the liver parasite burdens, evaluated by using the stauber method, indicated that the atovaquone loaded nanocapsules were significantly more effective than the free drug trypanosomiasis, another deadly disease caused by the parasite trypanosoma cruzi was also challenged by using nanoparticles of polyalkylcyanoacrylate as a targeted delivery system for nifurtimox the drugloaded nanoparticles significantly increased hotels soma san francisco trypanocidal activity compared with the empty one the use of poly lacticcoglycolic acid nanoparticles for targeted oral drug delivery to the inflamed gut tissue in the inflammatory bowel disease was examined such a strategy of local drug delivery was considered to be a distinct improvement, compared with existing colon delivery devices for this disease efficacy of nanoparticles as carrier systems for antiviral agents in human immunodeficiency virusinfected human monocytesmacrophages was evaluated in vitro by bender et al in the same year, macrophage targeting of antivirals, eg azidothymidine, was evaluated in vivo as a promising strategy for aids therapy the authors, after analyzing the results, concluded that nanoparticles could be considered as hotels soma san francisco a promising drug targeting system for azidothymidine to the res organs they also hypothesized that an increase in drug availability at the sites containing abundant macrophages might allow a reduction in dosage in order to avoid systemic toxicity for targeted gene delivery, calcium phosphate nanoparticles were found to be a unique class of nonviral vectors, which hotels soma san francisco can serve as efficient and alternative dna carriers moreover, the surface of these nanoparticles was suitably modified by absorbing a highly adhesive polymer eg polyacrylic acid and these surface modified calcium phosphate nanoparticles were used in vivo to target genes specifically to the liver chitosandna nanoparticles were designed as gene carriers using a complex coacervation process hotels soma san francisco the transfection efficiency was found to be celltype dependent conjugation of peg on the nanoparticles allowed lyophilization without aggregation and without the loss of bioactivity the clearance in mice following intravenous administration was found to be slower than unmodified nanoparticles, with a higher deposition in kidney and liver use of sodium chloride modified silica nanoparticles hotels soma san francisco snap as a novel nonviral vector with a high efficiency of dna transfer into cells has already been reported previous gene transfer methods using nonviral vectors, such as liposomes or nanoparticles, resulted in relatively low levels to approx of gene expression snap showed a better efficiency about of dna transfection into cells, as well as a hotels soma san francisco better protection of dna against degradation intravenous andor intraabdominal administration of the snap to mice revealed the accumulation of snap in the cells of the brain, liver, spleen, lung, kidney, prostate and testis, without any pathological cell changes or mortality, suggesting that they passed through the bloodbrain, blood prostate and bloodtestis barriers spongelike alginate nanoparticles hotels soma san francisco were found to be a new potential system for the delivery of antisense oligonucleotides the aim of this study was to design a new antisense oligonucleotide on carrier system based on alginate nanoparticles, and to investigate its ability to protect on from degradation in the presence of serum from the results, such nanosponges were found to hotels soma san francisco be promising carriers for specific delivery of on to the lung, liver and spleen summary during the last few decades, numerous approaches have been explored to modify the biodistribution and bioavailability of the drugs by using carriers systems of colloidal dimensions, eg liposomes, micelles and nanoparticles from the various studies reported in the literature, it can hotels soma san francisco be concluded that the factors responsible for particle uptake are the particle size, their surface charge, surface hydrophobicity and the presence andor absence of surface ligands keeping these key factors in mind, the designing and production of polymeric nanoparticles has been investigated since the late s the major challenge in the development of particulate carriers hotels soma san francisco for targeting at specific body sites is the preparation of the particles of optimum size with hydrophilic surfaces so as to have long circulation time in the blood and escape from res scavenging the bodys res, mainly the kupffer cells in the liver usually take up polymeric nanoparticles with hydrophobic surface therefore, the residence time of hotels soma san francisco these nanoparticles in the blood is considerably small however, as it has been observed that nanoparticles such as other colloidal drug delivery systems, on intravenous injection, are rapidly sequestered and retained by the organs comprising the reticuloendothelial system res, so that the targeting of nanoparticles to res is a much simpler process than the targeting to hotels soma san francisco any other organ the major defense system of the body, ie the reticuloendothelial system or more correctly, the mononuclear phagocyte system can recognize any foreign elements here the injected nanoparticles through the opsonization process the kupffer cells macrophages of the liver and of course to a lesser extent, the macrophages of the spleen and the hotels soma san francisco circulating macrophages play an important role in removing the opsonized particles particle size and surface properties of the particles can modulate the process of particle capture particles that have large hydrophobic surface are efficiently coated with plasma components and are rapidly removed from circulation thus, injected nanoparticles are covered by plasma proteins immediately the larger particles hotels soma san francisco are trapped in the liver but the smaller ones can reach the general circulation and the modified surfaces can be directed to the inflammation sites, endothelial cells or spleen targeting, usually achieved by injecting nanoparticles in vivo, is mainly passive, although active targeting is being done very recently an excellent example of passive targeting is hotels soma san francisco the uptake of nanoparticles by the kupffer cells of liver in many cases, this targeting can be exploited to help treatment in disease conditions eg leishmaniasis and candiasis, where macrophages are directly involved in the disease processes for greater specificity, the active targeting of the nanoparticulate delivery systems can be achieved by attaching the targeting ligand, hotels soma san francisco appropriate to the receptors on the target cells, to the surface of the particle conjugate monoclonal antibodies and sugar residues are the possible ligands the hepatocytes in the liver is an important target site for some diseases such as hepatitis, as well as in gene therapy in gene therapy, the liver can be used as bioreactors hotels soma san francisco where the administered gene can be used to express the missing factors such as growth hormones and blood factors in the liver, the endothelial lining of the blood vessels sinusoids have gaps or fenestrations, through which nanoparticle can pass and there is no intact basement membranes below these fenestrations thus, the nanoparticles can have a hotels soma san francisco close interaction with the liver hepotocytes the size of the gap was estimated to be between nm and nm hence, recent work in the field has suggested that the size of the nanoparticles should be less than nm in diameter for better interaction with the hepatocytes the polymeric nanoparticles, besides being biocompatible and biodegradable and having hotels soma san francisco longer circulation time in blood, remain unaffected by circulating lipases that protect the drug from the bioenvironment in an attempt to acquaint the readers with the sequence of events that are associated with nanoparticulate drug delivery to the resassociated disorders, this chapter first identifies the reticuloendothelial systems res, discusses about the possible mechanisms of the uptake hotels soma san francisco of nanoparticles by them, and finally, updates the application of drugloaded nanoparticles in the chemotherapy of diseases associated with res moreover, this chapter contributes to the furtherance of our present knowledge in the area of targeting by suggesting that the composition, surface characteristics and the size of the delivery vesicles are the three important parameters hotels soma san francisco that must be considered when drawing a strategy for efficient delivery acknowledgment the authors gratefully acknowledge the financial support provided to mkbasu by the council of scientific and industrial research csir, government of india, in the form of the emeritus scientist scheme references moghimi sm and patel hm serum mediated recognition of liposomes by phagocytic cells hotels soma san francisco of the reticuloendothelial system � the concept of tissue specificity adv drug del rev moghimi sm and hunter ac recognition by macrophages and liver cells of opsonized phospholipid vesicles and phospholipid head groups pharm res ll absolom d opsonins and dysopsonins � an overview meth enzymol hostetter mk, krueger ra and schmelling dj the biochemistry hotels soma san francisco of opsonization central role of the reactive thiolester of the third component of complement} infect dis ryder kw jr, kaplan je and saba tm serum calcium and hepatic kupffer cell phagocytosis proc soc exp biol med moghimi sm and patel hm calcium as a possible modulator of kupffer cell phagocytic function by regulating liver specific opsonic hotels soma san francisco activity bioehim biophys acta moghimi sm and patel hm serum opsonins and phagocytosis of saturated and unsaturated phospholipid liposomes bioehim biophys acta sinha j, raay b, das n, medda s, garai s, mahato sb and basu mk bacopasaponin c critical evaluation of antileishmanial properties in various delivery modes drug del sarkar s, mandal s, sinha j, hotels soma san francisco mukhopadhyay s, das n and basu mk quercetin critical evaluation as an antileishmanial agent in vivo, f drug targ lala s, pramanick s, mukhopadhyay s, bandyopadhyay s and basu mk harmine evaluation of its antileishmanial properties in various delivery systems j drug targ stolnik s, heald cr, neal j, garnett mc, davis ss, ilium l, hotels soma san francisco purkis sc, barlow rj and gellert pr polylactide polyethylene glycol micellerlike particles as potential drug carriers production, colloidal properties and biological performance j drug targ roser m, fischer d and kissel t surface modified biodegradable albumin nano and microspheres ii effect of surface charges on in vitro phagocytosis and biodistribution in rats eur j pharm biopharm hotels soma san francisco luck m, paulke br, schroder w, blunk t and muller rh analysis of plasma protein adsorption on polymeric nanoparticles with different surface characteristics } biomed mater res moghimi sm, porter cjh, muir is, ilium l and davis ss nonphagocytic uptake of intravenously injected microspheres in rat spleen influence of particle size and hydrophilic coating biochem hotels soma san francisco biophys res commun moore a, weissleder r and bogdanov a jr uptake of dextrancoated monocrys talline iron oxides in tumor cells and macrophages j magn reson imaging leroux jc, gravel p, balant l, volet b, anner bm, allemann e, doelker e and gurny r internalization of polyd,llactic acid nanoparticles by isolated human leucocytes and analysis hotels soma san francisco of plasma proteins adsorbed onto the particles ] biomed mater res bazile dv, ropert c, huve p, verrecchia t, marland m, frydman a, veillard m and spenlehauer g body distribution of fully biodegradable [c]poly lactic acid nanoparticles coated with albumin after parenteral administration to rats biomaterials norman me, williams p and ilium l influence of block hotels soma san francisco copolymers on the adsorption of plasma proteins to microspheres biomaterials armstrong ti, moghimi sm, davis ss and ilium l activation of the mononuclear phagocyte system by poloxamine its implication for targeted drug delivery pharm res vandorpe j, schact e, dunn s, hawley a, stolnik s, davis ss, garnett mc, davies mc and ilium l longcirculating hotels soma san francisco biodegradable polyphosphazene nanoparticles surfacemodified with polyphosphazenepolyethylene oxide copolymer biomaterials neal jc, stolnik s, garnett mc, davis ss and ilium l modification of the copolymers poloxamer and poloxamine can effect the physical and biological properties of surfacemodified nanospheres pharm res ilium l, jacobson lo, muller lh, ��� e and davis ss surface characteristics and the interaction of hotels soma san francisco colloidal particles with mouse peritoneal macrophages biomaterials moghimi sm, hawley ae, christy nm, gray t, ilium l and davis ss surface engineered nanospheres with enhanced drainage into lymphatics and uptake by macrophages of the regional lymph nodes febs lett l hawley ae, ilium l and davis ss lymph node localization of biodegradable nanospheres surface modified with hotels soma san francisco poloxamer and poloxamine block copolymers febs lett hawley ae, ilium l and davis ss preparation of biodegeradable, surface engineered plga nanospheres with enhanced lymphatic drainage and lymph node uptake pharm res ilium l, church ae, butterworth md, arien a, whetstone j and davis ss development of systems for targeting the regional lymph nodes for diagnostic hotels soma san francisco imaging in vivo behavior of colloidal pegcoated magnetite nanospheres in the rat following interstitial administration pharm res porter cj, moghimi sm, ilium l and davis ss the poly oxyethy lenepolyoxypropylene block copolymer poloxamer selectively redirects intravenously injected microspheres to sinusoidal endothelial cells of rabbit bonemarrow febs lett l araujo l, lodenberg r and kreuter j influence hotels soma san francisco of the surfactant concentration on the body distribution of nanoparticles} drug targ chen y, xou z, zheng d, xia k, zhao y, liu t, long z and xia j sodium chloride modified silica nanoparticles as a nonviral vector with a high efficiency of dna transfer into cells curr gene ther medda s, jaisankar p, manna rk, pal b, giri vs and basu mk phospholipid microspheres a novel delivery mode for targeting antileishmanial agent in experimental leishmaniasis } drug targ cui z, hsu ch and mumper rj physical characterization and macrophage cell uptake of mannancoated nanoparticles drug dev ind phar tyagi r, lala s, verma ak, nandy ak, mahato sb, maitra a hotels soma san francisco and basu mk targeted delivery of arjunglucoside i using surface hydrophilic and hydrophobic nanocarriers to combat experimental leishmaniasis } drug targ pandey r, zahoor a, sharma s and khuller gk nanoparticle encapsulated antitubercular drugs as a potential oral drug delivery system against murine tuberculosis tuberculosis pandey r and khuller gk subcutaneous nanoparticlebased antitubercular chemotherapy in an hotels soma san francisco experimental model antimicrob chemother pandey r, sharma a, zahoor a, sharma s, khuller gk and prasad � poly dl lactidecoglycolide nanoparticlebased inhalable sustained drug delivery system for experimental tuberculosis j antimicrob chemother fusai t, boulard y, durand r, paul m, bories c, rivollet d, astier a, houin r and deniau m ultrastructural changes in parasites hotels soma san francisco induced by nanoparticlebound pentamidine in a leishmania majormouse model parasite espuelus ms, legrand p, loiseau pm, bories c, barratt g and irache im in vitro antileishmanial activity of amphotericin � loaded in poly epsiloncaprolactone nanospheres j drug targ veerareddy pr, vobalaboina v and nahid a formulation and evaluation of oil inwater emulsions of piperine in visceral hotels soma san francisco leishmaniasis pharmazie cauchetier e, paul m, rivollet d, fessi h, astier a and deniau m therapeutic evaluation of free and nanocapsuleencapsulated atovaquone in the treatment of murine visceral leishmaniasis ann troy med parasitol gonzalezmartin g, merino i, rodriguezcabezas mn, torres m, nunez r and osuna a characterization and trypanocidal activity of nifurtimoxcontaining and empty nanoparticles of hotels soma san francisco polyethyl cyanoacrylates ] pharm pharmacol lamprecht a, ubrich n, yamamoto h, schafer u, takeuchi h, maincent p, kawashima y and lehr cm biodegradable nanoparticles for targeted drug delivery in treatment of inflammatory bowel disease j pharmacol exp ther bender ar, von briesen h, kreuter j, duncan ib and rubsamenwaigmani h efficiency of nanoparticles as a hotels soma san francisco carrier system for antiviral agents in human immunodeficiencyvirus infected human monocytesmacrophages in vitro antimicrob agents chemother lodenberg r and kreuter j macrophage targeting of azidothymidine a promising strategy for aids therapy aids res hum retroviruses roy i, mitra a, moitra a and mozumdar s calcium phosphate nanoparticles as novel nonviral vectors for targeted gene delivery int} pharm mao hq, roy k, troungle vl, janes ka, lin ky, wang y, august jt and leong kw chitosandna nanoparticles as gene carriers synthesis, characterization and transfection efficiency j control rel aynie i, vauthier c, chacun h, fattal e and couvreur p spongelike alginate nanoparticles as a new potential system for the delivery of antisense hotels soma san francisco oligonucleotides antisense nucleic acid drug del delivery of nanoparticles to the cardiovascular system banart khaw introduction nanoparticles have become one of the highly desirable drug delivery vehicles in recent years, not only due to the capacity but also due to their longevity most nanoparticles in use today are solid nanoparticles their applications in biological systems have hotels soma san francisco both advantages as well as adverse effects however, biocompatible nanoparticles such as liposomes or micells have circumvented some of these adverse consequences hood et al reported the use of lipid based nanoparticles nm, targeted with organic avp ligands, to target the endothelium of tumor vasculature to induce antiangiogenesis, following the delivery of mutant raf gene in , hotels soma san francisco kralj and pavelic wrote, the main interest currently lie in improving diagnostic methods and in developing better drug delivery systems to improve disease therapy relative to the application of nanotechnology the current chapter will be restricted to review of the application of nanoparticles, primarily nanolipid vesicles subsequently referred to by the original name, liposomes, to hotels soma san francisco the cardiovascular system, from diagnostic to therapeutic applications including novel cell membrane lesion sealing to gene delivery targeting the myocardium with immunoliposomes the interest in the use of nanoparticles, such as liposomes, for targeting the cardiovascular system has increased dramatically in recent years the first application of nontarget specific liposomes for localization in experimental myocardial infarction hotels soma san francisco was reported by caride et al they showed that plain cationic liposomes localized in the infarct better than neutral or anionic liposomes however, the first targeted delivery of liposomes in cardiovascular application was reported by us in although the exact size of the immunoliposomes used in that study was not determined, both multilamellar and unilamellar immunoliposomes hotels soma san francisco were generated these liposomes were targetspecific and were demonstrated to be able to target cardiac myosin, exposed to the extracellular milieu following experimental acute myocardial infarction using the canine model, inill labeled antimyosin immunoliposomes were demonstrated to localize in the infarct by gamma scintigraphic imaging, after catheter infusion of the immunoliposomes into the infarcted region hotels soma san francisco this study demonstrated the first potential application of liposomes as targeted nanolipid vesicles for the delivery of various pharmaceuticals despite this potential for in vivo targeted drug delivery, it was observed that these immunoliposomes also had high nontarget organ activities in vivo organs such as the liver and bone with high reticuloendothelial distribution were prime nontarget hotels soma san francisco organs for nonspecific immunoliposomes sequestration therefore, we reasoned that if the antimyosin immunoliposomes were made to mimic normal cells such as lymphocytes, then these modified immunoliposomes might circumvent the affinity for the reticuloendothelial system to mimic normal cells, sialoglycoprotein, fetuin, was attached to liposomes by either glutaraldehyde crosslinkage or cholate dialysis method in the presence hotels soma san francisco or absence of immunoglobulins although the initial studies were tantalizing, unequivocal demonstration of this phenomenon was not achieved the only clear cut advantage of sialoglycoprotein modified liposomes over plain liposome in mice was the increase retention of the liposome in the blood of mice at min post intravenous administration � vs � injected dose per gram, hotels soma san francisco respectively subsequently, klibanov and coworkers developed a method to prolong in vivo circulation time of liposomes, by coating liposomes with polyethyleneg lycol torchilin et al applied this method of polyethyleneglycol protection from sequestration by the reticuloendothelial system on antimyosin immunoliposomes, and demonstrated that pegantimyosinimmunoliposome with mole peg had slower blood clearance than pegantimyosin immunoliposomes with mol hotels soma san francisco peg or just immunoliposomes the half life t of immunoliposomes in rabbits with experimental acute myocardial infarction was min, whereas the t of pegcoated immunoliposomes at mole peg was about min hrs min and mol peg was min this increase in circulation time enabled enhanced targeting of radiolabeled pegimmunoliposomes in acute myocardial infarcts the maximum hotels soma san francisco ratio of infarct to normal tissue for plain liposomes was about , whereas that of mol pegimmunoliposomes was and that of mole pegimmunoliposomes was at hrs post intravenous liposome or immunoliposomes delivery the reduction in the uptake ratio of molepeg immunoliposomes is consistent with higher blood activity at the time of sacrifice than with mole pegimmunoliposomes if hotels soma san francisco the experiments were carried on longer, absolute uptake in the infarct, as well as the ratios of infarcted tissue to normal with mole pegimmunoliposomes should become greater than the values for the mole pegimmunoliposomes torchilin et al later reported that size also affected the targeting potential of pegmodified immunoliposomes in rabbits with experimental myocardial infarction hotels soma san francisco it appeared that small pegmodifiedantimyosin immunoliposomes of about nm diameter size had the highest accumulation of the intravenously administered immunoliposomes in the target � injected dose per gram of tissue � sd nonspecific uptake of the same pegantimyosin immunoliposomes in normal myocardium was only � idg unmodified plain liposomes and pegmodified plainliposomes had � and � hotels soma san francisco idg respectively in the infarcted myocardium normal myocardial activity was respectively � and � antimyosinliposome injection resulted in � and � idg localization in the infarct and normal myocardium respectively this resulted in the target to normal myocardial activity ratios of � for plain liposomes, � for pegplain liposomes, � for antimyosin liposomes and � for hotels soma san francisco pegantimyosin immunoliposomes the lower target to nontarget ratio of peg antimyosin immunoliposomes, relative to antimyosinimmunoliposomes in the infarct, is due to the higher blood activity of the former at hrs post intravenous administration of liposome preparations � vs idg respectively when larger liposome preparations nm diameter were used, it was observed that plain liposomes had hotels soma san francisco the same infarct localization activity � as the small plain liposomes however, modification with antimyosin, or with both antimyosin and peg, resulted in lower target activity , respectively but similar background activity and respectively it was reasoned that the lower targeting potential with the larger immunoliposomes was due to the limitation of these larger nanoparticles to extravasate hotels soma san francisco into the extracellular interstitial matrices, even though the blood activities at hrs were similar larger pegimmunoliposomes = and small peg immunoliposomes = � the larger pegmodified plain liposomes appeared to have similar nontarget organ activity as the smaller pegliposomes the mechanism of nonspecific accumulation with pegmodified plainliposomes may be related to blood activity that allowed longer hotels soma san francisco contact with nontarget tissues when peg modified large and small liposomes were used and respectively both large and small liposomes had tis of to min when they were modified with antimyosin, the tis were also similar between min small pegmodified liposomes had a ti of min, whereas larger pegmodified liposomes or small and large pegimmunoliposomes hotels soma san francisco had tis min it appears that this increase in blood circulation activity raised the background activity, as well as the absolute target activity, when pegmodified antimyosin immunoliposomes were used it was concluded that for diagnostic applications where high target to nontarget activity is desirable, immunoliposomes would be the best candidate for use however, in therapeutic applications hotels soma san francisco where absolute concentration of the targeting agent determines the efficacy of the intervention, small pegimmunoliposomes would be preferable however, large pegimmunoliposomes may also be useful due to the larger payload capacity of the larger liposomes, despite their lower target activity other nanoparticletargeting of the cardiovascular system although nanoparticles have been used as targeting agents for hotels soma san francisco tumors, blood and lymphatic vessels, the ultimate utility of such agents in the cardiovascular system is just beginning, even though the first in vivo demonstration of the feasibility of immunoliposomeapplication in the cardiovascular system was reported in recently, lanza and colleagues reported targeting of antiproliferative drugs, such as doxorubicin and paclitaxel, to the vascular smooth muscle hotels soma san francisco cells in cell cultures with a magnetic resonance imaging nanoparticle contrast agent in this study, the investigators prepared perfluorcarbon nanoparticles containing gadoliniumdtpabisoleate in surfactant comixture of lecithin and cholesterol the resultant nanoparticles had a mean diameter of nm these nanoparticles were targeted using a three step procedure initial targeting was achieved with biotinylated monoclonal antibody to hotels soma san francisco tissue factor tf, followed by administration of avidin that bound to biotin the third step consisted of administration of biotinylated nongaseous, lipidencapsulated, perflurocarbon emulsion nanoparticles loaded with doxorubicin or paclitaxel the study showed that tftargeted doxorubicin or paclitaxel loaded nanoparticles were more efficient antiproliferative agents than control targeted or nontargeted nanoparticles without drug loading the same group also showed that in vivo targeting with antifibrin antibody enabled visualization of the fibrin clots in canine femoral arteries by intravascular ultrasound imaging another application of targeted nanoparticles in the cardiovascular system was reported by spragg et al their study showed that immunoliposomes sporting monoclonal antibody specific for an extracellular domain of eselectin targeted hotels soma san francisco human umbilical vein endothelial cells huvec only after activation of these cells with recombinant human interleukin localization of the targeted immunoliposomes was to fold higher in il activated huvec than in unactivated ones other investigators have also shown that targeting of other adhesion molecules, such as icam, with antibodies to icam was feasible in vitrou echogenic hotels soma san francisco immunoliposomes targeted with antibodies to icam, vcam, fibrin and tissue factors have recently been reported for imaging of atheroma in yucatan mini swine model of endothelial denudation by intravascular ultrasound imaging expression of icam, vcam and tissue factor, as well as fibrin deposition, were visualized within min of antibodytargeted echogenic immunoliposomes administration novel application of hotels soma san francisco nanolmmunoliposomes although most of the applications of nanoparticle size immunoliposomes in the cardiovascular system have been in imaging or targeted drug delivery, in , we reported a novel application of antimyosin immunoliposomes for cell membrane lesion sealing of hypoxic cardiocytes in this application, we reasoned that cell membrane lesions that develop in myocardial injury and ischemia in hotels soma san francisco vivo or hypoxia in vitro result in irreversible myocyte death however, if these cell membrane lesions were sealed prior to extensive loss of intracellular contents, and hypoxia or ischemia is removed, then the injured cells, with the lesions now sealed with a membrane sealing agent, should be able to remain viable and undergo membrane repair hotels soma san francisco this hypothesis is demonstrated in fig the agent of cell membrane lesion sealing was proposed to be antimyosin immunoliposomes the concept of cell membrane lesion sealing as a repair mechanism is not exclusive to our hypothesis many fig diagrammatic representation of the process of cell membrane lesion sealing with antimyosin immunoliposomes csil cell with saicclamnd dismption hotels soma san francisco anchoring ml i ewoseij myosin tlirougli miitittiim tosnm cells, including mammalian cells, undergo rapid selfsealing of the ruptured cell membrane this is an innate property of many cells that responds to exposure to higher physiological concentration of ca in the extracytoplasmic environment when lesions develop in the cell membrane, utilizing intracellular membrane vesicles such as lysosomes hotels soma san francisco and endosomes to seal the lesions this innate mechanism, although highly useful, is not sufficient when development of cell membrane lesions is more extensive viability cpm isorniuxia ii � pl in our initial report, embryonic cardiocytes in tissue culture were used to demonstrate the role of antimyosinimmunoliposomes as cytoskeletalantigen specific immunoliposomes csils for sealing of hotels soma san francisco cell membrane lesions induced by vigorous process of induction of hypoxia cells x in sterile ml culture flasks with or without csils were flushed with n gas for min vigorously into the media dislodging the cells the caps were closed tight and the flasks were incubated in a �c c incubator for hrs the viability of hotels soma san francisco the cells were either assessed by trypan blue exclusion method or by [h]thymidine uptake, after an additional hrs of normoxic culture of the experimental cultures figure left and right shows that the viability of hypoxic cells treated with immunoliposomes csils � by trypan blue exclusion or x cpm by [h]thymidine uptake was not significantly different hotels soma san francisco from the viability of normoxic cultured controls � or � x cpm respectively, whether viability was assessed by the dye exclusion or [h]thymidine uptake method viability of the csil treated cells was significantly greater than the viability of hypoxic embryonic cardiocytes treated with plain liposomes plhypoxia, � or � x cpm, iggliposomes igglhypoxia, � , or hypoxia hotels soma san francisco alone � or � x fig left panel viability of controls viability f h] thymidine uptake trypan ehue uptake hypoxia hypoxia hypoxia fig viability of hypoxic cardiocytes treated with immunoliposomes il, plain liposomes pl, iggliposomes iggl and normoxic and hypoxic conditions determined by trypan blue dye exclusion left panel or with tritiated thymidine uptake criteria right hotels soma san francisco panel by the dye exclusion method was higher than by [h]thymidine uptake assessment fig right panel although the pattern is similar, the absolute difference may be due to the less stringent approach for the assessment viability by the trypan blue method inclusion of rhodamine labeled lipids into the formulation of the immunoliposomes, enabled visualization of hotels soma san francisco the attachment of liposomes on the surface of hypoxic cardi���tes in culture by epifluorescence fig or by confocal microscopy fig only hypoxic cells treated with rhodamine liposomes showed epifluorescence fig , left, whereas pl treated cells showed no epifluorescence fig , right similarly, confocal micrographs showed that there were discrete regions of epifluorescence, the diameter of which corresponded hotels soma san francisco to those of the liposomes nm assessment of the number of intact immunoliposomes fig epifluorescent micrographs of hypoxic hc cardiocytes treated with rhodamine antimyosin immunoliposomes left and rhodamineplain liposomes right fig, black and white confocal micrograph showing localization of intact liposomes left pseudocolor of another confocal micrograph showing a pink hue underlying structures which appear to hotels soma san francisco be individual liposomes the bars represent im on hypoxic cardiocytes with normal cell morphology resulted in � liposomes per cardiocytes fig , left however, there also appears to be diffused fluorescence in the cell membrane, indicative of the incorporation of the fluorescent lipids from the liposomes into the cell membrane fig , right, probably resulting from the hotels soma san francisco fusion of the immunoliposome membrane with that of the cell membrane the incorporation of the fluorescent lipid from the immunoliposomes to the cardiocytes is not due to the action of lipid transferases, since there are no transferases in the culture medium however, in in vivo situations, such transfer of liposomal lipids to normal cell membrane lipid hotels soma san francisco bilayer could occur preservation of myocardial viability by cell membrane lesion sealing with csils was also feasible in adult intact hearts in this study, immunoliposomes and control liposomes had an average diameter of � nm isolated adult rat cdi hearts were perfused with oxygenated krebshenseleit bicarbonate buffer at �c, after instrumentation on a langendorff perfusion hotels soma san francisco apparatus hearts were perfused under constant pressure of mm hg each heart was immersed in nacl solution maintained at �� and was paced at beatsmin hz the left ventricular enddiastolic pressure was set at mm hg, utilizing a waterfilled balloontipped catheter attached to a pressure transducer the baseline hemodynamic parameters were recorded using a stripchart recorder hotels soma san francisco after min of stabilization period global ischemia was induced by decreasing the perfusion pressure to zero within seconds then, a ml aliquot of freshly prepared mg ngpe modified antimyosin immunoliposomes csils, mg ngpe modified nonspecific iggliposomes iggl or placebo pbs was infused at various times of global ischemia various preparations of liposomes or placebo were administered hotels soma san francisco into the aorta via a threeway stopcock placed cm above the aorta, enabling administration of various agents without turning on the perfusion pump this process enabled maintenance of global ischemia for the duration of the ischemic period in all studies, a total global ischemia was maintained for min followed by reperfu sion for an additional hotels soma san francisco min during the reperfusion period, the end systolic and enddiastolic pressures were determined and the difference represented as left ventricular developed pressure lvdp lvdp of each heart was then compared with the baseline lvdp and lvdp of the baseline was determined when globally ischemic hearts were treated with csils at min of ischemia, the recovery of hotels soma san francisco function mean lvdp = � during reperfusion was near normal lvdp of sham operated hearts p = ns fig , left, and was highly and significantly greater than the lvdp of hearts treated with placebo � ,p = the total time function curve of the lvdp of hearts treated with csil at min of global ischemia was hotels soma san francisco � p = ns versus sham lvdp, but was greater than that of placebo treated hearts � , p = injury to hearts after min of global ischemia that were treated with csil or placebo, compared with sham operated heart by histochemical staining with nitroblue tetrazolium, is shown in fig , right nitroblue tetrazolium stains for dehydrogenase hotels soma san francisco enzyme activity and is seen as brown to purplish brown stained tissues these enzymes are lost following myocardial or cellular necrosis, resulting in no staining of the infarcted tissues seen in fig right, as light colored regions in the placebo treated hearts quantitative assessment also demonstrated that the size of the injury of csil treated hearts hotels soma san francisco � of total ventricles was the same as that of the sham operated hearts � ,p � ns i d so so time of reperfusion min e�pbs fig lvdp of globally ischemic or normal hearts treated with csil ?, sham operation and placebo o during reperfusion for min left panel, and the corresponding nitroblue tetrazolium chloride hotels soma san francisco stained heart slices showing normal myocardium stained brown and infarcted myocardium no staining, light color � oi if interventions were instituted almost immediately after the onset of global ischemia, then preservation of structure and function of the myocardium would be however, in reallife situations, time of initiation of injury to intervention cannot possibly be that short in most circumstances therefore, studies were also undertaken to determine whether there is a time dependency on myocardial function and structural preservation relative to csil administration thus, langendorff instrumented hearts were subjected to global ischemia as before, however, administration of csil or control nonspecific iggl was instituted at , and min of global ischemia reperfusion was instituted hotels soma san francisco at min and reperfusion sustained for min in globally ischemic hearts with csil administration at min of ischemia, return to near normal lvdp was achieved at min of reperfusion fig , top left panel when csil was administered at min of global ischemia, return to near normal function was at min of reperfusion fig , top right hotels soma san francisco panel however, when csil was administered at min of global ischemia, recovery of function was only � of baseline lvdp fig , bottom left panel, which was still greater than the lvdp of hearts injected with iggl � ,p = yet, the mean lvdp of all hearts treated with csil was statistically greater than the lvdp of hotels soma san francisco hearts treated with nonspecific iggl at corresponding times [fig bottom right period cramps and asprin panel] infarct sizes determined by computer planimetry of the nitroblue omin min nin min rrin min nin time of reperfusion min csil csil csil iggl iggl iggl ff sham �csil iggl t fbs treatments fig lvdp of globally ischemic adult rat hearts treated with csil ?, iggl ?