Optimal timing of prophylactic pegylated G-CSF after chemotherapy administration for patients with cancer: a systematic review and meta-analysis from Clinical Practice Guidelines for the use of G-CSF 2022.

INTRODUCTION: The timing of prophylactic pegylated granulocyte colony-stimulating factor (G-CSF) administration during cancer chemotherapy varies, with Day 2 and Days 3-5 being the most common schedules. Optimal timing remains uncertain, affecting efficacy and adverse events. This systematic review sought to evaluate the available evidence on the timing of prophylactic pegylated G-CSF administration. METHODS: Based on the Minds Handbook for Clinical Practice Guideline Development, we searched the PubMed, Ichushi-Web, and Cochrane Library databases for literature published from January 1990 to December 2019. The inclusion criteria included studies among the adult population using pegfilgrastim. The search strategy focused on timing-related keywords. Two reviewers independently extracted and assessed the data. RESULTS: Among 300 initial search results, only four articles met the inclusion criteria. A meta-analysis for febrile neutropenia incidence suggested a potential higher incidence when pegylated G-CSF was administered on Days 3-5 than on Day 2 (odds ratio: 1.27, 95% CI 0.66-2.46, p = 0.47), with a moderate certainty of evidence. No significant difference in overall survival or mortality due to infections was observed. The trend of severe adverse events was lower on Days 3-5, without statistical significance (odds ratio: 0.72, 95% CI 0.14-3.67, p = 0.69) and with a moderate certainty of evidence. Data on pain were inconclusive. CONCLUSIONS: Both Day 2 and Days 3-5 were weakly recommended for pegylated G-CSF administration post-chemotherapy in patients with cancer. The limited evidence highlights the need for further research to refine recommendations.

Comparison between a single dose of PEG G-CSF and multiple doses of non-PEG G-CSF: a systematic review and meta-analysis from Clinical Practice Guidelines for the use of G-CSF 2022.

BACKGROUD: Granulocyte colony-stimulating factor (G-CSF) is widely used for the primary prophylaxis of febrile neutropenia (FN). Two types of G-CSF are available in Japan, namely G-CSF chemically bound to polyethylene glycol (PEG G-CSF), which provides long-lasting effects with a single dose, and non-polyethylene glycol-bound G-CSF (non-PEG G-CSF), which must be sequentially administrated for several days. METHODS: This current study investigated the utility of these treatments for the primary prophylaxis of FN through a systematic review of the literature. A detailed literature search for related studies was performed using PubMed, Ichushi-Web, and the Cochrane Library. Data were independently extracted and assessed by two reviewers. A qualitative analysis or meta-analysis was conducted to evaluate six outcomes. RESULTS: Through the first and second screenings, 23 and 18 articles were extracted for qualitative synthesis and meta-analysis, respectively. The incidence of FN was significantly lower in the PEG G-CSF group than in the non-PEG G-CSF group with a strong quality/certainty of evidence. The differences in other outcomes, such as overall survival, infection-related mortality, the duration of neutropenia (less than 500/µL), quality of life, and pain, were not apparent. CONCLUSIONS: A single dose of PEG G-CSF is strongly recommended over multiple-dose non-PEG G-CSF therapy for the primary prophylaxis of FN.

Acute cytotoxic effects of silica microparticles used for coating of plastic blood-collection tubes on human periosteal cells.

Because of its simple operation, platelet-rich fibrin (PRF) is becoming more popular than the original form, platelet-rich plasma (PRP), in regenerative dentistry. PRF preparation requires plain glass blood-collection tubes, but not either anticoagulants or coagulation factors. However, such glass tubes designed for laboratory testing are no longer commercially available. Although several glass tubes specifically designed for PRF preparation are available, many clinicians prefer to obtain stably supplied substitutes, such as silica-coated plastic tubes produced by major medical device companies. The quality of PRF prepared by silica-coated tubes has not been assessed and we previously reported significant contamination of silica microparticles in the resulting PRF matrix and alerted clinicians against the use for PRF preparation. To further assess the biosafety of the silica microparticles, we presently examined their effects on human normal periosteal cells derived from alveolar bone. The periosteal cells were obtained from explant cultures of small periosteal tissues obtained from healthy donors. Silica microparticles were obtained from silica-coated tubes and added to cell cultures. Cellular responses were monitored using a tetrazolium assay, phase-contract inverted microscopy, an immunofluorescence method, and scanning electron microscopy. Silica microparticles adsorbed onto the cell surface with seemingly high affinity and induced apoptosis, resulting in significant reduction of cell proliferation and viability. These findings suggest that silica microparticles contained in plastic tubes for the purpose of blood coagulation are hazardous for various cell types around sites where silica-contaminated PRF matrices are implanted.

Responses of promyelocytic leukemia HL60 cells as an inflammatory cell lineage model to silica microparticles used to coat blood collection tubes.

BACKGROUND: The preparation of platelet-rich fibrin (PRF) requires glass blood collection tubes, and thus, the shortage or unavailability of such tubes has driven clinicians to search for suitable substitutes, such as silica-coated plastic tubes. However, we have previously demonstrated the cytotoxicity of silica microparticles (MPs) used in plastic tubes to cultured human periosteal cells. To further establish the effects of silica MPs on inflammation, we examined silica MP-induced changes in a human promyelocytic cell model in vitro. METHODS: Human promyelocytic HL60 cells were used either without chemical induction or after differentiation induced using phorbol myristate acetate (PMA) or dimethyl sulfoxide. HL60 cells, osteoblastic MG63, and Balb/c mouse cells were treated with silica MPs, and their surface ultrastructure and numbers were examined using a scanning electron microscope and an automated cell counter, respectively. Differentiation markers, such as acid phosphatase, non-specific esterase, and CD11b, were visualized by cytochemical and immunofluorescent staining, and superoxide dismutase (SOD) activity was quantified. RESULTS: Regardless of SOD activity, silica cytotoxicity was observed in MG63 and Balb/c cells. At sub-toxic doses, silica MPs slightly or moderately upregulated the differentiation markers of the control, PMA-induced monocytic, and dimethyl sulfoxide-induced granulocytic HL60 cells. Although SOD activity was the highest (P < 0.05) in PMA-induced cells, a silica-induced reduction in cell adhesion was observed only in those cells (P < 0.05). CONCLUSIONS: Silica MP contamination of PRF preparations can potentially exacerbate inflammation at implantation sites. Consequently, unless biomedical advantages can be identified, silica-coated plastic blood collection tubes should not be routinely used for PRF preparations.

Axenfeld-Rieger anomaly and Axenfeld-Rieger syndrome: clinical, molecular-cytogenetic, and DNA array analyses of three patients with chromosomal defects at 6p25.

Clinical phenotypes of and genetic aberrations in three unrelated Japanese patients with Axenfeld-Rieger anomalies and various accompanying malformations of systemic organs are described. GTG-banded chromosome analysis showed terminal deletions of the short arm of chromosome 6 in two patients and an inversion, inv(6)(p25q14), in the other. FISH and DNA array analyses revealed that the two patients with deletions had 5.0-5.7 Mb and 6.6 Mb 6p terminal deletions, respectively, and FOXC1 was apparently deleted in both patients. In the other patient, the inversion breakpoint at 6p25 was estimated to be in or very close to the FOXC1 locus, but DNA array analysis did not reveal a deletion around the breakpoint. Common extraocular findings in these patients included broad forehead, brachycephaly, hypertelorism, downslanting palpebral fissures, small anteverted nose, and cardiac defects. Two patients also exhibited autistic characteristics. The two patients with deletions exhibited poor muscle tone and developmental delays. Most of these extraocular findings were similar to those found in previous patients with FOXC1 mutations and distinct from those found in patients with PITX2 mutations, who frequently develop umbilical and dental anomalies. We suggest that the psychomotor retardation is a clinical manifestation associated with a deletion of multiple contiguous genes in the 6p terminus and that this phenomenon is similar to the 6p25 deletion syndrome. Understanding the relationship between genetic lesions and the spectrum of extraocular findings in patients with Axenfeld-Rieger anomalies may lead to better clinical management.

Hemoglobin encapsulation in vesicles retards NO and CO binding and O2 release when perfused through narrow gas-permeable tubes.

Intravenous administration of cell-free Hb induces vasoconstriction and circulatory disorders, presumably because of the intrinsic affinities to endogenous nitric oxide (NO) and carbon monoxide (CO) as vasorelaxation factors and because of the facilitated O(2) release that might induce autoregulatory vasoconstriction. We examined these gas reactions when Hb-containing solutions of four kinds were perfused through artificial narrow tubes at a practical Hb concentration (10 g/dl). Purified Hb solution, polymerized bovine Hb (Poly(B)Hb), encapsulated Hb [Hb-vesicles (HbV), 279 nm], and red blood cells (RBCs) were perfused through a gas-permeable narrow tube (25 microm inner diameter) at 1 mm/s centerline velocity. The level of reactions was determined microscopically based on the visible-light absorption spectrum of Hb. When the tube was immersed in NO and CO atmospheres, both NO binding and CO binding of deoxygenated Hb (deoxy-Hb) and Poly(B)Hb in the tube was faster than those of HbV and RBCs, and HbV and RBCs showed almost identical binding rates. When the tube was immersed in a N(2) atmosphere, oxygenated Hb and Poly(B)Hb showed much faster O(2) release than did HbV and RBCs. Poly(B)Hb showed a faster reaction than Hb because of the lower O(2) affinity of Poly(B)Hb than Hb. The diffusion process of the particles was simulated using Navier-Stokes and Maxwell-Stefan equations. Results clarified that small Hb (6 nm) diffuses laterally and mixes rapidly. However, the large-dimension HbV shows no such rapid diffusion. The purely physicochemical differences in diffusivity of the particles and the resulting reactivity with gas molecules are one factor inducing biological vasoconstriction of Hb-based oxygen carriers.

Amino group PEGylation of bovine lactoferrin by linear polyethylene glycol-p-nitrophenyl active esters.

PEGylation, the covalent attachment of polyethylene glycol (PEG) to pharmaceutical proteins, is regarded as an extremely useful procedure to generate protein drugs with intensified therapeutic properties. We examined the amino group modification of bovine lactoferrin (bLF) with linear PEG-p-nitrophenyl active esters. At pH 5.0, we specifically observed the formation of mono-PEGylated bLF in high yields. PEG-conjugation reactions advanced slowly and reached a steady state by 48 h in a buffer at pH 5.0. The hydrolysis half-lives of 5-kDa and 30-kDa PEG-p-nitrophenyl active esters at pH 5.0 were estimated to be approximately 117 and 136 h, respectively. The slow reaction and hydrolysis rates of PEG-p-nitrophenyl active esters may contribute to the formation of mono-PEGylated bLF that could not be obtained by PEGylation with linear N-hydroxysuccinimide (NHS) activated PEG.

PEGylated lactoferrin enhances its hepatoprotective effects on acute liver injury induced by D-galactosamine and lipopolysaccharide in rats.

Polyethylene glycol (PEG) is attached to proteins in order to increase their half-life in circulation and reduce their immunogenicity in vivo. The present study was conducted to examine whether two different sizes of PEGylated bovine lactoferrin (40k- and 20k-PEG-bLf) would enhance the protective effect of native bLf on liver injury induced by D-galactosamine (GalN) and lipopolysaccharide (LPS) in rats. The treatment of PEGylated bLf more remarkably prevented the elevation of serum levels of hepatic enzyme markers and inhibited inflammatory and hemorrhagic changes and hepatic apoptosis induced by GalN/LPS than native bLf. The treatment of PEGylated bLf more significantly inhibited the increased concentration of proinflammatory cytokines (TNF-alpha and IL-6) in serum caused by GaIN/LPS, and enhanced anti-inflammatory cytokine (IL-10) production more than native bLf. PEGylated bLf decreased serum levels of nitric oxide (NO) more than native bLf. These results indicate that PEGylated bLf inhibits more significantly the induction of inflammatory mediators such as cytokines and NO than native bLf, resulting in the enhancement of its prevention of fulminant liver failure induced by GalN/LPS in rats. The present study provided evidence that PEGylated bLf may offer a novel alternative therapy for the prevention of acute hepatic failure through its anti-inflammatory and immunomodulatory properties.

nal-IRI+5-FU/LV versus 5-FU/LV in post-gemcitabine metastatic pancreatic cancer: Randomized phase 2 trial in Japanese patients.

BACKGROUND: In the NAPOLI-1 phase 3 trial, liposomal irinotecan (nal-IRI) +5-fluorouracil/leucovorin (5-FU/LV) significantly increased mPFS versus 5-FU/LV (3.1 vs. 1.5 months [unstratified HR = 0.56, p = 0.0001]) in patients with mPAC that progressed on prior gemcitabine-based therapy. This randomized phase 2 trial evaluated nal-IRI+5-FU/LV tolerability (Part 1), safety, and efficacy (Part 2; outcomes reported here) in Japanese patients with mPAC that progressed on gemcitabine-based therapy. METHODS: Patients were randomized 1:1 and stratified by KPS (70 and 80 vs. ≥90) and baseline albumin (≥4.0 g/dl vs. <4.0 g/dl). Primary endpoint was PFS; secondary endpoints were ORR, DCR, OS, TTF, CA19-9 response, and QoL. The ITT population comprised all randomized patients. RESULTS: Patient characteristics differed between nal-IRI+5-FU/LV (n = 40) and 5-FU/LV (n = 39) arms, including baseline hepatic lesions (63% vs. 51%), stage IV disease at diagnosis (78% vs. 51%), and post-study anticancer therapy (55% vs. 72%). Investigator-assessed mPFS increase with nal-IRI+5-FU/LV was clinically meaningful and statistically significant versus 5-FU/LV (2.7 vs. 1.5 months, HR = 0.60). Independently assessed mPFS showed similar trends (1.7 vs. 1.6 months, HR = 0.79). mOS was 6.3 months with nal-IRI+5-FU/LV and not reached with 5-FU/LV. ORR increased significantly with nal-IRI+5-FU/LV versus 5-FU/LV (18% vs. 0, rate difference 17.5). Commonly reported grade ≥3 treatment-emergent AEs were decreased neutrophil count (37% vs. 3%), decreased white blood cell count (20% vs. 0), and diarrhea (17% vs. 3%). CONCLUSIONS: In conclusion, clinically meaningful and statistically significant gains in investigator-assessed PFS and ORR were observed with nal-IRI+5-FU/LV versus 5-FU/LV in Japanese patients, with no new or unexpected safety signals. (Clinicaltrials.gov ID: NCT02697058).

Peculiar flow patterns of RBCs suspended in viscous fluids and perfused through a narrow tube (25 microm).

Red blood cells (RBCs) generally deform to adopt a parachute-like, torpedo-like, or other configuration to align and flow through a capillary that is narrower than their major axis. As described herein, even in a narrow tube (25 microm) with diameter much larger than that of a capillary, flowing RBCs at 1 mm/s align axially and deform to a paraboloid shape in a viscous Newtonian fluid (505 kDa dextran medium) with viscosity of 23.4-57.1 mPa.s. A high-speed digital camera image showed that the silhouette of the tip of RBCs fits a parabola, unlike the shape of RBCs in capillaries, because of the longer distance of the RBC-free layer between the tube wall and the RBC surface ( approximately 8.8 microm). However, when RBCs are suspended in a "non-Newtonian" viscous fluid (liposome-40 kDa dextran medium) with a shear-thinning profile, they migrate toward the tube wall to avoid the axial lining, as "near-wall-excess," which is usually observed for platelets. This migration results from the presence of flocculated liposomes at the tube center. In contrast, such near-wall excess was not observed when RBCs were suspended in a nearly Newtonian liposome-albumin medium. Such unusual flow patterns of RBCs would be explainable by the principle; a larger particle tends to flow near the centerline, and a small one tends to go to the wall to flow with least resistance. However, we visualized for the first time the complete axial aligning and near-wall excess of RBCs in the noncapillary size tube in some extreme conditions.

Mechanism of flocculate formation of highly concentrated phospholipid vesicles suspended in a series of water-soluble biopolymers.

Polyethylene glycol-modified vesicles (liposomes) encapsulating hemoglobin (HbV) are artificial oxygen carriers that have been developed as a transfusion alternative. The HbV suspension in an albumin solution is nearly Newtonian; other biopolymers, hydroxyethyl starch (HES), dextran (DEX), and modified fluid gelatin, induce flocculation of HbVs through depletion interaction and render the suspensions as non-Newtonian. The flocculation level increased with hydrodynamic radius (R(h)) or radius of gyration (R(g)) of series of HES or DEX with different molecular weights at a constant polymer concentration (4 wt %). However, the flocculation level differed markedly among the polymers. A crowding index (C(i)) representing the crowding level of a polymer solution is defined as (excluded volume of one polymer) x (molar concentration) x Avogadro's number, using R(h) or R(g). All polymers' flocculation level increases when C(i) approaches 1: when the theoretical total excluded volumes approach the entire solution volume, the excluded HbV particles are forced to flocculate.

Lactoferrin conjugated with 40-kDa branched poly(ethylene glycol) has an improved circulating half-life.

PURPOSE: We developed a lactoferrin conjugate by modifying bovine lactoferrin (bLF) with a 40-kDa branched poly(ethylene glycol) (PEG) molecule (designated 40 k-PEG-bLf), and we evaluated its in vitro activities and pharmacokinetic properties. MATERIALS AND METHODS: We prepared 40k-PEG-bLf by amino conjugation with N-hydroxysuccinimide-activated PEG. This conjugate was purified by cation exchange chromatography and its in vitro biological activities, such as iron binding, anti-inflammatory effects, and resistance to proteolytic enzymes were investigated. In vivo pharmacokinetics analyses, were also performed to examine the rate of clearance from the plasma in rats. RESULTS: The 40k-PEG-bLf conjugate was fully active in iron binding and exhibited 97.1 +/- 5.5% (mean +/- S.E., n = 6) of the original anti-inflammatory activity. The in vitro peptic susceptibility of 40 k-PEG-bLf revealed that the proteolytic half-life increased at least 6-fold that of unmodified LF. This PEGylated conjugate demonstrated a plasma half-life that was 8.7-fold longer than that of the unmodified bLF in rats. CONCLUSIONS: The 40k-PEG-bLf exhibited improved in vitro bioactivity and stability and enhanced pharmacokinetic properties as compared to those of the unmodified bLF and the 20 k-PEG-bLf conjugate, which was recently developed by PEGylation of bLF with a 20-kDa branched PEG [Nojima Y. et al. Bioconjugate Chem. 19:2253-2259 (2008)].

Development of poly(ethylene glycol) conjugated lactoferrin for oral administration.

Lactoferrin (LF) is an iron-binding glycoprotein that possesses multifunctional biological activities. Recent reports from clinical trials suggest that LF is potentially effective as a therapeutic protein against cancer and gangrene. However, pharmaceutical proteins such as LF are unstable in vivo. Therefore, to improve stability, we developed mono-PEGylated bovine LF (20k-PEG-bLf) with branched 20 kDa (2 x 10 kDa) poly(ethylene glycol) (PEG). We examined in vitro activities such as iron binding, IL-6 cell based assay, and resistance to a proteolytic enzyme in artificial gastric fluid. The 20k-PEG-bLf protein was fully active in iron binding and exhibited 69.6 +/- 2.9% (mean +/- S.E., n = 6) of the original anti-inflammatory activity. The proteolytic half-life increased 2-fold over that of unmodified LF. In vivo pharmacokinetic analyses were performed to examine absorption from the intestinal epithelium and serum clearance. Direct administration of 20k-PEG-bLf (30 mg/kg) into rat stomachs demonstrated that the amount of absorption from the intestinal tract increased approximately 10-fold relative to unmodified LF. Intravenous injection of the protein (1 mg/kg) revealed that 20k-PEG-bLf prolongs serum half-life by approximately 5.4-fold, and that the area under the curve (AUC) was increased approximately 9.2-fold compared to that of unmodified LF. PEGylation improved the physical and pharmacokinetic properties of bovine LF. This is the first report on the use of bioconjugation of LF for the development of a promising oral pharmaceutical agent.

[Mucositis].

Mucositis is a significant dose-limiting factor associated with cancer chemotherapy and radiotherapy. For exact management, an early diagnosis and precise evaluation are surely required. A basis of the prevention and care of stomatitis is maintaining cleanliness and moisture in the mouth. The medical treatment plans of oral mucositis are a measure against infection, and prevention against symptoms, and are restoration of a tissue damage, and treatment to sharp pain. However, there is no still established prevention method. As for the present condition, in the clinical practice, there are many portions depending on experiential knowledge. In this paper, it outlined including Empiric therapy about measures of oral mucositis.

Intersubjective action-effect binding: eye contact modulates acquisition of bidirectional association between our and others' actions.

In everyday social life, we predict others' actions in response to our own actions. Subsequently, on the basis of these predictions, we control our actions to attain desired social outcomes and/or adjust our actions to accommodate the anticipated actions of the others. Representation of the bidirectional association between our and others' actions, that is, intersubjective action-effect binding, could make such intersubjective action control easier and smoother. The present study investigated not only whether or not intersubjective action-effect binding was acquired but also whether or not eye contact modulated it. Experiment 1 showed that after a repeated experience during which participants' finger movements triggered a target female individual's mouth gesture, observing the target's mouth gestures came to automatically trigger the participants' finger movements. Experiments 2 and 3 revealed that this effect was not observed when the target's gaze direction was averted (Experiment 2) or when the target's eyes were closed (Experiment 3) throughout the acquisition phase. These results indicate that intersubjective action-effect binding occurs and that an ostensive signal, that is, eye contact modulates it.

Interstitial deletion of 13q14.13-q32.3 presenting with Arima syndrome and bilateral retinoblastoma.

A patient with a large deletion of the distal part of the long arm of chromosome 13 showed severe psychomotor retardation, a characteristic face, nystagmus, retinopathy, cystic kidney disease, and brain malformation with molar tooth sign and cerebellar vermis hypoplasia, a phenotype typical of Arima syndrome. This patient also had bilateral retinoblastoma. Fluorescent in situ hybridization and single-nucleotide-polymorphism genotyping microarray demonstrated an interstitial deletion of 54 Mbp, ranging from 13q14.13 to 13q32.3 and involving the RB1 gene. This patient is the first case of Arima syndrome, or a Joubert syndrome-related disorder, that showed linkage to chromosome 13q.

PEGylated lactoferrin enhanced its hepatoprotective effects on acute liver injury induced by carbon tetrachloride in rats.

Polyethylene glycol (PEG) is attached to proteins in order to increase their half-life in circulation and reduce their immunogenicity in vivo. The present study was conducted to examine whether two different sizes of PEGylated bovine lactoferrin (40k-PEG-bLf and 20k-PEG-bLf) would enhance the protective effect of native bLf on liver injury induced by carbon tetrachloride (CCl(4)) in rats. Silymarin, a known hepatoprotective drug was used as a positive control. Compared to native bLf, the treatment of PEGylated bLf more markedly prevented the elevation of serum levels of hepatic enzyme markers and inhibited fatty degeneration and the hepatic necrosis induced by CCl(4). 40k-PEG-bLf showed a more significant suppressive effect on CCl(4)-induced hepatic injury than 20k-PEG-bLf. The treatment with PEGylated bLf elevated serum SOD activity reduced by CCl(4) more significantly than native bLf. 40k-PEG-bLf enhanced serum SOD activity more significantly than 20k-PEG-bLf. Immunohistochemical study showed that the PEGylation of bLf enhanced its intracellular transportation to hepatocytes. The increases in intracellular transportation of the PEGylated bLf in order were: 40k-PEG-bLf>20k-PEG-bLf>native bLf. These findings suggested that the mechanism of the enhancement of hepatoprotective effect by PEGylated bLf was associated with an increase in the intracellular transportation of PEGylated bLf in hepatocytes.

The pH-dependent formation of PEGylated bovine lactoferrin by branched polyethylene glycol (PEG)-N-hydroxysuccinimide (NHS) active esters.

PEGylation is an established method for improving the pharmacokinetic properties and pharmacodynamic effects of therapeutic proteins. Amino conjugation by N-hydroxysuccinimide activated polyethylene glycol (PEG-NHS) represents the most common modification of the target proteins. In this study, we compared the reaction velocities of pH-dependent preparations of bovine lactoferrin modified with branched PEG-NHS conducted at pH 7.4 to pH 9.0. PEGylation reaction rates were dependent on pH value, but not on PEG molecular mass. At pH 7.4, reactions advanced gradually and reached steady state by 2 h, whereas at pH 9.0, reactions advanced very quickly and reached steady state within 10 min. Hydrolysis half-life of PEG-NHS exceeded 120 min at pH 7.4, but was below 9 min at pH 9.0. Thus, the pH value is one of the most important factors to obtain the optimal condition for PEGylation by NHS esters.

Encapsulation of concentrated hemoglobin solution in phospholipid vesicles retards the reaction with NO, but not CO, by intracellular diffusion barrier.

One physiological significance of the red blood cell (RBC) structure is that NO binding of Hb is retarded by encapsulation with the cell membrane. To clarify the mechanism, we analyzed Hb-vesicles (HbVs) with different intracellular Hb concentrations, [Hb](in), and different particle sizes using stopped-flow spectrophotometry. The apparent NO binding rate constant, k(on)('(NO)), of HbV at [Hb](in) = 1 g/dl was 2.6 x 10(7) m(-1) s(-1), which was almost equal to k(on)((NO)) of molecular Hb, indicating that the lipid membrane presents no obstacle for NO binding. With increasing [Hb](in) to 35 g/dl, k(on)('(NO)) decreased to 0.9 x 10(7) m(-1) s(-1), which was further decreased to 0.5 x 10(7) m(-1) s(-1) with enlarging particle diameter from 265 to 452 nm. For CO binding, which is intrinsically much slower than NO binding, k(on)('(CO)) did not change greatly with [Hb](in) and the particle diameter. Results obtained using diffusion simulations coupled with elementary binding reactions concur with these tendencies and clarify that NO is trapped rapidly by Hb from the interior surface region to the core of HbV at a high [Hb](in), retarding NO diffusion toward the core of HbV. In contrast, slow CO binding allows time for further CO-diffusion to the core. Simulations extrapolated to larger particles (8 mum) showing retardation even for CO binding. The obtained k(on)('(NO)) and k(on)('(NO)) yield values similar to those reported for RBCs. In summary, the intracellular, not extracellular, diffusion barrier is predominant due to the rapid NO binding that induces a rapid sink of NO from the interior surface to the core, retarding further NO diffusion and binding.

Rheological properties of hemoglobin vesicles (artificial oxygen carriers) suspended in a series of plasma-substitute solutions.

Hemoglobin vesicles (HbV) or liposome-encapsulated Hbs are artificial oxygen carriers that have been developed for use as transfusion alternatives. The extremely high concentration of the HbV suspension (solutes, ca. 16 g/dL; volume fraction, ca. 40 vol %) gives it an oxygen-carrying capacity that is comparable to that of blood. The HbV suspension does not possess a colloid osmotic pressure. Therefore, HbV must be suspended in or co-injected with an aqueous solution of a plasma substitute (water-soluble polymer), which might interact with HbV. This article describes our study of the rheological properties of HbV suspended in a series of plasma substitute solutions of various molecular weights: recombinant human serum albumin (rHSA), dextran (DEX), modified fluid gelatin (MFG), and hydroxylethyl starch (HES). The HbV suspended in rHSA was nearly Newtonian. Other polymers-HES, DEX, and MFG-induced HbV flocculation, possibly by depletion interaction, and rendered the suspensions as non-Newtonian with a shear-thinning profile (10(-4)-10(3) s(-1)). These HbV suspensions showed a high storage modulus (G') because of the presence of flocculated HbV. However, HbV suspended in rHSA exhibited a very low G'. The viscosities of HbV suspended in DEX, MFG, and high-molecular-weight HES solutions responded quickly to rapid step changes in shear rates of 0.1-100 s(-1) and a return to 0.1 s(-1), indicating that flocculation is both rapid and reversible. Microscopically, the flow pattern of the flocculated HbV that perfused through microchannels (4.5 microm deep, 7 microm wide, 20 cmH2O applied pressure) showed no plugging. Furthermore, the time required for passage was simply proportional to the viscosity. Collectively, the HbV suspension viscosity was influenced by the presence of plasma substitutes. The HbV suspension provides a unique opportunity to manipulate rheological properties for various clinical applications in addition to its use as a transfusion alternative.