Staphylococcus aureus Decolonization of Mice With Monoclonal Antibody Neutralizing Protein A.

Staphylococcus aureus persistently colonizes the nasopharynx of about one-third of the human population, a key risk factor for community- and hospital-acquired invasive infections. Current strategies for S. aureus decolonization include topical and systemic administration of antibiotics, which is associated with selection for antibiotic resistance and posttreatment recolonization. Using a mouse model for S. aureus colonization, we show here that systemic administration of a recombinant monoclonal antibody neutralizing staphylococcal protein A (SpA) can stimulate antibacterial immunoglobulin G and immunoglobulin A responses and promote S. aureus decolonization. These results suggest that antibody neutralizing SpA, a B-cell superantigen, may also be useful for S. aureus decolonization in humans.

Staphylococcal Protein A Induces Leukocyte Necrosis by Complexing with Human Immunoglobulins.

One of the defining features of Staphylococcus aureus is its ability to evade and impair the human immune response through expression of staphylococcal protein A (SpA). Herein, we describe a previously unknown mechanism by which SpA can form toxic immune complexes when in the presence of human serum, which leads to the loss of human leukocytes. Further, we demonstrate that these toxic complexes are formed specifically through SpA's interaction with intact human IgG and that, in the presence of purified IgG Fab and Fc fragments, SpA shows no such toxicity. The mechanism of action of this toxicity appears to be one mediated by necrosis and not by apoptosis, as previously hypothesized, with up to 90% of human B cells rapidly becoming necrotic following stimulation with SpA-IgG complexes. This phenomenon depends on the immunoglobulin binding capacity of SpA, as a nonbinding mutant of SpA did not induce necrosis. Importantly, immune sera raised against SpA had the capacity to significantly reduce the observed toxicity. An unprecedented toxic effect of SpA-IgG complexes on monocytes was also observed, suggesting the existence of a novel mechanism independent from the interaction of SpA with the B cell receptor. Together, these data implicate SpA in inducing indiscriminate leukocyte toxicity upon formation of complexes with IgG and highlight the requirement for vaccination strategies to inhibit this mechanism. IMPORTANCE Staphylococcus aureus is one of the largest health care threats faced by humankind, with a reported mortality rate within the United States greater than that of HIV/AIDS, tuberculosis, and viral hepatitis combined. One of the defining features of S. aureus as a human pathogen is its ability to evade and impair the human immune response through expression of staphylococcal protein A. Herein, we show that SpA induces necrosis in various immune cells by complexing with human immunoglobulins. Vaccination of mice with a nontoxigenic SpA mutant induced sera capable of inhibiting this mechanism. These observations shed new light on the toxic mechanisms of this key staphylococcal virulence factor and on protective modalities of SpA-based vaccination.

Toxicity and Pharmacokinetic Profile for Single-Dose Injection of ABY-029: a Fluorescent Anti-EGFR Synthetic Affibody Molecule for Human Use.

PURPOSE: ABY-029, a synthetic Affibody peptide, Z03115-Cys, labeled with a near-infrared fluorophore, IRDye® 800CW, targeting epidermal growth factor receptor (EGFR) has been produced under good manufacturing practices for a US Food and Drug Administration-approved first-in-use human study during surgical resection of glioma, as well as other tumors. Here, the pharmacology, phototoxicity, receptor activity, and biodistribution studies of ABY-029 were completed in rats, prior to the intended human use. PROCEDURES: Male and female Sprague Dawley rats were administered a single intravenous dose of varying concentrations (0, 245, 2449, and 24,490 µg/kg corresponding to 10×, 100×, and 1000× an equivalent human microdose level) of ABY-029 and observed for up to 14 days. Histopathological assessment of organs and tissues, clinical chemistry, and hematology were performed. In addition, pharmacokinetic clearance and biodistribution of ABY-029 were studied in subgroups of the animals. Phototoxicity and ABY-029 binding to human and rat EGFR were assessed in cell culture and on immobilized receptors, respectively. RESULTS: Histopathological assessment and hematological and clinical chemistry analysis demonstrated that single-dose ABY-029 produced no pathological evidence of toxicity at any dose level. No phototoxicity was observed in EGFR-positive and EGFR-negative glioma cell lines. Binding strength and pharmacokinetics of the anti-EGFR Affibody molecules were retained after labeling with the dye. CONCLUSION: Based on the successful safety profile of ABY-029, the 1000× human microdose 24.5 mg/kg was identified as the no observed adverse effect level following intravenous administration. Conserved binding strength and no observed light toxicity also demonstrated ABY-029 safety for human use.

Staphylococcal protein A immunoadsorption for Goodpasture's syndrome in four Chinese patients.

BACKGROUND: Removal of anti-glomerular basement membrane (anti-GBM) antibody by extracorporeal treatment including plasmapheresis or immunoadsorption (IA) has been considered an effective method in the treatment of Goodpasture's syndrome. In this study, we investigated the effect of IA on serum anti-GBM level and evaluated its clinical efficacy in patients with Goodpasture's syndrome. METHODS: Four patients with Goodpasture's syndrome were enrolled in this study. All patients presented with severe renal failure and needed hemodialysis. They were treated with staphylococcal protein A IA, together with intravenous pulse methylprednisolone therapy and mycophenolate mofetil (n=3) or intermittent intravenous cyclophosphamide (n=1). IA was administered for 10 cycles per session and 8-10 sessions as a course; a total of 30-60 L of plasma was regenerated in each course. RESULTS: Serum anti-GBM antibody level decreased to the normal range in 3 patients after 1 course of IA treatment and remained negative in 2 patients who were followed up for 4 months. In the other patient, serum anti-GBM antibody level decreased by 62.8% after the first course of IA treatment and decreased to the normal range after the second course of IA. Rebound of serum anti-GBM was found in all patients during the first 3 to 4 IA sessions. Pulmonary hemorrhage resolved in all patients, including 1 patient who was unresponsive to methylprednisolone pulse therapy. One of the 4 patients who required initial hemodialysis on admission could stop hemodialysis after IA, and the other 3 patients who had 100% glomerular crescents were on maintenance hemodialysis. CONCLUSIONS: IA could remove serum anti-GBM antibody and resolve pulmonary hemorrhage effectively; the efficacy of IA in improving renal function is markedly determined by the severity of renal damage.

Protein A-independent tumoricidal responses in dogs after extracorporeal perfusion of plasma over Staphylococcus aureus.

Protein A-positive or -negative Staphylococcus aureus preparations were used in an extracorporeal system to treat dogs with spontaneously occurring cancers. Tumor regression was seen in 4 of 7 dogs treated by reinfusion of plasma that had been incubated with protein A-positive S. aureus Cowan I strain (SAC). Therapy was associated with fever, liver enzyme abnormalities, and hypocomplementemia. Tumor response and toxicity could be diminished by more extensive washing of the SAC preparation. Tumor regression was also seen in 2 of 2 animals treated with protein A-negative S. aureus Wood strain 46. In addition, tumors regressed in 3 of 4 dogs treated with infusions of protein A-free saline extracts from S. aureus. These results suggest that the release of a non-protein A bacterial product contributes to tumor regression following incubation of plasma with S. aureus.

Effects of plasma treatment with purified protein A and Staphylococcus aureus Cowan I on spontaneous animal neoplasms.

Eleven dogs with spontaneous neoplasms were intensively treated with an immunoadsorption system consisting of a continuous flow centrifuge, Protein A-Sepharose columns, and a semi-automatic elution system. Despite consistent and substantial lowering of immunoglobulin G levels, tumor regression was noted in only one of 11 dogs. In contrast, infusion of small volumes of plasma after incubation with heat and formalin-treated Staphylococcus aureus Cowan I resulted in a tumoricidal response in five of six animals. These results suggest that tumor necrosis is probably not induced by Protein A-mediated removal of humoral "blocking" factors.

Antibody-directed liposomes: comparison of various ligands for association, endocytosis, and drug delivery.

We have developed and compared the cytotoxicity of methotrexate-gamma-aspartate encapsulated in several liposome formulations which bind mouse monoclonal antibody in order to define conditions for screening cell lines and antibodies for liposomal efficacy. Liposomes conjugated to Staphylococcus aureus Protein A were more potent than liposomes conjugated to either rabbit or affinity-purified goat anti-mouse immunoglobulin (Ig) when incubated with AKR/J SL2 cells sensitized with specific antibody. The antibody-directed Protein A liposomes were also 10-fold more potent than liposomes conjugated directly to specific antibody against the AKR/J SL2. We examined the effect of antibody specificity, concentration, and isotype on liposome-mediated drug delivery to AKR/J SL2 cells. The growth-inhibitory effect of the drug in the antibody-directed Protein A liposomes varied with the target antigen on the cell. The potency of the liposomes with a given antibody was proportional to their relative binding and endocytosis by the cells, and to the reactivity of the particular antibody with the cell as demonstrated by indirect immunofluorescence. The Protein A liposomes maintained maximal potency down to antibody concentrations as low as 1 microgram/ml with the anti-Thy 1.1-sensitized AKR/J SL2 cells, thus demonstrating the possible use of these liposomes for hybridoma screening. Use of isotype-switched variants of the anti-Thy 1.1 antibody with the AKR/J SL2 cells showed the superior efficacy of the IgG2a, IgG2b, and IgG3 isotypes to the IgG1 with the Protein A liposomes. The large differential potency of the free drug and the drug encapsulated in antibody-directed Protein A liposomes was maintained even at short incubation times, thus providing a system which may be useful for eradication of tumor cells from bone marrow in vitro.

Induction of antitumor immunity via intratumoral tetra-costimulator protein transfer.

Our group recently described a novel two-step Fc(gamma1) fusion protein transfer method, which entails the docking of Fc(gamma1) fusion proteins onto cells precoated with chemically palmitated protein A (pal-prot A). In the present study, we have adapted this protein transfer method, originally used in an ex vivo context, for in situ tumor cell engineering, and in so doing, we have evaluated its utility for the induction of antitumor immunity via combinatorial costimulator protein transfer on to tumor cell surfaces. The feasibility of "painting" cells with preformed conjugates of a murine B7-1 costimulator derivative, B7-1.Fc(gamma1), and pal-prot A in a single step was first established ex vivo. Next, B7-1.Fc(gamma1):pal-prot A transfer was accomplished in vivo by directly injecting the preformed conjugates into highly aggressive L5178Y-R lymphomas grown intradermally in syngeneic mice. The presence of cell surface-associated B7-1 epitopes on cells of the injected tumors was documented by flow cytometric analysis of cells recovered subsequently from the injected tumors. B7-1.Fc(gamma1), along with Fc(gamma1) fusion protein derivatives of three additional costimulators (Fc(gamma1).4-1BBL, CD48.Fc(gamma1), and Fc(gamma1).CD40L) geared toward a variety of immune effectors, were together preconjugated with pal-prot A and injected directly into tumor beds. Significantly, this "tetra-costimulator" combination, delivered intratumorally, induced complete tumor regression in approximately 45% of treated mice, whereas control injections of pal-prot A alone had no therapeutic effect. Furthermore, there was evidence for systemic antitumor immunity in that tumor-specific CTLs were detected in spleens recovered from cured mice, and these mice were uniformly protected against tumor rechallenge at distant tumor sites. Hence, combinatorial costimulator transfer, coupled to intratumoral delivery, may have special advantages for the induction of antitumor immunity.

Characterization of immune responses to experimental polyvalent subunit vaccines assembled in iscoms.

Immune responses to experimental polyvalent subunit vaccines assembled in a particulate adjuvant/delivery system, iscoms, are described. The fusion protein ZZ-M5 comprises structures of staphylococcal protein A (ZZ) and the Plasmodium falciparum malaria antigen Pf155/RESA (M5). MHC congenic mice were immunized with ZZ-M5 conjugated to iscoms containing human influenza virus antigen (flu ag, M5-flu-isc) or to iscom matrix (iscom particles without flu ag, M5-isc). Comparison of antibody and T-cell responses to M5-isc and M5-flu-isc demonstrated that the flu ag in M5-flu-isc exhibits carrier-related helper functions and that the assembly of immunogens in M5-flu-isc did not result in any apparent antigenic competition. In addition, assembly of ZZ-M5 and flu ag in iscoms induced an alteration of the IgG subclass profile of the antibody response to M5. The results suggest that assembly of immunogens in iscoms may be a useful approach to the design of subunit vaccines but that both quantitative and qualitative aspects of the immunogenic properties of such constructs should be scrutinized.

Effective capture of proteins inside living cells by antibodies indirectly linked to a novel cell-penetrating polymer-modified protein A derivative.

Antibodies against cytoplasmic proteins are useful tools that can control cellular function and clarify signaling mechanisms. However, it is difficult to capture proteins inside living cells, and thus appropriate methods for antibody delivery to the cytoplasm of living cells are required. Cell-penetrating materials, such as the TAT-peptide, have received attention for their ability to deliver various cargos into living cells. However, the direct modification of cargos with cell-penetrating materials is time-consuming and lacks versatility. Therefore, we conceived that protein A, which can bind to the fragment crystallizable region of an antibody, could indirectly link antibodies with cell-penetrating materials, creating an efficient and simple antibody delivery system. Here, we constructed a novel antibody delivery system using a cell-penetrating polymer-modified protein A derivative (CPP-pAd). Living cells treated with CPP-pAd/antibody complexes showed significantly higher antibody levels than those achieved with the commercially available reagent HVJ-E. Pre-treatment with sucrose prevented cellular uptake of the CPP-pAd/antibody complex, suggesting that the CPP-pAd/antibody internalization mechanism occurs through clathrin-dependent endocytosis. Interestingly, intracellularly delivered antibodies did not colocalize with endosome/lysosome markers, further suggesting that antibodies were delivered to the cytoplasm by escape from endosome/lysosome. Moreover, we observed that anti-nuclear pore complex antibodies, delivered to cells using CPP-pAd, localized to the nuclear membrane and inhibited nuclear factor κB dependent luciferase activity. Together, these results suggest that the antibodies delivered by CPP-pAd captured functional proteins, making CPP-pAd a promising strategy for effective capture of proteins inside living cells.

A rapid technique for measuring leukocyte chemotaxis in vivo.

A modification of connective tissue air bleb technique was used to develop a model system in inbred strains of mice for the study of chemoattractants in vivo. The method was developed using the well characterized n-formylated chemotactic peptide, f-Met-Leu-Phe, as the positive control. Injection of 0.1 ml of f-Met-Leu-Phe solutions from 10(-7) to 10(-10) M resulted in an influx of polymorphonuclear leukocytes within 2 h. Study of the kinetics of the response showed that the number of infiltrating cells reached a peak within 8 h and slowly declined over a 2-day period. The predominant infiltrating cell type during the first 24 h was the polymorphonuclear leukocyte. Between 24 and 48 h the polymorphonuclear leukocytes were replaced by monocytes. By utilizing an inbred mouse strain (DBA-1J and 2J) sufficient or deficient in C5 it was possible to distinguish compounds that were directly chemotactic from those that worked indirectly, or whose chemotactic potential could be enhanced by generation of the chemotactic complement split product C5a. The method was found to be technically simple, reproducible and semi-quantitative and represents a good model system to facilitate the comparison of chemotactic responses in vivo and in vitro.

Cardiopulmonary toxicity in patients with breast carcinoma during plasma perfusion over immobilized protein A. Pathophysiology of reaction and attenuating methods.

Tumoricidal reactions in dogs with spontaneous breast carcinoma occur after perfusion of plasma over protein A derived from Staphylococcus aureus and immobilized in collodion charcoal. When this treatment was extended to humans with breast cancer, hemodynamic and physiologic changes were noted. The evolution and spectrum of these reactions were evaluated during 47 plasma infusions in five patients. Initial treatment conditions consisting of rapid perfusion of plasma over high quantities of immobilized protein A were employed for 12 treatments in two patients. Within 30 minutes after treatments were begun, mean blood pressure, systemic vascular resistance, and stroke volume increased, as did heart rate, cardiac output, and rectal temperature; however, mean pulmonary artery pressure and total pulmonary resistance did not change. At 90 minutes, hypotension developed (lowest mean blood pressure was 59 +/- 14 mm Hg) that was associated with a decrease in systemic vascular resistance and total pulmonary resistance (536 +/- 66 and 146 +/- 44 dynes . second . cm-5, respectively). Cardiac output increased, tachycardia developed, stroke volume decreased, and rectal temperature increased. During the hypotensive phase, values of creatinine clearance and fractional excretion of sodium diminished. Noncardiogenic pulmonary edema appeared occasionally, with bronchospasm noted once. No hemodynamic changes were seen when saline solution was passaged over protein A immobilized in collodion charcoal or when autologous plasma was given without passage over protein A immobilized in collodion charcoal. Treatment conditions were modified by diminishing protein A quantity and plasma volume and slowing plasma perfusion rate, which resulted in significant attenuation of all cardiopulmonary responses. This report, then, defines for the first time the physiologic basis of the cardiopulmonary toxicity in humans after plasma perfusion over immobilized protein A.

In vivo follow-up of the cytotoxic effect of protein A--ricin toxin conjugate, on antibody-coated target cells.

Antibodies of the IgG class, specifically interacted with H-2 antigens of murine leukemia EL4 cells, were used to bind the ricin toxin covalently linked to protein A of Staphylococcus aureus. The toxin thus complexed, introduced in the cytoplasm by endocytosis, was able to kill the leukemic cells inoculated in animals. The interaction of immunotoxin with the leukemic cells was performed in vitro using one, two or three treatments and the cytotoxic effect on the target cells was followed up in vivo. The time interval between immunotoxin treatments was indicated by the membrane turn-over study of EL4 cells coated with specific antibodies in their monomeric form, complexed by protein A or interacted with protein A--ricin toxin conjugate. A proportion of 99.8% cells killed was obtained after three treatments.

Protein A of Staphylococcus aureus evokes Th1 type response in mice.

Protein A (PA) of Staphylococcus aureus is known to elicit several cytokines such as IFN gamma, TNF alpha and IL1. However, it has not been delineated yet as to which differentiation pathway lymphocytes follow after stimulation by PA. In this report, we attempted to collect such evidences. Cytokines, such as IFN gamma, IL2, IL4, IL6, IL10, TNF alpha, IL1alpha and IL1beta were measured in serum by ELISA. Our results show that 1 microg dose of PA stimulates the production of IFN gamma (115 +/- 5 pg/ml), TNF alpha (250 +/- 8 pg/ml) and IL1alpha (100 +/- 5 pg/ml) as compared to control levels of, 22 +/- 2, 20 +/- 2 and 35 +/- 3 pg/ml respectively whereas IL2 and IL1beta secretion were less (beyond the lower detection limit of the kit and 25 +/- 1 pg/ml, respectively) as compared to control (28 +/- 2 and 52 +/- 4 pg/ml, respectively). Larger dose of PA (10 microg) increases the expression of IL2 (75 +/- 3 pg/ml), TNF alpha (1380 +/- 120 pg/ml), IL1alpha (495 +/- 10 pg/ml) and IL1beta (110 +/- 7 pg/ml) as compared to controls described above. We also observed that 1 microg dose of PA decreases IL4, IL6 and IL10 secretion to 9 +/- 1, 10 +/- 1 and 10 +/- 2 pg/ml, respectively, whereas 10 microg dose also decreased them to 11 +/- 1, 12 +/- 2 and 30 +/- 4 pg/ml, respectively as compared to the background controls, i.e. 50 +/- 5, 50 +/- 2 and 215 +/- 9 pg/ml respectively. The ratio of IFN gamma to IL4 increased and the peak value at 4 h, came to 13 +/- 1 and 9.6 +/- 0.5 with 1 microg and 10 microg PA, respectively, which is an established parameter indicating a Th1 type response. Flow cytometry analysis of CD4+/CD8+ cells, and c-myc protein expression by splenocytes indicate that 1 microg dose of PA causes 2-fold increase of CD4+ cells with no change in CD8+ cells, and 10-fold increase in c-myc protein, whereas 10 microg dose increases CD4+ cells 4-fold, CD8+ cells 3-fold and c-myc protein 100-fold. The cell cycle data shows an induction of apoptosis in thymocytes and splenocytes with the large dose (10 microg), whereas the 1 microg dose does not show any apoptosis. This report indicates that a Th1 response is induced in mice, after PA inoculation at a dose of 1 microg animal. Thus, cytokine mediated therapeutic strategies should consider the fact that an induction of large concentration of some cytokines might become detrimental to the host.

Protection against 7,12-dimethylbenzanthracene-induced tumour initiation by protein A in mouse skin.

Protein A is an immunostimulating glycoprotein obtained from Staphylococcus aureus Cowan I. Its antitumour activity is proven in various tumour models. Its ability to provide protection against tumour initiation by the chemical carcinogen 7,12-dimethylbenzanthracene (DMBA) has been investigated in the present study using a mouse skin model of two-stage carcinogenesis. Protein A was administered intraperitoneally (1 microgram/animal 20 g body wt.) twice a week for 2 weeks, prior to initiation by DMBA. The promotion was performed by twice weekly applications of 12-O-tetradecanoyl phorbol-13-acetate (TPA) (3 or 5 micrograms/animal in 100 microliters acetone). Protein A provided significant protection to animals from DMBA-induced tumour initiation as was observed by the decrease in cumulative number of tumours, percent of animals developing tumours, number of tumours per animal and rate of tumour growth. Our data indicate that protein A has anticarcinogenic properties.

The B-cell targeted CTA1-DD vaccine adjuvant is highly effective at enhancing antibody as well as CTL responses.

A novel immunomodulating gene-fusion protein, CTA1-DD, has been developed which combines the ADP-ribosylating ability of cholera toxin (CT) with a dimer of an Ig-binding fragment, D, of Staphylococcus aureus protein A. The CTA1-DD adjuvant is non-toxic and greatly augmented T-cell-dependent and T-cell-independent responses to soluble admixed antigens after systemic as well as mucosal immunizations. CTL and antibody responses of all classes were increased by 10- to 100-fold above those observed in control mice immunized without adjuvant. CTA1-DD does not appear to form immune complexes or bind to soluble Ig following injection, but rather it binds directly to B-cells of all isotypes, including naïve IgD+ cells. No binding was observed to macrophages or dendritic cells, and immunizations in Fc gamma R-deficient mice demonstrated unaltered enhancing effects. As shown by inactive mutants, the CTA1-DD adjuvant is dependent on ADP-ribosyltransferase activity and requires the binding to Ig- via the DD moiety. The enhancing effect is associated with enlarged germinal centers, and binding of CTA1-DD to the B-cells strongly upregulates co-stimulatory molecules and counteracts apoptosis by inducing intracellular Bcl-2.

In vitro and in vivo stability of new multilamellar vesicles.

Factors affecting multilamellar vesicles transport to the blood compartment after oral administration to rats were evaluated first in vitro. A high entrapment of protein A was obtained when the vesicles were prepared by shearing a lyotropic lamellar phase composed of soybean phosphatidylcholine, cholesterol and polyoxyethylene alcohol (C12H25(OCH2CH2)4OH) as neutral detergent. In vitro tests showed that these vesicles (spherulites) were stabled in 50% of fetal calf serum, in acidic (pH 3) or basic (pH 10) buffers, in pancreatin but are partially lysed in 20mM bile salts. Oral administration of spherulites entrapping 111In-NTA in fasting rats showed a increase of radioacticivity in blood. This could be explained by passage of some spherulites in the enterocytes.

Inhibition of rat adenocarcinoma metastases by "Staphylococcus aureus" protein A.

Fischer F 344/CRBL female rats were injected intravenously with 10(6) syngeneic 13,762 adenocarcinoma cells, and daily doses of either 10 micrograms, 100 micrograms or 1 mg Staphylococcus aureus protein A were administered intraperitoneally on days 1 through 11. The animals were sacrificed on day 12, their lungs infused with Bouin's solution, and lung metastases counted. A significant reduction in the number of visible metastatic nodules was observed in the animals given 100 micrograms and 1 mg of Staphylococcus aureus protein A daily.

Protection against carbon-tetrachloride-induced lymphoid organotoxicity in rats by protein A.

In a previous study we described protection of rats from carbon tetrachloride (CCl4)-induced hepatotoxicity by protein A (PA). In the present report we described protection against CCl4-induced lymphoid organotoxicity in rats by PA. Our data indicate that CCl4 administration produced a significant increase in the number of total leukocytes and polymorphs in blood, a significant decrease in leukocyte count in bone marrow, and a significant loss in weight of spleen and adrenals. In the animals receiving PA prior to and after CCl4, these values were found to be near those of controls. Histological examination revealed thymocyte depletion in thymic lobe and degenerative changes of tissue in spleen of CCl4-exposed animals. PA treatment to CCl4-exposed animals exhibited improvement and recovery from the damage caused by CCl4. These observations throw a new light on the field of CCl4 toxicity to lymphoid organs and their protection by PA.

Biodistribution and gastrointestinal drug delivery of new lipidic multilamellar vesicles.

Encapsulation of therapeutic molecules in a new noncationic multilamellar vector (Spherulites), composed of phosphatidylcholine, cholesterol, and polyoxyethylene alcohol, is described here. Spherulites with entrapped drugs were prepared by shearing a phospholipidic lyotropic lamellar phase using a recently discovered method. The average size of these vesicles is approximately 300 nm. Our formulation did not show cytotoxicity to human cells and could be used as a drug delivery system. Our previous experiments showed that this new multilamellar vector is stable in many different buffers such as serum, acidic or basic buffers, and enzymatic buffers and may deliver drugs in vivo. We describe two ways of administration for drug delivery. The tissue biodistribution of radiolabeled Spherulites entrapping 125I protein A was studied after intravenous injection in Wistar rats using the major organs of the body. Approximately 70% of the radioactivity was found in the spleen 60 min after injection and about half this percentage was found in the liver. By 6 hr, only 52% remained in the spleen. The other tissues accumulated <30% of the dose throughout the duration of the study. On the other hand, oral administration of Spherulites, entrapping111 In-NTA, in fasting rats showed a significant increase of radioactivity in blood.