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Nucleic acid vaccines have become a revolutionary technology to give a fast, safe, cost-effective and efficient response against viral infections, such as SARS-CoV-2 or Human papillomavirus (HPV). However, to ensure their effectiveness, the development of adequate methods to protect, carry, and deliver nucleic acids is fundamental. In this work, nanoparticles (NPs) of chitosan (CS)-tripolyphosphate (TPP)-plasmid DNA (pDNA) were thoroughly modulated and characterized, by measuring the charge and size through dynamic light scattering (DLS) and morphology by scanning electron microscopy (SEM). Stability, cytotoxicity and cellular uptake of NPs were also evaluated. Finally, the effect of polyplexes on the expression of HPV E7 antigen in human fibroblast and RAW cells was investigated through polymerase chain reaction (PCR) and real-time PCR. The results showed NPs with a spherical/oval shape, narrow size distribution 20 mV) and good stability after one month of storage at 4 °C in formulation buffer or when incubated in culture medium and trypsin. In vitro studies of NPs cytotoxicity revealed that the elimination of formulation buffers led to an improvement in the rate of cell viability. The E7 antigen transcription was also increased for NPs obtained with high pDNA concentration (60 μg/mL). The analyzed CS-TPP-pDNA polyplexes can offer a promising vehicle for nucleic acid vaccines, not only in the prevention or treatment of viral infections, but also to fight emergent and future pathogens.

In this study, we developed a novel solid lipid nanoparticle (SLN) formulation for drug delivery of small hydrophilic cargos to the retina. The new formulation, based on a gel core and composite shell, allowed up to two-fold increase in the encapsulation efficiency. The type of hydrophobic polyester used in the composite shell mixture affected the particle surface charge, colloidal stability, and cell internalization profile. We validated SLNs as a drug delivery system by performing the encapsulation of a hydrophilic neuroprotective cyclic guanosine monophosphate analog, previously demonstrated to hold retinoprotective properties, and the best formulation resulted in particles with a size of ±250 nm, anionic charge > −20 mV, and an encapsulation efficiency of ±60%, criteria that are suitable for retinal delivery. In vitro studies using the ARPE-19 and 661W retinal cell lines revealed the relatively low toxicity of SLNs, even when a high particle concentration was used. More importantly, SLN could be taken up by the cells and the release of the hydrophilic cargo in the cytoplasm was visually demonstrated. These findings suggest that the newly developed SLN with a gel core and composite polymer/lipid shell holds all the characteristics suitable for the drug delivery of small hydrophilic active molecules into retinal cells.

This paper presents a way to characterize the strength of bioinspired gecko-adhesives. These are the most promising mechanisms, so far, which enable controlled adhesion (i.e. turning it on and off). They emulate the forces present in geckos’ fingertips to stick temporarily to surfaces without any preconditioning and keeping them intact. Moreover, they do not need of any specific environment to work, which makes them specially useful for space applications such as grippers, or robots capable of climbing the outer surface of a space vehicle to perform inspections or repairs. To further enhance their adhesion strength, a high voltage electrostatic field is usually applied. However, while these adhesives tend to perform very well on smooth surfaces, the nefarious effects of surface roughness are yet uncharacterized. In this paper, we developed a statistical method to characterize those effects based on the concept of effective surface defects, and without need of a yet intractable direct measurement. In particular, controlled surface defects on adhesives themselves were statistically analyzed through a sample of over 2000 experiments; matching them with the statistical results. Through this process, the validity of the analysis lead to a way of calculating effective surface defects on substrates by considering the dual problem. Because this method does not require substrates to have any specific surface conditions, its use might be extended to study adhesion on more challenging surface morphologies such as that of kapton thermal covers, which often appear on space vehicles.

Conventional bivalent IgG antibodies targeting a subgroup of receptors of the TNF superfamily (TNFSF) including fibroblast growth factor-inducible 14 (anti-Fn14) typically display no or only very limited agonistic activity on their own and can only trigger receptor signaling by crosslinking or when bound to Fcγ receptors (FcγR). Both result in proximity of multiple antibody-bound TNFRSF receptor (TNFR) molecules, which enables engagement of TNFR-associated signaling pathways. Here, we have linked anti-Fn14 antibodies to gold nanoparticles to mimic the “activating” effect of plasma membrane-presented FcγR-anchored anti-Fn14 antibodies. We functionalized gold nanoparticles with poly-ethylene glycol (PEG) linkers and then coupled antibodies to the PEG surface of the nanoparticles. We found that Fn14 binding of the anti-Fn14 antibodies PDL192 and 5B6 is preserved upon attachment to the nanoparticles. More importantly, the gold nanoparticle-presented anti-Fn14 antibody molecules displayed strong agonistic activity. Our results suggest that conjugation of monoclonal anti-TNFR antibodies to gold nanoparticles can be exploited to uncover their latent agonism, e.g., for immunotherapeutic applications.

Cancer is the second leading cause of death in the world and is often untreatable. Protein-based therapeutics, such as immunotherapeutics, show promising results in the fight against cancer, resulting in their market share increasing every year. Unfortunately, most protein-based therapeutics suffer from fast degradation in the blood, making effective treatment expensive, causing more off-target effects (due to the high doses necessary), and often require repeated injections to stay within the correct therapeutic range. Encapsulation of these proteins inside nanocarriers is prompted to overcome these problems by enhancing targeted drug delivery and, thus, leading to a less frequent administration and lower required dose. However, most current protein encapsulation methods show very low loading capacities (LC). This leads to even more expensive treatments and might pose a further risk for the patient caused by systemic toxicity against high concentrations of the carrier material. We investigated and optimized protein nanoprecipitation as a method to obtain a high protein LC and encapsulation efficiency (EE) inside poly(lactic-co-glycolic acid; PLGA) nanoparticles via a simple two-step process. In this work, we used model proteins to investigate the influence of various parameters such as precipitation solvent, addition speed, and protein concentration on protein activity. Our work is a critical step towards the high-loading encapsulation of immunotherapeutics.

Immunotherapy is considered as a new pillar of cancer treatment. However, the application of some promising immunotherapeutic antibodies, such as antibodies against certain immune-stimulatory receptors of the TNF receptor superfamily (TNFRs), including CD40, 41BB, CD27 and anti-fibroblast growth factor-inducible 14 (anti-Fn14), are limited due to their low bioactivity. It has been previously shown that the bioactivity of such anti-TNFR antibodies could be improved by crosslinking or attachment to the plasma membrane by interaction with Fcγ receptors (FcγR). Both result in the proximity of multiple antibody-bound TNFR molecules, which allow for the activation of proinflammatory signaling pathways. In this work, we have grafted antibodies on gold nanoparticles to simulate the “activating” effect of FcγR-bound, and thus plasma membrane-presented anti-TNFR antibodies. We have developed and optimized the method for the preparation of gold nanoparticles, their functionalization with poly-ethylene glycol (PEG) linkers, and grafting of antibodies on the surface. We showed here that antibodies, including the anti-Fn14 antibody PDL192, can be successfully attached to nanoparticles without affecting antigen binding. We hypothesize that conjugation of monoclonal anti-TNFR antibodies to the inorganic nanoparticles is a promising technique to boost the efficacy of these immunotherapeutic antibodies.

Intramuscular delivery is one of the main routes for DNA vaccines administration. However, it requires large amounts of the DNA to be administered and external stimulation to encourage the internalization of the DNA. In this work, we consider alternative routes for less invasive administration and develop drug delivery systems (DDS) for intranasal administration. Chitosan polyplexes using sodium tripolyphosphate (TPP) as a crosslinker were prepared using the ionic gelation method. Our method allowed preparation of nanoparticles with the size below 50 nm, which is at least two times lower than previously reported sizes. Moreover, despite the small sizes, we obtained DNA encapsulation efficiencies of about 70%. Parameters that may affect the encapsulation efficiency were investigated, including different TPP-chitosan ratios and concentrations of DNA. We found that encapsulation efficiency of DNA inside the particles decreases with the increasing TPP-chitosan ratio. Moreover, increasing the DNA concentration leads to a higher encapsulation efficiency. Small (

High-throughput emulsion electrospinning is a technology that can enable practical nanofiber application for drug delivery. Core-shell structure of the electrospun fibers allows the encapsulation of the active pharmaceutical ingredients (APIs), protects their activity, and controls their release rate. However, electrospinning using high flow rates usually requires high electric fields that may negatively affect the activity of the biomolecules. Moreover charged APIs tend to migrate to the surface of the fibers during the electrospinning process leading to the high burst release. That is disadvantageous when long-term sustained release is needed. We have investigated the influence of the electrospinning parameters such as distances between the electrode and collector and the applied voltages to both, activity of the encapsulated proteins and their burst release. We have also tested the influence of number of the stabilizers, e.g. trehalose, pluronic, polyvinylpyrrolidone, on their ability to preserve the protein activity, and the influence of the different molecular weights of polyvinyl alcohol on the ability to sustain the release. Our results demonstrate the importance of the water phase composition to both activity and release and are critical for further understanding of the processes taking place during the emulsion electrospinning.

Nowadays, an electrospinning process is one of the most promising technique to produce nanofibers. The popularity of this method is still significant, due to its simplicity and relatively low cost. Moreover, many electrospun compounds can be used in numerous applications in different areas, for instance medical and pharmaceutical fields, textile engineering, environmental remediation. However, a design of an efficient electrospinning system remains challenging, due to high number of parameters strongly affecting fibers properties and productivity of their formation. Lately, a great attention is given to development of needleless configurations, characterized by multi-jets system, thanks to their enhanced output. Indeed, a surface of spinneret, where the liquid jet formation is initiated, plays crucial role to improve the process. The electric field profile of the spinning electrode directly influences the size and uniformity of the produced fibers and should be strongly considered in further development. Our study presents a comprehensive theoretical analysis of the most effective needleless electrospinning configurations and compares their electrostatic features. Our extensive analysis was prepared in COMSOL Multiphysics software, known as a great tool to describe in details physical phenomena and it can strongly help to select the proper electrospinning set up, depending on desired results.

Among the most promising technologies for sustained drug delivery systems are core-shell nanofibres prepared by electrospinning. However, the most common method for the production of those, coaxial electrospinning, suffers from extremely low flow rates limiting the practical applications of such fibres. Emulsion electrospinning, on the other hand, enables the use of the high-throughput needle-less electrospinning devices for the production of the core-shell nanofibres with active pharmaceutical ingredients embedded and protected in their core. The development of such drug delivery systems for the applications in bone regeneration is further challenged by the need for the inorganic additives meant to stimulate the regeneration of the bone. The main objective of this work is to develop a high-throughput electrospinning method for the production of hybrid (organic-inorganic) and bioactive scaffolds needed for bone regeneration. We demonstrate the importance of the formulation, e.g., presence of surfactants, on the stability of the emulsion, and thus on electrospinning. Our work is an important step forward towards the high-throughput production of complex multi-material scaffolds for sustained drug delivery.

The development of microstructure (gecko-like) adhesives has focused almost solely on their adhesive strength. However, for practical applications, especially in real-world environments, the adhesive's long-term performance is arguably equally important. One impediment to long-term viability is the adhesive's susceptibility to contamination. It is a challenge to develop an adhesive that can both adhere to a substrate while not becoming contaminated with dust and debris. In response, this paper experimentally explores the effect of modulus of elasticity, work of separation, and work of adhesion (adhesion energy) on the shear stress and particle detachment capabilities of wedge-shaped, directional microstructured adhesives. Particle removal is evaluated using both noncontact cleaning methods (centripetal force and electrostatic particle repulsion) and a dry contact cleaning method (load-drag-unload test). Results show that for a material with a high work of separation, high elastic modulus, and low work of adhesion, it is possible to create a microstructured adhesive with both high shear stress strength and low adhesion to dust particles. Results also show that, for dry contact cleaning, shear stress recovery mostly stems from particle rolling and not particle sliding. Moreover, shear test results show that augmenting the microstructured adhesive with electrostatic adhesion can reduce the negative effects on adhesion of a high elastic modulus materials' conformability to a substrate by providing a preload to the microstructured elements. Last, this paper is the first to report on a electrostatic/gecko-like adhesive that uses its electrostatic elements for both adhesion and dust repulsion; they were reported separately before.

Cancer is one of the leading causes of death in the world and protein therapeutics play an important role in combating this disease. Novel nanocarriers are needed for optimal delivery, enhanced therapeutic effect, and protection of proteins. Poly Lactic-co-Glycolic Acid (PLGA) nanoparticles are commonly used, since they are non-toxic, biodegradable, and allow for the sustained release of the active pharmaceutical ingredient (API). Accurate quantification of the therapeutic inside these nanocarriers is essential for further development and precise in vivo experiments, especially for determining the correct therapeutic dose. Bicinchoninic acid (BCA) assay is one of the most popular methods of protein quantification, known for its low sensitivity to common surfactants. However, large discrepancies between published results are often observed, with determined protein encapsulation efficiencies (EE) varying from 20 to 80%. We investigate the interference of excipients or the combination of excipients, on accurate EE determination of PLGA nanoparticles using the micro BCA assay. The EE was determined using multiple methods: by measuring the un-encapsulated protein (indirect approach) and directly by extracting the protein using sodium hydroxide and dimethyl sulfoxide. We show differences between the methods, highlight the most common pitfalls, and show the importance of using correct standards in assessing EE.

One of the constraints in the design of dry switchable adhesives is the compliance trade-off: compliant structures conform better to surfaces but are limited in strength due to high stored strain energy. In this work we study the effects of bending compliance on the shear adhesion pressures of hybrid electrostatic/gecko-like adhesives of various areas. We reaffirm that normal electrostatic preload increases contact area and show that it is more effective on compliant adhesives. We also show that the gain in contact area can compensate for low shear stiffness and adhesives with high bending compliance outperform stiffer adhesives on substrates with large scale roughness.

International audience; The exploitation of soft conducting polymer-based actuators suffers from two main shortcomings: their short life cycle and the reproducibility of the fabrication techniques. The short life cycle usually results from the delamination of the components due to stresses at the interface during the actuation. In this work, to achieve strong adhesion to poly(3,4- ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS) electrodes, the wetting properties of the surface of a polyvinylidene fluoride (PVDF) membrane are improved using argon-plasma-induced surface polymerization of poly(ethylene glycol) monomethyl ether methacrylate (PEGMA). Hybrid membranes are created with hydrophilic PVDF-graft-PEGMA outer surfaces and hydrophobic bulk. The width of each layer is controlled by spray coating, as it allows for the deposition of the reaction precursor to a certain depth. Subsequently, a PEDOT:PSS water solution fills the pores of the functionalized part of the membrane and a mixing layer between PEDOT:PSS and PVDF is created. We also show that PVDF-graft-PEGMA copolymers play an important role in binding the membrane to the electrodes and that direct mechanical interlocking in the pores can further improve the adhesion. Finally, PEDOT:PSS/PVDF-graft-PEGMA/PEDOT:PSS actuators are made by simple solution casting. They are capable of producing high strains of 0.6% and show no signs of delamination after more than 150 h or 104 actuation cycles. Furthermore, the preservation of the hydrophobic membrane in between two PEDOT:PSS layers increases the resistance between them from 0.36 Ω to 0.16 MΩ, thus drastically modifying the power dissipation of the actuators.

Microstructured (sometimes referred to as gecko-like) adhesives have numerous advantages over flat films, especially for practical applications on non-ideal surfaces that may be uneven or contaminated with dust. However, due to interdependence among material surface and bulk properties, the best material to fabricate such adhesives is still unknown. In this work, we analyzed eleven commercially available silicone elastomers to evaluate their use as flat and microstructured adhesives to address multiple material related questions that may impact the choice of the 'best' material for microstructured dry adhesives. To illustrate the applicability of the measured properties to modeling microstructured surfaces, we use stalk-shaped microstructures, whose contact mechanics are well understood. We demonstrate that there is no correlation between the adhesion strength of flat and microstructured adhesives; while bulk dissipation is the most important factor influencing the adhesion strength of flat elastomers, after microstructurization, interface toughness becomes more important. Therefore, microstructured elastomers loaded with high surface energy additives may demonstrate higher adhesion than their flat counterparts. We also compare the adhesion of flat and microstructured silicone elastomers on rough substrates. In this case, we show that while flat elastomer adhesion decreases with increasing substrate roughness, microstructured silicone adhesion actually increases with increasing roughness up to 0.19 [Formula: see text]m. This is the first time an increase in adhesion strength on rough surfaces is reported for materials stiffer than 1.0 MPa.

Bonding silicone elastomers is one of the largest challenges in microfabrication. Various strategies have been suggested, such as surface activation by oxygen plasma and bonding using partially cured elastomers. However, there is no universal method capable of bonding various commercially available elastomers. In this paper, we evaluate silicone crosslinker and catalyst as bonding enhancers. We demonstrate that these materials can be used to bond multiple commercially available elastomers, even a week after their moulding. Moreover, we demonstrate the applicability of such bonding enhancers to the fabrication of electrostatic/gecko-like adhesives. We show that increase in the crosslinking density at the interface may enhance the adhesion strength of such devices by reducing the shear compliance. This is the first time the use of these bonding enhancers is reported.

During cyclic actuation, conducting polymer based artificial muscles are often creeping from the initial movement range. One of the likely reasons of such behaviour is unbalanced charging during conducting polymer oxidation and reduction. To improve the actuation reversibility and subsequently the long time performance of ionic actuators, we suggest using spray-coated carbon nanotube (CNT) carpets on the surface of the conducting polymer electrodes. We show that carbon nanotubes facilitate a conducting polymer redox reaction and improve its reversibility. Consequently, in the long term, charge accumulation in the polymer film is avoided leading to a significantly improved lifetime performance during cycling actuation. To our knowledge, it is the first time a simple solution to an actuator creeping problem has been suggested.

Tri-layer electroactive bending polymeric artificial muscles generally exhibit no overshoot during open-loop step response with a non-zero initial slope when the output is the vertical position of the bending sample tip. We propose to identify such a bending step contraction by a nonlinear system derived from a linear first order system in the form: where the parameters k, T and r depend on the u-control voltage. We show the relevance of this approach for identifying the step-response of a PEDOT:PSS/PVDF/ionic liquid actuator developed at the laboratory. As a consequence, we try to show that a linear PI-controller, including voltage constraints, is a simple and an efficient approach for a closed-loop control of the bending actuator.

International audience; Inkjet printing is a key technology in the field of defined polymer deposition as well as in fabrication of strain sensors. It is also one of the most promising alternatives to prevalent fabrication of conducting polymer actuators. Nevertheless, inkjet printed actuators were not yet realised due to rheological properties of conducting polymer solutions that challenge jetting and the complex solution–membrane interactions, that lead to poor adhesion or uncontrolled infiltration. In order to enable this fabrication method, hybrid hydrophilic-hydrophobic polyvinylidene fluoride (PVDF) ultrafiltration membranes were used. They ensure good adhesion with printed poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) layer. Furthermore, PEDOT:PSS compositions that have liquid properties suitable for printing but do not cause uncontrolled infiltration through the membrane were identified. This allowed the realisation of the first inkjet printed PEDOT:PSS based ionic actuators working in air.

Anisotropic, gecko-inspired, microstructured adhesives are one of the most promising solutions for many applications in robotics and biomedical applications that require controllable adhesives to grip flat surfaces. In such adhesives, normal adhesion is negligible when loaded solely in the normal direction, but becomes available when the adhesive is loaded in shear first. However, much remains to be learned regarding the friction and failure mechanisms of microstructures loaded in shear. In response, we analysed the load–displacement profiles of wedge-shaped microstructured adhesives comprised of nine different silicone elastomers and their mixtures loaded in shear. The results show that the friction profile depends on at least three factors related to material properties: interfacial adhesion strength in the normal direction (work of separation), elastic modulus and the sample's imperfections (e.g. contamination, misalignment and moulding errors). Moreover, the work of separation influences the maximum friction load such that for materials with the same elastic modulus, the strongest interfacial adhesion yields the lowest friction force. To explain this, we suggest that strongly adhering materials will lead to a macroscopic frictional sliding of the array rather than previously reported stick-slip behaviour.

International audience; Despite a growing interest in conducting polymer based actuators, a robust fabrication technique is still needed. We suggest a fabrication method that allows fast production of conducting polymer actuators from commercially available polyvinylidene flouride membranes (PVDF) and a PEDOT/PSS solution. In order to improve adhesion of those materials, Ar plasma induced grafting is used to create hydrophilic surfaces of up to 40 μm. Hydrophilic PVDF-graft-polyethylene glycol allows diffusion of PEDOT/PSS in the pores of the membranes, while hydrophobic middle layers prevent short circuits. In this way, soft actuators can be fabricated by consequent drop casting and drying of conducting polymer.