Novel gene delivery systems: complexes of fusigenic polymer-modified liposomes and lipoplexes.

We have previously developed the succinylated poly(glycidol)-modified liposome which becomes fusigenic under weakly acidic condition. In this report, we describe that complexation of this pH-sensitive, fusigenic liposome with a lipoplex consisting of 3beta-(N-(N',N'-dimethylaminoethane) carbamoyl)cholesterol, dioleoylphosphatidylethanolamine and plasmid DNA gives efficient gene delivery systems. In this study, we prepared the complexes, which are termed SucPG-complexes, with a positively or negatively charged surface by mixing the lipoplex with varying amounts of the SucPG-modified liposomes. The positively charged SucPG-complexes either bearing or not bearing a cell-specific ligand, transferrin, could transfect HeLa cells efficiently. In contrast, the negatively charged complexes hardly transfected the cells when transferrin was not conjugated to them. However, the negatively charged SucPG complexes bearing transferrin exhibited high transfection ability against HeLa and K562 cells, indicating that this gene delivery was achieved through their binding to the cellular receptors. These transferrin-attached, negatively charged complexes retained the high transfection ability in the presence of serum. Thus, this negatively charged complex may be useful as nonviral vectors in vivo.

Solvent effect on helicity induction of zinc bilinone bearing a chiral auxiliary at the helix terminal.

Solvent effects on helicity induction in zinc bilinone (ZnBL) derivatives bearing chiral auxiliaries at their 19-positions were investigated by using circular dichroic spectroscopy and (1)H NMR experiments. In ZnBLs 1 and 2, which possess (R)-2-methyl-1-phenylpropyloxy and (R)-1-phenylethyloxy groups at their 19-positions, respectively, the efficiency of helicity induction was significantly affected by employed solvents (78-95% de in 1 and 33-89% de in 2). The free energy changes of the P-M interconversion of 1 and 2 were linearly in proportion to reduction in polarizability of solvents: lower polarizability of solvents led to better efficiency of helicity induction in 1 and 2. With the support of the (1)H NMR study in addition to the molecular modeling previously reported, it was indicated that the solvophobic van der Waals interaction between the alkyl group in the chiral auxiliary and the A-ring of the bilinone skeleton in the preferred conformer plays a crucial role in determining the efficiency of helicity induction in 1 and 2.

Synthesis of polyamidoamine dendrimers having poly(ethylene glycol) grafts and their ability to encapsulate anticancer drugs.

Polyamidoamine dendrimers having poly(ethylene glycol) grafts were designed as a novel drug carrier which possesses an interior for the encapsulation of drugs and a biocompatible surface. Poly(ethylene glycol) monomethyl ether with the average molecular weight of 550 or 2000 was combined to essentially every chain end of the dendrimer of the third or fourth generation via urethane bond. The poly(ethylene glycol)-attached dendrimers encapsulating anticancer drugs, adriamycin and methotrexate, were prepared by extraction with chloroform from mixtures of the poly(ethylene glycol)-attached dendrimers and varying amounts of the drugs. Their ability to encapsulate these drugs increased with increasing dendrimer generation and chain length of poly(ethylene glycol) grafts. Among the poly(ethylene glycol)-attached dendrimers prepared, the highest ability was achieved by the dendrimer of the fourth generation having the poly(ethylene glycol) grafts with the average molecular weight of 2000, which could retain 6.5 adriamycin molecules or 26 methotrexate molecules/dendrimer molecule. The methotrexate-loaded poly(ethylene glycol)-attached dendrimers released the drug slowly in an aqueous solution of low ionic strength. However, in isotonic solutions, methotrexate and adriamycin were readily released from the poly(ethylene glycol)-attached dendrimers.

Role of apoptosis in the closure of neonatal ductus arteriosus.

Apoptosis regulates the remodeling of tissue during embryonic development by eliminating unwanted cells and structures. The present study investigated smooth muscle cell (SMC) proliferation and apoptosis in the neonatal ductus arteriosus (DA) during closure. In the DA of 39 swine neonates and 5 autopsy human neonates, apoptosis was detected using in situ end-labeling and electron microscopy, and proliferation was evaluated using proliferating cell nuclear antigen. In swine, apoptosis of SMC was first observed at 24h after birth. After 48h, both apoptosis and proliferation quickly increased and became most prominent at 3 days, mainly in the intima and inner media. From 5 days, both apoptosis and proliferation quickly disappeared, and were present to a minor extent at the 2 weeks after birth. During these processes, there was no sign of inflammation or necrosis. In humans, apoptosis was found in tissue specimens obtained from 2 term neonates who died at 1 and 5 days after birth. These findings suggest that SMC contribute to the functional closure of the DA by active constriction, and soon after, they switch to proliferation and apoptosis, which may contribute to the anatomical closure of the DA.

Design of fusogenic liposomes using a poly(ethylene glycol) derivative having amino groups.

As a novel fusogenic liposome, we designed liposomes modified with poly(glycidol) having beta-alanine residues, which is a poly(ethylene glycol) derivative with positively charged groups. The polymer-modified liposomes of egg yolk phosphatidylcholine (EYPC) and dioleoylphosphatidylethanolamine (DOPE) were prepared by reverse phase evaporation. Fusion of the polymer-modified liposomes with anionic liposomes consisting of phosphatidic acid and DOPE was investigated. Fusion ability of the polymer-modified liposomes increased with increasing amount of the polymer fixed on the liposome. Also, inclusion of DOPE was necessary for the generation of the fusion ability of the polymer-modified liposomes. CV1 cells treated with the polymer-modified DOPE/EYPC liposomes containing calcein displayed diffuse fluorescence, suggesting that calcein was introduced into the cytoplasm. In contrast, only punctual fluorescence was observed in the cells treated with the polymer-modified EYPC liposomes containing calcein, indicating that calcein remained in the endosome and/or lysosome. In addition, COS1 cells were transfected efficiently by treatment with the polymer-modified EYPC/DOPE liposomes containing pSV2cat plasmid, whereas the transfection was not induced by treatment with the polymer-modified EYPC liposomes. Close correlation between fusion ability of the polymer-modified liposomes and their ability to deliver their contents to the cytoplasm implies that membrane fusion plays an important role in the liposome-mediated cytoplasmic delivery.

Temperature-controlled interaction of thermosensitive polymer-modified cationic liposomes with negatively charged phospholipid membranes.

To obtain cationic liposomes of which affinity to negatively charged membranes can be controlled by temperature, cationic liposomes consisting of 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol and dioleoylphosphatidylethanolamine were modified with poly(N-acryloylpyrrolidine), which is a thermosensitive polymer exhibiting a lower critical solution temperature (LCST) at ca. 52 degrees C. The unmodified cationic liposomes did not change its zeta potential between 20-60 degrees C. The polymer-modified cationic liposomes revealed much lower zeta potential values below the LCST of the polymer than the unmodified cationic liposomes. However, their zeta potential increased significantly above this temperature. The unmodified cationic liposomes formed aggregates and fused intensively with anionic liposomes consisting of egg yolk phosphatidylcholine and phosphatidic acid in the region of 20-60 degrees C, due to the electrostatic interaction. In contrast, aggregation and fusion of the polymer-modified cationic liposomes with the anionic liposomes were strongly suppressed below the LCST. However, these interactions were enhanced remarkably above the LCST. In addition, the polymer-modified cationic liposomes did not cause leakage of calcein from the anionic liposomes below the LCST, but promoted the leakage above this temperature as the unmodified cationic liposomes did. Temperature-induced conformational change of the polymer chains from a hydrated coil to a dehydrated globule might affect the affinity of the polymer-modified cationic liposomes to the anionic liposomes.

Temperature-dependent interaction of thermo-sensitive polymer-modified liposomes with CV1 cells.

Egg yolk phosphatidylcholine liposomes modified with a copolymer of N-acryloylpyrrolidine and N-isopropylacrylamide having a lower critical solution temperature at ca. 40 degrees C were prepared and an effect of temperature on their interaction with CV1 cells was investigated. The unmodified liposomes were taken up by the cells approximately to the same extent after 3 h incubation at 37 and 42 degrees C. In contrast, uptake of the polymer-modified liposomes by CV1 cells decreased slightly at 37 degrees C but increased greatly at 42 degrees C, compared to the unmodified liposomes. Proliferation of the cells was partly prohibited by the incubation with the unmodified liposomes encapsulating methotrexate at 37 and 42 degrees C. The treatment with the polymer-modified liposomes containing methotrexate at 37 degrees C hardly effected the cell growth. However, the treatment at 42 degrees C inhibited the cell growth completely. It is considered that the highly hydrated polymer chains attached to the liposome surface suppressed the liposome-cell interaction below the lower critical solution temperature of the polymer but the dehydrated polymer chains enhanced the interaction above this temperature. Because interaction of the polymer-modified liposomes with cells can be controlled by the ambient temperature, these liposomes may have potential usefulness as efficient site-specific drug delivery systems.

Temperature sensitization of liposomes using copolymers of N-isopropylacrylamide.

To obtain liposomes which release the contents in response to ambient temperature, liposomes modified with copolymers of N-isopropylacrylamide with varying lower critical solution temperatures have been designed. Poly(N-isopropylacrylamide-co-acrylamide)s with various compositions were synthesized by free-radical copolymerization. The lower critical solution temperature of the polymer increased with increasing acrylamide content in the polymer. Poly(N-isopropylacrylamide-co-acrylamide-co-N, N-didodecylacrylamide)s were also prepared via the same method as the thermosensitive polymers having anchor groups to the liposome membrane. Calcein-loaded dioleoylphosphatidylethanolamine/egg yolk phosphatidylcholine (6:4, w/w) liposomes were coated with these polymers by incubating the liposomes with aqueous solutions of the polymers. The liposomes hardly released the contents below the lower critical solution temperature of the polymer, but the release was greatly enhanced above that temperature. The liposomes were also made from a mixture of the same lipids and the polymer. The liposome revealed a more drastic release property than the liposomes prepared by the incubation with the polymer solution, because the polymer chains were bound on both surfaces of the membrane. The close relationship between lower critical solution temperatures of the polymers and temperature regions where enhancement of the release from the polymer-fixed liposomes demonstrates that the release was triggered by alteration of the polymers from a hydrophilic state to a hydrophobic state occurring at their lower critical solution temperatures.

Improvement of temperature-sensitivity of poly(N-isopropylacrylamide)-modified liposomes.

As a novel temperature-sensitive drug delivery system, we have designed liposomes coated with poly(N-isopropylacrylamide), which exhibits a coil-globule transition at 32-35 degrees C. In a previous study [ K. Kono, H. Hayashi, T. Takagishi, J. Control. Release 30 (1994) 69-75], it was shown that the release of contents from the polymer-coated liposomes is enhanced above the transition temperature of the polymer but the release is not controlled completely by the conformational change of the polymer. In this study, to improve temperature-sensitivity of the poly(N-isopropylacrylamide)-modified liposomes, the influence of lipid composition on the temperature-sensitivity of the liposomes was investigated. A copolymer of N-isopropylacrylamide and N, N-didodecylacrylamide was synthesized by free radical copolymerization. While the copolymer was insoluble in water, it exhibited a coil-globule transition around 28 degrees C when incorporated into an egg yolk phosphatidylcholine membrane. The copolymer-modified egg yolk phosphatidylcholine/dioleoylphosphatidylethanolamine liposomes were prepared by sonication or reverse phase evaporation. Release of calcein from the copolymer-modified liposomes was very slow below the transition temperature of the copolymer, whereas the release was enhanced above the transition temperature. Incorporation of dioleoylphosphatidylethanolamine into liposome membranes enhanced release greatly above the transition temperature of the copolymer. Fluorometric measurements using 8-anilino-1-naphthalenesulfonate and 1,6-diphenyl-1,3,5-hexatriene suggested that the copolymer decreases membrane fluidity of the liposomes near and above the transition temperature of the copolymer and that interaction between the copolymer and the liposome membranes is enhanced by inclusion of dioleoylphosphatidylethanolamine.

Thermosensitive polymer-modified liposomes that release contents around physiological temperature.

To obtain temperature-sensitive liposomes which release their contents around the physiological temperature, we designed dioleoylphosphatidylethanolamine liposomes modified with copolymers of N-isopropylacrylamide and acryloylpyrrolidine. Copolymers of acryloylpyrrolidine and N-isopropylacrylamide, which exhibit a lower critical solution temperature around the physiological temperature, were prepared by free radical copolymerization using azobis(isobutyronitrile) as the initiator. The copolymers with anchors to the liposome membrane were obtained by using N, N-didodecylacrylamide as an additional comonomer. The copolymer having the anchor group at the terminal of the polymer chain was also synthesized by copolymerization of these monomers in the presence of 2-aminoethanethiol and subsequent conjugation of N, N-didodecyl succinamic acid to the terminal amino group of the copolymer. Calcein-loaded dioleoylphosphatidylethanolamine liposomes modified with these copolymers were prepared and release of the contents from these liposomes was investigated. It was found that the release from these copolymer-modified liposomes was promoted around and above the lower critical temperature of the copolymer. Also, the liposomes modified with the terminal anchor-type copolymer released the contents more drastically responding to a small temperature change than the liposomes modified with random copolymers containing N,N-didodecylacrylamide units as the anchor.

Temperature-dependent associating property of liposomes modified with a thermosensitive polymer.

Novel temperature-sensitive liposomes having surface properties that can be controlled by temperature were designed as liposomes coated with poly(N-isopropylacrylamide), which exhibits a hydrated coil to dehydrated globule transition at ca. 32 degrees C. To obtain the polymer with anchoring groups to the liposome, a copolymer of N-isopropylacrylamide and octadecyl acrylate (99:1, mol/mol) was synthesized by radical copolymerization. The copolymer revealed the transition near 30 degrees C. Liposomes made from various phospholipids were prepared by sonication and coated with the copolymer. When dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine were used as liposome lipids, remarkable aggregation and fusion of the copolymer-modified liposomes took place between the transition temperature of the copolymer and the gel-liquid-crystalline transition temperature (Tc) of the lipid membranes. However, above the Tc, association between the liposomes was much less significant, although the copolymer is still hydrophobic. In the case of the copolymer-modified dilauroylphosphatidylcholine liposome, the membrane of which takes on a liquid-crystalline state under the experimental conditions, association between the liposomes also hardly occurred even when the copolymer became hydrophobic. On the other hand, below the transition temperature of the copolymer, the copolymer-modified liposomes were very stable and aggregation of the liposomes was not observed, irrespective of membrane lipid. Results obtained in this study demonstrate that the copolymer chains fixed on the surface of the liposome with a solid membrane promote aggregation and fusion of the liposomes by hydrophobic interactions between the copolymer chains and/or between the copolymer chains and the liposome membranes above the transition temperature of the copolymer.

Cytoplasmic delivery of calcein mediated by liposomes modified with a pH-sensitive poly(ethylene glycol) derivative.

Previously, as a new type of pH-sensitive liposome, we prepared egg yolk phosphatidylcholine (EYPC) liposomes bearing succinylated poly(glycidol), that is a poly(ethylene glycol) derivative having carboxyl groups, and showed that fusion ability of the liposomes increases under weakly acidic and acidic conditions (Kono, K., Zenitani, K. and Takagishi, T. (1994) Biochim. Biophys. Acta 1193, 1-9). In this study, we examined intracellular delivery of a water-soluble molecule, calcein, mediated by the succinylated poly(glycidol)-modified liposomes. When CV-1 cells, an established line of African green monkey kidney cells, were incubated with bare EYPC liposomes containing calcein at 37 degrees C, only weak and vesicular fluorescence of calcein was observed by using a fluorescence microscope. In contrast, the cells treated with the polymer-modified liposomes containing calcein displayed more intensive and diffuse fluorescence, indicating that calcein was transferred into the cytoplasm. Uptake of the polymer-modified liposomes by the cells was shown to decrease slightly as amount of the polymer fixed on the liposome increases. However, the fluorescence of calcein observed in the liposome-treated cell was, on the contrary, enhanced as amount of the polymer fixed on the liposome increases, indicating that the liposome modified with a higher amount of the polymer transfers its content into cytoplasm more efficiently after internalization into the cell. Fusion assay by resonance energy transfer using N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine and lissamine rhodamine B-sulfonylphosphatidylethanolamine suggested occurrence of fusion between the polymer-modified liposomes and endosomal and/or lysosomal membranes. Moreover, the liposome with a higher polymer content revealed higher percent fusion after internalization into the cell. These results imply that the polymer-modified liposomes transfer the content into the cytoplasm by fusing with the endosomal membrane after internalization into the cells through an endocytic pathway.

[Pharmacokinetic and clinical evaluation of cefozopran in premature and newborn patients].

Cefozopran (SCE-2787, CZOP), which is already on the market with a variety of approved indications in infectious diseases for adult patients, was administered to premature and newborn patients to evaluate the pharmacokinetics and the clinical efficacy. 1. Pharmacokinetics CZOP was intravenously administered at doses of 10.0 mg/kg, 21.4 mg/kg and 40.0 mg/kg to premature and newborn patients, and the blood concentrations and urinary excretion rate were examined. The blood CZOP concentrations were 31.7 and 65.5 micrograms/ml at 30 minutes after administration of 10.0 mg/kg and 40.0 mg/kg, respectively. The elimination half life was 1.78 hours and 2.31 hours, and the urinary recovery was 110.7% and 53.7% within 6 hours after administration, respectively. In the patient given 21.4 mg/kg, the blood CZOP concentration was 36.4 mg/kg at 1 an hour after administration and the elimination half life was 3.97 hours. The urinary recovery was 29.6% within 5 hours after administration. 2. Clinical results The clinical efficacy was evaluated in 19 patients and judged "good" or better in 13 of them with the efficacy rate or 68.4%. The bacteriological response was evaluated in 10 patients from whom Gram-positive cocci of S. aureus (6 strains), S. pneumoniae (1 strain) and E. faecalis (1 strain) and Gram-negative bacilli of H. influenzae (2 strains) and E. coli (2 strains) were isolated as possible causative organisms. With exception of 1 strain each of S aureus and H influenzae, which were not tested after the treatment with CZOP, all of these strains were found to be eradicated. 3. Adverse drug reactions (ADRs) of signs and symptoms and abnormal alterations of laboratory test values. Safety evaluation was made in 24 patients. ADRs of signs and symptoms were recognized in none of them. As abnormal alterations of laboratory test values, increased eosinophils in 3 patients, elevated GOT in one and elevated GPT in one were recognized. These results indicate that CZOP is a drug useful for treatment of infections in premature and newborn patients.

Temperature-controlled release property of phospholipid vesicles bearing a thermo-sensitive polymer.

As a novel temperature-sensitive liposome, dioleoylphosphatidylethanolamine vesicles bearing poly(N-isopropylacrylamide), which shows a lower critical solution temperature (LCST) near 32 degrees C, were designed. Poly(N-isopropylacrylamide) having long alkyl chains which are anchors to the lipid membranes was prepared by radical copolymerization of N-isopropylacrylamide and octadecyl acrylate using azobisisobutyronitrile as the initiator. The copolymer obtained revealed the LCST at about 30 degrees C in an aqueous solution. Dioleoylphosphatidylethanolamine vesicles coated with the copolymer was prepared and release property of the copolymer-coated vesicles was investigated. While release of calcein encapsulated in the copolymer-coated vesicles was limited below 30 degrees C, the release was drastically enhanced between 30 and 35 degrees C. Complete release from the vesicles was achieved within several seconds at 40 degrees C. This temperature-controlled release property of the vesicles can be attributable to stabilization and destabilization of the vesicle membranes induced by the copolymer fixed on the vesicles below and above the LCST, respectively. Moreover, the fluorometric measurement using dioleoyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethan ola mine suggested that the extensive release of calcein observed above the LCST is resulted from the bilayer to HII phase transition of the vesicle membranes. Since LCST of the copolymer is controllable, these vesicles might have potential usefulness as a drug delivery system with high temperature-sensitivity.

Evaluation of pulmonary artery histopathologic findings in congenital heart disease: an in vitro study using intravascular ultrasound imaging.

OBJECTIVES: This study aimed to 1) compare in vitro intravascular ultrasound images of human pulmonary arteries with corresponding histologic sections, and 2) correlate the relation between intravascular ultrasound findings and Heath-Edwards pathologic grade of pulmonary vascular changes. BACKGROUND: The pathologic assessment of the pulmonary vascular bed is essential for diagnosis and management of congenital heart disease with pulmonary hypertension. METHODS: We evaluated and compared intravascular ultrasound images with histologic findings at identical sites in 40 pulmonary artery segments from 17 autopsy studies: group 1 = 7 patients with pulmonary hypertension (Heath-Edwards grade I to V, 20 segments); group 2 = 10 patients without cardiopulmonary disease (20 segments). RESULTS: In group 2, the pulmonary artery wall echo consisted of a single layer. In group 1, 1) all segments of pulmonary arteries from patients with pulmonary hypertension showed a three-layered appearance; 2) in patients with mild pulmonary hypertension (Heath-Edwards grades I and II), intravascular ultrasound demonstrated increased thickness of the echoluscent zone due to medial hypertrophy with no intimal reaction; 3) patients with severe pulmonary hypertension (Health-Edwards grade III or higher) had intravascular ultrasound findings of increased medial thickness and a bright inner layer from intimal hyperplasia; 4) percent wall thickness derived from intravascular ultrasound showed a significant correlation with that determined by histologic examination (r = 0.89, p = 0.0001, n = 20). CONCLUSIONS: Changes observed with intravascular ultrasound imaging correlate well with histopathologic grade. Thus, intravascular ultrasound may have significant utility in the evaluation of pulmonary vascular morphology in patients with pulmonary hypertension.

Expression of the histamine H1 receptor gene in relation to atherosclerosis.

Histamine in serum and arterial tissue contributes to the pathogenesis of atherosclerosis and the formation of coronary artery vasospasm. As the effect of histamine at a given site will be mediated by its specific receptors, we investigated by in situ hybridization and Northern blot analysis the expression and localization of human histamine H1 receptor mRNA in the arterial wall and in cultured human aortic intimal smooth muscle cells (SMC) and immortalized SMC (ISS10) and endothelial cells (SE1). In situ hybridization showed that SMC and endothelial cells expressed H1 receptor mRNA in vivo and that the expression was increased in SMC in the thickened intima of atherosclerotic foci in both the aorta and coronary artery. By Northern blot analysis, we also detected histamine H1 receptor mRNA in cultured SMC, ISS10, and SE1 and found that platelet-derived growth factor stimulated SMC to increase their expression of the mRNA in vitro. These results suggest that up-regulation of histamine H1 receptor expression by platelet-derived growth factor plays an important role in the initiation and progression of cardiovascular diseases.

Effect of angiotensin II and thromboxane A2 on the production of matrix metalloproteinase by human aortic smooth muscle cells.

Angiotensin II (AGII) and thromboxane A2 (TXA2), potent vasoconstrictors, augmented the production of the precursor of tissue procollagenase/promatrixmetalloproteinase-1 (proMMP-1) and DNA synthesis in cultured human aortic smooth muscle cells (SMC) significantly compared with that in untreated SMC. Moreover, AGII and TXA2 stimulated hydrolysis of phosphoinositides and subsequent formation of inositol triphosphate (IP3), leading to an increase in the intracellular free Ca2+ concentration. These results suggest that the production of proMMP-1 increased by AGII and TXA2 in intimal SMC in relation to cell proliferation plays a role in arterial reconstruction in vascular diseases.

Novel pH-sensitive liposomes: liposomes bearing a poly(ethylene glycol) derivative with carboxyl groups.

Three kinds of succinylated poly(glycidol)s were synthesized as poly(ethylene glycol) derivatives having carboxyl groups by the reaction of poly(glycidol) with varying amounts of succinic anhydride in dimethylformamide. These polymers promoted fusion of egg-yolk phosphatidylcholine liposome more intensively with decreasing content of carboxyl groups at pH 7.4, although the extent of fusion was limited. However, the polymer with 56% of succinylated residues induced fusion of the liposome much more strongly at pH 4.0. Egg-yolk phosphatidylcholine liposomes bearing the succinylated poly(glycidol) which was combined with long alkyl chains as anchors to the liposomal membrane were prepared. The leakage of calcein entrapped in the inner aqueous phase of the liposomes was slight at pH 7.4. However, the leakage increased with decreasing pH. The turbidity measurement and the fusion assay indicate that the liposomes bearing the polymer fuse more intensively with decreasing pH and with increasing amount of the polymer bound to the liposomes.

Fusion activity of an amphiphilic polypeptide having acidic amino acid residues: generation of fusion activity by alpha-helix formation and charge neutralization.

A sequential polypeptide, poly(Glu-Aib-Leu-Aib) (Aib represents 2-aminoisobutyric acid), was synthesized and the pH-dependence of fusogenic activity of the polypeptide was studied. The polypeptide was designed to take amphiphilic structure upon the formation of alpha-helix. Circular dichroism spectra of the polypeptide showed a negative Cotton effect with double minima, indicative of an alpha-helical conformation. The alpha-helix content was increased with lowering pH and/or increasing the ionic strength. It was found that the polypeptide induces remarkable leakage of calcein from egg-yolk phosphatidylcholine (EYPC) vesicles loaded in the inner aqueous phase with lowering pH and/or increasing ionic strength. The polypeptide caused fusion of EYPC liposomes and dipalmitoylphosphatidylcholine liposomes more strongly with decreasing pH. Moreover, two distinct increases of fusogenic activity of the polypeptide were observed near pH 6.0 and below pH 4.5. The former corresponds to the midpoint of pH-dependent change in helical content of the polypeptide and the latter the pKa of the gamma-carboxyl group of glutamic acid. These results indicate that elevation of the fusogenic activity of the polypeptide is related to the increase in two factors, alpha-helix content and hydrophobicity.