Self-assembling polycation/DNA complexes represent a net charge, although the major band on SDS-PAGE copromising synthetic vector for gene delivery. However, migrates with albumin. Serum albumin binds to pLL/DNA despite considerable versatility and transfectional activity complexes in vitro, forming a ternary pLL/DNA/albumin in vitro, such materials are quickly eliminated from the complex which regains some ethidium bromide fluorbloodstream following intravenous injection (plasma ␣ halfescence and fails to move during agarose electrophoresis. life typically less than 5 min). For targeted systemic delivAlbumin also causes increased turbidity of complexes, and ery a more prolonged plasma circulation of the vector is reduces their zeta potential to the same level (−16 mV) essential. Here we have examined factors contributing to as is measured in serum. We propose that rapid plasma rapid elimination of poly(L-lysine) (pLL)/DNA complexes elimination of polycation/DNA complexes results from their from the bloodstream, and implicate the binding of proteins binding serum albumin and other proteins, perhaps due to to the polyelectrolyte complexes as a likely cause for their aggregation and phagocytic capture or accumulation of the blood clearance. pLL/DNA complexes reisolated from ternary complexes in fine capillary beds. serum associate with several proteins, depending on their

Gene therapy for systemic diseases requires intravenous administration, but existing vectors are not suitable for systemic delivery, often showing rapid elimination from the bloodstream that restricts potential transfection sites to "first-pass" organs. To develop long-circulating vectors, here we have compared polyplexes containing DNA and poly-L-lysine (PLL) or polyethylenimine (PEI), surface-modified with either monovalent polyethylene glycol (PEG) or multivalent copolymers of N-(2-hydroxypropyl)methacrylamide (PHPMA), correlating their biophysical properties with their distribution following intravenous injection. A key difference between the two types of coating is the introduction of lateral stabilization by surface attachment of multivalent PHPMA, in addition to the steric stabilization provided by both types of polymers. The alpha-half-life for bloodstream clearance of polycation/DNA polyplexes (typically <5 minutes in mice) could be extended using multivalent PHPMA coating to >90 minutes. We found that the dose administered, as well as the amount and molecular weight of the coating PHPMA, had important effects on circulation properties. Multivalent PHPMA coating allows, for the first time, considerably extended circulation time using polyplex systems-a prerequisite for systemic gene delivery.

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Cationic polymer/DNA complexes are widely used for gene delivery, although the influence of the cationic polymer on the biophysical properties of the resulting complex is poorly understood. Here, several series of cationic polymers have been used to evaluate the influence of structural parameters on properties of DNA complexes. Parameters studied included the length of side chain, charge type (primary versus tertiary and quaternary), polymer molecular weight, and charge spacing along the polymer backbone. Cationic polymers with short side chains (such as polyvinylamine) formed small complexes, resistant to destabilization by polyanions, with low surface charge, limited transfection activity, and efficient intranuclear transcription. Conversely, cationic polymers with long side chains (e.g., poly[methacryloyl-Gly-Gly-NH-(CH(2))(6)-NH(2))] showed inefficient complex formation, high positive surface charge, and better transfection activity. The effects of molecular weight varied between polymers, for example, low molecular weight poly(L-lysine) produced relatively small complexes, whereas low molecular weight poly[2-(trimethylammonio)ethyl methacrylate chloride] produced large aggregates. Polymers containing quaternary ammonium groups showed efficient complex formation but poor transfection. Finally, spreading charges widely on the polymer structure inhibited their ability to condense DNA. In summary, to achieve small, stable complexes, the use of cationic polymers with short side chains bearing primary amino groups is suggested.

Objective-Invasion of uterine spiral arteries by extravillous trophoblasts in the first trimester of pregnancy results in loss of endothelial and musculoelastic layers. This remodeling is crucial for an adequate blood supply to the fetus with a failure to remodel implicated in the etiology of the hypertensive disorder preeclampsia. The mechanism by which trophoblasts induce this key process is unknown. This study gives the first insights into the potential mechanisms involved. Methods and Results-Spiral arteries were dissected from nonplacental bed biopsies obtained at Caesarean section, and a novel model was used to mimic in vivo events. Arteries were cultured with trophoblasts in the lumen, and apoptotic changes in the endothelial layer were detected after 20 hours, leading to loss of endothelium by 96 hours. In vitro, coculture experiments showed that trophoblasts stimulated apoptosis of primary decidual endothelial cells and an endothelial cell line. This was blocked by caspase inhibition and NOK2, a FasL blocking antibody. NOK2 also abrogated trophoblast-induced endothelial apoptosis in the vessel model. Key Words: apoptosis Ⅲ endothelium Ⅲ trophoblast Ⅲ pregnancy Ⅲ arteries R emodeling of the uterine arteries is a key event in early pregnancy. In the first trimester of pregnancy, a subpopulation of fetal trophoblast cells, the extravillous trophoblast, invade the uterine wall (interstitial invasion) and its blood vessels (endovascular invasion) as far as the myometrial segments. In the uterine spiral arteries, the trophoblasts interdigitate between the endothelial cells (ECs), replacing the endothelial lining and most of the musculoelastic tissue in the vessel walls. This creates a high-flow, low-resistance circulation that increases maternal blood flow to the placental villi at the maternal-fetal interface. Conclusions-ExtravillousData suggest that trophoblasts bind to and migrate along the luminal surfaces of the endothelium and transiently coexist on the walls of partially modified spiral arteries before replacing the endothelium. 1,2 Little is known as to how these processes are regulated in normal pregnancies; however, their pivotal importance in the establishment and maintenance of a successful pregnancy is illustrated when they fail to occur or occur to a significantly reduced extent. Defective remodeling of the spiral arteries is associated with pregnancies complicated by preeclampsia and intrauterine growth restriction (IUGR) 3 and is proposed to lead to an overall state of oxidative stress or fluctuations in oxygen concentrations analogous to hypoxia-reperfusion within the placental environment. 4 Preeclampsia and IUGR are responsible for considerable perinatal mortality and morbidity and carry health implications in adult life, including increased risk of hypertension, heart disease, and diabetes. 5 The importance of interactions between trophoblasts and the vascular cells of the spiral arteries, which may account for these differences in remodeling, have yet to be determined in normal or compli...

The concept of steric stabilization was utilized for self-assembling polyelectrolyte poly-L-lysine/DNA (pLL/DNA) complexes using covalent attachment of semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). We have examined the effect of coating of the complexes with pHPMA on their physicochemical stability, phagocytic uptake in vitro, and biodistribution in vivo. The coated complexes showed stability against aggregation in 0.15 M NaCl and reduced binding of albumin, chosen as a model for the study of the interactions of the complexes with plasma proteins. The presence of coating pHPMA had no effect on the morphology of the complexes as shown by transmission electron microscopy. However, results of the study of polyelectrolyte exchange reactions with heparin and pLL suggested decreased stability of the coated complexes in these types of reactions compared to uncoated pLL/DNA complexes. Coated complexes showed decreased phagocytic capture by mouse peritoneal macrophages in vitro. Decreased phagocytosis in vitro, however, did not correlate with results of in vivo study in mice showing no reduction in the liver uptake and no increase in the circulation times in the blood. We propose that the rapid plasma elimination of coated pLL/DNA complexes is a result of binding serum proteins and also of their low stability toward polyelectrolyte exchange reactions as a consequence of their equilibrium nature.

Binding of serum proteins to polyelectrolyte gene delivery complexes is thought to be an important factor limiting bloodstream circulation and restricting access to target tissues. Protein binding can also inhibit transfection activity in vitro. In this study a multivalent reactive hydrophilic polymer has been used to inhibit protein binding. This polymer is based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA) bearing pendent oligopeptide (Gly-Phe-Leu-Gly) side chains terminated in reactive 4-nitrophenoxy groups (8.6 mol%). The polymer reacts with the primary amino groups of poly(L-lysine) (pLL) and produces a hydrophilic coating on the surface of pLL⅐DNA complexes (as measured by fluorescamine). The resulting pHPMA-coated complexes show a decreased surface charge (from ؉14 mV for pLL⅐DNA complexes to ؊25 mV for pHPMA-modified complexes) as measured by potential analysis. The pHPMA-coated complexes also show a slightly increased average diameter (approximately 90 nm compared with 60 nm for pLL⅐DNA complexes) as viewed by atomic force and transmission electron microscopy and around 100 nm as viewed by photon correlation spectroscopy. They are completely resistant to protein interaction, as determined by turbidometry and SDS-polyacrylamide gel electrophoresis analysis of complexes isolated from plasma, and show significantly decreased nonspecific uptake into cells in vitro. Spare reactive ester groups can be used to conjugate targeting ligands (e.g. transferrin) on to the surface of the complex to provide a means of tissue-specific targeting and transfection. The properties of these complexes therefore make them promising candidates for targeted gene delivery, both in vitro and potentially in vivo.Successful introduction of DNA into target cells in vivo could unleash many strategies of gene therapy that promise considerable therapeutic advances against a wide range of diseases (1-6). Presently, however, nearly all of these strategies are severely limited by inefficient delivery and expression of therapeutic DNA (7). There are two broad problems; first, the efficiency of transgene expression in target cells and tissues is usually low or of inadequate duration. Second, possibilities for delivery of genes to target cells in vivo are limited to local administration either by direct injection into the target tissue or its blood supply (8 -10) or by introduction into an appropriate body compartment (11). There are presently no plausible options for systemic delivery of genes following intravenous injection.Viruses comprise the most efficient means to introduce transgenes into cells, but often their immunogenic and inflammatory properties combine with safety concerns to limit their realistic clinical application (12, 13). We are therefore seeking to develop nonviral systems, with limited immunogenicity, suitable for targeted delivery of genes in vivo. Distribution kinetics of nonviral vectors based on cationic lipids or on cationic polymers have previously been examined in vivo; however, they are invariably cleared ...

Skeletal muscle undergoes a progressive age-related loss in mass and function. Preservation of muscle mass depends in part on satellite cells, the resident stem cells of skeletal muscle. Reduced satellite cell function may contribute to the age-associated decrease in muscle mass. Here, we focused on characterizing the effect of age on satellite cell migration. We report that aged satellite cells migrate at less than half the speed of young cells. In addition, aged cells show abnormal membrane extension and retraction characteristics required for amoeboid-based cell migration. Aged satellite cells displayed low levels of integrin expression. By deploying a mathematical model approach to investigate mechanism of migration, we have found that young satellite cells move in a random ''memoryless'' manner , whereas old cells demonstrate superdiffusive tendencies. Most importantly, we show that nitric oxide, a key regulator of cell migration, reversed the loss in migration speed and reinstated the unbiased mechanism of movement in aged satellite cells. Finally, we found that although hepatocyte growth factor increased the rate of aged satellite cell movement, it did not restore the memoryless migration characteristics displayed in young cells. Our study shows that satellite cell migration, a key component of skeletal muscle regeneration, is compromised during aging. However, we propose clinically approved drugs could be used to overcome these detrimental changes.