3556
Biomacromolecules 2010, 11, 3556–3562
Versatile Polymer Microspheres for Injection Therapy: Aspects of Fluoroscopic Traceability and Biofunctionalization Ketie Saralidze,*,† Menno L. W. Knetsch,† Cees van der Marel,‡ and Leo H. Koole† Department of Biomedical Engineering/Biomaterials Sciences, Faculty of Health, Medicine, and Life Sciences, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands, and Department of Surface and Thin Film Analysis, Mi Plaza Materials Analysis, Philips Research, High Tech Campus 4, 5656 AE Eindhoven, The Netherlands Received September 1, 2010; Revised Manuscript Received October 5, 2010
Synthesis and characterization of a series of novel microspheres featuring (i) radiopacity (i.e., clear fluoroscopic traceability) and (ii) an outer surface exposing aldehyde groups are reported. The aldehydes allowed us to tether proteins onto the particles’ surface under mild conditions, under which the protein conformation and, hence, structural motifs for biorecognition are preserved. Essential monomer building blocks were (i) 4-iodobenzoyl2-oxo-ethylmethacrylate (4-IEMA) for radiopacity and (ii) propenal for surface tethering of proteins. The particles demonstrated good X-ray visibility and cytocompatibility. Procedures to couple proteins onto the surface were optimized using fluorescent bovine serum albumin (FITC-BSA) or collagen (FITC-collagen). Furthermore, radiopaque microparticles with unlabeled bovine collagen type I were produced. The presence of immobilized collagen was verified with narrow-scan X-ray photoelectron spectroscopy. Fibroblasts readily adhere to and grow on the collagen-modified surfaces, whereas this was much less the case for the unmodified controls. The results led us to suggest that immobilized nondenatured collagen may transform filler particles from passive spaceoccupying objects to particles that cross-talk with surrounding tissues.
Introduction A variety of modern therapies is based on polymeric particles with dimensions in the 50-500 µm range. Generally, such particles are first suspended in a more or less viscous medium (e.g., a collagen suspension). Such formulations can be injected easily and accurately, for example, by reconstructive or cosmetic surgeons to treat acne scars,1,2 to correct wrinkles,3,4 to augment lips,5 or to treat facial lypoathrophy.6,7 Applications outside the domain of reconstructive/cosmetic surgery are rapidly gaining importance. Some examples are injectable microspheres to support unilateral vocal cord paralysis,8 to manage vesicoureteral reflux,9,10 or to improve intrinsic sphincter deficiency, which is the major cause of urinary stress incontinence.11-13 While the clinical performance of commercial products generally ranges from “acceptable” to “excellent”,12-16 it is clear that further technical developments of the biomaterials can provide a basis for greater success in various therapies. One prominent improvement strategy concerns traceability of injected microspheres in situ. Several research groups, including our own, have developed stable and biocompatible radiopaque polymer microspheres, which can be detected via computed tomography (CT) or other radiographic techniques, both during and after the injection procedure.17-22 Another improvement strategy relates to biofunctionalization of injectable microspheres. Evidently, introduction of bioactivity at the surface of the microspheres could result in a profitable interaction of the particles with their tissue environment. For instance, thrombin (the key enzyme in blood coagulation) has been immobilized at the surface of injectable microspheres. It could be shown that exposure of such particles to blood almost immediately initiates
coagulation.23 This may be useful under some critical circumstances, for example, to quickly stop excessive internal bleeding or to rapidly induce coagulation in an (cerebral) aneurysm. The aim of the present study was to develop a preparative route to injectable microspheres that are intended as fillers and bulking agents. These spheres possess both radiopacity and the possibility to tether proteins for biorecognition, or enzymes for bioactivity, to the surface. We sought mild immobilization conditions under which the structural integrity and bioactivity of vulnerable proteins would be preserved. Note that attachment of thrombin in our previous work was achieved through the rather harsh EDC/NHS-activated ester method.23,24 We describe the synthesis of a set of new radiopaque polymeric microspheres that expose aldehyde groups at their surface. It is well-known that immobilized aldehydes allow for a variety of surface modification reactions under mild conditions.25 To achieve this goal, we engaged mixtures of four different vinylic monomers (methylmethacrylate (MMA), propenal (acrolein), 2-[4-iodobenzoyl]-oxo-ethylmethacrylate (4-IEMA), and tetraethyleneglycol dimethacrylate (TEGDMA)) in suspension polymerization reactions. The resulting microspheres differed in the surface density of aldehyde groups and the level of radiopacity. All particles allowed facile immobilization of bovine collagen type I under relatively mild conditions (pH 9.6). Physical properties of the new radiopaque and collagen-carrying (and hence biorecognizable) microspheres are described, as well as phenomena occurring upon contacting the particles with cells in vitro. The potential utility of injectable microspheres with an exterior surface of immobilized collagen is briefly discussed.
Experimental Section * To whom correspondence should be addressed. Phone: +31 43 3881272. Fax: +31 43 3881725. E-mail:
[email protected]. † Maastricht University. ‡ Philips Research.
Materials. Chemicals were purchased from Sigma/Aldrich/Fluka (Zwijndrecht, The Netherlands), Acros (Landsmeer, The Netherlands), or Invitrogen (Breda, The Netherlands). Methylmethacrylate (MMA)
10.1021/bm1010273 2010 American Chemical Society Published on Web 10/20/2010
Polymer Microspheres for Injection Therapy was distilled at atmospheric pressure and stored at -20 °C. The monomer 4IEMA was synthesized from 4-iodobenzoyl chloride as described previously.24 Propenal, benzoyl peroxide (BPO), tetraethylene glycol dimethacrylate (TEGDMA), poly(vinyl alcohol) (PVA; M.W. 86000; 99-100% hydrolyzed), poly(ethylene glycol (PEG; MW 1000), poly(vinyl pyrrolidone) (PVP; K-23-32; MW 58000) were used as purchased. Cell culture medium (DMEM-F12), fetal bovine serum (FBS), and antibiotics were purchased Invitrogen (Breda, The Netherlands). MTT and dinitrophenyl-hydrazine were from Acros, Cyquant reagent from Invitrogen. The FITC-labeled forms of collagen and albumin were from Sigma. Buffer compositions are 0.2 M carbonate buffer prepared from 0.2 M Na-bicarbonate and 0.2 M Na-hydrogencarbonate, mixed to pH 9.6. Phosphate-buffered saline (PBS) is 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 in 1 L distilled water, and set to pH 7.4. Synthesis. Microspheres were prepared as described previously.26 One procedure (10% propenal; entry 4 in Table S1) is described in full detail as a typical example. PVA (1.25 g), PEG (1.50 g), and PVP (0.25 g) were dissolved in 100 mL of distilled water. The solution was magnetically stirred and heated to 85 °C. A reactant mixture with the following composition was prepared: MMA (3.87 g, 38.65 mmol), propenal (0.43 g, 7.67 mmol), 4-IEMA (1.70 g, 4.72 mmol), TEGDMA (0.34 g, 1.03 mmol), and benzoylperoxide (120 mg, 0.49 mmol). This mixture was added dropwise to the heated and stirred aqueous PVAPEG/PVP solution. Stirring and heating were continued for 6 h. When stirring was stopped, immediate settling of particles was observed. The supernatant was decanted carefully. The particles were allowed to cool to room temperature, washed with water (3×), and lyophilized. Yield: 5.45 g, (91%). To prepare the microspheres with a high content of propenal (70 and 100% propenal; entries 8 and 9 in Table S1), more BPO was required to initiate the suspension polymerization reaction, and obtain acceptable yields. X-ray Visibility. A single layer of microspheres with a diameter between 100 and 400 µm was deposited on a piece of 3 M Scotch tape. This specimen was compared for X-ray visibility to a porcine rib, which was cleaned from all soft tissue. Imaging was performed with a Phoenix/X-ray nano-CT system, operating at 70 kV. Analysis of Surface Aldehyde Groups. Aldehyde groups at the surface of the microspheres were analyzed through reaction with dinitrophenyl-hydrazine (DNPH).27 For this a 20 µg/mL solution of DNPH in ethanol was mixed with 100 mg spheres (the exact mass was determined for each reaction) and the reaction was allowed to run for 20 h. UV absorbance (at 348 nm) of the supernatant was measured prior to and after the reaction. From the extinction difference, the amount of DNPH coupled to the microspheres could be calculated. The microspheres turned bright yellow during the reaction, but this coloration was not suited for quantification. Coupling of Fluorescently Labeled Proteins. Microspheres (100 mg, all percentages of propenal) were washed twice with 50 mM carbonate buffer, pH 9.6. Then, incubation with 1 mL of (1) 10 mg/ mL FITC-BSA or (2) 0.8 mg/mL FITC-collagen I in 50 mM carbonate buffer, pH 9.6, followed (16 h at room temperature). In the case of FITC-collagen, the protein solution was first heat-treated at 80 °C for 10 min and rapidly cooled to room temperature. After 3 h of incubation, the microspheres were washed until no more significant fluorescence was detected in the wash. As a control, microspheres without propenal were used to determine the noncovalently bound protein on the surface. First, the microspheres were analyzed by fluorescence microscopy using a Nikon Eclipse 3600, equipped with a Coolsnap CCD camera. Thereafter, microspheres with FITC-albumin were digested with trypsin (0.5 mg/mL in PBS with 0.53 mM EDTA) for 16 h at 37 °C. The liberated protein was quantified by determining release fluorescence using a Gemini XS fluorometer, and comparison to a standard curve of trypsin-digested FITC-BSA. Microspheres with covalently attached FITC-collagen I were digested with collagenase (1 mg/mL in PBS pH
Biomacromolecules, Vol. 11, No. 12, 2010
3557
7.4) for 16 h at 37 °C. Quantification of digested collagen was performed similarly to FITC-BSA from microspheres, as described above. Collagen Coupling. To 500 mg of washed microspheres, 5 mL of 0.8 mg/mL bovine collagen I in 50 mM carbonate buffer, pH 9.6, was added. The collagen solution was heated to 80 °C for 10 min prior to incubation with microspheres. After 16 h, the particles were treated with 10 mg/mL NaCNBH3 for 2 h at room temperature. The microspheres were washed extensively in carbonate buffer and PBS and subsequently stored in PBS at 4 °C until further use. X-ray Photoelectron Spectroscopy. XPS analyses of microspheres to verify the presence of collagen on the surface have been carried out in a Quantera SXM from Ulvac-PHI (Q2). Microspheres were freezedried before XPS analysis. The measurements were performed using monochromatic Al KR radiation; during the measurements, the angle between the axis of the analyzer and the surface of the sample holder was 45°. Taking into account the fact that the diameter of the spheres is on average 200 µm and the measurements were performed with a measurement spot of only 50 µm positioned precisely at the center of the spheres, we conclude that the information depth is approximately 6 nm. By means of wide-scan measurements, the elements present at the surface have been identified. The chemical state and the atomic concentrations of the elements present are determined from narrowscan measurements. Standard sensitivity factors were used to convert peak areas to atomic concentrations. As a result of this, it is possible that the concentrations may be subject to a systematic error, which is not expected to be higher than 20%. Cytotoxicity. Mouse fibroblasts (3T3) were inoculated in 96-well plates at a density of 1000 cells/well. After incubation for 1 day at 37 °C, extracts of microspheres in culture medium were added. These extracts were obtained by incubating 200 mg spheres in 4 mL of medium for 2 days at 37 °C. The cells were allowed to proliferate for 3 days, and the number of viable cells was determined by addition of 0.5 mg/mL MTT in culture medium. After a further incubation of 1 h, the medium was aspirated and the formed blue formazan crystals were dissolved in isopropanol, and the absorbance at 570 nm was determined. For a direct contact cytotoxicity assay, cells were grown in 24-well plates up to approximately 50% confluency. Then microspheres shortly soaked in culture medium were added. After 5 days of incubation at 37 °C, photographs were taken using a standard digital camera on a Leica DM-IL microscope. Cell Adhesion. 3T3 cells were grown to confluency in 24-well plates. Microspheres with or without covalently coupled collagen were shortly soaked in culture medium and added to the cell layers and incubated at 37 °C for times indicated. In this setup, cells may actively migrate onto the spheres and attach to them. For quantification, microspheres were carefully removed from the cell layers (microscopic evaluation of the cell layer showed no significant damage of the remaining cell layer) and washed twice in culture medium. The last bit of medium was removed using a syringe equipped with a cut of 0.5 mm needle, and the spheres were frozen at -80 °C. The amount of attached cells was determined using the CyQuant reagent according to the manufacturer’s instructions. Alternatively, cells were grown on coverslips (12 mm diameter), and microspheres were carefully placed on the semiconfluent layers. After 24 or 48 h of incubation on the cell layers, the medium was aspirated and the coverslips could be removed from the culture dishes. The coverslips with microspheres lying on the cells were put on a microscope slide and put at an angle of approximately 45°. To study the adherence of the microspheres to the cells, medium was flushed over the coverslips at 20 mL · min-1. The resulting coverslips were (i) photographed macroscopically, (ii) observed microscopically, and (iii) fixated in 2.5% glutaraldehyde in PBS for more than 1 h at 4 °C, dehydrated with an ethanol series, dried in air, and prepared for observation with a scanning electron microscope (Philips XL30 SEM system).
3558
Biomacromolecules, Vol. 11, No. 12, 2010
Saralidze et al.
Figure 1. Typical scanning electron micrographs of the iodine-containing and aldehyde-exposing polymer microspheres of this study, prepared with 10 mol % propenal (A) or 20% propenal (B) in the monomer mixture. (C) Histograms showing the size distribution of the microspheres with 10 or 20 mol % propenal.
Results and Discussion Radiopaque and aldehyde-carrying microspheres were prepared in nine different compositions, as is compiled in Table S1. All syntheses proceeded smoothly, except for the compositions with a high content of propenal (>50%, entries 8 and 9 in Table S1). In these cases, the suspension polymerization only became successful upon increasing the concentration of the initiator by a factor of 3-4 (Table S1). In all cases, we obtained smooth and perfectly spherical particles with an average diameter in the range of 150-250 µm; this was verified with scanning electron microscopy (Figure 1A,B). The average diameter for microspheres with 10 or 20 mol % propenal were found to be 195 ( 39 and 186 ( 34 µm, respectively. Their size disctribution shown in Figure 1C demonstrates that over 90% of the microspheres are in the range of 150-250 µm. Most likely, this size distribution of the particles could be adapted to specific wishes, for example, by changing the stirring parameters (speed, size, and geometry of the reaction flask, etc.). This was not explored in this study. All syntheses were run with 4.72 mmol of the iodine-containing monomer 4-IEMA, which resulted in an iodine content of 10% (by mass) in all cases. It is our experience that this iodine content generally leads to sufficient fluoroscopic visibility, at least for microparticles with a diameter > ∼200 µm. This is, of course, dependent on the application. For the augmentation of soft tissues, the fluoroscopic visibility of the microspheres of this study would be sufficient. For injection therapies in more complex environments (e.g., periurethral soft tissues close to the sphincter muscle), higher contrast levels will probably be necessary. To illustrate this point, we recorded X-ray images of a single layer of three types of microspheres with a diameter of approximately 200 µm (Figure 2): (i) microspheres of PMMA (poly(methylmethacrylate)), which is a radiolucent material; (ii) microspheres containing 10% (by mass) of covalently coupled iodine; and (iii) microspheres of this study containing 20% (by mass) of covalently coupled iodine. These three specimens were partly placed against a bony background (a porcine rib; Figure 2). The PMMA particles are invisible, as expected. The 10% iodine particles are nicely discernible, but only outside the overlap zone
Figure 2. X-ray image of the microspheres of this study, partially against a bone background (porcine rib). Microspheres were glued as a single layer onto a piece of Scotch tape (15 × 15 mm; top image). Left square: microspheres of poly(methylmethacrylate); these particles are invisible as the material is transparent to X-radiation. Middle square: microspheres of this study containing 10% (by mass) iodine. Right square: microspheres of this study containing 20% (by mass) iodine.
with the bone. The 20% iodine particles are clearly visible, also, in the overlap area. The presence of aldehyde groups at the surface of the microspheres could be verified with the classic Brady test.27 This method is based on the reactivity of 2,4-dinitrophenylhydrazine (2,4-DNPH) toward aldehydes or ketones. The reaction yields 2,4-dinitrohydrozones, which are usually stable structures with a bright yellow or red color. In our case, the hydrozone is immobilized at the surface of the microspheres, as is depicted schematically in Figure 3. Indeed, we observed that all our microspheres turned bright yellow after the treatment with 2,4DNPH. Table S2 shows the quantification of the aldehyde groups, exposed at the surface of the microspheres. The number of surface aldehyde groups initially increases linearly with the propenal content of the microspheres (up to 30% propenal content). The maximum amount of surface aldehydes of 437.5 ( 3.6 nmol/g spheres is reached at 70% propenal. Cytotoxicity. In view of the intended applications, the microspheres must be absolutely nontoxic to cells. All our microspheres were tested for in vitro cytotoxicity by incubating
Polymer Microspheres for Injection Therapy
Figure 3. Schematic representation of the Brady reaction, involving an immobilized aldehyde. Note that an immobilized hydrozone is formed, which explains depletion of 2,4-DNPH from the supernatant and the intense yellow coloration of the particles, as the reaction is ongoing.
either extracts of microspheres or microspheres directly with mouse fibroblasts (3T3 cells). Extracts from microspheres with low aldehyde contents (entries 1-7 in Table S1) were nontoxic (Figure 4A). When these spheres were incubated in direct contact with mouse fibroblasts, no toxicity could be determined either (Figure 4B). Microspheres with higher aldehyde content (entries 8 and 9 in Table S1) were found to be cytotoxic, which may be due to unreacted propenal (LD50 ) 46 mg/kg) or to BPO or BPO decomposition products; additional amounts of BPO had to be used in the synthesis of these particles (vide supra). Clearly, this information disqualifies the microspheres with high aldehyde contents (entries 8 and 9 in Table S1) for further use. Coupling of Fluorescently Labeled Proteins. We first explored protein immobilization onto our microspheres with FITC-labeled albumin. As shown in Figure 5B, the particles became strongly fluorescent; extensive washing of the particles
Biomacromolecules, Vol. 11, No. 12, 2010
3559
did not diminish their fluorescent nature. On the other hand, treatment with trypsin resulted in loss of fluorescence. These observations, taken together, strongly suggest that the immobilization procedure was successful. Quantification of the proteolysis products showed that an optimal composition of the spheres, for albumin attachment, could be observed (Figure 5A). Microspheres based on 10-30% propenal had most FITClabeled albumin attached to their surface. The fact that higher densities of aldehyde groups on the surface did not result in increased albumin coupling may be somewhat surprising. This could be explained by the fact that multipoint attachment of albumin on the surface is more likely than attachment of additional protein from solution. The reaction of a soluble protein on the solid surface is less likely than the reaction of the free amino groups of an already coupled protein already in the vicinity of the aldehyde groups on the surfaces. Also, the space for additional protein molecules on the surface may become too small, causing steric hindrance for additional albumin molecules. When studying the coupling of FITC-labeled collagen, very similar results were obtained. The data presented in Figure 6A show a narrower distribution of covalent protein coupling to the different propenal microspheres. But again the spheres with 10-20% propenal turned out to have the most suitable composition for protein attachment. Treatment of these microspheres with collagenase removed most of the fluorescence (Figure 6B), demonstrating that the bound collagen still had a more or less native conformation that could be recognized by this specific protease.
Figure 4. (A) Cell viability of cells incubated with extracts from the range of microspheres. (B) Light microscopy of cells cultured in direct contact with the microspheres of this study. Control incubations were without any material or with cytotoxic latex. The scale bar represents 100 µm in all photographs.
Figure 5. (A) Quantification of attachment of FITC-labeled bovine serum albumin (BSA) to the series of microspheres with increasing aldehyde content. (B) Fluorescence micrographs from 20% propenal microspheres with (top) and without (bottom) FITC-BSA.
3560
Biomacromolecules, Vol. 11, No. 12, 2010
Saralidze et al.
Figure 6. (A) Quantification of FITC labeled collagen type I to the series of microspheres with increasing aldehyde content. (B) Fluorescence micrographs of 20% propenal microspheres with FITC collagen (top), after treatment with collagenase (middle), and the control spheres (bottom).
Figure 7. (A) XPS survey spectra, measured on two microspheres (20% propenal) of this study. Top spectrum: after surface modification with unlabeled collagen I. Bottom spectrum: control, that is, without surface modification. For clarity, the top spectrum has been shifted upward. Note the presence of a small extra peak at 398 eV in the top spectrum (asterisk). This peak, shown in detail in the narrow scan (B), is due to nitrogen, which is introduced through immobilization of collagen within the particle’s outermost surface layer.
XPS Analysis. The results of the XPS experiments are compiled in Table S3. Note that the experiments were done in duplo, that is, spectra of two microspheres + collagen and of two control microspheres were recorded. The surface modification is found to introduce nitrogen in the outermost surface layers of the microspheres (1.4 and 1.9% for the modified particles vs 0.0 and 0.6% for the controls). This was expected, because the control material is essentially devoid of nitrogen, while collagen of course contains nitrogen. Hence, the XPS data provide additional proof for the presence of the protein after the surface modification reaction. The surface concentrations of C are slightly larger for the collagen-modified microspheres in comparison with the controls. For O, the opposite was found. Noteworthy, iodine was detected at the surface of all microspheres. This reveals that the collagen blanket is thin (
