Advances in anti-invasive fungal drug delivery systems / 浙江大学学报·医学版

Currently, the first-line drugs for invasive fungal infections (IFI), such as amphotericin B, fluconazole and itraconazole, have drawbacks including poor water solubility, low bioavailability, and severe side effects. Using drug delivery systems is a promising strategy to improve the efficacy and safety of traditional antifungal therapy. Synthetic and biomimetic carriers have greatly facilitated the development of targeted delivery systems for antifungal drugs. Synthetic carrier drug delivery systems, such as liposomes, nanoparticles, polymer micelles, and microspheres, can improve the physicochemical properties of antifungal drugs, prolong their circulation time, enhance targeting capabilities, and reduce toxic side effects. Cell membrane biomimetic drug delivery systems, such as macrophage or red blood cell membrane-coated drug delivery systems, retain the membrane structure of somatic cells and confer various biological functions and specific targeting abilities to the loaded antifungal drugs, exhibiting better biocompatibility and lower toxicity. This article reviews the development of antifungal drug delivery systems and their application in the treatment of IFI, and also discusses the prospects of novel biomimetic carriers in antifungal drug delivery.

Ginsenoside Rg_3 based liposomes target delivery of dihydroartemisinin and paclitaxel for treatment of triple-negative breast cancer / 中国中药杂志

Ginsenoside Rg_3, an active component of traditional Chinese medicine(TCM), was used as the substitute for cholesterol as the membrane material to prepare the ginsenoside Rg_3-based liposomes loaded with dihydroartemisinin and paclitaxel. The effect of the prepared drug-loading liposomes on triple-negative breast cancer in vitro was evaluated. Liposomes were prepared with the thin film hydration method, and the preparation process was optimized by single factor experiments. The physicochemical properties(e.g., particle size, Zeta potential, and stability) of the liposomes were characterized. The release behaviors of drugs in different media(pH 5.0 and pH 7.4) were evaluated. The antitumor activities of the liposomes were determined by CCK-8 on MDA-MB-231 and 4T1 cells. The cell scratch test was carried out to evaluate the effect of the liposomes on the migration of MDA-MB-231 and 4T1 cells. Further, the targeting ability of liposomes and the mechanism of lysosome escape were investigated. Finally, H9c2 cells were used to evaluate the potential cardiotoxicity of the preparation. The liposomes prepared were spheroid, with uniform particle size distribution, the ave-rage particle size of(107.81±0.01) nm, and the Zeta potential of(2.78±0.66) mV. The encapsulation efficiency of dihydroartemisinin and paclitaxel was 57.76%±1.38% and 99.66%±0.07%, respectively, and the total drug loading was 4.46%±0.71%. The accumulated release of dihydroartemisinin and paclitaxel from the liposomes at pH 5.0 was better than that at pH 7.4, and the liposomes could be stored at low temperature for seven days with good stability. Twenty-four hours after administration, the inhibition rates of the ginsenoside Rg_3-based liposomes loaded with dihydroartemisinin(70 μmol·L~(-1)) and paclitaxel on MDA-MB-231 and 4T1 cells were higher than those of the positive control(adriamycin) and free drugs(P<0.01). Compared with free drugs, liposomes inhibited the migration of MDA-MB-231 and 4T1 cells(P<0.05). Liposomes demonstrated active targeting and lysosome escape. In particular, liposomes showed lower toxicity to H9c2 cells than free drugs(P<0.05), which indicated that the preparation had the potential to reduce cardiotoxicity. The findings prove that ginsenoside Rg_3 characterized by the combination of drug and excipient is an ideal substitute for lipids in liposomes and promoted the development of innovative TCM drugs for treating cancer.

Amplification of urea detection based on pH-sensitive liposomes

BACKGROUND: This study aimed to develop an amplification method of urea detection based on pHsensitive liposomes. RESULTS: The urease covalently immobilized on the magnetic particles and the pH-sensitive liposomes encapsulating ferricyanide were added to the cyclic-voltammeter cell solution where urea was distributed. The conversion of urea into carbonic acid seemed to induce a pH decrease that caused a reduction in the electrostatic repulsion between the headgroups of weakly acidic 1,2-dipalmitoyl-sn-glycero3-succinate. The reduction induced the liposomes to release potassium ferricyanide that was encapsulated inside. The effects of urea concentration and pH value were investigated. A specific concentration (0.5 mg/mL) of the urea solution was set to observe the response. The activity of urease was reversible with respect to the pH change between 7 and 5. The sensitivity of this detection was almost identical to the comparable techniques such as an enzyme-linked immunosorbent assay and a field-effect transistor. CONCLUSIONS: In summary, the methodology developed in this study was feasible as a portable, rapid, and sensitive method.

Effects of artepillin C on model membranes displaying liquid immiscibility

It has been hypothesized that the therapeutic effects of artepillin C, a natural compound derived from Brazilian green propolis, are likely related to its partition in the lipid bilayer component of biological membranes. To test this hypothesis, we investigated the effects of the major compound of green propolis, artepillin C, on model membranes (small and giant unilamelar vesicles) composed of ternary lipid mixtures containing cholesterol, which display liquid-ordered (lo) and liquid-disordered (ld) phase coexistence. Specifically, we explored potential changes in relevant membrane parameters upon addition of artepillin C presenting both neutral and deprotonated states by means of small angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and confocal and multiphoton excitation fluorescence microscopy. Thermotropic analysis obtained from DSC experiments indicated a loss in the lipid cooperativity of lo phase at equilibrium conditions, while at similar conditions spontaneous formation of unilamellar vesicles from SAXS experiments showed that deprotonated artepillin C preferentially located at the surface of the membrane. Time-resolved experiments using fluorescence microscopy showed that at doses above 100 µM, artepillin C in its neutral state interacted with both liquid-ordered and liquid-disordered phases, inducing curvature stress and promoting dehydration at the membrane interface.

Ectasia ductal mamaria en niños, a propósito de un caso clínico: A case report / Mammary duct ectasia in children

Introducción: La telorragia es un síntoma poco frecuente en pacientes pediátricos, la causa más frecuente en esta población es la ectasia ductal mamaria (EDM), que es una afección benigna y autolimitada, caracterizada por la dilatación del conducto mamario, fibrosis e inflamación periductal. Objetivo: Presentar un caso de EDM, para facilitar el rápido reconocimiento por parte de los médicos, y evitar estudios y tratamientos agresivos. Caso clínico: Lactante de sexo masculino de 6 meses de edad, sano, alimentado por lactancia materna exclusiva; consultó por un nódulo retroareolar derecho y telorragia unilateral. Se realizó una ecografía Doppler que mostró una lesión multiquística, sugerente de una EDM. Se planteó tratamiento expectante y acudió a control a los 6 meses con excelente evolución. Conclusiones: La EDM es la principal causa de telorragia en niños, corresponde a una afección benigna, y la resolución generalmente es espontánea, antes de los 9 meses. Por lo que su conocimiento es de gran relevancia para el adecuado diagnóstico y manejo de estos pacientes.

Introduction: Bloody nipple discharge is an infrequent symptom during childhood. The most common cause in this population is mammary duct ectasia (MDE), which is a benign and self-limiting condition, that is characterized by dilatation of the mammary ducts, fibrosis and periductal inflammation. Objective: Report of a case of MDE in order to improve physicians’ diagnosis accuracy and avoid aggressive studies and treatments. Case report: Six-months old male healthy infant, exclusively breastfeeded, that visited our clinic with a lump beneath his right nipple and bloody discharge from the same nipple. An ultrasound was performed which showed a multicystic lesion suggestive of MDE. Watchful waiting was decided as treatment, with good evolution after six months of follow up. Conclusions: The MDE is the leading cause of bloody discharge in pediatric population, being a benign condition that resolves spontaneously before nine months. The knowledge of this condition is essential so as to accurately diagnose and treat it.

The role of C-terminus carbohydrate-binding domain of Vibrio cholerae haemolysin/cytolysin in the conversion of the pre-pore β-barrel oligomer to a functional diffusion channel.

Background & objectives: Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa pore-forming toxin (PFT) secreted by O1 El Tor and non-O1 strains. The purified toxin, which contains two C-terminus carbohydrate-binding domains in addition to the cytolytic domain at the core, causes lysis of a wide spectrum of eukaryotic cells at picomolar concentrations, apoptogenesis of intestinal and immune cells and accumulation of fluid in rabbit ligated ileal loop. Therefore, it may potentially complement the action of cholera toxin (CT) in diarrheagenic strains that do not produce CT. We showed earlier that β1-galactosyl-terminated glycoconjugates are strong inhibitors of its pore-forming activity, though carbohydrates are not functional receptors of VCC. Here, we investigate how the 15 kDa C-terminus β-prism lectin domain contributed to pore formation in erthrocytes. Methods: VCC was isolated from the culture supernatant of late log phase grown bacteria and purified to homogeneity by chromatography. The 50 kDa truncated variant was generated by restricted proteolysis. Liposome was prepared by sonication of a suspension of phospholipids and calceine release assay was done by spectrofluorometric monitoring of the released dye trapped in liposome. Formation of β-barrel oligomers in erythrocyte stroma was monitored by scanning electron microscopy. Results: Proteolytic truncation of the C-terminus β-prism lectin domain decreased hemolytic activity of the toxin by ~800-fold without causing a significant change in pore-forming activity toward synthetic lipid vesicles devoid of incorporated glycoproteins/glycolipids. Truncation at the C-terminus did not impair membrane-binding or assembly to the oligomeric pore. Interpretation & conclusions: Our data indicated that the C-terminus domain played a critical role in translocation of the pre-pore oligomeric assembly from the cell surface or lipid-water interface to the hydrocarbon core of the membrane bilayer, signaling the formation of functional diffusion channels.

Marcado con 99m de liposomas convencionales y su evaluación biológica / 99mTc-labeling and biological evaluation of conventional liposomes

Introducción. Los liposomas son sistemas supramoleculares autoensamblables preparados ad hoc, compuestos de fosfolípidos y colesterol, diseñados para transporte de fármacos o radionucleidos. El 99mTc es el radionucleido más empleado por sus propiedades físicas apropiadas para la adquisición de imágenes y estudios en pacientes en el área de medicina nuclear (emisor gamma puro con E = 140 KeV , t1/2 = 6 horas). Objetivo. Evaluar la potencialidad de liposomas convencionales marcados con 99mTc como agente de diagnóstico de procesos tumorales. Método. La composición estudiada es: fosfatidilcolina, dioleilfosfatidiletanolamina y colesterol (1.1:0.4:1 relación molar). Se utilizó como agente reductor SnF2, en diferentes cantidades para optimizar el marcado. La pureza radioquímica y eficiencia de marcado se evaluaron con sistemas cromatográficos ITLC-SG FM NaCl 0,9 por ciento, ITLC-SG FM Piridina:ácido acético:agua (3:5:1.5 v/v). Se adquirieron imágenes a 1 h post inyección de los liposomas en ratones sanos y portadores de tumor mamario espontáneo. Resultados. El liposoma marcado mostró estabilidad durante 24 h, siendo la cantidad óptima de reductor 138 ug. La mejor captación en tumor fue a 1 h post administración del radiofármaco en los estudios centellográficos. Conclusiones. El método optimizado permite obtener liposomas marcados con 99mTc en forma sencilla, eficiente y estable. Estos radiofármacos mostraron un comportamiento fiscoquímico y biológico adecuado como agentes de diagnóstico tumoral requiriéndose estudios posteriores para su confirmación.

Background. Liposomes are self-assembled supramolecular systems, composed by phospholipids and cholesterol, designed for the transportation of drugs or radionuclides. Physical properties of 99mTc (pure gamma emitter with E = 140 KeV, t1/2= 6 hours) makes it useful for scintigraphic imaging. Purpose. The goal of this study was to evaluate 99mTc labeled conventional liposomes as potential diagnostic agents for malignant lesions. Methods. The studied liposome composition was hosphatidylcholine: dioleilphosphatidylcholine: cholesterol (1.1:0.4:1molar rate). In order to optimize the labeling, SnF2 was used as reducing agent. Radiochemical purity and labeling efficiency were evaluated using ascending thin layer chromatography. Scintigraphy images were obtained at 1 hour post-injection of labeled liposomes to healthy mice and with spontaneous breast tumors. Results. Labeled liposomes showed stability during 24 hours, being 138 ug the optimal amount of reducing agent for the technique used. Conclusions. The described method allows the production of 99mTc labeled liposomes in a simple, efficient and stable way. The radiopharmaceutical showed a physicochemical and biological behavior that allows its potential use as a tumor imaging agent, although further studies are required to confirm this issue.

Liposomal delivery of Mycobacterium leprae antigen(s) with murabutide and Trat peptide inhibits Fas-mediated apoptosis of peripheral blood mononuclear cells derived from leprosy patients.

Protective immunity against intracellular pathogen Mycobacterium leprae is dependent on the activation of T cells. Repeated stimulation of T cells by M. leprae antigens MLCwA (M. leprae total cell wall antigen) and ManLAM (mannose capped lipoarabinomannan) may lead to apoptosis in leprosy patients. In the present study, inhibition of the Fas-induced apoptosis of peripheral blood mononuclear cells of leprosy patients was investigated using above M. leprae antigen(s), in combination with immunomodulators murabutide (MB) and a Trat peptide in particulate form (liposome). Incubation of the cells with particulate mode of antigen presentation led to both decreased percentage of propidium iodide (PI) positive cells and T cells expressing Fas-FasL, as well as decreased caspase-8/-3 activities in the lepromatous patients, thereby inhibiting apoptosis, while converse was true with stimulation with soluble antigen. Concurrently, there was an upregulation of antiapoptotic protein Bcl-X(L) in the lepromatous patients, thereby inhibiting apoptosis. Thus, the liposomal formulation of antigen promoted proliferation of anergized T cell by inhibiting apoptosis through decreased expression of death receptors and caspase activities and increased expression of anti-apoptotic protein Bcl-X(L) in these patients.

Preparation and cytotoxicity of cisplatin-containing liposomes

We encapsulated cisplatin into stealth pH-sensitive liposomes and studied their stability, cytotoxicity and accumulation in a human small-cell lung carcinoma cell line (GLC4) and its resistant subline (GLC4/CDDP). Since reduced cellular drug accumulation has been shown to be the main mechanism responsible for resistance in the GLC4/CDDP subline, we evaluated the ability of this new delivery system to improve cellular uptake. The liposomes were composed of dioleoylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethyleneglycol 2000 (DSPE-PEG2000) and were characterized by determining the encapsulation percentage as a function of lipid concentration. Among the different formulations, DOPE/CHEMS/DSPE-PEG liposomes (lipid concentration equal to 40 mM) encapsulated cisplatin more efficiently than other concentrations of liposomes (about 20.0 percent, mean diameter of 174 nm). These liposomes presented good stability in mouse plasma which was obtained using a 0.24-M EDTA solution (70 percent cisplatin was retained inside the liposomes after 30 min of incubation). Concerning cytotoxic effects, they are more effective (1.34-fold) than free cisplatin for growth inhibition of the human lung cancer cell line A549. The study of cytotoxicity to GLC4 and GLC4/CDDP cell lines showed similar IC50 values (approximately 1.4 æM), i.e., cisplatin-resistant cells were sensitive to this cisplatin formulation. Platinum accumulation in both sensitive and resistant cell lines followed the same pattern, i.e., approximately the same intracellular platinum concentration (4.0 x 10-17 mol/cell) yielded the same cytotoxic effect. These results indicate that long-circulating pH-sensitive liposomes, also termed as stealth pH-sensitive liposomes, may present a promising delivery system for cisplatin-based cancer treatment. This liposome system proved to be able to circumvent the cisplatin resistance, whereas...

Self-assembled surfactant nano-structures important in drug delivery: a review.

Role of self assembled structures as a vehicle is significant over the years. Their applications have been found for all routes of drug delivery. These micro and nano structures are containers loaded with drugs, ideal for targeted and sustained release of the drug. Drug efficacy depends on the drug loaded into the vehicle, temperature, drug solubility, pH, release characteristics, additives and most significantly, the vehicle morphology. This in turn suggests that the same vehicle cannot be used with high efficiency for all types of drugs and locations where the drug delivery has to take place. The status of various self assembled structures and their applications in drug delivery is reviewed in this communication.

Delivery of Photosensitizers for Photodynamic Therapy / 대한소화기학회지

Photodynamic therapy (PDT) has been used to treat several types of cancer, and comprises intravascular administration of photosensitizer, uptake by cancer cells, and followed by irradiation of light of appropriate wavelength. Although PDT takes advantage of relative retention of photosensitizer by cancer cells, effective delivery of photosensitizing drugs is of great concern. Several delivery strategies have been employed in PDT. Photosensitizers can be delivered either by passive carriers such as liposomes, micelles, and polymeric particles, or by active targeting using cancer cell-directed ligands or antibodies. Although well-studied colloidal carriers effectively deliver photosensitizer to tumor cells, they are taken up by mononuclear phagocytic system. Delivery system using polymers is an attractive alternative to colloidal carriers, in which hydrophobic drugs are chemically or physically loaded to polymers. Though there are several steps to be solved, targeted delivery system utilizing receptors or antigens abundantly expressed on cancer cell theoretically provides a great deal of advantages over passive system. Selective uptake of photosensitizers by cancer cells may greatly enhance therapeutic efficacy as well as minimizing adverse effects resulting from accumulation in normal tissue. This review discusses various strategies for photosensitizer delivery that have been investigated to date.

Biophysical studies on the liposome-albumin system.

The potential use of liposomes as a delivery system is still limited by the poor understanding of their interaction mechanisms with biological media. In the present work, interaction between bovine albumin (BA) and liposomes was studied using phase transition and dielectric measurements as well as solubilization process using non-ionic detergent octylglucoside (OG). After liposomes were incubated with diluted and concentrated BA, phase transition, characterizing the liposome membrane exhibited a shift towards higher temperatures, together with initiation of multiple phase transitions. The relaxation time of liposome membrane molecules also increased in a concentration-dependent manner. The solubilization profiles of incubated samples also showed remarkable changes, especially in beginning of solubilization stages. Moreover, amount of detergent needed to completely solubilize membrane was also increased. It was concluded that BA significantly altered the physical state of liposome membrane, which may be attributed to BA interaction with liposomes surface and/or by its incorporation within the bilayer membrane.

Effect of Antisense TGF-beta1 Oligodeoxynucleotides in Streptozotocin-Induced Diabetic Rat Kidney

Transforming growth factor (TGF)-beta1 is an important fibrogenic factor that is involved in the pathogenesis of diabetic nephropathy. We evaluated the effect of circular antisense TGF-beta1 oligodeoxynucleotides (ODNs) on the TGF-beta1 expression in the rat mesangial cell culture and in streptozotocin (STZ)-induced diabetic rats. Circular antisense TGF-beta1 ODNs were found to be stable in rat serum, significantly decreasing TGF-beta1 mRNA expression compared with linear antisense ODNs in the rat mesangial cell culture. Circular antisense TGF-beta1 ODNs were introduced into the tail vein of normal rats using hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer method and were confirmed to be delivered effectively into the kidney, liver, lungs, and spleen. To inhibit the overexpression of TGF-beta1 in diabetic kidneys, we introduced circular antisense TGF-beta1 ODNs into the STZ-induced diabetic rats. On day 13 after circular antisense TGF-beta1 ODNs injection, TGF-beta1 mRNA and protein expression markedly decreased and urinary TGF-beta1 excretion rate also dropped in the circular antisense TGF-beta1 ODNs-treated diabetic rats. These results suggest that circular antisense TGF-beta1 ODNs may be a useful tool for developing new therapeutic application for progressive diabetic nephropathy.

Vectores lipidicos. Nuevas estrategias aplicadas a la terapia génica / Lipid vectors. New strategies for gene therapy

A diferencia del resto de las moléculas biológicas, los fosfolípidos son capaces de autoensamblarse espontáneamente. Con ellos es relativamente simples generar estructuras selladas extremadamente estables, de tamaño, forma y empaquetamiento controlables, llamadas liposomas. En este artículo revisaremos el uso de liposomas para generar vectores que mejoren los procesos de transfección en células eucarioticas, tanto in vivo como in vitro. Empleando vectores lipídicos, es potencialmente posible enviar selectivamente un segmento de AND a cualquier sitio del cuerpo, forzarlo a ingresar al interior celular y aun controlar el destino intracelular de la carga transportada. La clave del éxito de la transfección por medio de vectores lipídicos radica en que protegen mecánicamente al AND de la degradación plasmática, ofreciendo a la vez la oportunidad de controlar su biodistribuición, independientemente del tamaño del segmento de AND que se quiera expresar. Asimismo, son no carcinogénicos y pobremente inmunogénicos. Los avances en la química de sintésis de lípidos permitirán construir vectores cada vez más eficientes, que capitan con los altos niveles de transfección de los vectores virales, sumado a las ventajas de extrema versatilidad, facilidad de preparación y bioseguridad propias de la moléculas autoensamblables.

Novel methods for the encapsulation of meglumine antimoniate into liposomes

The antimonial drug, meglumine antimoniate, was successfully encapsulated in dehydration-rehydration vesicles and in freeze-dried empty liposomes (FDELs). High encapsulation efficiencies (from 28 to 58 percent) and low weight ratios of lipids to encapsulated antimony (from 1:0.15 to 1:0.3) were achieved. These formulations, contrary to those obtained by conventional methods, can be stored as intermediate lyophilized forms and reconstituted just before use. The efficacy of FDEL-encapsulated meglumine antimoniate was evaluated in hamsters experimentally infected with Leishmania chagasi. A significant reduction of liver parasite burdens was observed in animals treated with this preparation, when compared to control animals treated with empty liposomes. In contrast, free meglumine antimoniate was found to be inefficient when administered at a comparable dose of antimony. This novel liposome-based meglumine antimoniate formulation appears to be promising as a pharmaceutical product for the treatment of visceral leishmaniasis.

Liposomes: from biophysics to the design of peptide vaccines

Liposomes (lipid-based vesicles) have been widely studied as drug delivery systems due to their relative safety, their structural versatility concerning size, composition and bilayer fluidity, and their ability to incorporate almost any molecule regardless of its structure. Liposomes are successful in inducing potent in vivo immunity to incorporated antigens and are now being employed in numerous immunization procedures. This is a brief overview of the structural, biophysical and pharmacological properties of liposomes and of the current strategies in the design of liposomes as vaccine delivery systems

Caracterización de vesículas liposomales. 1. Efecto de la composición fosfolipídica sobre el volumen atrapado / Characterization of liposomes vesicles. 1.Effect of phospholipid composition on entrapped volume

La incidencia de la composición lipídica sobre el volumen atrapado de liposomas del tipo vesículas multilaminares grandes (MLV), almacenados durante 18 días bajo condiciones de refrigeración y en atmósfera libre de oxígeno ha sido examinada utilizando un nuevo método para la determinación del volumen de atrapamiento de solución acuosa de colorante amarillo FD&C No. 5 al 5 por ciento p/v, el método incluye centrifugación y espectrometría. Las MLVs fueron preparadas por el método clásico de evaporación de solvente descrito originalmente por Bangham y col. en 1965. Los resultados experimentales muestran que las vesículas con composición variable de colesterol poseen un volumen de atrapamiento de 4.9 a 15.7 µL/mg de lecitina

Immunoreactivity of mycobacterial 70 kDa protein in different physico-chemical forms.

The immunoreactivity of 70 kDa culture filtrate protein of Mycobacterium tuberculosis H37 Ra has been examined as such and by increasing its hydrophobicity through its conjugation to stearic acid ester (70 kDa-FAester). The cell mediated immune responses produced by 70 kDa-FAester encapsulated in phosphatidylcholine (PC) liposomes were significantly higher particularly at the 3rd week post immunization (wk p. im.) than that produced by the 70 kDa protein in PC liposomes, whereas humoral immune responses were non-significant. Further, these immune responses were comparable to that elicited by 70 kDa protein complexed with Freund's incomplete adjuvant (70 kDa-FIA). Results of this study suggest that changes in physicochemical nature of 70 kDa protein influences both the humoral and cellular immunoreactivity.

Interaction of pulmonary surfactant-associated protein (SP-A) with surfactant lipids.

A pulmonary surfactant-associated protein complex with components of 36, 32 and 28 kDa was isolated from human lung homogenates and reassembled with surfactant lipids prepared as small unilamellar liposomes. The role of divalent cations in the assembly of this recombinant lipoprotein complex was studied by monitoring the changes in turbidity, intrinsic tryptophanyl fluorescence and surface activity. The protein-facilitated lipid aggregation was promoted on addition of 5 to 20 mM Ca2+. Intrinsic fluorescence measurements on SP-A (28-36 kDa) indicated that the tryptophan side chains were in a relatively hydrophobic environment, that the wavelength of maximum fluorescence emission and also the relative fluorescence, were changed upon the binding of lipid. Tryptophanyl fluorescence of the lipoprotein assembly was quenched as indicated by a reduction in the effective Stern-Volmer constant. These results suggest that Ca2+ lipid-protein interactions are involved in the structure and function of extracellular lung surfactant assembly.