectively. In turn, the relative adsorption of ConA is described by the values 0.252 +/- 0.016, 0.217 +/- 0.014, 0.222 +/- 0.016, 0.046 +/- 0.006, 0.116 +/- 0.008, and 0.006 +/- 0.002, respectively. Low dispersions of fluorescence intensity around average values indicate homogeneous distribution of adsorbed proteins. The introduced approach enables a fast and easy way not only to quantify the relative amount of bound proteins but also to characterize quantitatively the organization of their surface distribution, as demonstrated for patchlike protein adsorption onto the polymer blend surface.

Structure evolution in layers of polymer blend nanoparticles.

The early stages of phase evolution, not available for nanometer polymer blend films spin-cast from solutions of incompatible mixtures, have been examined for films prepared from nanoparticles of deuterated polystyrene/ poly(methyl methacrylate) blends (1:1 mass fraction of dPS/PMMA) with PS-PMMA diblock copolymer additives. The initial phase arrangement, confined to the size of nanoparticles, has provided the homogeneity of the initial film composition. The early stages of structure formation, promoted by annealing and traced with atomic and lateral force microscopy (AFM, LFM) as well as secondary ion mass spectroscopy (SIMS), resulted in bilayers, observed commonly for as-prepared solvent-cast blends. The initiated capillary instability of the upper dPS-rich layer depended on copolymer additives, which enhanced the lateral structures pinning the dewetting process.