Critical micelle concentration and particle size determine adjuvanticity of cyclic lipopeptides.

Cyclic lipopeptides such as surfactin and polymyxin have potent mucosal adjuvant properties. Cyclic lipopeptides are tensioactive compounds but the relationship between adjuvanticity and surface activity is unknown. Here, we show that the critical micelle concentration (cmc) of surfactant and particle size of the surfactant-protein complex are important determinants of cyclic lipopeptide adjuvanticity. We found that the diameter of cyclic lipopeptide-ovalbumin (OVA) complex particles was significantly larger than that in the solutions of OVA alone at cyclic lipopeptide concentrations above the cmc. OVA-specific antibody titers in mice immunized intranasally with OVA and a cyclic lipopeptide at concentrations above its cmc were significantly higher than those in mice immunized with OVA plus the same dose of the cyclic lipopeptide but administered with formulations in which cyclic lipopeptide concentration was below the cmc. Thus, the concentration of the cyclic lipopeptide in the formulation at immunization, but not its overall dose, was critical for its adjuvanticity. Furthermore, two types of aggregates, the cyclic lipopeptide simplex micelles and the cyclic lipopeptide-OVA complex micelles, were found in formulations with SF concentrations above its cmc. Degranulation of mast cells exposed to SF simplex micelles was more pronounced when SF concentration was above the cmc. In conclusion, our study showed that surface activity properties, such as the cmc and the size of surfactant-protein complex contribute to the adjuvanticity of cyclic lipopeptides. Our study proposes a novel idea that cmc is a key parameter for tensioactive adjuvants. This article is protected by copyright. All rights reserved.

Preparation of Transdermal Gel Containing l-Theanine for the Potential Treatment of Premenstrual Syndrome: A Preclinical Study.

Background: Premenstrual syndrome (PMS) is experienced by many women who suffer from either its psychological or physical symptoms. Current treatment is limited to symptomatic therapy or oral contraceptives. On the other hand, l-theanine, which has a relaxant effect, has been reported to be useful for PMS, but its short half-life when administered orally makes it less effective. Permeability and properties of transdermal gel containing l-theanine were evaluated as a preclinical study of PMS symptoms relief formulation. Materials and Methods: Lyogel composed of stearic acid, stearyl alcohol, and propylene glycol was selected. The ratio of these components and the preparation method were investigated. Permeation of Strat-M membranes was evaluated by using Franz cells (in vitro). Moreover, lyogel was applied to institute of cancer research mice's backs for 10 days to examine the permeability of l-theanine. Results: l-Theanine solution did not permeate the Strat-M membrane at all in the permeation study, but lyogel allowed l-theanine to permeate. When the composition of lyogel was 4.4:11.1:296 (mmol) for stearic acid, stearyl alcohol, and propylene glycol, l-theanine absorption through Strat-M membrane was better. In skin permeation study using mice, l-theanine was detected in the serum, that is, it was proven that l-theanine penetrated the skin. Conclusion: The preparation of transdermal gels contained l-theanine was investigated as a preclinical study. The skin permeability of semisolid formulations of hydrophobic ointments, hydrophilic ointments, oily creams, creams, and lyogel containing theanine was compared and found that lyogel was the best. The composition of lyogel was also studied to obtain a formulation with good application comfort. Although it is suggested that this lyogel could be tested in clinical studies to determine whether it is effective for relief of PMS symptoms, lyogel may be suitable as an easy-to-use l-theanine-containing formulation for women that can relieve PMS symptoms.

Change in the character of liposomes as a drug carrier by modifying various polyethyleneglycol-lipids.

When liposomes, as a superior drug carrier, are injected intravenously, active liposomes as medicines require polyethyleneglycol (PEG) as a modification tool around the liposomal membrane. PEG modification of a liposome forms a fixed aqueous layer, and the trapping by cells of the reticuloendothelial system (RES) is avoided. Hence, PEG-modified liposomes have long circulation in the bloodstream, and passive targeting to tumors has been achieved by PEG modification. We have been studying the passive targeting by liposomes with the expectation of more usefulness. It was proved a correlation between the PEG molecular weight of PEG-modified liposomes and blood circulation time and antitumor effect, too. Liposomes modified by PEG2000 were useful for uptake into tumor cells. We thought that the re-uptake in the liposomal membrane also increased accumulation. Moreover, it was proved that mixing two different PEGs to modify the liposome surface gives a bigger fixed aqueous layer thickness (FALT) around the liposome, giving the liposome strong antitumor activity. Then, we designed a novel PEG-lipid, 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-PEG (different double arms PEG; DDA-PEG), which had two different PEGs (2000 and 500) in one molecule. One of the innovative characteristics of DDA-PEG-modified liposome (DDAL) is that it heightens the contact ability with tumor cells. DDAL may be an effective DDS carrier for solving various PEG dilemmas. It was observed that passive targeting by PEG-modified liposomes had different characteristics by changing PEG length, anchor type or those combination. Thus, it should be applied to liposomes suitable for various diseases.

The relationship between the chemical structure, physicochemical properties, and mucosal adjuvanticity of sugar-based surfactants.

The relationship between the chemical structure, physicochemical properties, and mucosal adjuvanticity of sugar-based surfactants (SBSs) has not been sufficiently elucidated. Thus, in the present study, we systematically analyzed 11 SBSs for mucosal adjuvanticity. Ovalbumin (OVA)-specific antibody titers were measured in mice immunized intranasally with OVA plus SBS. We found that four SBSs (trehalose monododecanoate, sucrose monododecanoate, n-dodecyl-α-d-maltopyranoside, and n-dodecyl-ß-d-maltopyranoside) exhibited the most potent adjuvanticity. We identified the following associations between chemical structure and adjuvanticity: 1) OVA-specific antibody titer increased with an increasing number of carbon atoms in the alkyl chain; 2) the adjuvanticity was not affected by the type of sugar or bond between the sugar and alkyl chain; and 3) SBSs with rigid structures exhibited less adjuvanticity. The relationship between physicochemical properties and adjuvanticity was as follows: 1) SBSs exhibited adjuvanticity above the critical micelle concentration and 2) in the SBSs with potent adjuvanticity, the diameter of the SBS-OVA complex was 70-75 nm. Our study indicates evidence for the direct involvement of chemical structure and physicochemical properties in determining adjuvanticity in SBSs.

Suitability of Polymyxin B as a Mucosal Adjuvant for Intranasal Influenza and COVID-19 Vaccines.

Polymyxin B (PMB) is an antibiotic that exhibits mucosal adjuvanticity for ovalbumin (OVA), which enhances the immune response in the mucosal compartments of mice. Frequent breakthrough infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants indicate that the IgA antibody levels elicited by the mRNA vaccines in the mucosal tissues were insufficient for the prophylaxis of this infection. It remains unknown whether PMB exhibits mucosal adjuvanticity for antigens other than OVA. This study investigated the adjuvanticity of PMB for the virus proteins, hemagglutinin (HA) of influenza A virus, and the S1 subunit and S protein of SARS-CoV-2. BALB/c mice immunized either intranasally or subcutaneously with these antigens alone or in combination with PMB were examined, and the antigen-specific antibodies were quantified. PMB substantially increased the production of antigen-specific IgA antibodies in mucosal secretions and IgG antibodies in plasma, indicating its adjuvanticity for both HA and S proteins. This study also revealed that the PMB-virus antigen complex diameter is crucial for the induction of mucosal immunity. No detrimental effects were observed on the nasal mucosa or olfactory bulb. These findings highlight the potential of PMB as a safe candidate for intranasal vaccination to induce mucosal IgA antibodies for prophylaxis against mucosally transmitted infections.

Beneficial effects by intake of Euphausiacea pacifica on high-fat diet-induced obesity.

Obesity is a major health problem showing increased incidence in developed and developing countries. We examined the effect of Euphausia pacifica (E. pacifica) (Pacific Krill) on high-fat diet (HFD)-induced obesity in C57BL/6 mice. No significant differences were observed in average food intake between the HFD and HFD with E. pacifica group, or the low-fat diet (LFD) and LFD with E. pacifica group for 18 weeks. The increased ratio of body weight in the HFD containing E. pacifica group was significantly reduced, being 10% lower than that with HFD group in the 18th week (HFD, 298.6±18.8% vs. HFD with E. pacifica, 267.8±16.2%; p<0.05), while the ratio for the LFD containing E. pacifica group was reduced by 4% compared with LFD group (LFD, 244.2±11.6% vs. LFD with E. pacifica, 234.1±18.0%). There were no effects of E. pacifica on total cholesterol levels in serum and liver, whereas the supplement of E. pacifica tended to decrease triglyceride levels in the HFD groups. The leptin level in serum was significantly decreased in the HFD group (p<0.01) by E. pacifica. The adipocyte area (1926±1275 µm(2)) in the HFD containing E. pacifica group was significantly reduced by 20% (p<0.001) compared with the HFD group. These results suggested that E. pacifica supplementation in the diet is beneficial for the prevention of HFD-induced obesity.

The Basic Study of Liposome in Temperature-Sensitive Gel at Body Temperature for Treatment of Peritoneal Dissemination.

Peritoneal dissemination is a disease that is difficult to treat surgically because it is widely scattered and proliferates in the abdominal cavity. It is a challenge that even if the drug is administered directly into the abdominal cavity, it rapidly disappears from the abdominal cavity, and the therapeutic effect is not optimal, as expected. In this study, for a liposomal paclitaxel in temperature-sensitive gel that is a suspension before administration and a gel after intraperitoneal administration, the antitumor effect of this formulation was evaluated. Temperature-sensitive gels were prepared using methylcellulose, sodium citrate, and macrogol 4000 and mixed with liposomal paclitaxel. Liposomal paclitaxel containing temperature-sensitive gel in the body was administered into the peritoneal cavity of a mouse model of peritoneal dissemination; the number of cells was significantly reduced compared to a paclitaxel solution of liposomal paclitaxel. These results showed that the liposome in temperature-sensitive gel inhibited cell proliferation in the abdominal cavity. This formulation can be administered easily at room temperature, and it gels and remains in the abdominal cavity for a long period, resulting in a more substantial effect than the existing drug.

A systematic assessment of the relationship between synthetic surfactants and mucosal adjuvanticity.

Intranasal immunization with surfactants as vaccine adjuvants enhances protective immunity against invasive mucosal pathogens. However, the effects of surfactants and their adjuvanticity on mucosal immune responses remain unclear. Comparison of the mucosal adjuvanticity of 20 water-soluble surfactants from the four classes based upon the polarity composition of the hydrophilic headgroup revealed that the order of mucosal adjuvanticity was as follows: amphoteric > nonionic > cationic > anionic. Within the same class, each surfactant displayed different adjuvanticity values. Analysis of the diameter and ζ-potential of amphoteric surfactant-OVA complexes and their surface physicochemical properties revealed that the diameter was approximately 100 nm, which is considered suitable for immune induction, and that the ζ-potential of the anionic surfactant-OVA complexes was exceedingly negative. The increase in the number of carbon atoms in the hydrophobic tailgroups of the amphoteric surfactant resulted in an increase in the OVA-specific Ab titers. Our findings demonstrate that amphoteric surfactants exhibit potent mucosal adjuvanticity and highlight the importance of the number of carbon atoms in the tailgroups and the diameter and ζ-potential of the complexes when designing mucosal adjuvants.

[Utility and improvement of liposome injections AmBisome for clinical use].

We have studied AmBisome, which is amphotericin B containing liposomes, used in the treatment of mycosis. Any potential problems regarding the liposome formulation remain unknown because it is a new dosage form. AmBisome is useful in that it can decrease adverse reactions while maintaining a therapeutic effect. However, it is doubtful whether AmBisome is suitable in a busy clinical environment. For example, this formulation should be filtered with an established filter. We then carried out a questionnaire survey involving medical staff (doctors and nurses) to clarify the problems regarding the liposome formulation and its practical utilization. Most doctors were satisfied with the effect of AmBisome, but about 90% of nurses who had used the preparation answered that it was troublesome to use the filter. On the other hand, only 40% of doctors understood how to use the filter when AmBisome was made up; 14% of nurses also did not know how to use the filter. We thought this was because they were not shown how to use it; actually, 30% of doctors were shown the preparation method by medical representatives (MR), although no nurse received an explanation. The residual rate of amphotericin B on 100 used filter pieces differed 50-fold between the minimum and maximum values. AmBisome is effective as an antifungal agent. However, pharmaceutical companies must liaise with medical staff for its effective clinical use. We hope that such companies will have exchanges with medical staff to develop safe and simple medicines, and that liposomes will be effective for many patients.

Phototoxicity of coproporphyrin as a novel photodynamic therapy was enhanced by liposomalization.

This study attempted the liposomalization of coproporphyrin I (CPI) with hydrophobic properties. Liposomalization of CPI was not successful at any pH when using lactate buffer. In contrast, when using 9% sucrose/10mM phosphate buffer (pH 7.8), CPI liposomes (Lipo-CPI) and polyethyleneglycol (PEG) modified liposomes (PEG-CPI) were prepared with a high entrapment ratio of CPI and small particle size. Plasma CPI concentration at 6h after PEG-CPI injection were 6.5-fold greater than that after the injection of Lipo-CPI. In tumors, the CPI concentration was higher after PEG-CPI injection than after Lipo-CPI or CPI solution. Therefore, PEG-CPI was likely to increase blood circulation and achieve greater accumulation of CPI in the tumor. When loaded into tumor cells, photosensitizers generate singlet oxygen during laser irradiation, resulting in the induction of necrosis in the cells. The order of magnitude of CPI tumor cells uptake was PEG-CPI>Lipo-CPI>CPI solution. Thus, the PEG modification of CPI liposomes improved its tumor cell uptake. Furthermore, it is likely that the order of the ability to produce singlet oxygen was PEG-CPI [symbol: see text] Lipo-CPI>CPI solution. The cytotoxicity of PEG-CPI was significantly greater than the other formulations, suggesting that the cytotoxicity reflected the CPI concentration in tumor cells. In conclusion, PEG-CPI was confirmed to show effective tissue distribution, elevated CPI concentration in the tumor cells, to produce singlet oxygen, and cytotoxicity by PDT.

[The Collaboration between Clinical Therapy and Academic Study to Benefit Patients].

　Cancer chemotherapy-induced stomatitis may spread throughout a patient's entire oral cavity and decrease the patient's QOL. The therapy for stomatitis at Iwate Medical University Hospital (IMUH) includes dental treatment before chemotherapy, in addition to oral rinses or cryotherapy as a preventative measure. However, in our survey of doctors and nurses, the responses of patients "satisfied" with the present approach for stomatitis treatment reached only 5.1%. Therefore, we attempted treatment using an indomethacin spray, prepared as a hospital preparation, with pre-approval of the ethics committee and based on a previous report of its positive effect on patients at another hospital. We observed that the indomethacin spray succeeded in decreasing chemotherapy-induced oral pain, and its effect was maintained for 2 h in patients at IMUH. The ratio of female patients who rated indomethacin spray as good was higher than that of males. Comments from some patients included a complaint that the nozzle of the injection tip was too short; thus we increased the length of the nozzle from 2 to 7 cm. At present, indomethacin spray is being used to treat stomatitis patients at IMUH. Indeed, the indomethacin spray has been used since October 2011. It was used on 34 patients in 2016. In this review, we describe the collaboration between IMUH and the basic application of studies in our university laboratory.

Dual-effect liposomes with increased antitumor effects against 67-kDa laminin receptor-overexpressing tumor cells.

This study sought to evaluate the antitumor effects of and elucidate the mechanisms underlying (-)-epigallocatechin-3-O-gallate (EGCG) and polyethyleneglycol (PEG)-modified liposomes. EGCG functions as a target ligand of the 67-kDa laminin receptor (67LR), which is expressed on high-grade tumor cells. An EGCG derivative was synthesized for binding to the end of PEG. Doxorubicin (DOX)-loaded EGCG-PEG-modified liposome (EPL) significantly decreased tumor size in mice bearing high 67LR-high-expressing tumors. Caspase-3 activity, which indicates induction of apoptosis, was also elevated only in the EPL group. The importance of PEG for the antitumor effects of EGCG was noted, as soluble EGCG did not accumulate at a sufficient concentration to exert an apoptotic effect. Moreover, EPL significantly increased caspase-8 activity, suggesting that EPL-induced apoptosis occurred due to caspase-8 activity induced following the binding of EGCG to 67LR as a cell-death ligand. In conclusion, EPL appear to have superior antitumor activity against high 67LR-expressing tumor cells, as the liposomes had dual effects, namely antitumor effects due to the loaded DOX and apoptosis induced by the bound EGCG.

Study on liposomalization of zinc-coproporphyrin I as a novel drug in photodynamic therapy.

Photodynamic therapy (PDT) with a photosensitizer and laser irradiation has been shown to have potential effects in cancer chemotherapy. However, the commercial drug clinically gave many problems due to the poor solubility of the photosensitizer in water and the photosensitivity as an adverse reaction of PDT. We have examined best condition on the liposomalization of Zn-complexed coproporphyrin I (ZnCPI) as novel photosensitizer. The difference of pH in buffer significantly changed the ZnCPI entrapped ratio. The entrapped ratio of ZnCPI in PBS(-) buffer was 10.8+/-0.3%, whereas, these levels in some lactate buffer (below pH 5.0) increased. The change between the molecular form<=>ionic form of ZnCPI was occurred due to the change of the pH of buffer, and the amount of ZnCPI in the liposomal membrane changed. The difference of this level was considered to be contributed by the change of zeta potentials. Next, we examined the effect of the different pH of the buffer in liposomal preparation on the ZnCPI distribution in each tissue after each liposome administration. At 2 and 6h post-injection of ZnCPI liposome (pH 4.6), the ZnCPI concentration in the plasma of Ehrlich ascites carcinoma bearing mice was shown to be higher compared to that in other groups. The ZnCPI concentrations in the tumor after 2 and 6h of ZnCPI liposome (pH 4.6) treatment were shown to be higher than that in other groups. In conclusion, it is considered that the ZnCPI liposome (pH 4.6) had the effective antitumor activity with laser irradiation without the adverse reactions.

Effect of Liposomes with Different Double Arms Polyethyleneglycol on Hepatic Metastasis Model Mice and Evaluation Using a Fluorescent Imaging Device.

BACKGROUND: We have previously reported the synthesis of a novel polyethyleneglycol (PEG) lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (DDA-PEG). This study aimed to clarify the anti-metastatic effect and localization of DDA-PEG-modified liposomes on a murine hepatic metastasis model. METHODS: M5076 ovarian sarcoma cells were inoculated for hepatic metastasis model mice. The accumulation of liposomes in the tumor and metastatic sites was detected by fluorescent imaging device. In metastasis study, doxorubicin (DOX) loaded DDA-PEG-modified liposome (DDA-LDOX) was injected. Alexa Fluor 790 NHS Ester loaded DDA-PEG-modified liposomes were used to detect fluorescence intensity at metastatic sites when visualized topically using a fluorescence imaging device. RESULTS: DDA-PEG-modified liposomes accumulated at the sites of hepatic metastasis but not in the normal hepatocytes. Furthermore, the DDA-LDOX inhibited metastasis in this model. The survival time of M5076 ovarian sarcoma bearing mice in DDA-LDOX group was longer than those in control, DOX solution and the other PEG-modified liposomal DOX groups, and the survival ratio in DDALDOX group remained 66.7% until 60 days after treatment. CONCLUSION: It is expected that the DDA-PEG-modified liposomes will extensively contribute in clinical practice as a superior drug carrier because this liposomes proved to be effective against metastasis.

The phototoxicity of photofrin was enhanced by PEGylated liposome in vitro.

In recent years, photodynamic therapy (PDT) with a photosensitizer and laser has been given attention, especially for the treatment of superficial cancers, such as lung, gastric, bladder and cervical cancer. In this study, in order to enhance the efficacy of PDT, photofrin liposome (PF-Lip) was prepared with dimyristoylphosphatidylcholine, dimyristoylphosphatidylglycerol and cholesterol. Polyethyleneglycol modified photofrin liposome (PF-PEG-Lip) was prepared by modification of PF-Lip with monomethoxypolyethyleneglycol-2.3-dimyristoylglycerol. PF-Lip and PF-PEG-Lip entrapped with photofrin with 81.0+/-5.9 and 81.2+/-9.2%, respectively. The particle size of each liposome was 114.3+/-5.7nm (PF-Lip) and 118+/-3.5nm (PF-PEG-Lip), respectively. It was suggested that PEGylated liposomes has no effect on the trapping ratio of PF and particle size. Phototoxicity was enhanced by liposomalization, especially PEG-modification. However, PF-PEG-Lip inhibited the uptake of photofrin into tumor cells. The amount of singlet oxygen from photofrin solution (PF-sol) and each liposome was PF-PEG-Lip=PF-Lip>PF-sol. The photofrin release revel of PF-PEG-Lip was lower than that of PF-Lip. In conclusion, the phototoxicity of PF-PEG-Lip was significantly higher than that of PF-sol or PF-Lip. It is expected that formation of a fixed aqueous layer on the liposome membrane by PEGylation physically changed it into the stable state of PF-PEG-Lip.

Characterization and cytotoxicity of mixed polyethyleneglycol modified liposomes containing doxorubicin.

Liposomes are recognized as one of the useful drug carriers, but have many problems to overcome before their clinical application. Liposomes, bonding peculiarly with serum protein (opsonization), are taken up by reticuloendothelial system (RES) cells in the liver and spleen. It is known that polyethyleneglycol (PEG) modification of the liposome surface induces the formation of a fixed aqueous layer around the liposomes due to the interaction between the PEG-polymer and water molecule, and thus prevents the attraction of opsonins. Namely, PEG-modified liposomes are able to escape trapping by the RES cells, and have a prolonged circulation time. In this study, the effects of different anchors with the same PEG molecular weight on the cell uptake and cytotoxicity of mixed PEG-modified liposomal doxorubicin (DOX) were examined. The fixed aqueous layer thickness (FALT) of liposomes covered with mixtures of PEG-molecules which differ in their chain length were increased, compared to that of the single PEG2000-modified liposome. Mixed PEG-modification of liposomes with different anchors (PEG2000-(1-monomethoxypolyethyleneglycol-2,3-distearoylglycerol (DSG): cholesterol (CHO)=1:1)-modified liposome) led to an increase in the FALT, compared to that of each single PEG-modification. The uptake of DOX into Ehrlich ascites carcinoma cells by the liposomes covered with PEG-CHO was higher than the other liposomes. Thus, liposomes covered with PEG-DSG and PEG-CHO have an enhanced cytotoxicity. In conclusion, it was confirmed that mix-modified liposomes using PEG-lipid with different anchors were superior.

Effect of Mixing Time and Storage Condition on Characterization of Heparinoid Admixtures with Corticosteroids.

In dermatologic therapy, several external preparations formulated as ointments or creams are prescribed. And they are often admixture to improve patient compliance. In this study, we prepared admixtures of moisturizer with steroids and examined their usability and the amount of principal agent in formulations, particularly focusing on the moisturizer content. Four heparinoid semisolid formulations were selected: Hirudoid® soft ointment 0.3% (Formulation A) and 3 generic agents [(Besoften® oil-based cream 0.3% (Formulation B), Kuradoido® ointment 0.3% (Formulation C), and Hepadaerm ointment 0.3% (Formulation D)], and Antebate® ointment 0.05% (Formulation E) were used as steroids. Formulation A and B are water-in-oil emulsions, and Formulation C and D are oil-in-water emulsions. Admixtures looked like to be mixed uniformly by visual observation. In the examination of heparinoid amount, admixture A+E and B+E were mixed uniformly. On the other hand, admixture C+E was remarkable un-uniformly. It was speculated that the emulsification of formulation C was broken. The phenomenon was supported by the result of malleability. After 8 weeks storage, the heparinoid ratio in each formulation could be expressed as follows: Admixture B≥Admixture A>Admixture C=Admixture D. A suitable storage temperature was 4°C. The results of physicochemical data analysis reveal the formulations composed of water-in-oil cream, i.e., Formulation A and Formulation B, to be the optimal choices for mixing with steroid ointments. Mixing time and storage conditions may be optimized to solve pharmaceutical problems. Moreover, understanding the emulsion type and character of semisolid formulations can expand the range of formulation options.

Enhanced antitumor activity of different double arms polyethyleneglycol-modified liposomal doxorubicin.

The effect of polyethyleneglycol (PEG)-modified liposome as a drug carrier has been demonstrated clinically. We designed and synthesized a novel PEG-lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (different double arms PEG, DDA-PEG) which had two PEG chains of 500 and 2000 in one molecule to develop more useful PEG-modified liposome. DDA-PEG-modified liposomal doxorubicin (DDA-LDOX(7.5)) had the biggest fixed aqueous layer thickness (FALT) compared with other PEG-lipid-modified liposomes even if the added amount was a few. It was thought that FALT was the indication of blood circulation time. In DOX uptake in tumor cells, DDA-LDOX(7.5) group increased the DOX concentration in tumor cells because it had contact ability with tumor cells. Hence, DDA-LDOX(7.5) which has long circulation time in the bloodstream and contact ability with tumor cells, also had a strong antitumor effect on mice bearing M5076 ovarian sarcoma cells which were DOX low sensitive cells according to the expression of multidrug resistance protein. Furthermore, this liposome maintained a high DOX concentration in a tumor for a long time. These results indicated that the useful antitumor effect of DDA-LDOX(7.5) against M5076 ovarian sarcoma cells is a promising DDS carrier for therapies against drug resistant tumors.

[Preparation of liposome using egg yolk].

In this study, 162 students in the 6 year Pharmacy Program at the School of Pharmacy, Iwate Medical University were asked to prepare liposomal preparations using chicken egg yolk and to evaluate their properties with the aim of developing novel liposomes. High-purity lecithins are generally used for preparing liposomes but they are expensive. On the other hand, egg yolk has various components, including lecithin and cholesterol, which are important components for the formation of liposomes, so it was hypothesized that liposomes prepared from egg yolk may participate in the formation of cell membrane in chicks. Both liposomes from egg yolk (YL) and from lecithin (LL) exhibited Malthesian crosses using a polarizing microscope and multilamellar vesicles were observed, confirming that liposomal preparations from egg yolk were useful. The particle size of YL was about 100 nm with one peak. Furthermore, the YL are believed to be viable under different conditions because the particle size did not change when they were prepared in buffers having different pH values. The results of these experiments indicate that liposomal preparations from egg yolk can serve as natural materials, although some obstacles remain. This is a unique approach for carrying out practical training in our 6 year pharmaceutical science program.

Correlation of fixed aqueous layer thickness around PEG-modified liposomes with in vivo efficacy of antitumor agent-containing liposomes.

Liposomes are recognized as useful drug carriers, but have some problems to overcome. Liposomes are easily opsonized with serum proteins (opsonization) and taken up by the reticuloendothelial system (RES) cells, such as spleen and liver. Polyethyleneglycol (PEG) modification on the liposomal membrane forms a fixed aqueous layer and thus prevents opsonization and uptake by the RES. Our research indicates clearly that the electrical potential distributions near the membrane surfaces were different between doxorubicin (DOX)-containing liposomes with and without a PEG coating. Moreover, the value of the fixed aqueous layer thickness (FALT) around the liposome, formed by PEG modification, correlates with the circulation time and antitumor effect in a murine model. In this review, we introduce the observation that measurement of FALT as a physical characteristics is a useful method for demonstrating the antitumor effect of antitumor agent-containing PEG-modified liposomes. The use of this technique may preclude the performance of certain in vivo experiments. Our approach using FALT enables the rapid and reliable development of PEG-modified liposome formulations.