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Chapter 1 Artificial Oxygen Carriers

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Artificial Oxygen Carriers

ABSTRACT

The development of artificial systems that can substitute blood transfusions in restoring oxygen homeostatic concentration has attracted the scientific community attention in the past decades. Many drawbacks are infact associated with commonly used blood transfusion, including the fewness of donors, the presence of blood antigens and the risk of infection. The two principal types of oxygen carrying products under commercial development are basically Perfluorocarbons and Hemoglobin (Hb) based oxygen carriers (HBOC). Since Hb is a fragile and reactive natural multifunctional hemoprotein whose behaviour is substantially modified when it is no longer enclosed within the red blood cells, many drawbacks mostly associated with Hb oxidation frequently occur in the development of Hb based oxygen carriers. For this reason the use of cellular HBOC in the development of artificial oxygen carriers appears to be advantageous thanks to the possibility of co-encapsulating reducing agents and enzymes aimed at minimising oxidative phenomena.

1.1 Introduction

Several diseases are related to heme and hemoglobin disorders. Gene mutations result in a group of hereditary diseases termed hemoglobinopathies, among which the most common are sickle-cell disease and thalassemia. Decreased levels of hemoglobin and heme synthesis lead to symptoms of anaemia, whereas alterations of heme metabolic pathways generate porphyrias syndromes.

Medical interest toward Hemoglobin (Hb) is related to the possibility of its administration as blood substitute to re-establish oxygen homeostasis in tissues. At present, blood transfusions are mostly applied for this purpose, but the need of the right type of blood and its short shelf life are still serious problems to be overcome. In this case, the use of autologous transfusions prevents the need of cross-matching and, although autologous transfusions are considered the safest, they are not always

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feasible because they may cause perioperative anaemia and are more expensive than allogenic transfusions [Vanderlinde, 2002; Rao, 2002; Pape, 2007].

The development of oxygen carriers is particularly indicated in the case of urgent need of oxygen delivery to tissues and to solve the above mentioned problems related to blood transfusions. The ideal oxygen carrier would deliver oxygen, not transmit disease, not have immunosuppresive effects, would have less strict storage requirements than for human blood, would not need cross-matching, would be available at reasonable costs, be easy to administer, and able to reach all areas of human body, including ischemic tissues [Chang, 1997; Sakai, 2000; Ness, 2007]]. The potential clinical application of oxygen carriers covers several diseases such as sickle-cell anaemia, autoimmune hemolytic anaemia, infarction, surgery and traumas. Their administration would also be accepted in Jehovah's Witness community without violation of the doctrine on blood [Cothren, 2004]. Furthermore, it is known from more than 50 years, that solid tumors are characterized by hypoxia. Hypoxic cell results more resistant to standard chemotherapy and radiotherapy, are more invasive and metastatic, resistant to apoptosis and genetically instable. Nowadays, some oxygen carrier products are experimentally and clinically investigated as cancer chemo- and radio-sensitizing agents [Yu, 2007].

1.2 Hemoglobin

Hb is the physiological oxygen-transport metalloprotein present in red blood cells, in mammals and other animals. Hb primary function is to bind oxygen that diffuses into the bloodstream from the lungs, transport it to outlying tissues and release it mainly for aerobic respiration. Hb molecule has a tetrameric structure (64 kDa, 3.2 nm average radius), made of two  and  globin chains. Each unity has a molecular weight of 16 kDa and contains a prosthetic moiety. The prosthetic group is constituted by an iron atom conjugated to a porphyrinic ring (Heme), buried in a hydrophobic pocket, and capable of carrying one oxygen molecule [Alayash, 2004]. During the, fetal, neonatal and adult stages of life, two main forms of Hb which differ in globin sequence have been identified: in adults 96–98% is Hb A, while during embryonic life and early neonatal period Hb F, which has a higher oxygen affinity, is mainly present.In the firsts stages of the embryo development, Hb is synthesized in the yolk sac, while from about the 10th–12th week of gestation, there is Hb synthesis in the liver and the spleen with the production of fetal and, later, adult Hb. At a later stage of

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development the bone marrow takes over as the main site of Hb synthesis and, in adult life, bone marrow erythroblasts synthesize both Hb A and the minor Hb [Fantoni, 1981]. Following the two-state model of Monod et al. [Monod, 1965] Hb can be mainly in two conformations, different by a tridimensional point of view. The two conformations, called T and R, are strictly related to oxygen binding to Heme group. While the deoxy protein prefers a ‘tense’ state (T), the binding of the ligand determines the shift of the allosteric equilibrium towards the relaxed state (R) [Lukin, 2004].

The binding of oxygen by the protein is cooperative: as Hb binds successive oxygen molecules, the oxygen affinity of each subunit increases. This is caused by changes in the tetrameric structure of the protein after the binding of the ligand. This behaviour is also reflected into a sigmoidal, rather than hyperbolic oxygen dissociation curve. Moreover, the transition between R-T state is facilitated by the reversible binding of 2,3-diphosphoglycerate (DPG), which acts as an allosteric effector. When DPG is bounded to Hb, its oxygen affinity is reduced and, as consequence, oxygen can be easily offloaded. At tissues level, oxygen can be used as substrate for aerobic metabolism, while carbonic anhydride, a waste of tissues metabolism, can bind to the protein, generating carbamino-Hb, which is then transported to the lungs. At lungs level Hb shift back to the R-state because of the local conditions which promote the dissociation of the DPG and, as a consequence, the release of carbonic anhydride and the binding of the oxygen to the protein [Kim, 2004].

The link between Hb and oxygen is essential for the protein homeostatic function. Anyway, Hb can be easily oxidized by oxygen itself to Methemoglobin (Fe2+ to Fe3+) generating the superoxide anion [Shikama, 2003] and other reactive oxygen species (ROS).

As a consequence Heme iron can reacts with so produced H2O2 both in its ferrous

(Fe2+) and ferric (Fe3+) forms; however, the oxidative processes proceed quite differently. For example, oxidation of Fe2+ by H2O2 results in the formation of ferryl

Heme (Fe4+) without generation of a protein or porphyrin-associated free radical. The remaining solution of H2O2 is oxidized by Fe4+ giving O2 and Fe3+. At this point

the porphyrin ring may be degraded in a peroxidative cycle as O2 is generated and

trapped in the Heme pocket [Nagababu 2000, Nagababu 2004]. Alternatively, Fe3+ in the presence of H2O2 oxidizes to oxoferryl (Fe4+O) and a protein- or prosthetic

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group-associated free radical is generated from the two-electron reduction of H2O2 toH2O

[Jia 2007].

The spontaneous oxidation of Fe+2 is called autoxidation and within the red blood cells is generally maintained at 3% of total Hb by MetHb reductase system, including superoxide dismutase and catalase.

1.2.1 Hb Physicochemical Characterization

The analysis of Hb optical and physicochemical properties appears very useful both in clinical and research activity. It is well known that many Hb-associated diseases affect the protein structural properties, as well as the maintenance of the protein physicochemical features is the key point in developing Hb based therapeutic systems.

1.2.1.1 Hb UV Spectra Properties

The forms in which Hb actually exists in blood are called Hb derivatives. The most common Hb derivatives are Oxy-Hb (Hbox) and Deoxy-Hb (Hbdox), which represent oxygen bound HEME and ligand-free HEME Hb respectively. Others Hb derivatives are represented by Carboxy-Hb (HbCO), SulfHb (HbS), MetHb and Ferryl-Hb, which represent Carbon monoxide or sulphur bound Hb, HEME-Fe3+-Hb and HEME-Fe4+ -Hb respectively. -HbS is the only -Hb derivative in which no interaction between the ligand and HEME iron is observed. Morel and Chang [Morel, 1967] showed that Hb-sulphur interaction occurs to one or both of a pair of E pyrrole carbon atoms at the periphery of the porphyrin ring, either as a sulfhydryl group or as a three-membered ring episulfide structure. HEME iron oxidation state changes and the binding of ligands to the protein determines differences in the Hb UV/VIS spectrum of absorbance; this property is commonly used in research and clinical field to characterize Hb samples.

In Table 1 UV-Vis absorbance characteristic peaks regarding Hb derivatives are reported.

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Hb Derivative Soret Region Peaks (nm)

Visible Region Peaks (nm) Hbox 415 541. 577 Hbdox 430 555 HbCO 419 540, 569 MetHb 405 500, 631 Ferryl-Hb 418 545. 580 HbS-ox 420 622 HbS-dox 423 619

Table 1 Hb Derivatives UV-Vis Absorbance Spectra Peaks

1.2.1.2 Hb Circular Dichroism Absorption Spectrum

Alteration regarding Hb secondary structure can be investigated by analysing its Circular Dichroism absorption spectrum.

As reported in Equation 1, Circular Dichroism (CD) is the difference in the absorption (A) of left-handed circularly polarised light (L-CPL) and right-handed circularly polarised light (R-CPL) and occurs when a molecule contains one or more chiral chromophores (light-absorbing groups) .

A() = A()LCPL - A()RCPL

Equation 1 Circular Dichroism

CD spectrum may exhibit both positive and negative peaks and is usually reported in

units of ellipticity which is represented by the T symbol and is measured in

millidegrees (mdeg). 

The over mentioned technique is commonly used to study chiral molecules of all types and sizes, but it is in the study of large biological molecules where it finds its most important applications.

Since differences in the absorption of left-handed polarized light versus right-handed polarized light arise from structural asymmetry, the absence of regular structure results in zero CD intensity, while a molecular ordered structure results in a characteristic CD spectrum.

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Proteins secondary structure can be investigated by CD spectroscopy in the "far-UV" spectral region (190-250 nm). Each of the three basic secondary structures of a polypeptide chain (helix, sheet, and random coil) shows a characteristic CD spectrum. In the far-UV region the chromophore is the peptide bond, and CD signal results strong for - helix, weak for -sheets and around zero for random coil [Cantor 1980]. In Figure 1 the CD spectra of “all D”, “all E” and “all random coil” forms of poly-L-lysine are reported [Townend, 1966].

Figure 1D-helix(D E-sheet(E  and random coil r), CD Spectrum of Poly-L-lysine

Each protein has a unique CD spectrum which corresponds to its native folding. Therefore CD technique is a powerful tool to detect eventually occurring alterations in the protein secondary structure, generally associated to abnormal functional properties. Moreover, CD spectroscopy is widely applied to determine proteins secondary structures. The development of mathematical models, based on both algorithms and neural networks, allows for the prediction of proteins folding, in terms of D-helix, E-sheet, E-turn and random coil percentage, without requiring inputs of structural information from X-ray diffraction analysis [Sreerama, 1993; Stokkum, 1990].

Indeed, CD analysis is a common mean to investigate the persistence of Hb functionality in Hb based oxygen carriers. The secondary structure of functional Hb and the corresponding CD spectrum are reported in Figure 2 [Jia, 2007; Kabsch, 1983].

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Figure 2 Human Hb Circular Dichroism Spectrum and Secondary Structure Composition

1.2.1.3 Hill Coefficient Determination

In order to determine the maintenance of Hb cooperative properties in oxygen binding, the application of Hill equation (Equation 2) is commonly used.

log

log

50

1

log

n

pO

2



n

p



T

T

Equation 2 Hill Equation

In this equation n represents the Hill coefficient, while p50 is the oxygen tension needed to saturate 50% of binding sites and T is the fraction of ligand binding sites filled. The Hill coefficient reflects the cooperative activity between Hb subunits. Hill coefficient of 2.7 reflects a cooperative tetrameric Hb; otherwise, for a non-cooperative Hb Hill coefficient value is 1 [Alayash, 2004].

To calculate Hb cooperativity, the Hill plot is made by Graphing log [ /(1- )] versus log PO2. The Hill plot for non-cooperative binding, will be a straight line with slope =

1. In such a plot, the abscissa value (the value of log PO2) corresponding to log [ /(1 -

)] = 0 will equal log P50. When Hb first begins binding (at low PO2), its Hill plot

has a slope >1, corresponding to the weak-binding state (large P50). As binding progresses, the curve switches over to approach another, parallel line that describes the strong-binding state (small P50). The Hill coefficient is represented by calculating the slope of the line that connects the above mentioned parallel lines (Figure 3).

Secondary Structure Percentage D-helix E-sheet E-turn Random Coil 76 0 11 13

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Figure 3 Hill Plot [Drees, 2000]

In addition to the estimation of Hill coefficient regarding Oxygen-Hemoglobin interaction, the protein ability of cooperatively binding carbon monoxide is commonly investigated both in clinical and research field [Widdopp, 2002]. As reported by Anderson in 1968, a Hill coefficient value of 2.34 reflects a cooperative binding between Hb and Carbon monoxide in dilute Hb solutions (Hb concentration 4.5 x10-3 M and 2.34 x 10-7).

1.3 Artificial Oxygen Carriers

In the early seventies, the unfeasibility to administer free Hb as oxygen therapeutic due to its short half life, abnormal high oxygen affinity and the incoming of serious side effects, namely malaise, abdominal pain, hemoglobinuria, and renal toxicity was observed [Rabiner, 1970].

The toxic effects of Hb infusion were correlated to the breakdown of the tetramer into dimers in the bloodstream, each dimer containing an  and a  subunit [Chang, 1997]. As alternatives to the administration of free Hb, the use of synthetic oxygen carriers based on fluorocarbons (FBOCs) or Hb based oxygen carriers (HBOCs) has captured the attention of scientists [Remy, 1999; Chang, 2004].

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1.3.1 FluoroCarbons

FBOCs are oil-like chemicals presenting high gas solubility. As reported by Clark and Gollan [Clark, 1966] a mouse can sustain life if submerged in a FBOC solution, equilibrated with oxygen. To be used as blood substitutes, FBOCs are generally emulsified in aqueous media using different surfactant agents in order to improve their dispersion in plasma-like solutions. Anyway, the presence of severe side effects derived by the use of FBOCs (reduction in platelet activity, and immunogenic response [Riess, 2001]) determined an increasing attention of the scientific community in developing both acellular and cellular HBOCs systems.

1.3.2 Acellular Hemoglobin-Based Oxygen Carriers (HBOCs)

Acelullar HBOCs consist of solutions of purified Hb, usually modified in order to avoid the breakdown of the tetramer into the bloodstream. The acellular HBOCs include intramolecularly crosslinked Hb [Chattarje, 1986; Przybelski, 1997], polymerized Hb [Kasper, 1996; Gould, 1998], polymer-conjugated Hb [Iwashita, 1991; Caron, 1998], and recombinant cross-linked Hb [Murray, 1995]. Cross-linking of Hb was investigated also for the preparation of stable microcapsule made with Hb itself. Terephthaloylchloride was applied as crosslinking agent, allowing for the incorporation of inositol hexaphosphate and glucose, and followed by stabilization through glutaraldehyde. In this work, the 5-µm diameter microcapsules were able to ensure oxygen transfer, but suffered rapid lysis by proteases [Lèvy, 1982].

Generally, chemical modifications on Hb molecule are associated to a low oxygen affinity and changes in the cooperativity and in the binding to carbon dioxide and chloride, which are normal allosteric modifiers of Hb oxygen affinity [Colombo, 1982; Nagababu, 2002]. Moreover, the major side effects reported in the case of the administration of acellular HBOCs are their action as plasma expander and induced hypertension [Moncada, 1991; Doherty, 1998]. It has been assumed that modified Hb could easily enter into the interstitial space and there acts as a sink in binding and removing the nitric oxide needed for maintaining the normal tone of smooth muscles [Chang, 2004].

Non-hypertensive effect on Hb solution can be generated by enhancing the molecular size of the protein [Hu, 2007]. Hb pegylation is considered a promising way to achieve this objective [Rohlfs, 1998; Winslow, 1998]. The conjugation of Hb and

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polyethylene glycol (PEG) chains is known to enhance the viscosity of Hb solutions, resulting in a vasodilation effect, also caused by an enhanced nitric oxide (NO) release from endothelium [Tsai, 1998; Li, 2006]. This effect can be reduced by using a pegylated Hb S-nitrosylated on Cys-93,which appears promising in the recovering of the NO-like bioactivity of the artificial oxygen carrier [Asanuma, 2007]. Since it was demonstrated that the hypertensive effect is also related to abnormal oxygen offloading at tissue level [Winslow, 2000; McCarthy, 2001], the elevated oxygen affinity detected on pegylated Hb is assumed to prevent vasoconstriction [Acharya, 2005].

Several chemical strategies have been developed to prepare PEGprotein conjugates and involve the formation of covalent bonds on protein amino, carboxy or thiol groups [Veronese, 2001]. Regarding Hb-PEG conjugation, there is evidence that the functional properties of the modified protein are a direct consequence of surface decoration [Manjula, 2005], independent of both the site and pegylation chemistry [Hu, 2005]. Presently, the 2-imminothiolane (IMT) reaction is themost commonly used [Iafelice, 2007]. In this two-step reaction the protein is first activated by IMT at amino group level, then maleimido-PEG (MAL-PEG) reacts with the activated protein at IMT thiols group, generating MAL-PEG-Hb. Moreover, the length of PEG chains seems not to affect the functional properties of pegylated Hb. Although PEG chains of 5,10 and 20 kDa have been tested, 5 kDa PEG molecules are the most widely used [Manjula, 2003]. Presently, pegylated-Hb represents the intermediate between acellular and cellular HbOCs, having a protein core, hydration layer and a surrounding PEG shell. Among MAL-PEG-Hb, Hemospan has recently been applied for clinical trials. Hemospan is Human Hb conjugated with 7-8 MAL-PEG units, 5kDa average molecular weight each [Vandergriff, 2009]. Phase-I and Phase-II trials have shown few side effects, such as hypertension phenomena and gastrointestinal discomforts, after Hemospan administration [Björkholm, 2005]; anyway an increased risk for myocardal infarction has been recently highlighted as a consequence of the administration of different type of HBOCs including Hemospan [Nayanson, 2008].

1.3.3 Cellular Hemoglobin-Based Oxygen Carriers (HBOCs)

Cellular HBOCs consist of Hb molecules encapsulated inside oxygen carriers of different nature, aimed at mimicking red blood cells features. Cellular HBOCs advantages consist of protecting the surrounding tissues and blood components from

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direct contact with potentially toxic tetrameric Hb, avoiding Hb colloidal osmotic effect, prolonging Hb circulation half life and not requiring the direct modification of Hb molecule [Fournier, 1999]. Additionally, with the application of nanotechnology it is possible to achieve sub-micron sized oxygen carrier and thus ensure oxygen availability to all body compartments [Wright, 1996; Brig, 53].

Basically, two types of cellular HBOCs have been studied: liposome systems [Philips, 1999; Arifin, 2003]and polymeric micro/nanoparticles systems [Meng, 2004; Arifin, 2005].

Phospholipid vesicles or liposomes encapsulating purified and concentrated human Hb (HbV) have been investigated as a transfusion alternative. It has been shown that the cellular structure of HbV prevents direct contact of Hb with the blood components and the endothelial lining, shielding cells from the side effects of Hb molecules. Moreover, liposomes with 200–250 nm diameter were found to have longer vascular retention in respect to the 1- to 5-µm diameter liposomes [Chang, 1999; Tsuchida, 2006].Microcirculatory observations showed that the cellular structure of HbV is important to control reactions with endothelium-derived vasorelaxation factors. Animal studies of extreme hemodilution and resuscitation from hemorrhagic shock attest the sufficient oxygen transporting capacity of HbV [Ogata, 2000; Contaldo, 2005]. Studies of biodistribution and metabolism revealed that HbVs are eventually captured in the reticuloendothelial system [Awasthi, 2004a; Awasthi, 2004b], and degraded within 1 week [Tsuchida, 2006; Sakai, 2004].

However, it is known that liposomal lipids may undergo peroxidation. This process modifies the physical properties of the membranes, including its permeability to different solutes and the packing of lipids and proteins into the membranes, which in turn, influences their function [Schnitzer, 2007]. A close interdependence between Hb oxidation and lipid peroxidation, which can be limited by using saturated lipids (e.g. saturated phosphatidylcoline) and freshly prepared hemolysate was shown [Szebeni, 1984].Moreover, the stability of the Hb encapsulated into liposomes is affected by an interaction between protein and negatively charged phospholipids, which in turn brings to Hb denaturation. The presence of cholesterol in the lipid bilayer reduces the interaction with the phospholipids and enhances protein stability inside the HbV [Szebeni, 1985].

Surface modification of the HBOCs, such as the application of pegylated matrices, is generally performed to further improve the colloidal stability and the intravascular

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persistence and of the oxygen carrier, once administered [Sakai, 2000b]. Although Hb loaded liposome with a PEG grafted surface suspended in human serum albumin reached preclinical trials [Buehler, 2004], PEG conjugation to liposome suffers of physical limits for the occurrence of phase separation [Antonietti, 2003] and may not effectively prevent complement activation [Moghimi, 2003]. There is evidence that the concomitant presence of PEG and 1,5-O-dihexadecyl-N-succinyl-Lglutammate (DHSG), on the surface and into HbV membrane respectively, leads to a higher biocompatibility of the HBOC with plasma. This HbV has mo major influence on the extrinsic or intrinsic coagulation activity, on the kallikrein-kinin cascade and on complement consumption [Abe, 2007; Sakai, 2008].

Recently HbV have been applied in in-vivo tests using rats as animal models; hemorrhagic shock induced rats were treated with HbV, highlighting HbV ability to act as artificial oxygen carriers until autologous blood volume and oxygen-carrying capacity are restored [Taguchi, 2009]. Moreover Yamamoto [Yamamoto, 2009] demonstrated that systemic administration of HbV elevates tumor tissue oxygen tension, enhancing the cytotoxic activity of radiotherapy in solid tumor treatment. The use of biopolymers for the development of HBOCs has been extensively investigated due to the broad variety of commercially available polymers, the possibility of developing new synthetic or hybrid polymers with tailored characteristic and the availability of nanotechnologies for the production of polymeric nanoparticles for biomedical applications.

Compared to liposomes, polymeric nanoparticles are less affected by protein leakage and the introduction of antiopsinizing moieties (e.g. polyethylene glycol, heparin) on particle surface guarantees longer blood half lives [Chauvierre, 2004; Zhao, 2007]. Polyethylene glycol (PEG), poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and their copolymers (PLGA) are the most applied polymers in the development of polymeric cellular HBOCs [Przybelski, 1997; Yu, 1996; Chang, 2004]. The employment of other types of polymers, such as heparin poly(isobutylcyanoacrylate) copolymers [Chauvierre, 2004], monomethoxy poly(ethylene glycol)-block-poly-D,L-lactide (PEG-PLA) [Meng, 2004], poly(N-isopropylacrylamide) [Patton, 2005], poly(butadiene)-poly(ethylene glycol) (PBD-PEO) amphiphilic diblock copolymers [Arifin, 2005], poly(-caprolactone) (PCL) and poly(-caprolactone-ethylene glycol) [Zhao, 2007], has been reported in literature.

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In the development of polymeric HBOCs, several particle structures have been investigated, such as microcapsules [El-Gybali, 2004], nanocapsules [Yu, 1996], polymersomes [Arifin, 2003], hydrogel nanoparticles [Patton, 2006], and porous particles [Zhao, 2007], as well as the coating of nanoparticles surface with Hb instead of incapsulating it [Chauvierre, 2004].

The loading of Hb into polymeric nano-micro systems has been performed by using different techniques. The most applied techniques based on preformed polymers are water/oil/water (w/o/w) double emulsion technique [Meng, 2004], solvent diffusion/evaporation [Zhao, 2007], and self assembly of amphiphilic diblock copolymers [Arifin, 2005]. Regarding the in situ polymerization techniques, the most recently developed is the interfacial polymerization (IP) or polycondensation technique. In IP, the condensation of the polymer at the oil/water interface is initiated in the presence of an oil-soluble monomer and leads to the formation of an ultrathin synthetic polymer film around microdroplets [El-Gybali, 2004]. The simultaneous cross-linking and loading of Hb into polymeric nanoparticles has been also investigated by means of UV-induced photopolymerization of acrylamide, N,N-methylenebis-(acrylamide) and poly(N-isopropylacrylamide)-Hb [Patton, 2006]. Hb content in nanoparticles and Encapsulation Efficiency (EE) strictly depends upon the polymer matrix and the selected preparation method. A content of 7.8% of human Hb with respect to copolymer dry mass was recorded on stealth nanoparticles based on heparinpoly (isobutylcyanoacrylate) copolymers. This result was explained with the occurrence of Hb adsorbance onto nanoparticles surface instead of being incorporated inside the polymeric matrix [Chauvierre, 2004].

Nano-scaled self-assembled polymersomes based on poly(butadiene)-poly(ethylene glycol) (PBD-PEG) with molecular weights of 22,000–12,600, 5000–2300, 2500– 1300, and 1800–900 g mol1 showed an EE ranging between 2.7 and 12.2%. The highest EE values were recorded with 1300 g mol1 and 2300 g mol1 PEG [Arifin, 2005]. An extremely high EE 93.1%, has been observed when loading bovine Hb into monomethoxyPEG-block-poly(D,L-lactide) microspheres obtained by w/o/w double emulsion technique. The authors highlighted a correlation between the molecular weight of monomethoxyPEG (MPEG) blocks and the EE. In particular, it was noticed that copolymers containing MPEG2000 led to more effective entrapment of bovine Hb than those with MPEG5000 [Meng, 2004]. Stimuli responsive Hb loaded nanosystems have recently been investigated [Patton, 2005; Patton, 2006; Yin, 2009].

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The attention has been focused on poly (N-isopropyl acrylamide) and poly(acrylamide) hydrogel based HbOCs as temperature-sensitive and pH-responsive oxygen carriers. These hydrogel nanoparticles displayed different swelling and shrinking rate in response to physiological changes in temperature, reflected also on zeta potential values, oxygen affinity, and cooperativity while maintaining colloidal stability. Temperature-sensitive oxygen carriers should ideally target hypoxic tissues as a result of physiological drops in tissues temperature. They observed that nanoscale hydrogel particles swelled as the temperature decreased from 40 to 29 °C, which suggests expansion of the hydrogel matrix and reduced resistance to oxygen transport. In the case of pH-responsive poly(acrylamide) hydrogel particles, the oxygen carrier would target tissues with low oxygen tensions elicited by decreased physiological pH. Recently new polymeric nanoparticles based on polysaccharide-poly(alkyl cyanoacrylate) have been investigated in the preparation of cellular HBOCs [Baudin-Creuza, 2008], highlighting promising results in terms of encapsulated Hb functionality, biocompatibility and stealth properties.

Moreover surface charged polymeric Hb loaded nanoparticles were prepared using PEG-PLA polymeric matrix modified with cationized cetyltrimethylammonium bromide and anionized sodium dodecyl sulphate. In this work, results showed the achievement of polymeric nanoparticles possessing hypothetic long circulation time and suitable physicochemical features to be used as artificial oxygen carriers [Xu, 2009].

1.3.3.1 Procedures to Avoid Methemoglobin Formation

Methemoglobin (MetHb) accumulation continues to occur inside cellular HbOCs due to autooxidation of the ferrous hemes of Hb [Sugawara, 2003], oxidation of oxyhemes by vascular nitric oxide [Hrinczenkoa, 2000] and peroxidative interaction with the phospholipids of the liposome membrane [Szebeni, 1986]. The reduction of metHb in red blood cells is performed by NADH-cytochrome b5 [Abe, 1979]reducing systems, and directly by glutathione and ascorbic acid. The oxidized agents are then reduced again by corresponding reductase. Moreover, superoxide dismutase catalase system reduces the formation of active oxygen species. Since these chemicals and reducing systems are completely removed in Hb purification process [Sakai, 1993], studies for the reduction of MetHb formation have been performed. Mainly three strategies have

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been approached, in order to limit the formation of MetHb during cellular HBOCs preparation, after administration in vivo or during storage.

The first strategy concerns the coencapsulation of reducing enzymes belonging to the system naturally present in red blood cells. The earliest encapsulation of the contents of red blood cells, including Hb and enzymes, inside microscaled artificial membranes was reported by Chang in 1957 [Chang, 1988]. Biodegradable polylactide and copolymers with PEG (PLA-PEG) were applied for the coencapsulation of superoxide dismutase catalase, MetHb reductase and Hb into nanosystems. In vitro studies evidenced the diffusion of small hydrophilic molecules, glucose and reducing agents into the nanocapsules. In addition, long-term follow up in vivo has been completed and the results showed that PEG-PLA nanocapsules containing Hb did not elicit any changes in biochemistry, enzymes, or histology [Yu, 1996; Chang, 2000; Chang, 2002]. Prolonged oxygen-carrying ability and reduced MetHb formation in vivo were observed also by coencapsulating lipopolysaccharide-free catalase and Hb into 250 nm lipid vesicles [Teramura, 2003].

The second strategy implies the preparation of nanoparticles in the presence of reducing agents such as ascorbic acid, glutathione and methylene blue. Ascorbic acid, flavin and 5-hydroxyanthranilic acid show very rapid reduction in vitro, but their use is limited by a prompt undesired decomposition in air. Moreover, under aerobic conditions, thiol autoxidation produces active oxygen species that adversely promote metHb formation. On the other hand, under anaerobic conditions, thiols act as reductants for metHb and as oxygens scavengers [Sakai, 2000a; Sakai, 1994]. The metHb reduction by thiols proceeds as a pseudo-first-order reaction in a nitrogen atmosphere. Thiol effectiveness order resulted in cysteine>homocysteine>glutathione>acetylcysteine. The thiol group dissociates to a thiolate anion, and one electron transfers from it to metHb, resulting in the production of deoxyHb and disulfide. On the contrary, their order based on the apparent rate constants of oxidation by oxygen is cysteine>homocysteine>acetylcysteine >glutathione. Since suitable reductant should possess a small rate of autoxidation but a high efficiency of MetHb reduction, their order based on the applicability in HBOC formulations resulted as homocysteine>glutathione>cysteine>acetylcysteine. The addition of homocysteine to HBOCs has hence been adopted and resulted in an efficient reduction of MetHb formation after in vivo administration [Sakai, 2000; Takeoka, 1997]. It is also reported that, in case of lipogel particles encapsulating

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bovine Hemoglobin (BHb) synthesized via photopolymerization liposomal reactors, MetHb formation was reduced by 61% by co-encapsulating N-acetylcysteine [Patton, 2005]. Recently the system formed by ascorbic acid-glutathione has been addressed to be very effective in preventing Hb oxidation, highlighting a superior antioxidant activity if compared with ascorbic acid or glutathione alone [Simoni, 2009].

Considering the well-known reaction of Horseradish Peroxidase (HRP) in theH2O2

assay method and the presence of a ferric Heme in both HRP and MetHb, the co-encapsulation of Hb and MetHb with L-tyrosine as H2O2 elimination system was

investigated. A remarkable inhibitory effect on MetHb formation after in vivo administration of the lipid vesicles was observed [Atoji, 2006].

A third approach is aimed at limiting MetHb formation during the preparation of HBOC. It involves the inactivation and lock of Hb into a stable conformation by saturation with carbon monoxide (HbCO). This procedure is generally applied during the purification of Hb from blood, where the protein is subsequently reconverted into its oxygenated form by irradiation with visible light of liquid film of Hb under O2

[Sakai, 1994; Atoji, 2006]. This methodology has been extended to the preparation of HBOCs by loading HbCO into the vesicle and reactivating the porphyrine groups by treatment with -radiations [Sakai, 2000; Akama, 96].

Recently, the preparation of porous Hb loaded nanoparticles was investigated as HBOCs. The particles were prepared by modified double emulsion and solvent diffusion/evaporation methods with poly -caprolactone) (PCL) and poly(-caprolactone-ethylene glycol) (PCL-PEG). In agreement with previous studies [Meng, 2004], it was observed that the procedure utilizing dichloromethane (DCM)/ethyl acetate (EA) as a solvent with an unsaturated external aqueous phase yielded the highest EE (87.4%) with a small mean particle size (153 nm). The formation of porous channels was attributed to the diffusion of the solvent and the pore-connecting efficiency reached 87.5%when PCL-PEG was employed.

The authors assume that the connected porous channels provide an inand-out exchange not only for oxygen but also for recycling systems, glucose and reductants, solving the problem of MetHb formation. Anyhow, the diffusion mechanisms of various molecules are yet unknown [Zhao, 2007].

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1.3.3.2 Cellular HBOCs Physicochemical Properties

To be used as artificial oxygen carriers, cellular HBOCs must posses characteristic features in terms of size and surface properties, in order to reach every district of the body and to present a functional half-life once in the bloodstream.

Some of the most widely used micro/nanoparticles characterization technique are reported below.

Size Determination (Granulometry in Suspension)

One of the quickest and highly reproducible way of determining particles dimension is light scattering based granulometry in suspension. In general the scattering of light may be thought as the redirection of light that takes place when an electromagnetic (EM) wave (i.e. an incident light ray) encounters an obstacle; the majority of light scattered is then emitted at the identical frequency of the incident light (Figure 4).

Figure 4 Light Scattering [Chu, 1970]

Since light scattering has a direct dependence on particles molecular weight and size (Lord Rayleigh, Fraunhofer and Gustav Mie theories), it is possible to estimate particles size by means of light scattering analysis of the analyte solution [Hahn 2009]. Diameter distribution of nanoparticles can be thus expressed in terms of volume percent (V%) or number percent (N%). V% distribution is evaluated on the total volume occupied by the entire nanoparticles population, while N% indicates the percentage of particles having the same size with respect to the total population of nanoparticles in the sample.

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Morphological Analysis

To obtain information about particles morphology, the use of scanning electron microscopy (SEM) appears very useful. In SEM technique a focused beam of high-energy electrons interacts with the surface of the analysed sample, which is generally coated with a thin layer of conducting material, such as carbon, gold, or some other metal or alloy. The signals that derive from electron-sample interactions reveal information about the sample, including external morphology (texture) and orientation of its structure [Goldstein, 2003; Reimer, 1998; Egerton, 2005].

Zeta Potential Measurements

Particles surface properties are strictly associated both to their physicochemical stability and to their half-life once in-vivo. Particles surface charge and hydrophobicity are known to determine the amount and type of adsorbed blood components once the system is injected the main stream, influencing the in-vivo fate of nanoparticles. The binding of opsonins, called opsonization, acts as a bridge between nanoparticles and the recognition and sequestration by the Reticulo-endothelial system (RES), constituted by a cells such as macrophages and macrophage precursors, specialized endothelial cells lining the sinusoids of the liver, spleen, and bone marrow, and reticular cells of lymphatic tissue and bone marrow. A general trend delineated in literature is that there is an increase in protein adsorption with increasing surface charge and hydrophobicity [Piras, 2009].

Zeta potential is a physical property that almost all particles in solution posses. In aqueous environment the liquid phase surrounding particles can be divided mainly in two layers; Stern Layer is the inner layer where ions are strictly associated with particle surface, while the Diffuse Layer is the outer layer where ions interaction with particles becomes weaker . The potential measured at the Stern-Diffuse layer is considered as Zeta potential [Delgado, 2005] (Figure 5).

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Figure 5 Zeta Potential [Hunter, 1981]

Zeta potential is measured by applying an electric field across the dispersion. Particles within the dispersion with a zeta potential will migrate toward the electrode of opposite charge with a velocity proportional to the magnitude of the zeta potential. This velocity is measured using the technique of laser Doppler anemometry. The frequency shift or phase shift of an incident laser beam caused by these moving particles is measured as the particle mobility, and this mobility is converted to the zeta potential by inputting the dispersant viscosity, and the application of the

Smoluchowski or Huckel theories [Hunter, 1981].

1.4 Conclusion

Although blood transfusions are safer than ever, the risk of infection is still present due to rare pathogens or new emerging infectious diseases which are not revealed by means of the commonly used clinical screenings; moreover, bloodlettings cannot satisfy the increasing blood demand. Many systems devoted to overcome these drawbacks are currently under investigations, anyway physiological side effects and abnormalities in Hb functionality are commonly observed in most of the systems produced. For these reasons the development of more effective and safer oxygen therapeutics is still a current issue among the scientific community.

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Figura

Table 1 Hb Derivatives UV-Vis Absorbance Spectra Peaks
Figure 1 D -helix( D E -sheet( E  and random coil r), CD Spectrum of  Poly-L-lysine
Figure 2  Human Hb Circular Dichroism Spectrum and Secondary Structure  Composition
Figure 3 Hill Plot [Drees, 2000]
+3

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