K. A. Dawson, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland Fax: +353 1 716 2415 Tel: +353 1 716 2447 E-mail: email@example.com T. Cedervall, Department of Biophysical Chemistry, Lund University, Box 124, SE-22100 Lund, Sweden Fax: +46 46 222 4116 Tel: +46 46 222 8240 E-mail: firstname.lastname@example.org
In a biological environment, nanoparticles immediately become covered by an evolving corona of biomolecules, which gives a biological identity to the nanoparticle and determines its biological impact and fate. Previous efforts at describing the corona have concerned only its protein content. Here, for the first time, we show, using size exclusion chromatography, NMR, and pull-down experiments, that copolymer nanoparticles bind cholesterol, triglycerides and phospholipids from human plasma, and that the binding reaches saturation. The lipid and protein binding patterns correspond closely with the composition of high-density lipoprotein (HDL). By using fractionated lipoproteins, we show that HDL binds to copolymer nanoparticles with much higher specificity than other lipoproteins, probably mediated by apolipoprotein A-I. Together with the previously identified protein binding patterns in the corona, our results imply that copolymer nanoparticles bind complete HDL complexes, and may be recognized by living systems as HDL complexes, opening up these transport pathways to nanoparticles. Apolipoproteins have been identified as binding to many other nanoparticles, suggesting that lipid and lipoprotein binding is a general feature of nanoparticles under physiological conditions.
Nanoparticles entering any biological fluid will immediately be covered by a corona of biomolecules. The corona confers biological identity to the nanoparticles, as it is this that interacts with the cellular machinery, thereby determining the nanoparticle destiny in and impacts on organisms. Previously, research has focused on the protein composition of the corona, but many other biomolecules, such as carbohydrates, nucleic acids, and lipids, can also be contained in the corona and play important biological roles that are different from those of proteins. It is therefore essential to extend the characterization of the corona to include other biomolecules, as each plays a vital role in cellular functionality and signaling. Complete characterization of the nanoparticle biomolecule corona may be the key to classifying nanoparticle risk and predicting impacts.
The nanoparticles used in this study are copolymers of N-isopropylacrylamide (NIPAM) and N-t-butylacrylamide (BAM) at four ratios (85 : 15, 75 : 25, 65 : 35, and 50 : 50) and three sizes (70, 120 and 200 nm in diameter). These nanoparticles are well characterized, and can be prepared monodisperse with defined size and hydrophobicity (through the NIPAM/BAM ratio), making them highly suitable as model nanoparticles in biophysical and biochemical studies. In addition, these polymer particles have been suggested as drug delivery vessels with potential for controlled release . The identity of proteins in the hard corona on these nanoparticles is well established, and several biophysical parameters, including stoichiometries, exchange rates, and enthalpies of the interactions, have been determined [2–4]. Surprisingly, most of the identified proteins are apolipoproteins and other proteins associated with lipoprotein particles. Serum albumin is also present, but has a much higher dissociation rate and lower affinity than the apolipoproteins, and will therefore be replaced over time by apolipoproteins in plasma. The selective binding of apolipoproteins raises many interesting questions that this article begins to address. For instance, it is not known whether the nanoparticles bind only the apolipoproteins or the complete lipoprotein particles, and, if the latter is true, whether a specific lipoprotein particle is selectively targeted. Additionally, we are interested in knowing whether the binding is mediated by proteins, lipids, or both, and whether the bound protein or lipoprotein particle retains its receptor binding and enzymatic activity.
Lipids in blood are transported via lipoprotein particles, of eight to several hundred nanometers in diameter, containing lipids and proteins. Triglycerides and cholesterol esters are found in the core of these lipoprotein particles, surrounded by proteins and a monolayer of phospholipids. The proteins in the lipoprotein particles are mainly apolipoproteins with a range of structural and functional properties. The different classes of lipoprotein particles can be distinguished from one another by the composition of apolipoproteins and lipids. HDL is a heterogeneous population of lipoprotein particles . Apolipoprotein A-I is the main protein component of HDL. In blood, apolipoprotein A-I recruits phospholipids and cholesterol to form discoidal HDL particles that mature into larger, spherical HDL particles. Apolipoprotein A-II and apolipoprotein E are present in subfractions of HDL. Additionally, proteins involved in lipid metabolism are also associated with HDL (but not with other lipoprotein particles), including lecithin:cholesterol acyltransferase, cholesterol ester transfer protein, and paraoxinase, which are involved in cholesterol metabolism and transport.
Here we have investigated the binding to copolymer nanoparticles of both lipids and proteins from whole plasma and from isolated lipoprotein fractions, using size exclusion chromatography, gel electrophoresis, NMR, and enzymatic assays. We have investigated in detail the lipid and protein binding pattern, and the results imply that the copolymer nanoparticles bind HDL lipoprotein particles.
Lipid binding to nanoparticles determined by size exclusion chromatography
Size exclusion chromatography was carried out to establish whether lipids are associated with copolymer nanoparticles, as shown in Fig. 1. This technique has been previously used to determine interactions of proteins with nanoparticles, as the elution behavior of the proteins is shifted upon interaction with the nanoparticles . Copolymer nanoparticles with composition 50 : 50 NIPAM:BAM and diameters of 120 or 200 nm were incubated with plasma, pelleted by centrifugation, washed, and finally redispersed in buffer before being loaded onto a Sephacryl S-1000 SF column. The presence of cholesterol in the eluted fractions was determined by an enzymatic assay. The results (Fig. 1) show that cholesterol coelutes with nanoparticles of different size at their respective elution volumes. This clearly shows that plasma cholesterol associates with the copolymer nanoparticles.
Lipids identified by NMR spectroscopy after extraction
NMR spectroscopy was used to detect and identify the lipids bound to the copolymer nanoparticles in plasma. NIPAM/BAM 50 : 50 copolymer nanoparticles were mixed with plasma, and unbound plasma lipids were removed by repeated washing/centrifugation steps. Lipids were then extracted from the nanoparticles using chloroform/methanol extraction, and analyzed by NMR spectroscopy (Fig. 2A). A substantial amount of nanoparticles ended up in the chloroform phase, disturbing the spectra and making quantification difficult. However, 1H 1D-NMR spectra of lipids extracted from nanoparticles incubated in plasma consistently showed signals specific for cholesterol at 0.68 p.p.m. and for triglyceride at 4.1–4.3 p.p.m., as shown in Fig. 2A. Lipids extracted directly from plasma (Fig. 2B) gave the same peaks as in the spectrum of lipids extracted from nanoparticles. The peaks were confirmed by reference spectra and through 2D total correlation spectroscopy (TOCSY) NMR experiments. In humans, about 80% of the phospholipid content in lipoprotein complexes is phosphatidylcholine, which could be identified in the extract from plasma from the nitrogen-attached methyl groups visible at 3.4 p.p.m., but not in the extract from nanoparticles incubated in plasma. However, as we have shown that lipids bind to the nanoparticles (both by size exclusion chromatography and by enzymatic assay), an enzymatic kit for detection of phosphatidylcholine was used to verify that phosphatidylcholine is indeed bound to the nanoparticle pellet before extraction. The kit detected phosphatidylcholine in the pellet of 50 : 50 NIPAM/BAM but not in the pellet of the less hydrophobic 65 : 35 NIPAM/BAM. The lower affinity for the less hydrophobic nanoparticles correlates well with the results from protein, cholesterol and triglyceride binding studies, where binding is seen only with the more hydrophobic 50 : 50 particles, and serves as a negative control (see below). This means that phospholipids are adsorbed onto the nanoparticle but are not detected in the NMR spectrum of the extract, probably as a result of being bound to the nanoparticles also in the chloroform phase and therefore not being visible, owing to slow tumbling.
Surface characteristics are important for lipid binding
The surface hydrophobicity of the copolymer nanoparticles can be varied by changing the ratio of the two comonomers. After incubation in plasma and repeated washing/centrifugation, considerable amounts of cholesterol were identified in the pellets of 50 : 50 NIPAM/BAM nanoparticles (Fig. 3A). In comparison, the less hydrophobic 65 : 35 NIPAM/BAM nanoparticles bound very little cholesterol (Fig. 3A). This shows that the amount of lipids bound to the nanoparticles is dependent on the hydrophobicity of the nanoparticle surface. This behavior is similar to the hydrophobicity dependence in protein binding reported previously . The hydrophobicity dependence was also confirmed in NMR experiments on extracts from 65 : 35 NIPAM/BAM nanoparticles, showing much lower or no signals from cholesterol and triglyceride as compared with extracts from 50 : 50 NIPAM/BAM nanoparticles. The same behavior was seen for phospholipid binding as described in the previous paragraph. Copolymer nanoparticles of even lower hydrophobicity (75 : 25 and 85 : 15 NIPAM/BAM) were also tested, and the amount of bound cholesterol was at background level (data not shown). However, these nanoparticles disperse more readily in aqueous buffers, which makes any detailed comparison difficult. The low amount of lipids bound to the less hydrophobic nanoparticles serves as a good negative control for the lipid binding detected on the 50 : 50 NIPAM/BAM copolymer nanoparticles.
Lipid binding is surface area dependent
Copolymer nanoparticles (50 : 50 NIPAM/BAM) of two sizes, 120 and 200 nm in diameter, were incubated in plasma, and the bound lipids were separated from free lipids by repeated centrifugation and washing. The two nanoparticles produce very similar pellets, but the surface area is 1.7 times greater in the 120 nm nanoparticle pellet than in the 200 nm nanoparticle pellet. One milligram of nanoparticles corresponds to approximately 1.1 × 1012 (1.8 × 10−12 mol) 120 nm particles or 2.4 × 1011 (4.0 × 10−13 mol) 200 nm particles. A comparison of the amount of bound lipids in a nonsaturated system shows that 0.5 mg of 120 nm nanoparticles binds about 1.4–1.9 times more cholesterol and triglycerides than 0.5 mg of 200 nm nanoparticles, as shown in Table 1. Consequently, the amount of lipids bound depends on the total surface area rather than on the pellet volume or the number of nanoparticles. In control experiments using plasma without nanoparticles, only minute amounts of lipids are detected, and these are subtracted from the values reported in Table 1. An additional factor that may influence the cholesterol binding is the surface curvature/particle size, as the naturally occurring lipoprotein complexes range in size from 8–10 nm to 100 nm, and the amount of cholesterol associated with each differs, as shown in Table 1.
Table 1. Amount and ratio ± standard deviation of lipids on 50 : 50 NIPAM/BAM copolymer particles with two different diameters and at two plasma concentrations. One milligram of nanoparticles contains approximately 1.8 × 10−3 nmol of 120 nm particles or 4.0 × 10−4 nmol of 200 nm particles, and the surface area is 1.7 times larger in 1 mg of 120 nm particles than in 200 nm particles.
Cholesterol (nmol·mg−1 particles)
Triglyceride (nmol·mg−1 particles)
Molar ratio of cholesterol/ triglyceride
120 nm 50 : 50, 33% plasma
11.1 ± 1.6
3.4 ± 0.1
3.2 ± 0.6
120 nm 50 : 50, 67% plasma
17.9 ± 0.9
6.1 ± 0.6
2.9 ± 0.4
200 nm 50 : 50, 33% plasma
5.9 ± 0.1
2.5 ± 0.3
2.4 ± 0.3
200 nm 50 : 50, 67% plasma
11.0 ± 0.4
4.0 ± 0.4
2.7 ± 0.4
Lipid binding by copolymer nanoparticles reaches saturation
If nanoparticles bind discrete lipoprotein particles, the binding may reach saturation. This was tested in experiments with increasing amounts of plasma added to a constant amount of 200 nm 50 : 50 NIPAM/BAM nanoparticles. The cholesterol levels were determined by enzymatic assay, as shown in Fig. 3B. After a steep increase of the amount of bound cholesterol, a plateau was reached at about 40% plasma, indicating saturation. This is approximately the same percentage of plasma as that at which protein saturation was reached in protein adsorption experiments at similar particle concentrations, suggesting a coupled binding behavior . At saturation, there is approximately 20 nmol cholesterol per mg 200 nm nanoparticles, which corresponds to 50 000 cholesterol or cholesterol ester molecules per nanoparticle, or 60 μg HDL per mg nanoparticles (assuming 3.08 wt% cholesterol and 17.6 wt% cholesterol ester in HDL). Using a radius of 5 nm and a density of 1.14 g·mL−1 for HDL, this can be estimated to be 400 HDL molecules per 200 nm particle, or one-quarter of the theoretical maximum coverage in one layer. The same experiments were performed with 70 and 120 nm nanoparticles, but complete saturation could not be reached, owing to the larger particle surface area. Fewer nanoparticles in each sample will lead to small pellets that are difficult to handle in a reproducible way.
The cholesterol/triglyceride ratio is increased in the nanoparticle lipid corona
The molar ratio of cholesterol and triglyceride bound to the nanoparticles was established for the 120 and 200 nm 50 : 50 NIPAM/BAM nanoparticles at two plasma concentrations (Table 1). To ensure that there were no differences in the experimental routine, the pellets were split into two equal parts in the last wash before the cholesterol and triglyceride levels were measured. The cholesterol/triglyceride molar ratios varied between 2.4 and 3.2, but were within experimental error for all conditions. The cholesterol/triglyceride molar ratio measured in the same plasma was 1.5. Thus, the cholesterol/triglyceride ratio was increased by a factor of 2 following interaction with and binding to the nanoparticles as compared with the ratio in plasma, indicating that specific lipoprotein particles were targeted. The specificity is further analyzed in Table 2, where the ratios from Table 1, after conversion to mass ratios, are compared to ratios for the different lipoprotein classes. The approximate amount of protein was determined by comparing bound apolipoprotein A-I with known amounts of apolipoprotein A-I by SDS/PAGE, as shown in Fig. S1 Table 2 also shows the apolipoprotein pattern estimated from SDS/PAGE and compares it with the different lipoprotein classes.
Table 2. Protein and lipid composition of lipoprotein particles, and the biomolecule corona around the 200 nm 50 : 50 NIPAM/BAM nanoparticles following incubation in plasma. The lipoprotein compositions are from several references collected by LipidBank (http://lipidbank.jp).
Mass ratio of protein, cholesterol, and triglyceride
Mass percentage of apolipoprotein contributions
Bound protein and lipids from purified lipoprotein particle fractions
Three fractions of lipoprotein particles − chylomicrons + very low density lipoprotein (VLDL), low-density lipoprotein (LDL), and HDL – were obtained from human plasma by ultracentrifugation in a salt gradient. The HDL fraction was further fractionated into HDL and very high-density lipoprotein (VHDL) fractions. The proteins in the final four fractions were visualized by SDS/PAGE (Fig. 4A, lanes 1–4), and the proteins bound to 50 : 50 NIPAM/BAM 200 nm copolymer nanoparticles from each lipoprotein particle fraction are shown in Fig. 4A, lanes 5–8. The main proteins in the chylomicron + VLDL fraction (Fig. 4A, lane 1) were (in size order) apolipoprotein B-100 and/or apolipoprotein B-48 (apolipoprotein B-100 is not separated from its truncated variant apolipoprotein B-48 in this system), human serum albumin (HSA), apolipoprotein E, and apolipoprotein A-I. The same proteins were present on the nanoparticles from this fraction (Fig. 4A, lane 5), but the relative apolipoprotein E and apolipoprotein A-I as compared with apolipoprotein B-100 were much greater on the nanoparticles, indicating preferential binding of apolipoprotein A-1 and apolipoprotein E. In the LDL fraction (Fig. 4A, lane 2), the main protein was apolipoprotein B-100, as expected, but visible amounts of albumin, apolipoprotein E and apolipoprotein A-I were also present. The relative amount of apolipoprotein B-100 was much less on the copolymer nanoparticles incubated in the LDL fraction (Fig. 4A, lane 6), indicating that lipoprotein particles with apolipoprotein E or apolipoprotein A-I preferentially bind to the copolymer nanoparticles. In the HDL and VHDL fractions (Fig. 4A, lanes 3 and 4), the major proteins were apolipoprotein A-I and HSA. On the copolymer nanoparticles incubated in the HDL and VHDL fractions (Fig. 4A, lanes 7 and 8), apolipoprotein A-I dominated.
Previous studies of proteins bound to the 50 : 50 NIPAM/BAM copolymer nanoparticles in human plasma did not identify apolipoprotein B in the hard corona . Here we observed, in small amounts, apolipoprotein B on nanoparticles incubated in the lipoprotein fractions in which apolipoprotein B is the dominating protein (chylomicrons, VLDL, and LDL). The ratio of apolipoprotein B to apolipoprotein A-I was significantly lower on the nanoparticles relative to the respective lipoprotein fraction, indicating that apolipoprotein B-100 or LDL bind to the copolymer nanoparticles with lower affinity than apolipoprotein A-I or HDL. Size exclusion chromatography was used to further study this competition. NIPAM/BAM 50 : 50 200 nm copolymer nanoparticles were mixed with lipoprotein particle fractions and, after a washing step, particles and their bound proteins were loaded onto a Sephacryl S-1000 column. The lipoprotein particles did not affect the elution volume of the nanoparticles, and free LDL and HDL clearly eluted separately from the nanoparticles (Fig. 4B). Eluted nanoparticles were pelleted, and the associated proteins were separated by SDS/PAGE (Fig. 4C). Only apolipoprotein A-I was present on the eluted copolymer nanoparticles mixed with the LDL fraction, indicating that apolipoprotein A-I–HDL has a much greater binding affinity than apolipoprotein B-100–LDL for the copolymer nanoparticles. As expected, only apolipoprotein A-I was recovered from the nanoparticles after mixing with the HDL or VHDL fractions. No proteins could be seen in SDS/PAGE from the nanoparticles incubated with the chylomicron–VLDL fractions, probably because the amounts bound to the nanoparticles were too small. In all cases of lipoprotein particles binding to the copolymer nanoparticles, apolipoprotein A-I was identified, suggesting that the binding is mediated by apolipoprotein A-1, although a similar role for apolipoprotein A-IV and apolipoprotein E cannot be excluded.
The relative amounts of cholesterol on the nanoparticles mixed with the HDL or LDL fractions were determined after gel filtration. The volume of HDL or LDL fraction mixed with the nanoparticles was the same in each experiment, which means that there was 6.5 times more cholesterol available in the samples incubated with the LDL fraction than in those incubated with the HDL fraction. Nevertheless, there was 1.5 times more cholesterol on the eluted nanoparticles mixed with HDL than on the nanoparticles mixed with LDL, as shown in Fig. 4D. This shows that the nanoparticles bind both proteins and lipids with high specificity, supporting the conclusion that HDL rather than LDL binds to the copolymer nanoparticles.
This is, to our knowledge, the first time that lipids have been detected in the biomolecular corona surrounding nanoparticles in human plasma. Moreover, we have found that intact HDL particles bind to nanoparticles. We show, with three different approaches, that the plasma lipids, cholesterol and triglycerides, are present on 50 : 50 NIPAM/BAM copolymer nanoparticles incubated in plasma. First, cholesterol elutes together with nanoparticles in size exclusion chromatography, and the elution position of cholesterol depends on the size of the nanoparticles. Second, nanoparticles preincubated in plasma were extracted with a mixture of chloroform and methanol. In the extract, cholesterol and triglyceride were detected with NMR spectroscopy. Third, cholesterol, phospholipids and triglyceride were detected by enzymatic assay on nanoparticles following incubation with human plasma and separation of unbound lipids by centrifugation. The amount of bound lipids depends on the surface area presented by the nanoparticles and not on the pellet size following centrifugation. Less hydrophobic nanoparticles (65 : 35, 75 : 25 and 85 : 15 NIPAM/BAM) bind no or minute amount of lipids in any of these methods, and therefore provide an excellent negative control.
Lipoprotein particles can be distinguished from one another by the identity and amount of proteins, and by the amount and ratio of cholesterol and triglycerides. We have previously characterized the protein profile and shown that it includes apolipoproteins and enzymes . The identified apolipoproteins and enzymes found in that study correspond to the proteins found mainly in HDL and chylomicrons, which further strengthens the present results. Furthermore, the protein/cholesterol and protein/triglyceride ratios correspond well with the ratios in HDL (Table 2), but not with the ratios in larger lipoprotein particles. The protein/triglyceride and cholesterol/triglyceride ratios are in the lower range, implying that a small number of triglyceride-rich lipoprotein particles, like chylomicrons, also bind to the nanoparticles. Chylomicrons, like HDL, contain apolipoprotein A-I, which is identified as the main candidate for mediating binding of lipoprotein complexes to the nanoparticles. In experiments with plasma fractions enriched in different lipoprotein classes, the 50 : 50 NIPAM/BAM nanoparticles show high specificity for apolipoprotein A-I and bind lipids from the plasma fraction enriched in HDL with higher affinity than those from the plasma fraction enriched to LDL. In conclusion, the results strongly suggest that the 50 : 50 NIPAM/BAM copolymer nanoparticles in plasma are associated with intact lipid-loaded apolipoprotein A-I-containing lipoprotein particles, preferentially HDL. Apolipoproteins have also been detected on other nanoparticles, e.g. polystyrene, solid lipid nanoparticles, and carbon nanotubes, raising the possibility that binding of intact lipoprotein particles extends to other classes of nanoparticles [6–15].
We may speculate a little on the role of the lipoprotein biomolecular corona in determining the destiny of nanoparticles that enter the bloodstream. The size of the nanoparticles used in this study is of the same order as that of large lipoprotein particles, and there is a possibility that the relative curvature between the nanoparticles and lipoproteins favors the binding of small HDL over larger lipoproteins, although no such tendency can be seen in the results in Table 2. An interesting aspect of the equal size of the nanoparticles and the lipoproteins is, however, that it may open the door to the lipoprotein transport system for the lipoprotein-coated nanoparticles. Lipoprotein particles are known to bind selectively to receptors expressed in organs and cells. There are several receptors described that mediate lipid transport and endocytosis of LDL. In the field of nanomedicine, this has led to numerous publications exploring the possibility of using LDL in drug delivery systems [16,17]. The most common receptor is scavenger receptor class B type 1 (SR-BI), which mediates the bidirectional lipid transfer between VLDL, LDL and HDL and cells . SR-BI is expressed mainly in the liver and steroidogenic glands, but also in brain, intestine, and placenta, and in cells such as macrophages and endothelial cells . Another possible receptor is cubilin, which has been shown to bind apolipoprotein A-I and HDL, and to mediate endocytosis of HDL [18,19]. Cubilin is expressed in the proximal tubule in kidney and in epithelial cells in yolk sac and intestine [20,21]. As the copolymer nanoparticles bind HDL, it is possible that there will be receptor-mediated uptake and enrichment of the nanoparticles in organs and cells rich in SR-BI and cubillin. Thus, the discovery that nanoparticles can bind intact lipoprotein complexes offers a new window on nanomedicine, as nanoparticles may also hitch a lift on existing cellular lipidic transport pathways. We are currently investigating the biological fate of these lipoprotein-binding nanoparticles in vitro.
We have, for the first time, detected lipids in the biomolecular corona surrounding nanoparticles and characterized the lipid binding. The interaction with lipoprotein particles is highly specific, and several experimental findings suggest that the copolymer nanoparticles (50 : 50 NIPAM/BAM) bind complete and intact lipoprotein particles with high specificity for HDL. It is possible that lipoprotein particle binding is a common feature of nanoparticles in a general sense, which makes the mechanism of the binding and the implications for nanoparticle fate and impacts in vivo important topics for future study.
NIPAM/BAM copolymer nanoparticles of diameter 70, 120 and 200 nm and with several different ratios of the comonomers (85 : 15, 25 : 75, 65 : 35 and 50 : 50 NIPAM/BAM) were synthesized in the presence of SDS as described previously, although higher SDS concentrations were used in the present work, resulting in similarly sized nanoparticles . The procedure for the synthesis was as follows: 2.8 g of monomers (in the appropriate w/w ratio) and 0.28 g of crosslinker (N,N-methylenebisacrylamide) was dissolved in 190 mL of MilliQ water with either 0.8 g of SDS (for the 70 nm nanoparticles) or 0.32 g of SDS (for the 200 nm nanoparticles), and degassed by bubbling with N2 for 30 min. Polymerization was induced by adding 0.095 g ammonium persulfate initiator in 10 mL of MilliQ water and heating at 70 °C for 4 h . The nanoparticles were extensively dialyzed against MilliQ water for several weeks, with the water being changed daily, until no traces of monomers, crosslinker, initiator or SDS could be detected by proton NMR (spectra were acquired in D2O using a 500 MHz Varian Inova spectrometer). The nanoparticles were freeze-dried and stored in the refrigerator until used.
Human plasma, and buffers
Blood was drawn from a healthy individual into tubes with EDTA or heparin, and centrifuged at 14 000 g for 30 min. The supernatants from several vials were combined, and aliquots of 400 μL were stored at – 80 °C. Before each experiment, plasma aliquots were centrifuged at 14 000 g to remove possible aggregates.
Enzymatic determination of triglycerides and cholesterol
Copolymer nanoparticles were dispersed on ice in NaCl/Pi/EDTA (10 mm phosphate, 150 mm NaCl, pH 7.5, 1 mm EDTA) and mixed with various amounts of plasma. After 1 h of incubation on ice, the mixtures were heated to 23 °C to promote aggregation of the nanoparticles. The samples were centrifuged at 14 000 g for two minutes, and the nanoparticle pellets were saved and washed three times with NaCl/Pi/EDTA. The amount of bound triglycerides was measured by adding 150 μL of a 4 : 1 mixture of Free Glycerol Reagent (Sigma, Stockholm, Sweden) and Triglyceride Reagent (Sigma) to the pellets. The nanoparticle pellets were dispersed, and incubated at 37 °C for about 30 min. After incubation, the nanoparticles were pelleted by centrifugation at 14 000 g for two minutes, and the absorbance of the supernatant was measured at 495 nm. The reagents cleave the fatty acids from the triglycerides, and the amount of free glycerol is quantified and used as a reporter of the initial amount of triglycerides, so a standard curve of glycerol was used in each experiment. The amount of bound cholesterol was determined using an Amplex®Red Cholesterol kit (Invitrogen, Stockholm, Sweden). The nanoparticle pellets were resuspended in 50 μL of cholesterol kit reaction buffer and 50 μL of the cholesterol kit working solution. After 30 min of incubation at 37 °C, the nanoparticles were pelleted by centrifugation at 14 000 g for two minutes, and the fluorescence of the supernatant was measured. The cholesterol kit determines the concentration of total cholesterol, including the part that is present in the lipoprotein particle core as esters with fatty acids.
Size exclusion chromatography of nanoparticles and bound plasma lipids
Copolymer nanoparticles of 0.5 mL (10 mg·mL−1) were mixed with 0.4 mL of NaCl/Pi/EDTA or with 0.4 mL of human plasma, and incubated on ice. After 1 h, the samples were heated to 23 °C to promote aggregation of nanoparticles, allowing pelleting by centrifugation. The pellets were washed with 1 mL of NaCl/Pi/EDTA and dispersed in 0.5 mL of NaCl/Pi/EDTA on ice. The mixture was loaded onto a 30 × 1.5 cm Sephacryl S-1000 SF column and eluted with NaCl/Pi/EDTA at a flow rate of 0.8 mL·min−1, and 1.7 mL fractions were collected. The elution profile of the nanoparticles was obtained by recording the scattering at 280 nm in a UV spectrometer after aggregation of the nanoparticles at 37 °C. To obtain the elution profiles of cholesterol or triglyceride, the nanoparticles in each fraction were pelleted by centrifugation at 14 000 g for two minutes, and the amount of lipid was determined by enzymatic assays as described above.
In the experiments with fractionated lipoprotein particles, 1 mL of copolymer nanoparticles (10 mg·mL−1) was mixed with 0.5 mL of lipoprotein particles, and incubated and washed as described above. The mixture was loaded onto a 95 × 1.5 cm Sephacryl S-1000 column and eluted with NaCl/Pi/EDTA. To analyze the bound proteins, 2 mL fractions from each nanoparticle and lipoprotein fraction were spun down, and the proteins were desorbed by SDS/PAGE loading buffer and separated by SDS/PAGE (15% gel).
Extraction of lipids and detection by NMR
Plasma (800 μL) was incubated with 800 μL of 10 mg·mL−1 200 nm 50 : 50 copolymer nanoparticles in NaCl/Pi/EDTA on ice for 1 h, and then for 30 min at room temperature. The nanoparticles were then harvested by centrifugation at 14 000 g for two minutes, and washed three times with 800 μL of NaCl/Pi/EDTA. Plasma (800 μL) or nanoparticle pellets, resuspended in 800 μL of NaCl/Pi, were extracted against 1.2 mL of 0.5 m KH2PO4, 6 mL of CHCl3, and 2 mL of MeOH. The chloroform phase was evaporated, and the remaining material was dissolved in 600 μL of deuterated (99.8%) chloroform. 1H/1D spectra were recorded at 25 °C using a 600 MHz Varian Unity Inova spectrometer. The chemical shift was referenced to the residual chloroform signal (δ 7.26).
Enzymatic determination of phosphatidylcholine
Plasma (200 μL) was incubated with 200 μL of 10 mg·mL−1 120 nm 50 : 50 or 65 : 35 copolymer nanoparticles in NaCl/Pi/EDTA on ice for 1 h, and then for 30 min at room temperature. The nanoparticles were then harvested by centrifugation at 14 000 g for two minutes, and washed three times with 400 μL of NaCl/Pi/EDTA. The pellet was analyzed with the kit Phospholipids B no. 990-54009E (Wako, Neuss, Germany) in a reaction volume of 1.5 mL.
Purification of lipoprotein particle fractions from plasma
Lipoprotein particle fractions were purified as described by Schumaker and Puppione . Lipemic citrate plasma was ultracentrifuged repeatedly at 147 000 g in an Optimal L-70K Beckman centrifuge with a Ti 701 rotor, for 25 h at 12 °C. Before each centrifugation, the density was adjusted with 5 m NaCl and saturated NaBr, both containing 0.04% EDTA. After each centrifugation step, a lipoprotein fraction was collected from the top of the centrifuge tubes. The corresponding densities from which the fractions were collected are: 1.0068, 1.068, 1.21 and 1.25 g·mL−1 for chylomicron + VLDL, LDL, HDL, and VHDL, respectively. All fractions were dialyzed against NaCl/Pi/EDTA. The cholesterol and the triglyceride concentrations were determined to be 1.5 and 6.8 mm in the chylomicron + VLDL fraction, 15 and 2.6 mm in the LDL fraction, and 2.4 and 0.73 mm in the HDL fraction, respectively. In the VHDL fraction, the cholesterol concentration was 0.073 mm, but the triglyceride concentration was too low to be determined.
This work was funded in part by the EU FP6 projects NanoInteract (NMP4-CT-2006-033231), BioNanoInteract SRC and SIGHT (IST-2005-033700-SIGHT), the Swedish Research Council (VR), and Science Foundation Ireland.