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Langmuir 2008, 24, 11790-11802
Mannose-Based Molecular Patterns on Stealth Microspheres for Receptor-Specific Targeting of Human Antigen-Presenting Cells Uta Wattendorf,† Ge´raldine Coullerez,‡ Janos Vo¨ro¨s,§ Marcus Textor,‡ and Hans P. Merkle*,† Institute for Pharmaceutical Sciences, ETH Zurich, 8093 Zurich, Switzerland, Laboratory for Surface Science and Technology, ETH Zurich, 8093 Zurich, Switzerland, and Laboratory for Biosensors and Bioelectronics, ETH Zurich, 8092 Zurich, Switzerland ReceiVed April 7, 2008. ReVised Manuscript ReceiVed July 22, 2008 The targeting of antigen-presenting cells has recently gained strong attention for both targeted vaccine delivery and immunomodulation. We prepared surface-modified stealth microspheres that display various mannose-based ligands at graded ligand densities to target phagocytic C-type lectin receptors (CLRs) on human dendritic cells (DCs) and macrophages. Decoration of microspheres with carbohydrate ligands was achieved (i) by electrostatic surface assembly of mannan onto previously formed adlayers of poly(L-lysine) (PLL) or a mix of PLL and poly(L-lysine)-graftpoly(ethylene glycol) (PLL-PEG), or (ii) through assembly of PLL-PEG equipped with small substructure mannoside ligands, such as mono- and trimannose, as terminal substitution of the PEG chains. Microspheres carrying mannoside ligands were also studied in combination with an integrin-targeting RGD peptide ligand. Because of the presence of a mannan or PEG corona, such microspheres were protected against protein adsorption and opsonization, thus allowing the formation of specific ligand-receptor interactions. Mannoside density was the major factor for the phagocytosis of mannoside-decorated microspheres, although with limited efficiency. This strengthens the recent hypothesis by other authors that the mannose receptor (MR) only acts as a phagocytic receptor when in conjunction with yet unidentified partner receptor(s). Analysis of DC surface markers for maturation revealed that neither surface-assembled mannan nor mannoside-modified surfaces on the microspheres could stimulate DC maturation. Thus, phagocytosis upon recognition by CLRs alone cannot trigger DC activation toward a T helper response. The microparticulate platform established in this work represents a promising tool for systematic investigations of specific ligand-receptor interactions upon phagocytosis, including the screening for potential ligands and ligand combinations in the context of vaccine delivery and immunomodulation.
1. Introduction Efficient recognition of invading pathogens is a prime role of antigen-presenting cells (APCs). Therefore, targeting of receptors on APCs, being involved in pathogen recognition, have gained much attention for both vaccine delivery and immunomodulation.1 Among the many APC receptors, such as Toll-like receptors (TLRs), C-type lectins (CLRs), scavenger receptors (SRs) or immunoglobulin receptors (FcRs), especially TLRs and CLRs have been extensively studied for their capacity to recognize pathogen associated molecular patterns (PAMPs).2,3 PAMPs are invariant structures that are unique to pathogens and include diverse molecular structures such as lipopolysaccharides (LPS), mannans, aldehyde-derivatized proteins, and bacterial DNA, among others.4,5 One may hypothesize that the cross-talk between a vaccine delivery system decorated with PAMP-like motives and APC receptors could be a strategy to affect its recognition and uptake by APCs as well as the type and extent of APC maturation. Here we challenge this hypothesis using stealth microparticles with mannose-enriched surface coatings. Microparticles of about 1 to 10 µm in diameter are logical targeting * To whom correspondence should be addressed. E-mail: hmerkle@ pharma.ethz.ch. † Institute for Pharmaceutical Sciences. ‡ Laboratory for Surface Science and Technology. § Laboratory for Biosensors and Bioelectronics.
(1) Gogola´k, P.; Re´thi, B.; Hajas, G.; Rajnavo¨lgyi, E´. J. Mol. Recognit. 2003, 16, 299–317. (2) Proudfoot, O.; Apostolopoulos, V.; Pietersz, G. A. Mol. Pharm. 2007, 4, 58–72. (3) Cambi, A.; Figdor, C. G. Curr. Opin. Cell Biol. 2003, 15, 539–546. (4) Medzhitov, R.; Janeway, C. A., Jr Science 2002, 296, 298–300. (5) Clark, R.; Kupper, T. J. InVest. Dermatol. 2005, 125, 629–637.
systems for vaccines and immunomodulators, as such particles undergo rapid phagocytosis by APCs, an exclusive feature of phagocytes such as dendritic cells (DCs) and macrophages (MΦs). In contrast to other internalization mechanisms, phagocytosis appears to be especially suitable, as it is the main internalization mechanism for pathogens in this size range. The CLR family comprises a number of pathogen recognition receptors (PRRs) that bind to carbohydrate ligands in the presence of Ca2+ ions.6 Targeting CLRs on APCs was identified as a promising strategy for immunomodulation, as documented by several studies showing that immune responses were enhanced or modified by coupling mannose type ligands, such as mannose moieties or mannan, to proteins or particulates.7-12 However, most of these approaches used either soluble protein with covalently coupled ligands, or ligand-decorated liposomal carriers. Thus, there were multiple opportunities for ligand-receptor interactions, e.g., through proteinic epitopes or opsonization of the carrier. Hence, it is often difficult to specify in detail whether an observed effect on phagocytosis or immunomodulation is (6) Van Vliet, S. J.; Gruen, C. H.; Van Kooyk, Y. In Protein-Carbohydrate Interactions in Infectious Diseases; Bewley, C., Ed.; Royal Society of Chemistry: Cambridge, U.K., 2006; pp 106-124. (7) Toda, S.; Ishii, N.; Okada, E.; Kusakabe, K. I.; Arai, H.; Hamajima, K.; Gorai, I.; Nishioka, K.; Okuda, K. Immunology 1997, 92, 111–117. (8) Cui, Z.; Han, S.-J.; Huang, L. Pharm. Res. 2004, 21, 1018–1025. (9) Jain, S.; Vyas, S. P. J. Liposome Res. 2006, 16, 331–345. (10) Apostolopoulos, V.; Pietersz, G. A.; Tsibanis, A.; Tsikkinis, A.; Drakaki, H.; Loveland, B. E.; Piddlesden, S. J.; Plebanski, M.; Pouniotis, D. S.; Alexis, M. N.; McKenzie, I. F.; Vassilaros, S. Breast Cancer Res. 2006, 8, R27. (11) White, K.; Rades, T.; Kearns, P.; Toth, I.; Hook, S. Pharm. Res. 2006, 23, 1473–1481. (12) Hattori, Y.; Kawakami, S.; Lu, Y.; Nakamura, K.; Yamashita, F.; Hashida, M. J. Gene Med. 2006, 8, 824–834.
10.1021/la801085d CCC: $40.75 2008 American Chemical Society Published on Web 09/12/2008
Targeting APCs Using Surface-Modified Microspheres
exclusively due to a specific ligand-receptor interaction, or results from a combination of more than a single recognition signal. Furthermore, most appoaches do not, or not exclusively, target a phagocytic mechanism. Therefore, the aim of this work was to develop a versatile microparticulate platform that will allow the study of phagocytic recognition signals of APCs in more detail, displaying various ligands on the surface of a stealth microsphere. Poly(ethylene glycol) (PEG) coatings of appropriate architecture are commonly used to prepare microspheres with stealth surfaces that are repellent to serum protein adsorption and cellular recognition.13 It is the uncharged and highly hydrophilic character of such a PEG corona and its low toxicity and immunogenicity that makes it most suitable for biomedical applications.14 Negatively charged surfaces can be conveniently coated with a PEG corona by electrostatic adsorption of poly(L-lysine)-graftpoly(ethylene glycol) copolymers, here further denoted as PLLPEG, consisting of a polycationic PLL backbone whose side chain amino groups are partly grafted with PEG chains.15,16 The degree of PEG substitution of such polymers is indicated by the grafting ratio g, which is defined as the number of L-lysine monomers divided by the number of PEG side chains. Because of its positive charge at physiological pH, the PLL backbone adsorbs strongly onto negatively charged substrates through electrostatic interaction, forming a less than 10 nm thick adlayer of PLL-PEG stretching its PEG chains out perpendicularly to the surface.16,17 In fact, we recently showed that polystyrene (PS) microspheres which were surface coated with PLL(20)-[3.5]PEG(2), i.e., a copolymer with a 20 kDa PLL backbone, 2 kDa PEG side chains, and a grafting ratio of approximately 3.5, were efficiently shielded from both serum protein adsorption and recognition by phagocytes.18,19 It has been further shown that the PEG side chains of PLLPEG can be terminally functionalized with bioligands, such as adhesion peptides or biotin, while retaining the repellent character of its adlayer to unspecific protein adsorption.20-22 Most recently, terminal substitution of the PEG side chains has been also demonstrated for selected carbohydrates.23 Here we investigate two prototype microsphere formulations for their capacity to induce receptor-mediated phagocytosis in APCs, the first coated with mixtures of PLL and PLL-PEG and then functionalized with oligomannose by adsorption of mannan, a polymannoside PAMP from S. cereVisiae (Figure 1).24 Mannan is assumed to be the major antigen of yeast cell walls25 and a well-established multivalent ligand for the receptors TLR4 and (13) Wattendorf, U.; Merkle, H. P. J. Pharm. Sci. DOI: 10.1002/jps.21350. Published Online: Feb 27, 2008. http://dx.doi.org/10.1002/jps.21350. (14) Harris, J. M.; Chess, R. B. Nat. ReV. Drug DiscoVery 2003, 2, 214–221. (15) Kenausis, G. L.; Vo¨ro¨s, J.; Elbert, D. L.; Huang, N.; Hofer, R.; RuizTaylor, L.; Textor, M.; Hubbell, J. A.; Spencer, N. D. J. Phys. Chem. B 2000, 104, 3298–3309. (16) Huang, N.-P.; Michel, R.; Vo¨ro¨s, J.; Textor, M.; Hofer, R.; Rossi, A.; Elbert, D. L.; Hubbell, J. A.; Spencer, N. D. Langmuir 2001, 17, 489–498. (17) Pasche, S.; De Paul, S. M.; Vo¨ro¨s, J.; Spencer, N. D.; Textor, M. Langmuir 2003, 19, 9216–9225. (18) Wattendorf, U.; Koch, M. C.; Walter, E.; Vo¨ro¨s, J.; Textor, M.; Merkle, H. P. Biointerphases 2006, 1, 123–133. (19) Wattendorf, U.; Kreft, O.; Textor, M.; Sukhorukov, G. B.; Merkle, H. P. Biomacromolecules 2008, 9, 100–108. (20) Huang, N.-P.; Vo¨ro¨s, J.; De Paul, S. M.; Textor, M.; Spencer, N. D. Langmuir 2002, 18, 220–230. (21) VandeVondele, S.; Vo¨ro¨s, J.; Hubbell, J. A. Biotechnol. Bioeng. 2003, 82, 784–790. (22) Mu¨ller, M.; Vo¨ro¨s, J.; Csu´cs, G.; Walter, E.; Danuser, G.; Merkle, H. P.; Spencer, N. D.; Textor, M. J. Biomed. Mater. Res., Part A 2003, 66A, 55–61. (23) Coullerez, G.; Seeberger, P. H.; Textor, M. Macromol. Biosci. 2006, 6, 634–647. (24) Vinogradov, E.; Petersen, B.; Bock, K. Carbohydr. Res. 1998, 307, 177– 183. (25) Domer, J. E.; Garner, R. E. NATO ASI Ser., Ser. H 1991, 53, 415–430.
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DC-SIGN (dendritic cell-specific ICAM-3 grabbing nonintegrin).26,27 For the second prototype, we coated microspheres with stealth adlayers of mannoside-substituted PLL-PEG. We hypothesized that the presentation of mannoside ligands linked to the surface of microspheres at high densities via flexible PEG chains could be a strategy to mimic high molecular weight mannose-based PAMPs with synthetic small molecular ligands. Aspects that will be covered in this work are (i) the formulation of the two microparticulate prototypes, (ii) the availability of the surface-exposed mannose-based ligands to lectin binding, (iii) the capacity of the formed adlayers to repel protein adsorption, (iv) the ζ-potentials of such particles, and, in particular, (v) the specific recognition and phagocytosis of the microparticulates by both DCs and MΦs as APCs; further, (vi) potential effects of mannoside substitutions on APC maturation will be looked at. Although specific recognition of the two prototype microspheres could be demonstrated, we will reject the paradigm that, by themselves, mannose-based molecular patterns on microparticulates are recognized as PAMP-like motives.
2. Experimental Section 2.1. Materials. PLL(20)-g[3.5]-PEG(2), PLL(20)-g[4.0]-PEG(2)/PEG(3.4)-RGD(8%) and PLL(20)-g[3.7]-PEG(2)/PEG(3.4)RDG(9%) were obtained from SurfaceSolutionS GmbH (Zurich, Switzerland) and consisted of a PLL backbone of 20 kDa with grafted PEG chains of 2 kDa and a grafting ratio (which specifies the total number of lysine monomers divided by the number of PEG side chains) of g ) 3.5-4.0. The notation used for ligand-modified copolymers PLL-g-PEG(2)/PEG(3.4)-ligand(x%) indicates the percentage of ligand covalently coupled to 3.4 kDa PEG side chains relative to the total number of PEG chains as determined by NMR. The full sequences of the coupled peptides were N-acetylGCRGYGRGDSPG-amide (biologically active peptide) and Nacetyl-GCRGYGRDGSPG-amide (inactive control). For simplicity and readability of this paper, the polymers are henceforth abbreviated as PLL-PEG, PLL-PEG-RGD and PLL-PEG-RDG, respectively. As microparticulates we used negatively charged, carboxylated polystyrene (PS) microspheres (5 µm diameter) from Micromod Partikeltechnologie GmbH, Rostock, Germany. All chemicals used in this study were of analytical grade (from Fluka, Buchs, Switzerland) unless otherwise specified. Ultrapure water (NANOpure DiamondTM, Skan AG, Allschwil, Switzerland) was used for buffer preparation. 2.2. SynthesisofMannoside-SubstitutedPLL-PEGCopolymers. Sulfhydryl-tri(ethylene glycol) derivatized mannose and branched R(1,3)-R(1,6)-trimannose were prepared by methods analogous to those described in the literature by Ratner et al.28 and were kindly provided by Riccardo Castelli of Prof. Seeberger’s laboratory at ETH Zurich. Mannose elongated with a sulfhydryl-propyl linker was synthesized by allylation reaction as described by Houseman et al.29 The mannoside ligand (1.2 equiv) dissolved in 50 mM sodium tetraborate buffer at pH 8.5 (
