Thermosensitive liposomes are major drug delivery carriers, which enable targeting of drugs and burst release of the drugs from the liposomes at the site of action by applying a local heat stimulation above body temperature. Although the burst release is significant for a one-shot high-rate release of drugs at the target site, this type of release has a limited sustained action of the drugs. In this study, we report the alkali-encapsulating thermosensitive liposomes enabling environment pH regulation by sustained continuous cargo release at human body temperature. The liposomes encapsulating alkalis successfully neutralized the environmental acids for hours by releasing the alkalis and prevented acid erosion of hydroxyapatite matrix. Taken together, the present liposomes are effective for the sustained release of cargo at body temperature, specifically the alkali-encapsulating liposomes can be a preventing agent for dental caries in the oral cavity. The sustained release under endogenous body heat characteristics of thermosensitive liposomes showcased in this study can also be extended for prolonged intravenous drug exposure from targeted liposomal drug nanotherapeutics in the near future.

This study reports a nano-capsule based chemical release system capable of in vivo drug dosing. We have successfully designed thermosensitive liposomes which release the cargo at desired temperature in different released modes (single bolus, sustained slow, stepwise releases). The system was capable of delivering drug with minimal dosage repeatedly, which is suitable for brief actuator halting in insect platform control. In addition, liposomes encapsulating glucose injected to the insect automatically release fuel to boost the endurance and operating time of the insect-computer hybrid system.

Thermosensitive fluorescent dyes can convert thermal signals into optical signals as a molecular nanoprobe. These nanoprobes are playing an increasingly important part in optical temperature sensing and imaging at the nano- and microscale. However, the ability of a fluorescent dye itself has sensitivity and accuracy limitations. Here we present a molecular strategy based on self-assembly to overcome such limitations. We found that thermosensitive nanovesicles composed of lipids and a unique fluorescent dye exhibit fluorescence switching characteristics at a threshold temperature. The switch is rapid and reversible and has a high signal to background ratio (>60), and is also highly sensitive to temperature (10-22%/°C) around the threshold value. Furthermore, the threshold temperature at which fluorescence switching is induced, can be tuned according to the phase transition temperature of the lipid bilayer membrane forming the nanovesicles. Spectroscopic analysis indicated that the fluorescence switching is induced by the aggregation-caused quenching and disaggregation-induced emission of the fluorescent dye in a cooperative response to the thermotropic phase transition of the membrane. This mechanism presents a useful approach for chemical and material design to develop fluorescent nanomaterials with superior fluorescence sensitivity to thermal signals for optical temperature sensing and imaging at the nano- and microscales.

Nanocapsules present a promising platform for delivering chemicals and biomolecules to a site of action in a living organism. Because the biological action of the encapsulated molecules is blocked until they are released from the nanocapsules, the encapsulation structure enables triggering of the topical and timely action of the molecules at the target site. A similar mechanism seems promising for the spatiotemporal control of signal transduction triggered by the release of signal molecules in neuronal, metabolic, and immune systems. From this perspective, nanocapsules can be regarded as practical tools to apply signal molecules such as neurotransmitters to intervene in signal transduction. However, spatiotemporal control of the payload release from nanocapsules persists as a key technical issue. Stimulus-responsive nanocapsules that release payloads in response to external input of physical stimuli are promising platforms to enable programmed payload release. These programmable nanocapsules encapsulating neurotransmitters are expected to lead to new insights and perspectives related to artificial extracellular synaptic vesicles that might provide an experimental and therapeutic strategy for neuromodulation and nervous system disorders.

Aggregation of liposomal platelet substitutes with activated platelets is the primary endpoint to estimate hemostatic potential. Although light transmission aggregometry is a "gold standard" in assessing platelet aggregation in vitro, this method is less specific and sensitive when tested using liposomal platelet substitutes. In the current study, a new method is developed to evaluate the function of platelet substitutes. By labeling liposomes with a fluorescent dye, DiD, we evaluated their ability to target platelet aggregates using a fluorescence microscope. By incorporating an image-based 96 microtiter microplate, this method was optimized by varying the final lipid concentrations and washing times and validated using unmodified liposomes (e.g., L550 with 0 mol% of carboxylic headgroup lipid; L551 with 9 mol% of carboxylic headgroup lipid) and modified liposomes (e.g., H12-L551 with 9 mol% of carboxylic headgroup lipid and 0.3 mol% of dodecapeptide). Our results showed that 200 μM of H12-L551 liposomes and four washes represent optimal conditions for quantitative fluorescence imaging. This method allowed users to qualitatively observe the fluorescently labeled liposomes involved in platelet aggregates. The imaging analysis tool was sufficiently sensitive to quantitatively determine the significantly enhanced delivery of the modified liposomes to platelet aggregates. This enhancement was achieved using dodecapeptide, which specifically binds to activated platelets. This robust and high-throughput method enables the evaluation of liposome function and should facilitate the development of platelet substitutes with a greater ability to target platelet aggregates.

This paper reports the on-demand artificial muscle relaxation using a thermosensitive liposome encapsulating γ-aminobutyric acid (GABA) inhibitory neurotransmitter. Muscle relaxation is not feasible in principle, although muscle contraction can be easily induced by electrical stimulation. Herein, thermosensitive liposomes (phase transition temperature = 40 °C) were synthesized to encapsulate GABA and were injected into a leg of a living beetle. The leg was wrapped around by a Ni-Cr wire heater integrated with a thermocouple to enable the feedback control and to manipulate the leg temperature. The injected leg was temporarily immobilized by heating it up to 45 °C. The leg did not swing even by electrically stimulating the leg muscle. Subsequently, the leg recovered to swing. The result indicates that GABA was released from liposomes and fed to the leg muscle, enabling temporal muscle relaxation.

Chloroquine was among the first of several effective drug treatments against malaria until the onset of chloroquine resistance. In light of diminished clinical efficacy of chloroquine as an antimalarial therapeutic, there is potential in efforts to adapt chloroquine for other clinical applications, such as in combination therapies and in diagnostics. In this context, we designed and synthesized a novel asymmetrical squaraine dye coupled with chloroquine (SQR1-CQ). In this study, SQR1-CQ was used to label live Plasmodium falciparum (P. falciparum) parasite cultures of varying sensitivities towards chloroquine. SQR1-CQ positively stained ring, mature trophozoite and schizont stages of both chloroquine⁻sensitive and chloroquine⁻resistant P. falciparum strains. In addition, SQR1-CQ exhibited significantly higher fluorescence, when compared to the commercial chloroquine-BODIPY (borondipyrromethene) conjugate CQ-BODIPY. We also achieved successful SQR1-CQ labelling of P. falciparum directly on thin blood smear preparations. Drug efficacy experiments measuring half-maximal inhibitory concentration (IC50) showed lower concentration of effective inhibition against resistant strain K1 by SQR1-CQ compared to conventional chloroquine. Taken together, the versatile and highly fluorescent labelling capability of SQR1-CQ and promising preliminary IC50 findings makes it a great candidate for further development as diagnostic tool with drug efficacy against chloroquine-resistant P. falciparum.

脂質分子の自発的分子集合により形成される閉鎖小胞体(リポソーム)は、タンパク質、核酸、低分子薬物などを微粒子として体内に投与するキャリアとして使われ、人工血液や薬物送達システム(DDS)への応用においてその安全性や効果が実証されている。温度に応答した薬物放出機能をもつ温度応答性リポソームは、熱刺激により薬物動態を制御するナノデバイスとして次世代DDSへの応用が期待される。本稿では、温度応答性リポソームの設計原理とDDSへの応用について概説する。温度応答性リポソームをDDS技術として生体内で安全・効果的に機能させるには、特定細胞や組織の標的化、微小空間の正確な温度制御と温度モニタリング、薬物動態や薬物放出挙動の可視化などの新しい技術開発と並行した進展が重要となる。(著者抄録)

In this paper, we describe the newly designed liposomes modified with amphiphilic far-red squaraine dye and folic acid for its application in folate receptor-targeted bioimaging. Enhanced intracellular uptake of the engineered liposomes has been demonstrated on SKOV-3 ovarian cancer cells.

Optically switchable fluorescent molecule is useful for developing photo-functional materials. However, most of the conventional fluorescent molecule is in a class of "always-on" type, which does not possess a fluorescence switching function. In this work, switchable fluorescent nanoparticles based on self assembly of amphiphilic derivative of rhodamine B is synthesized and evaluated for its optical properties. The fluorescent nanoparticles functioned as a photo-switchable and self-erasable nanoprobe by reversible intramolecular ring opening cyclization of the rhodamine B derivative in dispersion and freestanding hydrogel. (C) 2016 Elsevier B.V. All rights reserved.

Thermosensitive lipid-based nanoparticle (liposome) is a key platform for the controlled release of functional molecules by regional heating. Temperature monitoring in a three-dimensional matrix is necessary to conduct controlled heating at the targeted region. Currently, conventional liposomes do not possess a function for temperature monitoring. Herein, an extended concept for temperature monitoring using the liposomes was examined using near-infrared (NIR) laser-induced heating of water in hydrogel. The temperature distribution in hydrogel by photothermal conversion can now be traced by fluorescence in real time, with the use of liposomes that release the fluorescence cargo at different threshold temperatures. The liposome platform, equipped with temperature-sensing capability, extend the concept of temperature monitoring for temperature-triggered drug release, as well as thermotherapy.

Micro/nano-bubbles are practical nanomaterials designed to increase the gas content in liquids. We attempted to use oxygen micro/nano-bubble dispersions as an oxygen-rich liquid as a means for total liquid ventilation. To determine the oxygen content in the bubble dispersion, a new method based on a spectrophotometric change between oxy- and deoxy-hemoglobin was established. The oxygen micro/nano-bubble dispersion was supplied to an experimental total ventilation liquid in anesthetic rats. Though the amount of dissolving oxygen was as low as 6 mg/L in physiological saline, the oxygen content in the oxygen micro/nano-bubble dispersion was increased to 45 mg/L. The positive correlation between the oxygen content and the life-saving time under liquid ventilation clearly indicates that the life-saving time is prolonged by increasing the oxygen content in the oxygen micro/nano-bubble dispersion. This is the first report indicating that the oxygen micro/nano-bubbles containing a sufficient amount of oxygen are useful in producing oxygen-rich liquid for the process of liquid ventilation.

Control of cargo release from nanoscale carriers is an important technology for maximizing the benefits of nanoparticulate drug delivery systems. Herein, we attempt to trigger the release of cargo from liposomes by photothermal conversion of water with a 980 nm near-infrared (NIR) laser. This study examined liposomes of two types formulated by 1,2-dipalmitory-sn-glycero-3-phosphocholine (DPPC) or a mixture of DPPC/cholesterol with an anionic lipid and PEG-lipid as stabilizers encapsulating calcein as a cargo at different ionic strengths. Liposome formulation encapsulating a hypertonic solution with a lipid membrane shows that a gel to liquid-crystalline phase transition at around 40 degrees C effectively released the cargo from liposomes at temperature above 40 degrees C with NIR irradiation. Our proof of concept has been further demonstrated in a cancer cell with monitoring the actual "intracellular temperature" using a fluorescent thermosensor. Intracellular thermometry revealed that it was not until the intracellular temperature reached around 40 degrees C by NIR irradiation that the release of the cargo started gradually, showing good agreement with the result from the extracellular in vitro study. This targeted release of cargo from thermosensitive liposomes based on a photothermal effect using a NIR laser offers a potent nanoscale platform for the on-demand release of drugs in intracellular space with local hyperthermia. The intracellular thermometry facilitates the quantitative monitoring and control of the hyperthermia at the cellular level.

Bone marrow is a key element in the diagnosis of disorders of erythropoiesis, including anemia, and a potential target in their treatment. However, because efficient delivery of diagnostic and therapeutic agents to bone marrow is difficult, such delivery is achieved by administering drugs in large quantities that often have adverse effects. Here, we achieved selective delivery of recombinant human erythropoietin (rHuEPO) to bone marrow, via its encapsulation in liposomes with l-glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5-dihexadecyl ester (SA) (liposome-EPO). The result, in a rabbit model of renal anemia, was a beneficial effect on hematopoiesis, better than with rHuEPO alone. Also, we determined that liposome-EPO delivery to bone marrow depended on specific uptake by bone marrow macrophages because of the presence of SA. These results indicate both that liposome-EPO is a new, promising erythropoietin-stimulating agent and that liposomes with SA have potential for diagnostic and therapeutic applications in diseases originating from bone marrow.

The capsular structure of liposomes is attractive for encapsulation of pharmaceuticals to develop nanoparticle-based drug delivery systems. Here unique liposomes which are modified with succinic acid on their surface arereviewed at a point of view of drug delivery carriers. The negatively charged carboxyl group of the succinic acid onthe surface of the liposomes contributes to facilitate the formation of the unilamellar liposomes which have superiorability to efficiently encapsulate the pharmaceuticals. Interestingly, we found that the liposomes modified with suc-cinic acid are highly distributed in the bone marrow through the intravenous injection. These characteristics of thesurface–modified liposomes with succinic acid would be applicable for the drug delivery system targeting bone mar-row.

Background: Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt) in the arginine head group.Methods: Cationic lipids were hydrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine (TM) 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine (TM) 2000.Results: We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt) that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular. Arginine-based cationic liposomes with HCl-salt showed the highest transfection efficiency in PC-12 cells. However, arginine-based cationic liposomes with TFA salt showed the highest transfection efficiency in HeLa cells, regardless of the presence of serum, with very low associated cytotoxicity.Conclusion: The gene delivery efficiency of amino acid-based cationic assemblies is influenced by the amino acids (ie, arginine or lysine) present as the hydrophilic head group and their associated counterions.

The delivery of specific genes into neurons offers a potent approach for treatment of diseases as well as for the study of neuronal cell biology. Here we investigated the capabilities of cationic amino acid based lipid assemblies to act as nonviral gene delivery vectors in primary cultured neurons. An arginine-based lipid, Arg-C3-Glu2C14, and a lysine-based lipid, Lys-C3-Glu2C14, with two different types of counterion, chloride ion (Cl-) and trifluoroacetic acid (TFA-), were shown to successfully mediate transfection of primary cultured neurons with plasmid DNA encoding green fluorescent protein. Among four types of lipids, we optimized their conditions such as the lipid-to-DNA ratio and the amount of pDNA and conducted a cytotoxicity assay at the same time. Overall, Arg-C3-Glu2C14 with TFA- induced a rate of transfection in primary cultured neurons higher than that of Lys-C3-Glu2C14 using an optimal weight ratio of lipid-to-plasmid DNA of 1. Moreover, it was suggested that Arg-C3-Glu2C14 with TFA- showed the optimized value higher than that of Lipofectamine2000 in experimental conditions. Thus, Arg-C3-Glu2C14 with TFA- is a promising candidate as a reliable transfection reagent for primary cultured neurons with a relatively low cytotoxicity.

BACKGROUND: Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt) in the arginine head group. METHODS: Cationic lipids were hydrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000. RESULTS: We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt) that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular. Arginine-based cationic liposomes with HCl-salt showed the highest transfection efficiency in PC-12 cells. However, arginine-based cationic liposomes with TFA salt showed the highest transfection efficiency in HeLa cells, regardless of the presence of serum, with very low associated cytotoxicity. CONCLUSION: The gene delivery efficiency of amino acid-based cationic assemblies is influenced by the amino acids (ie, arginine or lysine) present as the hydrophilic head group and their associated counterions.

Intravenous injection of liposomes into pigs reportedly induces anaphylactoid reactions at a small dose, resulting in circulatory disorder. Hemoglobin vesicles (HbVs) are artificial oxygen carriers encapsulating Hb solution in liposomes. It is not known how pigs respond to HbV injection. We aimed to analyze the cardiopulmonary responses to small injections of HbV and empty vesicle (EV) and compare them with a conventional liposome (CL) with a different lipid composition containing phosphatidylglycerol (PG). PG is known to induce an anaphylactoid reaction in pigs. Nine male miniature pigs were used for HbV, EV, and CL injections. The anesthetized pig received 0.05 and 0.5 mL/kg of a test fluid for the first and second injection with a 70 min interval. Results show that CL repeatedly induced significant increases in systemic and pulmonary arterial pressures and vascular resistances and decreases in heart rate and cardiac output (CO). HbV and EV at the first injection-induced pulmonary hypertension, with significantly smaller changes in systemic arterial pressure and CO. No remarkable response was visible at the second injection in spite of a larger dosage. Only CL repeatedly induced thrombocytopenia, leukocytopenia, and plasma thromboxane B(2) increase resulting from complement activation, although HbV and EV showed smaller changes. Transmittance electron micrograph of pulmonary intravascular macrophages (PIMs) showed phagocytosis of HbV, indicating the possibility that nonspecific phagocytosis by PIMs relates to the responses observed after the first injection. HbV does not induce a significant anaphylactoid reaction in pigs compared with CL because of the different lipid composition.

The hemoglobin-vesicle (HbV) is an artificial oxygen carrier encapsulating a concentrated hemoglobin solution in a phospholipid vesicle (liposome). During or after transporting oxygen, macrophages capture HbVs in the reticuloendothelial system (RES) with an approximate circulation half-life of 3 days. Animal studies show transient splenohepatomegaly after large doses, but HbVs were completely degraded, and the components were excreted in a few weeks. If a blood substitute is used for emergency use until red blood cell transfusion becomes available or for temporary use such as a priming fluid for an extracorporeal circuit, then one option would be to remove HbVs from the circulating blood without waiting a few weeks for removal by the RES. Using a mixture of beagle dog whole blood and HbV, we tested the separation of HbV using a centrifugal Fresenius cell separator and an ultrafiltration system. The cell separator system separated the layers of blood cell components from the HbV-containing plasma layer by centrifugal force, and then the HbV was removed from plasma phase by the ultrafiltration system. The HbVs (250-280 nm) are larger than plasma proteins (< 22 nm diameter) but smaller than blood cell components (> 3 µm). The size of HbVs is advantageous to be separated from the original blood components, and the separated blood components can be returned to circulation.

リポソームは細胞型人工酸素運搬体としての応用のほか,各種薬物や遺伝子の運搬体としての応用が期待されている.人工酸素運搬体に利用されるリポソームは大量投与を想定して開発され,薬物キャリアーとして少量投与で使用される血中滞留性リポソームとは粒子径や脂質組成などで異なる特徴を有している.このリポソームにヘモグロビンを内包させた人工酸素運搬体(Hb小胞体)を大量投与(680mg lipid/kg body weight)すると,主に肝臓,脾臓,骨髄に捕捉され,2〜3日程度の半減期で血中から消失する.一方,薬物送達の対象となる少投与量(15mg lipid/kg body weight)では,興味深いことに骨髄に集中した体内分布が観測される.この体内動態特性により,骨髄に薬剤を効率的に運搬できる薬物送達システムとしての新しい応用が期待できる.(著者抄録)

Liposomes reportedly accumulate in monophagocytic systems (MPSs), such as those of the spleen. Accumulation of considerable amounts of liposome in a MPS can affect immunologic response. While developing a liposomal oxygen carrier containing human hemoglobin vesicle (HbV), we identified its suppressive effect on the proliferation of rat splenic T cells. The aim of this study was to elucidate the mechanism underlying that phenomenon and its effect on both local and systemic immune response. For this study, we infused HbV intravenously at a volume of 20% of whole blood or empty liposomes into rats, removed their spleens, and evaluated T cell responses to concanavalin A (Con A) or keyhole limpet hemocyanin (KLH) by measuring the amount of [(3)H]thymidine incorporated into DNA. Cells that phagocytized liposomal particles were sorted using flow cytometry and analyzed. Serum anti-KLH antibody was measured after immunizing rats with KLH. Results showed that T cell proliferation in response to Con A or KLH was inhibited from 6 h to 3 days after the liposome injection. Direct cell-to-cell contact was necessary for the suppression. Both inducible nitric-oxide synthase and arginase inhibitors restored T cell proliferation to some degree. The suppression abated 7 days later. Cells that trapped vesicles were responsible for the suppression. Most expressed CD11b/c but lacked class II molecules. However, the primary antibody response to KLH was unaffected. We conclude that the phagocytosis of the large load of liposomal particles by rat CD11b/c+, class II immature monocytes temporarily renders them highly immunosuppressive, but the systemic immune response was unaffected.

Electrostatic interaction is an important secondary force affecting the structure, stability, and function of lipid vesicles (liposomes). For this study, a negatively charged lipid with carboxylic acid was mixed with phospholipid to produce anionic vesicles. The electrostatics of the carboxylated anionic vesicle (ca. 200 nm diameter) was determined and correlated with entrapment capacity of the vesicles. Correlative analysis revealed the zeta potential of the vesicles as a factor quantitatively affecting the entrapment capacity for a water-soluble marker, in which the entrapment capacity reached its maximum level in less than -30 mV of zeta potential. Transmission electron microscopy (TEM) revealed that the vesicles with high entrapment capacity are composed of a unilamellar membrane. This finding is expected to be useful for efficient encapsulation of water-soluble pharmaceuticals within vesicles.

BACKGROUND & AIMS: Recently, we described a novel surface-modified lipid vesicle formulation (liposome) that had very high targeting to bone marrow in normal rabbits. Because the bone marrow is the site of hematopoiesis, bone marrow-targeted drug-delivery systems have many potential applications. In this study we investigated whether these bone marrow-targeted vesicles are also similarly effective for bone marrow targeting in rhesus monkeys, a primate animal model that is more relevant to humans. MATERIALS & METHODS: The preformed vesicles encapsulating 30 mM glutathione were labeled with technetium-99m ((99m)Tc) for scintigraphic imaging. The vesicles were 216 +/- 21 nm in diameter with a negative surface charge composed of DPPC, cholesterol, anionic amphiphile and poly(ethylene glycol)-DSPE (1:1:0.2:0.013 molar ratio). RESULTS: The whole-body images of rhesus monkeys receiving intravenous (99m)Tc vesicles revealed high uptake of the (99m)Tc vesicles in bone marrow. Based on image analysis, we estimated that approximately 70% of the injected dose of the (99m)Tc vesicles was taken up by the bone marrow. CONCLUSION: This finding increases the feasibility of using this bone marrow-specific drug-delivery system for clinical applications.

Hb-vesicles (HbV) are artificial O(2) carriers encapsulating concentrated Hb solution (35 g/dL) with a phospholipid bilayer membrane (liposome). The concentration of the HbV suspension is extremely high ([Hb] = 10 g/dL) and it has an O(2) carrying capacity that is comparable to that of blood. HbV is much smaller than RBC (250 vs. 8000 nm), but it recreates the functions of RBCs; (i) the slower rate of O(2) unloading than Hb solution; (ii) colloid osmotic pressure is zero; (iii) the viscosity of a HbV suspension is adjustable to that of blood; (iv) HbV is finally captured by and degraded in RES; (v) co-encapsulation of an allosteric effector to regulate O(2) affinity; (vi) the lipid bilayer membrane prevents direct contact of Hb and vasculature; (vii) NO-binding is retarded to some extent by an intracellular diffusion barrier, and HbV does not induce vasoconstriction. (viii) Both RBC and HbV can be a carrier of not only O(2) but also exogenous CO. However, HbV has limitations such as a shorter functional half-life when compared with RBCs. On the other hand, the advantages of HbV are that it is pathogen-free and blood-type-antigen-free; moreover, it can withstand long-term storage of a few years, none of which can be achieved by the RBC transfusion systems.

Polymer-conjugated nanoparticles are an important technology to control the stability, safety, and efficacy in drug delivery systems. Herein, we investigate self-organized mixed assemblies of a lipophilic drug candidate, curcumin (Cm), and a poly(oxyethylene) cholesteryl ether (PEG-Chol). Cm was assembled together with PEG-Chol to form nano-sized assemblies (around 10 nm) of assumed micelles. In contrast with the rapid decomposition of free Cm due to the hydrolysis, the Cm was highly stabilized in the nanoparticles, especially at below 40 mol% Cm. Cell viability assay revealed that the cytotoxic activity of the Cm/PEG-Chol nanoparticles against myeloma cells is higher than those of free Cm in a comparison at 1 microM. On the other hand, both the Cm/PEG-Chol nanoparticles and PEG-Chol micelles had significant cytotoxicity to the myeloma cells at 5 microM. Taken together, the present Cm/PEG-Chol system offers a stable nanoparticle encapsulating Cm which can be injected as a liquid. Cm and vehicle micelles will damage the cancer cells cooperatively.

Hemoglobin-vesicles (HbV) or liposome-encapsulated hemoglobin (LEH) are artificial oxygen carriers that mimic the cellular structure of RBCs. In contrast to other liposomal products containing antifungal or anticancer drugs, one injection of HbV in place of a blood transfusion is estimated as equivalent to a massive dose, such as several hundred milliliters or a few liters of normal blood contents. The fluid must therefore contain a sufficient amount of Hb, the binding site of oxygen, to carry oxygen like blood. Encapsulation of Hb can shield various toxic effects of molecular Hbs. On the other hand, the liposomal structure, surface property, and the balance between the stability for storage and blood circulation and instability for the prompt degradation in the reticuloendothelial system must be considered to establish an optimal transfusion alternative.

Hemoglobin (Hb, Mw: 64 500) and albumin (Mw: 66 500) are major protein components in our circulatory system. On the basis of bioconjugate chemistry of these proteins, we have synthesized artificial O(2) carriers of two types, which will be useful as transfusion alternatives in clinical situations. Along with sufficient O(2) transporting capability, they show no pathogen, no blood type antigen, biocompatibility, stability, capability for long-term storage, and prompt degradation in vivo. Herein, we present the latest results from our research on these artificial O(2) carriers, Hb-vesicles (HbV) and albumin-hemes. (i) HbV is a cellular type Hb-based O(2) carrier. Phospholipid vesicles (liposomes, 250 nm diameter) encapsulate highly purified and concentrated human Hb (35 g/dL) to mimic the red blood cell (RBC) structure and eliminate side effects of molecular Hb such as vasoconstriction. The particle surface is modified with PEG-conjugated phospholipids, thereby improving blood compatibility and dispersion stability. Manipulation of physicochemical parameters of HbV, such as O(2) binding affinity and suspension rheology, supports the use of HbV for versatile medical applications. (ii) Human serum albumin (HSA) incorporates synthetic Fe(2+)porphyrin (FeP) to yield unique albumin-based O(2) carriers. Changing the chemical structure of incorporated FeP controls O(2) binding parameters. In fact, PEG-modified HSA-FeP showed good blood compatibility and O(2) transport in vivo. Furthermore, the genetically engineered heme pocket in HSA can confer O(2) binding ability to the incorporated natural Fe(2+)protoporphyrin IX (heme). The O(2) binding affinity of the recombinant HSA (rHSA)-heme is adjusted to a similar value to that of RBC through optimization of the amino acid residues around the coordinated O(2).

Blood transfusion systems have greatly benefited human health and welfare. Nevertheless, some problems remain: infection, blood type mismatching, immunological response, short shelf life, and screening test costs. Blood substitutes have been under development for decades to overcome such problems. Plasma component substitutes have already been established: plasma expanders, electrolytes, and recombinant coagulant factors. Herein, we focus on the development of red blood cell (RBC) substitutes. Side effects hindered early development of cell-free hemoglobin (Hb)-based oxygen carriers (HBOCs) and underscored the physiological importance of the cellular structure of RBCs. Well-designed artificial oxygen carriers that meet requisite criteria are expected to be realized eventually. Encapsulation of Hb is one idea to shield the toxicities of molecular Hbs. However, intrinsic issues of encapsulated Hbs must be resolved: difficulties related to regulating the molecular assembly, and management of its physicochemical and biochemical properties. Hb-vesicles (HbV) are a cellular type of HBOC that overcome these issues. The in vivo safety and efficacy of HbV have been studied extensively. The results illustrate the potential of HbV as a transfusion alternative and promise its use for other clinical applications that remain unattainable using RBC transfusion.

Hemoglobin vesicle (HbV) is an artificial oxygen carrier that encapsulates solution of purified and highly concentrated (ca. 38 g dL(-1)) human hemoglobin. Its exceptionally high concentration as a liposomal product (ca. 40% volume fraction) achieves an oxygen-carrying capacity comparable to that of blood. We use small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) to investigate the hierarchical structures and dynamics of HbVs in concentrated suspensions. SAXS data revealed unilamellar shell structure and internal density profile of the artificial cell membrane for Hb encapsulation. The SAXS intensity of HbV at scattering vector q > 0.5 nm(-1) manifests dissolution states of the encapsulated Hbs in the inner aqueous phase of the vesicle having ca. 240 nm diameter. The peak position as well as the height and width of static structure factor of Hb before and after encapsulation are almost identical, demonstrating the preserved protein-protein interactions in the confined space. To overcome multiple scattering from turbid samples, we employed thin layer-cell DLS combined with the so-called bruteforce and echo techniques, which allows us to observe collective diffusion dynamics of HbVs without dilution. A pronounced slowdown of the HbV diffusion and eventual emergence of dynamically arrested state in the presence of high-concentration plasma substitutes (water-soluble polymers), such as dextran, modified fluid gelatin, and hydroxylethyl starch, can be explained by depletion interaction. A significantly weaker effect of recombinant human serum albumin on HbV flocculation and viscosity enhancement than those induced by other polymers is clearly attributed to the specificity as a protein; its compact structure efficiently reduces the reservoir polymer volume fraction that determines the depth of the attractive potential between HbVs. These phenomena are technically essential for controlling the suspension rheology, which is advantageous for versatile clinical applications.

Liposome encapsulated hemoglobin is a long-sought goal in Japan, where the product is called hemoglobin vesicles (HbV), which distinguishes this product from the one developed primarily in the US, whose designation is liposome-encapsulated Hb (LEH). HbV is the result of a long series of studies in which the size of the vesicles, including the number of lipid layers, the surface composition and materials co-encapsulated have been optimized. HbV is produced by an extrusion process that has commercial potential, although at this time the product has not yet been produced in quantities sufficient for clinical trials. Sterilization of the hemoglobin, prior to encapsulation, is performed using heat, and antioxidants are co-encapsulated to retard hemoglobin oxidation. Oxygen affinity is regulated to any desired P50 by co-encapsulation of allosteric effectors, and this group has contributed important studies on the effect of different P 50 on oxygen delivery to tissues by HbV. The product is claimed to be stable when stored for up to 2 years. A commercial effort has been launched in Japan, and it is hoped that HbV could be in human clinical trials within the next few years. This chapter summarizes the characteristics of the preparation process of HbV based on the sciences of molecular assembly to induce their excellent performances. © 2006 Elsevier Ltd. All rights reserved.

Considering the physiological significance of the cellular structure of a red blood cell (RBC), it may be reasonable to mimic its structure for designing a hemoglobin (Hb)-based oxygen carrier. In this chapter, we have summarized the characteristics and performances of Hb-vesicles (HbV) that have been developed on the basis of molecular assembly. Collaborative in vitro and in vivo studies have revealed sufficient safety and efficacy of HbV.

Since the discovery of a red-colored saline solution of a heme derivative that reversibly binds and releases oxygen (1983), significant efforts have been made to realize an oxygen infusion as a red cell substitute based on the sciences of both molecular assembling phenomena and macromolecular metal complexes. The authors have specified that hemoglobin (Hb)-vesicles (HbV) and recombinant human serum albumin-hemes (rHSA-heme) would be the best systems that meet the clinical requirements. (A) Hb is rigorously purified from outdated, donated red cells via pasteurization and ultrafiltration, to completely remove blood type antigen and pathogen. The HbV encapsulates thus purified concentrated Hb solution with a phospholipid bimolecular membrane (diameter, 250 nmø), and its solution properties can be adjusted comparable with blood. Surface modification of HbV with a water-soluble polymer ensures stable dispersion state and storage over a year at 20°C. In vivo tests have clarified the efficacy for extreme hemodilution and resuscitation from hemorrhagic shock, and safety in terms of biodistribution, metabolism in reticuloendothelial system (RES), clinical chemistry, blood coagulation, etc. The HbV does not induce vasoconstriction thus maintains blood flow and tissue oxygenation. (B) rHSA is now manufactured in Japan as a plasma-expander. The rHSA can incorporate eight heme derivatives (axial base substituted hemes) as oxygen binding sites, and the resulting rHSA-heme is a totally synthetic O2-carrier. Hb binds endothelium-derived relaxation factor, NO, and induces vasoconstriction. The rHSA-heme binds NO as Hb does, however, it does not induce vasoconstriction due to its low pI (4.8) and the resulting low permeability across the vascular wall (1/100 of Hb). A 5%-albumin solution possesses a physiologic oncotic pressure. Therefore, to increase the O2-transporting capacity, albumin dimer is effective. Albumin dimer can incorporate totally 16 hemes with a regulated oncotic pressure. The rHSA-heme is effective not only as a red cell substitute but also for oxygen therapeutics (e.g. oxygenation for tumor). Significant efforts have been made to produce HbV and rHSA-heme with a facility of Good Manufacturing Practice (GMP) standard, and to start preclinical and finally clinical trials. Copyright © 2005 John Wiley & Sons, Ltd.

ヘモグロビン(Hb)を濃度高くリン脂質小胞体の内水相に内包したヘモグロビン小胞体(HbV)が人工酸素運搬体として開発され,赤血球と同等の酸素運搬機能と安全性が動物投与試験から明らかにされた.界面活性剤deca(oxyethylene)dodecyl ether(C12E10))でHbVを溶解してリポポリサッカライド(LPS)を遊離させた後,リムルステスト試薬と混合し,ゲル化反応を比濁時間分析法によって解析する方法を検討した.0.1EU/mLまでの検出限界を得ることができ,添加回収法によりその妥当性を検証することができた

The intervesicular aggregation of phospholipid vesicles is induced by the addition of water-soluble polymers such as poly(ethylene glycol), dextran, etc. due to the interaction between the vesicular surface and the water-soluble polymers. The interaction can be expressed by the critical molecular weight (M(c)) of the water-soluble polymers for the aggregation of vesicles. The surface modification of vesicles with glycolipids (O-1,O-5-bis(octadecyl) N-maltooligonoyl-L-glutamate) accelerates the aggregation of vesicles induced by dextran; therefore, M(c) significantly decreased due to the surface modification. No dependence of phospholipid concentration and dextran concentration in an aqueous phase on the M(c) indicates that dextran does not act as a cross-linking agent among the vesicles. A clear dependence of the density of the saccharide chains on the vesicular surface on the M(c) suggests that dextran should adsorb on the surface of the vesicles by the interaction with the oligosaccharide chains on the surface and cause vesicular aggregation. A lower critical solution temperature was observed for this kind of interaction, and the critical temperature was controlled by changing the molecular weight of dextran.