Hemoglobin-loaded ZIF-8 nanoparticles functionalized with human serum albumin as stealth, stable, and biocompatible oxygen carriers

Abstract

Hemoglobin-based oxygen carriers (HBOCs) offer a promising alternative to transfusions with donor red blood cells (RBCs), particularly in emergency and battlefield settings where blood availability and storage pose significant challenges. However, the clinical translation of HBOCs has been hindered by issues related to structural instability, immune clearance, and impaired hemoglobin (Hb) functionality. To address these limitations, we developed a next-generation HBOC by encapsulating Hb within zeolitic imidazolate framework-8 (ZIF-8) nanoparticles (NPs) (Hb@ZIF-8 NPs) and functionalizing the surface with a covalently bound layer of human serum albumin (HSA)-the most prevalent protein in human plasma. This strategy-employing a poly-l-lysine bridging step and glutaraldehyde crosslinking-resulted in HSA-coated Hb@ZIF-8 NPs with high Hb loading, enhanced colloidal stability in physiologically relevant media, and reduced opsonin adsorption. Compared to PEGylated controls, HSA-coated Hb@ZIF-8 NPs demonstrated superior stealth properties, including minimal IgG binding and preserved dysopsonin (i.e., bovine serum albumin) association. Spectroscopic analyses and oxygen dissociation measurements confirmed that encapsulated Hb retained oxygen-binding and -release capabilities with cooperative behavior. Furthermore, cytotoxicity assays in macrophage cultures revealed improved biocompatibility relative to previously reported ZIF-8-based HBOCs. These findings highlight the potential of HSA-functionalized Hb@ZIF-8 NPs as a safe and effective platform for oxygen delivery, supporting their further development for transfusion medicine and acute care applications.

Scheme 1. Schematic illustration of Hb-loaded ZIF-8 NPs (Hb@ZIF-8 NPs) synthesized from zinc ions (Zn²⁺), 2-methylimidazole (HmIm), Hb, and poly(ethylene glycol) (PEG), followed by coating with poly-l-lysine (PLL) and human serum albumin (HSA). Coating of Hb@ZIF-8 NPs with HSA protects against nonspecific protein adsorption and recognition by the immune system, while preserving the oxygen delivery function of Hb. The NPs are referred to as Hb@ZIF-8/PLL/HSA_XL and Hb@ZIF-8/PLL_XL/HSA_XL NPs, where the subscript ‘XL’ denotes crosslinking with glutaraldehyde (GA) following PLL or HSA deposition.

Fig. 1. (a) Cryo-SEM micrograph and corresponding size histogram of Hb-loaded ZIF-8 NPs (Hb@ZIF-8 NPs). Zeta (ζ)-potential, encapsulation efficiency (EE) and loading content (LC) are also reported. (b) (i) ζ-potential measurements of Hb@ZIF-8 NPs after sequential deposition of poly-l-lysine (PLL), human serum albumin (HSA) and crosslinking with glutaraldehyde (GA) in MQ and HEPES buffer at varying concentrations, pH conditions, and with or without NaCl. Buffers marked with a red asterisk indicate disassembly of the NPs, while double red asterisks denote NP agglomeration. (ii) Photographic images of Hb@ZIF-8 NPs coated with PLL, HSA and crosslinked with GA after two washes in: MQ (orange), 25 mM HEPES pH 7.4 with NaCl (blue), 25 mM HEPES pH 8.5 with NaCl (green), 25 mM HEPES pH 7.4 (purple), 25 mM HEPES pH 8.5 (red), 10 mM HEPES pH 7.4 (yellow) and 10 mM HEPES pH 8.5 (dark blue). PLL and HSA were used at concentrations of 2 mg mL⁻¹, and GA was applied at a 1 : 10 molar ratio relative to Hb. All NPs were incubated with GA for 4 h.

Fig. 2. (a) (i) Zeta (ζ)-potential measurements of Hb-loaded ZIF-8 NPs (Hb@ZIF-8 NPs) incubated with increasing concentrations of poly-l-lysine (PLL). (ii) ζ-potential measurements of PLL-coated Hb@ZIF-8 NPs incubated with increasing concentrations of human serum albumin (HSA), along with photographic images of the NP solutions at each HSA concentration after the final wash. The coating was conducted for 10 min in 10 mM HEPES buffer at pH 8.5. Data points marked with a red asterisk indicate HSA concentrations that resulted in NP disassembly. (b) ζ-potential measurements of PLL- and HSA-coated Hb@ZIF-8 NPs (i.e., Hb@ZIF-8/PLL/HSA NPs) and after incubation with 1 mM glutaraldehyde (GA) in MQ for different time intervals. The crosslinked NPs are denoted as Hb@ZIF-8/PLL/HSA_XL NPs, where the subscript ‘XL’ indicates HSA crosslinking with GA after deposition, and O/N stands for overnight incubation.

Fig. 3. (a) (i) Zeta (ζ)-potential measurements of Hb-loaded ZIF-8 NPs (Hb@ZIF-8 NPs) following deposition of poly-l-lysine (PLL) and crosslinking at various Hb : glutaraldehyde (GA) molar ratios (i.e., Hb@ZIF-8/PLL_XL NPs), where the subscript ‘XL’ indicates PLL crosslinking with GA after deposition. (ii) Hb loss after the deposition of human serum albumin (HSA) onto Hb@ZIF-8/PLL_XL NPs. (iii) Photographic images taken after the first centrifugation step, showing HSA deposition onto Hb@ZIF-8/PLL_XL NPs crosslinked at different Hb : GA molar ratios. (b) (i) ζ-potential measurements of the stepwise assembly of HSA-coated Hb@ZIF-8/PLL_XL NPs (i.e., Hb@ZIF-8/PLL_XL/HSA_XL NPs), where the subscript ‘XL’ indicates GA crosslinking after both PLL and HSA deposition. Several Hb : GA molar ratios were tested for HSA crosslinking. (ii) Hb loss after HSA crosslinking at different Hb : GA molar ratios and (iii) photographic images taken after the first centrifugation step, showing Hb@ZIF-8/PLL_XL/HSA_XL NPs crosslinked at different Hb : GA molar ratios. (c) Oxygen released per mg of Hb from Hb@ZIF-8/PLL/HSA_XL and Hb@ZIF-8/PLL_XL/HSA_XL NPs at different Hb : GA molar ratios.

Fig. 4. (a) Table with zeta (ζ)-potential measurements and encapsulation efficiency (EE) of poly-l-lysine (PLL)- and human serum albumin (HSA)-coated Hb-loaded ZIF-8 NPs (i.e., Hb@ZIF-8/PLL/HSA_XL and Hb@ZIF-8/PLL_XL/HSA_XL NPs). Data are presented as mean values. (b) Cryo-SEM micrograph and corresponding size distribution histogram of Hb@ZIF-8/PLL_XL/HSA_XL NPs. (c) FTIR spectra of free Hb, Hb-loaded ZIF-8 NPs (i.e., Hb@ZIF-8 NPs), PLL, HSA, Hb@ZIF-8/PLL/HSA_XL and Hb@ZIF-8/PLL_XL/HSA_XL NPs. The subscript ‘XL’ indicates crosslinking with glutaraldehyde following PLL and/or HSA deposition.

Fig. 5. Cumulative Hb release in various physiologically relevant media after incubation of: (a) bare Hb-loaded ZIF-8 NPs (Hb@ZIF-8 NPs) and poly-l-lysine (PLL)- and human serum albumin (HSA)-coated Hb@ZIF-8 NPs, denoted as (b) Hb@ZIF-8/PLL/HSA_XL and (c) Hb@ZIF-8/PLL_XL/HSA_XL NPs, where the subscript ‘XL’ indicates crosslinking with glutaraldehyde following PLL and/or HSA deposition.

Fig. 6. Fluorescein 5(6) – Isothiocyanate (FITC)-labelled bovine serum albumin (BSA-FITC) and Immunoglobin G (IgG-FITC) adsorbed onto increasing concentrations of Hb-loaded ZIF-8 NPs (Hb@ZIF-8 NPs) coated with poly-l-lysine (PLL)- and human serum albumin (HSA), or polyethylene glycol (PEG)-coated Hb@ZIF-8 NPs (Hb@ZIF-8/PEG NPs). The PLL- and HSA-coated NPs are denoted as Hb@ZIF-8/PLL/HSA_XL and Hb@ZIF-8/PLL_XL/HSA_XL NPs, where the subscript ‘XL’ indicates glutaraldehyde crosslinking following PLL and/or HSA deposition.

Fig. 7. (a) UV-vis spectra of free Hb, Hb-loaded ZIF-8 NPs (Hb@ZIF-8 NPs) coated with poly-l-lysine (PLL)- and human serum albumin (HSA) after treatment with compressed air or sodium dithionite (SDT) to generate oxygenated (oxyHb) and deoxygenated (deoxyHb) forms, respectively. (b) Oxygen dissociation curve showing the oxygen saturation as a function of the partial oxygen pressure (pO₂) along with the p50 (pO₂ at which Hb is 50% saturated with oxygen) and Hill coefficient (n_H) values of free Hb and the different NPs, with statistical significance indicated (***p < 0.001). The NPs are denoted as Hb@ZIF-8/PLL/HSA_XL and Hb@ZIF-8/PLL_XL/HSA_XL NPs, where the subscript ‘XL’ indicates glutaraldehyde crosslinking following PLL and/or HSA deposition.

Fig. 8. Normalized cell viability (nCV) of RAW 264.7 cells following incubation for 24 h with increasing concentrations of poly-l-lysine (PLL)- and human serum albumin (HSA)-coated Hb-loaded ZIF-8 NPs. The dashed line indicates the 70% viability threshold defined by ISO 10993-5 for classifying materials as non-cytotoxic. The NPs are denoted as Hb@ZIF-8/PLL_XL/HSA_XL NPs where the subscript ‘XL’ indicates glutaraldehyde crosslinking following PLL and HSA deposition.