We report the design of a novel microcapsule platform for in situ pH sensing and photothermal heating, which involves the encapsulation of pH-responsive polymer-coated molybdenum disulfide (MoS) nanosheets (NSs) in microcapsules with an aqueous core and a semipermeable polymeric shell. The MoS NSs were functionalized with pH-responsive polymers having fluorescent groups at the distal end to provide pH-sensitive Förster resonance energy transfer (FRET) effect. The pH-responsive polymers were carefully designed to produce a dramatic change in the polymer conformation, which translated to a change in the FRET efficiency near pH 7.0 in response to subtle pH changes, enabling the detection of cancer cells. The pH-sensitive MoS NSs were microfluidically encapsulated within semipermeable membranes to yield microcapsules with a uniform size and composition. The microcapsules retained the MoS NSs without leakage while allowing the diffusion of small ions and water through the membrane. At the same time, the membranes excluded adhesive proteins and lipids in the surrounding media, protecting the encapsulated MoS NSs from deactivation and enabling in situ pH monitoring. Moreover, the encapsulated MoS NSs showed high-performance photothermal heating, rendering the dual-functional microcapsules highly suitable for cancer diagnosis and treatment.

Highlights d Directed evolution of nanobodies that potently neutralize SARS-CoV-2 d CDR-swapping mutagenesis facilitates large affinity and activity improvements d Nanobody binding to RBD competes with ACE2 and two classes of neutralizing mAbs d Neutralizing nanobodies display drug-like biophysical properties

The COVID-19 pandemic continues to be a severe threat to human health, especially due to current and emerging SARS-CoV-2 variants with potential to escape humoral immunity developed after vaccination or infection. The development of broadly neutralizing antibodies that engage evolutionarily conserved epitopes on coronavirus spike proteins represents a promising strategy to improve therapy and prophylaxis against SARS-CoV-2 and variants thereof. Herein, a facile multivalent engineering approach is employed to achieve large synergistic improvements in the neutralizing activity of a SARS-CoV-2 cross-reactive nanobody (VHH-72) initially generated against SARS-CoV. This synergy is epitope specific and is not observed for a second high-affinity nanobody against a non-conserved epitope in the receptor-binding domain. Importantly, a hexavalent VHH-72 nanobody retains binding to spike proteins from multiple highly transmissible SARS-CoV-2 variants (B.1.1.7 and B.1.351) and potently neutralizes them. Multivalent VHH-72 nanobodies also display drug-like biophysical properties, including high stability, high solubility, and low levels of non-specific binding. The unique neutralizing and biophysical properties of VHH-72 multivalent nanobodies make them attractive as therapeutics against SARS-CoV-2 variants.