Biocompatible and biodegradable polymersome encapsulated hemoglobin: a potential oxygen carrier

This work describes the development of polymersome-encapsulated hemoglobin (PEH) self-assembled from biodegradable and biocompatible amphiphilic diblock copolymers composed of poly(ethylene oxide) (PEO), poly(caprolactone) (PCL), and poly(lactide) (PLA). In the amphiphilic diblock, PEO functions as the hydrophilic block, while either PCL or PLA can function as the hydrophobic block. PEO, PCL, and PLA are biocompatible polymers, while the last two polymers are biodegradable. PEH dispersions were prepared by extrusion through 100 nm pore radii polycarbonate membranes. In this work, the encapsulation efficiency of human and bovine hemoglobin (hHb and bHb) in polymersomes was adjusted by varying the initial concentration of Hb. This approach yielded Hb loading capacities that were comparable to values in the literature that supported the successful resuscitation of hamsters experiencing hemorrhagic shock. Moreover, the Hb loading capacities of PEHs in this study can also be tailored simply by controlling the diblock copolymer concentration. In this study, typical Hb/diblock copolymer weight ratios ranged 1.2-1.5, with initial Hb concentrations less than 100 mg/mL. The size distribution, Hb encapsulation efficiency, oxygen affinity (P 50), cooperativity coefficient (n), and methemoglobin (metHb) level of these novel PEH dispersions were consistent with values required for efficient oxygen delivery in the systemic circulation. Taken together, our results demonstrate the development of novel PEH dispersions that are both biocompatible and biodegradable. These novel dispersions show very good promise as therapeutic oxygen carriers.     

Design of polypeptide-functionalized polystyrene microspheres

In this contribution, the principle of spontaneous surface segregation has been applied for the preparation of polypeptide-functionalized polystyrene microspheres. For that purpose, an amphiphilic diblock copolymer was introduced in the mixture styrene/divinylbenzene and polymerized using AIBN as initiator. During the polymerization, cross-linked particles were obtained in which the diblock copolymer was encapsulated. The amphiphilic diblock copolymers used throughout this study contain a hydrophilic polypeptide segment, either poly(L-lysine) or poly(L-glutamic acid) and a hydrophobic polystyrene block. After 4 h of polymerization, rather monodisperse particles with sizes of approximately 3-4 microm were obtained. Upon annealing in hot water, the hydrophilic polypeptides migrate to the interface, hence, either positively charged or neutral particles were obtained when poly(L-lysine) is revealed at the surface and exposed to acidic or basic pH, respectively. On the opposite, negatively charged particles were achieved in basic pH water by using poly(L-glutamic acid) as additive. The surface chemical composition was modified by changing the environment of the particles. Thus, exposure in toluene provoked a surface rearrangement, and due to its affinity, the polystyrene block reorients toward the interface.     

Reversible protection of Cys-93(β) by PEG alters the structural and functional properties of the PEGylated hemoglobin

As a potential hemoglobin (Hb)-based oxygen carrier (HBOC), the PEGylated Hb has received much attention for its non-nephrotoxicity. However, PEGylation can adversely alter the structural and functional properties of Hb. The site of PEGylation is an important factor to determine the structure and function of the PEGylated Hb. Thus, protection of some sensitive residues of Hb from PEGylation is of great significance to develop the PEGylated Hb as HBOC. Here, Cys-93(β) of Hb was conjugated with 20 kDa polyethylene glycol (PEG20K) through hydrazone and disulfide bonds. Then, the conjugate was modified with PEG5K succinimidyl carbonate (PEG5K-SC) using acylation chemistry, followed by removal of PEG20K Hb with hydrazone hydrolysis and disulfide reduction. Reversible conjugation of PEG20K at Cys-93(β) can protect Lys-95(β), Val-1(α) and Lys-16(α) of Hb from PEGylation with PEG5K-SC. The autoxidation rate, oxygen affinity, structural perturbation and tetramer instability of the PEGylated Hb were significantly decreased upon protection with PEG20K. The present study is expected to improve the efficacy of the PEGylated Hb as an oxygen therapeutic.     

PEG-SS-PPS: reduction-sensitive disulfide block copolymer vesicles for intracellular drug delivery

Under appropriate conditions, block copolymeric macroamphiphiles will self-assemble in water to form vesicles, referred to as polymersomes. We report here polymersomes that can protect biomolecules in the extracellular environment, are taken up by endocytosis, and then suddenly burst within the early endosome, releasing their contents prior to exposure to the harsh conditions encountered after lysosomal fusion. Specifically, block copolymers of the hydrophile poly(ethylene glycol) (PEG) and the hydrophobe poly(propylene sulfide) (PPS) were synthesized with an intervening disulfide, PEG17-SS-PPS30. Polymersomes formed from this block copolymer were demonstrated to disrupt in the presence of intracellular concentrations of cysteine. In cellular experiments, uptake, disruption, and release were observed within 10 min of exposure to cells, well within the time frame of the early endosome of endolysosomal processing. This system may be useful in cytoplasmic delivery of biomolecular drugs such as peptides, proteins, oligonucleotides, and DNA.     

Non-conservative surface decoration of hemoglobin: influence of neutralization of positive charges at PEGylation sites on molecular and functional properties of PEGylated hemoglobin

High hydrodynamic volume, high viscosity and high colloidal osmotic pressure (COP) of PEGylated hemoglobin (Hb) have been suggested to neutralize the vasoactivity of acellular Hb. Consequences of non-conservative PEGylation (positive charge of the amino groups at the PEGylation sites is neutralized) using succinimidyl-ester of propionic acid PEG5K on the properties of PEGylated Hb have now been investigated. Non-conservative PEGylation of Hb leads to a much higher increase in the COP and viscosity of Hb than conservative extension arm facilitated (EAF) PEGylation of Hb. Introduction of alphaalpha-fumaryl crosslinking decreased the COP of non-conservative PEGylated Hb by stabilization of interdimeric interactions. Compared to the EAF-PEGylated alphaalpha-fumaryl Hb, non-conservative PEGylated product shows a comparable COP and higher viscosity. Conservative PEGylation of alphaalpha-fumaryl Hb by reductive alkylation chemistry does not increase the COP to this level, but enhanced the molecular volume and viscosity comparable to EAF-PEGylated product. Thus, the molecular properties of PEGylated Hb can be fine tuned using different PEGylation platforms and provide a unique opportunity for the design of second generation PEGylated Hbs.     

Photopolymerization of bovine hemoglobin entrapped nanoscale hydrogel particles within liposomal reactors for use as an artificial blood substitute

Lipogel particles encapsulating bovine hemoglobin (BHb) were synthesized via photopolymerization of poly(N-isopropylacrylamide) (pNIPA) and poly(acrylamide) (pAAm) monomers within liposomal reactors. Nanoscale hydrogel particles (NHPs) encapsulating bovine hemoglobin, which represent a hybrid between acellular and cellular hemoglobin based oxygen carriers, were formed upon solubilization of the lipid bilayer of lipogel particles encapsulating BHb. Lipogels and NHPs encapsulating BHb constitute a new class of blood substitute that prevents both dissociation of hemoglobin (Hb) and in vivo exposure of acellular Hb, while allowing oxygen transport through the polymer matrix. pNIPA and pAAm particles encapsulating BHb displayed oxygen affinities ranging from 9.9 +/- 1.9 to 14.4 +/- 0.1 mmHg for lipogels, methemoglobin levels ranging from 9.3 +/- 3.7% to 26.0 +/- 5.0% for lipogels and NHPs, and encapsulation efficiencies ranging from 34.2 +/- 3.4% to 97.4 +/- 15.8% for lipogels and NHPs. Interestingly, the methemoglobin level of pNIPA particles was reduced 61% by coencapsulating the reducing agent, N-acetylcysteine. Fractionation and light scattering results showed that lipogels and NHPs were spherical and exhibited narrow size distributions. The colloidal osmotic pressure of pNIPA and pAAm lipogels ranged from 3.71 +/- 0.02 to 206.87 +/- 0.42 mmHg, depending on UV-irradiation time, type of buffer, and polymer composition. These results demonstrate that hemoglobin can be encapsulated within hydrogel based particles for use as an artificial blood substitute.     

Determination of size distribution and encapsulation efficiency of liposome-encapsulated hemoglobin blood substitutes using asymmetric flow field-flow fractionation coupled with multi-angle static light scattering

In this study, we investigated the size distribution, encapsulation efficiency, and oxygen affinity of liposome-encapsulated tetrameric hemoglobin (LEHb) dispersions and correlated the data with the variation in extruder membrane pore size, ionic strength of the extrusion buffer, and hemoglobin (Hb) concentration. Asymmetric flow field-flow fractionation (AFFF) in series with multi-angle static light scattering (MASLS) was used to study the LEHb size distribution. We also introduced a novel method to measure the encapsulation efficiency using a differential interferometric refractive index (DIR) detector coupled to the AFFF-MASLS system. This technique was nondestructive toward the sample and easy to implement. LEHbs were prepared by extrusion using a lipid combination of dimyristoyl-phosphatidylcholine, cholesterol, and dimyristoyl-phosphatidylglycerol in a 10:9:1 molar ratio. Five initial Hb concentrations (50, 100, 150, 200, and 300 mg Hb per mL of buffer) extruded through five different membrane pore diameters (400, 200, 100, 80, and 50 nm) were studied. Phosphate buffered saline (PBS) and phosphate buffer (PB) both at pH 7.3 were used as extrusion buffers. Despite the variation, extrusion through 400-nm pore diameter membranes produced LEHbs smaller than the pore size, extrusion through 200-nm membranes produced LEHbs with diameters close to the pore diameter, and extrusion through 100-, 80-, and 50-nm membranes produced LEHbs larger than the pore sizes. We found that the choice of extrusion buffer had the greatest effect on the LEHb size distribution compared to either Hb concentration or extruder membrane pore size. Extrusion in PBS produced larger LEHbs and more monodisperse LEHb dispersions. However, LEHbs extruded in PB generally had higher Hb encapsulation efficiencies and lower methemoglobin (metHb) levels. The choice of extrusion buffer also affected how the encapsulation efficiency correlated with Hb concentration, extruder pore size, and the metHb level. The most optimum encapsulation efficiency and amount of Hb entrapped were achieved at the highest Hb concentration and the largest pore size for both extrusion buffers (62.38% and 187.14 mg Hb/mL of LEHb dispersion extruded in PBS, and 69.98% and 209.94 mg Hb/mL of LEHb dispersion extruded in PB). All LEHbs displayed good oxygen-carrying properties as indicated by their P(50) and cooperativity coefficients. LEHbs extruded in PB had an average P(50) of 23.04 mmHg and an average Hill number of 2.29, and those extruded in PBS had average values of 27.25 mmHg and 2.49. These oxygen-binding properties indicate that LEHbs possess strong potential as artificial blood substitutes. In addition, the metHb levels in PB-LEHb dispersions are significantly low even in the absence of antioxidants such as N-acetyl-L-cysteine.     

Site-specific PEGylation of hemoglobin at Cys-93(beta): correlation between the colligative properties of the PEGylated protein and the length of the conjugated PEG chain

Increasing the molecular size of acellular hemoglobin (Hb) has been proposed as an approach to reduce its undesirable vasoactive properties. The finding that bovine Hb surface decorated with about 10 copies of PEG5K per tetramer is vasoactive provides support for this concept. The PEGylated bovine Hb has a strikingly larger molecular radius than HbA (1). The colligative properties of the PEGylated bovine Hb are distinct from those of HbA and even polymerized Hb, suggesting a role for the colligative properties of PEGylated Hb in neutralizing the vasoactivity of acellular Hb. To correlate the colligative properties of surface-decorated Hb with the mass of the PEG attached and also its vasoactivity, we have developed a new maleimide-based protocol for the site-specific conjugation of PEG to Hb, taking advantage of the unusually high reactivity of Cys-93(beta) of oxy HbA and the high reactivity of the maleimide to protein thiols. PEG chains of 5, 10, and 20 kDa have been functionalized at one of their hydroxyl groups with a maleidophenyl moiety through a carbamate linkage and used to conjugate the PEG chains at the beta-93 Cys of HbA to generate PEGylated Hbs carrying two copies of PEG (of varying chain length) per tetramer. Homogeneous preparations of (SP-PEG5K)(2)-HbA, (SP-PEG10K)(2)-HbA, and (SP-PEG20K)(2)-HbA have been isolated by ion exchange chromatography. The oxygen affinity of Hb is increased slightly on PEGylation, but the length of the PEG-chain had very little additional influence on the O(2) affinity. Both the hydrodynamic volume and the molecular radius of the Hb increased on surface decoration with PEG and exhibited a linear correlation with the mass of the PEG chain attached. On the other hand, both the viscosity and the colloidal osmotic pressure (COP) of the PEGylated Hbs exhibited an exponential increase with the increase in PEG chain length. In contrast to the molecular volume, viscosity, and COP, the vasoactivity of the PEGylated Hbs was not a direct correlate of the PEG chain length. There appeared to be a threshold for the PEG chain length beyond which the protection against vasoactivity is decreased. These results suggest that the modulation of the vasoactivity of Hb by PEG could be a function of the surface shielding afforded by the PEG, the latter being a function of the disposition of the PEG chain on the protein surface, which in turn is a function of the length of the PEG chain. Thus, the biochemically homogeneous PEGylated Hbs described in the present study, surface-decorated with PEG chains of appropriate size, could serve as potential candidates for Hb-based oxygen carriers.     

Autoxidation of the site-specifically PEGylated hemoglobins: role of the PEG chains and the sites of PEGylation in the autoxidation

The PEGylated hemoglobin (Hb) has been evaluated as a potential blood substitute. In an attempt to understand the autoxidation of the PEGylated Hb, we have studied the autoxidation of the PEGylated Hb site-specifically modified at Cys-93(beta) or at Val-1(beta). PEGylation of Hb at Cys-93(beta) perturbed the heme environment and increased the autoxidation rate of Hb, which is at a higher level than that caused by PEGylation at Val-1(beta). The perturbation of the heme environment of Hb is attributed to the maleimide modification at Cys-93(beta) and not due to conjugation of the PEG chains. However, the PEG chains enhance the autoxidation and the H 2O 2 mediated oxidation of Hb. Accordingly, the PEG chains are assumed to increase the water molecules in the hydration layer of Hb and enhance the autoxidation by promoting the nucleophilic attack of heme. The autoxidation rate of the PEGylated Hb does not show an inverse correlation with the oxygen affinity. The H 2O 2 mediated structural loss and the heme loss of Hb are increased by maleimide modification at Cys-93(beta) and further decreased by conjugation of the PEG chains. The autoxidation of the PEGylated Hbs is attenuated significantly in the plasma, possibly due to the presence of the antioxidant species in the plasma. This result is consistent with the recent suggestion that there is no direct correlation between the in vitro and in vivo autoxidation of the PEGylated Hb. Therefore, the pattern of PEGylation can be manipulated for the design of the PEGylated Hb with minimal autoxidation.     

Synthesis of novel folic acid-functionalized biocompatible block copolymers by atom transfer radical polymerization for gene delivery and encapsulation of hydrophobic drugs

Two synthetic routes to folic acid (FA)-functionalized diblock copolymers based on 2-(methacryloyloxy)ethyl phosphorylcholine [MPC] and either 2-(dimethylamino)ethyl methacrylate [DMA] or 2-(diisopropylamino)ethyl methacrylate [DPA] were explored. The most successful route involved atom transfer radical polymerization (ATRP) of MPC followed by the tertiary amine methacrylate using a 9-fluorenylmethyl chloroformate (Fmoc)-protected ATRP initiator. Deprotection of the Fmoc groups produced terminal primary amine groups, which were conjugated with FA to produce two series of novel FA-functionalized biocompatible block copolymers. Nonfunctionalized MPC-DMA diblock copolymers have been previously shown to be effective synthetic vectors for DNA condensation; thus, these FA-functionalized MPC-DMA diblock copolymers appear to be well suited to gene therapy applications based on cell targeting strategies. In contrast, the FA-MPC-DPA copolymers are currently being evaluated as pH-responsive micellar vehicles for the delivery of highly hydrophobic anticancer drugs.     

Oxidized mono-, di-, tri-, and polysaccharides as potential hemoglobin cross-linking reagents for the synthesis of high oxygen affinity artificial blood substitutes

Various oxidized mono/di/tri/poly saccharides were studied as potential hemoglobin (Hb) cross-linkers in order to produce oxygen carriers with high oxygen affinities (low P(50)'s) and high molecular weights (therefore lower macromolecular diffusivities compared to tetrameric Hb). Such physical properties were desired to produce polymerized hemoglobins (PolyHbs) with oxygen release profiles similar to that of human blood, as was demonstrated in work by Winslow (1). In this present study, bovine hemoglobin was cross-linked with a variety of oxidized (ring-opened) saccharides, which resulted in cross-linked Hb species ranging in size from 64 to 6400 kDa (depending on the particular oxidized saccharide used in the reaction) and P(50)'s ranging from 6 to 15 mmHg. A parallel synthetic approach was used to synthesize these carbohydrate-hemoglobin conjugates, and asymmetric flow field-flow fractionation (AFFF) coupled with multi-angle static light scattering (MASLS) was used to measure the absolute molecular weight distribution of these PolyHb dispersions. Cross-linking reactions were conducted at two pHs (6 and 8), with larger cross-linked Hb species produced at pH 8 (where hydrolysis was most likely to occur between glycosidic bonds linking adjacent saccharide rings) rather than at pH 6. The largest molecular weight species formed from these reactions consisted of Hb cross-linked with ring-opened lactose, maltose, methylglucopyranoside, sucrose, trehalose, and 15 kDa and 71 kDa dextran at high pH (pH 8). The most promising Hb cross-linker was methylglucopyranoside, which resulted in very large cross-linked Hb species, with low P(50)'s and lower methemoglobin (metHb) levels compared to the other Hb cross-linking reagents.     

Methoxy poly(ethylene glycol)-block-poly(delta-valerolactone) copolymer micelles for formulation of hydrophobic drugs

Six amphiphilic diblock copolymers based on methoxy poly(ethylene glycol) (MePEG) and poly(delta-valerolactone) (PVL) with varying hydrophilic and hydrophobic block lengths were synthesized via a metal-free cationic polymerization method. MePEG-b-PVL copolymers were synthesized using MePEG with Mn = 2000 or Mn = 5000 as the macroinitiator. 1H NMR and GPC analyses confirmed the synthesis of diblock copolymers with relatively narrow molecular weight distributions (Mn/Mw = 1.05-1.14). DSC analysis revealed that the melting temperatures (Tm) of the copolymers (47-58 degrees C) approach the Tm of MePEG as the PVL content is decreased. MePEG-b-PVL copolymer aggregates loaded with the hydrophobic anti-cancer drug paclitaxel were found to have effective mean diameters ranging from 31 to 970 nm depending on the composition of the copolymers. A MePEG-b-PVL copolymer of a specific composition was found to form drug-loaded micelles of 31 nm in diameter with a narrow size distribution and improve the apparent aqueous solubility of paclitaxel by more than 9000-fold. The biological activity of paclitaxel formulated in the MePEG-b-PVL micelles was confirmed in human MCF-7 breast and A2780 ovarian cancer cells. Furthermore, the biocompatibility of the copolymers was established in CHO-K1 fibroblast cells using a cell viability assay. The in vitro hydrolytic and enzymatic degradation of the micelles was also evaluated over a period of one month. The present study indicates that the MePEG-b-PVL copolymers are suitable biomaterials for hydrophobic drug formulation and delivery.     

Purification of hemoglobin from red blood cells using tangential flow filtration and immobilized metal ion affinity chromatography

Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter.     

Polymersomes: a new multi-functional tool for cancer diagnosis and therapy

Nanoparticles are being developed as delivery vehicles for therapeutic pharmaceuticals and contrast imaging agents. Polymersomes (mesoscopic polymer vesicles) possess a number of attractive biomaterial properties that make them ideal for these applications. Synthetic control over block copolymer chemistry enables tunable design of polymersome material properties. The polymersome architecture, with its large hydrophilic reservoir and its thick hydrophobic lamellar membrane, provides significant storage capacity for both water soluble and insoluble substances (such as drugs and imaging probes). Further, the brush-like architecture of the polymersome outer shell can potentially increase biocompatibility and blood circulation times. A further recent advance is the development of multi-functional polymersomes that carry pharmaceuticals and imaging agents simultaneously. The ability to conjugate biologically active ligands to the brush surface provides a further means for targeted therapy and imaging. Hence, polymersomes hold enormous potential as nanostructured biomaterials for future in vivo drug delivery and diagnostic imaging applications.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells.

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 +/- 1.14 . 10(5) and (2.2 +/- 1.2) . 10(5) spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethylmaleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Extension Arm Facilitated PEGylation of Hemoglobin: Correlation of the Properties with the Extent of PEGylation

Human hemoglobin (Hb) conjugated with six copies of PEG-5K is nonhypertensive. The hexaPEGylated Hb exhibits molecular size homogeneity in spite of the chemical heterogeneity with respect to the sites of conjugation (Manjula et al., 2005). In the present study, Hb conjugated with an average of 4, 6, 8 and 10 copies of PEG-5K chains have been generated using the extension arm facilitated PEGylation protocol. Except for the tetraPEGylated Hb, all the other products exhibit molecular size homogeneity. The molecular, colligative and functional properties of PEG-Hb conjugates have been correlated with the extent of PEGylation. The results imply that six copies of PEG-5K chains are accommodated on Hb without significant crowding on the molecular surface. As more copies of PEG-5K chains are conjugated to form octa and deca PEGylated Hb, the PEG-chains conjugated appear to undergo transition from a mushroom (compact) to a brush-like conformation (extended conformation) with a concomitant decrease in the propensity of the molecule to transition from oxy to deoxy conformation in the presence of allosteric effectors. The viscosity and the colloidal osmotic pressure of Hb increase with the number of the PEG-chains conjugated in an exponential fashion. The composition of the PEGylated Hb generated appears to be controlled by (i) high reactivity of thiol groups of the extension arms on Hb with maleimide-PEG, (ii) increase in the viscosity of the reaction mixture as the level of PEGylation increases and (iii) increased resistance induced by the PEG-shell of PEGylated Hb to accommodate more PEG-chains as the level of PEGylation increases. Potential implications of extent of PEGylation on the oxygen delivery by PEG-Hb conjugate in vivo have been discussed.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Encapsulation of hemoglobin inside liposomes surface conjugated with poly(ethylene glycol) attenuates their reactions with gaseous ligands and regulates nitric oxide dependent vasodilation

Acellular hemoglobin (Hb)-based O2 carriers (HBOCs) are being investigated as red blood cell (RBC) substitutes for use in transfusion medicine. However, commercial acellular HBOCs elicit both vasoconstriction and systemic hypertension which hampers their clinical use. In this study, it is hypothesized that encapsulation of Hb inside the aqueous core of liposomes should regulate the rates of NO dioxygenation and O2 release, which should in turn regulate its vasoactivity. To test this hypothesis, poly(ethylene glycol) (PEG) conjugated liposome-encapsulated Hb (PEG-LEHs) dispersions were prepared using human and bovine Hb. In this study, the rate constants for O2 dissociation, CO association, and NO dioxygenation were measured for free Hb and PEG-LEH dispersions using stopped-flow UV-visible spectroscopy, while vasoactivity was assessed in rat aortic ring strips using both endogenous and exogenous sources of NO. It was observed that PEG-LEH dispersions had lower O2 release and NO dioxygenation rate constants compared with acellular Hbs. However, no difference was observed in the CO association rate constants between free Hb and PEG-LEH dispersions. Furthermore, it was observed that Hb encapsulation inside vesicles prevented Hb dependent inhibition of NO-mediated vasodilation. In addition, the magnitude of the vasoconstrictive effects of Hb and PEG-LEH dispersions correlated with their respective rates of NO dioxygenation and O2 release. Overall, this study emphasizes the pivotal role Hb encapsulation plays in regulating gaseous ligand binding/release kinetics and the vasoactivity of Hb.     

Formation of core-shell type biodegradable polymeric micelles from amphiphilic poly(aspartic acid)-block-polylactide diblock copolymer

Poly(aspartic acid)-block-polylactide diblock copolymers (PAsp-b-PLAs) having both hydrophilic and hydrophobic segments of various lengths were synthesized. These PAsp-b-PLA diblock copolymers formed polymeric micelles consisting of a hydrophobic PLA core and a hydrophilic, pH-sensitive PAsp shell in aqueous solution. The effects of the segment length of both the PLA and the PAsp portions and the pH of the solution on the shapes and sizes of the PAsp-b-PLA polymeric micelles were investigated. The results indicated a balance between the effects of electrostatic repulsion, hydrogen bonding in the PAsp shell layer, and hydrophobic interactions in the PLA core determine the sizes of the PAsp-b-PLA polymeric micelles. Moreover, the PAsp-b-PLA polymeric micelles did not possess any cytotoxic activity against L929 fibroblast cells. The obtained polymeric micelle should be useful for biodegradable biomedical materials such as drug delivery vehicle.     

Monomolecular assembly of siRNA and poly(ethylene glycol)-peptide copolymers

In this work, we design and investigate the complex formation of highly uniform monomolecular siRNA complexes utilizing block copolymers consisting of a cationic peptide moiety covalently bound to a poly(ethylene glycol) (PEG) moiety. The aim of the study was to design a shielded siRNA construct containing a single siRNA molecule to achieve a sterically stabilized complex with enhanced diffusive properties in macromolecular networks. Using a 14 lysine-PEG (K14-PEG) linear diblock copolymer, formation of monomolecular siRNA complexes with a stoichiometric 1:3 grafting density of siRNA to PEG is realized. Alternatively, similar PEGylated monomolecular siRNA particles are achieved through complexation with a graft copolymer consisting of six cationic peptide side chains bound to a PEG backbone. The hydrodynamic radii of the resulting complexes as measured by fluorescence correlation spectroscopy (FCS) were found to be in good agreement with theoretical predictions using polymer brush scaling theory of a PEG decorated rodlike molecule. It is furthermore demonstrated that the PEG coating of the siRNA-PEG complexes can be rendered biodegradable through the use of a pH-sensitive hydrazone or a reducible disulfide bond linker between the K14 and the PEG blocks. To model transport under in vivo conditions, diffusion of these PEGylated siRNA complexes is studied in various charged and uncharged matrix materials. In PEG solutions, the diffusion coefficient of the siRNA complex is observed to decrease with increasing polymer concentration, in agreement with theory of probe diffusion in semidilute solutions. In charged networks, the behavior is considerably more complex. FCS measurements in fibrin gels indicate complete dissociation of the diblock copolymer from the complex, while transport in collagen solutions results in particle aggregation.