Morphology, binding behavior and MR-properties of paramagnetic collagen-binding liposomes

Morphology, binding behavior and MR-properties of

paramagnetic collagen-binding liposomes

Citation for published version (APA):

Sanders, H. M. H. F., Stijkers, G. J., Mulder, W. J. M., Huinink, H. P., Erich, S. J. F., Adan, O. C. G.,

Sommerdijk, N. A. J. M., Merkx, M., & Nicolay, K. (2009). Morphology, binding behavior and MR-properties of

paramagnetic collagen-binding liposomes. Contrast Media and Molecular Imaging, 4(2), 81-88.

https://doi.org/10.1002/cmmi.266

DOI:

10.1002/cmmi.266

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Published: 01/01/2009

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Received: 27 June 2008, Revised: 9 December 2008, Accepted: 9 December 2008, Published online in Wiley InterScience: 2009

Morphology, binding behavior and

MR-properties of paramagnetic

collagen-binding liposomes

H. M. H. F. Sanders

*, G. J. Strijkers

, W. J. M. Mulder

, H. P. Huinink

,

S. J. F. Erich

, O. C. G. Adan

, N. A. J. M. Sommerdijk

, M. Merkx

and K. Nicolay

Collagen is an important component of the extracellular matrix (ECM) and plays an important role in normal tissue maturation and in pathological processes such as atherosclerosis and myocardial infarction. The diagnostics of the latter diseases using MRI could strongly benefit from the use of collagen-specific contrast agents. The current study aimed to develop a bimodal liposomal MR contrast agent that was functionalized with CNA35, a collagen adhesion protein of the Staphylococcus aureus bacterium. The liposomes were characterized in terms of CNA35 protein conjugation and loading. The overall morphology was assessed with DLS and cryo-TEM, while cryo-TEM tomography was used to visualize the protein coverage of the liposomes. The binding properties of the contrast agent were investigated using a fluorescence assay based on the rhodamine content of the liposomes. The bulk relaxivity was determined using regular relaxometry while the MR-properties of liposomes in their bound state were studied using NMR depth profiling. This CNA35 functionalized contrast agent and the set ofin vitro experiments we performed indicate the potential of this technology forin vivo molecular imaging of collagen. Copyright # 2009 John Wiley & Sons, Ltd.

Keywords: collagen; molecular imaging; paramagnetic liposomes; contrast-enhanced MRI; CNA35; cryo-TEM

1.

INTRODUCTION

Collagen is the major constituent of the extracellular matrix and responsible for maintaining the structural integrity of organs and tissues. Next to its crucial role in normal tissue maturation, collagen is also involved in wound and fracture healing and in important pathological processes such as atherosclerosis and myocardial infarction (1–5). In atherosclerosis, collagen is considered to be a major indicator for plaque stability (6–9). About 60% of the total protein content in atherosclerotic plaques is collagen, mainly consisting of type I and III (1,8,10). Collagen is of major importance in all stages of plaque development and may even be involved in retention of low-density lipoprotein, calcification and in the promotion of atherogenesis (6,10). In general, plaques with a more stable phenotype have a high collagen content, which may ultimately lead to arterial stenosis (1,7). Instable plaques suffer from a collagen deficit due to excessive breakdown by matrix metalloproteinases, making these plaques prone to rupture (1,7).

The formation of collagen is a key event during tissue remodeling after myocardial infarction (MI). Cardiac infarction is a frequently occurring cardiovascular event, caused by athero-sclerosis. Over the past decades early mortality has considerably decreased due to the success of acute intervention strategies (11). Patients surviving this acute phase of myocardial infarction enter a phase of cardiac wound healing, ultimately resulting in the formation of scar tissue. However, in the long term a major complication is heart failure resulting from inadequate healing. During the first phases of wound healing collagen levels are

strongly elevated and remain significantly higher throughout the process (3,4,11,12). Imaging collagen level and distribution in the early stages after infarction could serve as an important diagnostic tool to detect eminent heart failure or likelihood of rupture of the cardiac wall (13).

MRI is a noninvasive imaging modality capable of generating high-resolution images with excellent soft-tissue contrast. Collagen detection by MRI has been pioneered by Navon and (www.interscience.wiley.com) DOI:10.1002/cmmi.266

* Correspondence to: H. M. H. F. Sanders, Eindhoven University of Technology, Biomedical NMR, n-laag, room B2.01, PO Box 513, 5600 MB Eindhoven, The Netherlands.

E-mail: h.m.h.f.sanders@tue.nl

a H. M. H. F. Sanders, G. J. Strijkers, K. Nicolay

Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands

b H. M. H. F. Sanders, N. A. J. M. Sommerdijk

Soft Matter Cryo-TEM research unit, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands

c W. J. M. Mulder

Translational and Molecular Imaging Institute, Department of Radiology, Mount Sinai School of Medicine, New York, NY, USA

d H. P. Huinink, S. J. F. Erich, O. C. G. Adan

Transport in Permeable Media, Department of Applied Physics, Eindhoven University of Technology, Eindhoven, The Netherlands

e M. Merkx

Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands

1

coworkers using a method based on double quantum coher-ences, which are formed due to interactions of water protons with collagen (14). This elegant technique is dependent on the presence of structurally organized collagen. However, the active collagen remodeling that occurs in many pathologies causes a considerable fraction of the collagen fibers to be disorganized, which limits the applicability of this technique. Therefore, we propose MRI detection of collagen using a collagen specific contrast agent in the present study.

Recently, Caravan and coworkers reported a collagen specific MRI contrast agent consisting of 3 Gd-DTPA moieties bound to a peptide with a moderate affinity for collagen type I (15,16). Using this contrast agent they were able to discriminate scar tissue formed after myocardial infarction from viable myocardium and blood. This study illustrates that the high collagen concentration present in many tissues make it an attractive molecular target for MR imaging, especially since MRI suffers from a relatively low sensitivity (15,16).

In order to deal with the potential problem of low MRI detection sensitivity, potent contrast agents, such as those based on pegylated liposomes carrying a high payload of gadolinium ions, can be used (16,17). These particles have a high relaxivity per particle and can be equipped with a targeting ligand to yield a target specific contrast agent. The in vivo use of liposomes is mainly directed towards vascular targets, although liposomes have been shown to extravasate into diseased tissues, like tumors (18,19), atherosclerotic plaques (20,21) and infarcted myocardium (22–24). The circulation time of liposomes can be increased by pegylation, enabling ample time for targeting, which may help to overcome their potentially slow tissue penetration. Specific delivery of large targeted contrast agents (
50 nm), i.e. iron oxide particles and liposomes, to extracellularly located apoptic cells was shown in both tumor (25) and myocardial infarction (26,27). In this study we report on the development of a collagen-specific MRI contrast agent by functionalization of Gd-containing liposomes with the collagen-binding protein CNA35 (28). This stable and structurally well-characterized 35 kDa protein is known to bind different types of collagen with a similar affinity (28,29). Fluorescently labeled CNA35 was shown to be a versatile probe for optical imaging of collagen, both in the field of tissue engineering (30,31) and for ex vivo imaging of atherosclerosis (9). The latter study showed that Oregon green-labeled CNA35 was able to bind to collagen in plaques in vivo, whereas collagen in healthy vessels was hardly targeted. This observation is attributed to the leaky endothelium at sites of atherosclerotic plaques.

Here, the structural and MRI-properties of CNA35-func-tionalized liposomes were investigated in great detail using a variety of experimental approaches. Firstly the particles were subjected to an in-depth cryo-TEM analysis to investigate the liposomal structure and the protein distribution at the surface of the particles. Furthermore the protein loading per liposome was determined. Secondly, the efficacy of the targeting ligand, CNA35, was assessed using a fluorescence assay. Thirdly the MR-properties of the CNA35-functionalized liposomes in bulk solution and, more importantly, in their bound state were investigated. With this aim experiments on three-dimensional collagen structures and collagen surfaces were carried out at 6.3 T. Lastly, the influence on the longitudinal relaxation of bulk water protons by collagen-associated CNA35-functionalized liposomes was quantified using a technique called high-resolution NMR depth profiling (32). Using this range of

techniques we were able to show that CNA35-functionalized liposomes have great potential for MRI-based molecular imaging of collagen.

2.

RESULTS AND DISCUSSION

CNA35 was covalently linked to liposomes comprising gadolinium-DTPA-bis-stearyl-amide (Gd-DTPA-BSA), phosphocholine (DSPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-{methoxy[poly(ethylene glycol)]-2000} (PEG2000-DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-{maleimide[poly(ethylene glycol)]-2000} (Mal-PEG2000-DSPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-PE) with a molar ratio of 0.25:0.37:0.33:0.025:0.025:0.001. With that aim, the CNA35 protein was first treated with N-succinimidyl S-acetylthioacetate (SATA) to introduce free thiols at the distal end of its lysine residues and subsequently allowed to react with the maleimide-labeled pegylated lipid in the liposomal preparation. Using a protein determination the amount of coupled protein was determined to be 1.58 nmol of CNA35 per 1mmol lipid. Assuming a surface area of 0.6 nm2per lipid and unilaminar liposomes with a diameter of 200 nm (circa 420,000 lipids per liposome), approximately 660 copies of CNA35 were estimated to be present per liposome (33). A ratio of 1 CNA35 to 15 Mal-PEG2000-DSPE molecules was used, which would correspond to 700 proteins per liposome, therefore the coupling efficiency was around 95%.

SDS-PAGE analysis was carried out to verify actual coupling of the proteins to the maleimide-functionalized lipids (Fig. 1). The trace for the CNA35 protein itself displayed a single band at a molecular weight of around 35 kDa, in agreement with the

Figure 1. SDS–PAGE stained with Coomassie blue. Lane A: MW marker (10, 20, 25, 37, 50, 75 and 100 kDa); lane B: unfunctionalized liposomes; lane C: CNA35-functionalized liposomes; and lane D: CNA35. *CNA35 conjugated to 1, 2 or 3 lipids from bottom to top. **CNA35 with a molecular weight of 35 kDa. A smear is observed in the samples with high lipid contents. This figure is available in colour online at www. interscience.wiley.com/journal/cmmi

H. M. H. F. SANDERS ET AL.

molecular weight of CNA35. Unfunctionalized liposomes gave rise to a smear of background signal up to 25 kDa. The CNA35-functionalized liposomes resulted, next to the lipid background, in three bands higher than 35 kDa. Since no band at 35 kDa was observed, it can be concluded that all CNA35 had been lipidated. We infer that the three bands originated from CNA35 covalently bound to either one, two or three lipids. The estimated weight increments indeed correspond to the molecular weight of mal-PEG2000-DSPE. The finding that a substantial amount of CNA35 was conjugated with two or three lipid moieties is consistent with fluorescent labeling experiments, which also showed conjugation of multiple amine-reactive fluorescent groups per protein (data not shown). A recent mass spectrometry study showed that most of the reactive lysines are concentrated at the C-terminus of CNA35, explaining why fluorescent labeling does not perturb collagen binding activity (29,30).

2.1.2. Characterization

The hydrodynamic diameter of the CNA35-functionalized liposomes was determined with dynamic light scattering experiments. In addition we used this technique to investigate possible aggregate formation. The experiments revealed a single peak in intensity mode with a Z-average of 194 nm and a poly dispersity index of approximately 0.1, indicative of a narrow size distribution and thus the absence of aggregation (data not shown).

To characterize these protein-functionalized liposomal prep-arations in more detail, cryo-TEM was performed. High-resolution cryo-TEM images (Fig. 2, left) demonstrated the uniformity of the vesicles. The preparations predominantly contained unilamellar spherical vesicles with a narrow size distribution, while very few multilamellar vesicles were observed. Non-vesicular structures, like disks or micelles, were not observed in any of the pre-parations investigated with cryo-TEM. The surface distribution of the proteins conjugated to the vesicles was visualized by cryo electron tomography. The typical three-dimensional reconstruc-tion, shown in Fig. 2 (right), displayed a homogeneous distribution of CNA35 (shown in blue) on the liposomes (in red). The proteins were located outside of the bilayer, suggesting that they are available for binding with collagen. Furthermore the tomograms did not indicate the presence of protein aggregates, which could lead to a reduced collagen binding activity of the contrast agent.

2.2. Quantification of collagen binding affinity

Having established the amount of CNA35 conjugation and the proper orientation of the collagen-binding domains at the exterior of the liposomal surface, we next determined the collagen affinity of the liposomes by means of a fluorescence binding assay. Several concentrations of liposomes were incubated on a surface of rat tail collagen type I deposited on the bottom of a 96-well plate. Figure 3 shows the amount of well-associated rhodamine fluorescence as a function of liposome concentration, assuming unilaminar vesicles of 200 nm in diameter (420 000 lipids per liposome). The binding curve could be fitted with an apparent dissociation constant Kd¼ 2.8  0.3 nM. Hardly any binding was observed for

unfunc-tionalized liposomes under the same conditions. The dissociation constant of liposomes is approximately 200-fold lower than that of fluorescently labeled CNA35, previously determined to be 0.5mM (30). The apparent collagen-binding affinity of

CNA35-functionalized liposomes is affected by several factors: the number of active CNA35 binding domains, the fraction of liposome–conjugated CNA35 involved in binding and finally possible multivalent interactions. As described earlier, most CNA35 domains on the liposome were believed to be fully capable of binding. However, the fraction of CNA35 per liposome involved in the actual binding and the importance of multivalent interactions are presently unknown. To demonstrate binding specificity, unfunctionalized and CNA35 functionalized liposomes were incubated on a surface that had been pre-incubated and thus was saturated with CNA35, resulting in no significant binding based on the fluorescent read-out (data not shown).

2.3. MRI-properties of CNA35-functionalized liposomes 2.3.1. Three-dimensional collagen matrices

As a next step, the MRI-properties of CNA35-functionalized liposomes were characterized in bulk solution and, more importantly, in their bound state. The CNA35-functionalized particles exhibited an ionic bulk relaxivity of 4.6 and 2.6 mM1s1

per gadolinium chelate at 1.4 and 6.3 T, respectively (T¼ 293 1 K). Three-dimensional bovine collagen type I matrices with an average pore size of 100–200mm were used to verify whether CNA35-functionalized liposomes were capable of

Figure 2. Left: cryo-TEM image of unfunctionalized liposomes. Right: three-dimensional tomogram of CNA35-functionalized liposomes recon-structed from a series of TEM images taken at different angles. The lipid bilayer is shown in red and the individual CNA35 proteins are shown in blue.

Figure 3. Fluorescence binding assay of liposome binding to rat tail collagen type I. CNA35-conjugated liposomes in HBS showed a distinct pattern of saturated binding, whereas incubation with unfunctionalized liposomes in HBS only led to very low levels of fluorescence. The solid lines are fits to the experimental data using a one site binding model.

3

lowering the T1relaxation time of water protons near collagen

significantly. A T1-map was obtained using multiple spin echo MRI

acquisitions with a varying repetition time. In the T1-map shown

in Figure 4, next to the holders (black circles), three collagen matrices incubated with buffer (A), unfunctionalized liposomes (B) and CNA35-functionalized liposomes (C) can be observed. Incubation with 10 nM CNA35-functionalized liposomes

(saturated binding) resulted in a substantial decrease of T1from

2550 50 to 1650  50 ms. Incubation with the same concen-tration of unfunctionalized liposomes resulted in no significant T1

reduction.

Although collagen binding of CNA35-functionalized liposomes was clearly observed in this system, a drawback was that the collagen location and spatial distribution was not well defined. Furthermore, influences of partial volume effects could not be estimated, making quantification of the relaxivity of liposomes in their bound state difficult.

2.3.2. Targeting to a two-dimensional collagen surface

In order to establish a more controlled experiment to assess the MR properties of collagen-associated CNA35-functionalized liposomes, the 96-well plates that were also used for the fluorescence assay were subjected to MRI measurements on a 6.3 T scanner. Since the collagen layer was presumed to be very thin a large partial volume effect was expected. Compared with the three-dimensional collagen matrices, however, this time the influence of the partial volume effect could be taken into account. A T1-map was obtained using an inversion recovery

sequence. The results are depicted in Figure 5 showing cross-sections through four wells. As controls, a collagen-coated well incubated with HEPES buffered saline (HBS) at pH 7.4 [Fig. 5(A)] and a well incubated with unfunctionalized liposomes (B) were used. Both controls showed no noticeable T1reduction

near the bottom as compared with the T1 of bulk water (ca

2500 ms).

Wells C and D were incubated with 5 and 25 nM CNA35-functionalized liposomes, respectively, and clearly showed a

strong T1reduction in the order of 1000 ms at the location of the

collagen coating. At certain places two pixels were enhanced, corresponding to a thickness of 120mm. The two wells at the bottom had a different fluorescence level (0.3 vs 0.5 a.u. for 5 and 25 nMliposomes, respectively (Fig. 5), which was associated with

a clear T1 reduction in both cases. Although the wells had a

rectangular shape, they seemed slightly skewed on the T1-map.

This was most likely caused by a susceptibility induced distortion, which also led to an apparent T1shortening near some of the

vertical edges of the wells.

2.3.3. High-resolution NMR depth profiling

From the results shown in Figure 5 the T1of wells incubated with

5 and 25 nMof CNA35-functionalized liposomes were essentially

the same; although based on fluorescence, a difference in T1at

the bottom of the well was expected. Therefore we investigated these wells with the use of high-resolution NMR depth profiling (32). With this method a high-resolution T1 profile in one

dimension perpendicular to the bottom of the well was recorded using a saturation recovery experiment. Using extremely high gradients (36.4 T m1) a spatial resolution of 5mm was achieved. The saturation recovery experiment data were fitted with a single parameter. The only free variable in this fit is the surface relaxation rate, which quantified the influence of a surface on the longitudinal relaxation rate of the magnetization. We published a detailed report about this technique recently, for details see Huinink et al. (32). The surface relaxation rate was useful in assessing the local MR contrast properties of the targeted CNA35-functionalized liposomes and hence their potential utility as a molecular imaging agent in biomedical applications. The surface relaxation rates of a collagen-coated well, a collagen-coated well incubated with unfunctionalized liposomes and a collagen-coated well incubated with CNA35-functionalized liposomes were determined. With that aim, one-dimensional T1-profiles were measured perpendicular to

the bottom of the well, as a function of the distance to the bottom of the well (Fig. 6). For all wells the T1of the bulk solution

Figure 4. T1-map of bovine collagen type I matrices incubated with (A) HBS, (B) unfunctionalized liposomes in HBS (
10 nMliposomes) and (C)

CNA35-functionalized liposomes in HBS (
10 nMliposomes). Following

incubation with liposomes, the samples were extensively washed with HBS as detailed in the materials and methods. On the right the pseudo color scale for T1-values (from 1.0 to 3.5 s) is shown. MRI data were collected at 6.3 T.

Figure 5. T1-map generated with an inversion recovery sequence show-ing four collagen-coated wells that were incubated with: (A) HBS, pH 7.4; (B) 25 nMunfunctionalized liposomes in HBS; (C) 5 nMCNA35-liposomes in

HBS; and (D) 25 nMCNA35-functionalized liposomes in HBS. The incu-bations were followed by extensive washing to remove the nonbound paramagnetic liposomes. Subsequently the wells were filled with HBS. Measurements were performed at 6.3 T.

H. M. H. F. SANDERS ET AL.

was 2800 130 ms and T1for the unincubated collagen-coated

well remained almost unchanged down to the bottom. For the well incubated with CNA35-functionalized liposomes, a strong shortening of the longitudinal relaxation T1was found near the

collagen surface. T1 dropped from 2.8 s to about 0.7 s over a

distance of approximately 120mm. A T1-shortening of about

1000 ms, localized to two pixels (
120 mm), was observed in the two-dimensional T1-maps shown in Figure 5. Taking partial

volumes effects into account, a similar T1-shortening profile

might have caused the observed T1-reduction in these

experiments. The surface relaxation rate of the CNA35-functionalized liposomes containing 25 mol% Gd-DTPA-BSA was estimated to be 4.0 0.3  105m s1. The surface relaxation rate of the control well, i.e. not incubated with paramagnetic liposomes, was 0.2 0.2  105m s1, whereas the surface relaxation rate of unfunctionalized liposomes was 0.5 0.2  105m s1, indicative of little nonspecific binding. These results correlated very well with the fluorescence measurements.

To investigate whether surface relaxation rate was linearly dependent on the local liposome concentration, the surface relaxation rates of five wells with different fluorescence intensities were measured [Fig. 7(A)]. The fluorescence signal was assumed to correlate to liposome coverage of the surface. Indeed, the data showed a linear relation between fluorescence and surface relaxation rate. In addition we prepared liposomes that had different percentages of gadolinium lipid (0, 5, 10, 15, 20 and 25% mol/mol) incorporated to investigate whether an optimal amount of gadolinium inclusion in the liposomes could be found. Characterization of these liposomes indicated that they are similar to 25% mol/mol version, in terms of size and protein loading. Fluorescence measurements (data not shown) demon-strated that the same amount of liposomes had been bound in all cases. Therefore, we assumed saturated and equal liposomal coverage of the surface, resulting in surfaces enriched with 0, 5, 10, 15, 20 or 25% gadolinium liposomes. A linear trend was observed over the complete range [Fig. 7(B)], indicating that the higher the local concentration of gadolinium, the higher the surface relaxation rate. The surface relaxation rate in case

of liposomes without gadolinium was 0.6 0.2  105m s1 [Fig. 7(B)], which was somewhat higher than the value of 0.2 0.2  105m s1 found for the untargeted surface [see Fig. 7(A)]. This difference revealed that surface coverage with nonparamagnetic liposomes somewhat affected the local MR properties as well. A control well, without any collagen deposited at the bottom and not incubated with liposomes, displayed a surface relaxation rate of 0.2 0.2  105m s1. Unexpectedly, collagen did not seem to influence surface relaxation rate significantly, while coverage with nonparamagnetic liposomes did.

3.

SUMMARY

We successfully prepared CNA35-functionalized liposomes with a diameter of approximately 200 nm. The protein conjugation efficiency was 95% and using a fluorescence assay it was shown that the collagen-binding properties of CNA35 were preserved. We showed that CNA35 was rather evenly distributed over

Figure 6. T1as function of distance from the bottom of collagen-coated wells (x¼ 0 corresponds to the bottom) measured at 1.41 T using the one-dimensional high-resolution NMR depth profiling method. Incubations were done with HBS (blue), unfunctionalized liposomes (red) or CNA35-functionalized liposomes (black). The spatial resolution of the one-dimensional profiles was 5mm.

Figure 7. (A) Surface relaxation rate as function of liposome-associated fluorescence. It was assumed that the fluorescence signal linearly depends on the liposome coverage of the surface. Solid line: linear fit of the data (R2¼ 0.98). (B) Surface relaxation rate as a function of the molar fraction of Gd-DTPA-BSA in the liposomes. Based on fluorescence measurements the surface coverage with liposomes was assumed to be the same and fully saturated. Solid line: linear fit of the data (R2

¼ 0.94).

the liposomal surface using a sophisticated tomographic cryo-TEM approach. The nano-molar binding constant of this nanoparticulate formulation of CNA35 makes this contrast agent valuable for molecular imaging of collagen.

The MRI contrast properties of the CNA35-functionalized liposomes were investigated using several in vitro experiments. Three-dimensional bovine collagen type I matrices showed a significant T1 lowering upon incubation with

CNA35-functionalized liposomes. High-resolution NMR depth profiling revealed a strong increase of surface relaxation rate upon binding of CNA35-functionalized liposomes. Exploiting this technique a linear relationship between the surface longitudinal relaxivity and the liposome coverage of the collagen-coated surface was found. This suggests that NMR depth profiling allows future quantitative comparisons of collagen binding contrast agents, which enables comparison between different contrast agent carriers conjugated to CNA35. This approach will help us identify the most suitable candidate for in vivo molecular MRI studies of, for example, atherosclerosis or myocardial infarction. In conclusion, we have developed a liposomal collagen-specific contrast agent that binds to collagen and obtained detailed information about the morphology, binding- and MR-properties of this contrast agent.

4.

EXPERIMENTAL

1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-{methoxy [poly(ethylene glycol)]-2000} (PEG2000-DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-{maleimide[poly(ethylene glycol)]-2000} (Mal-PEG2000-DSPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-PE) were obtained from Avanti Polar Lipids (Alabaster, AL). Gadolinium–diethylene triamine pentaacetic acid-bis(stearylamide) (Gd-DTPA-BSA) was purchased from Gateway Chemical Technology (St Louis, MO, USA). N-Succinimidyl-S-acetylthioacetate was obtained from Sigma Chemical Co. (St Louis, MO, USA), and HEPES was obtained from Merck (Darmstadt, Germany). All other chemicals were of analytical grade or the best grade available. Polycarbonate filters for liposome extrusion and Costar 8-well strip plates were from Costar (Cambridge, MA, USA).

4.2. CNA-expression

Vector pQE30CNA35 coding for the collagen binding part of the A-domain of Staphylococcus aureus collagen adhesin (amino acids 30-344) fused to an N-terminal His-tag was a kind gift from Dr Magnus Ho¨o¨k (Texas A&M University, USA). The plasmid was transformed into E. coli BL21(DE3) (Novagen, Nottingham, UK). The recombinant expression and purification of CNA35 were carried out as described previously (30).

4.3. Preparation of paramagnetic CNA35-functionalized liposomes

Liposomes containing Gd-DTPA-BSA, DSPC, cholesterol, PEG2000-DSPE and Mal-PEG2000-DSPE mixed at a molar ratio of 0.25:0.37:0.33:0.025:0.025 were prepared by lipid film hydration according to the procedure described by Mulder

et al. (16). For fluorescence detection of the liposomes, 0.1 mol% of rhodamine-PE was also included. CNA35 was coupled to the liposomes by a sulfhydryl–maleimide coupling method as reported previously (16). CNA35 was reacted with succinimidyl S-acetylthioacetate in a molar ratio of 1:8 for 1 h in an NaHCO3

buffer at pH 8.0. Unreacted SATA was removed by using a Vivaspin concentrator with a MWCO of 10 kDa. Subsequently deacetylation was conducted with a hydroxyl amine (0.5M

hydroxyl amine, 1MHEPES, 32 mMEDTA, pH 7.0) solution for 1 h.

Modified CNA35 was then allowed to react with preformed liposomes at 48C overnight in HBS (20 mM HEPES, 135 mM

NaCl, pH 7.4). Uncoupled CNA35 was separated from CNA-functionalized liposomes by ultracentrifugation, twice, at 358 000 g for 45 min at 48C (Beckmann Optima L-90 K, rotor type 70.1 Ti). The supernatant was removed and the liposomal pellet was dissolved in HBS, yielding a final concentration of 25 mMlipid. This solution was stored under N2at 48C in the dark.

The above procedure was also used to obtain liposomes with different molar fractions of Gd-DTPA-BSA. Liposomes were prepared containing 0, 5, 10, 15, 20 and 25% Gd-DTPA-BSA which was compensated for by increasing the DSPC content, while the other components, PEG-DSPE, Mal-PEG-DSPE and cholesterol, were not altered.

4.4. Protein determination

The total amount of CNA35 coupled to the liposomes was determined using a modification of the Bradford method (34). The modification only involved the preparation of the calibration line. A calibration curve was prepared using a range of CNA35 concentrations and a fixed amount of unfunctionalized lipo-somes. The lipid concentration in the samples was adjusted to the same level as was used in the calibration curves.

4.5. Cryo-TEM

Cryo transmission electron microscopy (Cryo-TEM) was per-formed in low-dose mode using a Gatan cryo-holder operating at ca 1708C and an FEI Titan Krios TEM equipped with a field emission gun (FEG) operating at 300 kV. Images were recorded using a 2 k 2 k Gatan CCD camera equipped with a post column Gatan energy filter (GIF) at an angle of 08. The sample vitrification procedure was carried out using an automated vitrification robot, viz. a FEI VitrobotTM _{Mark III. The Quantifoil grids were made}

hydrophilic with a surface plasma treatment using a Cressington 208 carbon coater operating at 5 mA for 40 s prior to the sample preparation and vitrification.

For cryo-electron tomography a sample was prepared contain-ing streptavidin–gold (6 nm), which was purchased from Aurion. A tilt series of 187 images from 708 to þ708 was recorded using the FEI Explore 3D software (settings: Saxton scheme set for an interval of 18: I/I60¼ 1.6, Dfocus ¼ 2 mm). The tomographic

reconstruction was performed using the Tom Toolbox software package employing the weighted back projection algorithm. Additional image analysis was performed using Amira version 3.1.1.

4.6. Dynamic light scattering

The average hydrodynamic radius of the liposomes was determined using a Malvern ZetaSizer Nano S at a temperature H. M. H. F. SANDERS ET AL.

of 258C. The concentration of the solution was 50 mMof lipids in

an HBS (20 mMHEPES, 135 mMNaCl) buffer at pH 7.4.

4.7. Fluorescence-based collagen binding assay

Wells of an eight-well strip plate (Corning, Schiphol-Rijk, the Netherlands) were incubated overnight at 48C with 50 mL of 55mg/mL rat-tail collagen type I (C7661, Sigma-Aldrich) in HBS. Next, nonbound proteins were aspirated from the wells using an automated Wellwash AC plate washer (Thermo, Breda, the Netherlands) and the wells were rinsed three times with 300mL HBS at 208C. Wells were blocked with 250 mL of 5% (w/v) milk powder in HBS for 3 h at 208C, aspirated and again rinsed three times with 300mL HBS. In some control experiments this step was followed by incubation with 50mL of a solution containing 10 mM

CNA35 for 3 h at 208C. Next, 50 mL of a solution of CNA35-functionalized liposomes in HBS was added to each well and incubated for 3 h at 208C. Wells were aspirated and washed 10 times with 300mL HBS. Fluorescence was subsequently measured using a Fluoroskan Ascent FL plate reader (excitation: 578 nm; emission: 620 nm). The data was fitted using a one-site binding model, with the formula Y¼ Bmax X/(Kdþ X).

4.8. High-resolution NMR depth profiling

The surface relaxation rate determination was performed as described before (32). The thickness of the bottom of clear, flatbottom Costar eight-well strip-plates (Corning, Schiphol-Rijk, the Netherlands) was reduced to approximately 100mm using a milling cutter (Deckel, Veenendaal, Netherlands) followed by subsequent polishing (Struers, Knuth Rotor).

The micro-imaging NMR setup (1.41 T) was based on the so-called GARField method with a single linear gradient of 36.4 T m1. The saturation recovery T1 sequence used in our

experiments consisted of four saturation pulses with an average pulse length of 0.7ms, corresponding to an average flip angle of 708, spaced with an average time of tS¼ 1 ms. The saturation

recovery sequence is given by (tS– 708)4–tr– 908 – te– 908 – te–

echo, in whichtS,trand 2teare the time between the saturation

pulses, the time to allow the magnetization to recover and the echo time, respectively. The echo time, 2te, was set at 200ms,

giving a spatial resolution of 5mm in our setup. For each recovery time tr, which was varied in 20 steps between 0 and 10 s,

4096 echoes were acquired.

4.9. MRI-measurements on well plates

The same eight-strip-well plates used for the fluorescence-based collagen-binding assay were imaged on a 6.3 T scanner (Bruker-Biospin, Ettlingen, Germany) with a 9.5 cm horizontal bore. An inversion recovery sequence, with 12 inversion times ranging from 12 to 5000 ms and two averages, was used to obtain a T1-map with a field of view of 3 3 cm2and a slice thickness of

1 mm, perpendicular to the bottom of the wells. A matrix size of 512 512 yielded an in-plane spatial resolution of 60  60 mm2. Total acquisition time was approximately 10 h and the measure-ments were performed at room temperature, T¼ 293  1 K. 4.10. MRI of three-dimensional-collagen matrices

Three-dimensional bovine collagen matrices (catalog no. 354613) were purchased from BD Biosciences. The matrices were hydrated in a solution of HBS (pH 7.4), unfunctionalized

liposomes or CNA35-functionalized liposomes (5 mM lipid).

Transient compression of the matrices was applied to speed up the homogeneous hydration of the matrices. After an incubation of 15 min at room temperature, T¼ 293  1 K, the matrices were transferred to a fresh HBS (pH 7.4) solution and subsequently compressed 10 times, in order to promote the removal of unbound contrast agent. After 1 min, the matrices were transferred to a fresh HBS solution and again compressed ten times, followed by the same procedure for the third and final time. Next, the matrices were placed in a custom made holder and imaged using the above mentioned 6.3 T-scanner. Eight spin echo measurements, with a varying repetition time (ranging from 236.4 to 6000 ms) were done and a pixel by pixel T1map was

calculated using Mathematica (Wolfram Research, Inc.). In all scans the TE was 10.2 ms, slice thickness 0.5 mm, FOV 2.56 2.56 cm2and matrix size 256 256, yielding an in-plane resolution of 100mm2.

Acknowledgements

The authors would like to thank the group of Professor W. Baumeister (Max Planck Institute of Biochemistry, Martinsried, Germany) and in particular Dr S. Nickel, for guidance in the reconstruction of the tomography series. Furthermore, the authors would like to thank Sanne Reulen and Monica Breurken for useful discussions and experimental help with respect to the production of CNA35. This study was funded in part by the BSIK program entitled Molecular Imaging of Ischemic Heart Disease (project number BSIK03033) and by the EC - FP6-project DiMI, LSHB-CT-2005-512146. This study was performed in the frame-work of the European Cooperation in the field of Scientific and Technical Research (COST) D38 Action Metal-Based Systems for Molecular Imaging Applications.

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