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Anal. Chem. 2010, 82, 4337–4343

TOF-Secondary Ion Mass Spectrometry Imaging of Polymeric Scaffolds with Surrounding Tissue after in Vivo Implantation Leendert A. Klerk,† Patricia Y. W. Dankers,‡,§ Eliane R. Popa,§ Anton W. Bosman,| Marjolein E. Sanders,⊥ Kris A. Reedquist,⊥ and Ron M. A. Heeren*,† FOM Institute AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands, Institute for Complex Molecular Systems, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands, Department of Pathology and Medical Biology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands, SupraPolix Research Center, Horsten 1, 5612 AX Eindhoven, The Netherlands, and Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands Supramolecular polymeric materials are of increasing interest for the use as drug delivery carriers. A thorough insight in the biocompatibility and the degradation of these materials in vivo are of fundamental importance to further their development and application in medical practice. Molecular imaging techniques are powerful tools that enable the elucidation of molecular distributions in and around such polymer implants. A supramolecular polymeric hydrogel was implanted under the renal capsule to study its biocompatibility with TOF-SIMS. This results in a molecular cartography of the polymer implant combined with the cellular signature of the implantation environment. In this experiment, molecular signals are observed from cells that are involved in the biological response to the implant, e.g., macrophages. These molecular signatures are compared with macrophage standards cultured in different polarization environments. On the basis of this comparison, information can be acquired on the various macrophage differentiations that are connected to different stages in the foreign body response. Mass spectrometric imaging techniques offer the opportunity to visualize different histological phenomena in a single experiment without the need for specific immunohistochemical markers. Cellular infiltration into the polymer is visualized, offering a clear view on both biological and polymer features in a single imaging experiment. Biodegradable polymers are of high interest for medical applications in the fields of tissue engineering and drug delivery. In tissue engineering, bioactive scaffolds can be designed that are slowly degraded by the body while their function is taken over by newly generated tissue. Drug delivery systems are designed to locally deliver active pharmaceutical components at the place * To whom correspondence should be addressed. Phone: +31 20 7547100. Fax: +31 20 7547290. E-mail: [email protected]. † FOM Institute AMOLF. ‡ Eindhoven University of Technology. § University Medical Center Groningen. | SupraPolix Research Center. ⊥ University of Amsterdam. 10.1021/ac100837n  2010 American Chemical Society Published on Web 05/12/2010

in the body where their function is needed while other parts of the body are exposed to a much lower dose. Drug delivery systems are also applied to elongate the effective dose level of the drug in the body. Both these issues are important in the design of more effective medical treatments.1 In the development of biomaterials, thorough investigation of how the material used behaves in vivo is crucial. Undesired influence of the material itself on the body should be limited, and the biomaterial should not cause any adverse side effects. It is, therefore, important to thoroughly assess the reaction of the body under influence of the drug carrier. Also, in-depth insight into the possible changes that the physiological environment induces in the carrier material is needed. A novel approach to the controlled release of drugs is the use of supramolecular polymeric biomaterials. The use of supramolecular functionality in polymeric scaffolds has been shown to be very promising as a carrier for tissue engineering2,3 and drug delivery4 systems. Supramolecular polymers are macromolecules in which the monomers are held together via directed noncovalent interactions or in which polymers are cross-linked through these interactions. One way of achieving noncovalent binding is through multiple hydrogen bond interactions. The combination of multiple hydrogen bonds in one binding site is known to lead to cooperatively strong, yet dynamic, binding. The most obvious example of cooperative hydrogen bonding from nature is the interaction between the two strands of deoxyribonucleic acid (DNA). The use of hydrogen bond interactions for drug delivery has numerous advantages over classical approaches. Unlike diffusion driven release, the release of a noncovalently bound drug is controlled through the strength of the supramolecular interaction, which can (1) Ratner, B. D., Hoffmann, A. S., Schoen, F. J., Lemons, J. E., Eds. Biomaterials science: an introduction to materials in medicine; Academic Press: San Diego, CA, 1996. (2) Dankers, P. Y. W.; Harmsen, M. C.; Brouwer, L. A.; Van Luyn, M. J. A.; Meijer, E. W. Nat. Mater. 2005, 4, 568–574. (3) Dankers, P. Y. W.; van Leeuwen, E. N. M.; van Gemert., G. M. L.; Spiering, A. J. H.; Harmsen, M. C.; Brouwer, L. A.; Janssen, H. M.; Bosman, A. W.; van Luyn, M. J. A.; Meijer, E. W. Biomaterials 2006, 27, 5490–5501. (4) Dankers, P. Y. W.; Huizinga-van der Vlag, A.; van Gemert, G. M. L.; Petersen, A. H.; Meijer, E. W.; Janssen, H. M.; Bosman, A. W.; van Luyn, M. J. A.; Popa, E. R., Biomaterials to be submitted for publication.

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be controlled by choosing different derivatization methods of the drug or the carrier. Covalent binding of the drug to the carrier, on the other hand, restricts the possibility of mixing multiple different drugs in one delivery carrier due to synthetic restrictions. This makes, for instance, the addition of proteins difficult. Supramolecular binding allows mixing of carrier material, drugs, peptides, proteins, labeling molecules, and any other components after the synthesis, in solution or melt.2 This modular approach offers opportunities that allow the tuning of material properties and pharmacological functionality. Polymer hydrogels have long been a drug delivery carrier of great interest.5 In this paper, we analyze a hydrogel drug delivery carrier that is composed of elongated polymer chains cross-linked by the quadruple hydrogen bonding ureidopyrimidinone (UPy) group.6 An important consideration in the design of biodegradable polymers for use in medical applications is the interaction of implanted material with the host immune system. Macrophages play a key role in the foreign body response, and earlier studies have indicated that macrophages with distinct functional capacities are involved in different steps of the foreign body response to implanted polymers.7 Following extravasation into tissue surrounding the implant, peripheral blood monocytes differentiate into mature macrophages in response to local environmental cues. Depending on the cues received, mature macrophages can promote inflammation and tissue destruction (M1 macrophages), orchestrate tissue remodeling, wound healing, and angiogenesis (M2 macrophages), or suppress immune responses (M2c macrophages). Macrophages displaying these distinct properties can be generated by exposure of monocytes to polarizing stimuli in vitro, recognized by unique cell surface marker expression patterns and cytokine response profiles.8,9 To visualize the different stages in the foreign body response around the hydrogel implant, it is necessary to be able to discriminate between these types of macrophages. However, for identification of the type or types of macrophages involved, multiple markers have to be identified quantitatively, which is not possible using current immunohistochemical methods. Another restriction of immunohistochemical methods is that the polymer cannot be studied simultaneously, and therefore, the analysis is limited to the biological part of the system. Here, we explore the capabilities of imaging mass spectrometry (MS) to fill the gap in our understanding about the foreign body reaction on the polymer implant, using direct molecular imaging. We use time-of-flight secondary ion mass spectrometry (TOF-SIMS) as an imaging tool to investigate the foreign body response based on mass spectrometrically pinpointed molecules. TOF-SIMS imaging reveals molecular distributions at the cellular length scale.10-15 It is also

widely applied in the analysis of polymeric surfaces and polymers in the biomedical field.16-19 In the study presented in this paper, these application fields are combined by imaging both biological tissue at the cellular length scale and a polymer implant, in a single imaging experiment. Here, we performed TOF-SIMS imaging on rat renal tissue following polymer hydrogel implantation and compared observed molecular distributions with those identified in purified macrophages generated in vitro under defined polarizing conditions. In a single experiment, we were able to simultaneously visualize cellular distributions and the degradation of the polymer. Spatial information is obtained on the molecules that are involved in the foreign body reaction. This local mass spectrometric information is compared with macrophage standards, cultured under different polarization conditions, giving information on the macrophage phenotypes present. This demonstrates the potential of TOF-SIMS and MS imaging in general, for studying the foreign body response in vivo. It provides a wealth of position-related molecular information on the interaction of the living system with a polymer implant.

(5) Kim, S. W.; Bae, Y. H.; Okano, T. Pharm. Res. 1992, 9, 283–290. (6) Sijbesma, R. P.; Beijer, F. H.; Brunsveld, L.; Folmer, B. J. B.; Hirschberg, J. H. K. K.; Lange, R. F. M.; Lowe, J. K. L.; Meijer, E. W. Science 1997, 278, 1601–1604. (7) Rı´hova´, B. Adv. Drug Delivery Rev.: Targetable Drug Carriers 1996, 21, 157–176. (8) Mantovani, A.; Sica, A.; Locati, M. Eur. J. Immunol. 2007, 37, 14–16. (9) Mosser, D. M.; Edwards, J. P. Nat. Rev. Immunol. 2008, 8, 958–969. (10) Parry, S.; Winograd, N. Anal. Chem. 2005, 77, 7950–7957. (11) Altelaar, A. F.; Luxembourg, S. L.; McDonnell, L. A.; Piersma, S. R.; Heeren, R. M. Nat. Protoc. 2007, 2, 1185–1196. (12) McDonnell, L. A.; Piersma, S. R.; Altelaar, A. F. M.; Mize, T. H.; Luxembourg, S. L.; Verhaert, P. D. E. M.; van Minnen, J.; Heeren, R. M. A. J. Mass Spectrom. 2005, 40, 160–168.

(13) Parry, S. A.; Kurczy, M. E.; Fan, X.; Halleck, M. S.; Schlegel, R. A.; Winograd, N. Appl. Surf. Sci.: Proc. Sixteenth Int. Conf. Secondary Ion Mass Spectrom., SIMS XVI 2008, 255, 929–933. (14) Fletcher, J. S.; Rabbani, S.; Henderson, A.; Blenkinsopp, P.; Thompson, S. P.; Lockyer, N. P.; Vickerman, J. C. Anal. Chem. 2008, 80, 9058–9064. (15) Fletcher, J. S.; Lockyer, N. P.; Vaidyanathan, S.; Vickerman, J. C. Anal. Chem. 2007, 79, 2199–2206. (16) Lee, J.-W.; Gardella, J. A. Anal. Chem. 2003, 75, 2950–2958. (17) Shard, A. G.; Davies, M. C.; Tendler, S. J. B.; Jackson, D. E.; Lan, P. N.; Schacht, E.; Purbrick, M. D. Polymer 1995, 36, 775–779. (18) Chiarelli, M. P.; Proctor, A.; Bletsos, I. V.; Hercules, D. M.; Feld, H.; Leute, A.; Benninghoven, A. Macromolecules 1992, 25, 6970–6976. (19) Braun, R. M.; Cheng, J.; Parsonage, E. E.; Moeller, J.; Winograd, N. Anal. Chem. 2006, 78, 8347–8353.

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EXPERIMENTAL SECTION Sample Preparation. Polymer hydrogels were implanted under the renal capsule of rats. The hydrogels were produced in collaboration with SupraPolix B.V., Eindhoven, The Netherlands, and composed of supramolecular ureido-pyrimidinone (UPy)6 modified polymers in 0.9% NaCl. These supramolecular polymers were synthesized in a comparable manner as described before for UPy-based thermoplastic elastomers.3 They consist of poly(ethylene glycol) (Mn ) 6000 g/mol) (PEG) and polycaprolactone (Mn ) 1250 g/mol) (PCL) precursor polymers in a 9:1 w/w % ratio chain extended with a UPy moiety, resulting in a 5% (w/w) hydrogel. These UPy groups form noncovalent crosslinks between polymer chains through quadruple hydrogen bonding and allow supramolecular binding of functional additives through the same quadruple hydrogen bonding moieties.2 All animal procedures were approved by the committee for care and use of laboratory animals of the University of Groningen and performed according to governmental and international NIH guidelines on animal experimentation. The hydrogels were implanted in Fischer rats (F344, Harlan) under the renal capsule by making a small incision. Through this incision, a pocket was created using a blunt needle in which the polymer hydrogel was implanted. The rats were sacrificed, and the kidneys were harvested 15 days after implantation. Half of the kidney was used for histological studies and embedded in paraffin. The other half was snap frozen in liquid nitrogen and stored at -80 °C for later analysis using imaging MS.

Figure 1. Schematic and histological view of the kidney capsule, fibrous capsule, and cortex after implantation of the hydrogel. The general effect after 15 days of implantation is shown with a PAS staining, and the presence of macrophages is shown with an ED1 staining. (A) The contralateral control kidney and (B) the experimental kidney with the hydrogel are depicted. The kidney capsule is indicated with Kc, and the fibrous capsule is indicated with Fc. The position where the hydrogel was implanted is indicated with h, the cortex is indicated with C. In (B), the kidney capsule and the outer fibrous capsule cannot be seen in the picture. Scale bar ) 100 µm.

Cryo-sections (10 µm thick) of the polymer-tissue samples were made using a Leica CM1900 cryo-microtome at -20 °C. Sections were stored at -80 °C until analysis. The sample was brought to room temperature under dry conditions in a vacuum desiccator. The samples were then coated with 1 nm of gold using a Quorum Technologies SC7640 sputter coater equipped with a FT7607 quartz crystal microbalance stage and a FT7690 film thickness monitor. After gold coating, the samples were introduced into the mass spectrometer. The limited amount of sample preparation needed for TOF-SIMS circumvents biases introduced by extensive sample treatment. TOF-SIMS analysis was performed using a TRIFT II mass spectrometer (Physical Electronics, Eden Prairie, MN) equipped with a Au primary ion gun. 20 keV Au+ primary ions were selected for analysis. SIMS spectra were measured both in the positive and the negative secondary ion mode with a secondary ion energy of 9 keV. Histological Staining. Staining of 5 µm zinc-fixed paraffinembedded sections were performed as follows. These sections were not taken adjacent to the sections that were measured with MS imaging but from the other half of the same kidney or from a different animal with the exact same implant. They nevertheless give a good indication of the histology of the implantation site. Periodic acid-Schiff (PAS) staining was performed to evaluate the general renal morphology (15 min of 1% periodic acid, 30 min of Schiff’s reagent (Merck), 5 min of hematoxylin at 37 °C, and 10 s of 70% ethanol with 1% hydrochloric acid). ED1 staining was done to visualize the presence of macrophages, which indicates an inflammatory response (Figure 1). Antigen retrieval was carried out on dewaxed paraffin-embedded sections by incubation in 0.1 M Tris-buffer pH 9.0 at 80 °C for 18 h. Aspecific staining was blocked with 2% bovine serum albumin (Sigma) for 30 min. Endogenous peroxidase was blocked with 1% hydrogen peroxide (Merck) for 30 min. The sections were incubated with the mouse antirat CD68 (1:200; anti-ED1; Serotec) for 1 h and with rabbitantimouse-peroxidase (1:100; Dako) for 30 min. Color development

was performed using 3,3-diaminobenzidine tetrachloride (brown; Sigma). Sections were counterstained with hematoxylin (blue; Merck). Micrographs of tissue slices were taken on a Leica DMLB microscope with Leica DC300 camera and Leica QWin 2.8 software. These methods offer an excellent view on the specific labeled components but no information on the exact molecular composition and no view on the polymer. The sections that were used for mass spectrometry were imaged using a Leica DM-RX microscope equipped with a Nikon DMX 1200 CCD camera or using a flatbed scanner without prior staining to make overlays with the MS images. Comparison with in Vitro Polarized Macrophages. Macrophages were polarized in vitro and analyzed with TOF-SIMS to obtain the molecular signature of different macrophage phenotypes. Human monocytes were used and stimulated by six different polarization agents, given in Table 1. Monocytes were isolated from human blood, donated by a healthy volunteer. These cells were cultured for 6 days with the addition of recombinant human (rh) granulocyte/macrophage colony stimulating factor (rh-GM-CSF; 5 ng/mL), rh macrophage colony stimulating factor (rh-MCSF; 25 ng/mL), rh-inteferon γ(rhIFNγ; 20 ng/mL), rh-interleukin-10 (rh-IL-10; 10 ng/mL) (R&D Systems, Abingdon, UK), lipopolysaccharide (LPS; 1ug/mL) from E.coli (Sigma, Zwijndrecht, Netherlands), or rh tumor necrosis factor (rh-TNF; 10 ng/mL) (Biosource, Invitrogen, Breda, Netherlands). After culturing, the cells were washed with sucrose (300 mmol/L) to remove salts. Cytospinning was performed at 550 rpm for 2 min on indium tin oxide (ITO) coated glass slides using 40 000 cells per sample. Sample slides were prewetted for 1 min at 550 rpm using sucrose solution. The human macrophage samples were measured with and without 1 nm Au coating. Only the Au-coated samples are considered for comparison with the implantation study to get the most reliable comparison with the Au-coated samples that were used for tissue implant analysis. The peaks that are related to the Analytical Chemistry, Vol. 82, No. 11, June 1, 2010

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Table 1. Overview of the Applied Macrophage Stimuli and Their Effects polarization stimulus macrophage colony-stimulating factor (M-CSF) granulocyte macrophage colony-stimulating factor (GM-CSF) interferon-gamma (IFN-γ) interleukin-10 (IL-10) lipopolysaccharides (LPS) tumor necrosis factor-R (TNF-R)

effect growth factor, promotes differentiation of monocytes into proinflammatory M1 macrophages growth factor, promotes differentiation of monocytes into proinflammatory M1 macrophages alarm signal, released when “foreign body” is detected, promotes M1 macrophage polarization anti-inflammatory cytokine, promotes differentiation into anti-inflammatory, regulatory macrophages, inhibits proinflamatory cytokine production; stimulates differentiation into regulatory macrophages (M2 type) toll-like receptor ligand, promotes secretion of proinflammatory cytokines, macrophage polarization dependent upon cofactors a proinflammatory factor that stimulates phagocytosis; stimulates macrophages into dendritic cells, M1 type of cells

Au coating (e.g., Au adducts) were excluded from analysis by comparison with the non-Au-coated samples, when doing peakassignment in the Au-coated sample. TOF-SIMS analysis was performed using the same conditions as for the implantation samples. Statistical Data Analysis. Statistical data analysis was used as an exploratory tool for the analysis of the imaged implantation samples to cope with the unknown complexity of the samples. Principal component analysis (PCA) and PCA with VARIMAX optimization20 were both used for this purpose, using in-house made tools in MatLab (version 7.0.4, R14, SP2, The MathWorks, Natick, MA) and the in-house built “ChemomeTricks” package.21 Another reason to use methods to retrieve correlated peaks in the data set is related to the fact that polymers do not typically show up as a single peak or a combination of a few peaks but rather as a large series of peaks,22 which results in a much lower single peak signal/noise ratio. Therefore, it is very hard to reveal polymer localizations by simply plotting the ion image of one polymer-specific peak. The use of all the polymer specific signal results in a much more contrast-rich image. This is also the case for correlated biologically relevant peaks. Score plots that are a result of PCA are, therefore, used for visualization as they reflect the combination of relevant spectral features. RESULTS Simultaneous Imaging of Hydrogel and Tissue. After implantation, harvesting, and preparation of the samples, the 10 µm thick cryo-sections were analyzed both at a large field of view with low image resolution and at a small field of view with high image resolution. The large-area image yields an overview of the implantation site. PCA with VARIMAX optimization was done on this data set, using the mass range between 200 and 1000 Da. This gives an overview of correlated peaks in the (quasi-)molecular and large-fragment ion region and, therefore, gives the most distinctive spectrum (Figure 2b). The distributions based on the PCA results provide a clear image of the different areas that can be characterized. Several conclusions can be drawn from this image (Figure 2a). Cholesterol, with peaks at m/z 369 [M - OH]+ and m/z 383 [M - H]+, is a highly abundant cell membrane (20) Klerk, L. A.; Broersen, A.; Fletcher, I. W.; van Liere, R.; Heeren, R. M. A. Int. J. Mass Spectrom.: Imaging Mass Spectrom. Spec. Issue 2007, 260, 222– 236. (21) Eijkel, G. B.; Kaletas, B. K.; Van Der Wiel, I. M.; Kros, J. M.; Luider, T. M.; Heeren, R. M. A. Surf. Interface Anal. 2009, 41, 675–685. (22) Klerk, L. A. Imaging mass spectrometry of polymeric systems, Ph. D. thesis, Utrecht University (2009), Utrecht, The Netherlands.

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component that localizes in the tissue region. The polymer is seen in the subcapsular pocket and is identified by its characteristic distribution of peaks with a 44 Da interval and the mass of a PEG monomer unit, as well as its 16 Da (for O loss or the difference between K+ and Na+ cationized species) and 14 Da (CH2) loss pattern (see Figure 2c). Adding up the O and two CH2 intervals gives 44 Da, the mass of one PEG monomer unit (CH2CH2O). There is also a 4 or 2 Da deviation from the predominant peaks visible, attributed to different degrees of unsaturation, for instance in one of the end groups. End group analysis was not possible due to the unknown cationization agent. A colocalization of various lipids is seen within the polymer area as well as just outside the pocket. The presence of lipids in the polymer region indicates cellular infiltration or at least biological activity within the polymer. The presence of lipids outside the kidney capsule is attributed to adipose tissue. The signal in this region is very high because the lipids form a thin film on the ITO substrate, which enhances the ionization efficiency due to its conductivity and higher stopping power.23 Some silicone (most probably polydimethylsiloxane, PDMS) was localized on one side of the implantation pocket, as concluded from the silicone-specific peaks at m/z 73 and m/z 147. We attribute this to the tools that were used during the surgical implantation procedure, most probably the needle used for making a subcapsular pocket. Silicones are a common lubricant and biocompatible and have a very low surface energy which makes them spread easily. Their abundance as a contaminant in operating rooms is an obvious consequence. Moreover silicones are easily ionized in SIMS. To get a clearer view on cell infiltration into the polymer material, SIMS imaging was performed at high resolution, using a 360 µm field of view. To see enough image detail at this resolution, also, the higher intensity low-mass fragment peaks are considered because (quasi-)molecular and large fragment ions often give too low intensity when very small areas are measured. In the positive SIMS mode, this high resolution view (Figure 3a) shows a choline-predominant region (m/z 104 for the choline headgroup [CH3)3NCH2CH2OH]+), a polymer-related distribution (indicated by m/z 45 for the PEG monomer [C2H5O]+), and a localization for cholesterol-derived compounds (m/z 369, which corresponds to [M - OH]+, which is a quasi-molecular ion of cholesterol but could also be related to one of its derivatives). Cholesterol-related signals are high in the fibrous (23) Postawa, Z. Appl. Surf. Sci. 2004, 231-232, 22–28.

Figure 2. (a) Large area image of the hydrogel implant under the renal capsule of a rat, 15 days after implantation. Various localizations are indicated, based on PCA+VARIMAX results (see spectra in (b)) and in the overlay with an optical microscope image. The presence of lipids inside the polymer area shows cellular infiltration in the drug delivery carrier. Some smearing artifact is visible at the bottom region of the polymer. The respective spectral results are given in (b). The first and second PCs gave noninformative distributions. PC 3 shows signal for various lipids, including diacyl glycerols (m/z 550-610), PC 4 shows cholesterol ((M-OH)+ at m/z 369.4 and M+ at m/z 385.4), PC 5 shows silicone contamination (C7H21O2Si3+ at m/z 221.1, further identified from low-mass peaks in the corresponding region), PC 7 shows the polymer distribution, readily recognized from the m/z 44 spacing between the peaks, which exactly corresponds to the mass of one PEG unit. (c) Shows the PEG distribution in detail with characteristic 16 Da (K+ and Na+ difference or O loss) and 14 Da (CH2 loss) intervals. The change from 0.2 to 0.3 values is due to binning down and, thus, rounding to 0.1 Da.

tissue on the exterior side of the kidney and sparsely distributed within the polymer region. Interestingly, the cholesterol signal partially coincides with the PEG signal. The PEG also does not seem to take up the major part of the implant but rather be distributed within newly formed tissue, possibly as a degradation product. Compound overlap as discussed here is often observed in imaging MS. This is usually indicated by the complementary use of the primary colors red, green, and blue for no more than three specific peaks or principal components. Overlap then results in the generation of additive color combinations such as purple in Figure 3b. When assessing the negative ion SIMS images (Figure 3b), a remarkable distribution is seen for m/z 465.32. It shows up as a granularly distributed signal within the implantation site and complements a distribution at m/z 124.01 (a peak which could not be identified). The compound that the m/z 465.32 signal results from is most probably cholesterol sulfate, which has a theoretical mass of 465.304 Da for [M - H]+. This is confirmed (Figure 3c) by the colocalization of the signal at m/z 96.97 and m/z 95.98, which both are due to sulfate-related ions (HSO4- and SO4-, respectively) but could, at the obtained resolving power, also be due to phosphate ions (H2PO4- and HPO4-, respectively). The fact that the distribution at m/z 78.96, which is due to phosphate (PO3-) and cannot be related to sulfate, anticorrelates with the distribution of cholesterol sulfate, confirms that the compound at 465.32 is indeed a sulfate. On the basis of this, a confident assignment of this peak can be made. Furthermore, cholesterol sulfate is

known to be abundant in healthy kidney tissue (also seen in the investigated control kidney in this study, see next subsection).24,25 Its function is not fully understood, but suggestions are made about regulating enzyme activity and playing a role in cell adhesion and differentiation as well as signal transduction.26 In earlier studies, cholesterol sulfate was detected in the kidney and skin of patients with Fabry disease using TOF-SIMS.27 When considering the localization of the several components in the positive and negative secondary ion images, several interesting observations can be made. First of all, the PEG signal colocalizes with the cholesterol sulfate signal to a large extent. This implies that a polymer-related signal coincides with a tissuerelated signal which means that either of these components interchange at a dimension that cannot be seen at this resolution (a few micrometers) or there is PEG-related material present inside the cholesterol sulfate containing cells. Furthermore, the cholesterol derived signal (at m/z 369) coincides with the cholesterol sulfate signal as expected but at a different intensity profile. This is due to the fact that m/z 369 results from a number of cholesterol derivatives, including cholesterol and cholesterol (24) Iwamori, M.; Moser, H. W.; Kishimoto, Y. Biochim. Biophys. Acta 1976, 441, 268–279. (25) Strott, C. A.; Higashi, Y. J. Lipid Res. 2003, 44, 1268–1278. (26) Christie, W. W. The lipid library; American Oil Chemists’ Society, Urbana, IL, 2009. (27) Touboul, D.; Roy, S.; Germain, D. P.; Chaminade, P.; Brunelle, A.; Lapre´vote, O. Int. J. Mass Spectrom.: Imaging Mass Spectrom. Spec. Issue 2007, 260, 158–165.

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Figure 3. High resolution image of the hydrogel implant under the renal capsule. MS images are shown in the overlay with an image taken with an optical microscope. The image (a), recorded in positive ion mode SIMS, shows distribution for the main cholesterol-derived fragment (red, m/z 369.35, [M - OH]+), PEG (green, seen as the monomer-derived fragment at m/z 45.03 [C2H5O]+, also seen at m/z 133, [C6H13O3]+), and choline (blue, m/z 104.11, [(CH3)3NCH2CH2OH]). The high cholesterol signal from the substrate is due to ionization enhancement by ITO, which is a conductor with high primary ion stopping power. The image (b), recorded in negative ion mode SIMS, shows sulfate (blue, 96.97 Da, HSO4-), which exactly coincides with a compound at 465.32 Da (red) and an unassigned compound at 124.02 Da (green). The compound at 465.32 is most likely cholesterol sulfate which has a theoretical mass of 465.304 for (M - H)- and is confirmed by complementing information from phosphate and sulfate-selective images in (c), which shows a comparison between different phosphate and sulfate related peaks. The purple color indicates overlap of sulfate (blue) and cholesterol sulfate (red). The correspondence of m/z 96 with m/z 465.32 in (b) but not with m/z 79 proves that the compound at m/z 465.32 contains sulfate and not phosphate. Scale bar in (c) ) 100 µm.

sulfate. Also, the abundance of choline, largely anticorrelated with cholesterol-related signal, is quite striking as choline is an abundant compound in most biological tissues. Therefore, further analysis, including MS/MS-based determination of the assigned 4342

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compounds is necessary for firm conclusions on the foreign body response mechanism. Comparison between Macrophage Standards and the Hydrogel Implant. Macrophage standards were imaged using TOF-SIMS. In these images, the cellular region was selected for a region of interest (ROI) analysis. The ROI was determined based on molecular signature (the presence of biological molecules and the absence of substrate-specific peaks) and morphology. The ROI spectra of these cell aggregates were plotted and phenotypespecific peaks were extracted. Many peaks show high intensity in multiple macrophage types, but some peaks were specific for only one or two types of macrophage polarizations. On the basis of these more unique peaks, an estimation can be made of the type of macrophages that are present in the tissue samples. The most intense and specific peaks are listed in the Tables S-1 and S-2 (Supporting Information). A comparison is also made with the rat kidney standard, to determine the presence of the apparent compounds in the bare rat kidney. This gives an indication of what peaks, and eventually what compounds, can be expected for the different macrophage polarization. When assessing the peaks that were measured in the differently polarized macrophage standards, a few specific peaks can be picked out. First of all, a peak at m/z 465.3 (cholesterol sulfate) in negative ion mode is highly abundant in the M-CSF stimulated cells as well as in the control kidney and present at low concentration in the IFN-γ and IL-10 stimulated cells. The highly specific m/z 281.2 (oleic acid) in the implantation region, indicates activity as observed in the IFN-γ stimulated cells (Figure 4). Oleic acid is also found in the control kidney but at much lower concentration, which is confirmed by a low signal from the kidney tissue region in the implantation sample. In the positive ion mode, m/z 157.9 is found, which is highly specific for IFN-γ and LPS stimulated macrophages. When plotting the distribution of this signal in the implantation sample (Figure 4), high abundance is seen in the implantation region and low abundance is seen in the kidney region, which correlates with its low abundance in the control kidney. This, once again, indicates toward macrophages that are similar to the IFN-γ polarized cells. However, although partially coincident with the oleic acid (m/z 281.2) and m/z 339.1 distributions, the distributions do not completely overlap. A reliable assignment can, therefore, not be given at this point. Another peak, at m/z 383.3, which is most probably resulting from a D-type vitamin, shows a very distinct distribution along the edge of the implantation site, at the fibrous capsule region. This peak was seen at high intensity in the M-CSF-stimulated cells but also in the IFN-γ and LPS stimulated cells. Its distribution partially corresponds to the cholesterol sulfate distribution, but there is no exact overlap. The numerous cross-correlations between the various peaks assessed shows the complexity of the immune response in general and after polymer implantation, in particular. Although no definite distinction between different macrophage phenotypes can be made in tissue at this point, these experiments show the potential of delivering insight in different molecular mechanisms. The images resulting from the implantation study show how TOF-SIMS imaging can be used for distinguishing several domains in the foreign body response.

infiltration was shown in the polymer. This adds a novel dimension to the investigation of these types of samples. Issues related to the dissolution of the polymer during immunohistochemical staining procedures, that often lead to a loss of the polymer, are not encountered when using TOF-SIMS. Therefore, direct evidence is obtained on the presence of active cells within the polymer hydrogel. Macrophages were polarized using different stimuli. This resulted in mass spectra with phenotype-specific peaks, but a reliable classification is so far not possible. To do this, a more extensive study is needed. To make a conclusive comparison with the implantation study, the use of rat macrophages instead of human macrophages is needed to exclude any species-specific differences.28 Once a reliable profile for the different macrophage phenotypes is available, more definite observations can be made for the implantation samples. Recent developments in TOF-SIMS instrumentation,14,29 allowing MS/MS analysis of TOF-SIMS generated ions, can further solidify the assignment of the observed peaks, e.g., the sulfate and phosphate peaks. MALDI-TOF imaging can also contribute to the analysis of biological components but has the disadvantage of not being able to detect the polymer due to its high molecular weight and extensive supramolecular crosslinking. For biological molecules, MS/MS analysis using MALDI generated ions will contribute to the complete understanding of the sample. These results and the opportunities provided for further research show the enormous potential TOF-SIMS imaging has in the analysis of implanted biomaterials. The independence of specific antibodies and the ability to distinguish cellular as well as synthetic components based on their molecular profile allows us to do explorative research on the foreign-body response. Also, it enables one to study the drug distribution in the carrier as well as the degradation of polymeric materials in vivo, in a single ex vivo imaging experiment. We expect that the drug release can be monitored by visualizing the drug distribution in the tissue around the implant at different time points. The simultaneous visualization of native, introduced, and induced molecules allows one to further assess the reaction of the body on foreign materials and the possible changes in the material following implantation. This adds a new dimension to biomaterials research, allowing access to molecular information that can currently not be obtained otherwise.

Figure 4. Distribution at the gel implantation site of compounds that were found in the macrophage standards. The sample images show the same region with different magnification. The table gives the relative intensities for the selected peaks as they were found in the macrophage samples. A full list is available in Tables S-1 and S-2 in the Supporting Information.

DISCUSSION AND OUTLOOK This study shows that TOF-SIMS is a very powerful technique for studying samples in which both synthetic polymers and biological activity play a role. Both the polymeric implant and the biological activity can be imaged in a single measurement. Cellular (28) Strauss-Ayali, D.; Conrad, S. M.; Mosser, D. M. J. Leukocyte Biol. 2007, 82, 244–252. (29) Piehowski, P. D.; Carado, A. J.; Kurczy, M. E.; Ostrowski, S. G.; Heien, M. L.; Winograd, N.; Ewing, A. G. Anal. Chem. 2008, 80, 8662–8667.

ACKNOWLEDGMENT The authors acknowledge project funding from the Strategic Research Fund (SRF) of ICI plc. The imaging facilities at AMOLF are part of a research program of the “Stichting voor Fundamenteel Onderzoek der Materie (FOM)”, which is financially supported by the “Nederlandse organisatie voor Wetenschappelijk Onderzoek (NWO). Gaby van Gemert (SupraPolix B.V., Eindhoven, The Netherlands) is acknowledged for the synthesis of the hydrogels. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 31, 2010. Accepted May 5, 2010. AC100837N Analytical Chemistry, Vol. 82, No. 11, June 1, 2010

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