Tailoring Thermoresponsive Poly(N-Isopropylacrylamide) Toward Sensing Perfluoroalkyl Acids

Widespread distribution of poly- and perfluoroalkyl substances (PFAS) in the environment combined with concerns for their potentially negative health effects has motivated regulators to establish strict standards for their surveillance. The United States Environmental Protection Agency issued a cumulative domestic threshold of 70 ppt for water supplies, and this bar is even lower in some local districts and other countries. Monitoring PFAS consequently requires sensitive analytical equipment to meet regulatory specifications, and liquid chromatography with tandem mass spectroscopy (LC/MS/MS) is the most common technique used to satisfy these requirements. Though extremely sensitive, the instrument is often burdened by pretreatment regimens, sedentation, and user proficiency barriers that encumber or limit its effectiveness. As an alternative, polymeric strategies for detecting PFAS are promising candidates for funneling recognition, transduction, and receptor elements into a single sensing platform to overcome some of the hurdles affecting LC/MS/MS. Toward this end, poly(N-isopropylacrylamide) (PNIPAM), an extensively studied thermoresponsive polymer, is a hydrogel with tailorable swelling properties dependent upon its polymeric composition and surrounding media. This polymer holds a lower critical solution temperature (LCST) around 32 °C that marks its transition from a relatively hydrophilic, swollen state to a hydrophobic, collapsed state once heated, and prior research indicates that surfactants such as sodium dodecyl sulfate can heavily influence the temperature at which this transition occurs and the ultimate swelling ratio for crosslinked hydrogels. Two particularly concerning fluorosurfactants, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), were hypothesized to act similarly to their non-fluorinated analogs by augmenting the swelling of PNIPAM in a dose-dependent manner. The effect of these fluoropollutants on PNIPAM was therefore studied to identify 1) if PFOS and PFOA would have an appreciable effect on the swelling behavior of varying PNIPAM morphologies, 2) if the swelling response could be enhanced by adding functional comonomers into the PNIPAM backbone, and 3) if the swelling behavior could be outfitted with Förster resonance energy transfer (FRET)-compatible dyes to signal the contaminants’ concentration. As such, crosslinked PNIPAM hydrogels were functionalized with fluorinated comonomers to induce fluorine-fluorine attraction amongst the polymers and their analytes to strengthen their recognition capability and microgels were equipped with FRET-capable dyes to achieve a fluorescent transduction motif indicative of the contaminants’ presence. Results indicated that PFOS augments the swelling of PNIPAM hydrogels significantly while PFOA causes microgels to collapse at temperatures below their innate LCST. FRET primarily replicated swelling observations as expected for the distance-mediated fluorescent phenomenon. Though the fluoropollutants generated appreciable swelling perturbations at concentrations within the micromolar range, additional functionalization is necessary to exploit the molecular-level interactions between PNIPAM and target fluorosurfactants to yield detection limits within the range needed for environmental applications

A Nanoparticle/enzyme System For The Simultaneous Detection And Decontamination Of Organophosphates

The need for a direct visual response system for the detection of organophosphorus compounds stems from the continued threat and use of these toxic agents in military and terrorist conflicts. The development of an enzyme-inhibitor triggered release system allows for direct visual detection with high specificity. Mesoporous silica nanoparticles (MSNs) have physical features that make them attractive as scaffolds for the construction of these systems, such as pore diameters (20-500 Á) that can be synthetically controlled, large surface areas (300-1500 m2g-1), large pore volumes, chemical inertness, stability at elevated temperatures, and surfaces that can be easily functionalized. In our studies, the dye Congo Red was loaded into the pores of MSNs, which were then capped by tethering an enzyme (organophosphorus hydrolase (OPH) or acetylcholinesterase (AChE)) to the external surfaces of MSNs through a competitive inhibitor (diethyl 4-aminobenzyl phosphonate (DEABP) or tacrine, respectively). OPH has been extensively studied for its ability to hydrolyze a wide range of organophosphorus compounds, rendering them non-toxic. AChE has been commonly used for organophosphate detection resulting from its sensitivity to phosphorylation. Upon addition of organophosphorus compounds to suspensions of the modified MSNs, the enzymes detached from the MSN surface, releasing the dye and providing a visual confirmation of organophosphate presence. Enzyme kinetics were studied using 31P NMR or UV-Visible spectroscopy; Congo Red release was also monitored by UV-Visible spectroscopy. The system was sensitive and specific for organophosphorus compounds both in phosphate-buffered saline and in human serum. The rate of dye release directly correlated with the rate of organophosphorus conversion for OPH and the rate of phosphorylation for AChE

Preparation of nanoparticles by the self-organization of polymers consisting of hydrophobic and hydrophilic segments: Potential applications

AbstractThis review describes the preparation of core-corona type polymeric nanoparticles and their applications in various technological and biomedical fields. Over the past two decades, we have studied the synthesis and clinical applications of core-corona polymeric nanoparticles composed of hydrophobic polystyrene and hydrophilic macromonomers. These nanoparticles were utilized as catalyst carriers, carriers for oral peptide delivery, virus capture agents, and vaccine carriers, and so on. Moreover, based on this research, we attempted to develop novel biodegradable nanoparticles composed of hydrophobic poly(γ-glutamic acid) (γ-PGA) derivatives (γ-hPGA). Various model proteins were efficiently entrapped on/into the nanoparticles under different conditions: encapsulation, covalent immobilization, and physical adsorption. The encapsulation method showed the most promising results for protein loading. It is expected that biodegradable γ-hPGA nanoparticles can encapsulate and immobilize various biomacromolecules. Nanoparticles consisting of hydrophobic and hydrophilic segments have great potential as multifunctional carriers for pharmaceutical and biomedical applications, such as drug, protein, peptide or DNA delivery systems

Skin-Integrated wearable systems and implantable biosensors: a comprehensive review

Biosensors devices have attracted the attention of many researchers across the world. They have the capability to solve a large number of analytical problems and challenges. They are future ubiquitous devices for disease diagnosis, monitoring, treatment and health management. This review presents an overview of the biosensors field, highlighting the current research and development of bio-integrated and implanted biosensors. These devices are micro- and nano-fabricated, according to numerous techniques that are adapted in order to offer a suitable mechanical match of the biosensor to the surrounding tissue, and therefore decrease the body’s biological response. For this, most of the skin-integrated and implanted biosensors use a polymer layer as a versatile and flexible structural support, combined with a functional/active material, to generate, transmit and process the obtained signal. A few challenging issues of implantable biosensor devices, as well as strategies to overcome them, are also discussed in this review, including biological response, power supply, and data communication.This research was funded by FCT- FUNDAÇÃO PARA A CIÊNCIA E TECNOLOGIA, grant numbers: PTDC/EMD-EMD/31590/2017 and PTDC/BTM-ORG/28168/2017

Synthesis and Applications of Smart Glycopolymers and Their Interactions with Lectins

The last two decades have seen an urgent need to find more efficient methods with minimal health impact to diagnose and treat common diseases. Recent research on drug and gene delivery by polymeric vectors has shown promising results because polymers provide excellent drug protection whilst showing low toxicity both in vitro and in vivo. Nevertheless, targeted delivery is a major characteristic that polymeric vectors often lack. Glycopolymers, however, can form specific interactions with lectins by mimicking biological interactions. These interactions are responsible for numerous biological mechanisms and can be exploited to achieve targeted delivery of medicines. Herein, different polymerisation techniques for the synthesis of glycopolymers with different architectures and properties are explored. First, the preparation of star shaped glycopolymers as a way to increase drug encapsulation and lectin binding is discussed. Thereafter, reducible glycopolymers prepared via step growth polymerisation are investigated as potential gene transporters. Furthermore, the interactions between glycopolymers and a lectin are measured under physiological conditions and the results are discussed. Finally, recent progress on glycopolymers and the gaps that still need to be addressed are discussed

Hydrogel microparticles for biosensing

Due to their hydrophilic, biocompatible, and highly tunable nature, hydrogel materials have attracted strong interest in the recent years for numerous biotechnological applications. In particular, their solution-like environment and non-fouling nature in complex biological samples render hydrogels as ideal substrates for biosensing applications. Hydrogel coatings, and later, gel dot surface microarrays, were successfully used in sensitive nucleic acid assays and immunoassays. More recently, new microfabrication techniques for synthesizing encoded particles from hydrogel materials have enabled the development of hydrogel-based suspension arrays. Lithography processes and droplet-based microfluidic techniques enable generation of libraries of particles with unique spectral or graphical codes, for multiplexed sensing in biological samples. In this review, we discuss the key questions arising when designing hydrogel particles dedicated to biosensing. How can the hydrogel material be engineered in order to tune its properties and immobilize bioprobes inside? What are the strategies to fabricate and encode gel particles, and how can particles be processed and decoded after the assay? Finally, we review the bioassays reported so far in the literature that have used hydrogel particle arrays and give an outlook of further developments of the field. Keywords: Hydrogel; Biosensor; Microparticle; Multiplex assayNovartis Institutes of Biomedical Research (Presidential Fellowship)Novartis Institutes of Biomedical Research (Education Office)National Cancer Institute (U.S.) (Grant 5R21CA177393-02)National Science Foundation (U.S.) (Grant CMMI-1120724)Institute for Collaborative Biotechnologies (Grant W911NF-09-0001)United States. Army Research Offic

Recent Advances in the Development of Biomimetic Materials

: In this review, we focused on recent efforts in the design and development of materials with biomimetic properties. Innovative methods promise to emulate cell microenvironments and tissue functions, but many aspects regarding cellular communication, motility, and responsiveness remain to be explained. We photographed the state-of-the-art advancements in biomimetics, and discussed the complexity of a "bottom-up" artificial construction of living systems, with particular highlights on hydrogels, collagen-based composites, surface modifications, and three-dimensional (3D) bioprinting applications. Fast-paced 3D printing and artificial intelligence, nevertheless, collide with reality: How difficult can it be to build reproducible biomimetic materials at a real scale in line with the complexity of living systems? Nowadays, science is in urgent need of bioengineering technologies for the practical use of bioinspired and biomimetics for medicine and clinics

Ionic-liquid-based approaches to improve biopharmaceuticals downstream processing and formulation

The emergence of biopharmaceuticals, including proteins, nucleic acids, peptides, and vaccines, revolutionized the medical field, contributing to significant advances in the prophylaxis and treatment of chronic and life-threatening diseases. However, biopharmaceuticals manufacturing involves a set of complex upstream and downstream processes, which considerably impact their cost. In particular, despite the efforts made in the last decades to improve the existing technologies, downstream processing still accounts for more than 80% of the total biopharmaceutical production cost. On the other hand, the formulation of biological products must ensure they maintain their therapeutic performance and long-term stability, while preserving their physical and chemical structure. Ionicliquid (IL)-based approaches arose as a promise alternative, showing the potential to be used in downstream processing to provide increased purity and recovery yield, as well as excipients for the development of stable biopharmaceutical formulations. This manuscript reviews the most important progress achieved in both fields. The work developed is critically discussed and complemented with a SWOT analysis.publishe

Rational Design of Responsive Prodrug-based Nanomedicines for Antitumor Therapy

A major issue of conventional chemotherapy is the lack of selective delivery which results in high systemic exposure and severe side effects. The limitations of small molecular weight drugs for cancer therapy prompted the development of diverse nanocarrier systems for targeted drug delivery. The impressive progress in nanomaterial science and increased understanding of the nano-bio interface allowed the progression of these systems. However, the targeted delivery remains challenging due to obstacles that are encountered during the drug delivery process, particularly, in the tumor microenvironment (TME). (Multi)stimuliresponsive nanocarriers have the potential to overcome the faced barriers by taking advantage of altered pathological characteristics in the TME and/or intracellular signals. The motivation of the presented work was to incorporate rational design features into novel responsive nanomedicines to address the limitations of conventional chemotherapeutic drugs and tackle issues of current drug delivery systems (DDS). For this purpose, prodrugs of the chemotherapeutic agent doxorubicin (Dox) were combined with three nanocarrier designs including polymer-drug conjugates, a nanoemulsion (NE), and nanogels (NGs). The Dox prodrugs comprised cleavable motifs which introduce a responsiveness towards endogenous stimuli into the nanocarriers. The nanocarrier architectures with different sizes and compositions were evaluated in terms of controlled drug release, drug-carrier compatibility, carrier degradability, and transport restrictions in the TME, all of which are important aspects for an efficient delivery process. The choice of the cleavable linkage strongly affects the specificity of the desired responsive behavior. To address this aspect, Dox prodrugs with pH- or protease-cleavable bonds were evaluated regarding their impact on the intracellular drug release. Activatable fluorescence probes were utilized to follow the drug release from polymer-drug conjugates in real-time. This assessment of the linker formed the basis for the rational design of two prodrug-based nanomedicines with adjusted cleavage properties. First, a pH-sensitive Dox prodrug was entrapped in a NE to form a DDS with explicit intracellular drug release. The second design was based on dual-responsive nanogels as multistage delivery systems with specific extracellular response to protease and acid-mediated intracellular payload release. Initially, we evaluated the impact of the cleavable linkage on the drug release using theranostic polymer conjugates (TPC) with activatable fluorescence probes. The TPC represent model DDS that consist of dendritic polyglycerol (dPG) as polymeric carrier labeled with an indodicarbocyanine (IDCC) dye that quenches the fluorescence of Dox, conjugated through a cleavable linker. Cleavage of the conjugates was mediated either by acidic pH or protease activity. By tracking the fluorescence recovery in a cell-based microplate assay, we were able to obtain characteristic release profiles of Dox for different cell lines. Here, the pH-cleavable linker was found to be cleaved mainly intracellularly, whereas the protease-sensitive system suffered from extracellular drug release. The intracellular release was crucial to treat multidrug-resistant cells and overcome their resistance mechanisms. It can be highlighted that the modular synthetic approach, combined with the cell-based assay, has potential to extend the common in vitro methods to evaluate DDS performance. The results of this study motivated us to develop a pH-sensitive Dox prodrug (C16-Dox) to efficiently dissolve the drug in the nanodroplets of an oil/water NE. By attaching a hydrophobic alkyl chain (C16), Dox was provided with an amphiphilic character for increased drug-carrier compatibility. pH-sensitive properties of the prodrug allowed the intracellular release of the drug from the NE by recovering the hydrophilic parent drug. The new formulation of Dox (NE-C16-Dox) was compared with free Dox in a murine breast cancer model. Enhanced delivery to tumor tissue and reduction of systemic toxicity allowed the administration of a higher Dox dose in the NE formulation as compared to the free drug. The high dosage significantly inhibited the primary tumor growth and prevented the formation of distant lung metastasis without signs of side effects. The improved chemotherapeutic index compared to free Dox indicates that NE-C16-Dox is a promising formulation for breast cancer treatment At last, we combined protease- and pH-sensitive moieties into a multistage nanocarrier to enhance drug transport in tumor tissue. Matrix metalloproteinase (MMP)-sensitive NGs (pNGs) were developed which consists of a dPG scaffold crosslinked with a fluorogenic peptide. The crosslinker integrates biodegradability to the nanocarrier mediated by proteases in the TME. The intrinsic reporter moiety of the crosslinker allowed us to study the influence of different pNG compositions on the degradation profile in detail. One pNG candidate was chosen to conjugate the therapeutic drug Dox through a pH-sensitive linkage to dPG. The degradable multistage pNGs demonstrated deeper penetration into multicellular tumor spheroids (MCTS) as compared to their non-degradable counterparts. Hence, the triggered size reduction of the pNGs by enzymatic degradation facilitated the infiltration of the nanocarrier into dense tissue and thereby promoted the delivery of the therapeutic cargo

Multifunctional and Stimuli-Responsive Polymersomes for Biomedical Applications

The demand for multifunctional nanocontainers possessing both recognition ability and responsive nature is increasing greatly because of their high potential in various biomedical applications. The engineering of such smart nanovesicles is useful to enhance the efficiency of many therapeutic and diagnostic tools that have the applicability in targeted drug delivery systems as well as designing sensing devices or conducting selective reactions as nanoreactors in the scope of nanobiotechnology. For this purpose, this study demonstrates the formation of multifunctional and stimuli-responsive polymersomes comprising various abilities including pH and light sensitivity as well as many reactive groups with sufficient accessibility to be used as smart and recognitive nanocontainers. The fabrication included several steps starting from the synthesis of azide and adamantane terminated block copolymers, which were then self-assembled to prepare the polymersomes with the corresponding functional groups for the subsequent post-conjugations at the vesicle periphery. The accessible and sufficiently reactive groups were quantitatively proven when UV and IR cleavable NVOC protected amino groups as well as β-cyclodextrin molecules were conjugated to the pre-formed polymersomes through click chemistry and strong host-guest complexations. The gained light responsivity with the aid of successful NVOC attachment enabled further selective photochemical reactions triggered either by UV or NIR light leading to liberated amine groups on the polymersome surface. Therein, these released amino groups were further conjugated with a model fluorescent compound as mimicking the attachment of biorecognition elements to see the direct picture of the applicability. To realize this concept in a more localized and selective way as well as to avoid the possible side effects of UV light, the NIR-light induced photochemical reactions and further dye coupling were performed when polymersomes were immobilized onto solid substrates. This fixation was achieved by adapting the host-guest chemistry into this part and conjugating the adamantane decorated polymersomes onto β-cyclodextrin coated substrates. Several investigations including adhesion behavior, pH sensitivity and mechanical properties of the established multifunctional polymersomes under liquid phase have been performed. It has been found that the polymersome shape is highly dependent on the attractive forces of the substrate and needs to be optimized to avoid the flattening of the vesicles. For these optimization steps, different conditions were investigated including the decrease of cyclodextrin amount and additional surface passivation with PEG molecules on the solid substrates. Besides, the calculated Young’s and bending modulus of the polymersome membrane from AFM measurements showed a robust but still flexible “breathable” membrane which is an important criterion for the applicability of these smart and stable vesicles. In addition, the hosting ability as well as diffusion limits and sufficient membrane permeability of the polymersomes were observed by encapsulating gold nanoparticles as a smart cargo and doxorubicin molecules as an anticancer drug. In conclusion, the established multifunctional polymersomes are highly versatile and thus present new opportunities in the design of targeted and selective recognition systems which is highly interesting for various applications including development of microsystem devices, design of chemo/biosensors, and also for conducting enhanced, combined therapy in the field of drug delivery