Synthesis, Hemoglobin Encapsulation and Biorthogonal PEGylation in Hierarchically Porous UiO-66 Nanoparticles for...

Hemoglobin-based oxygen carriers (HBOCs) are an important platform for oxygen delivery to mitigate hypoxia in several biomedical contexts. However, free hemoglobin (Hb) is unstable and vasoactive outside red blood cells. Here, we present a reproducible, scalable step-by-step protocol for fabricating hierarchically porous UiO-66 nanoparticles (HP-UiO-66 NPs) that encapsulate Hb and are stabilized by bioorthogonal polyethylene glycol (PEG) shells. Hierarchical mesoporosity is introduced by dodecanoic acid modulation and subsequent acid washing, yielding HP-UiO-66-NH₂ NPs with preserved crystallinity and combined type I/IV N2 isotherms. Surface amines are converted to azides (HP-UiO-66-N₃ NPs), enabling catalyst-free strain-promoted alkyne-azide cycloaddition (SPAAC) to produce Hb@HP-UiO-66-PEG NPs. The workflow comprises: i) defect engineering of UiO-66-NH₂ NPs, ii) modulator removal, iii) azide installation, iv) Hb loading in buffered aqueous media, and v) SPAAC-mediated PEGylation. Critical experimental parameters, standard failure modes, and troubleshooting strategies are detailed at each stage to enhance inter-laboratory reproducibility. The protocol is amenable to gram-scale production of HP-UiO-66-NH₂ NPs per batch. Encapsulation efficiencies of ~35% (loading content ~24 wt%) for Hb are obtained under mild conditions, with redispersible NPs with a hydrodynamic diameter of ~140 nm and low polydispersity after PEGylation. Comprehensive physicochemical characterization verifies the integrity of the framework, mesoporosity, successful surface chemistry, and a diffuse PEG corona up to ~12 nm wide. Functional assays show reversible oxygenation/deoxygenation of encapsulated Hb, indicating retained oxygen-transporting capacity. PEGylation markedly enhances colloidal and structural stability in saline, HEPES, and serum, preventing phosphate-induced degradation observed for non-PEGylated counterparts. By integrating detailed troubleshooting guidance, video-assisted visualization of critical steps, and scale-up considerations, this modular protocol provides a robust and accessible route to metal-organic framework-based HBOCs. The strategy is readily adaptable to other biomacromolecules or enzymes requiring protective yet accessible microenvironments for therapeutic delivery.