Antibody desolvation with sodium chloride and acetonitrile generates bioactive protein nanoparticles.

About 30% of the FDA approved drugs in 2021 were protein-based therapeutics. However, therapeutic proteins can be unstable and rapidly eliminated from the blood, compared to conventional drugs. Furthermore, on-target but off-tumor protein binding can lead to off-tumor toxicity, lowering the maximum tolerated dose. Thus, for effective treatment therapeutic proteins often require continuous or frequent administration. To improve protein stability, delivery and release, proteins can be encapsulated inside drug delivery systems. These drug delivery systems protect the protein from degradation during (targeted) transport, prevent premature release and allow for long-term, sustained release. However, thus far achieving high protein loading in drug delivery systems remains challenging. Here, the use of protein desolvation with acetonitrile as an intermediate step to concentrate monoclonal antibodies for use in drug delivery systems is reported. Specifically, trastuzumab, daratumumab and atezolizumab were desolvated with high yield (∼90%) into protein nanoparticles below 100 nm with a low polydispersity index (<0.2). Their size could be controlled by the addition of low concentrations of sodium chloride between 0.5 and 2 mM. Protein particles could be redissolved in aqueous solutions and redissolved antibodies retained their binding activity as evaluated in cell binding assays and exemplified for trastuzumab in an ELISA.

Modulation of Chitosan-TPP Nanoparticle Properties for Plasmid DNA Vaccines Delivery.

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 <180 nm and positive zeta potentials (>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.

Anti-Fn14 Antibody-Conjugated Nanoparticles Display Membrane TWEAK-Like Agonism.

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.

Efficient Delivery of Hydrophilic Small Molecules to Retinal Cell Lines Using Gel Core-Containing Solid Lipid Nanoparticles.

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.

Evaluation of silicone elastomers as structural materials for microstructured adhesives.

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.

Evaluation of Material Properties for Practical Microstructured Adhesives: Low Dust Adhesion and High Shear Strength.

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.

Understanding the influence of silicone elastomer properties on wedge-shaped microstructured dry adhesives loaded in shear.

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.

Spray-coated carbon nanotube carpets for creeping reduction of conducting polymer based artificial muscles.

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.

Hybrid PVDF/PVDF-graft-PEGMA Membranes for Improved Interface Strength and Lifetime of PEDOT:PSS/PVDF/Ionic Liquid Actuators.

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 10(4) 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.