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Maintenance of membrane phospholipid asymmetry. Lipid-cytoskeletal interactions or lipid pump?

Two models for the mechanism of maintenance of lipid asymmetry in erythrocytes are considered: binding of internal lipids to cytoskeletal proteins, and pumping of internal lipids from the outside to the inside of the cell. Analysis of the kinetics of lipid internalization suggests that the first model is more likely, and that the apparent pumping of lipids represents the activity of an ATP-dependent lipid flip/flop catalyst.

Cell Membrane↗

Intestinal effects of the products of lipid digestion on gastric electrical activity in the cat. Possible involvement of vagal intestinal receptors sensitive to lipids.

The relationship between the lipid content of the intestinal lumen and gastric motor activity was studied in anesthetized cats. For this purpose the electromyographic activity was recorded in the antrum whereas the small intestine (duodenum and first part of jejunum or ileum) was perfused with various solutions including calcium propionate, tributyrin, sodium caprylate, potassium oleate, mixtures containing linoleic acid, monolein, triolein, mixture of triglycerides, and glycerol. Long-chain lipids and glycerol both induced a decrease in the rate of basal antral activity. This effect was found to depend on the state of lipid hydrolysis (triglycerides, monoglycerides, and fatty acids) and was most marked with monolein, linoleic acid, and glycerol. With the middle-chain lipids used, these effects were only slight; short-chain lipids did not induce any visible changes in gastric activity. All these effects were prevented by cervical bivagotomy. It was concluded that the two types of lipid-sensitive vagal receptors, which we recently showed to exist in the small intestine, trigger an inhibitory enterogastric reflex that contributes to the regulation of gastric emptying.

Animals↗

Respective role of plasma nonesterified fatty acid oxidation and total lipid oxidation in lipid-induced insulin resistance.

To investigate the respective role of nonesterified fatty acids (NEFA) oxidation and total lipid oxidation in lipid-induced insulin resistance, we measured the response of glucose metabolism to insulin in normal subjects without (control study) or with either heparin (heparin study) or triglycerides (TG) emulsion (Ivelip study) infusion. Three-step euglycemic-mild-hyperinsulinemic clamp studies were performed. Lipid and glucose metabolism were studied using indirect calorimetry and [6,6-2H2]glucose and [1-13C]palmitate infusions. NEFA concentration and turnover and oxidation rates were decreased by insulin in the control study, but were maintained during the heparin and Ivelip infusion studies. Total lipid oxidation was decreased similarly in the control and heparin studies, but was increased during the Ivelip infusion. Stimulation of glucose oxidation and utilization by insulin was reduced in the Ivelip study, but not in the heparin study. Thus, peripheral insulin resistance was observed in the presence of a combined increase in total lipid and NEFA oxidation, but not during an isolated increase in NEFA oxidation. On the other hand, insulin-induced inhibition of glucose production was impaired in both the heparin and Ivelip studies. We conclude that total lipid oxidation is a major determinant of peripheral insulin resistance, whereas hepatic insulin resistance could be induced even by a moderate increase in NEFA availability.

Adult↗

Lipid-cell interactions. Liposome adsorption and cell-to-liposome lipid transfer are mediated by the same cell-surface sites.

The competitive behavior of solid vs. fluid liposomes in liposome-to-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding, transfer and 31P-NMR experiments have demonstrated that: (i) solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substances; (ii) fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; (iii) fluid liposomes that escape lysis dissociate from the cell, taking away cell lipid molecules. The latter process underlies the mechanism of cell-to-fluid liposome lipid transfer. In contrast, no lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface. Moreover, cell-induced aggregation of both fluid and solid freshly added liposomes is also inhibited by preincubation of the cells with either solid or fluid liposomes. Thus, different types of interaction of both fluid and solid liposomes with the cell are mediated by the same (or closely related) sites on the cell surface.

Adsorption↗

Lipid provisioning of turtle eggs and hatchlings: total lipid, phospholipid, triacylglycerol and triacylglycerol fatty acids.

Lipid composition of eggs and hatchlings was studied in painted, snapping and Blanding's turtles from western Nebraska. The average total lipid proportions of the egg yolk, post-embryonic yolk and hatchling soma dry masses were high in painted turtles (29.80%, 42.16% and 14.18%, respectively) relative to snapping and Blanding's turtles (egg yolk < 14%, postembryonic yolk < 17%, hatchling soma < 2%). The proportion of total egg yolk lipid used during development varied among species (painted turtles, x = 45.4%; snapping turtles, x = 68.9%; Blanding's turtles, x = 86.6%). Total lipid data are consistent with patterns reported for turtle species whose hatchlings overwinter in the nest (e.g., painted turtles) vs those whose hatchlings emerge in the fall (e.g., snapping and Blanding's turtles). In all species, the total lipid quantity of egg and hatchling components was primarily triacylglycerol (> 63%), an energy storage form. Predominant triacylglycerol fatty acids in eggs and hatchlings were 16:0, 16:1 and 18:1, and concentration changes of some yolk fatty acids occurred during development. The average phospholipid (a membrane form) quantity of the egg and hatchling components was relatively low in painted turtles (< 2% of the total lipid). In snapping and Blanding's turtles, the relatively large phospholipid proportion of the post-embryonic yolks (approximately 7%) could potentially contribute more to post-embryonic growth in these species than the phospholipids of painted turtle post-embryonic yolks.

Animals↗

The relationship between the acute changes in the systemic inflammatory response, lipid soluble antioxidant vitamins and lipid peroxidation following elective knee arthroplasty.

BACKGROUND AND AIMS: The aim of the present study was to examine the time course of the change in the concentrations of plasma retinol, alpha-tocopherol, lutein, lycopene, alpha-carotene, beta-carotene (antioxidant vitamins) and malondialdehyde (lipid peroxidation) in patients following elective knee arthroplasty. METHODS: Patients (n=20) who underwent an elective knee arthroplasty, had venous blood samples withdrawn pre-operatively and at 12, 24, 48, 72 and 168 h after the start of surgery for the analysis of circulating concentrations of C-reactive protein, albumin, cholesterol, triglycerides and malondialdehyde and also the lipid soluble antioxidants vitamins. RESULTS: Over the study period of 0-168 h there was a significant increase in circulating C-reactive protein concentrations (peak 48 h, P<0.001) and a significant fall in albumin, cholesterol and triglyceride concentrations (trough 48 h, P<0.001). Malondialdehyde concentrations fell by approximately 35% (P<0.001) during the study period. However, they did not alter significantly over the study period when adjusted for triglyceride (P=0.309). The plasma concentrations of retinol, alpha-tocopherol, lutein, lycopene, alpha-carotene and beta-carotene all fell (P0.001). When adjusted for cholesterol, the reductions in plasma concentrations of alpha-tocopherol, lutein, lycopene, and beta-carotene in the post-operative period were no longer statistically significant. CONCLUSIONS: The results of the present study indicate that, in apparently healthy subjects undergoing an acute inflammatory insult, circulating lipid soluble vitamin antioxidants are transiently reduced. However, when corrected for lipids the concentrations were similar to those of baseline. In the absence of an increase in lipid peroxidation such falls in plasma concentrations of lipid soluble vitamins are unlikely to be a reliable measure of status.

Aged↗

Development and evaluation of nitrendipine loaded solid lipid nanoparticles: influence of wax and glyceride lipids on plasma pharmacokinetics.

Nitrendipine is an antihypertensive drug with poor oral bioavailability ranging from 10 to 20% due to the first pass metabolism. For improving the oral bioavailability of nitrendipine, nitrendipine loaded solid lipid nanoparticles have been developed using triglyceride (tripalmitin), monoglyceride (glyceryl monostearate) and wax (cetyl palmitate). Poloxamer 188 was used as surfactant. Hot homogenization of melted lipids and aqueous phase followed by ultrasonication at temperature above the melting point of lipid was used to prepare SLN dispersions. SLN were characterized for particle size, zeta potential, entrapment efficiency and crystallinity of lipid and drug. In vitro release studies were performed in phosphate buffer of pH 6.8 using Franz diffusion cell. Pharmacokinetics of nitrendipine loaded solid lipid nanoparticles after intraduodenal administration to conscious male Wistar rats was studied. Bioavailability of nitrendipine was increased three- to four-fold after intraduodenal administration compared to that of nitrendipine suspension. The obtained results are indicative of solid lipid nanoparticles as carriers for improving the bioavailability of lipophilic drugs such as nitrendipine by minimizing first pass metabolism.

Administration, Oral↗

Mixtures of cationic lipid O-ethylphosphatidylcholine with membrane lipids and DNA: phase diagrams.

Ethylphosphatidylcholines are positively charged membrane lipid derivatives, which effectively transfect DNA into cells and are metabolized by the cells. For this reason, they are promising nonviral transfection agents. With the aim of revealing the kinds of lipid phases that may arise when lipoplexes interact with cellular lipids during DNA transfection, temperature-composition phase diagrams of mixtures of the O-ethyldipalmitoylphosphatidylcholine with representatives of the major lipid classes (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, cholesterol) were constructed. Phase boundaries were determined using differential scanning calorimetry and synchrotron x-ray diffraction. The effects of ionic strength and of DNA presence were examined. A large variety of polymorphic and mesomorphic structures were observed. Surprisingly, marked enhancement of the affinity for nonlamellar phases was observed in mixtures with phosphatidylethanolamine and cholesterol as well as with phosphatidylglycerol (previously reported). Because of the potential relevance to transfection, it is noteworthy that such phases form at close to physiological conditions, and in the presence of DNA. All four mixtures exhibit a tendency to molecular clustering in the gel phase, presumably due to the specific interdigitated molecular arrangement of the O-ethyldipalmitoylphosphatidylcholine gel bilayers. It is evident that a remarkably broad array of lipid phases could arise in transfected cells and that these could have significant effects on transfection efficiency. The data may be particularly useful for selecting possible "helper" lipids in the lipoplex formulations, and in searches for correlations between lipoplex structure and transfection activity.

1,2-Dipalmitoylphosphatidylcholine↗

Organization and dynamics of pyrene and pyrene lipids in intact lipid bilayers. Photo-induced charge transfer processes.

The dynamics of fluorescence quenching and the organization of a series of pyrene derivatives anchored in various depths in bilayers of phosphatidylcholine small unilamellar vesicles was studied and compared with their behavior in homogeneous solvent systems. The studies include characterization of the environmental polarity of the pyrene fluorophore based on its vibronic peaks, as well as the interaction with three collisional quenchers: the two membrane-soluble quenchers, diethylaniline and bromobenzene, and the water soluble quencher potassium iodide. The system of diethylaniline-pyrene derivatives in the membrane of phosphatidylcholine vesicles was characterized in detail. The diethylaniline partition coefficient between the lipid bilayers and the buffer is approximately 5,800. Up to a diethylaniline/phospholipid mole ratio of 1:3 the perturbation to membrane structure is minimal so that all photophysical studies were performed below this mole ratio. The quenching reaction, in all cases, was shown to take place in the lipid bilayer interior and the relative quenching efficiencies of the various probe molecules was used to provide information on the distribution of both fluorescent probes and quencher molecules in the lipid bilayer. The quenching efficiency by diethylaniline in the lipid bilayer was found to be essentially independent on the length of the methylene chain of the pyrene moiety. These findings suggest that the quenching process, being a diffusion controlled reaction, is determined by the mobility of the diethylaniline quencher (with an effective diffusion coefficient D approximately 10(-7) cm2 s-1) which appears to be homogeneously distributed throughout the lipid bilayer. The pulsed laser photolysis products of the charge-transfer quenching reaction were examined. No exciplex (excited-complex) formation was observed and the yield of the separated radical ions was shown to be tenfold smaller than in homogenous polar solutions. The decay of the radical ions is considerably faster than the corresponding process in homogenous solutions. Relatively high intersystem crossing yields are observed. The results are explained on the basis of the intrinsic properties of a lipid bilayer, primarily, its rigid spatial organization. It is suggested that such properties favor ion-pair formation over exciplex generation. They also enhance primary geminate recombination of initially formed (solvent-shared) ion pairs. Triplet states are generated via secondary geminate recombination of ion pairs in the membrane interior. The results bear on the general mechanism of electron transfer processes in biomembranes.

Calorimetry, Differential Scanning↗

New fluorescent octadecapentaenoic acids as probes of lipid membranes and protein-lipid interactions.

The chemical and spectroscopic properties of the new fluorescent acids all(E)-8, 10, 12, 14, 16-octadecapentaenoic acid (t-COPA) and its (8Z)-isomer (c-COPA) have been characterized in solvents of different polarity, synthetic lipid bilayers, and lipid/protein systems. These compounds are reasonably photostable in solution, present an intense UV absorption band (epsilon(350 nm) approximately 10(5) M(-1) cm(-1)) strongly overlapped by tryptophan fluorescence and their emission, centered at 470 nm, is strongly polarized (r(O) = 0.385 +/- 0.005) and decays with a major component (85%) of lifetime 23 ns and a faster minor one of lifetime 2 ns (D,L-alpha-dimyristoylphosphatidylcholine (DMPC), 15 degrees C). Both COPA isomers incorporate readily into vesicles and membranes (K(p) approximately 10(6)) and align parallel to the lipids. t-COPA distributes homogeneously between gel and fluid lipid domains and the changes in polarization accurately reflect the lipid T(m) values. From the decay of the fluorescence anisotropy in spherical bilayers of DMPC and POPC it is shown that t-COPA also correctly reflects the lipid order parameters, determined by 2H NMR techniques. Resonance energy transfer from tryptophan to the bound pentaenoic acid in serum albumin in solution, and from the tryptophan residues of gramicidin in lipid bilayers also containing the pentaenoic acid, show that this probe is a useful acceptor of protein tryptophan excitation, with R(O) values of 30-34 A.

1,2-Dipalmitoylphosphatidylcholine↗

Influence of the lipid composition on the organization of skin lipid model mixtures: an infrared spectroscopy investigation.

The polymorphism of the lipids of the stratum corneum (SC), the top layer of the epidermis, has a fundamental impact on the permeability properties of the skin barrier. In this work, we have examined by infrared spectroscopy the thermal behavior of model mixtures involving ceramide, palmitic acid and cholesterol, the three main components of the SC lipids, to gain a refined description of the participation of the various lipid species in the different phases observed as a function of temperature. The results show that below 40 degrees C ceramide, cholesterol and palmitic acid exist mainly in crystalline domains and the lipidic species show very limited miscibility. Between 40 and 50 degrees C, a transition from the crystalline to a liquid ordered (lo) phase occurs and it involves ceramides, cholesterol and palmitic acid. When the mixture has a high cholesterol content, this lo phase is stable up to 75 degrees C. For low cholesterol content, the mixtures undergo a second transition toward a more disordered phase which is likely not lamellar. The formation of these phases is critically dependent on the lipid composition and, therefore, it is likely that composition changes of SC lipids affect the phase behavior and, consequently, the skin barrier properties.

Animals↗

Gender differences in exercise-induced changes in sex hormone levels and lipid peroxidation in athletes participating in the Hawaii Ironman triathlon. Ginsburg-gender and exercise-induced lipid peroxidation.

BACKGROUND: Exercise reduces the risk of coronary heart disease in men and women but paradoxically, may promote free-radical formation, lipid peroxidation and vascular tissue injury. In this study, we assessed whether exercise-induced oxidative stress similarly affected men and women who participated in the Hawaii Ironman triathlon. METHODS AND RESULTS: Fifty-seven athletes (38 males) who completed the triathlon (3.9 km swim, 180.2 km bike, 42.2 km run) participated in this study. Blood samples were obtained 2 days before and immediately after the triathlon for the measurement of lipids, antioxidants and sex hormones and for the assessment of the susceptibility of plasma lipids to peroxidation. Lipid changes after exercise were similar for men and women. However, the susceptibility of plasma lipids to peroxidation was reduced by 61% (P < 0.001) in men and only 14% (P = NS) in women postrace. These changes were not associated with the supplemental use or levels of antioxidants. In addition, in men there was an increase of 58% in the antioxidant sex hormone estradiol and a decrease of 58% in testosterone (P < 0.001) postrace. No significant changes were noted for these two hormones in women. CONCLUSIONS: There are significant gender-specific differences in the susceptibility of lipids to peroxidation and in changes in estradiol and testosterone levels as a result of ultra-endurance exercise. These changes may in part explain the salutary effect of exercise on the development of vascular disease.

Adult↗

The effects of menopausal status and exercise training on serum lipids and the activities of intravascular enzymes related to lipid transport.

The study purpose was to compare the effect of exercise training on serum lipid and apolipoprotein concentrations and the activities of intravascular enzymes related to lipid transport in previously untrained eumenorrheic, premenopausal (PRM) women (n = 21; mean age, 36 +/- 3 years) and estrogen-free postmenopausal (POM) women (n = 16; mean age, 68 +/- 8 years). Subjects trained at a progressive intensity and duration (50% to 75% maximal O2 consumption [VO2max], 200 to 300 kcal/session) 4 d/wk for 12 weeks. Before and after training, VO2max, body weight, relative body fat, and fasting blood samples were obtained following 2 weeks on a standardized diet designed to maintain body weight and during the early follicular stage for the PRM group. Blood samples were analyzed for serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), the cholesterol content of the HDL3 subfraction, apolipoprotein (apo)A-I and apoB, lipoprotein(a), and the activity of lecithin:cholesterol acyltransferase (LCAT). Total and hepatic triglyceride lipase activity (HTGLA) were determined from plasma samples obtained after heparin administration. The cholesterol content of the low-density lipoprotein (LDL) and HDL2 subfractions and endothelial-bound lipoprotein lipase activity (LPLA) were calculated. A two (group) x two (time) multivariate ANOVA (MANOVA), with repeated measures for time indicated that the exercise-induced changes in physiological measurements, serum lipid or apolipoprotein concentrations, or enzyme activities did not differ between groups. Serum concentrations of TC, LDL-C, and HDL3 cholesterol, TG, and apo A-I and apoB were higher in POM women compared with the PRM group (P < .05 for all). For the combined groups, body weight and relative body fat did not change with training, but VO2max increased an average of 18.5% (P < .05). LPLA, HTGLA, and LCAT activity were unaltered with exercise training. Except for a small but significant decrease in HDL-C (-5.5%) and an elevation in apoB (4.3%; P < .05 for both), the concentrations of serum lipids and apolipoproteins did not change over the training period. We conclude that in previously untrained women, menopausal status does not influence the exercise training response of serum lipids or apolipoproteins or activities of intravascular enzymes related to lipid transport.

Adult↗

Purification and properties of a lipid transfer particle from Bombyx mori: comparison to the lipid transfer particle from Manduca sexta.

A lipid transfer particle (LTP) was purified from the hemolymph of the silkworm Bombyx mori. Like other insect LTPs, the B. mori LTP is a very high density lipoprotein containing 21% lipid and three apoproteins of mass approximately 350 kDa, approximately 85 kDa, and approximately 60 kDa. B. mori LTP catalyzes the exchange of lipids between different density class lipoproteins found in adult hemolymph and between adult lipoproteins and vitellogenin. However, in no case was net lipid transfer observed. Manduca sexta LTP also catalyzed exchange of lipids, but not net transfer of lipids, between different density class lipoproteins found in adult hemolymph.

Animals↗

Site-directed mutagenesis of surfactant protein A reveals dissociation of lipid aggregation and lipid uptake by alveolar type II cells.

Surfactant protein A (SP-A) binds to dipalmitoylphosphatidylcholine (DPPC) and induces phospholipid vesicle aggregation. It also regulates the uptake and secretion of surfactant lipids by alveolar type II cells. We introduced the single mutations Glu195-->Gln (rE195Q), Lys201-->Ala (rK201A) and Lys203-->Ala (rK203A) for rat SP-A, Arg199-->Ala (hR199A) and Lys201-->Ala (hK201A) for human SP-A, and the triple mutations Arg197, Lys201 and Lys203-->Ala (rR197A/K201A/K203A) for rat SP-A, into cDNAs for SP-A, and expressed the recombinant proteins using baculovirus vectors. All recombinant proteins avidly bound to DPPC liposomes. rE195Q, rK201A, rK203A, hR199A and hK201A function with activity comparable to wild type SP-A. Although rR197A/K201A/K203A was a potent inducer of phospholipid vesicle aggregation, it failed to stimulate lipid uptake. rR197A/K201A/K203A was a weak inhibitor for lipid secretion and did not competed with rat [125I]SP-A for receptor occupancy. From these results, we conclude that Lys201 and Lys203 of rat SP-A, and Arg199 and Lys201 of human SP-A are not individually critical for the interaction with lipids and type II cells, and that Glu195 of rat SP-A can be replaced with Gln without loss of SP-A functions. This study also demonstrates that the SP-A-mediated lipid uptake is not directly correlated with phospholipid vesicle aggregation, and that specific interactions of SP-A with type II cells are involved in the lipid uptake process.

1,2-Dipalmitoylphosphatidylcholine↗

Liquid chromatographic analysis of sebum lipids and other lipids of medical interest.

A technique is described for the high-pressure liquid chromatographic (HPLC) analysis of sebum lipid classes. The lipid present in sebum are separated by gradient elution HPLC from a microparticulate silica column and detected using a moving-wire detector. The system described can be linked to a computer. Quantitation can be carried out by comparing peak areas obtained with those of an internal standard. Peak trapping for further investigations of the separated components, for example by gas chromatography-mass spectrometry, is very easy. Sebum lipids are separated into the following lipid classes: hydrocarbons and squalene, cholesterol esters and wax esters, fatty acids as their methyl esters, triglycerides, 1,3-diglycerides, 1,2-diglycerides, free cholesterol, monoglycerides and other polar materials. Besides to sebum, the method has been successfully applied to other lipid mixtures, such as serum lipids. Examples of other applications are shown.

Chromatography, High Pressure Liquid↗

Improved high-performance liquid chromatographic method for the separation and quantification of lipid classes: application to fish lipids.

An improved straight-phase HPLC method for the separation and quantification of lipid classes is described. Two binary gradient solvent systems were used, one for polar and one for neutral lipids, and detection was performed with a light-scattering detector. The developed HPLC methods were highly reproducible and allowed base-line separation of all investigated polar lipid classes (phosphatidic acid, diphosphatidylglycerol. phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylinositol and lysophosphatidylcholine) and neutral lipid classes (triacylglycerol, free fatty acid, diacylglycerol, cholesterol and monoacylglycerol) except of cholesterol ester and wax ester. Application of the chromatographic systems demonstrated that the methods are suitable for quantitative analysis of the major lipid classes present in lipid extracts from livers and eggs of Atlantic salmon (Salmo salar).

Animals↗

Effect of lipid matrix and size of solid lipid nanoparticles (SLN) on the viability and cytokine production of macrophages.

Solid lipid nanoparticles (SLN) interact with mononuclear cells following intravenous injection. Little is known about the interaction of SLN with these cells, including cytotoxic effects and a possible up-regulation of pro-inflammatory cytokines. Therefore, we investigated the influence of lipid matrix, concentration, and size of SLN on murine peritoneal macrophages (mphi). mphi were incubated with SLN consisting of different lipid matrices and coated with the same surfactant. Cytotoxicity as assessed by MTT test was found to be concentration-dependent and was dramatically influenced by the lipid matrix. Marked cytotoxic effects were observed when cells were incubated with SLN consisting of stearic acid (STE) or dimethyl-dioctadecylammonium bromide (DDA) at concentrations of 0.01%, whereas SLN consisting of triglycerides, cetylpalmitate or paraffin did not exert major cytotoxic effects at the same concentrations. Cytotoxic effects were most likely caused by products of enzymatic degradation including free stearic acid. Analysis of cytokine production by mphi following incubation with SLN revealed concentration-dependent decreases in IL-6 production. These decreases seemed to be associated with cytotoxic effects. IL-12 and TNF-alpha production was neither detected in supernatants of mphi treated with SLN at any concentration nor in those of untreated cells. The size of SLN did neither affect cytotoxicity of SLN nor resulted in induction or digression of cytokine production by mphi. In conclusion, results of the present study revealed that the nature of the lipid matrix and the concentration of SLN dramatically impact cytotoxicity of SLN on mononuclear cells. Lipid matrices of SLN should therefore be carefully chosen and tested for later intravenous use.

Animals↗