Accurate medical recordkeeping is important for personal and public health. Conventional forms of on-patient medical information, such as medical alert bracelets or finger-markings, may compromise patient privacy because they are readily visible to other people. Here, the development of an invisible, temporary, and easily deployable on-patient medical recordkeeping system is reported. Information is stored in unique patterns of spatially distributed near-infrared (NIR) fluorescent quantum dots (QDs), which are delivered to the skin using dissolvable microneedle arrays. The patterns are invisible to the naked eye but detectable with an infrared camera, which can extract information with >98% accuracy using automated pattern recognition software. By encapsulating NIR QDs in an FDA-approved biodegradable polymer, biodegradation rates can be tuned so that the encoded medical information can be conveyed in both a spatial and temporal manner, with some components fading within 100 days and others persisting for 6 months. This may be particularly useful for administering a series of vaccinations or treatments by indicating if enough time has passed for the patient to receive the next dose. Importantly, this system contains no personal information, does not require connection to a centralized database, and is not visible to the naked eye, ensuring patient privacy.

Decentralized manufacture of thermostable mRNA vaccines in a microneedle patch (MNP) format could enhance vaccine access in low-resource communities by eliminating the need for a cold chain and trained healthcare personnel. Here we describe an automated process for printing MNP Coronavirus Disease 2019 (COVID-19) mRNA vaccines in a standalone device. The vaccine ink is composed of lipid nanoparticles loaded with mRNA and a dissolvable polymer blend that was optimized for high bioactivity by screening formulations in vitro. We demonstrate that the resulting MNPs are shelf stable for at least 6 months at room temperature when assessed using a model mRNA construct. Vaccine loading efficiency and microneedle dissolution suggest that efficacious, microgram-scale doses of mRNA encapsulated in lipid nanoparticles could be delivered with a single patch. Immunizations in mice using manually produced MNPs with mRNA encoding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain stimulate long-term immune responses similar to those of intramuscular administration. Automated fabrication of microneedle patch mRNA vaccines for COVID-19 may improve vaccine access.

Layer-by-layer (LbL) self-assembly is an attractive method for the immobilization of macromolecules at interfaces. Integrating proteins in LbL thin films is however challenging due to their polyampholyte nature. Recently, we developed a method to integrate lysozyme into multilayers using protein-polyelectrolytes complexes (PPCs). In this work, we extended this method to a wide range of protein-polyelectrolyte combinations. We demonstrated the robustness and versatility of PPCs as building blocks. LL-37, insulin, lysozyme, and glucose oxidase were complexed with alginate, poly(styrenesulfonate), heparin, and poly(allylamine hydrochloride). The resulting PPCs were then LbL self-assembled with chitosan, PAH, and heparin. We demonstrated that multilayers built with PPCs are thicker compared to the LbL self-assembly of bare protein molecules. This is attributed to the higher mass of protein in the multilayers and/or the more hydrated state of the assemblies. PPCs enabled the self-assembly of proteins that could otherwise not be LbL assembled with a PE or with another protein. Furthermore, the results also show that LbL with PPCs enabled the construction of multilayers combining different proteins, highlighting the formation of multifunctional films. Importantly, we show that the adsorption behavior and thus the multilayer growth strongly depend on the nature of the protein and polyelectrolyte used. In this work, we elaborated a rationale to help and guide the use of PPCs for protein LbL assembly. It will therefore be beneficial to the many scientific communities willing to modify interfaces with hard-to-immobilize proteins and peptides.

Correction for ‘Hybrid chemoenzymatic heterogeneous catalyst prepared in one step from zeolite nanocrystals and enzyme–polyelectrolyte complexes’ by Margot Van der Verren et al., Nanoscale Adv., 2021, DOI: 10.1039/d0na00834f.

The immobilization of proteins to impart specific functions to surfaces is topical for chemical engineering, healthcare and diagnosis. Layer-by-Layer (LbL) self-assembly is one of the most used method to immobilize macromolecules on surfaces. It consists in the alternate adsorption of oppositely charged species, resulting in the formation of a multilayer. This method in principle allows any charged object to be immobilized on any surface, from aqueous solutions. However, when it comes to proteins, the promises of versatility, simplicity and universality that the LbL approach holds are unmet due to the heterogeneity of protein properties. In this review, the literature is analyzed to make a generic approach emerge, with a view to facilitate the LbL assembly of proteins with polyelectrolytes (PEs). In particular, this review aims at guiding the choice of the PE and the building conditions that lead to the successful growth of protein-based multilayered self-assemblies. (C) 2020 Elsevier B.V. All rights reserved.

The immobilization of proteins to impart specific functions to surfaces is topical for chemical engineering, healthcare and diagnosis. Layer-by-Layer (LbL) self-assembly is one of the most used method to immobilize macromolecules on surfaces. It consists in the alternate adsorption of oppositely charged species, resulting in the formation of a multilayer. This method in principle allows any charged object to be immobilized on any surface, from aqueous solutions. However, when it comes to proteins, the promises of versatility, simplicity and universality that the LbL approach holds are unmet due to the heterogeneity of protein properties. In this review, the literature is analyzed to make a generic approach emerge, with a view to facilitate the LbL assembly of proteins with polyelectrolytes (PEs). In particular, this review aims at guiding the choice of the PE and the building conditions that lead to the successful growth of protein-based multilayered self-assemblies.

The combination of heterogeneous catalysts and enzymes, in so-called hybrid catalysts, is an attractive strategy to effectively run chemoenzymatic reactions. Yet, the preparation of such bifunctional materials remains challenging because both the inorganic and the biological moieties must be integrated in the same solid, while preserving their intrinsic activity. Combining an enzyme and a zeolite, for example, is complicated because the pores of the zeolite are too small to accommodate the enzyme and a covalent anchorage on the surface is often ineffective. Herein, we developed a new pathway to prepare a hybrid catalyst built from glucose oxidase and TS-1 zeolite. Such hybrid material can catalyze the in situ formation of H2O2, which is subsequently used by the zeolite to trigger the epoxidation of allylic alcohol. Starting from an enzymatic solution and a suspension of zeolite nanocrystals, the hybrid catalyst is obtained in one step, using a continuous spray drying method. While enzymes are expectedly unable to resist the conditions used in spray drying (temperature, shear stress, etc.), we leverage on the preparation of “enzyme-polyelectrolyte complexes” (EPCs) to increase the enzyme stability. Importantly, the use of EPCs also appears to prevent enzyme leaching and to stabilize the enzyme against pH changes. We show that the one-pot preparation by spray drying gives access to hybrid catalysts with unprecedented performance in the targeted chemoenzymatic reaction. Interestingly, the hybrid catalyst performs much better than the two catalysts operating as separate entities. We anticipate that this strategy could be used as an adaptable method to prepare other types of multifunctional materials.

Controlling the sequence in which macromolecules interact allows creating self-assemblies that only exist because of the construction path that was followed. Because structure correlates with function, exploring paths of self-assembly holds the potential to confer various functionalities to the assembly. In this paper, lysozyme (Lyz) was used as a templating agent in the layer-by-layer (LbL) self-assembly of poly(allylamine hydrochloride) (PAH) with poly(styrenesulfonate) (PSS). To do so, Lyz was complexed with PSS, and the resulting complex was alternately adsorbed with PAH. By carrying the LbL assembly at low pH and in pure water, PAH adsorption causes the subsequent release of Lyz. The result is that even though it was used for assembly, only a very small fraction of Lyz is present in the resulting film. Using quartz crystal microbalance with dissipation monitoring mounted with a humidity module, the swelling/deswelling behavior and the composition of these polyelectrolyte multilayers (PEMs) were investigated. It appears that such PEMs obtained via a complex adsorption sequence are much thicker and trapped in a nonequilibrium state. Their dehydration is irreversible as it causes their collapse into denser PEMs. After dehydration, the PEMs share a common composition with the ones constructed without the use of Lyz as a templating agent. Fitting the Flory-Huggins equation to the data suggests that both PEM types separate into a polymer-rich phase and a water-rich phase when exposed to increasing water vapor pressure. Nevertheless, the presence of a swelling hysteresis in the PEMs templated with Lyz suggests that they possess a different internal structure or that trace amounts of Lyz affect the polymer relaxation kinetics. The use of proteins as templating elements thus allows to construct PEMs that are thick, highly hydrated, and gel-like at the nanoscale level rather than thin dense PE blends, which is topical for biomedical applications.

The deconstruction of self-assemblies based on proteins and polyelectrolytes (PEs) and the subsequent release of intact proteins require either a switch from attractive to repulsive mode or particular PE properties (degradability, responsiveness, or differential affinity). Here, an interfacial self-assembly made of three charged species, i.e., a strong polyacid complexed with a protein and a weak polybase, is shown to self-reorganize upon a shift in pH. When the pH takes a value that is one pH unit lower than the pKa of the weak polybase, the two PEs associate, thereby releasing the protein. The disassembly thus relies on associative forces rather than on the alteration of the protein-PE coupling strength. Hence, it allows the release of a protein using two simple PEs. The method is illustrated for lysozyme, which recovered up to half of its initial bioactivity after release. In contrast, a control self-assembled film that could not reorganize maintained only about 21% of the protein bioactivity after disassembly. This versatile approach is valuable for drug delivery devices and biomaterials as it allows the release of large numbers of active protein molecules.

Layer-by-layer (LbL) assembly is an attractive method for protein immobilization at interfaces, a much wanted step for biotechnologies and biomedicine. Integrating proteins in LbL thin films is however very challenging due to their low conformational entropy, heterogeneous spatial distribution of charges, and polyampholyte nature. Protein-polyelectrolyte complexes (PPCs) are promising building blocks for LbL construction owing to their standardized charge and polyelectrolyte (PE) corona. In this work, lysozyme was complexed with poly(styrenesulfonate) (PSS) at different ionic strengths and pH values. The PPCs size and electrical properties were investigated, and the forces driving complexation were elucidated, in the light of computations of polyelectrolyte conformation, with a view to further unravel LbL construction mechanisms. Quartz crystal microbalance and atomic force microscopy were used to monitor the integration of PPCs compared to the one of bare protein molecules in LbL assemblies, and colorimetric assays were performed to determine the protein amount in the thin films. Layers built with PPCs show higher protein contents and hydration levels. Very importantly, the results also show that LbL construction with PPCs mainly relies on standard PE-PE interactions, independent of the charge state of the protein, in contrast to classical bare protein assembly with PEs. This considerably simplifies the incorporation of proteins in multilayers, which will be beneficial for biosensing, heterogeneous biocatalysis, biotechnologies, and medical applications that require active proteins at interfaces.

A standard method of protein immobilization is proposed, based on the use of protein-polyelectrolyte complexes (PPCs) as building blocks for layer-by-layer assembly. Thicker multilayers, with a higher polyelectrolyte fraction, are obtained with PPCs compared to single protein molecules. Biological activity is not only maintained, but specific activity is also higher, as demonstrated for lysozyme-poly(styrene sulfonate) complexes. This is attributed to the more hydrated state of the assemblies. This new method of protein immobilization opens up perspectives for biotechnology and biomedical applications.

The immunostimulation capacity of most vaccines is enhanced through antigen adsorption on aluminum hydroxide (AH) adjuvants. Varying the adsorption conditions, i.e. pH and ionic strength (I), changes the antigen adsorbed amount and therefore the ability of the vaccine to stimulate the immune system. Vaccine formulations are thus resulting from an empirical screening of the adsorption conditions. This work aims at studying the physicochemical effects of adjusting the ionic strength of commercial AH adjuvant particles suspensions with sodium chloride (NaCl). X-ray photoelectron spectroscopy data show that AH particles surface chemical composition is neither altered by I adjustment with NaCl nor by deposition on gold surfaces. The latter result provides the opportunity to use AH-coated gold surfaces as a platform for advanced surface analysis of adjuvant particles, e.g. by atomic force microscopy (AFM). The morphology of adjuvant particles recovered from native and NaCl-treated AH suspensions, as studied by scanning electron microscopy and AFM, reveals that AH particles aggregation state is significantly altered by NaCl addition. This is further confirmed by nitrogen adsorption experiments: I adjustment to 150mM with NaCl strongly promotes AH particles aggregation leading to a strong decrease of the developed specific surface area. This work thus evidences the effect of NaCl on AH adjuvant structure, which may lead to alteration of formulated vaccines and to misinterpretation of data related to antigen adsorption on adjuvant particles.

Aluminum hydroxide (AH) salts are the most widely used adjuvants in vaccine formulation. They trigger immunogenicity from antigenic subunits that would otherwise suffer from a lack of efficiency. Previous studies focusing on antigen AH interaction mechanisms, performed with model proteins, suggested that electrostatic interactions and phosphate -hydroxyl ligand exchanges drive protein adsorption on AH. We however recently evidenced that NaCl, used in vaccine formulation, provokes AH particle aggregation. This must be taken into account to interpret data related to protein adsorption on AH. Here, we report on the successful development and use of a stable AH-coated surface to explore the mechanisms of protein adsorption by means of ultrasensitive surface analysis tools. Bovine serum albumin (BSA) adsorption was studied at different pHs and ionic strengths (I) using quartz crystal microbalance. The results show that protein adsorption on the AH adjuvant cannot be explained solely by electrostatic interactions and ligand exchanges. Hence, a higher adsorption was observed at pH 3 compared to pH 7, although AH and BSA respectively undergo repulsive and attractive electrostatic interactions at these pH values. Almost no effect of I on adsorption was moreover noted at pH 7. These new developments and observations not only suggest that other mechanisms govern protein adsorption on AH but also offer a new platform for the study of antigen adsorption in the context of vaccine formulation. Immobilizing particles on QCM sensors also enriches the range of applications for which QCM can be exploited, especially in colloid science.