Method Article

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

DOI:

10.3791/70445

⸱

May 8th, 2026

 , 
1Department of Health Technology, Technical University of Denmark

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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 (<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'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 (ζ)-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.

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Protocol

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1. Hb extraction (Expected duration: 2 days)

NOTE: The extraction of Hb from commercially available bovine blood (SSI Diagnostica A/S, Hillerød, DK) is conducted following our previously reported protocol49.

  1. Transfer bovine blood (typically 10 mL per extraction) into a 50 mL conical centrifuge tube. Add an equal volume of 0.9% (w/v) saline solution to dilute the blood and reduce viscosity. Gently invert several times until a homogeneous suspension is obtained.
  2. Centrifuge the diluted blood at 1,500 x g for 20 min at 4 °C. After centrifugation, a clear yellow supernatant (plasma) appears above a dense red pellet (RBCs). If complete pelleting is not achieved, continue centrifugation as necessary.
  3. Carefully decant and discard the plasma layer without disturbing the pellet. Add fresh saline (10 mL), resuspend the RBC pellet by gentle inversion, and repeat the centrifugation step under identical conditions. Perform two washing cycles in total to remove plasma proteins and platelets.
    NOTE: The washing process is crucial for obtaining pure RBCs free of plasma contaminants that could interfere with the downstream toluene extraction.
  4. After the second wash, discard the supernatant and resuspend the cleaned RBC pellet in a mixture of toluene, ultrapure water, and saline at the volume ratio of 1:0.4:1. For example, mix 10 mL toluene, 4 mL ultrapure water, and 10 mL saline in a 50 mL separatory funnel before adding the pellet.
  5. Seal the funnel and invert gently several times to mix the phases. Place the funnel at 4 °C overnight (~12-16 h) to allow complete phase separation. The toluene phase extracts the lipid membranes, leaving a clear aqueous phase enriched in Hb.
  6. The following day, carefully collect the lower aqueous layer using the stopcock of the separatory funnel. This layer appears transparent and dark red.
  7. Transfer the aqueous Hb solution into centrifuge tubes and spin at 8,000 x g for 20 min at 4 °C to remove any remaining cell debris or emulsified material.
  8. Filter the supernatant through a diatomaceous earth frit using a vacuum filtration setup. Apply gentle vacuum only (avoid high negative pressure that can cause foaming or protein denaturation).
  9. Collect the filtrate containing stroma-free Hb. Store this stock on ice if used immediately, or aliquot (~1 mL) and freeze at −80 °C for long-term use.
    NOTE: Hb aliquots may be stored frozen for several months without detectable degradation.
  10. Quantify Hb concentration using a bicinchoninic acid protein assay according to the manufacturer's protocol, using pure bovine Hb as a calibration standard. Adjust final Hb stock to the desired working concentration (typically 20 mg mL-1 in HEPES buffer, pH 7.4).
    NOTE: Expect a clear, red Hb solution free of cellular material.
  11. Optional: measure the UV-vis absorption spectra (375-700 nm) of extracted Hb to assess its oxygen binding and releasing ability. Record the initial UV-vis spectrum. Add sodium dithionite and purge with N2 gas for 10 min to release oxygen, and record the spectrum of deoxygenated Hb. Subsequently, purge with air for 10 min to allow reoxygenation and re-record the spectrum.
    NOTE: The presence of characteristic Soret and Q-bands of oxygenated (~412 nm for Soret band; ~541 and ~577 nm for Q-bands) and deoxygenated (~430 nm for Soret band; ~557 nm for Q-band) Hb after purging with air and N2, respectively, indicates that Hb remains functional (Supplementary Figure 1).
    CAUTION: Handle toluene with care in a fume hood while wearing appropriate personal protective equipment, including a lab coat, safety glasses, and gloves. In the present study, all toluene-containing waste liquids were collected and disposed of as organic chemical liquid waste in designated containers (Group C), labeled "toluene and mixture," in accordance with the guidelines of the Danish Ministry of the Environment. The corresponding waste-disposal documentation is provided in Supplementary File 1.

2. Fabrication of HP-UiO-66-NH2 NPs (Expected duration: 3 days)

CAUTION: HCl (37%) is corrosive; handle inside a fume hood with a lab coat, acid-resistant gloves, and appropriate eye protection. Add acid slowly and avoid inhalation of vapors.

  1. Weigh ZrCl4 (400 mg, 1.715 mmol) into a 250 mL round-bottom flask. Slowly add DMF (80 mL) and ultrasonicate for 5 min to completely dissolve ZrCl4. Add DA (12 g, 60 mmol) and ultrasonicate for an additional 30 min until a clear, homogeneous solution is obtained. Measure the total solution volume using a graduated cylinder and add DMF as needed to adjust the final volume to 100 mL. DA acts as a competitive modulator, introducing controlled lattice defects.
    NOTE: Add DMF slowly when dissolving ZrCl4, as the coordination process is exothermic and may cause slight fuming; perform all operations in a fume hood. Account for the volume increase caused by the incorporation of DA by using an initial volume of 80 mL DMF to dissolve ZrCl4 and DA, and adjust the final volume to 100 mL only after complete dissolution to ensure accurate concentration control.
  2. Add 2-amino-1,4-benzenedicarboxylic acid (BDC-NH2, 155 mg, 0.857 mmol) to the same flask and sonicate for an additional 30 min. Continue until a homogeneous pale yellow solution is obtained.
  3. Transfer the mixture into a 150 mL Teflon-lined autoclave or round-bottom pressure vessel suitable for solvothermal synthesis. Seal tightly and place in a preheated muffle furnace at 120 °C for 18 h. Do not disturb the reactor during heating to avoid uncontrolled nucleation.
  4. After the reaction, turn off the furnace and allow the vessel to cool naturally to room temperature (~2 h). Observe a fine yellow suspension appearing uniformly throughout the medium.
  5. Collect the product by centrifugation (11,000 x g, 10 min, room temperature) and discard the supernatant. Wash the pellet with DMF (100 mL) three times using the same centrifugation conditions to remove excess ligand and unreacted DA. If the product is not completely pelleted, extend the centrifugation time as necessary.
  6. Prepare an acid removal solution consisting of DMF (100 mL) containing 0.5 mL concentrated HCl (37%). Immerse the washed pellet completely in this solution and heat it in a muffle furnace at 90 °C for 12 h. This step removes the DA modulator, creating mesopores within the structure.
  7. After 12 h, centrifuge (11,000 x g, 5 min, room temperature) and discard the supernatant. Repeat the acid treatment using a fresh batch of DMF containing 0.5 mL HCl to ensure complete removal of DA. If the product is not completely pelleted, extend the centrifugation time as necessary.
  8. Wash the pellet sequentially with DMF, acetone, and ethanol (100 mL, 11,000 x g, 5 min, room temperature) to remove any residual acid and solvent. If the precipitate is not completely pelleted, extend the centrifugation time as necessary. Between each wash, gently redisperse the pellet by vortexing or mild sonication.
  9. Dry the product in a vacuum oven (140 °C) overnight after ambient drying inside a fume hood. The final materials (HP-UiO-66-NH2 NPs) appear as fine yellow powder. Expect a typical yield of ~428 mg.
  10. Confirm that PXRD peaks at 2θ = 7.3°, 8.4°, 25.7° remain sharp (Figure 2A), indicating retention of crystalline UiO-66 topology. Verify complete removal of DA by 1H NMR after alkaline (2 M KOH in D2O) digestion (no methylene peaks at 1.2 ppm) (Figure 2B).
  11. Characterize porosity by N2 adsorption-desorption at 77 K50. The curve displays a combined Type I/IV isotherm with a hysteresis loop (Figure 2C), confirming mesopore formation.
    NOTE: Incomplete high-temperature drying or insufficient sample mass leads to inaccurate porosity measurements using N2 adsorption-desorption isotherms; repeat the vacuum-drying process at 140 °C and ensure the sample weight exceeds 100 mg if the Brunauer−Emmett−Teller (BET) area is < 1,000 m2 g-1.
  12. Optional: assess NPs' morphology using SEM. HP-UiO-66-NH2 NPs exhibit spherical morphology with a narrow size distribution of ~100 nm (Figure 2D).
    NOTE: Expect HP-UiO-66-NH2 NPs possessing both micro- and mesopores, suitable for subsequent Hb encapsulation.
  13. The synthesis of HP-UiO-66-NH2 NPs can be scaled up by proportionally increasing all reagents and solvents used in steps 2.1 - 2.9 and performing multiple parallel solvothermal reactions. By maintaining the same molar ratios, concentrations, reaction temperature, and reaction time, and increasing reagent and solvent quantities by 1.5-fold, two or more 150 mL Teflon-lined autoclaves or round-bottom pressure vessels can be operated simultaneously, enabling gram-scale production of HP-UiO-66-NH2 NPs (Figure 3).
    NOTE: Handle all organic solvents inside a fume hood while wearing a lab coat, acid-resistant gloves, and appropriate eye protection. Collect all DMF-containing waste liquids and dispose of them as organic chemical liquid waste in designated containers (Group C) labeled "DMF and mixture" in accordance with the guidelines of the Danish Ministry of the Environment. Waste solutions containing trace amounts of ZrCl4 and HCl were handled accordingly. The corresponding waste-disposal documentation is provided in Supplementary File 1.

3. Azide functionalization of HP-UiO-66-NH2 NPs (HP-UiO-66-N3 NPs) (Expected duration: 1 day)

CAUTION: Trimethylsilyl azide (TMSN₃) and tert-butyl nitrite (tBuONO) are toxic and volatile. Conduct all steps in a fume hood wearing a lab coat, nitrile gloves, and eye protection.

  1. Accurately weigh out HP-UiO-66-NH2 NPs (50 mg) into a 25 mL round-bottom flask. Add THF (10 mL) and ultrasonicate for 5 min to fully disperse the particles. The suspension turns opaque but uniform.
    NOTE: Uniform dispersion of NPs after sonication is critical for effective contact with reactants. If the dispersion is incomplete, increase the solution volume and repeat sonication.
  2. Place the flask in an ice-water bath (0 °C) on a magnetic stirrer set to 400 rpm. Using a glass syringe, add tBuONO (0.4 mL) dropwise over 5 min while maintaining the temperature below 5 °C.
  3. Immediately add TMSN₃ (0.35 mL) dropwise under the same cold conditions. A mild effervescence may appear, indicating nitrosation progress. Wrap the bath with aluminum foil to protect the reaction from light..
  4. Continue stirring the reaction mixture overnight (~16 h) in the ice-water bath to allow complete azide substitution of amine groups on the MOF surface.
  5. Remove the ice bath and allow the suspension to reach room temperature. Centrifuge (11,000 x g, 5 min, room temperature) to collect the modified NPs. If the precipitate is not completely pelleted, extend the centrifugation time as necessary.
  6. Wash the pellet thoroughly with THF (10 mL) three times, collecting it by centrifugation (11,000 x g, 5 min, room temperature) after each wash. Follow this with three additional washes using acetone (10 mL), centrifuging under the same conditions, to remove residual reagents and by-products. If the precipitate is not completely pelleted, extend the centrifugation time as necessary.
  7. Dry the product (HP-UiO-66-N3 NPs) using an oven (140 °C) under vacuum overnight after ambient drying inside a fume hood. Expect a typical yield of ~45 mg.
  8. Verify the chemical transformation by FTIR spectroscopy: a sharp asymmetric azide stretch appears at ~2122 cm-1 (Figure 4A). The band intensity correlates with surface substitution efficiency.
    NOTE: Incomplete conversion can impair subsequent click conjugation. If the azide band is weak, repeat the functionalization using 2-fold quantities of tBuONO and TMSN₃. Expect azido-terminated HP-UiO-66-N3 NPs with preserved crystallinity and mesoporous architecture (Figure 4B-D), ready for Hb encapsulation and SPAAC PEGylation.
    CAUTION: Collect all waste liquids containing TMSN₃, including mixtures with other reagents (e.g., tBuONO and THF), in designated Group O hazardous waste containers in accordance with the guidelines of the Danish Ministry of the Environment. Clearly label all containers to indicate the presence of TMSN₃ and its mixtures. Relevant waste disposal documentation is provided in Supplementary File 1.

4. Hb Loading and Quantification (Expected duration: 2 days)

  1. Transfer HP-UiO-66-N3 NPs (5 mg) into a 2 mL centrifuge tube followed by ~2 min sonication in 0.5 mL of HEPES buffer (20 mM, pH 7.4). Add 0.5 mL of HEPES buffer containing Hb (20 mg mL-1) and gently invert the tube several times to fully disperse the NPs.
  2. Place the tube on a thermoshaker and incubate at room temperature (22-25 °C) under 1,000 rpm shaking for 2 h.
  3. After incubation, centrifuge 13,500 x g for 5 min at room temperature to pellet the resulting Hb@HP-UiO-66-N3 NPs. Collect the supernatant carefully for quantification of unentrapped Hb. If the precipitate is not completely pelleted, extend the centrifugation time as necessary.
  4. Wash the pellet gently with ultrapure water (1 mL) by pipetting up and down to remove loosely adsorbed Hb, and repeat the wash twice. Carefully collect the supernatant of each wash for quantification of unentrapped Hb. Resuspend the final pellet in 0.5 mL ultrapure water for characterization. Keep samples in refrigerator (4 °C) to prevent oxidation.
  5. Determine unentrapped Hb in the collected supernatants using the sodium lauryl sulfate (SLS)-Hb assay51. Pipette 10 µL of each supernatant into a 96-well microplate and add 100 µL of SLS reagent (0.6 mg mL-1 in ultrapure water). Incubate 5 min at room temperature in the dark, then read absorbance at 412 nm using a UV-vis spectrophotometer.
  6. Prepare a standard curve using Hb stock solutions (0-10 mg mL-1) in HEPES buffer to convert absorbance values to concentration.
  7. Calculate encapsulation efficiency (EE) and loading content (LC) as:
    EE (%) = [(initial Hb - supernatant Hb)/initial Hb] × 100
    LC (%) = [(encapsulated Hb)/(encapsulated Hb + NP mass)] × 100
  8. To scale up the Hb encapsulation, incubate 100 mg of NPs with 20 mL of Hb solution (10 mg mL⁻¹). To assess the porosity after encapsulation, immerse the resulting NPs in ethanol for 24 h, then dry overnight in a vacuum oven at 140 °C, and record the N2 adsorption-desorption isotherm to evaluate porosity. The red color of the powder indicates successful encapsulation of Hb (Figure 5A), and a marked decrease in N2 uptake confirms the successful filling of mesopores with Hb (Figure 5B).
  9. Measure ζ-potential using a Zetasizer Nano ZS. The surface charge decreases from ~+40 mV (HP-UiO-66-N3 NPs) to ~+20 mV (Hb@HP-UiO-66-N3 NPs), consistent with Hb adsorption (Figure 5C).
  10. Optional: acquire SEM micrograph (Figure 5D) and PXRD diffractogram (Figure 5E) to ensure morphological integrity and structural stability.
  11. Record UV-vis spectra (375-700 nm) to assess reversible oxygen binding of the NPs. Add sodium dithionite and purge with N2 for 10 min to release oxygen, record the spectrum, then purge with air for 10 min to allow re-binding, and record the spectrum again (Figure 5F).
    NOTE: The presence of characteristic Soret and Q-bands of oxygenated (~412 nm for Soret band; ~541 and ~577 nm for Q-bands) and deoxygenated (~430 nm for Soret band; ~557 nm for Q-band) Hb after purging with air and N2, respectively, indicates that Hb remains functional.
  12. Expect uniform red Hb@HP-UiO-66-N3 NPs containing functionally active Hb with reversible oxygenation behavior.

5. Fabrication of Hb@HP-UiO-66-PEG NPs: PEGylation via SPAAC (Expected duration: 2 days)

  1. Prepare a 2 mL centrifuge tube containing Hb@HP-UiO-66-N₃ NPs (10 mg) dispersed in 0.5 mL ultrapure water. Add DBCO-PEG (Mw ~2,000, 20 mg in 0.5 mL ultrapure water) to achieve a 2:1 (w/w) ratio of PEG to NPs. Gently vortex until fully mixed.
  2. Incubate the mixture on a thermoshaker at room temperature and 1,000 rpm for 1 h, then transfer to a refrigerator (4 °C) for overnight reaction (~16 h). This catalyst-free SPAAC click reaction covalently couples DBCO to the azide groups, forming stable triazole linkages.
  3. Centrifuge at 12,000 x g for 5 min at room temperature to pellet the PEGylated Hb@HP-UiO-66-PEG NPs. If the precipitate is not completely pelleted, extend the centrifugation time as necessary. Discard the supernatant containing unreacted PEG.
  4. Wash the pellet three times with ultrapure water (2 mL each by pipetting up and down) under identical centrifugation conditions. This ensures the removal of free PEG chains and small molecular by-products.
  5. Resuspend the purified Hb@HP-UiO-66-PEG NPs in 2 mL ultrapure water and store at 4 °C.
    The colloid appears homogeneous and slightly opalescent, indicating good dispersion stability.
  6. Confirm successful PEGylation by FTIR spectroscopy (Figure 6A): disappearance of the azide band at 2122 cm-1 and emergence of PEG-derived methylene (−CH2−) and ether bond (−O−) signals at 2883 and 1097 cm-1, respectively.
  7. Verify by 1H NMR (Figure 6B) after alkaline digestion in 2 M KOH/D2O; PEGylated samples show an additional signal at δ = 3.53 ppm corresponding to the oxyethylene protons of PEG.
  8. Measure hydrodynamic diameter and polydispersity index (PDI) by DLS (Figure 6C).
    An increase from ~115 nm (PDI ~0.1) to ~140 nm (PDI ~0.17) confirms surface coating with a thin polymer layer.
  9. Optional: measure surface ζ-potential using a Zetasizer Nano ZS to verify the negligible effect of surface PEGylation on the surface charge (Figure 6D). A minor change after PEGylation (from ~+20 mV to ~+17 mV) indicates minimal impact on surface charge.
  10. Observe morphology by TEM (Figure 6E). TEM reveals a diffuse ~10-12 nm rim around the particle edge representing the PEG corona.
  11. Perform oxygen-release assay using an oxygen electrode (Figure 6F) to show that Hb function remains intact after PEGylation, similar to the non-PEGylated form. Expect a stable, reddish-brown-dispersed Hb@HP-UiO-66-PEG NPs exhibiting strong colloidal stability and retained oxygen-binding capacity, suitable for long-term physiological testing.
  12. Optional: conduct oxygen dissociation curves (ODCs) of free Hb and Hb encapsulated in Hb@HP-UiO-66-PEG NPs to identify the partial oxygen pressure at 50% Hb oxygen saturation (p50) and Hill coefficient (Hill-n) (Figure 7).

6. Assessment of NPs stability (Expected duration: 7 days)

  1. Dispense Hb@HP-UiO-66-PEG NPs (~5 mg) into seven 2 mL centrifuge tubes. Add 2 mL of each medium: ultrapure water, saline (0.9% NaCl), HEPES (20 mM), mouse serum, human plasma, Dulbecco's modified Eagle's medium−high glucose (DMEM), and phosphate-buffered saline (PBS). Ensure that the centrifuge tube caps are tightly closed to prevent medium evaporation during incubation. CD-1 mouse serum and human plasma are purchased from Innovative Research (Novi, MI, US).
  2. Incubate the samples at 37 °C in a thermoshaker at 1,000 rpm for up to 7 days.
  3. After 3 and 7 days, collect particles by centrifugation (12,000 g, 10 min, room temperature), wash once with ultrapure water, and dry for PXRD (Figure 8A). If the precipitate is not completely pelleted, extend the centrifugation time as necessary.
  4. At 0, 24, 72, and 168 h, withdraw aliquots (~50 µL) and dilute them 20-fold for DLS analysis (size and PDI) (Figure 8B).
  5. Optional: prepare SEM samples after 48 h in PBS to visually check morphology (Figure 8C).
    NOTE: Diluting the NPs suspension (in PBS) with ultrapure water is essential for SEM analysis as it reduces background interference from buffer components and facilitates clear imaging of the NPs.
  6. Compare to non-PEGylated Hb@HP-UiO-66-N3 NPs under identical conditions; rapid disappearance of characteristic crystalline reflections and emergence of broad amorphous scattering features indicate instability without PEG coating.
    NOTE: PEGylated samples remain structurally intact after 7 days in all media, while non-PEGylated ones decompose within 24 h in PBS or DMEM.

7. Physicochemical characterization (Expected duration: ~15 days)

NOTE: This section describes the analytical methods required to confirm NPs' morphology, composition, porosity, and Hb integrity. These characterization techniques collectively validate the reproducibility of the synthesis and modification processes.

  1. PXRD
    1. Dry ~5 mg of each NP sample in a freezer drier overnight before measurement to remove adsorbed moisture.
    2. Load the sample onto a flat zero-background silicon holder and gently flatten using a clean glass slide to form a thin, uniform layer.
      NOTE: Avoid pressing too hard, which can induce preferred orientation effects.
    3. Record PXRD patterns using a benchtop diffractometer equipped with Cu Kα radiation (λ = 1.5418 Å). Set scanning parameters to 2θ = 4-50°, step size = 0.02°, dwell = 1 s per step.
    4. Compare the obtained patterns with the simulated UiO-66 structure generated using Mercury software based on the previously reported crystal data31. The presence of sharp peaks at 7.3°, 8.4°, and 25.7° indicates crystalline integrity.
  2. N2 Adsorption-Desorption (BET Surface Area and Pore Size Distribution)
    1. Degas ~100 mg of each NP sample at 140 °C under vacuum for 24 h using a Micromeritics Smart VacPrep system with purging ultra-high-purity N2.
    2. Immediately transfer the degassed samples to the analysis port of a Micromeritics Tristar surface area analyzer. Record N2 isotherms at 77 K within a relative pressure range (P/P₀) of 0-1.
    3. Compute surface areas using the BET method following the extended Rouquerol criteria52. Calculate pore size distribution via the non-local density functional theory implemented in the Micromeritics software.
    4. Generate the adsorption/desorption curves and pore size distribution plots using Origin 2022. Import the adsorption/desorption data into the software, with relative pressure (P/P₀) on the X-axis and the adsorbed volume of N2 on the Y-axis.
    5. Plot the pore size distribution data with pore width as the X-axis and incremental pore volume as the Y-axis. Present the results as "Line + Symbol" graphs. Perform all analyses using the default software settings.
  3. FTIR Spectroscopy
    1. Grind ~5 mg of each dried sample into a fine powder using a clean agate mortar. Place the powder on the ATR crystal of an ALPHA-P Bruker spectrometer53.
    2. Collect spectra in the 4,000-400 cm-1 range with 2 cm-1 resolution.
    3. Identify characteristic peaks: Appearance of N3 stretch at ~2122 cm-1 confirms azide formation (Figure 4A). After PEGylation, observe −CH2− and C-O-C bands at ~2883 cm-1 and ~1097 cm-1, respectively, along with the disappearance of N3 stretch (Figure 6A). Expect the disappearance of the azide band and emergence of PEG-derived methylene (−CH2−) and ether bone (−O−) signals confirm the completion of the SPAAC reaction.
    4. Plot the FTIR curves using Origin 2022. Import the raw data into the software with X (wavenumber) and Y (transmittance) values. Generate graphs in "Line" format using the software's default settings.
  4. 1H NMR
    1. Add ~10 mg of NPs to 0.5 mL of 2 M KOH in D2O placed in a glass vial, and heat at 80 °C for 1-2 h to digest the framework.
    2. Centrifuge (12,000 x g, 10 min, room temperature) to spin down the undissolved residues. Transfer the supernatant to a 5 mm NMR tube.
    3. Record 1H NMR spectra on a 400 MHz Bruker Avance spectrometer.
    4. Assign the characteristic chemical shifts, including the aromatic protons of BDC-NH2 (δ = 6-8 ppm) and PEG signals (δ = ~3.53 ppm, Figure 6B), using the software of MestReNova. Analyze the NMR data using the software's NMR processing module with its default settings.
  5. SEM
    1. Deposit ~5 µL drop of NP suspension onto a silicon wafer; allow to air-dry in a dust-free cabinet. If charging occurs, sputter-coat with 5 nm gold.
    2. Image on a FEI Quanta 200 SEM at 10-20 kV accelerating voltage.
    3. Analyze the images using ImageJ. Import the original TIFF images with embedded scale information into the software and adjust brightness and contrast solely for visualization. Do not apply any additional processing that might affect the dimensional information. Generate the scale bars using the software's Analyze function.
  6. TEM
    1. Place ~5 µL of NP dispersion on a lacey-carbon Cu grid (300 mesh). Blot excess with filter paper and dry at ambient conditions.
    2. Examine under a FEI Tecnai T20 G2 TEM at 200 kV.
    3. Analyze the images using ImageJ. Image processing follows the procedures described in Section 7.5.3.
  7. DLS and ζ-Potential
    1. Dilute NPs ~20-fold in ultrapure water for size and PDI analysis; for ζ-potential, dilute in HEPES buffer (20 mM, pH 7.4).
    2. Measure using a Zetasizer Nano ZS at 25 °C. At least three independent samples (n = 3) are prepared, and each sample is measured in triplicate.
    3. Plot the DLS and ζ-potential data using Origin 2022. Present the intensity-weighted particle size distribution as "Line + Symbol" graphs and ζ-potential results as "Column" graphs. Report the data as mean ± standard deviation (n = 3). Perform all analyses using the software's default settings.
  8. UV-vis Spectroscopy for Hb functionality
    1. Disperse 3 mg of free Hb or a corresponding amount of NPs (i.e., containing an equivalent amount of encapsulated Hb) in 3 mL ultrapure water. Transfer the suspensions to a quartz cuvette. Record UV-vis spectra from 375-700 nm using a UV-2600 UV−vis spectrophotometer.
    2. Add a pinch of sodium dithionite to the Hb solution or NP suspension and purge with N2 for 10 min to deoxygenate the sample. Record the shift of the Soret band from 412 to 430 nm and the merging of Q-bands. Reoxygenate the sample by air bubbling for 10 min to observe reversal (Figure 5F and Supplementary Figure 1).
    3. Plot the data using Origin 2022. Present the spectra as "Line" graphs, with wavelength on the X-axis and absorbance on the Y-axis.
  9. Quantification of released oxygen
    1. Disperse 1 mg of free Hb or a corresponding amount of Hb@HP-UiO-66-N3 and Hb@HP-UiO-66-PEG NPs (containing an equivalent amount of encapsulated Hb) in 600 µL ultrapure water. Transfer the suspensions into 2 mL glass vials and seal them tightly with rubber caps.
    2. Carefully insert the needle probe of the oxygen electrode through the rubber cap into the suspension to monitor the dissolved oxygen level using a needle-type O2 meter.
    3. Quickly add K3[Fe(CN)6] solution (10 wt% in 100 µL ultrapure water) to the suspension using a syringe needle and continuously record the change in oxygen level.
    4. Calculate the released oxygen by subtracting the maximum value observed after K3[Fe(CN)6] addition from the initial value before its addition. Normalize the obtained values to the oxygen released from the same amount of free Hb (Figure 6F) using Origin 2022. Measure three independent samples (n = 3) for free Hb or each NP.
      NOTE: Hb@HP-UiO-66-N3 and Hb@HP-UiO-66-PEG NPs show comparable oxygen-release, each releasing ~60% of the oxygen released by free Hb.
  10. Measurement of ODCs and calculation of p50 and Hill-n
    1. Prepare free bovine Hb or Hb@HP-UiO-66-PEG NPs at a final Hb concentration of ~1 mg mL-1 in 3 mL HEPES-saline buffer (20 mM HEPES, 0.9% (w/v) NaCl, pH 7.4). Transfer the sample into a quartz cuvette equipped with a magnetic stirring bar.
    2. Place the cuvette into a Hemox analyzer. Set the measurement temperature to 37 °C and allow the system to equilibrate under continuous stirring until a stable baseline is achieved.
    3. Record ODCs by stepwise deoxygenation over a partial pressure range of 1-148 mmHg. Use the pre-installed Hemox analyzer software to measure ODCs and calculate p50 and Hill-n using the default analysis settings. Measure three independent samples (n = 3) for each sample.
    4. Export the processed data and plot the ODCs using Origin 2022 in the default "Line" graph format.

8. Reproducibility and statistical analysis

  1. Perform all procedures described in this protocol at least three independent times. Report results as typical outcomes observed across these repetitions. Maintain the key experimental parameters, such as reaction times and temperatures, within a reasonable range to ensure reproducibility and reliability for other users.
  2. Present structural characterization (PXRD, N2 adsorption-desorption, FTIR, 1H NMR, SEM, UV-vis) as representative data for qualitative confirmation without performing statistical analysis.
  3. Report quantitative measurements (DLS, ζ-potential, p50, Hill-n) as mean ± SD (n ≥ 3). Determine statistical significance using unpaired two-tailed Welch's t-test or one-way ANOVA, as appropriate. Indicate significance levels as ns (no significant), * = p < 0.05, ** = p < 0.005, *** = p < 0.001, **** = p < 0.0001.

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Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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...

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Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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...

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Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Authors declare no competing financial interest.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
2-amino-1,4-benzenedicarboxylic acid Merck Life Science A/S (Søborg, DK)381071-25G
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acidMerck Life Science A/S (Søborg, DK)H3375-100G
Acetone (99%)VWR International (Søborg, DK)23J244030
Bicinchoninic acid protein assay Thermo Fisher Scientific (Roskilde, DK)23235
Bovine blood SSI Diagnostica A/S (Hillerød, DK)77667
Centrifuge tube
Centrifuger Thermo Fisher Scientific Inc (Massachusetts, US)75009903Megafuge ST Plus series 
Deuterium oxideMerck Life Science A/S (Søborg, DK)151882
Dibenzocyclooctyne-polyethylene glycolBroadPharmBP-23949
Dodecanoic acid Merck Life Science A/S (Søborg, DK)L556-1KG
Dulbecco’s modified eagle’s medium−high glucose Merck Life Science A/S (Søborg, DK)11965092
Ethanol (100%)VWR International (Søborg, DK)24L044013
Fourier transform infrared spectroscopy Bruker (Billerica, US)
Hemox analyzer TSC Scientific Crp (PA, USA)
Human citrated plasmaTebuBioSER-PLP25
Hydrochloric acid (37%)Merck Life Science A/S (Søborg, DK)320331-500ML
ImageJ softwareNational Institutes of Health (USA)Version 1.47t
Lacey-carbon Cu grid (300 mesh)Agar Scientific Ltd., (Stansted Essex, UK)AGS166-3H
Magnetic stirrerIKA-Werke GmbH & Co. KG (,DE)Ident. No.: 0010011496
MestReNova sofewareMestrelab Research (Spain)Version 12
Micromeritics Tristar surface area analyzerThermo Fisher Scientific IncASAP 2020 Plus
Mini centrifugeEppendorf Nordic (Hørsholm, DK)Catalog No. 5452000010
Mouse serumInnovative Research (Novi, MI, US)IGMSCD1SER10ML
Muffle furnace Nabertherm GmbH (Bremen, DE)Muffle furnace LE 6/11
N,N′-dimethylformamide (99.9%)VWR International (Søborg, DK)250131a008
Nanodrop 2000c equipment Thermo Fisher
Nuclear magnetic resonance Bruker (Billerica, US)
O2 meter PreSens (Regensburg, Germany)
Origin 2022 softwareOriginLab Corporation (MA, USA)Version 2022 
Oxygen sensorPreSens (Regensburg, DE)OXY-1 ST trace
Phosphate buffered saline Merck Life Science A/S (Søborg, DK)P4417-50TAB
PlatereaderTecan Group Ltd.  (Männedorf, CH)
Potassium hexacyanoferrateMerck Life Science A/S (Søborg, DK)60299-100G
Potassium hydroxideMerck Life Science A/S (Søborg, DK)221473-500G
Powder X-ray diffraction Malvern Panalytical Ltd., (Malvern, UK)Model: Malvern Panalytical Aeris Research 600W XRD for powder measurements (Released 2020)
Round bottom pressure vessel Merck Life Science A/S (Søborg, DK)SYNP170004-1EA
Scanning electron microscopy FEI Company (Hillsboro, US)Dual Beam FEI Helios Nanolab 600
sodium cloride Merck Life Science A/S (Søborg, DK)71376
Sodium dithionite Merck Life Science A/S (Søborg, DK)71699-250G
Sodium dodecyl sulfate Merck Life Science A/S (Søborg, DK)L4509-100G
Synthware™ round bottom pressure vesselMerck Life Science A/S (Søborg, DK)SYNP170003
Tert-butyl nitrite Merck Life Science A/S (Søborg, DK)20370-100ml-F
Tetrahydrofuran (99.7%)VWR International (Søborg, DK)201022A004
ThermoshakerGrant Instruments (Cambridge) Ltd (UK)PHMT-PSC18 (01012015020119)
Transmission electron microscopy FEI Company (Hillsboro, US)FEI Tecnai T20
Trimethylsilyl azide Merck Life Science A/S (Søborg, DK)155071-10G
TSC HEMOX DAQ SystemTSC Scientific Crp (PA, USA)Hemox analyzer software 
Ultrosound sonicator Branson Ultrasonics Corporation (Danbury, US)SKU: 2410240003
UV-Vis spectrophotometer Shimadzu (Kyoto, JP)
Vacuum oven Thermo Fisher Scientific Inc (Massachusetts, US)
Zetasizer Nano ZSMalvern Panalytical Ltd., (Malvern, UK)Serial NO.MAL1188530
Zirconium(IV) chlorideMerck Life Science A/S (Søborg, DK)357405-100G

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Hierarchical PorosityBioorthogonal ChemistryStrain Promoted CycloadditionMetal Organic FrameworksOxygen CarriersColloidal Stability

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