Current antiretroviral drugs used to prevent or treat human immunodeficiency virus type 1 (HIV-1) infection are not able to eliminate the virus within tissues or cells where HIV establishes reservoirs. Hence, there is an urgent need to develop targeted delivery systems to enhance drug concentrations in these viral sanctuary sites. Macrophages are key players in HIV infection and contribute significantly to the cellular reservoirs of HIV because the virus can survive for prolonged periods in these cells. In the present work, we investigated the potential of the lipid-based Neutraplex nanosystem to deliver anti-HIV therapeutics in human macrophages using the human monocyte/macrophage cell line THP-1. Neutraplex nanoparticles as well as cationic and anionic Neutraplex nanolipoplexes (Neutraplex/small interfering RNA) were prepared and characterized by dynamic light scattering. Neutraplex nanoparticles showed low cytotoxicity in CellTiter-Blue reduction and lactate dehydrogenase release assays and were not found to have pro-inflammatory effects. In addition, confocal studies showed that the Neutraplex nanoparticles and nanolipoplexes are rapidly internalized into THP-1 macrophages and that they can escape the late endosome/lysosome compartment allowing the delivery of small interfering RNAs in the cytoplasm. Furthermore, HIV replication was inhibited in the in vitro TZM-bl infectivity assay when small interfering RNAs targeting CXCR4 co-receptor was delivered by Neutraplex nanoparticles compared to a random small interfering RNA sequence. This study demonstrates that the Neutraplex nanosystem has potential for further development as a delivery strategy to efficiently and safely enhance the transport of therapeutic molecules into human monocyte-derived macrophages in the aim of targeting HIV-1 in this cellular reservoir.
In vitro cytotoxicity assays are essential tools in the screening of engineered nanomaterials (NM) for cellular toxicity. The resazurin live cell assay is widely used because it is non-destructive and is well suited for high-throughput platforms. However, NMs, in particular carbon nanotubes (CNT) can interfere in assays through quenching of transmitted light or fluorescence. We show that using the resazurin assay with time-point reading of clarified supernatants resolves this problem. Human lung epithelial (A549) and murine macrophage (J774A.1) cell lines were exposed to NMs in 96-well plates in 200 μL of media/well. After 24 h incubation, 100 μL of supernatant was removed, replaced with resazurin reagent in culture media and aliquots at 10 min and 120 min were transferred to black-wall 96-well plates. The plates were quick-spun to sediment the residual CNTs and fluorescence was top-read (λEx=540 nm, λEm=600 nm). The procedure was validated for CNTs as well as silica nanoparticles (SiNP). There was no indication of reduction of resazurin by the CNTs. Stability of resorufin, the fluorescent product of the resazurin reduction was then assessed. We found that polar CNTs could decrease the fluorescence signal for resorufin, possibly through oxidation to resazurin or hyper-reduction to hydroxyresorufin. This effect can be easily quantified for elimination of the bias. We recommend that careful consideration must be given to fluorimetric/colorimetric in vitro toxicological assessments of optically/chemically active NMs in order to relieve any potential artifacts due to the NMs themselves.
To examine the role that lipoprotein charge plays in cholesterol metabolism in vivo, we characterized the effects of an intravenous injection of 40 μmol of an uncharged phospholipid (phosphatidylcholine, PC) or an anionic phospholipid (phosphatidylinositol, PI) into fasted rabbits. PC injection had a negligible effect on lipoprotein charge and composition, similar to that observed in a saline-injected animal. In contrast, PI injection caused a significant increase in the net negative surface charge of all lipoproteins after only 10 min, followed by a gradual return to normal by 24 h. Lipoprotein compositional analysis showed that PI caused a significant increase of cholesteryl ester (CE) and cholesterol (FC) in the VLDL pool by 3 h, with no changes in VLDL-triglyceride content. While the bulk of the plasma CE was located in the HDL pool in the PC-injected animals, in the PI animals, VLDL became the major CE storage compartment. No major changes in the levels or composition of HDL or LDL were evident over the 24-h turnover period. Co-injection of [3H]FC revealed a 30-fold greater rate of clearance of the labeled cholesterol from the PI-injected rabbit plasma. In addition, the rate of cholesterol esterification by lecithin:cholesterol acyltransferase was almost completely inhibited in the PI animals. In summary, a bolus injection of PI into rabbits appears to enhance the mobilization of cellular sterol and promote a rapid clearance of both FC and CE from the plasma compartment. The data show that lipoprotein charge can affect cholesterol transport and that this process can be selectively manipulated. —Stamler, C. J., D. Breznan, T. A-M. Neville, F. J. Viau, E. Camlioglu, and D. L. Sparks. Phosphatidylinositol promotes cholesterol transport in vivo. J. Lipid Res. 2000. 41: 1214–1221. To examine the role that lipoprotein charge plays in cholesterol metabolism in vivo, we characterized the effects of an intravenous injection of 40 μmol of an uncharged phospholipid (phosphatidylcholine, PC) or an anionic phospholipid (phosphatidylinositol, PI) into fasted rabbits. PC injection had a negligible effect on lipoprotein charge and composition, similar to that observed in a saline-injected animal. In contrast, PI injection caused a significant increase in the net negative surface charge of all lipoproteins after only 10 min, followed by a gradual return to normal by 24 h. Lipoprotein compositional analysis showed that PI caused a significant increase of cholesteryl ester (CE) and cholesterol (FC) in the VLDL pool by 3 h, with no changes in VLDL-triglyceride content. While the bulk of the plasma CE was located in the HDL pool in the PC-injected animals, in the PI animals, VLDL became the major CE storage compartment. No major changes in the levels or composition of HDL or LDL were evident over the 24-h turnover period. Co-injection of [3H]FC revealed a 30-fold greater rate of clearance of the labeled cholesterol from the PI-injected rabbit plasma. In addition, the rate of cholesterol esterification by lecithin:cholesterol acyltransferase was almost completely inhibited in the PI animals. In summary, a bolus injection of PI into rabbits appears to enhance the mobilization of cellular sterol and promote a rapid clearance of both FC and CE from the plasma compartment. The data show that lipoprotein charge can affect cholesterol transport and that this process can be selectively manipulated. —Stamler, C. J., D. Breznan, T. A-M. Neville, F. J. Viau, E. Camlioglu, and D. L. Sparks. Phosphatidylinositol promotes cholesterol transport in vivo. J. Lipid Res. 2000. 41: 1214–1221. The factors that regulate cholesterol flux to the liver are as yet poorly understood but are thought to involve two distinct systems: a cellular sterol regulatory system and an intravascular transport system. The classic view has been that excess extrahepatic cholesterol can be transported in high density lipoprotein (HDL) particles to the liver for excretion (1Glomset J.A. The plasma lecithin:cholesterol acyltransferase reaction. (Review).J. Lipid Res. 1968; 9: 155-167Google Scholar). HDL has been shown to be able to adsorb cholesterol and cholesteryl esters (CE) from cell membranes (2Phillips M.C. Gillotte K.L. Haynes M.P. Johnson W.J. Lund-Katz S. Rothblat G.H. Mechanisms of high density lipoprotein-mediated efflux of cholesterol from cell plasma membranes.Atherosclerosis. 1998; 137: S13-S17Google Scholar, 3de la Llera-Moya M. Rothblat G.H. Connelly M.A. Kellner-Weibel G. Sakr S.W. Phillips M.C. Williams D.L. Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface.J. Lipid Res. 1999; 40: 575-580Google Scholar, 4Graf G.A. Connell P.M. van der Westhuyzen D.R. Smart E.J. The class B, type I scavenger receptor promotes the selective uptake of high density lipoprotein cholesterol ethers into caveolae.J. Biol. Chem. 1999; 274: 12043-12048Google Scholar). Studies suggest that this efflux of sterol may be closely regulated by the cell and may involve specific cell surface domains called caveolae (5Fielding P.E. Fielding C.J. Plasma membrane caveolae mediate the efflux of cellular free cholesterol.Biochemistry. 1995; 34: 14288-14292Google Scholar). HDL is thought to transport sterol either directly or indirectly to the liver and clearance appears to involve specific cell-surface receptors called scavenger receptor-BI (SR-BI) (6Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor (see comments).Science. 1996; 271: 518-520Google Scholar). The direct pathway describes the undeviating delivery of this lipid to the liver, which is thought to account for approximately 30% of hepatic uptake of CE in rabbits (7Goldberg D.I. Beltz W.F. Pittman R.C. Evaluation of pathways for the cellular uptake of high density lipoprotein cholesterol esters in rabbits.J. Clin. Invest. 1991; 87: 331-346Google Scholar). The second, indirect, pathway constitutes the major hepatic sterol uptake route in rabbits (7Goldberg D.I. Beltz W.F. Pittman R.C. Evaluation of pathways for the cellular uptake of high density lipoprotein cholesterol esters in rabbits.J. Clin. Invest. 1991; 87: 331-346Google Scholar). The sterol is initially transferred from HDL to the rapidly turning over VLDL remnant lipoprotein pool, from which it is then cleared by the liver (8Fielding C.J. Fielding P.E. Molecular physiology of reverse cholesterol transport.J. Lipid Res. 1995; 36: 211-228Google Scholar, 9Tall A.R. An overview of reverse cholesterol transport.Eur. Heart J. 1998; 19: A31-A35Google Scholar). Intravascular sterol transport is thought to primarily involve the actions of two different plasma proteins: lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP). The consensus view has been that LCAT may form a concentration gradient to move sterol into and through the plasma compartment by promoting the conversion of free cholesterol (FC) to CE on HDL particles (10Jonas A. Lecithin:cholesterol acyltransferase.in: Gotto Jr., A.M. Plasma Lipoproteins. Elsevier, Amsterdam1987: 299-333Google Scholar). CETP may then promote this lipid flux by moving the newly formed CE from HDL to the apoB-containing lipoprotein pool (11Tall A.R. Plasma lipid transfer proteins.J. Lipid Res. 1986; 27: 361-367Google Scholar, 12Lagrost L. The role of cholesteryl ester transfer protein and phospholipid transfer protein in the remodeling of plasma high-density lipoproteins.Trends Cardiovasc. Med. 1997; 7: 218-224Google Scholar). Studies have shown that both the rate of FC esterification and the transfer of these CE into VLDL are enhanced, both in a postprandial lipemic state (13Francone O.L. Gurakar A. Fielding C. Distribution and functions of lecithin:cholesterol acyltransferase and cholesteryl ester transfer protein in plasma lipoproteins. Evidence for a functional unit containing these activities together with apolipoproteins A-I and D that catalyzes the esterification and transfer of cell-derived cholesterol.J. Biol. Chem. 1989; 264: 7066-7072Google Scholar, 14Mowri H.O. Patsch J.R. Ritsch A. Fwöger B. Brown S. Patsch W. High density lipoproteins with differing apolipoproteins: Relationships to postprandial lipemia, cholesteryl ester transfer protein, and activities of lipoprotein lipase, hepatic lipase, and lecithin:cholesterol acyltransferase.J. Lipid Res. 1994; 35: 291-300Google Scholar) and also in some hyperlipidemic states (12Lagrost L. The role of cholesteryl ester transfer protein and phospholipid transfer protein in the remodeling of plasma high-density lipoproteins.Trends Cardiovasc. Med. 1997; 7: 218-224Google Scholar, 15Tall A. Granot E. Brocia R. Tabas I. Hesler C. Williams K. Denke M. Accelerated transfer of cholesteryl esters in dyslipidemic plasma. Role of cholesteryl ester transfer protein.J. Clin. Invest. 1987; 79: 1217-1225Google Scholar, 16Dobiasova M. Stribrna J. Sparks D.L. Pritchard P.H. Frohlich J.J. Cholesterol esterification rates in very low density lipoprotein- and low density lipoprotein-depleted plasma. Relation to high density lipoprotein subspecies, sex, hyperlipidemia, and coronary artery disease.Arterioscler. Thromb. 1991; 11: 64-70Google Scholar). This CETP-mediated enhanced transfer of CE into VLDL was originally thought to be a result of a reciprocal mass exchange of CE and triglyceride between HDL and VLDL (11Tall A.R. Plasma lipid transfer proteins.J. Lipid Res. 1986; 27: 361-367Google Scholar). More recent work has shown, however, that both the presence of preferred triglyceride (TG)-enriched substrates and end products of lipolysis can influence the actions of CETP and enhance transfer into VLDL (12Lagrost L. The role of cholesteryl ester transfer protein and phospholipid transfer protein in the remodeling of plasma high-density lipoproteins.Trends Cardiovasc. Med. 1997; 7: 218-224Google Scholar). Other work has revealed that the electrostatic properties of plasma lipoproteins can also affect the interlipoprotein transfer of lipid by CETP (17Nishida H.I. Arai H. Nishida T. Cholesterol ester transfer mediated by lipid transfer protein as influenced by changes in the charge characteristics of plasma lipoproteins.J. Biol. Chem. 1993; 268: 16352-16360Google Scholar, 18Masson D. Athias A. Lagrost L. Evidence for electronegativity of plasma high density lipoprotein-3 as one major determinant of human cholesteryl ester transfer protein activity.J. Lipid Res. 1996; 37: 1579-1590Google Scholar). Investigations with LCAT have shown that this enzyme is also sensitive to charge properties of HDL (19Jonas A. Daehler J.L. Wilson E.R. Anion effects on the reaction of lecithin-cholesterol acyltransferase with discoidal complexes of phosphatidylcholines. apolipoprotein A-I cholesterol.Biochim. Biophys. Acta. 1986; 876: 474-485Google Scholar, 20Sparks D.L. Frank P.G. Neville T.A. Effect of the surface lipid composition of reconstituted LPA-I on apolipoprotein A-I structure and lecithin:cholesterol acyltransferase activity.Biochim. Biophys. Acta. 1998; 1390: 160-172Google Scholar). While an increase in the protein-dependent negative charge on HDL was shown to be associated with a stimulation of LCAT, increased anionic lipid content in HDL had an inhibitory effect on the enzyme (20Sparks D.L. Frank P.G. Neville T.A. Effect of the surface lipid composition of reconstituted LPA-I on apolipoprotein A-I structure and lecithin:cholesterol acyltransferase activity.Biochim. Biophys. Acta. 1998; 1390: 160-172Google Scholar). All lipoprotein classes exhibit a net negative charge and studies have shown that this charge is due to both the apolipoprotein composition of the lipoprotein and its content of anionic lipids (specifically phosphatidylinositol (PI)) (21Sparks D.L. Phillips M.C. Quantitative measurement of lipoprotein surface charge by agarose gel electrophoresis.J. Lipid Res. 1992; 33: 123-130Google Scholar, 22Davidson W.S. Sparks D.L. Lund-Katz S. Phillips M.C. The molecular basis for the difference in charge between pre-beta- and alpha-migrating high density lipoproteins.J. Biol. Chem. 1994; 269: 8959-8965Google Scholar). Experiments in this laboratory have shown that interfacially active enzymes are affected by lipoprotein charge and that HDL metabolism in vivo is directly affected by the charge on HDL particles (23Braschi S. Neville T.A. Vohl M.C. Sparks D.L. Apolipoprotein A-I charge and conformation regulate the clearance of reconstituted high density lipoprotein in vivo.J. Lipid Res. 1999; 40: 522-532Google Scholar). The data suggest that lipoprotein charge may affect cholesterol metabolism in vivo. The present study examines the effects of the anionic lipid, PI, on lipoprotein charge and cholesterol metabolism in a rabbit. We show that this lipid: 1) increases the negative surface charge of all the plasma lipoproteins, 2) increases VLDL cholesterol content, and 3) stimulates a sterol flux through the plasma compartment. The study shows that cholesterol metabolism in vivo can be manipulated by altering the charge characteristics of plasma lipoproteins. [3H]cholesterol was purchased from Mandel NEN Life Science Producers (Guelph, ON). 1-Palmitoyl-2-oleoyl phosphatidylcholine (PC) and phosphatidylinositol (PI) were obtained from Avanti Polar Lipids (Birmingham, AL). Silica Gel Impregnated Glass Fiber Sheets (ITLC™ SG) were obtained from Gelman Sciences (Ann Arbor, MI) and silica gel 60 plates were obtained from EM Science (Gibbstown, NJ). All other reagents were of analytical grade. PI and PC vesicles were prepared by drying 40 μmol of each lipid to completion in a 12 × 75 mm culture tube under N2. The lipids were solubilized in 3 mL of 50 mm sodium phosphate, pH 7.2, 150 mm sodium chloride (PBS) by sonication for 1 min at constant duty cycle. The vesicles were incubated at 37°C for 10 min and then sonicated at a high output for 4 min in 10°C water bath under N2. Samples were centrifuged for 5 min at 3,000 rpm to remove any particulate titanium. Male New Zealand white rabbits (3.5–4.0 kg) were fasted for 12 h prior to the experiment and remained fasted until after the final time point sample was taken. Rabbits had free access to water during this time. A catheter was inserted into the marginal ear vein and blood samples were collected into tubes containing 7.5% (K3) EDTA solution at the desired time points. A pre-injection blood sample was taken and the vesicle solution of either PI (n = 4), or PC (n = 2) or saline (n = 1) was injected into the marginal ear vein. A sample of blood was removed at 10 and 30 min, 1, 3, 6, and 24 h after the injection of the lipid vesicles. All blood samples were placed on ice and then centrifuged at 3,000 rpm for 15 min at 4°C to separate the plasma. In order to ensure that LCAT was inhibited in the stored plasma, iodoacetamide (150 mm) was added to plasma samples (24Guerin M. Dolphin P.J. Chapman M.J. A new in vitro method for the simultaneous evaluation of cholesteryl ester exchange and mass transfer between HDL and apoB-containing lipoprotein subspecies. Identification of preferential cholesteryl ester acceptors in human plasma.Arterioscler. Thromb. 1994; 14: 199-206Google Scholar). In order to determine the rate of clearance of cholesterol from the rabbit, a radioactive tracer was added to the vesicle preparations prior to injection into the rabbit. Two hundred μCi [3H]FC was dried in a 12 × 75 mm culture tube with 40 μmol of PI or POPC. Three ml of PBS was added to the dried lipids and the mixture was sonicated as described above. In some studies, to verify that PI did not affect the incorporation/clearance of the tracer, the tracer was combined with 1 mg of PC and 3 mL of PBS and vesicles were prepared as described above. This tracer/vesicle preparation was injected and then, 5 min later, the PI or PC vesicles were injected and blood was sampled as described. Lipoprotein fractions were isolated by sequential ultracentrifugation (VLDL+IDL, d < 1.019 g/mL; LDL, d 1.019–1.063 g/mL; and HDL, d 1.063–1.21 g/mL) and lipoprotein lipid composition (total cholesterol, FC, and TG concentrations) was determined enzymatically using kits from Roche Diagnostic (Laval, PQ). An aliquot of each lipoprotein was dialyzed into PBS and surface charge characteristics were determined by electrophoresis on pre-cast 0.5% agarose gels (Beckman, Paragon Lipo Kit) (21Sparks D.L. Phillips M.C. Quantitative measurement of lipoprotein surface charge by agarose gel electrophoresis.J. Lipid Res. 1992; 33: 123-130Google Scholar). Protein concentrations were determined using the Lowry method as modified by Markwell et al. (25Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal. Biochem. 1978; 87: 206-210Google Scholar). PI concentration was determined after lipid extraction in chloroform and methanol by high performance thin-layer chromatography on silica gel 60 plates using a solvent system composed of chloroform–methanol–ammonia, 65:35:5 (v/v/v). Lipids were charred with a 10% copper sulfate (w/v), 8% phosphoric acid (w/w) solution and PI concentration was quantified from a standard curve. To determine the amount of 3H associated with FC and CE, a fraction of each lipoprotein (20 μL) and whole plasma (40 μL) was extracted in ethanol and hexane and the lipids were separated by thin-layer chromatography on ITLC™ SG plates using a solvent system composed of hexane–diethyl ether–acetic acid, 90:10:1 (v/v/v). The effect of PI on LCAT activity was examined in incubations of plasma samples that were not treated with iodoacetamide. Four hundred μL of plasma at each time point was incubated for 30 min with 10 μCi of [3H]FC on filter paper discs (16Dobiasova M. Stribrna J. Sparks D.L. Pritchard P.H. Frohlich J.J. Cholesterol esterification rates in very low density lipoprotein- and low density lipoprotein-depleted plasma. Relation to high density lipoprotein subspecies, sex, hyperlipidemia, and coronary artery disease.Arterioscler. Thromb. 1991; 11: 64-70Google Scholar) at 37°C and the reaction was terminated with the addition of 2 mL ethanol. Reaction products were extracted in hexane and the amount of 3H associated with CE and FC was determined by thin-layer chromatography as described above. Figure 1 illustrates the effect of injection of PI and PC vesicles on the estimated surface potential of HDL, LDL, and VLDL. While injection of PC or saline (not shown) had no significant effect on lipoprotein charge, injection of PI vesicles caused all lipoprotein fractions to migrate further on the agarose gel, indicative of an increased negative surface charge. This increased negative charge inlipoproteins from the PI-injected rabbit reached a peak early in the time course and then returned to normal by 24 h. The HDL fraction exhibited a background surface potential of −12.2 mV, prior to the PI injection and then reached a peak negative charge of −18.0 mV 10 min after the PI injection. Similarly, the VLDL fraction had an initial surface potential of −8.8 mV, which increased to −11.2 mV after 10 min. LDL charge peaked somewhat later; LDL had an initial surface potential of −3.9 mV, and peaked 60 min after the PI injection at −8.0 mV. Plasma lipoprotein compositions of ultracentrifugally isolated lipoproteins sampled before and after the bolus injection of phospholipid were determined. The protein concentrations of the HDL fractions remained relatively constant after injection of the different lipids, while the protein concentrations in the LDL and VLDL fractions increased slightly in both the PC and PI injected animals over time (Table 1). The HDL and LDL fractions exhibited no significant change in either TG/protein, CE/protein, or FC/protein ratios after injection of PI. However, injection of PC or saline was associated with a small reduction in HDL CE and FC and a concomitant increase in these lipids in LDL (Table 1). In PI-injected animals, HDL and LDL-PI concentrations increased from being below detection levels to 33.3 and 3.6 nmol/ml plasma, respectively, by 10 min post injection. The PI concentration in VLDL increased to 1.4 nmol/ml by 30 min and then all PI concentrations decreased to undetectable background levels by 3 h. VLDL fractions from the PI-injected rabbit showed a significant change in FC/protein and CE/protein ratios (Fig. 2). While the FC/protein ratios in VLDL increased after both PC and PI injections, the magnitude of increase was 3-fold greater in the PI-injected animal and peaked 3 h post injection (Fig. 2A). Figure 2B shows a similar 3-fold increase in VLDL-CE, which also peaked 3 h after the injection. These increases in VLDL-CE shifted the relative plasma CE storage location from the HDL pool to the VLDL pool (65–30% and 10–50% of total plasma CE, respectively). This was followed by a subsequent return to near normal VLDL compositional values by 24 h. No other changes in VLDL composition were observed in PI-injected rabbits and there was only a slight increase in CE and TG in the VLDL pools after injection of PC or saline.TABLE 1.Effect of phospholipid injection on lipoprotein lipid and protein concentrationsCE/ProteinFC/ProteinTG/ProteinProteinmg/mg proteinmg/dlPC InjectionHDLt = 00.23 ± 0.020.06 ± 0.010.12 ± 0.0157.4 ± 2.2t = 30.14 ± 0.020.04 ± 0.010.17 ± 0.0243.9 ± 0.7t = 240.11 ± 0.010.04 ± 0.010.20 ± 0.0146.9 ± 1.7LDLt = 03.1 ± 0.80.41 ± 0.052.7 ± 0.21.7 ± 0.1t = 39.2 ± 0.50.87 ± 0.103.3 ± 0.31.4 ± 0.1t = 245.8 ± 0.30.33 ± 0.072.6 ± 0.23.1 ± 0.1VLDLt = 00.22 ± 0.020.03 ± 0.011.6 ± 0.15.7 ± 0.3t = 30.44 ± 0.080.23 ± 0.023.4 ± 0.16.3 ± 0.1t = 240.53 ± 0.050.32 ± 0.013.8 ± 0.29.4 ± 0.2PI InjectionHDLt = 00.63 ± 0.070.13 ± 0.010.29 ± 0.0326.6 ± 2.4t = 30.63 ± 0.120.15 ± 0.010.36 ± 0.0531.8 ± 3.3t = 240.70 ± 0.060.16 ± 0.030.42 ± 0.0629.3 ± 2.6LDLt = 01.8 ± 0.30.43 ± 0.021.6 ± 0.33.5 ± 0.1t = 31.8 ± 0.30.47 ± 0.031.7 ± 0.25.3 ± 0.2t = 242.0 ± 0.20.49 ± 0.032.0 ± 0.18.8 ± 0.5VLDLt = 00.34 ± 0.040.03 ± 0.023.5 ± 0.22.9 ± 0.1t = 31.50 ± 0.30.79 ± 0.064.4 ± 0.22.6 ± 0.1t = 240.70 ± 0.20.50 ± 0.042.3 ± 0.25.4 ± 0.2Phosphatidylinositol (PI) and phosphatidylcholine (PC) vesicles (40 μmol) were injected into fasting rabbits. Plasma was sampled at various times (t) and lipoproteins were isolated ultracentrifugally. Lipoprotein triglyceride (TG) cholesterol (FC), and cholesteryl ester (CE) concentrations (mg/mg protein) were determined as described and are representative of two injections of PC and four injections of PI in rabbits. Protein concentrations (mg/dl) were determined as described and values are representative of two injections of PC and four injections of PI in rabbits. Open table in a new tab Phosphatidylinositol (PI) and phosphatidylcholine (PC) vesicles (40 μmol) were injected into fasting rabbits. Plasma was sampled at various times (t) and lipoproteins were isolated ultracentrifugally. Lipoprotein triglyceride (TG) cholesterol (FC), and cholesteryl ester (CE) concentrations (mg/mg protein) were determined as described and are representative of two injections of PC and four injections of PI in rabbits. Protein concentrations (mg/dl) were determined as described and values are representative of two injections of PC and four injections of PI in rabbits. Figure 3 compares the clearance of [3H]FC at each time point for rabbits injected with PI or PC vesicles. It is evident that the radioactive FC in plasma rapidly fell to approximately 15% of the initial dose after only 1 h in the rabbit injected with PI. In comparison, the rabbit injected with PC still contained approximately 90% of the injected radioactivity after 1 h. The PC-injected rabbit took more than 6 h to clear [3H]FC to a baseline level comparable to that of an animal injected with PI. The half-life of [3H]FC in the PI-injected rabbits (0.3 h) was approximately 30-fold shorter than that for the PC-injected rabbits (8.4 h). To determine whether the tracer clearance was affected by co-injection of [3H]FC with different lipids, experimentswere undertaken where [3H]FC was complexed with a small amount (1 mg) of PC, injected into the rabbit, and then PI vesicles were injected 5 min later. This resulted in the same rapid clearance of the tracer as was seen with the co-injection of PI and [3H]FC (data not shown). Figure 4 shows the amount of newly formed [3H]CE remaining in the plasma after the injection of [3H]FC incorporated into either PC or PI vesicles. Initial 10-min levels of [3H]CE were >6 times lower in the PI-injected rabbit, as compared to the PC-injected rabbit. The levels of [3H]CE in the PI-injected rabbit began to rise after 1 h and eventually rose 4-fold by 24 h. In the PC-injected rabbit, the level of [3H]CE fell to approximately 10% of the injected dose by 6 h and remained constant until 24 h. It is notable that after 24 h, there was almost twice the amount of [3H]CE in the plasma of the PI-injected rabbit, relative to that of the PC-injected animal. Figure 5A and B illustrates the distribution of [3H]FC and [3H]CE in each lipoprotein fraction. In the PC-injected rabbit, [3H]FC was predominantly found in the LDL and HDL fractions at the early time points (Fig. 5A). By 6 h, the amount of [3H]FC in LDL and HDL fell and paralleled a major increase in the VLDL [3H]FC content. In contrast, in the PI-injected animal, most of the [3H]FCwas found in the VLDL fraction at the early time points, while by 24 h, [3H]FC was approximately equally distributed through all the lipoprotein fractions. Newly synthesized [3H]CE distribution in the different lipoprotein fractions showed quite different patterns in the PI- and PC-injected animals (Fig. 5B). In the PC-injected rabbits, [3H]CE was enriched in the HDL pool and depleted in the VLDL pool. However, in the PI-injected rabbits, [3H]CE levels in HDL were reduced by almost 50% relative to the PC animals, and paralleled an equivalent increase of [3H]CE in the VLDL pool. The effect of PC or PI injections on the rate of cholesterol esterification by LCAT was measured in individual plasma samples from the various time points. No major change in LCAT activity was observed after injection of PC vesicles (Fig. 6). In contrast, Fig. 6 shows that endogenous LCAT activity was significantly inhibited (18% of normal) 10 min after injection of PI. PI injection reduced the fractional rates of cholesterol esterification from 45% to 8%per hour. Six hours after the PI vesicle injection, LCAT activity returned to approximately 75% of normal. Studies have shown that alterations in the composition of lipoprotein particles can affect their charge and structural properties and that these characteristics will influencelipoprotein remodelling and plasma lipid metabolism (26Patsch J.R. Prasad S. Gotto Jr., A.M. Patsch W. High density lipoprotein2. Relationship of the plasma levels of this lipoprotein species to its composition, to the magnitude of postprandial lipemia, and to the activities of lipoprotein lipase and hepatic lipase.J. Clin. Invest. 1987; 80: 341-347Google Scholar, 27De Rijke Y.B. Biessen E.A.L. Vogelezang C.J.M. Van Berkel T.J.C. Binding characteristics of scavenger receptors on liver endothelial and Kupffer cells for modified low-density lipoproteins.Biochem. J. 1994; 304: 69-73Google Scholar, 28La Belle M. Blanche P.J. Krauss R.M. Charge properties of low density lipoprotein subclasses.J. Lipid Res. 1997; 38: 690-700Google Scholar). In general, lipoprotein charge is primarily due to the specific apolipoprotein composition of the lipoprotein class (22Davidson W.S. Sparks D.L. Lund-Katz S. Phillips M.C. The molecular basis for the difference in charge between pre-beta- and alpha-migrating high density lipoproteins.J. Biol. Chem. 1994; 269: 8959-8965Google Scholar, 29Chappey B. Myara I. Benoit M.O. Mazière C. Mazière J.C. Moatti N. Characteristics of ten charge-differing subfractions isolated from human native low-density lipoproteins (LDL). No evidence of peroxidative modifications.Biochim. Biophys. Acta. 1995; 1259: 261-270Google Scholar, 30Chauhan V. Wang X. Ramsamy T. Milne R.W. Sparks D.L. Evidence for lipid-dependent structural changes in specific domains of apolipoprotein B100.Biochemistry. 1998; 37: 3735-3742Google Scholar). Our studies have shown that this protein-dependent charge is governed by the conformation of the apolipoprotein and therefore directly affected by different uncharged lipids that are able to modify the protein conformation (31Sparks D.L. Lund-Katz S. Phillips M.C. The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles.J. Biol. Chem. 1992; 267: 25839-25847Google Scholar, 32Sparks D.L. Davidson W.S. Lund-Katz S. Phillips M.C. Effects of the neutral lipid content of high density lipoprotein on apolipoprotein A-I structure and particle stability.J. Biol. Chem. 1995; 270: 26910-26917Google Scholar). In addition, lipoprotein charge is also affected by its content of charged molecules, predominantly anionic lipids such as PI and non-esterified free fatty acids (NEFA) (22Davidson W.S. Sparks D.L. Lund-Katz S. Phillips M.C. The molecular basis for the difference in charge between pre-beta- and alpha-migrating high density lipoproteins.J. Biol. Chem. 1994; 269: 8959-8965Google Scholar). PI is an anionic lipid found in all classes of lipoproteins and accounts for approximately 4% of the total phospholipid (PL) in HDL (22Davidson W.S. Sparks D.L. Lund-Katz S. Phillips M.C. The molecular basis for the difference in charge between pre-beta- and alpha-migrating high density lipoproteins.J. Biol. Chem. 1994; 269: 8959-8965Google Scholar). Little is known of what affects or regulates the amount of PI in the different lipoprotein classes. In contrast, numerous factors can affect
Here, we have described the dataset relevant to the A549 cellular proteome changes after exposure to either titanium dioxide or carbon black particles as compared to the non-exposed controls, "Proteomic changes in human lung epithelial cells (A549) in response to carbon black and titanium dioxide exposures" (Vuong et al., 2016) [1]. Detailed methodologies on the separation of cellular proteins by 2D-GE and the subsequent mass spectrometry analyses using MALDI-TOF-TOF-MS are documented. Particle exposure-specific protein expression changes were measured via 2D-GE spot volume analysis. Protein identification was done by querying mass spectrometry data against SwissProt and RefSeq protein databases using Mascot search engine. Two-way ANOVA analysis data provided information on statistically significant A549 protein expression changes associated with particle exposures.
The likelihood of environmental and health impacts of silicon dioxide nanoparticles (SiNPs) has risen, due to their increased use in products and applications. The biological potency of a set of similarly-sized amorphous SiNPs was investigated in a variety of cells to examine the influence of physico-chemical and biological factors on their toxicity. Cellular LDH and ATP, BrdU incorporation, resazurin reduction and cytokine release were measured in human epithelial A549, human THP-1 and mouse J774A.1 macrophage cells exposed for 24 h to suspensions of 5–15, 10–20 and 12 nm SiNPs and reference particles. The SiNPs were characterized in dry state and in suspension to determine their physico-chemical properties. The dose-response data were simplified into particle potency estimates to facilitate the comparison of multiple endpoints of biological effects in cells. Mouse macrophages were the most sensitive to SiNP exposures. Cytotoxicity of the individual cell lines was correlated while the cytokine responses differed, supported by cell type-specific differences in inflammation-associated pathways. SiNP (12 nm), the most cytotoxic and inflammogenic nanoparticle had the highest surface acidity, dry-state agglomerate size, the lowest trace metal and organics content, the smallest surface area and agglomerate size in suspension. Particle surface acidity appeared to be the most significant determinant of the overall biological activity of this set of nanoparticles. Combined with the nanoparticle characterization, integration of the biological potency estimates enabled a comprehensive determination of the cellular reactivity of the SiNPs. The approach shows promise as a useful tool for first-tier screening of SiNP toxicity.
The cover image, by Marianne B. Ariganello et al., is based on the Research Article A matrix-assisted laser desorption ionization–time-of-flight–time-of-flight–mass spectrometry-based toxicoproteomic screening method to assess in vitro particle potencies, https://doi.org/10.1002/jat.3642
Two-way ANOVA results for the A549 protein spots that changed significantly due to particle exposures (n = 3). The SSP number corresponds to the identifier number that PDQuest used to identify the spot based on its coordinate in the gel. The number below Treatment main effect (Trt), Dose main effect (Dose) or interaction between Treatment and Dose (T x D) corresponds to the p-value, where the bolded number emphasized p-value
