Name: Synthesis, Hemoglobin Encapsulation and Biorthogonal PEGylation in Hierarchically Porous UiO-66 Nanoparticles for Oxygen Delivery Applications (Video) | JoVE Journal
Uploaded: 2026-05-08T13:10:00.000+00:00
Duration: 9 min 24 s
Description: Learn a scalable protocol for synthesizing hierarchically porous UiO-66 nanoparticles that encapsulate hemoglobin for oxygen delivery applications. This method enables gram-scale production of stable, PEGylated hemoglobin-based oxygen carriers.

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5 minutes

We gratefully acknowledge support from the European Research Council under the European Union&#39;s Horizon 2020 Research and Innovation Program (Grant No. 101002060).

Authors declare no competing financial interest.

This protocol presents a robust and reproducible route to synthesize, functionalize, and characterize HP-UiO-66 NPs designed for Hb encapsulation and oxygen-delivery applications. By adopting an inside-out engineering approach, this work establishes a generalizable framework for the design of MOF NPs for biomedical applications, including biomacromolecule and drug delivery. While the underlying material concept and functional performance have been reported previously, the present protocol significantly extends prior work...

Adequate oxygenation is critical for effective therapy and tissue healing across diverse biomedical settings1. In trauma and surgical hemorrhage, severe oxygen loss can be life-threatening, highlighting the need for safe blood substitutes when donor blood is unavailable2. In oncology, hypoxic tumor regions limit the efficacy of oxygen-dependent treatments, and supplemental oxygen delivery can markedly enhance therapeutic efficacy3,4,5,6. Oxygen also plays a central role in wound healing, where increased oxygen availability promotes angiogenesis and tissue repair7. Similarly, in ischemic conditions and organ transplantation, oxygen carriers can reduce hypoxic injury and improve tissue viability and recovery8,9.
Synthetic hemoglobin (Hb)-based oxygen carriers (HBOCs) represent a promising approach to mitigate hypoxia10. However, Hb outside of red blood cells (RBCs) is inherently unstable and rapidly dissociates into dimers that can cause nephrotoxicity, oxidative damage, and vasoconstriction due to nitric oxide scavenging11. To mitigate these challenges, encapsulation strategies have been developed to stabilize Hb within biocompatible nanocarriers such as liposomes12, polymersomes13, and polymeric nanoparticles (NPs)14,15,16,17. Although these systems enhance Hb stability, achieving high encapsulation efficiency while preserving native oxygen-binding function remains challenging18. In addition, oxidative conversion to methemoglobin and heme leakage can compromise oxygen-carrying performance19. Rapid clearance and premature Hb leakage further limit circulation time and clinical translation20.
Metal-organic frameworks (MOFs) have recently emerged as versatile nanoplatforms for drug and biomacromolecule delivery due to their crystalline architecture, large surface area, and tunable porosity21. Composed of metal nodes coordinated with organic linkers, MOFs enable molecular diffusion and structural customization, making them attractive for biomedical applications. However, most MOFs possess predominantly microporous cavities (&#60;2 nm), which severely restrict post-synthetic encapsulation of macromolecules such as proteins, nucleic acids, or enzymes22,23,24,25. One approach to overcome this limitation is in situ biomimetic mineralization, where biomolecules act as nucleation centers during MOF growth, as demonstrated for ZIF-8 and ZIF-9026,27,28. Despite its effectiveness, this strategy requires mild synthesis conditions, which restrict the range of compatible MOF chemistries29,30.
UiO-66, a zirconium-based MOF constructed from Zr6O4(OH)4 octahedral nodes and 1,4-benzenedicarboxylate (BDC) linkers, stands out for its exceptional chemical stability and biocompatibility31. Its robust framework resists decomposition in aqueous and physiological environments, making it ideal for biomedical applications32. Nevertheless, its intrinsic microporous structure limits its utility for biomacromolecule encapsulation. Hb, with a molecular dimension of ~5 nm, cannot be accommodated within the internal pores of pristine UiO-66. Correspondingly, only a limited number of studies have explored UiO-66 in Hb-related systems, in which the MOFs are primarily used for surface-based Hb sensing rather than for framework encapsulation33,34. As a result, UiO-66-based platforms for Hb encapsulation remain relatively underdeveloped.
Very recently, we introduced a hierarchically porous UiO-66 NP (HP-UiO-66 NP) that overcomes this pore size restriction by incorporating mesoporous domains capable of accommodating Hb, thereby extending the functional scope of UiO-66 for HBOC development35. Hierarchical porosity is introduced through defect engineering, specifically by employing long-chain monocarboxylic acids, such as dodecanoic acid (DA), as modulators during synthesis36,37. This modulator competes with the BDC ligand for coordination with Zr4+, leading to defect formation and enlarged mesopores. Subsequent removal of the modulator yields HP-UiO-66 NPs containing coexisting micro- and mesoporous domains, with tunable pore size through modulator concentration or chain length36.
Although the introduction of mesoporosity improves molecular accessibility, it may also compromise framework integrity in phosphate-containing buffers. Phosphate ions can coordinate with Zr clusters, leading to partial or complete structural disintegration38,39. Accordingly, surface stabilization strategies are essential to preserve colloidal and chemical stability under physiological conditions40. In general, MOF surface stabilization can be achieved through noncovalent interactions (e.g., van der Waals interaction and hydrogen bonding)41, coordination interactions with exposed metal sites42, or covalent attachment to organic linkers43. Among different linkers, polyethylene glycol (PEG) is particularly attractive due to its broad availability and minimal synthetic complexity44. Importantly, PEG surface modification, commonly referred to as PEGylation, confers several favorable properties for drug delivery systems, including excellent biocompatibility, reduced nonspecific protein adsorption, and reduced immune recognition and macrophage uptake45,46. Collectively, these effects reduce immunogenic responses and prolong systemic circulation47.
Traditional PEGylation techniques often rely on carbodiimide or maleimide chemistry, which typically require catalysts or reactive intermediates that can damage sensitive biomolecules, such as Hb. To circumvent this limitation, we employ strain-promoted alkyne-azide cycloaddition (SPAAC), a catalyst-free, bioorthogonal reaction between azide- and dibenzylcyclooctyne (DBCO)-functionalized species, enabling site-specific conjugation under mild aqueous conditions35,48. This approach ensures efficient surface modification without compromising encapsulated Hb function.
Building on our previously reported HP-UiO-66 NPs35, which focused on structural design and biofunctional validation, the present protocol emphasizes methodological transparency and reproducibility by providing a detailed, step-by-step experimental workflow with defined parameter ranges and critical handling considerations. Troubleshooting guidance is included for key stages of synthesis, Hb encapsulation, and PEGylation via SPAAC chemistry, ultimately yielding stable Hb@HP-UiO-66-PEG NPs (see Figure 1).
The workflow involves: (i) defect engineering using DA as a modulator to synthesize amino-modified HP-UiO-66-NH2 NPs; (ii) azide functionalization of surface amine groups; (iii) encapsulation of Hb under mild, aqueous conditions; (iv) covalent PEG conjugation through SPAAC.
For each major step, this protocol identifies common experimental challenges, such as particle aggregation, pore collapse, or inefficient surface functionalization, and provides practical troubleshooting strategies to improve inter-laboratory reproducibility. Importantly, this article extensively demonstrates that the synthesis of HP-UiO-66 NPs is readily scalable, enabling reliable gram-scale production per batch.
The final PEGylated nanocarriers exhibit high colloidal stability in saline, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and serum, while maintaining Hb&#39;s oxygen-binding and release capacity35. Analytical methods such as Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), powder X-ray diffraction (PXRD), nitrogen (N2) sorption, zeta (&#950;)-potential analysis, dynamic light scattering (DLS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) confirm chemical transformation, framework integrity, porosity, nanoscale morphology and successful surface functionalization.
By emphasizing reproducibility, scalability, and operational clarity, supported by video-based visualization of technically sensitive steps, this methodology provides a reproducible, robust, and accessible route to fabricate stable MOF-based oxygen carriers. Importantly, the approach is modular and readily adaptable to other biomacromolecules or enzymes that require a protected yet oxygen-permeable microenvironment to retain functionality. Accordingly, Hb@HP-UiO-66-PEG NPs represent a promising step toward the creation of HBOCs capable of delivering oxygen efficiently while resisting degradation in biological fluids.
From a practical perspective, this strategy can be extended to other biomacromolecules beyond Hb, provided that they possess dimensions below approximately 20 nm, corresponding to the maximum accessible mesopore size, and exhibit one or more of the following characteristics: (i) sensitivity to harsh chemical conditions, favoring mild synthesis and surface modification; (ii) a requirement for a hydrated, oxygen-permeable microenvironment to maintain functional activity; and (iii) susceptibility to phosphate-induced MOF degradation, necessitating surface stabilization. The approach relies on moderate defect densities to balance mesopore accessibility with framework integrity. When catalyst-free, site-specific surface modification is required to preserve biomolecule function, SPAAC-based PEGylation offers clear advantages. Consequently, this protocol is well-suited for efficient biomacromolecule loading, functional preservation, and long-term colloidal stability under physiological conditions, whereas substantially larger biomolecules or systems demanding extreme thermal or acid stability may require alternative MOF platforms.

<table><tbody><tr><td>2-amino-1,4-benzenedicarboxylic acid&amp;nbsp;</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>381071-25G</td><td></td></tr><tr><td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>H3375-100G</td><td></td></tr><tr><td>Acetone (99%)</td><td>VWR International (S&amp;oslash;borg, DK)</td><td>23J244030</td><td></td></tr><tr><td>Bicinchoninic acid protein assay&amp;nbsp;</td><td>Thermo Fisher Scientific (Roskilde, DK)</td><td>23235</td><td></td></tr><tr><td>Bovine blood&amp;nbsp;</td><td>SSI Diagnostica A/S (Hiller&amp;oslash;d, DK)</td><td>77667</td><td></td></tr><tr><td>Centrifuge tube</td><td></td><td></td><td></td></tr><tr><td>Centrifuger&amp;nbsp;</td><td>Thermo Fisher Scientific Inc (Massachusetts, US)</td><td>75009903</td><td>Megafuge ST Plus series&amp;nbsp;</td></tr><tr><td>Deuterium oxide</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>151882</td><td></td></tr><tr><td>Dibenzocyclooctyne-polyethylene glycol</td><td>BroadPharm</td><td>BP-23949</td><td></td></tr><tr><td>Dodecanoic acid&amp;nbsp;</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>L556-1KG</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified eagle&amp;rsquo;s medium&amp;minus;high glucose&amp;nbsp;</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>11965092</td><td></td></tr><tr><td>Ethanol (100%)</td><td>VWR International (S&amp;oslash;borg, DK)</td><td>24L044013</td><td></td></tr><tr><td>Fourier transform infrared spectroscopy&amp;nbsp;</td><td>Bruker (Billerica, US)</td><td></td><td></td></tr><tr><td>Hemox analyzer&amp;nbsp;</td><td>TSC Scientific Crp (PA, USA)</td><td></td><td></td></tr><tr><td>Human citrated plasma</td><td>TebuBio</td><td>SER-PLP25</td><td></td></tr><tr><td>Hydrochloric acid (37%)</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>320331-500ML</td><td></td></tr><tr><td>ImageJ software</td><td>National Institutes of Health (USA)</td><td></td><td>Version 1.47t</td></tr><tr><td>Lacey-carbon Cu grid (300 mesh)</td><td>Agar Scientific Ltd., (Stansted Essex, UK)</td><td>AGS166-3H</td><td></td></tr><tr><td>Magnetic stirrer</td><td>IKA-Werke GmbH &amp;amp; Co. KG (,DE)</td><td>Ident. No.: 0010011496</td><td></td></tr><tr><td>MestReNova sofeware</td><td>Mestrelab Research (Spain)</td><td></td><td>Version 12</td></tr><tr><td>Micromeritics Tristar surface area analyzer</td><td>Thermo Fisher Scientific Inc</td><td>ASAP 2020 Plus</td><td></td></tr><tr><td>Mini centrifuge</td><td>Eppendorf Nordic (H&amp;oslash;rsholm, DK)</td><td>Catalog No.&amp;nbsp;5452000010</td><td></td></tr><tr><td>Mouse serum</td><td>Innovative Research (Novi, MI, US)</td><td>IGMSCD1SER10ML</td><td></td></tr><tr><td>Muffle furnace&amp;nbsp;</td><td>Nabertherm GmbH (Bremen, DE)</td><td>Muffle furnace LE 6/11</td><td></td></tr><tr><td>N,N&amp;prime;-dimethylformamide (99.9%)</td><td>VWR International (S&amp;oslash;borg, DK)</td><td>250131a008</td><td></td></tr><tr><td>Nanodrop 2000c equipment&amp;nbsp;</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Nuclear magnetic resonance&amp;nbsp;</td><td>Bruker (Billerica, US)</td><td></td><td></td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt; meter&amp;nbsp;</td><td>PreSens (Regensburg, Germany)</td><td></td><td></td></tr><tr><td>Origin 2022 software</td><td>OriginLab Corporation (MA, USA)</td><td></td><td>Version 2022&amp;nbsp;</td></tr><tr><td>Oxygen sensor</td><td>PreSens (Regensburg, DE)</td><td>OXY-1 ST trace</td><td></td></tr><tr><td>Phosphate buffered saline&amp;nbsp;</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>P4417-50TAB</td><td></td></tr><tr><td>Platereader</td><td>Tecan Group Ltd.&amp;nbsp; (M&amp;auml;nnedorf, CH)</td><td></td><td></td></tr><tr><td>Potassium hexacyanoferrate</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>60299-100G</td><td></td></tr><tr><td>Potassium hydroxide</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>221473-500G</td><td></td></tr><tr><td>Powder X-ray diffraction&amp;nbsp;</td><td>Malvern Panalytical Ltd., (Malvern, UK)</td><td></td><td>Model: Malvern Panalytical Aeris Research 600W XRD for powder measurements (Released 2020)</td></tr><tr><td>Round bottom pressure vessel&amp;nbsp;</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>SYNP170004-1EA</td><td></td></tr><tr><td>Scanning electron microscopy&amp;nbsp;</td><td>FEI Company (Hillsboro, US)</td><td>Dual Beam FEI Helios Nanolab 600</td><td></td></tr><tr><td>sodium cloride&amp;nbsp;</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>71376</td><td></td></tr><tr><td>Sodium dithionite&amp;nbsp;</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>71699-250G</td><td></td></tr><tr><td>Sodium dodecyl sulfate&amp;nbsp;</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>L4509-100G</td><td></td></tr><tr><td>Synthware&amp;trade; round bottom pressure vessel</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>SYNP170003</td><td></td></tr><tr><td>Tert-butyl nitrite&amp;nbsp;</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>20370-100ml-F</td><td></td></tr><tr><td>Tetrahydrofuran (99.7%)</td><td>VWR International (S&amp;oslash;borg, DK)</td><td>201022A004</td><td></td></tr><tr><td>Thermoshaker</td><td>Grant Instruments (Cambridge) Ltd (UK)</td><td>PHMT-PSC18 (01012015020119)</td><td></td></tr><tr><td>Transmission electron microscopy&amp;nbsp;</td><td>FEI Company (Hillsboro, US)</td><td>FEI Tecnai T20</td><td></td></tr><tr><td>Trimethylsilyl azide&amp;nbsp;</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>155071-10G</td><td></td></tr><tr><td>TSC HEMOX DAQ System</td><td>TSC Scientific Crp (PA, USA)</td><td></td><td>Hemox analyzer software&amp;nbsp;</td></tr><tr><td>Ultrosound sonicator&amp;nbsp;</td><td>Branson Ultrasonics Corporation (Danbury, US)</td><td>SKU: 2410240003</td><td></td></tr><tr><td>UV-Vis spectrophotometer&amp;nbsp;</td><td>Shimadzu (Kyoto, JP)</td><td></td><td></td></tr><tr><td>Vacuum oven&amp;nbsp;</td><td>Thermo Fisher Scientific Inc (Massachusetts, US)</td><td></td><td></td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern Panalytical Ltd., (Malvern, UK)</td><td>Serial NO.MAL1188530</td><td></td></tr><tr><td>Zirconium(IV) chloride</td><td>Merck Life Science A/S (S&amp;oslash;borg, DK)</td><td>357405-100G</td><td></td></tr></tbody></table>

synthesis, hemoglobin encapsulation and biorthogonal pegylation in hierarchically porous uio-66 nanoparticles for oxygen delivery applications

Synthesis, Hemoglobin Encapsulation and Biorthogonal PEGylation in Hierarchically Porous UiO-66 Nanoparticles for Oxygen Delivery Applications

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-NH&#8322; NPs with preserved crystallinity and combined type I/IV N2 isotherms. Surface amines are converted to azides (HP-UiO-66-N&#8323; 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-NH&#8322; 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-NH&#8322; 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.

Successful synthesis of HP-UiO-66-NH2 NPs was verified by PXRD (Figure 2A), which exhibited the characteristic diffraction peaks at 2θ = 7.3°, 8.4°, and 25.7°, in good agreement with the simulated UiO-66 structure36. The slight peak broadening relative to the simulated pattern was attributed to the nanoscale dimensions of particles and confirmed preservation of crystalline integrity following synthesis54. Complete removal of DA was c...

This protocol details the synthesis, hemoglobin encapsulation, and surface PEGylation of hierarchically porous UiO-66 nanoparticles designed for oxygen delivery. The procedure combines defect engineering with strain-promoted biorthogonal conjugation to generate highly permeable and physiologically stable carriers suitable for biomedical applications.

Hemoglobin-based oxygen carriers (HBOCs) are an important platform for oxygen delivery to mitigate hypoxia in several biomedical contexts. However, free ...

synthesis-hemoglobin-encapsulation-biorthogonal-pegylation

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Learn a scalable protocol for synthesizing hierarchically porous UiO-66 nanoparticles that encapsulate hemoglobin for oxygen delivery applications. This method enables gram-scale production of stable, PEGylated hemoglobin-based oxygen carriers.

Synthesis, Hemoglobin Encapsulation and Biorthogonal PEGylation in Hierarchically Porous UiO-66 Nanoparticles for Oxygen Delivery Applications (Video) | JoVE Journal

Synthesis, Hemoglobin Encapsulation and Biorthogonal PEGylation in Hierarchically Porous UiO-66 Nanoparticles for Oxygen Delivery Applications

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