Acidic pH-induced changes in lipid nanoparticle membrane packing.

To enable the release of the encapsulated nucleic acids into the cytosol of targeted cells, the interaction of lipid nanoparticles (LNPs) with endosomes is critical. We investigated changes in the physicochemical properties of LNPs containing ionizable cationic lipids that were induced by acidic pH, which reflects the conditions in the maturation of endosomes. We prepared a LNP containing an ionizable cationic lipid. The laurdan generalized polarization values, which are related to the hydration degree of the lipid membrane interface and are often used as an indicator of membrane packing, decreased with a decrease in pH value, showing that the membrane packing was decreased under acidic conditions. Furthermore, the pH-induced variation increased with an increasing percentage of ionizable cationic lipids in the LNPs. These results indicated that electrostatic repulsion between lipid molecules at acidic pH decreased the packing density of the lipids in the LNP membrane. Reducing the order of lipids could be a trigger to form a non-bilayer structure and allow fusion of the LNPs with the membrane of maturing endosomes in an acidic environment. The LNPs were used to incorporate and transport small interfering RNA (siRNA) into cells for knockdown of the expression of ß-galactosidase. The knockdown efficiency of siRNA encapsulated in LNPs tended to increase with the ratio of KC2. These results, which demonstrate the underlying phenomena for the fusion of membranes, will help clarify the mechanism of the release of encapsulated nucleic acids.

Physicochemical Characterization of Liposomes That Mimic the Lipid Composition of Exosomes for Effective Intracellular Trafficking.

Exosomes mediate communication between cells in the body by the incorporation and transfer of biological materials. To design an artificial liposome, which would mimic the lipid composition and physicochemical characteristics of naturally occurring exosomes, we first studied the physicochemical properties of exosomes secreted from HepG2 cells. The exosome stiffness obtained by atomic force microscopy was moderate. Some liposomes were then fabricated to mimic the representative reported lipid composition of exosomes. Their physicochemical properties and cellular internalization efficiencies were investigated to optimize the cellular internalization efficiency of the liposomes. A favorable internalization efficiency was obtained by incubating HeLa cells with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Chol)/1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) (40/40/20 mol %) liposomes, which have a similar stiffness and zeta potential to exosomes. A dramatic increase in internalization efficiency was demonstrated by adding DOPS to simple DSPC/Chol liposomes. We found that DOPS had a more desirable effect on cellular internalization than its saturated lipid counterpart, 1,2-distearoyl-sn-glycero-3-phospho-l-serine. Furthermore, it was shown that the phosphatidylserine-binding protein, T-cell immunoglobulin mucin protein 4, was largely involved in the intracellular transfer of DSPC/Chol/DOPS liposomes. Thus, DOPS was a key lipid to provide the appropriate stiffness, zeta potential, and membrane surface affinity of the resulting liposome. Our results may help develop efficient drug carriers aiming to internalize active substances into cells.

The elution behavior of cyclosporine congeners in a developed HPLC system reflects the lipophilicity.

Recently, the development of cyclic peptide drugs has accelerated. To develop an analytical method to determine the physicochemical properties of these lipophilic drug candidates, we investigated the separation mechanism of cyclosporine congeners A, B, C, and D using HPLC with a column packed with 2-µm nonporous octadecylsilyl silica particles at high temperature. The four congeners were eluted with good repeatability in terms of retention time, peak area, and theoretical plate number. A difference of one amino acid in the eleven amino-acid sequence of the cyclosporine congeners was able to be recognized by our system within 4 min by isocratic elution, and the resolution was greater than 1.68. The calculated logP values of these congeners were well correlated with the retention factors with a correlation coefficient of 0.991. We could elucidate the separation mechanism of cyclosporine congeners on the high-temperature HPLC system. These results show that this method using HPLC on a column packed with 2-µm nonporous octadecylsilyl silica particles can be used for studying the lipophilicity of cyclosporine congeners.

Refining Calibration Procedures of Circular Dichroism Spectrometer to Improve Usability.

Circular dichroism (CD) is a technique used for conformational studies of peptides and proteins. We studied the specific calibration procedures of CD spectrometers based on procedures specified in the European Pharmacopoeia. We aimed to develop procedures to improve the usability of CD, in addition to reducing adverse effects on users' health. The use of ethanol instead of 1,4-dioxane as the solvent for isoandrosterone was examined. Both solvents yielded the same maximum value of +3.3 for molar CD. We also studied a two-point calibration method using (1S)-(+)-ammonium 10-camphorsulfonate instead of (1S)-(+)-10-camphorsulfonic acid, which is a hygroscopic compound. Both compounds yielded similar results and the values for (1S)-(+)-ammonium 10-camphorsulfonate of 2.39 ± 0.04 and -4.92 ± 0.06 at 290.5 and 192.5 nm, respectively, were within the criteria defined in the European Pharmacopoeia. The inter-laboratory repeatability was also acceptable. These studies provide specific procedures for calibrating CD spectrometers for drug development.

Effect of surface charge on the size-dependent cellular internalization of liposomes.

Here we report that the size dependence of cellular internalization of liposomes differs depending on the surface charge. We prepared liposomes of various lipid compositions ranging from 100 to 200 nm size. It was found that cationic liposomes composed of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) were most effectively internalized into cells when their mean particle sizes were around 180 nm. When their size was reduced to around 90 nm, the level of internalization reduced six-fold. Conversely, hydrogenated soy phosphatidylcholine (HSPC)/N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG2000-DSPE)/cholesterol(Chol) liposomes, HSPC/PEG2000-DSPE liposomes, and HSPC/Chol liposomes were most readily internalized when they were around 110 to 130 nm in mean particle size. Unlike DOPC/DOTAP liposomes the difference between the maximum and minimum levels of internalization was less than two-fold. It has been suggested that strong electrostatic interactions between cationic liposomes and the negatively charged plasma membrane affect the size dependence and optimal size range for internalization of liposomes. Size dependence of internalization should be carefully monitored for effective formulation development and quality control of liposome drug products.

[An elderly non-Hodgkin lymphoma patient with a massive tumor of the heart].

We report on an elderly patient with a malignant lymphoma forming a huge mass in the heart. An 82-year-old woman became aware of general fatigue and a cough in August 1999. Her right supraclavicular, bilateral axillary, and right inguinal lymph nodes were swollen. A hypodermical mass in the right frontal chest was detected. Her left axillary lymph node was biopsied. She was diagnosed as having non-Hodgkin lymphoma, diffuse large cell type, B-cell type. Computed tomography scans showed a markedly thickened right ventricular wall of the heart, swollen lymph nodes of the mediastinum, bilateral pleural effusions, and a tumor in the spleen. Lymphoma cells were found in the pleural effusion, and the lymphoma was diagnosed as clinical stage IV. Hypofunction of the heart, ejection fraction (EF) 49%, was demonstrated with transthoracic echocardiography. EF increased to 70% after 3 courses of chemotherapy with CHOP regimen. All lesions disappeared after 6 courses of chemotherapy were completed. After consolidative radiotherapy with a total dose of 37 Gy to the mediastinum and heart, bilateral pleural effusions, elevation of the patient's lactate dehydrogenase level and soluble IL-2 receptor value were recognized, which suggested relapse of the lymphoma, although histopathological confirmation could not be realized.

[Clinical study of individual TS-1 therapy for inoperable gastric cancer].

TS-1 is a novel oral anticancer drug that is a formation of 5-FU. It consists of tegafur, CDHP (which inhibits 5-FU degradation enzyme), and Oxo (which reduces gastrointestinal toxicities) for an increased anticancer effect. We applied individual TS-1 therapy in 22 cases (cs) of inoperable gastric cancer and studied the clinical and adverse effects. Patients were treated with daily oral administration of 80-100 mg TS-1 for 4 weeks, followed by a rest for 1 or 2 weeks. The response rate was found to be 27.3% (6/22) (PR: 6 cs, NC: 4 cs, PD: 10 cs, NE: 2 cs). Overall, the median survival time was 8.2 months and the one-year survival rate was 23.6%. By location, the response rate of the primary lesion was 27.3% (6/22), abdominal lymph node metastasis 18.8% (3/16), and liver metastasis 33.3% (4/12). There was no significant difference in the response rate by tissue type. A comparison by whether or not patients had undergone previous chemotherapy revealed a response rate of 37.5% (6/16) in patients who had undergone previous chemotherapy, and 0% (0/6) in those who had not. The prevalence of adverse effects was 68.2% (15/22), with the main adverse effects being myelosuppression, pigmentation and appetite loss. However, adverse effects with a grade of more than 3 occurred in only one case of neutropenia. We could observe the course of all patients on an outpatient basis.