Hemoglobin-based oxygen carrier

Abstract

The invention relates to a hemoglobin-based oxygen carrier where the carrier has a metal-organic framework to entrap hemoglobin and is provided as nanoparticles; as well as a method for preparing the nanoparticles. The invention further relates to a pharmaceutical comprising the hemoglobin-based oxygen carrier.

Description

[0001] TITLE: Hemoglobin-based oxygen carrier

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a hemoglobin-based oxygen carrier where the carrier has a metal-organic framework to entrap hemoglobin and is provided as a nanoparticle; as well as a method for preparing the nanoparticle. The invention further relates to a pharmaceutical comprising the hemoglobin-based oxygen carrier.

[0004] BACKGROUND OF THE INVENTION

[0005] The significance of blood in sustaining human life is undeniable, yet the reliance on donor blood for patient assistance poses significant challenges. In particular, red blood cells have a limited storage lifespan; and require cold storage and rigorous testing before use in transfusion. Synthetic oxygen carriers, in particular the so-called hemoglobin (Hb)- based oxygen carriers (HBOCs), have been explored in order to leverage the exceptional oxygen transport properties of native hemoglobin. HBOCs offer potential universality by lacking blood antigens, thereby enabling direct transfusion. Moreover, their preparation in sterile conditions and enhanced stability at ambient temperatures reduce disease transmission risks and improve their availability and transportability.

[0006] However, there remains the challenge of providing a safe HBOC that is both free of sideeffects in patients; that has oxygen carrying capacity comparable to natural blood; and where the risk of Hb oxidation to non-functional methemoglobin (metHb) is minimized. Furthermore, significant constraints on HBOCs must be met in terms of carrier particle size (diameter). Particles falling below the required size range risk leakage from blood vessels, which is considered to be an underlying cause of vasoconstriction, systemic hypertension, and oxidative tissue injury. Particles falling above the required size range risk phagocytosis and degradation. None of the HBOCs reported to date have been approved for human use in the USA or Europe.

[0007] Accordingly there remains a need for HBOCs that meet the above requirements for their use in medical treatment of subjects suffering from acute blood loss or anemia. The present invention satisfies this unmet need.

[0008] SUMMARY OF THE INVENTION

[0009] In a first embodiment, the invention provides a population of hemoglobin-based oxygen carrying nanoparticles, wherein each nanoparticle comprises:

[0010] (a) zeolite imidazole;

[0011] (b) poly(ethylene glycol); and (c) hemoglobin; wherein the nanoparticles are obtained by combining 2-methylimidazole, polyethylene glycol, hemoglobin and Zn2+ions in a single reaction vessel.

[0012] In a second embodiment, the invention provides a method of preparing the population of hemoglobin-based oxygen carrying nanoparticles of the first embodiment, comprising the steps of:

[0013] (a) providing an aqueous solution of 2-methylimidazole, poly(ethylene glycol), and hemoglobin;

[0014] (b) adding an aqueous solution of Zn(NOs)2 ■ 6H2O to the aqueous solution of (a) under constant stirring and incubating for a period of time;

[0015] (c) subjecting the aqueous solution produced in step (b) to a g force to sediment the nanoparticles therein,

[0016] (d) re-suspending the nanoparticles sedimented in step (c) in an aqueous liquid,

[0017] (e) optionally re-sedimenting the nanoparticles re-suspended in step (d) and repeating step (d).

[0018] In a third embodiment, the invention provides a pharmaceutical composition for use as a medicament, comprising the population of hemoglobin-based oxygen carrying nanoparticles according to the first embodiment.

[0019] In a fourth embodiment, the invention provides a pharmaceutical kit of parts comprising zinc nitrate hexahydrate, 2-methylimidazole, poly(ethylene glycol) and hemoglobin for preparation of the pharmaceutical composition for use as a medicament of the third embodiment.

[0020] In a fifth embodiment, the invention provides the use of PEG to increase the hemoglobin loading capacity of Metal-Organic Framework-based HBOC nanoparticles.

[0021] DESCRIPTION OF THE INVENTION

[0022] Brief description of the figures:

[0023] Figure 1. Cartoon illustrating A: the synthesis of hemoglobin (Hb)-loaded ZIF-8 NPs (Hb@ZIF-8 NPs) in one-pot by combining Hb, zinc ions (Zn2+), and 2-methylimidazole (Hmlm); B: the synthesis of hemoglobin (Hb)-loaded ZIF-8 NPs (HbPEG@ZIF-8 NPs or HbPVP@ZIF-8 NPs) in one-pot by combining Hb, zinc ions (Zn2+), 2-methylimidazole (Hmlm) and either poly(ethylene glycol) (PEG) or polyvinylpyrrolidone.

[0024] Figure 2. Properties of Hb@ZIF-8 NPs prepared using various Zn2+: HmIm molar ratios: (a) NPs' size: hydrodynamic diameter size (nm) versus Zn2+: HmIm molar ratio; b) NPs' zeta «)-potential (mV) versus Zn2+: HmIm molar ratio; c) NPs' % yield versus Zn2+: HmIm molar ratio; d) NPs' entrapment efficiency (EE) versus Zn2+: HmIm molar ratio; e) Photographic images of the Hb@ZIF-8 NPs after the first centrifugation step; f) Scanning electron microscopy images of Hb@ZIF-8 NPs prepared with three different Zn2+: HmIm molar ratios. The higher magnification images have scale bars of 1 pm. Yield (%) = (mass of freeze-dried Hb@ZIF-8 NPs) / (theoretical mass) x 100; EE (%) = (Hb entrapped in the Hb@ZIF-8 NPs) I (initial added Hb) x 100.

[0025] Figure 3. Graph showing absorbance readings at 280 nm versus Hb concentration for use as a standard curve.

[0026] Figure 4. Properties of Hb@ZIF-8 NPs prepared using a Zn2+: HmIm molar ratio of 1:4; 10 mg mL1of Hb; with or without a capping agent (PEG or PVP) provided at different concentrations: a) NPs' size: hydrodynamic diameter size (nm) versus capping agent concentration (mg mL1); b) NPs' zeta «)-potential versus capping agent concentration (mg mL-1); and c) NPs' entrapment efficiency (EE) versus capping agent concentration (mg mL1). d) Photographic images of the supernatants obtained after the first centrifugation step of Hb@ZIF-8 NPs prepared without or with different concentrations (mg mL-1) of PEG (top) or PVP (bottom).

[0027] Figure 5. Photographic images of Hb@ZIF-8 NPs prepared using a Zn2+: HmIm molar ratio of 1:4; 10 mg mL1of Hb; and when including a capping agent (PEG or PVP) at different concentrations, showing the resulting pelleted NPs.

[0028] Figure 6. Fourier-transform infrared spectra of unloaded ZIF-8 NPs, free Hb and Hb@ZIF-8 NPs fabricated without and with the addition of the capping agent: (a) PEG, and in (b) PVP. The NPs are termed HbxPEGy@ZIF-8 NPs or HbxPVPy@ZIF-8 NPs where x and y indicate the concentrations of Hb and capping agent, respectively, used for the assembly in mg mL-1. Some of the ZIF-8 and polymer (PEG / PVP) characteristic bands are indicated by standard and striped-tail arrows, respectively.

[0029] Figure 7. Powder X-ray diffraction (PXRD) patterns and scanning electron microscopy (SEM) images of Hb@ZIF-8 NPs prepared without and with the addition of the capping agents: PEG and PVP. Shown in a) a simulated PXRD pattern of sodalite ZIF-8 for reference. PXRD patterns and SEM images are shown for: b) Hbio@ZIF-8 NPs, c) HbioPEGy@ZIF-8 NPs prepared with different PEG concentrations and d) HbioPVPy@ZIF- 8 NPs prepared with different PVP concentrations. The NPs are termed HbxPEGy@ZIF-8 NPs or HbxPVPy@ZIF-8 NPs, where x and y indicate the concentrations in mg mL1of Hb and capping agent, respectively, used in NP assembly. The intensity (in arbitrary units, [a.u.]) of X-ray beams diffracted at a given angle reveals and describes the presence of a specific crystalline structure in the sample. The lack of peaks, in contrast, indicates a lack of crystalline phases, meaning that the sample is amorphous. The SEM images have scale bars of 1 pm.

[0030] Figure 8. Diffraction pattern of ZIF-8MetOH NPs and simulated pattern of ZIF-8. The dashed lines indicate the systematic shift in the peaks, evaluated to determine instrumental misalignments, which are independent of the sample. The inserted SEM image shows the ZIF-8MetOH NPs, wherein the white line indicates the diameter of one representative particle measuring 35 nm.

[0031] Figure 9. Properties of HbxPEPy@ZIF-8 NPs prepared using Zn2+: HmIm molar ratio of 1:4; Hb concentrations ranging from 0 to 50 mg mL-1; with or without PEG as capping agent: a) NPs' size (hydrodynamic diameter in nm) versus Hb concentration, b) NPs' zeta «)-potential versus Hb concentration; c) NPs' Hb entrapment efficiency (EE%) (shown as data points) and entrapped Hb concentration (mg mL-1) (shown as a histogram) versus Hb concentration used to prepare the NPs; and d) Photographic images of the NPs obtained after the first centrifugation step of HbxPEPy@ZIF-8 NPs prepared using increasing Hb concentrations (1 - 50 mg mL-1) with or without PEG.

[0032] Figure 10. Powder X-ray diffraction (PXRD) patterns and scanning electron microscopy (SEM) images of Hbx@ZIF-8 NPs prepared with or without PEG as capping agent. PXRD and SEM images are shown for: a) ZIF-8 NPs (including the simulated pattern of ZIF- CO3-1 (ZIF-C)); b) PEG2oo@ZIF-8 NPs (including the sodalite ZIF-8 polymorph pattern); c) Hbx@ZIF-8 NPs; and d) HbxPEG2oo@ZIF-8 NPs. The NPs are termed Hbx@ZIF-8 NPs or HbxPEGy@ZIF-8 NPs, where x and y indicate the concentrations of Hb and capping agent, respectively, used for NP assembly in mg mL-1. The SEM images have scale bars of 1 pm.

[0033] Figure 11. FTIR spectra of (a) Hb@ZIF-8 NPs; and (b) HbxPEG2oo@ZIF-8 NPs prepared with different Hb concentrations (i.e., 1, 10, 20 and 40 mg mL1). The spectra of ZIF-8, PEG, PEG2OO@ZIF-8 and free Hb are included for reference. Hb 's amide I (1649 cm-1) and II (1530 cm'1) bands are indicated by dashed lines. Some of the ZIF-8 and PEG characteristic bands are indicated by standard and striped-tail arrows, respectively.

[0034] Figure 12 (a) Circular dichroism (CD) spectra of free Hb and Hb@ZIF-8 NPs prepared with different Hb concentrations (i.e., 1, 10, 20 and 40 mg mL-1) and with (represented by dashed lines) or without (represented by solid lines) the addition of 200 mg mL1 PEG. Graphs showing b) and d) concentration of oxygen released (|iM) and c) and e) percentage of oxyhemoglobin (oxyHb) present in free Hb or in Hb@ZIF-8 NPs prepared using different Hb concentrations (i.e., 1, 10, 20 and 40 mg mL-1) and with (represented by dashed bars) or without (represented by solid bars) the addition of 200 mg mL1PEG. The oxyHb content has been calculated relative to the Hb stock (i.e., 4 or 6 mg mL-1) used to prepare the NPs and the free Hb control. Statistical analysis: ****p < 0.0001, ***p < 0.001, *p < 0.05.

[0035] Figure 13. Graph showing in (a) the % of met-hemoglobin (metHb%) content in free Hb and Hbx@ZIF-8 NP test samples right after preparation and following incubation for 4, 24 and 48 h at room temperature (RT) or 4 h at 37 °C (b) the increase in metHb% (AmetHb) calculated by substracting the metHb% right after preparation; where the Hbx@ZIF-8 NP samples were prepared using different Hb concentrations and with or without the addition of PEG. The NPs are termed Hbx@ZIF-8 NPs or HbxPEGy@ZIF-8 NPs, where x and y indicate the concentrations of Hb and PEG, respectively, used for the assembly in mg mL-1. Note: negative values are due to the subtraction of the metHb content of the Hb stock solution (i.e., solution used to prepare the NPs).

[0036] Figure 14. MetHb quantification method: (a) cartoon of 4 sample tubes wherein the contents of: tube 1 is Hb4oPEG2oo@ZIF-8 NPs; tube 2 is Hb4oPEG2oo@ZIF-8 NPs and KCN (to transform Hb or metHb into cyanoHb (CN-Hb) or cyanometHb (CN-MetHb) respectively; tube 3 is Hb4oPEG2oo@ZIF-8 NPs and K3[Fe(CN)6] (to transform all forms of Hb into metHb); and tube 4 is Hb4oPEG2oo@ZIF-8 NPs, K3[Fe(CN)6] and additionally KCN to stabilize the resulting metHb as CN-MetHb. The metHb quantification was repeated with control solutions of Sigma (bovine) Hb, and Hb stock sample. UV-vis spectra are shown for the contents from the tubes 1-4 for: (b) Sigma (bovine) Hb, (c) Hb stock and (d) Hb4oPEG2oo@ZIF-8 NPs. The difference in Abs 630-680 nm between tubes 1 and 2 corresponds to the native metHb from the sample; while the difference in Abs 630-680 nm between tubes 3 and 4 corresponds to the metHb produced by all forms of Hb.

[0037] Figure 15. Graph showing oxygen dissociation curves for human red blood cells (RBCs), bovine Hb and Hb4oPEG2oo@ZIF-8 NPs, plotted as percent oxygen saturation against oxygen partial pressure (pCh) in mmHg. The table shows the corresponding oxygen partial pressures at which Hb is 50% saturated with oxygen (p50) and the Hill coefficients (n). Abbreviations, terms and definitions:

[0038] Capping agent as defined and tested herein is either PEG or PVP used for one pot synthesis of a HBOC.

[0039] HBOC is a hemoglobin (Hb)-based oxygen carrier

[0040] MQ water is sterile, deionized and demineralized water.

[0041] Nanoparticle: as defined herein is a particle of matter that is between 100 and 1000 nanometers in diameter, as measured by the method of example 2.2.

[0042] RBC or RBCs are Red Blood Cell(s).

[0043] Room Temperature: is 18 to 25 degrees Celsius

[0044] SEM: is Scanning Electron Microscopy

[0045] Detailed description of the invention:

[0046] I: Metal-organic framework-based HBOC nanoparticles li: Composition of the metal-organic framework-based HBOC nanoparticles

[0047] In a first embodiment, the invention provides one or more NPs (e.g. a population of NPs) each NP comprising a zeolitic imidazole framework 8; PEG and Hb, wherein the NPs are assembled by bringing zinc ions (Zn2+), 2-methylimidazole (Hmlm), poly(ethylene glycol) (PEG), and Hb in contact in an aqueous solvent. Assembly of the NPs relies on a self-assembly process taking place in a single reaction vessel.

[0048] The self-assembly process takes place under mild reaction conditions, preferably using MQ water as a solvent at room temperature, where the Zn2+ions, Hmlm, PEG and Hb are preferably brought in contact by stirring.

[0049] The zinc ions (Zn2+) are preferably provided in the form of Zn(NOs)2 ■ 6H2O.

[0050] The PEG is -OH terminated having at least Mw 6000 Da; preferably a Mw of 6000 to 10,000 Da, for example Mw 6000 Da. The Zn2+ions (or Zn(NOs)2 ■ 6H2O) and Hmlm that are brought into contact in the aqueous solvent are provided in a molar ratio of less than 1.0: 10.0; preferably in a molar ratio of between 1: 1 and 1:9, for example in a molar ratio of 1.0: 1.0, 1.0: 2.0 1.0: 3.0, 1.0:4.0, 1.0: 5.0, 1.0: 6.0, 1.0: 7.0, 1.0:8.0 or 1.0:9.0; more preferably about 1.0:4.0.

[0051] The zinc ions (Zn2+) provided in the aqueous solvent for nanoparticle assembly preferably has a concentration of 5-20 mM, preferably 10 mM.

[0052] The Hmlm provided in the aqueous solvent for nanoparticle assembly preferably has a concentration of 10-500 mM, preferably 40 mM.

[0053] The PEG provided in the aqueous solvent for nanoparticle assembly has a concentration of at least 10 mg mL1PEG, for example from 10 to 300 mg mL1PEG, preferably from 100 to 300 mg mL1PEG, more preferably from 150 to 250 mg mL1PEG, such as 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300mg mL1PEG, for example about 200 mg mL1PEG.

[0054] The Hb provided in the aqueous solvent for encapsulation within the nanoparticle during assembly has a concentration of at least 1 mg mL1Hb; for example 1-100 mg mL1Hb; for example 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg mL1Hb; preferably from 10 to 50 mg mL1Hb; preferably about 40 mg mL1Hb. The Hb may be extracted for RBCs derived from a mammal, for example Bovine RBCs or Human RBCs. The synthesis of recombinant a- and p-hemoglobin chains and their assembly into tetrameric hemoglobin provides an alternative source of hemoglobin (as described by Villarreal et aL, 2008).

[0055] Preferably the aqueous solvent used herein is MQ water.

[0056] In summary, each NP has the composition HbxPEGy@ZIF-8 NP where x and y indicate the concentrations in mg mL1of Hb and PEG, respectively, used for NP assembly. For example, a NP of the invention having a composition Hb4oPEG2oo@ZIF-8 NP is defined herein as a NP prepared from a solution comprising 40 mg mL1Hb and 200 mg mL1PEG. Alternatively, a NP of the invention may have an HbxPEGy@ZIF-8 NP selected from among HbiPEGio@ZIF-8; HbiPEG2oo@ZIF-8; HbioPEGioo@ZIF-8; HbioPEG2oo@ZIF-8; and Hb4oPEGioo@ZIF-8, preferably Hb4oPEG2oo@ZIF-8 NP.

[0057] The NPs have a mean average hydrodynamic diameter of from 100 nm to 1000 nm; preferably from 100 nm to 500 nm, more preferably from 100 nm to 300 nm. lii: Properties of Metal-organic framework-based HBOC nanoparticles The properties of the HBOC NPs of the invention are the result of their one-pot method of assembly, which in turn, is dependent on the composition and the molar ratio of the starting components from which the NPs are assembled. The physical and functional properties the HBOC NPs of the invention and the contribution of the starting components used for their assembly to these properties is demonstrated herein by preparing and comparing HbxPEGy@ZIF-8 NP and HbxPVPy@ZIF-8 NPs of different composition.

[0058] Iii-1 Zn2+:HmIm molar ratio as a parameter for controlling the properties of HBOC NPs of the invention

[0059] The properties of Hbio@ZIF-8 NPs, prepared from a starting solution comprising five different Zn2+: HmIm molar ratios (1.0: 1.0, 1.0:2.0, 1.0:4.0, 1.0: 10.0, and 1.0:50.0) while maintaining constant concentrations of Zn2+(10 mM) and Hb (10 mg mL-1) in MQ water, are set out below:

[0060] The sizes of the resulting Hb@ZIF-8 NPs, measured as their hydrodynamic diameter were about 150 nm, only rising to 195 nm when the Zn2+: HmIm molar ratio was 1.0:50.0 (example 1.3.1). This size range meets the clinical requirements for an HBOC to achieve an extended half-life, which is a size in the range of 100 nm-1 pm. The morphology of the resulting Hbio@ZIF-8 NPs was spherical when the Zn2+: HmIm molar ratio was from 1.0: 1.0 to 1.0: 4.0 (example 1.6).

[0061] The ^-potential of all the resulting Hbio@ZIF-8 NPs was negative (about -5 mV to about -8 mV) (example 1.3.2). A negative charge is important for their intended use as HBOCs because it discourages serum protein adsorption and enhances circulation times of the NPs.

[0062] Yield of the resulting Hbio@ZIF-8 NPs (as determined herein; example 1.4) is a measure of the incorporation of the starting components into the assembled NPs. The use of lower Zn2+:HmIm molar ratios ensured sufficient yield, such as a ratio of no more than 1.0: 10.0, preferably a ratio of from 1.0: 1.0 to 1.0: 4.0.

[0063] Hb encapsulation efficiency (EE) of the resulting Hbio@ZIF-8 NPs (example 1.5) was demonstrated for NPs prepared using Zn2+: HmIm molar ratios of 1.0: 10.0 and lower. Since formation of the metal-organic framework of Hb@ZIF-8 NPs is mediated by Zn2+ions coordinating with the organic linker, the use of the lower Zn2+: Hmlm molar ratios ensures incorporation of the starting components and efficient Hb entrapment. Preferably the Zn2+: HmIm molar ratio is from 1.0: 1.0 to 1.0:4.0, since Hb entrapment is 60% when using a 1.0: 1.0 ratio, increasing to ~85% when using a 1.0:4.0 ratio.

[0064] Iii-2 PEG as a component for controlling the properties of HBOC NPs of the invention The properties of HbioPEGy@ZIF-8 NPs prepared from a starting solution comprising a Zn2+: HmIm molar ratio of 1.0:4.0, using Zn2+(10 mM) and Hb (10 mg mL1) in MQ water and including either 5, 10, 50, 100, or 200 mg mL1PEG, are set out below:

[0065] The size of the resulting HbioPEGy@ZIF-8 NPs, measured as their hydrodynamic diameter, was about 160 nm, only rising to 190 nm when using higher PEG concentrations, such as 200 mg mL1PEG, thereby preserving a size range for all said NPs that is compatible with their clinical use as a HBOC (example 2.2.1). SEM analysis demonstrated the spherical morphology of all the resulting HbioPEGy@ZIF-8 NPs (example 2.4).

[0066] The ^-potential of all the resulting HbioPEGy@ZIF-8 NPs was negative (about -7mV) (example 2.2.2), demonstrating that ^-potential of said NPs is independent of the PEG concentration used, where their negative ^-potential is important for their clinical use as a HBOC.

[0067] The Hb EE of all the resulting HbioPEGy@ZIF-8 NPs was over 90%, whereas the inclusion of PEG, such as 200 mg mL1PEG, further increased Hb EE by about 5% (example 2.2.4).

[0068] The chemical composition of the resulting HbioPEGy@ZIF-8 NPs, analyzed by FTIR spectroscopy, revealed bands consistent with the presence of the ZIF-8 framework and encapsulated Hb, as well as the inclusion of PEG in the assembled NPs (example 2.3). Powder X-ray diffraction analysis of the resulting HbioPEGy@ZIF-8 NPs, revealed that the characteristic sodalite (SOD) crystalline structure of the ZIF-8 framework is perturbed by Hb-loading, whereby Hbio@ZIF-8 NPs exhibit an amorphous structure. However, the inclusion of PEG, and in proportion to its concentration in the starting solution, serves to preserve or at least partially preserve the characteristic SOD structure of ZIF-8 in the Hb-loaded HbioPEGy@ZIF-8 NPs (example 2.4). These analyses provide the first demonstration that the SOD crystalline structure of Hb-loaded ZIF-8 NPs, prepared under aqueous mild conditions, is preserved by including PEG in their assembly. While not being bound by theory, it is proposed that preservation of this SOD crystalline structure can extend the functional properties of the NPs of the present invention and their use as HBOCs, for example allowing the incorporation of co-reagents (e.g. reducing agents or antioxidant enzymes) into the NPs.

[0069] Iii-3 The Hb-loading capacity and properties of HBOC NPs of the invention

[0070] The properties of HbxPEGy@ZIF-8 NP, prepared from a starting solution comprising a Zn2+: HmIm molar ratio of 1.0:4.0, with or without 200 PEG mg mL-1, loaded with Hb provided in the starting solution at concentration range of 0-50 mg mL-1; are set out below: The Hb EE of the resulting Hbx@ZIF-8 NPs was about 90%, with a maximum Hb entrapment of 18 mg mL1Hb for Hb2o@ZIF-8. The inclusion of PEG increased Hb entrapment efficiency and capacity considerably, for example to levels as high as 85% EE and ~34 mgmL1Hb (i.e., for Hb4oPEG2oo@ZIF-8 NPs) (example 3.2.3). Importantly, the native secondary structure of Hb encapsulated within the NPs of the invention is preserved (example 3.4.2), which is central to the preservation of Hb's functional properties.

[0071] Preservation, or at least partial preservation, of the characteristic sodalite (SOD) crystalline structure and spherical shape of the resulting HbxPEGy@ZIF-8 NPs is also dependent on the inclusion of PEG when the NPs are loaded at higher Hb concentrations (e.g. > 10 mg mL1Hb) (example 3.3.2).

[0072] The size of the resulting HbxPEGy@ZIF-8 NPs, measured as hydrodynamic diameter, was about 190 nm for unloaded NPs, declining to about 148 nm when loaded with Hb, with or without PEG (example 3.2.1). Hence, the size of NPs resulting from greatly increased Hb loading remained within a range compatible with their clinical use as a HBOC.

[0073] The ^-potential of the resulting HbxPEGy@ZIF-8 NPs, prepared with or without PEG, was reversed from positive to negative when the NPs were loaded with Hb, reaching a - potential of about -6 mV (example 3.3.2).

[0074] The resulting HbxPEGy@ZIF-8 NPs prepared with or without PEG and loaded using at least 20 mg mL1Hb, exhibit similar functional properties as free Hb with respect to oxygen release (example 3.4.4). Furthermore, since up to 95% of the bound oxygen in these NPs was in the form of OxyHb, almost all Hb entrapped within the NPs retains the full functionality of native Hb. The Hb entrapped within the NPs exhibited higher oxygen affinity, and a lower Hill coefficient when compared to free Hb or human RBCs, which would be generally considered compatible with their clinical use as HBOCs. Since the Hill coefficient of the entrapped Hb in the NPs was positive, it can be concluded that the tetrameric structure of Hb is preserved after the entrapment.

[0075] MetHb, which is formed when the iron atom of Hb's heme group is oxidized from the ferrous (Fe2+) to the ferric state (Fe3+), is unable to bind oxygen. In native RBCs, the oxidation into metHb is prevented or reverted by a set of antioxidant compounds (e.g., ascorbic acid, a-tocopherol, or ferritin) and enzymatic systems (e.g., metHb reductase, cytochrome B5 reductase or flavin reductases). Although the HBOCs of the present invention lack the natural oxidative-reductive machinery present in biological RBCs, the inclusion of PEG in the NPs greatly reduced metHb formation during prolonged storage (example 3.4.7). Indeed, the low residual levels of detected metHb can be eliminated by pre-treating the Hb with a reducing agent prior to Hb loading during assembly of the HBOCs of the invention.

[0076] In summary, the nanoparticles of the invention are characterized by high levels of Hb entrapment, reaching an exceptional level of 34 mg mL-1; and by having the key functional properties required for their use as HBOCs in clinical use. These key functional properties include their oxygen-releasing capabilities, low levels of metHb conversion at elevated Hb concentrations; low Hmlm content thereby promoting enhanced biocompatibility; and improved crystallinity of the assembled HbxPEGy@ZIF-8 NPs. These key functional properties are attributable to the composition and assembly of the HbxPEGy@ZIF-8 NPs of the invention, in particular to loading with high Hb concentrations (e.g. 40 - 50 mg Hb. mL-1), the molar ratio of Zn2+: HmIm and the presence of PEG in the aqueous solutions provided for their assembly.

[0077] II. Manufacture of Metal-organic framework-based HBOC nanoparticles

[0078] In a second embodiment, the invention provides a method for preparing nanoparticles (a population of nanoparticles) of the invention, wherein each NP comprising a zeolitic imidazole framework 8; PEG and Hb, and wherein the NPs are assembled by bringing zinc ions (Zn2+), 2-methylimidazole (Hmlm), poly(ethylene glycol) (PEG), and Hb in contact in an aqueous solvent.

[0079] The method is further suitable for preparing all embodiments of said NPs of the invention, as defined in section I.

[0080] The method of preparing the nanoparticles (population of nanoparticles) of the invention as further detailed in example 1, comprises the following steps:

[0081] (a) providing an aqueous solution of 2-methylimidazole (Hmlm), poly(ethylene glycol) (PEG), and hemoglobin (Hb);

[0082] (b) adding an aqueous solution of Zn(NOs)2 ■ 6H2O to the aqueous solution of (a) under constant stirring and incubating for a period of time;

[0083] (c) subjecting the aqueous solution produced in step (b) to a g force to sediment the nanoparticles therein,

[0084] (d) re-suspending the nanoparticles sedimented in step (c) in an aqueous liquid,

[0085] (e) optionally washing the re-suspended nanoparticles of step (d) by repeating steps (c) and (d); and (f) storing the recovered nanoparticles obtained in step (d) or (e) at a temperature of 4°C or below.

[0086] Preferably the Hmlm and Zn(NOs)2 ■ 6H2O in the aqueous solution produced in step (b) provide for a Zn2+: HmIm molar ratio of less than 1.0: 10.0; preferably in a molar ratio of between 1.0: 1.0 and 1.0:9.0, for example in a molar ratio of 1.0: 1.0, 1.0:2.0, 1.0:3.0, 1.0:4.0, 1.0:5.0, 1.0:6.0, 1.0:7.0, 1.0:8.0 or 1.0:9.0; more preferably about 1.0:4.0.

[0087] Preferably the PEG is -OH terminated having at least Mw 6000 Da; preferably a Mw of 6000 to 10,000 Da, for example Mw 6000 Da.

[0088] Preferably the PEG provided in the aqueous solvent of step (a) has a concentration of at least 10 mg mL1PEG, for example from 10 to 300 mg mL1PEG, preferably from 100 to 300 mg mL1PEG, more preferably from 150 to 250 mg mL1PEG, such as 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300mg mL1PEG, for example about 200 mg mL1PEG.

[0089] Preferably steps (a) and (b) are performed at room temperature, (such as about 18 - 25 °C).

[0090] Preferably the aqueous solution of 2-methylimidazole (Hmlm), poly(ethylene glycol) (PEG), and hemoglobin (Hb); and the aqueous solution of Zn(NOs)2 ■ 6H2O are prepared using MQ water as aqueous solvent.

[0091] III. Pharmaceutical composition comprising metal-organic framework-based HBOC nanoparticles

[0092] In a third embodiment, the invention provides a pharmaceutical composition comprising a population of NPs according to the invention as defined in section I, for use as a medicament, for example for the purpose of transfusion.

[0093] The pharmaceutical composition may be used in the treatment of a human suffering from a lack of RBCs, where the population of NPs of the present invention can be administered by transfusion to the subject without risk of cross-match-reactivity or transmission of infectious agents, if aseptic conditions are ensured.

[0094] The pharmaceutical composition comprises said population of NPs according to the invention suspended in a saline solution.

[0095] IV A pharmaceutical kit of parts In a fourth embodiment, the invention provides a kit of parts comprising 2- methylimidazole, poly(ethylene glycol), haemoglobin, and zinc nitrate hexahydrate for the preparation of a pharmaceutical composition comprising a population of NPs according to the invention as defined in section I, for use as a medicament, for example for the purpose of transfusion. Said 2-methylimidazole, poly(ethylene glycol), haemoglobin, and zinc nitrate hexahydrate may each be provided in the form of aqueous solutions in containers, or they may be provided in dry form in containers suitable for providing aqueous solutions thereof by the addition of an aqueous solvent, preferably MQ water.

[0096] The kit of parts is provided with instructions for bringing the 2-methylimidazole, poly(ethylene glycol), haemoglobin, and zinc nitrate hexahydrate in contact for the assembly of NPs of the invention, and the incorporation of the resulting HBOCs in a pharmaceutical composition for use as a medicament.

[0097] V Use of PEG to increase the Hb loading capacity of Metal-organic frameworkbased HBOC nanoparticles

[0098] In a fifth embodiment, the invention provides for the use of PEG to increase the Hb loading capacity of Metal-organic framework-based HBOC nanoparticles, in particular HBOCs wherein the Metal-organic framework is ZIF-8 as defined in Section I.

[0099] Preferably the PEG is -OH terminated having at least MW 6000 Da; preferably a MW of 6000 to 10,000 Da, for example MW 6000 Da.

[0100] The PEG is provided in an aqueous solvent for use in the preparation of the HBOC nanoparticles of the invention (as defined in section I), where preferably it is provided at a concentration of at least 10 mg mL1PEG, for example from 10 to 300 mg mL1PEG, preferably from 100 to 300 mg mL-1. PEG, more preferably from 150 to 250 mg mL-1. PEG, such as 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300mg mL1PEG, for example about 200 mg mL1PEG.

[0101] According to said fifth embodiment, the PEG may also include its use to reduce the conversion of OxyHb to MetHb entrapped within the HBOC nanoparticles of the invention by oxidation.

[0102] According to said fifth embodiment, PEG may also include its use to preserve, or at least partially preserve, the sodalite (SOD) crystalline structure of the ZIF-8 Metalorganic framework in the HBOC nanoparticles of the invention.

[0103] EXAMPLES Materials:

[0104] Bovine blood with citrate was purchased from SSI Diagnostica A / S (Hillerod, Denmark). Zinc nitrate hexahydrate (Zn(NOs)2 6H2O), 2-methylimidazole (Hmlm), ethylenediaminetetraacetic acid (EDTA) solution, tris(hydroxymethyl) aminomethane (TRIS), polyethylene glycol (PEG, -OH terminated, Mw 6000 Da), polyvinylpyrrolidone (PVP, Mw 10 000 Da), potassium hexacyanoferrate (III) (K3[Fe(CN)6]), potassium cyanide (KCN) and Hb from bovine blood in lyophilized powder form were obtained from Merck Life Sciences A / S (Soborg, Denmark). Methanol (MetOH) (> 99.9%) was acquired from VWR international (Soborg, Denmark). Hellmanex II was purchased form Hellma (Jena, Germany).

[0105] Ultrapure water (Mili-Q (MQ), gradient A 10 system, TOC < 4 ppb, resistance 18 MV cm, EMD Millipore) was used to prepare the 0.9% sodium chloride (NaCI) solution and TRIS 2 buffer (10 mM TRIS and 150 mM NaCI, pH 7.4).

[0106] Example 1: Preparation of metal-organic framework nanoparticles (Hb)-loaded ZIF-8 NPs (Hb@ZIF-8 NPs), were prepared from zinc ions (Zn2+), 2- methylimidazole (Hmlm) and Hb, employing MQ as a solvent. These NP components were combined in a one-pot reaction performed at RT that allows for the in-situ entrapment of Hb within the NPs. The steps used in preparation of the Hb@ZIF-8 NPs are set out below with reference to Figure 1A.

[0107] 1.1 Hb Extraction from Bovine Blood

[0108] Hb was extracted from bovine blood by hypotonic hemolysis according to Zijlstra et al., (1997). In brief, the blood was washed (3x, 2000g, 20 min, 4 °C) with a saline solution (0.9% NaCI 1: 1 v / v ratio) using a high-speed centrifuge (SL16R centrifuge, ThermoScientific, Hvidovre, Denmark). To lyse the RBCs, the obtained pellet was thoroughly mixed with MQ and toluene (1: 1: 0.4 volume ratio). The resulting mixture was stored overnight at 4 °C in a tapping flask to allow for the separation of the stroma- free Hb, which was collected, centrifuged (8000g, 20 min, 4 °C) and filtered with an ash-free paper. The Hb concentration was determined by ultraviolet-visible spectroscopy (UV-vis) by measuring the absorbance (Abs) at 280 nm using a Nanodrop (2000c, Thermo Fisher Scientific, Waltham, MA, USA). Aliquots of free Hb (2 mL) were prepared and stored at -80 °C for future use.

[0109] 1.2 Preparation of Hb@ZIF-8 NPs test samples

[0110] Hb@ZIF-8 NPs were prepared from aqueous solutions comprising Zn2+and Hmlm in the following molar ratios: 1.0: 1.0, 1.0:2.0, 1.0:4.0, 1.0: 10.0 and 1.0:50.0 of Zn2+:HmIm, while keeping constant the Zn(NOs)2 ■ 6H2O concentration at lOmM and the Hb concentration at 10 mg mL-1. The reaction was performed at RT.

[0111] In detail, Hmlm (10, 20, 40, 100 or 500 pL, 2 M Hmlm in MQ, for a 1.0: 1.0, 1.0:2.0, 1.0:4.0, 1.0: 10.0 and 1.0:50.0 Zn2+: HmIm molar ratio, respectively), Hb (108 pL, 185 Hb mg mL1in MQ) and MQ (to a final volume of 1 mL) were added to a 8 mL glass vial. Next, 1 mL of Zn(NOs)2 ■ 6H2O (20 mM in MQ) was added to the mixture under constant stirring (800 rpm). After 10 min, the mixture was transferred to a 2 mL Eppendorf tube for washing (3x, 12 000g, 5 min, 4 °C, MQ). The samples were stored at 4 °C for future use. The final volume of all the samples was 2 mL.

[0112] 1.3 Properties of Hb@ZIF-8 NPs test samples

[0113] 1.3.1 Nanoparticle size of Hb@ZIF-8 NPs test samples

[0114] The hydrodynamic size of the Hb@ZIF-8 NPs test samples was evaluated by NP tracking analysis (NTA) (ZetaView PMX120, Particle Metrix GmbH, Germany). Measurements from 11 different positions were performed with a sensitivity of 60, a frame rate of 30 and a shutter value of 100. At least 1000 NPs were traced in each sample.

[0115] An optimal NP size of about 150 nm (hydrodynamic diameter) was obtained for Hb@ZIF- 8 NPs test samples prepared using Zn2+: HmIm in a molar ratio of 1: 1, 1:2, 1:4, and 1: 10. At higher Zn2+: HmIm molar ratios (1:50), NP size increased to ~195 nm (Figure 2 (a)).

[0116] 1.3.2 ^-potential of Hb@ZIF-8 NPs test samples

[0117] The ^-potential of the Hb@ZIF-8 NPs test samples was evaluated using a Zetasizer nanoseries nano-ZS (Malvern Panalytical LTd., Malvern, UK).

[0118] A negative ^-potential was obtained for all Hb@ZIF-8 NPs test samples demonstrating that within the tested range of Zn2+: HmIm molar ratios the resulting NPs were characterized by negative ^-potentials (about -5mV to about -8mV). The negative - potential is attributed to the negative charge Hb has under the NP preparation conditions (pH > pl of 6.8). The negative ^-potential of the Hb@ZIF-8 NPs is an important property for ensuring their biocompatibility, circulation lifetime, and biodistribution (figure 2(b)).

[0119] 1.4 Nanoparticle yield of Hb@ZIF-8 NPs test samples

[0120] The yield was determined by freeze-drying a known volume of Hb@ZIF-8 NPs and calculated as follows: yield (%) = (mass of freeze-dried Hb@ZIF-8 NPs) / (theoretical mass) x 100. The theoretical mass was calculated considering the amounts of the chemicals added to prepare the Hb@ZIF-8 NPs.

[0121] Yield of Hb@ZIF-8 NPs declined with increasing Zn2+: HmIm molar ratios. Yields of 50% to 45% were observed at the lowest ratios (1.0: 1.0 to 1.0:4.0), but diminishing to ~14% at a 1.0: 10.0 ratio and dropping to ~0% at a 1.0: 50.0 ratio. At the higher ratios, insufficient Zn2+levels for coordination may lead to a loss of organic linker, leading to lower Hb entrapment, thereby accounting for the reduced yield (figure 2(c)).

[0122] 1.5 Encapsulation efficiency of Hb@ZIF-8 NPs test samples

[0123] The EE of the Hb@ZIF-8 NPs was determined after disassembling the Hb@ZIF-8 NPs with the chelating agent EDTA. Specifically, EDTA (10 pL, 50 mM) was added to 10 pL of Hb@ZIF-8 NPs. Then, depending on the starting Hb concentration of the samples, different dilutions were prepared (in MQ) to obtain Abs values at 280nm lying within the standard curve (figure 3), that correlates Hb concentration with Abs 280 nm from which the Hb content of the disassembled Hb@ZIF-8 NPs was derived.

[0124] The following formula was used:

[0125] EE (%) = (Hb entrapped in the Hb@ZIF-8 NPs) I (initial added Hb) x 100.

[0126] While the EE of Hb@ZIF-8 NPs prepared using the lowest Zn2+: HmIm molar ratio (1.0: 1.0) was 60%, and rose as high as ~85% when using a 1.0:4.0 ratio, any further increase in the ratio correlated with a decline in EE (figure 2(d)).

[0127] The optimal EE of Hb@ZIF-8 NPs prepared using Zn2+: HmIm molar ratio ranging from (1.0: 1.0) to (1.0:4.0) was confirmed by the absence of Hb in the supernatant following recovery of the NPs by centrifugation, as compared to the Hb seen in the supernatant of NPs prepared using Zn2+: HmIm molar ratio ranging from (1.0: 10.0) to (1.0: 50.0) (figure 2 (e)).

[0128] 1.6 Nanoparticle morphology of Hb@ZIF-8 NPs test samples

[0129] SEM imaging was performed using a Quanta FEG 250 Analytical Environmental SEM (FEI Company, Hillsboro, US). The Hb@ZIF-8 NPs were drop-casted onto a glass slide mounted on top of an adhesive carbon tab. Once dried, the surface of the samples was sputter coated with gold for 15 s at 20 mA using a Quorum Q150T system (Quorum Technologies Ltd., UK). Images were acquired using an Everhart-Thornley detector and an accelerating voltage of 10-20 kV. To estimate the size of the Hb@ZIF-8 NPs, 150 NPs were measured using Image!. Hb@ZIF-8 NPs prepared using a Zn2+: HmIm molar ratio of 1.0: 1.0; 1.0:2.0 and 1.0:4.0 exhibited a spherical morphology (figure 2 f)).

[0130] Example 2: Preparation and properties of metal-organic framework nanoparticles comprising a capping agent

[0131] Hb@ZIF-8 NPs prepared using a Zn2+: HmIm molar ratio of 1.0:4.0 ratio, characterized by a spherical morphology; a hydrodynamic diameter of ~150 nm; a negative - potential of -5 mV; a yield of 44%; and an EE at 85% was selected for evaluating the impact of including a capping agent in NP preparation.

[0132] 2.1 Preparation of Hb@ZIF-8 NPs test samples with PEG or PVP as capping agents

[0133] Hb@ZIF-8 NPs were prepared using a Zn2+: HmIm molar ratio of 1:4 ratio and 10 mg mL1of Hb (as described in example 1.1), but modified by the addition of a capping agent in increasing concentrations (i.e., 0, 5, 10, 50, 100 and 200 mg mL1PEG or PVP). In detail, Hmlm (40 pL, 2 M in MQ), Hb (108 pL, 185 mg mL1in MQ), capping agent (i.e., either 0, 40 or 400 pL, 50 mg mL-1 in MQ for a final concentration of 0, 5 and 10 mg mL1capping agent, respectively, or 83.3, 166.7, 333.3 pL, 1200 mg mL1in MQ, for a final concentration of 50, 100 and 200 mg mL1capping agent, respectively) and MQ (to a final volume of 1 mL) were combined in a 8 mL glass vial to form a mixture. While the mixture was stirred (800 rpm), 1 mL of Zn(NOs)2 ■ 6H2O (20 mM in MQ) was added. After 10 min, the test samples were collected, washed, and stored as described in example 1.1.

[0134] 2.2 Properties of Hb@ZIF-8 NPs test samples with PEG or PVP as capping agents

[0135] 2.2.1 Hydrodynamic diameter (measured as described in example 1.2): The inclusion of PEG in Hb@ZIF-8 NPs prepared according to 2.1, led to a slight increase in hydrodynamic diameter (~190 nm at 200 mg mL-1), while the inclusion of PVP did not notably alter size (~170 nm) over the range of tested capping agent concentrations (5, 10, 50, 100, and 200 mg.mL-1) (figure 4 (a)).

[0136] 2.2.2 Np ^-potential (measured as describe in example 1.3):

[0137] The inclusion of PEG in Hb@ZIF-8 NPs prepared according to 2.1, had no significant effect on the ^-potential of the resulting NPs, which was about -7 mV (figure 4 (b)).

[0138] 2.2.3 NP encapsulation efficiency:

[0139] The EE of the Hb@ZIF-8 NPs prepared with PVP or PEG was evaluated indirectly as the difference between the initial added Hb concentration and the Hb concentration in the supernatant after the first centrifugation step. The following formula was used: EE (%) = (initial added Hb - Hb in the supernatant) / (initial added Hb) x 100. The EE of the Hb@ZIF-8 NPs prepared without PVP or PEG was measured as described in example 1.5.

[0140] Encapsulation of Hb in Hb@ZIF-8 NPs prepared according to 2.1, had an EE of over 90%, which was slightly increased by the inclusion of PEG (~5% at 200 mg mL-1) while being decreased by inclusion of PVP, (Figure 4c). Photographic evidence of supernatants following centrifugation of Hb@ZIF-8 NPs with various capping agent concentrations (Figure 4d) revealed less red hue with increasing PEG, indicating more efficient Hb integration into the NPs, aligning with EE findings. Conversely, Hb@ZIF-8 NPs prepared with the highest PVP concentration (200 mg mL-1) displayed more red hue, suggesting less efficient Hb integration and decreased EE.

[0141] 2.3 Chemical structure of Hb@ZIF-8 NPs assembled with PEG or PVP as capping agents.

[0142] The chemical structure of Hb@ZIF-8 NPs (with PEG or PVP) was analyzed by FTIR spectroscopy as follows: Attenuated total reflectance -FTIR spectra were recorded using a Shimadzu IRAffinity-lS spectrophotometer (Shimadzu Corp., Kyoto, Japan). The Hb@ZIF-8 NPs in powder form were analyzed under ambient conditions. For each sample, 32 scans were accumulated in the transmittance mode and collected in the wavenumber range of 650-3500 cm-1. A resolution of 2 cm-1, mirror speed of 2 mm s-1, and square triangle apodization function were set during the measurements. Background spectra were collected before each acquisition and subtracted from the sample spectra (figure 6).

[0143] The spectrum of unloaded ZIF-8 NPs displayed anticipated bands from the Hmlm ligand comprising bands at 994 and 1142 cm-1, attributed to C-N stretching vibrations, and a band at 1574 cm-1related to the C=N stretching mode. Bands in the 1500-1350 cm-1range originated from the imidazole ring stretch, but the expected band at 421 cm-1from the Zn-N interaction could not be detected due to instrument noise in the far-IR. In the spectrum of free Hb, the key absorption bands detected were: an amide I band at 1649 cm-1(C=O stretching) and an amide II band at 1530 cm-1(in-plane N-H bending). A weaker band in the 3300-3500 cm-1range, known as amide A, from N-H stretching vibrations was also observed. These bands were consistently present in all Hb@ZIF-8 NPs regardless of PEG or PVP used (designated as HbxPEGy@ZIF-8 NPs and HbxPVPy@ZIF-8 NPs where x and y indicate the concentrations of Hb and capping agent, respectively, used for the assembly), confirming successful Hb encapsulation. In HbioPEG@ZIF-8 NPs, as PEG concentration increased, bands associated with the PEG polymer became more noticeable (indicated with striped-tail arrows) (figure 6 (a)). Thus, for HbioPEGio@ZIF-8 NPs, only a small shoulder could be detected at 1095 cm-1, which is linked to the C-O-C stretching of PEG'S aliphatic ether bonds. In contrast, a clearly marked band in this region can be observed upon increasing the PEG concentration to 200 mg mL1(i.e., HbioPEG2oo@ZIF-8 NPs). Moreover, the 2880 cm-1band from PEG’S aliphatic C-H stretching also becomes noticeable in a region of low overlap with the spectra of Hmlm and Hb. Surprisingly, as the content of PEG increases, the bands associated with ZIF-8 (indicated with standard arrows) also become more visible, which points towards an increased Hmlm content within the NPs.

[0144] The spectrum of PVP displays major bands at -2950-2920 cm-1(arising from the asymmetric CH2 stretching of the pyrrole ring and symmetric CH2 stretching of the chain), at 1651 cm-1(resulting from C-0 stretching) and at 1285 cm-1(from CH2 wagging and C-N stretching) (figure 6(b)). These bands overlap significantly with those displayed by Hb and Hmlm. Hence, for HbioPVP@ZIF-8 NPs prepared with the highest PVP concentrations (i.e., 100 and 200 mg mL-1), only very small dips (indicated with striped-tail arrows) within the 1410-1460 cm-1range can be observed.

[0145] 2.4 Crystalline structure and morphology of Hb@ZIF-8 NPs assembled with PEG or PVP as capping agents.

[0146] The crystalline structure of Hb@ZIF-8 NPs (with PEG or PVP) was analyzed by Powder X-ray diffraction (PXRD), using a Malvern Panalytical Aeris Research benchtop powder diffractometer (Malvern Instruments Ltd., Malvern, UK) with a Cu Ko X-ray source (40 kV, 15 mA, A = 1.5406 A). The diffraction patterns were obtained in the range of 5-35° 20, at a step size of 0.011° 20 and a time per step of 109.65 s. A 1 / 4° divergence slit, nickel beta-filter, 0.04 rad Soller slits, 9 mm anti-scatter slit, and a low beam knife were used during the measurements. Moreover, the sample stage was spun at a speed of 0.5 rev s’1. The freeze-dried samples were measured on zero-background holders made from single crystal silicon (PW1817 / 32, Panalytical) due to the limited amount of powder available. To ease handling and mounting, a few drops of ethanol were used to apply and spread the powder onto the holder's surface. The empty sample holders were also measured to account for the background signal introduced by the holder, air, and X-ray optics of the instrument. The software HighScore Plus by Malvern Panalytical was used to analyze the obtained diffraction patterns. The empty holder scans were subtracted from the corresponding sample scans. The position of the peaks was compared to those of the simulated ZIF-8 polymorph diffraction patterns, obtained from the Cambridge Structural Database of the Cambridge Crystallographic Data Centre, with deposition numbers: 864312 and 1032088 (Morris et al., 2012; and Basnayake et aL, 2015).

[0147] X-ray diffractograms of Hb@ZIF-8 NPs prepared without or with the incorporation of increasing concentrations of PEG and PVP are shown in figure 7a simulated PXRD pattern associated with the sodalite (SOD) crystal structure of standard ZIF-8 (l43m crystal system with unit cell dimension a = 17.0095 A at 298 K) is shown for reference (Figure 7a) (Morris et al., 2012).

[0148] Upon loading Hb in the absence of capping agent, the diffractogram of the Hb@ZIF-8 NPs assumes a new profile having a pronounced 'hump', with just a few peaks only detectable at very low intensity, consistent with a predominantly amorphous phase (Figure 4b). This is consistent with Hb disrupting the crystalline arrangement of Zn2+and Hmlm. Additionally, the position of the main diffraction peak that could be detected (i.e., at 10.9°) does not correspond with the characteristic diffraction peak of the (Oil) plane which, according to the simulated ZIF-8 pattern, should appear at 20 = 7.3°.

[0149] Surprisingly, the addition of PEG results in the appearance of the main diffraction peaks associated with the characteristic SOD structure of ZIF-8 (Figure 7c). The intensity (in arbitrary units) of the principal diffraction peaks at 20 = 7.3, 10.3, 12.6, 14.6, 16.3, and 18.0 ° corresponding to the (Oil), (002), (112), (022), (013) and (222) planes, respectively, increased relative to the amorphous hump as the PEG content of the NPs was raised. Note that the slight shift in the position of the diffraction peak towards lower values of 20 with respect to the simulated pattern was systematic (i.e., it was observed for all the peaks). This indicates that the shift is due to the measurement set-up (e.g., a small misalignment in the sample height) and not to differences in lattice distances. This conclusion was confirmed by the analysis of reference ZIF-8 NPs synthesized in MetOH under the same diffractometer settings, which presented the same systematic shift in all the reflections (figure 8).

[0150] The PXRD data indicates that PEG enhances the crystallization process, potentially by interacting with the ZIF-8 nuclei and promoting the growth of certain planes. The ability of PEG to aid in the formation of ZIF-8 could be a result of interactions between the repeat unit of ethoxyl groups in PEG and Zn2+, resulting in an accelerated formation of pre-nucleation clusters of ZIF-8 around PEG. The presence of PEG around the aforementioned clusters confers the HbxPEGy@ZIF-8 NPs with enhanced colloidal stability and dispersity. The data provides the first demonstration that the SOD crystalline structure of Hb-loaded ZIF-8 NPs, prepared under aqueous mild conditions, is preserved by including PEG in the assembled NPs.

[0151] In contrast, the diffractogram of the PVP-containing Hb@ZIF-8 NPs was consistent with an amorphous phase, similar to that obtained for Hbio@ZIF-8 NPs, as shown in Figure 7d. The PXRD data indicates that PVP does not preserve the characteristic SOD crystalline structure of ZIF-8 in PVP-containing Hb@ZIF-8 NPs. SEM of the respective Hb@ZIF-8 NPs prepared with or without capping agent, displayed a relatively spherical morphology with a diameter of 100-200 nm. The inclusion of PEG decreased NP diameter, such that the diameter of Hbio@ZIF-8 NPs decreased from ~215 nm to ~125 nm when PEG was introduced (i.e., 10 mg mL-1). A further size decrease to ~95 nm was detected for HbioPEG2oo@ZIF-8 NPs.

[0152] Example 3. Hb-loading of metal-organic framework nanoparticles comprising a capping agent

[0153] HbxPEGy@ZIF-8 NPs prepared using a Zn2+: HmIm molar ratio of 1.0:4.0 ratio, with or without PEG at 200 mg mL1, were used for the further optimization for use as a HBOC, in view of their crystalline structure and enhanced EE shown in example 2. The HbxPEGy@ZIF-8 NPs were tested for the maximum Hb concentration they could trap, when assembled with escalating Hb concentrations (0-50 mg mL-1).

[0154] 3.1 Preparation of HbxPEGy@ZIF-8 NPs test samples

[0155] HbxPEGy@ZIF-8 NP samples were prepared using a Zn2+: HmIm molar ratio of 1.0:4.0 ; with a range of Hb concentrations (0-50 mg mL-1); and with or without PEG (200 mg mL-1) (as described in example 2.1).

[0156] 3.2 Properties of the HbxPEG2oo@ZIF-8 NPs test samples prepared with different Hb concentrations.

[0157] 3.2.1 Hydrodynamic diameter (measured as described in example 2.1):

[0158] The concentration of Hb used in Hb@ZIF-8 NPs preparation did not significantly alter their hydrodynamic diameter (~150 nm). When PEG was included in HbxPEG2oo@ZIF-8 NPs preparation, the NP hydrodynamic diameter decreased when concentrations of Hb were 20 mg mL1or higher (figure 9(a)). Thus, while HbioPEG2oo@ZIF-8 NPs were 190 nm in diameter, the diameter of Hb2oPEG2oo@ZIF-8 NPs was reduced to 148m.

[0159] 3.2.2 NP ^-potential (measured as described in example 1.3): Hb-loading of both Hbx@ZIF-8 NPs and HbxPEG2oo@ZIF-8 NPs with as little as 1 mg mL-1Hb reversed the NP charge from positive (21.1 and 28.2 mV for un-loaded ZIF-8 NPs and un-loaded PEG2oo@ZIF-8 NPs, respectively) to negative (of ~-6 mV) (Figure 9b).

[0160] 3.2.3 NP encapsulation efficiency (measured as described in example 2.2.4):

[0161] The maximum concentration of entrapped Hb in Hbx@ZIF-8 NPs was studied by increasing the Hb concentration in the starting solution. At a Hb concentration of 20 mg mL-1, the amount of entrapped Hb was 18 mg mL1and the EE was 88%. Any further increase in Hb concentration resulted in a marked decrease in both the EE and the amount of entrapped Hb (figure 9c). However, the inclusion of PEG in the assembly of HbxPEG2oo@ZIF-8 NPs increased the Hb entrapment capacity, from ~20 mg mL1for Hb4o@ZIF-8 NPs to ~34 mg mL1for Hb4oPEG2oo@ZIF-8 NPs. Additionally, HbxPEG2oo@ZIF-8 NPs have an increased EE. For example, in the absence of PEG, EEs of 51 and 13% were obtained for Hb4o@ZIF-8 and Hbso@ZIF-8 NPs, respectively. Upon the addition of PEG, the EE increased to 85 and 66% for the same two initial Hb concentrations in the starting solution.

[0162] Images of the resulting HbxPEG2oo@ZIF-8 NPs test samples (i.e., NPs loaded with 5 - 40 mg mL1Hb) taken after the first centrifugation step, show transparent and colorless supernatants, in contrast to corresponding Hbx@ZIF-8 NPs (figure 9d). Furthermore, the NP yield, observed in terms of packed NP volume of the pellet, increased with Hb concentration, particularly in the presence of PEG. Together the data demonstrates that PEG significantly increased the maximum Hb loading capacity of the NPs (e.g., Hb4oPEG2oo@ZIF-8 NPs having an Hb content of ~34 mg mL1and EE 85%).

[0163] 3.3 Crystalline structure and morphology of Hbx@ZIF-8 NPs and HbxPEG2oo@ZIF-8 NPs test samples prepared with different Hb concentrations.

[0164] 3.3.1 The crystalline structure of NPs was analyzed by Powder X-ray diffraction (PXRD), as described in example 2.4.

[0165] 3.3.2 X-ray diffractograms and SEMs of Hbx@ZIF-8 NPs prepared with increasing concentrations of Hb, with and without PEG (at a 200 mg mL1concentration) and their Hb-free counterparts are shown in figure 10. Unloaded ZIF-8 NPs, synthesized in MQ without PEG, lack the expected rhombic dodecahedron shape typical of ZIF-8 (figure 10a), and SEM images thereof reveal a flat, flake-like structure with uneven NP sizes. Furthermore, the diffractogram differs from the anticipated SOD crystal structure of ZIF- 8 by having diffraction peaks that align with planes (110), (020), (120), (200), and (001), characteristic of a ZIF-CO3-1 (ZIF-C) polymorph (also depicted in figure 10a for comparison) (Basnayake et al., 2015). This outcome was expected since excess of organic ligand is required to obtain SOD ZIF-8 NPs with their distinctive rhombic dodecahedron shape in aqueous solution (here, a Zn2+: HmIm molar ratio of only 1:4 was used).

[0166] Surprisingly, the addition of PEG produces ZIF-8 NPs with the characteristic SOD crystalline structure, comprising the key reflections at 20 = 7.2, 10.2, 12.5, 14.5, 16.3, and 17.8° align with SOD ZIF-8's (Oil), (002), (112), (022), (013), and (222) planes (figure 10(b)). This confirms that crystalline SOD ZIF-8 NPs can be obtained under mild aqueous conditions with low Hmlm concentrations by including PEG during NP assembly. SEM micrographs of PEG@ZIF-8 NPs (figure 10b) exhibited a spherical shape and a more uniform diameter of ~150 nm (considerably smaller than their PEG-free counterparts).

[0167] A change in both the crystal structure and appearance was observed when the ZIF-8 NPs were loaded with Hb, regardless of whether PEG was added (Figure 10c). SEM images of Hbi@ZIF-8 NPs show a diverse mix of spherical, flake-like, and star-shaped particles, some quite large (>1 pm). This diversity in shapes might contribute to discrepancies with NTA measurements, which approximate an average diameter of ~190 nm. Based on their diffraction pattern, that aligns with the ZIF-C polymorph, Hb@ZIF-8 NPs assembled with low Hb concentrations maintain the same crystalline structure as their Hb-free counterparts. Their diffractogram, characterized by a hump, reveals the presence of an amorphous phase that dominates as the Hb concentration used in NP preparation increased (figure 10c). No diffractogram reflections were visible for NPs prepared at Hb concentrations greater than 10 mg mL-1; while SEM images of the resulting Hb@ZIF-8 NPs were rounder and smaller. Including PEG in NP assembly modified both crystal structure and shape of the resulting HbxPEG2oo@ZIF-8 NPs (figure lOd). At lower Hb concentrations, the diffractogram reflections matched the simulated SOD ZIF-8 pattern in terms of peak position and intensity. Specifically, key reflections at 20 = 7.3, 10.4, 12.7, 14.7, and 16.4° were detectable, especially at the lowest Hb concentration (1 mg mL-1). Corresponding SEM images of HbiPEG2oo@ZIF-8 NPs displayed the characteristic rhombic dodecahedron shape associated with this polymorph. Although increasing the Hb concentration led to a more dominant amorphous phase, the presence of PEG clearly preserved the crystalline structure of the resulting NPs (e.g. main SOD structure reflections remain detectable in Hb2oPEG2oo@ZIF-8 NPs when PEG was present, unlike its PEG-free counterpart).

[0168] FTIR analysis revealed Hmlm-associated bands in Hb2oPEG2oo@ZIF-8 NPs, whereas in the absence of PEG, these bands were only detected at the lowest Hb concentration (figure 11, grey arrows). This supports a role for PEG in the incorporation of Hmlm into the NP structure.

[0169] SEM images of HbxPEG2oo@ZIF-8 NPs showed spherical NPs of relatively uniform sizes, despite some aggregates visible at low magnification (~1 pm in diameter). Based on SEM analysis, HbxPEG2oo@ZIF-8 NPs were systematically smaller across all Hb concentrations (e.g., Hb4o@ZIF-8 NPs prepared without and with PEG had a diameter of 291 and 183 nm, respectively). In summary, the structural analyses demonstrate PEG'S capacity to boost and maintain the SOD crystalline structure of Hb@ZIF-8 NPs.

[0170] 3.4 Functional properties of Hbx@ZIF-8 NPs and HbxPEG2oo@ZIF-8 NPs test samples prepared with different Hb concentrations. 3.4.1. Analysis of Hb's secondary structure by Circular dichroism (CD). Far-UV CD spectra were acquired using a JASCO J-815 spectropolarimeter (JASCO, Essex, UK). Hb@ZIF-8 NPs were evaluated within 48 h after preparation using free Hb as a control. All the samples were dispersed in MQ at an estimated Hb concentration of 0.08 mg mL-1. The diluted samples were measured at RT in a 0.5 mm path length cell with an acquisition range of 190-260 nm and a step size of 1 nm. Five blank measurements (i.e., MQ) were performed before analysing each sample and eight spectra were acquired per sample. The cuvette was cleaned with a 2% aqueous solution of Hellmanex II in between samples. The software Spectra Analysis - Spectra Manager Version 2 from JASCO was used to post-process the CD data. For each sample, the eight spectra acquired were averaged. The corresponding five blank measurements were also averaged and subsequently subtracted from the sample's average spectrum. The resulting spectra were smoothed with a moving average of 5 points.

[0171] 3.4.2 Hb's secondary structure in Hbx@ZIF-8 NPs and HbxPEG2oo@ZIF-8 NPs test samples based on CD analysis.

[0172] The CD spectra of Hb@ZIF-8 NPs fabricated with varying Hb concentrations (1, 10, 20, and 40 mg mL-1) with and without PEG (200 mg mL-1) are shown in figure 12a. Free Hb is characterized by a CD spectrum comprising bands of a-helical structure: a positive band at 194 nm and negative bands at 209 and 221 nm (Greenfield et al 2006). Based on the similarity in helicity of the Hb@ZIF-8 and HbPEG@ZIF-8 NPs to free Hb, the secondary structure of Hb is maintained in most Hb@ZIF-8 NPs. Minor differences in amplitude between the spectra of NPs prepared with high Hb concentrations and free Hb likely stem from variations in the Hb concentration of the final suspensions. Nonetheless, the matching spectra shape indicates that the secondary structure of Hb is preserved upon encapsulation in the NPs.

[0173] 3.4.3 Quantification of Released Oxygen

[0174] The oxygen releasing ability of free Hb and Hb entrapped within Hb@ZIF-8 NPs and HbPEG@ZIF-8 NPs was evaluated by measuring the oxygen concentration (pM) before and after the addition of K3[Fe(CN)6]. The measurements were conducted within 8 h of NP preparation. Hb@ZIF-8 NPs, HbPEG@ZIF-8 NPs and free Hb (from the stock used to prepare the NPs) were diluted in MQ and added into a glass vial (600 pL, 4 and 6 mg mL-1of Hb). These Hb concentrations were obtained by preparing Hb@ZIF-8 NPs and HbPEG@ZIF-8 NPs with 1 mg mL1Hb (by scaling-up the protocol described in section 2.1 and increasing all volumes by a factor of 5). The Hb content was quantified via UV- vis, recording the Abs at 280 nm following the disassembly of the NPs with EDTA (as described in section 2.2.4). After sealing the vial with a rubber cap, a needle-type oxygen microsensor (PreSens, Regensburg, Denmark) was inserted into the solution. The oxygen concentration (pM) was monitored while 50 pL of a 10% K3[Fe(CN)6] aqueous solution were added and mixed by gently shaking the vial. The concentration of released oxygen was obtained by calculating the difference between the last reading before the addition of K3[Fe(CN)6] and the maximum value reached following incubation with K3[Fe(CN)6]. The relative fraction of oxyHb in the NPs with respect to that in the Hb stock was then calculated as: oxyHb (%) = (released oxygen from Hb@ZIF-8 NPs or HbPEG@ZIF8-NPs) / (released oxygen from free Hb) x 100.

[0175] 3.4.4 Oxygen release by Hbx@ZIF-8 NPs and HbxPEG2oo@ZIF-8 NPs

[0176] The oxygen concentration released from the Hbx@ZIF-8 NPs and HbxPEG2oo@ZIF-8 NPs was compared to free Hb using an oxygen electrode (as in 3.4.3) as shown in figure 12 b. Although Hbx@ZIF-8 NPs prepared with lower Hb concentrations (1 and 10 mg mL-1) released lower oxygen concentrations than free Hb, encapsulation did not appear to severely impact Hb's binding sites. Hbx@ZIF-8 NPs prepared with increasing Hb concentrations enhanced their functionality; such that Hbio@ZIF-8 NPs released ~73 pM oxygen which was only 15% lower than free Hb. Hb2o@ZIF-8 NPs and Hb4o@ZIF-8 NPs released the same oxygen concentration as free Hb, irrespective of the presence of PEG.

[0177] Since the percentage of oxyHb in Hb2o@ZIF-8 NPs and Hb4o@ZIF-8 NPs reached up to 95%, almost all Hb entrapped remained functional, (figure 12c). These results were confirmed when the analyses were repeated with Hbx@ZIF-8 NPs and HbxPEG2oo@ZIF-8 NPs resuspended at a higher Hb concentration of 6 mg mL1Hb (figure 12d and 12e. In summary, Hb2o@ZIF-8 NPs and Hb4o@ZIF-8 NPs, with or without PEG, retained outstanding oxygen-carrying abilities.

[0178] 3.4.5 Quantification of MetHb

[0179] First, the concentration of Hb within the different Hb@ZIF-8 NPs and HbPEG@ZIF-8 NPs was calculated as described in example 1.5. Then, the Abs readings at 542, 562 and 577 nm of free Hb and Hb@ZIF-8 NPs or HbPEG@ZIF-8 NPs (20 pL, 3.5 mg mL1of Hb) were measured right after preparation and after storing them in different conditions. The concentrations of oxygenated Hb (oxyHb), deoxygenated Hb (deoxyHb), metHb, and the metHb% were calculated using Benesch equations (Benesch et al., 1973) as follows: oxyHb (M) = (1.474 A577 - 0.6820 A562 - 0.5329 A542) x 104. deoxyHb (M) = (1.4749 A562 + 0.2141 A577 -1.1042 A542) x 104. metHb (M) = (4.5852 A542 - 0.8375 A562 - 3.7919 A577) x 104. metHb (%) = [metHb] / ([oxyHb] + [deoxyHb] + [metHb]) x 100.

[0180] For a more precise assessment of the metHb content in the optimized formulation (i.e., Hb4oPEG2oo@ZIF-8), a protocol reported by Arnaud et al. (2017) was followed. Briefly, the following content was added to four different Eppendorfs:

[0181] Eppendorf 1: 450 pL Hb4oPEG2oo@ZIF-8 NPs (3.5 mg mL1Hb) + 20 pL TRIS 2

[0182] Eppendorf 2: 450 pL Hb4oPEG2oo@ZIF-8 NPs (3.5 mg mL1Hb) + 10 pL KCN (10% in TRIS 2) + 10 pL TRIS 2

[0183] Eppendorf 3: 450 pL Hb4oPEG2oo@ZIF-8 NPs (3.5 mg mL1Hb) + 10 pL (K3[Fe(CN)6]) (10% in TRIS 2) + 10 pL TRIS 2

[0184] Eppendorf 4: 450 pL Hb4oPEG2oo@ZIF-8 NPs (3.5 mg mL1Hb) + 10 pL KCN (10% in TRIS 2) + 10 pL (K3[Fe(CN)6]) (10% in TRIS 2)

[0185] The content of the Eppendorfs was mixed carefully and after 10 min of incubation, 300 pL from each Eppendorf was transferred to four micro cuvettes. Subsequently, the Abs readings at 630 (Abseso) and 680 nm (Abseso) were recorded by Nanodrop using TRIS 2 as a blank. The metHb content was calculated as follows: metHb (%) = 100 x (A Abs(Eppendorf 1)- A Abs(Eppendorf 2)) / ( A Abs(Eppendorf 3)-A Abs(Eppendorf 4)) where A Abs= Abseso - Abseso. The Abseso was considered to subtract the background Abs.

[0186] The metHb% of free Hb (bovine Hb stock used to synthesize the Hb@ZIF-8 NPs) and metHb (Hb in powder form purchased from Sigma which is known to be metHb) were also estimated using this method and compared to the metHb% of Hb4oPEG2oo@ZIF-8 NPs.

[0187] 3.4.6. MetHb content of Hbx@ZIF-8 NPs and HbxPEG200@ZIF-8 NPs

[0188] The metHb percentage (metHb%) in free Hb solutions and Hb entrapped within the Hbx@ZIF-8 NPs assembled with increasing Hb concentrations (i.e., 10, 20 and 40 mg mL-1) and without (i.e., Hbx@ZIF-8 NPs) or the addition of PEG (i.e., HbxPEG2oo@ZIF-8 NPs) and then stored at RT (for 4, 24, and 48 h), or at 37 °C (for 4 h), was determined (figure 13a and 13b).

[0189] Immediately after preparation, the Hb@ZIF-8 NPs exhibit a similar metHb content to free Hb (~10%) (Figure 13a). In addition, the metHb content of both free Hb and Hb@ZIF-8 NPs, after incubation for 4h at RT, remained similar with that of the NPs right after preparation. To disregard discrepancies in the results stemming from using different Hb stocks for NP preparation, the increase in metHb content (i.e., AmetHb) was calculated by subtracting the metHb content of the Hb stock solution used to prepare the NPs. After 4 hours at RT, AmetHb of all NPs, with or without PEG, showed little increase (figure 13b); while both an elevated incubation temperature (37°C) and prolonged incubation led to an increase in metHb content in the tested NP samples. For instance, Hb2o@ZIF-8 NPs had 2%, 16%, and 27% more metHb after 4, 24, and 48 hours at RT, respectively. Conversion to metHb was faster at 37°C than RT, as illustrated by Hb4o@ZIF-8 NPs having 18% metHb when incubated at 37 °C compared to 2% metHb at RT. Surprisingly, the presence of PEG in the NPs reduced metHb formation, when loaded with higher Hb concentrations. For example, after 24 and 48 hours, Hb2o@ZIF-8 NPs had 16% and 27% AmetHb, while in Hb2oPEG2oo@ZIF-8 NPs the increase was of only 9% and 18%, respectively. This protective effect of PEG was even more pronounced in Hb4oPEG2oo@ZIF-8 NPs, whose AmetHb levels were only 6% and 22% after 24 and 48 h, respectively, as opposed to 23% and 37% for the PEG-free counterpart. This study reveals an important property of PEG in limiting Hb's autoxidation.

[0190] The metHb content of Hb4oPEG2oo@ZIF-8 NPs was 8.78 ± 1.45%, which is comparable to the 8.54 ± 0.77% found in the free Hb suspension used for their creation, as determined by the method illustrated in figure 14. This verified that Hb entrapment method within NPs of the invention (e.g. Hb4oPEG2oo@ZIF-8 NPs) does not promote Hb oxidation into metHb. Note that the metHb content detected in the Hb4oPEG2oo@ZIF-8 NPs (~8%) is a result of the high metHb content in the free Hb solution used for their synthesis rather than transformation in the NPs. The metHb levels in the starting Hb solution can be reduced by incubation with a reducing agent (e.g., ascorbic acid) prior to use in Hb loading.

[0191] 3.4.7. Oxygen Dissociation Curve (ODC) and Hill coefficient determination

[0192] The ODCs of human Hb inside the RBCs, free Hb (bovine Hb stock used to synthesize the Hb@ZIF-8 NPs) and Hb entrapped within Hb4oPEG2oo@ZIF-8 NPs were obtained at 37 °C using an Hemox analyzer (TSC Scientific Corp., New Hope, PA, USA). The different suspensions were diluted to an Hb concentration of 3 mg mL1in 0.9% NaCI. Then, the partial oxygen pressure (pO2) at which Hb is 50% saturated (p50) and Hill coefficient (n) were obtained using the pre-installed software (TCS HEMOX DAQ System).

[0193] 3.4.8. ODC and Hill coefficient of Hb entrapped in Hb4oPEG2oo@ZIF-8 NPs.

[0194] The oxygen-releasing capacity of the Hb entrapped within Hb4oPEG2oo@ZIF-8 NPs had a lower p50 value (5.5 mmHg) when compared to both free Hb (32 mmHg) and human Hb within native human RBCs (39 mmHg). Thus, the entrapped Hb had a correspondingly higher oxygen affinity (figure 15). Since HBOCs with lower p50 values may reduce vasoconstriction (e.g. 5-10 mmHg), the NPs tested herein would fall within this preferred range.

[0195] The Hill coefficient (n) of Hb entrapped in Hb4oPEG2oo@ZIF-8 NPs (n = 1.6) compared to free Hb (n = 1.8), and human Hb in RBCs (n = 2.1) showed a slight decrease (Figure 15). Since the Hill coefficient of the entrapped Hb was above 1, Hb binding to oxygen after encapsulation in ZIF-8 remained positively cooperative. Considering that cooperativity is a result of subunit-subunit interaction, it can be concluded that the tetrameric structure of Hb is preserved after the entrapment in Hb4oPEG2oo@ZIF-8 NPs.

[0196] Embodiments of the invention

[0197] A first embodiment provides a population of hemoglobin-based oxygen carrying nanoparticles, wherein each nanoparticle comprises:

[0198] (a) zeolite imidazole; (b) poly(ethylene glycol); and (c) hemoglobin; wherein the nanoparticles are obtained by combining 2-methylimidazole, polyethylene glycol, haemoglobin and Zn2+ ions.

[0199] A second embodiment provides the population of hemoglobin-based oxygen carrying nanoparticles of the first embodiment, wherein the nanoparticles are obtained by combining Zn2+ and 2-methylimidazole in a molar ratio of 1.0: 1.0 to 1.0: 10.0, preferably 1.0:4.0.

[0200] A third embodiment provides the population of hemoglobin-based oxygen carrying nanoparticles of the first or second embodiment, wherein the mean average hydrodynamic diameter of the nanoparticles is from 100 ;im to 500 |im.

[0201] A fourth embodiment provides the population of hemoglobin-based oxygen carrying nanoparticles of any one of the first to third embodiments, wherein the poly(ethylene glycol) is -OH terminated having at least MW 6000 Da; preferably a MW of 6000 to 10,000 Da.

[0202] A fifth embodiment provides the population of hemoglobin-based oxygen carrying nanoparticles of the fourth embodiment, wherein the poly(ethylene glycol) for obtaining the nanoparticles has a concentration of 5 - 200 mg mL-1, preferably 200 mg mL-1. A sixth embodiment provides the population of hemoglobin-based oxygen carrying nanoparticles of any one the first to fifth embodiments, wherein the haemoglobin for obtaining the nanoparticles has a concentration of 1 to 50 mg mL-1, preferably 40 mg mL-1.

[0203] A seventh embodiment provides the population of hemoglobin-based oxygen carrying nanoparticles of any one of the first to sixth embodiments, wherein the haemoglobin is derived from bovine red blood cells.

[0204] An eighth embodiment provides the population of hemoglobin-based oxygen carrying nanoparticles of any one of the first to seventh embodiments, wherein the nanoparticles have a negative ^-potential.

[0205] A ninth embodiment provides a method of preparing the population of hemoglobinbased oxygen carrying nanoparticles of any one of the first to eighth embodiments, comprising the steps:

[0206] (a) providing an aqueous solution of 2-methylimidazole, poly(ethylene glycol), and hemoglobin;

[0207] (b) adding an aqueous solution of Zn(NO3)2 ■ 6H2O to the aqueous solution of (a) under constant stirring and incubating for a period of time;

[0208] (c) subjecting the aqueous solution produced in step (b) to a g force to sediment the nanoparticles therein,

[0209] (d re-suspending the nanoparticles sedimented in step (c) in an aqueous liquid,

[0210] (e) re-suspending the nanoparticles sedimented by step (d) in an aqueous liquid and optionally repeating steps (c) and (d).

[0211] A tenth embodiment provides the method of preparing the population of hemoglobinbased oxygen carrying nanoparticles of the ninth embodiment, further comprising the step of introducing the nanoparticles of step (e) in a pharmaceutical composition.

[0212] An eleventh embodiment provides a pharmaceutical composition for use as a medicament, comprising the population of hemoglobin-based oxygen carrying nanoparticles of any one of the first to eighth embodiments.

[0213] A twelfth embodiment provides the pharmaceutical composition according embodiment eleven, for use in treatment of a human suffering from haemorrhagic shock or anaemia requiring transfusion. A thirteenth embodiment provides a pharmaceutical kit of parts comprising zinc nitrate hexahydrate, 2-methylimidazole, poly(ethylene glycol) and hemoglobin for preparation of the pharmaceutical composition for use as a medicament according to the eleventh or twelfth embodiment.

[0214] A fourteenth embodiment provides a use of PEG to increase the hemoglobin loading capacity of Metal-Organic Framework-based HBOC nanoparticles.

[0215] A fifteenth embodiment provides the use of PEG according to the fourteenth embodiment, wherein the Metal-Organic Framework of said nanoparticles is ZIF-8.

[0216] A sixteenth embodiment provides a method for treating a subject in need thereof by administering a pharmaceutical composition comprising the population of hemoglobinbased oxygen carrying nanoparticles according to any one of the first to eighth embodiments.

[0217] A seventeenth embodiment provides a method for treating a mammalian subject (e.g. human) suffering from haemorrhagic shock or anaemia requiring transfusion with the pharmaceutical composition comprising the population of hemoglobin-based oxygen carrying nanoparticles according to any one of the first to eighth embodiments.

[0218] References:

[0219] Basnayake, S. A.; Su, J.; Zou, X.; Balkus, K. J. Carbonate-Based Zeolitic Imidazolate Framework for Highly Selective CO 2 Capture. Inorg Chem 2015, 54 (4), 1816-1821. https: / / doi.org / 10.1021 / ic5027174.

[0220] Benesch, R. E.; Benesch, R.; Yung, S. Equations for the Spectrophotometric Analysis of Hemoglobin Mixtures. Anal Biochem 1973, 55 (1), 245-248. https: / / doi.org / 10.1016 / 0003-2697(73)90309-6.

[0221] Greenfield, N. J. Using Circular Dichroism Spectra to Estimate Protein Secondary Structure. Nat Protoc 2006, 1 {6), 2876-2890. https: / / doi.org / 10.1038 / nprot.2006.202.

[0222] Morris, W.; Stevens, C. J.; Taylor, R. E.; Dybowski, C.; Yaghi, O. M.; Garcia-Garibay, M. A. NMR and X-Ray Study Revealing the Rigidity of Zeolitic Imidazolate Frameworks. The Journal of Physical Chemistry C 2012, 116 (24), 13307-13312. https: / / doi.org / 10.1021 / jp303907p.

[0223] Villarreal, D. M., C. L. Phillips, A. M. Kelley, S. Villarreal, A. Villaloboz, P. Hernandez, J. S. Olson, D. P. Henderson. Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System. Applied and Environmental Microbiology 2008 Vol. 74, No. 18; https: / / doi.org / 10.1128 / AEM.01291-08

[0224] Zijlstra, W. G.; Buursma, A. Spectrophotometry of Hemoglobin: Absorption Spectra of Bovine Oxyhemoglobin, Deoxyhemoglobin, Carboxyhemoglobin, and Methemoglobin. Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology 1997, 118 (4), 743-749. https: / / doi.org / 10.1016 / S0305-0491(97)00230-7.

Claims

Claims:

1. A population of hemoglobin-based oxygen carrying nanoparticles, wherein each nanoparticle comprises:(a) zeolite imidazole;(b) hydroxy-terminated poly(ethylene glycol); and(c) hemoglobin; wherein the nanoparticles are obtained by self-assembly comprising combining 2-methylimidazole, hydroxy-terminated polyethylene glycol, haemoglobin and Zn2+ions in an aqueous solvent.

2. The population of hemoglobin-based oxygen carrying nanoparticles of claim 1, wherein the nanoparticles are obtained by combining Zn2+and 2- methylimidazole in a molar ratio of 1.0: 1.0 to 1.0: 10.0, preferably 1.0:4.0.

3. The population of hemoglobin-based oxygen carrying nanoparticles of claim 1 or 2, wherein the mean average hydrodynamic diameter of the nanoparticles is from 100 |im to 500 |im, as determined by NP tracking analysis.

4. The population of hemoglobin-based oxygen carrying nanoparticles of any one of claims 1-3, wherein the poly(ethylene glycol) is -OH terminated having at least Mw 6000 Da; preferably a Mw of 6000 to 10,000 Da.

5. The population of hemoglobin-based oxygen carrying nanoparticles of claim 4, wherein the poly(ethylene glycol) for obtaining the nanoparticles has a concentration of 5 - 200 mg mL-1, preferably 200 mg mL-1.

6. The population of hemoglobin-based oxygen carrying nanoparticles of any one of claims 1-5, wherein the haemoglobin for obtaining the nanoparticles has a concentration of 1 to 50 mg mL-1, preferably 40 mg mL-1.

7. The population of hemoglobin-based oxygen carrying nanoparticles of any one of claims 1-6, wherein the haemoglobin is derived from bovine red blood cells.

8. The population of hemoglobin-based oxygen carrying nanoparticles of any one of claims 1-7, wherein the nanoparticles have a negative ^-potential.

9. A method of preparing the population of hemoglobin-based oxygen carrying nanoparticles of any one of claims 1-8, comprising the steps:(a) providing an aqueous solution of 2-methylimidazole, hydroxy-terminated poly(ethylene glycol), and hemoglobin;(b) adding an aqueous solution of Zn(NOs)2 ■ 6H2O to the aqueous solution of (a) under constant stirring and incubating for a period of time;(c) subjecting the aqueous solution produced in step (b) to a g force to sediment the nanoparticles therein,(d) re-suspending the nanoparticles sedimented in step (c) in an aqueous liquid,(e) re-suspending the nanoparticles sedimented by step (d) in an aqueous liquid and optionally repeating steps (c) and (d).

10. The method of preparing the population of hemoglobin-based oxygen carrying nanoparticles of claim 9, further comprising the step of introducing the nanoparticles of step (e) in a pharmaceutical composition.

11. A pharmaceutical composition for use as a medicament, comprising the population of hemoglobin-based oxygen carrying nanoparticles of any one of claims 1 -8.

12. A pharmaceutical composition for use in treatment of a human suffering from haemorrhagic shock or anaemia requiring transfusion, comprising the population of hemoglobin-based oxygen carrying nanoparticles of any one of claims 1 -8.

13. A pharmaceutical kit of parts for use as a medicament comprising a composition comprising zinc nitrate hexahydrate, and a composition comprising 2-methylimidazole, hydroxy-terminated poly(ethylene glycol) and hemoglobin for preparation and administration of a self-assembled population of hemoglobin-based oxygen carrying nanoparticles .

14. Use of hydroxy-terminated poly(ethylene glycol) to increase the hemoglobin entrapment capacity of self-assembled Metal-Organic Framework-based HBOC nanoparticles obtained by self-assembly comprising combining 2- methylimidazole, hydroxy-terminated polyethylene glycol, haemoglobin and Zn2+ions in an aqueous solvent.

15. Use of hydroxy-terminated poly(ethylene glycol)according to claim 14, wherein the Metal-Organic Framework of said nanoparticles is ZIF-8.

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