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Despite the emergence of a rich literature on the rise of China in Latin America (LAC) since 2000, we are still grappling with this phenomenon. In this article we seek to theorize this expanding South–South relationship from two vantage points. First, from the perspective of China, we argue that, by necessity, the PRC has had to internationalize its development strategy in order to compensate for its serious natural resource deficit, feed the world's largest domestic population, and fuel the soon-to-be largest economy in the world. LAC has been just one slice of China's ‘go out’ strategy. Our second perspective probes the effect of China's entry into the region. Through the lens of development economics, we identify three separate political economy scenarios that have been accentuated within those countries that have the strongest economic ties with China. We rely on measures of institutional performance and macro-economic trends to illustrate the variable effects of China on LAC.

Distribuidora Importadora Michelle S.A. – DIMSA cuenta con una cartera de 1,250 clientes distribuidos entre: bodegas, mayoristas, minoristas, puestos de mercados, boticas, instituciones, etc.) Se dedica a la distribución y representación de los productos de consumos masivos, mayormente en el rubro de limpieza; tiene una fuerza de venta conformada por 09 Vendedores / Cobradores. aproximadamente, el 90.00% son al crédito y el 10% al contado; de ahí que resulte sumamente importante la elaboración de la presente tesis doctoral, que analiza la morosidad, la liquidez, la rentabilidad, las líneas de crédito, promedio dia de cobranza, etc. Los créditos son otorgados a 07, 14, 21 y 28 días, considerándose como moroso a aquel crédito que llega al día 30 y no es cancelado. En la presente tesis doctoral, se analiza la solvencia a corto plazo de Dimsa, con el objetivo de conocer cómo se encuentra financiada la empresa; es decir, como la política de crédito y cobranzas implementada genera la administración de la morosidad y el control del riesgo. De acuerdo a los resultados obtenidos, se puede afirmar que la política de ventas al crédito necesita ser analizada a fondo, procurando recuperarlos en el tiempo establecido, caso contrario afectará la rentabilidad y la liquidez de la empresa y el análisis efectuado, se concluye que la Morosidad no influye negativamente en la Liquidez de la empresa. Pero, sí influye negativamente en la Rentabilidad. Confirmando que, si la Morosidad se incrementa, la Rentabilidad disminuye. Distribuidora Importadora Michelle S.A. – DIMSA, It has a portfolio of clients between 1250 diatribuidos; wineries, wholesalers, retailers, market stalls, pharmacies, and institutions. Is dedicated to distribuition and representation of products of mass consumtion, mostly in the area of cleanings. Has a sales force made up of 9 dealers/collectors, approximately, 90% are to credit and 10% cash is why it is extremely important to the development of this doctoral thesis analyzes the default, liquidity, profitability, lines average days of collection credits are awarded credits 7,14,21 and 28 days delinquent considering as one who reaches the 30th and is not paid. The present thesis, the short term solvency of DIMSA is analized in order to know how the company is financed; ie how credit policy and implemented collections generates management of bad debts and risk control according to the results obtained suggest that the policy of credit sales needs to be analyzed thoroughly trying to retrieve a set time otherwise affect profitability and liquidity of the company and the analysis conducted concluded that delinquencies will not adversely affect the liquidity of the company but adversely affects the profitability, confirming tha if delinquency increases profitability decreases. Tesis

El presente artículo científico estudia al sistema EXPORTA FÁCIL como medio de envío postal hacia el exterior que tiene a bien promover las exportaciones de las micros y pequeñas empresas (MYPES) y, en efecto, contribuir al desarrollo de las mismas; durante el 2007 hasta el 2011. Como se conoce, el sector empresarial viene evolucionando constantemente y las particularidades afines a su propia actividad generan cambios estructurales que conllevan a los jefes de estado a desarrollar alternativas que contribuyan a facilitar y dinamizar las tareas de comercialización de las mismas; sea a nivel local, nacional e internacional a través de las exportaciones. En ese sentido estudios como el que se prevé efectuar en esta investigación, asienta su interés de identificar aspectos de la utilización de los envíos postales regulados por Aduana denominado Exporta Fácil. Si bien a nivel nacional y en distintas regiones y localidades del país, el sistema muestra frecuencia de envíos del que se podría entender que ha causado un efecto positivo en la comercialización al exterior por medio de esta herramienta de gestión logística y operativa a favor de las micros y pequeñas empresas (MYPES). Sin embargo nace la inquietud de poder identificar cuál ha sido el uso de esta herramienta en Loreto. Asimismo, el poder investigar determinará cuántas micros y pequeñas empresas (MYPES) han utilizado este sistema, qué productos han enviado, qué montos y a qué mercados, en el periodo 2007 - 2011. The scientific article studies the “Exporta Facil” system, as a mean of posting to abroad, which it has to promote the exportations of micros and small enterprises (MYPES) and, in fact, to contribute to the development of itself; during the period of 2007 until 2011. As it is known, the business sector comes to evolve constantly and the related particularities to its own activity making some structural changes that it takes to every head of state to develop alternatives that contributes to facilitate and boost the marketing tasks of themselves; either locally, national and international through out the exportations. In this sense, a study expects to make in this investigation, regarding to identify features of posting utilizations regulated by Customs Office named as Exporta Facil. While nationwide, different regions and places in this country, the system shows the frequencies of posting might understand to have caused a positive effect in the commercialization to abroad by mean of this tool of management logistic and operations in favor of the micros and small enterprises. However, the curiosity of power to identify was the use of this tool in Loreto. In addition, to investigate would determine how many micro and small enterprises (MSEs) have used this system, what products have been sent, what amounts and what markets in the period of 2007-2011. Tesis

Orientational photorefractive gratings in nematic liquid crystals (NLCs) on a poly(N-vinyl-carbazole) film dispersed with 5,10,15,20-tetra-phenylporphyrinatozinc across poly(vinyl alcohol) film were observed. The maximum diffraction efficiency of 0.3-0.5% was measured.

Liposomes and lipid-based drug delivery systems have been used extensively over the last decade to improve the pharmacological and therapeutic activity of a wide variety of drugs. More recently, this class of carrier systems has been used for the delivery of relatively large DNA and RNA-based drugs, including plasmids, antisense oligonucleotides and ribozymes. Despite recent successes in prolonging the circulation times of liposomes, virtually all lipid compositions studied to date are removed from the plasma compartment within 24h after administration by the cells and tissues of the reticuloendothelial system (RES). Plasma proteins have long been thought to play a critical role in this process but only a few efforts were made to evaluate the relevant importance of plasma protein-liposome interactions in the clearance process. Strategies to increase the bioavailability of liposomal drugs have included altering lipid compositions and charge, increasing lipid doses, and incorporating surface coatings. All of these modifications can influence membrane-protein interactions. In this article, we will focus on our experiences with liposome-blood protein interactions and how alterations in the chemical and physical properties of the carrier system influence the interactions with blood proteins and circulation times.

The recent clinical successes experienced by liposomal drug delivery systems stem from the ability to produce well-defined liposomes that can be composed of a wide variety of lipids, have high drug-trapping efficiencies and have a narrow size distribution, averaging less than 100 nm in diameter. Agents that prolong the circulation lifetime of liposomes, enhance the delivery of liposomal drugs to specific target cells, or enhance the ability of liposomes to deliver drugs intracellularly can be incorporated to further increase the therapeutic activity. The physical and chemical requirements for optimum liposome drug delivery systems will likely apply to lipid-based gene delivery systems. As a result, the development of liposomal delivery systems for systemic gene delivery should follow similar strategies.

Our recent in vivo studies have investigated the surface adsorption property of various circulating liposomes to blood proteins, and have related this property to liposome clearance behavior. In particular, we have investigated liposomes composed of different charged or neutral lipids, fatty acyl chain length and saturation, and cholesterol content. From these studies an apparent inverse relationship between the amount of blood protein that associates with large unilamellar vesicles and the circulation half-lives of the liposomes is observed, indicating that protein-mediated liposome clearance mechanisms are dominant. Furthermore, by comparing the protein profiles of rapidly cleared liposomes with liposomes exhibiting enhanced circulation times, key blood proteins have been identified and implicated in the clearance process.

Liposomal drug-delivery systems have come of age in recent years, with several liposomal drugs currently in advanced clinical trials or already on the market. It is clear from numerous pre-clinical and clinical studies that drugs, such as antitumor drugs, packaged in liposomes exhibit reduced toxicities, while retaining, or gaining enhanced, efficacy. This results, in part, from altered pharmacokinetics, which lead to drug accumulation at disease sites, such as tumors, and reduced distribution to sensitive tissues. Fusogenic liposomal systems that are under development have the potential to deliver drugs intracellularly, and this is expected to markedly enhance therapeutic activity. Advances in liposome design are leading to new applications for the delivery of new biotechnology products, such as recombinant proteins, antisense oligonucleotides and cloned genes.

Clusterin is a heterodimeric multifunctional protein expressed in a variety of tissues and cells. It forms high density lipid complexes in plasma and participates in the control of the lytic activity of the late complement complex (TCC, C5b-9). Together with vitronectin, clusterin binds to the nascent amphiphilic C5b-9 complex, rendering it water soluble and lytically inactive. To define the interactions that underlie the complement-inhibitory function of clusterin, we have examined the binding interactions between [125I]clusterin and the isolated components of the complex, C5b-6, C7, C8, and C9 and vitronectin. By using ligand blotting in the presence of Tween, specific binding of the labeled clusterin with C7, the beta-subunit of C8 and C9 was detected. Binding to C9 was competed by polymerized C9, but not by C8, C7, C6, and CD59, suggesting that the conformational change occurring during the hydrophilic-amphiphilic transition of C9 exposes the interaction site for clusterin. When thrombin-treated C9 was analyzed, clusterin was found to recognize the C9b fragment containing the hydrophobic membrane interaction segment. Both subunits of clusterin interact with C9 and are similarly potent in inhibiting C5b-9-mediated hemolysis and Zn+(+)-induced C9 polymerization. These results show that clusterin exerts its inhibitory effect by interacting with a structural motif common to C7, C8 alpha, and C9b.

The proteins associated with liposomes in the circulation of mice were analyzed in order to determine whether bound proteins significantly influence the fate of liposomes in vivo. Liposomes were administered intravenously via the dorsal tail vein of CD1 mice and were isolated from blood after 2 min in the absence of coagulation inhibitors using a rapid "spin column" procedure. Various negatively charged liposomes exhibiting markedly different clearance properties were studied; notably, these included liposomes containing 10 mol % ganglioside GM1 which has been previously shown to effectively limit liposomal uptake by the fixed macrophages of the reticuloendothelial system. The protein binding ability (PB; g of protein/mol of lipid) of the liposomes was quantitated and related to the circulation half-life (tau 1/2) of the liposomes. Liposomes having similar membrane surface charge imparted by different anionic phospholipids were found to exhibit markedly different protein binding potentials. Furthermore, PB values determined from the in vivo experiments were found to be inversely related to circulation half-lives. PB values in excess of 50 g of protein/mol of lipid were observed for rapidly cleared liposomes such as those containing cardiolipin or phosphatidic acid (tau 1/2 less than 2 min). PB values for ganglioside GM1-containing liposomes (tau 1/2 greater than 2 h) were significantly less (PB less than 15 g of total protein/mol of total lipid). PB values were also determined for liposomes recovered from in vitro incubations with isolated human serum; relative PB values obtained from these in vitro experiments were in agreement with relative PB values measured from in vivo experiments. PB values, therefore, could be a useful parameter for predicting the clearance behavior of liposomes in the circulation. Liposomes exhibiting increased PB values in vivo were shown by immunoblot analysis to bind more immune opsonins, leading to a higher probability of phagocytic uptake. Finally, based on results obtained using the in vitro system, it is suggested that the mechanism by which ganglioside GM1 prolongs the murine circulation half-life of liposomes is by reducing the total amount of blood protein bound to the liposomes in a relatively nonspecific manner.

In response to climate change, the Kyoto Protocol, and the need to reduce greenhouse gas emissions, researchers are looking to marine geological storage of CO2 as one of the most promising options. Marine geological storage of CO2 involves the capture of CO2 from major point sources (such as a power plant) and the transport of CO2 to storage sites in marine geological structures such as a deep sea saline aquifer. Since 2005, we have developed relevant technologies for marine geological storage of CO2 . Those technologies include possible storage site surveys and basic designs for CO2 transport and storage processes. To design a reliable CO2 marine geological storage system, we devised a hypothetical scenario and used a numerical simulation tool to study its detailed processes. The process of transport CO2 from the capture sites to the storage sites can be simulated with a thermodynamic equation of state. We compared and analyzed the relevant equation of state, including the Benedict-Webb-Rubin-Starling (BWRS), Peng-Robinson (PR), Peng-Robinson-Boston-Mathias (PRBM) and Soave-Redlich-Kwong (SRK) equations of state. To evaluate the predictive accuracy of the equation of state, we compare the results of numerical calculations with experimental reference data. In a supercritical state (above 31.1°C and 73.9bar), which corresponds to the thermodynamic conditions of CO2 reservoir sites, the BWRS, PR, and PRBM equations of state showed a good predictive capability. On the other hand, the SRK equation of state showed a high error rate of 300% in the supercritical state. This paper analyzes the major design parameters that are useful for constructing onshore and offshore CO2 transport systems. On the basis of a parametric study of the hypothetical scenario, we suggest relevant variation ranges for the design parameters, particularly the flow rate, diameter, temperature, and pressure. Using the hypothetical scenario, we also studied how the thermodynamic conditions of CO2 affect on the fluid flow behavior and thermal characteristics of a pipeline transport system. In summary, this paper presents our analysis and deductions of the major design parameters that are useful for constructing onshore and offshore CO2 transport systems.Copyright © 2009 by ASME

In order to facilitate the isolation of liposomes from blood components, we have developed a simple and rapid procedure combining chromatographic and centrifugal methods. This 'spin column' procedure was used to isolate liposomes from incubation mixtures with human serum or from the blood of CD1 mice after intravenous administration of liposomes. An advantage of this procedure is that processing times are fast (typically minutes) such that the isolation procedure can be done in the absence of chelators or other coagulation inhibitors which may affect protein/liposome interactions. Furthermore, several samples can be analyzed together and small sample volumes can be processed. In addition, we show that this spin column procedure can be employed to isolate large unilamellar vesicles averaging 100 nm in diameter from lipoproteins and plasma proteins. The applicability of this spin column procedure in studying protein/liposome interactions is demonstrated by quantitating the amount of human complement component C3 bound per liposome using a C3 competitive ELISA assay after incubation with human serum. The proteins associated with the recovered liposomes were further analyzed by conventional SDS-polyacrylamide gel electrophoresis. We show that egg phosphatidylcholine/cholesterol (55:45, mol/mol) or egg phosphatidylcholine/cholesterol/dioleoylphosphatidylserine (35:45:20, mol/mol) liposomes isolated from the circulation of CD1 mice within minutes of administration have distinct, complex profiles of associated proteins. By isolating circulating large unilamellar liposomes using the spin column method and characterizing the proteins associated with their membranes, this protein fingerprinting approach will expedite identifying protein interactions which affect liposome stability and clearance in vivo.

We have studied the complement-activating properties of liposomes. We show that surface charge is a key determinant of complement-activating liposomes. The nature of the charge, whether negative or positive, appears to dictate which pathway of the complement system is activated. Phosphatidylcholine:cholesterol (PC:CHOL, 55:45 mol/mol) liposomes were made to exhibit a positive or negative surface charge by the addition of cationic or anionic lipids, respectively. Normal human or guinea pig serum was incubated with liposomes, followed by determining the residual hemolytic activity of the serum as a measure of complement activation. Negatively charged liposomes containing phosphatidyl-glycerol, phosphatidic acid, cardiolipin, phosphatidylinositol, or phosphatidylserine activated complement in a Ca(2+)-dependent manner suggesting activation occurred via the classical pathway. Positively charged liposomes containing stearylamine or 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane activated complement via the alternative pathway. Neutral liposomes, PC:CHOL (55:45) and PC:CHOL:dipalmitoylphosphatidylethanolamine (35:45:20), failed to activate complement as measured by the hemolytic assays. We show that unsaturated liposomes are more potent complement activators than saturated liposomes and that 45 mol% cholesterol promotes complement protein-liposome interactions. Immunoblot analysis of phosphatidylglycerol-containing liposomes showed that C3b and C9 were associated with these liposomes. Thus, the complement consumption measured in the hemolytic assays represents active cleavage of the complement components and not passive adsorption to the liposome surface. These studies suggest that membranes composed of net charged phospholipids can activate the complement system. This observation underlines the importance in biologic membranes of complement regulatory proteins that protect normal cells from complement attack.

Liposomal nanoparticles (LNs) encapsulating therapeutic agents, or liposomal nanomedicines (LNMs), represent one of the most advanced classes of drug delivery systems, with several currently on the market and many more in clinical trials. During the past 20 years, a variety of techniques have been developed for encapsulating both conventional drugs and the new genetic drugs (plasmid DNA–containing therapeutic genes, anti-sense oligonucleotides, and small, interfering RNA [siRNA]) within LNs encompassing a very specific set of properties: a diameter centered on 100 nm, a high drug-to-lipid ratio, excellent retention of the encapsulated drug, and a long (> 6 hours) circulation lifetime. Particles with these properties tend to accumulate at sites of disease, such as tumors, where the endothelial layer is “leaky” and allows extravasation of particles with small diameters. Thus, LNs protect the drug during circulation, prevent it from reaching healthy tissues, and permit its accumulation at sites of disease. We will discuss recent advances in this field involving conventional anticancer drugs as well as gene-delivery, immunostimulatory, and gene-silencing applications involving the new genetic drugs. LNMs have the potential to offer new treatments in such areas as cancer therapy, vaccine development, and cholesterol management.

Introduction Over a 10-y period at our community hospital, more than 50% of women 40 y of age and younger underwent a mastectomy as first line breast cancer treatment. These results catapulted a study to identify personal and physical implications of a mastectomy and to determine if, in women of all ages, breast conservation therapy with close follow-up is a better alternative to mastectomy. Methods Six hundred eight women underwent a mastectomy for breast cancer from 1989 to 2005 at our teaching institution; 77% ( n = 120) of 156 successfully contacted women agreed to participate in the study, and 70% ( n = 84) of them completed a questionnaire. Results Most women discovered their breast cancer through mammography or self breast examination, 31% and 28%, respectively. Five patients were diagnosed at Stage 0, 35 at Stage 1, 26 at Stage 2, 8 at Stage 3, 1 at Stage 4, and 9 patients had an unknown stage of disease. Sixty-three patients primarily discussed their treatment plan with a surgeon; 80 were satisfied with the time spent discussing their treatment. Twenty-four patients underwent various reconstruction procedures; most (75%) were satisfied with their treatment and reconstruction choices. Conclusions Mastectomy as a treatment choice for breast cancer did not have the negative personal and physical outcome that we had predicted. Personal choice and a surgeon's advice were the primary influencing factors on the women's treatment choice of mastectomy. Adequate preoperative discussion time and a multimodality cancer team can be most helpful in providing comprehensive treatment options for all women with breast cancer.

It is well established that the circulation half-life of liposomes increases with increasing dose. This effect is commonly attributed to ‘saturation’ of the fixed and free macrophages of the reticuloendothelial system resulting in reduced clearance rates. However, it is also known that the clearance rate of liposomes is dependent on the amount of associated blood protein, leading to the possibility that dose-dependent increases in circulation lifetimes could be due to decreases in the amount of blood protein associated per liposome. In order to test this hypothesis, the protein binding and clearance properties of large unilamellar liposomes composed of distearoylphosphatidylcholine/cholesterol and egg phosphatidylcholine/dioleoylphosphatidic acid/cholesterol were examined in mice. Liposomes were injected over a dose range of 10 to 1000 mg lipid/kg body weight, and the circulation lifetime and liver and spleen accumulation monitored. As expected, longer circulation half-lives were observed at higher doses for both liposome compositions. However, it was also found that at higher liposome doses, significantly less protein was bound per liposome. The results indicate that there is a limited pool of blood proteins that is able to interact with liposomes of a given composition. At higher lipid doses these blood proteins are distributed over more liposomes resulting in lower protein binding values and longer circulation lifetimes.

The in vivo association of blood proteins with large unilamellar liposomes composed of saturated phosphatidylcholines was analyzed to determine the effect of membrane fluidity and hydrocarbon chain length on liposome-plasma protein interactions and liposome clearance. Liposomes composed of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and diarachidoylphosphatidylcholine (DAPC) were administered via the lateral tail vein of CD-1 mice and were subsequently isolated from the blood at 2 min postinjection. The protein binding ability (PB, grams of protein bound per mole total lipid) of the liposomes was quantified and related to their circulation half-lives. Liposomes composed of long-chain saturated phospholipids that exist in the gel (frozen) state at 39 degrees C (DPPC,DSPC and DAPC) bound large quantities of blood proteins, in excess of 48 g of protein per mole total lipid, and were found to be rapidly cleared from the circulation. The incorporation of cholesterol into DSPC liposomes resulted in significantly decreased PB values and enhanced circulation lifetimes for this lipid system. This cholesterol effect plateaued at 30 mol % cholesterol, corresponding to the loss of the gel-liquid crystalline phase transition, and resulted in PB values of 23-28 grams of protein per mole of total lipid. The types of blood proteins binding to DSPC liposomes were not significantly altered by the inclusion of cholesterol. This is the first demonstration of rapid clearance of neutral large unilamellar liposomes having high levels of bound protein.

Cationic liposomes provide a means to introduce genes into cells both ex vivo and in vivo. In the past few years their use has been described in several tissues, e.g. lungs, liver, endothelium, brain. In this study we evaluated a commercially available poly-cationic liposome formulation in delivering a reporter gene into cultured myogenic cell lines from mouse and rat, and primary fetal human myoblasts. We also examined the effect of serum on liposome-mediated transfection and designed a new procedure to enhance transfection efficiency, based on the pre-condensation of plasmid DNA with polylysine. Polylysine pre-condensation was particularly effective when transfecting the cells in the presence of serum, a finding that could be significant for in vivo transfections.

Liposomes recovered from the blood of liposome-treated CD1 mice were previously reported to have a complex protein profile associated with their membranes (Chonn, A., Semple, S.C., and Cullis, P.R. (1992) J. Biol. Chem. 267, 18759-18765). In this study, we have further characterized and identified the major proteins associated with very rapidly cleared large unilamellar vesicles. These liposomes contained phosphatidylcholine, cholesterol, and anionic phospholipids (phosphatidylserine, phosphatidic acid, or cardiolipin) that dramatically enhance the clearance rate of liposomes from the circulation. These anionic phospholipids are normally found exclusively in the interior of cells but become expressed when cells undergo apoptosis or programmed cell death, and thus, they are believed to be markers of cell senescence. Analysis of the proteins associated with these liposomes by SDS-polyacrylamide gel electrophoresis revealed that two of the major proteins associated with the liposome membranes are proteins with electrophoretic mobilities corresponding to M(r) of 66,000 and 50,000-55,000. The 66-kDa protein was identified to be serum albumin by immunoblot analysis. Using various biochemical and immunological methods, we have identified the 50-55-kDa protein as the murine equivalent of human beta 2-glycoprotein I. beta 2-glycoprotein I has a strong affinity for phosphatidylserine, phosphatidic acid, and cardiolipin inasmuch as the levels of beta 2-glycoprotein I associated with these anionic liposomes approach or even exceed those of serum albumin, which is present in serum at a concentration 200-fold greater than beta 2-glycoprotein I. Further, we demonstrate that the amount of beta 2-glycoprotein I associated with liposomes, as quantitated by an enzyme-linked immunosorbent assay, is correlated with their clearance rates; moreover, the circulation residency time of cardiolipin-containing liposomes is extended in mice pretreated with anti-beta 2-glycoprotein I antibodies. These findings strongly suggest that beta 2-glycoprotein I plays a primary role in mediating the clearance of liposomes and, by extension, senescent cells and foreign particles.

Serum complement opsonizes particles such as bacteria for clearance by the reticuloendothelial system. Complement has been reported to interact with liposomes and therefore may mediate the reticuloendothelial system clearance of liposomes. This study has used a rat serum model to define some of the characteristics of liposomes which modulate their ability to activate complement. Using functional hemolytic assays and C3/C3b crossed immunoelectrophoresis, we have demonstrated that liposomes activated rat complement in a dose-dependent manner with higher concentrations of liposomes activating higher levels of complement. The detection of complement activation required the inclusion of phospholipids bearing a net charge. Complement activation occurred via the classical pathway; no alternative pathway activation was detected. The presence of cholesterol contributed to complement activation in a dose-dependent manner. Phospholipid fatty acyl chain length did not influence complement activation while the introduction of unsaturated acyl chains markedly decreased levels of complement activation. Liposome size also influenced complement activation with 400 nm unilamellar vesicles more effectively activating complement than 50 nm vesicles for equivalent amounts of exposed lipid. These studies demonstrate that the composition of the liposome greatly affects the in vitro activation of rat serum complement and suggest that the biological half-life of liposomes in the circulation of rats may be altered by changing the liposome composition to reduce complement activation.

Alterations in the lipid composition of biological membranes can have dramatic effects on their ability to interact with soluble proteins. In a series of studies employing large unilamellar vesicles (LUVs) produced by an extrusion technique, we have characterized the influence of membrane components on protein-membrane interactions. Much of our understanding of how and why proteins interact with seemingly inert membrane surfaces stems mainly from studies involving one or two protein component systems. These studies, however, do not accurately reflect the interactions that occur in the complex biological milieu (reviewed by Horbett and Brash, 1987). There have been very few studies reported on the interactions of proteins with liposomal systems incubated with whole blood. There are two main reasons for this. First, the large majority of studies on the association of plasma proteins with liposomes in vitro have been performed employing multilamellar systems. Due to the variable lamellarity of liposomes of different lipid compositions, quantification of the amount of various proteins associated per liposome has not been possible. Second, convenient techniques have not been available for the isolation of liposomes, particularly LUVs, from blood components. We have recently described a rapid method for the isolation of well-defined large unilamellar liposomes from the blood of liposome-treated mice (Chonn et al., 1991). With such a procedure now available, we have started to biochemically and immunologically characterize the amount and type of proteins associated with liposomes exposed to the complex biological milieu.

The proteins associated with liposomes in the circulation of mice were analyzed in order to determine whether bound proteins significantly influence the fate of liposomes in vivo. Liposomes were administered intravenously via the dorsal tail vein of CD1 mice and were isolated from blood after 2 min in the absence of coagulation inhibitors using a rapid "spin column" procedure. Various negatively charged liposomes exhibiting markedly different clearance properties were studied; notably, these included liposomes containing 10 mol % ganglioside GM1 which has been previously shown to effectively limit liposomal uptake by the fixed macrophages of the reticuloendothelial system. The protein binding ability (PB; g of protein/mol of lipid) of the liposomes was quantitated and related to the circulation half-life (tau 1/2) of the liposomes. Liposomes having similar membrane surface charge imparted by different anionic phospholipids were found to exhibit markedly different protein binding potentials. Furthermore, PB values determined from the in vivo experiments were found to be inversely related to circulation half-lives. PB values in excess of 50 g of protein/mol of lipid were observed for rapidly cleared liposomes such as those containing cardiolipin or phosphatidic acid (tau 1/2 less than 2 min). PB values for ganglioside GM1-containing liposomes (tau 1/2 greater than 2 h) were significantly less (PB less than 15 g of total protein/mol of total lipid). PB values were also determined for liposomes recovered from in vitro incubations with isolated human serum; relative PB values obtained from these in vitro experiments were in agreement with relative PB values measured from in vivo experiments. PB values, therefore, could be a useful parameter for predicting the clearance behavior of liposomes in the circulation. Liposomes exhibiting increased PB values in vivo were shown by immunoblot analysis to bind more immune opsonins, leading to a higher probability of phagocytic uptake. Finally, based on results obtained using the in vitro system, it is suggested that the mechanism by which ganglioside GM1 prolongs the murine circulation half-life of liposomes is by reducing the total amount of blood protein bound to the liposomes in a relatively nonspecific manner.

The studies presented in this thesis were aimed at understanding the basis for the apparent recognition of liposomes as foreign particles by the reticuloendothelial system. This phenomenon represents a major problem to the development of optimal liposome targeted drug delivery systems. An understanding of the clearance process potentially will lead to the design of improved liposome carrier systems that display favorable and predictable pharmacokinetic properties. The studies fall into two main areas of investigation. The first area involved the study of the complement activating properties of liposomes composed of various lipid species. This is of interest because the activation of the complement system is known to provide the effector molecules that lead to opsonization and lysis of foreign particulates. It had not been previously determined whether liposomes composed of simple lipid species activate the complement system in the complex biological milieu. The studies presented here used a complement hemolytic assay to demonstrate that surface charge is a key determinant of complement-activating liposomes. The nature of the charge, whether negative or positive appeared to dictate which pathway of the complement system is activated. Negatively charged liposomes activated complement in a Ca2+ dependent manner suggesting that activation occurred via the classical pathway. Positively charged liposomes activated complement via the alternative pathway. Neutral liposomes failed to activate complement as measured by the hemolytic assays. Further, it was shown that unsaturated liposomes were more potent complement activators than saturated liposomes and that 45 mol% cholesterol promoted complement protein/liposome interactions. The relation between complement activation and clearance rate from the circulation was investigated. Although there appeared to be a direct relation between complement activation and clearance for liposomes composed of various anionic phospholipids, the relation appeared to break down for other liposomal systems. This suggests that other factors play a more important role in liposome clearance. The second area of investigation was directed at establishing an unambiguous role for proteins in mediating the clearance process in vivo. Liposomes were administered intravenously into CD1 mice and after various times the liposomes were recovered from the blood. The amount of total protein binding to the liposomes was quantitated using the bicinchoninic acid protein assay, and the proteins associated with the recovered liposomes were analyzed by SDS-polyacrylamide gel electrophoresis. In order to facilitate the isolation of liposomes from blood components, a simple and rapid procedure combining chromatographic and centrifugal methods was developed. This “spin column” procedure enabled the use of large unilamellar liposomes instead of multilamellar vesicles, thus making possible the measurement of parameters such as the amount of protein bound/vesicle. From these studies, it was established that the amount of protein bound to the liposomes was inversely related to the circulation half-life of the liposomes. The proteins associated predominantly with very rapidly cleared liposomes consisted of large amounts of opsonins including C3 fragments and IgG, as well as other proteins such as apolipoprotein H which as of yet has no recognized opsonic role. Furthermore, these studies demonstrated that the mechanism by which ganglioside GM1 prolongs the circulation half-life of liposomes was by reducing the total amount of blood protein bound to the liposomes in a relatively non-specific manner. These studies established unambiguously that the apparent differences in liposome clearance behavior observed in vivo is related to the amount and type of protein associated with the liposomes in vivo. This has important implications for the design of liposomes having stable properties in the circulation and for the design of biocompatible surfaces.

A wide range of liposome compositions have previously been examined in vivo for their ability to affect the uptake of liposomes into cells of the reticuloendothelial (RE, mononuclear phagocyte) system (Allen, T.M. and Chonn, A. (1987) FEBS Lett. 223, 42-46; Allen et al. (1989) Biochim. Biophys. Acta 981, 27-35). In this study we have examined the ability of cultured murine bone marrow macrophages to endocytose liposomes of various compositions and have looked for correlations between the in vivo and the in vitro observations. Compounds which substantially decreased RE uptake of liposomes in vivo, such as monosialoganglioside (GM1) and a novel synthetic lipid derivative of polyethyleneglycol (PEG-PE), also greatly decreased liposome uptake by bone marrow macrophages in a concentration-dependent manner. Lipids which increase bilayer rigidity, such as sphingomyelin (SM) and cholesterol (CHOL), decreased both in vivo and in vitro uptake of liposomes. Likewise, positive correlations were observed between the in vivo behavior of liposomes containing phosphatidylserine (PS) or various gangliosides and the ability of these liposomes to be taken up by bone marrow macrophages. Total liposome uptake by macrophages increased with incubation time at 37 degrees C while very little liposome association with the macrophages was observed at 4 degrees C. Liposome uptake increased with liposome concentration and for liposomes composed of egg phosphatidylcholine (PC) uptake plateaued at 40 nmol lipid per mg cell protein. There was an inverse correlation between liposome size of extruded large unilamellar vesicles and their uptake by macrophages.