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.     

Polymersome encapsulated hemoglobin: a novel type of oxygen carrier

Bovine hemoglobin (Hb) was encapsulated inside polymer vesicles (polymersomes) to form polymersome encapsulated Hb (PEH) dispersions. PEH particles are 100% surface PEGylated with longer PEG chains and possess thicker hydrophobic membranes as compared to conventional liposomes. Polymersomes were self-assembled from poly(butadiene)-poly(ethylene glycol) (PBD-PEO) amphiphilic diblock copolymers with PBD-PEO molecular weights of 22-12.6, 5-2.3, 2.5-1.3, and 1.8-0.9 kDa. The first two diblock copolymers possessed linear hydrophobic PBD blocks, while the later possessed branched PBD blocks. PEH dispersions were extruded through 100 and 200 nm pore radii membranes. The size distribution, Hb encapsulation efficiency, P(50), cooperativity coefficient, and methemoglobin (metHb) level of PEH dispersions were consistent with values required for efficient oxygen delivery in the systemic circulation. The influence of different molecular weight diblock copolymers on the physical properties of PEH dispersions was analyzed. PBD-PEO copolymers with molecular weights of 22-12.6 and 2.5-1.3 kDa completely dissolved in aqueous solution to form polymersomes, while the other two copolymers formed a mixture of solid copolymer precipitates and polymersomes. PEHs self-assembled from 22-12.6 and 2.5-1.3 kDa PBD-PEO copolymers possessed Hb loading capacities greater than PEG-LEHs, PEGylated actin-containing LEHs, and nonmodified LEHs, although their sizes were smaller and their hydrophobic membranes were thicker. The Hb loading capacities of these polymersomes were also higher than lipogel encapsulated hemoglobin particles and nanoscale hydrogel encapsulated hemoglobin particles. PEH dispersions exhibited average radii larger than 50 nm and exhibited oxygen affinities comparable to human erythrocytes. Polymersomes did not induce Hb oxidation. The interaction between Hb and the membrane of 2.5-1.3 kDa PBD-PEO polymersomes improved the monodispersity of these particular PEH dispersions. These results suggest that PEHs could serve as efficient oxygen therapeutics.     

Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes

Liposomes of defined size and homogeneity have been prepared by sequential extrusion of the usual multilamellar vesicles through polycarbonate membranes. The process is easy, reproducible, produces no detectable degradation of the phospholipids, and can double the encapsulation efficiency of the liposome preparation. Multilamellar vesicles extruded by this technique are shown by both negative stain and freeze-fracture electron microscopy to have mean diameters approaching the pore diameter of the polycarbonate membrane through which they were extruded. When sequentially extruded down through a 0.2 μm membrane, the resulting vesicles exhibit a very homogeneous size distribution with a mean diameter of 0.27 μm while maintaining an acceptable level of encapsulation of the aqueous phase.     

Hemoglobin vesicles containing methemoglobin and L-tyrosine to suppress methemoglobin formation in vitro and in vivo

Hemoglobin (Hb) vesicles have been developed as cellular-type Hb-based O(2) carriers in which a purified and concentrated Hb solution is encapsulated with a phospholipid bilayer membrane. Ferrous Hb molecules within an Hb vesicle were converted to ferric metHb by reacting with reactive oxygen species such as hydrogen peroxide (H(2)O(2)) generated in the living body or during the autoxidation of oxyHb in the Hb vesicle, and this leads to the loss of O(2) binding ability. The prevention of metHb formation by H(2)O(2) in the Hb vesicle is required to prolong the in vivo O(2) carrying ability. We found that a mixed solution of metHb and L-tyrosine (L-Tyr) showed an effective H(2)O(2) elimination ability by utilizing the reverse peroxidase activity of metHb with L-Tyr as an electron donor. The time taken for the conversion of half of oxyHb to metHb (T(50)) was 420 min for the Hb vesicles containing 4 g/dL (620 microM) metHb and 8.5 mM L-Tyr ((metHb/L-Tyr) Hb vesicles), whereas the time of conversion for the conventional Hb vesicles was 25 min by stepwise injection of H(2)O(2) (310 microM) in 10 min intervals. Furthermore, in the (metHb/L-Tyr) Hb vesicles, the metHb percentage did not reach 50% even after 48 h under a pO(2) of 40 Torr at 37 degrees C, whereas T(50) of the conventional Hb vesicles was 13 h under the same conditions. Moreover, the T(50) values of the conventional Hb vesicles and the (metHb/L-Tyr) Hb vesicles were 14 and 44 h, respectively, after injection into rats (20 mL/kg), confirming the remarkable inhibitory effect of metHb formation in vivo in the (metHb/L-Tyr) Hb vesicles.     

Extrusion of electroformed giant unilamellar vesicles through track-etched membranes

Unilamellar vesicle populations having a narrow size distribution and mean radius below 100 nm are preferred for drug delivery applications. In the present work, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was used to prepare giant unilamellar vesicles (GUVs) by electroformation and multilamellar vesicles (MLVs) by thin film hydration. Our experiments show that in contrast to MLVs, a single-pass extrusion of GUVs through track-etched polycarbonate membranes at moderate pressure differences is sufficient to produce small liposomes having low polydispersity index. Moreover, we observe that the drug encapsulating potential of extruded liposomes obtained from GUVs is significantly higher compared to liposomes prepared by extrusion of MLVs. Furthermore, our experiments carried out for varying membrane pore diameters and extrusion pressures suggest that the size of extruded liposomes is a function of the velocity of GUV suspensions in the membrane pore.     

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.     

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.     

Biomimetic Dissolution: A Tool to Predict Amorphous Solid Dispersion Performance

The presented study describes the development of a membrane permeation non-sink dissolution method that can provide analysis of complete drug speciation and emulate the in vivo performance of poorly water-soluble Biopharmaceutical Classification System class II compounds. The designed membrane permeation methodology permits evaluation of free/dissolved/unbound drug from amorphous solid dispersion formulations with the use of a two-cell apparatus, biorelevant dissolution media, and a biomimetic polymer membrane. It offers insight into oral drug dissolution, permeation, and absorption. Amorphous solid dispersions of felodipine were prepared by hot melt extrusion and spray drying techniques and evaluated for in vitro performance. Prior to ranking performance of extruded and spray-dried felodipine solid dispersions, optimization of the dissolution methodology was performed for parameters such as agitation rate, membrane type, and membrane pore size. The particle size and zeta potential were analyzed during dissolution experiments to understand drug/polymer speciation and supersaturation sustainment of felodipine solid dispersions. Bland-Altman analysis was performed to measure the agreement or equivalence between dissolution profiles acquired using polymer membranes and porcine intestines and to establish the biomimetic nature of the treated polymer membranes. The utility of the membrane permeation dissolution methodology is seen during the evaluation of felodipine solid dispersions produced by spray drying and hot melt extrusion. The membrane permeation dissolution methodology can suggest formulation performance and be employed as a screening tool for selection of candidates to move forward to pharmacokinetic studies. Furthermore, the presented model is a cost-effective technique.     

The pressure-dependence of the size of extruded vesicles

Variations in the size of vesicles formed by extrusion through small pores are discussed in terms of a simple model. Our model predicts that the radius should decrease as the square root of the applied pressure, consistent with data for vesicles extruded under various conditions. The model also predicts dependencies on the pore size used and on the lysis tension of the vesicles being extruded that are consistent with our data. The pore size was varied by using track-etched polycarbonate membranes with average pore diameters ranging from 50 to 200 nm. To vary the lysis tension, vesicles made from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine), mixtures of POPC and cholesterol, and mixtures of POPC and C(16)-ceramide were studied. The lysis tension, as measured by an extrusion-based technique, of POPC:cholesterol vesicles is higher than that of pure POPC vesicles whereas POPC:ceramide vesicles have lower lysis tensions than POPC vesicles.     

Heme structure of hemoglobin M Iwate [alpha 87(F8)His-->Tyr]: a UV and visible resonance Raman study

Heme structures of a natural mutant hemoglobin (Hb), Hb M Iwate [alpha87(F8)His-->Tyr], and protonation of its F8-Tyr were examined with the 244-nm excited UV resonance Raman (UVRR) and the 406.7- and 441.6-nm excited visible resonance Raman (RR) spectroscopy. It was clarified from the UVRR bands at 1605 and 1166 cm(-)(1) characteristic of tyrosinate that the tyrosine (F8) of the abnormal subunit in Hb M Iwate adopts a deprotonated form. UV Raman bands of other Tyr residues indicated that the protein takes the T-quaternary structure even in the met form. Although both hemes of alpha and beta subunits in metHb A take a six-coordinate (6c) high-spin structure, the 406.7-nm excited RR spectrum of metHb M Iwate indicated that the abnormal alpha subunit adopts a 5c high-spin structure. The present results and our previous observation of the nu(Fe)(-)(O(tyrosine)) Raman band [Nagai et al. (1989) Biochemistry 28, 2418-2422] have proved that F8-tyrosinate is covalently bound to Fe(III) heme in the alpha subunit of Hb M Iwate. As a result, peripheral groups of porphyrin ring, especially the vinyl and the propionate side chains, were so strongly influenced that the RR spectrum in the low-frequency region excited at 406.7 nm is distinctly changed from the normal pattern. When Hb M Iwate was fully reduced, the characteristic UVRR bands of tyrosinate disappeared and the Raman bands of tyrosine at 1620 (Y8a), 1207 (Y7a), and 1177 cm(-)(1) (Y9a) increased in intensity. Coordination of distal His(E7) to the Fe(II) heme in the reduced alpha subunit of Hb M Iwate was proved by the observation of the nu(Fe)(-)(His) RR band in the 441.6-nm excited RR spectrum at the same frequency as that of its isolated alpha chain. The effects of the distal-His coordination on the heme appeared as a distortion of the peripheral groups of heme. A possible mechanism for the formation of a Fe(III)-tyrosinate bond in Hb M Iwate is discussed.     

SENSORY PROPERTIES OF EXTRUDED CORN MEAL RELATED TO THE SPATIAL DISTRIBUTION OF PROCESS CONDITIONS

The quality determining factors of extruded products are affected by the temperature, shear and pressure generated by any input to the extruder during the short residence time (< 120s). Although the relationship of process history to measurable product qualities has been established, sensory qualities have not been well correlated to these process responses. Sensory attributes of extruded corn meal products were investigated and correlated to measured physical properties in this study. Corn meal was extruded in a twin screw extruder (Baker Perkins MPF 50/25; LD ratio 15:1) with step increases in screw speed from 200-400 rpm, and moisture from 16-22%. Principal component analysis (PCA) of main factors from sensory color, crispness, and adhesiveness was correlated to process torque, pressure and temperature. Spatial distribution of process response and product attributes showed crispness to be dependent on extrusion temperature. Porosity and adhesiveness were not correlated to any measured process response. PCA analysis identified significant differences in the effects of moisture and screw speed input to the extruder on product properties.     

Potential of peroxynitrite to promote the conversion of oxyhemoglobin to methemoglobin

Superoxide anion and NO can react to form the highly oxidizing species peroxynitrite (ONOO-)which can react directly with hemoglobin (Hb) even in the presence of physiological concentration CO:. Thisresearch was to determine the ONOO--mediated oxidation damage to the heme of oxyhemoglobin (oxyHb)under conditions expected in blood. Results showed that 8-10 mol ONOO- was needed to quickly andcompletely convert 1 mol oxyHb to methemoglobin (metHb). ONOO- (20-140 μM) caused raoid andextensive formation of metHb from oxyHb (50 μM) mainly occurring within first 5-20 min of incubation.The conversion efficiency reached 16%, 48%, 60%, 79% and 88% output of metHb after 90 min ofincubation at 0, 20, 40, 100, and 140 μM ONOO- respectively. 1 mM CO2 caused a small decrease in theability of ONOO- to oxidize oxyHb, and ONOO--promoted conversion of oxyHb to metHb increased whenpH decreased from 8.0 to 6.0. Relatively lower temperature in blood condition will inhibit this reaction insome degree. We postulate that ONOO- can mediate oxidation damage to the heme, and cause heme lossfrom the hydrophobic cavity of Hb when its concentration exceeded 90 μM. These results indicated thatONOO- could convert oxyHb to metHb under the conditions expected in blood, and this reaction wasregulated by CO2 concentration, reaction time, temperature and pH value.     

Prolonged oxygen-carrying ability of hemoglobin vesicles by coencapsulation of catalase in vivo

Hemoglobin (Hb) vesicles (particle diameter, ca. 250 nm) have been developed as Hb-based oxygen carriers in which a purified Hb solution is encapsulated with a phospholipid bilayer membrane. The oxidation of Hb to nonfunctional ferric Hb (metHb) was caused by reactive oxygen species, especially hydrogen peroxide (H(2)O(2)), in vivo in addition to autoxidation. We focused on the enzymatic elimination of H(2)O(2) to suppress the metHb formation in the Hb vesicles. In this study, we coencapsulated catalase with Hb within vesicles and studied the rate of metHb formation in vivo. The Hb vesicles containing 5.6 x 10(4) unit mL(-1) catalase decreased the rate of metHb formation by half in comparison with Hb vesicles without catalase. We succeeded in prolonging the oxygen-carrying ability of the Hb vesicle in vivo by the coencapsulation of catalase.     

Extrusion of pectin/starch blends plasticized with glycerol

The microstructural and thermal dynamic mechanical properties of extruded pectin/starch/glycerol (PSG) edible and biodegradable films were measured by scanning electron microscopy (SEM) and thermal dynamic mechanical analysis (TDMA). SEM revealed that the temperature profile (TP) in the extruder and the amount of water present during extrusion could be used to control the degree to which the starch was gelatinized. TDMA revealed that moisture and TP during extrusion and by inference the amount of starch gelatinization had little effect on the mechanical properties of PSG films. Furthermore, TDMA revealed that PSG films underwent a glass transition commencing at about −50°C and two other thermal transitions above room temperature. Finally, it was concluded that the properties of extruded PSG films were comparable to those cast from solution.     

Differences in coordination states of substituted tyrosine residues and quaternary structures among hemoglobin M probed by resonance Raman spectroscopy

Among the four types of hemoglobin (Hb) M with a substitution of a tyrosine (Tyr) for either the proximal (F8) or distal (E7) histidine in the α or β subunits, only Hb M Saskatoon (βE7Tyr) assumes a hexacoordinate structure and its abnormal subunits can be reduced readily by methemoglobin (metHb) reductase. This is distinct from the other three M Hbs. To gain new insight into the cause of the difference, we examined the ionization states of E7 and F8 Tyrs by UV resonance Raman (RR) spectroscopy and Fe–O(Tyr) bonding by visible RR spectroscopy. Hb M Iwate (αF8Tyr), Hb M Boston (αE7Tyr), and Hb M Hyde Park (βF8Tyr) exhibited two extra UV RR bands at 1,603 cm⁻¹ (Y8a′) and 1,167 cm⁻¹ (Y9a′) arising from deprotonated (ionized) Tyr, but Hb M Saskatoon displayed the UV RR bands of protonated (unionized) Tyr at 1,620 and 1,175 cm⁻¹ in addition to those of deprotonated Tyr. Evidence for the bonding of both ionization states of Tyr to the heme in Hb M Saskatoon was provided by visible RR spectroscopy. These results indicate that βE7Tyr of Hb M Saskatoon is in equilibrium between protonated and deprotonated forms, which is responsible for facile reducibility. Comparison of the UV RR spectral features of metHb M with that of metHb A has revealed that metHb M Saskatoon and metHb M Hyde Park are in the R (relaxed) structure, similar to that of metHb A, whereas metHb M Iwate, metHb M Boston and metHb M Milwaukee are in the T (tense) quaternary structure.     

Entrapment of ovalbumin into liposomes--factors affecting entrapment efficiency, liposome size, and zeta potential

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB + 0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential--the factors that are of great importance for the use of liposomes as drug carriers.     

Mechanical properties of vesicles. I. Coordinated analysis of osmotic swelling and lysis.

To determine how transmembrane osmotic gradients perturb the structure and dynamics of biological membranes, we examined the effects of medium dilution on the structures of osmolyte-loaded lipid vesicles. Our preparations were characterized by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) spectroscopies. Populations of Escherichia coli phosphatidylethanolamine (PE) or dioleoylphosphatidylglycerol (DOPG) vesicles prepared by the pH jump technique were variable and polymodal in size distribution. Complex and variable structural changes occurred when PE vesicles were diluted with hypotonic buffer. Such vesicles could not be used as model systems for the analysis of membrane mechanical properties. NaCl-loaded, DOPG vesicles prepared by extrusion through 100 nm (diameter) pores were reproducible and monomodal in size distribution and unilamellar, whereas those prepared by extrusion through 200-, 400-, or 600-nm pores were variable and polymodal in size distribution and/or multilamellar. Time and pressure regimes associated with osmotic lysis of extruded vesicles were defined by monitoring release of carboxyfluorescein, a self-quenching fluorescent dye. Corresponding effects of medium dilution on vesicle structure were assessed by DLS spectroscopy. These experiments and the accompanying analysis (Hallett, F.R., J. Marsh, B.G. Nickel, and J.M. Wood. 1993. Biophys. J. 64:000-000) revealed conditions under which vesicles are expected to reside in a consistently strained state.     

Different types of glutathionylation of hemoglobin can exist in intact erythrocytes

Glutathionylation of hemoglobin (Hb) was studied by incubation of intact human erythrocytes with 1 mM tert-butylhydroperoxide (tBHP). Electrophoresis of the membranes showed a time dependent increase of membrane-bound Hb alpha chain until 10 min, and immunoblotting study showed that membrane-bound Hb alpha chain reacted with anti-glutathione antibody only after 10 min. Concomitant with the Hb alpha chain, membrane associated actin, spectrin, and glyceraldehyde 3-phosphate dehydrogenase reacted with the antibody. Cytosolic Hb of the control erythrocytes reacted with anti-glutathione antibody. Together with our previous paper, the present study indicates that at least three different types of glutathionylation of Hb can exist in erythrocytes. The first type is a mixed disulfide bond between reduced glutathione (GSH) and normal Hb. The second type is a disulfide bond between the cysteine 93 of metHb beta chain and oxidized glutathione (GSSG), and the third type is a disulfide bond between the other cysteine residues of metHb alpha chain and/or metHb beta chain and GSSG.     

Determination of liposomal encapsulation efficiency using proton NMR spectroscopy

A rapid and simple approach using 1H NMR was developed for determination of liposomal encapsulation efficiency without the need for physical separation of entrapped and non-entrapped marker. Measurements were made using a marker (homocarnosine) with a pH-sensitive 1H chemical shift in the presence of a pH gradient across the phospholipid vesicle membrane, or by addition of the chemical shift reagent, thulium(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-(methylene phosphonic acid sodium salt) (TmDOTP5-). The measured encapsulation efficiencies for the liposomal dispersions prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) through extrusion using 50, 200 and 1000 nm polycarbonate membranes were found to be identical using the two different experimental approaches.     

Size and CT density of iodine-containing ethosomal vesicles obtained by membrane extrusion: Potential for use as CT contrast agents

Computed tomography (CT) is the primary non-invasive imaging technique used for most patients with suspected liver disease. In order to improve liver-specific imaging properties and prevent toxic effects in patients with compromised renal function, we investigated the encapsulation of iodine within ethosomal vesicles. As a first step in the development of novel contrast agents using ethosomes for CT imaging applications, iodine was entrapped within ethosomes and iodine-containing ethosomes of the desired size were obtained by extrusion using a polycarbonate membrane with a defined pore size. Ethosomes containing iodine showed a relatively high CT density, which decreased when they were extruded, due to the rupture and re-formation of the lipid bilayer of the ethosome. However, when a solution with a high iodine concentration was used as a dispersion media during the extrusion process, the decrease in CT density could be prevented. In addition, ethosomes containing iodine were taken up efficiently by macrophages, which are abundant in the liver, and these ethosomes exhibited no cellular toxicity. These results demonstrate that iodine could be entrapped within ethosomal vesicles, giving the ethosomes a relatively high CT density, and that the extrusion technique used in this study could conveniently and reproducibly produce ethosomal vesicles with a desired size. Therefore, ethosomes containing iodine, as prepared in this study, have potential as contrast agents with applications in CT imaging.