Mannose-based molecular patterns on stealth microspheres for receptor-specific targeting of human antigen-presenting cells.

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)- graft-poly(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.

PEGylation as a tool for the biomedical engineering of surface modified microparticles.

Microparticles are of considerable interest for drug delivery, vaccination and diagnostic imaging. In order to obtain microparticles with long circulation times, or to provide the prerequisite for tissue specific targeting through decoration with suitable ligands, their surfaces need to be modified such that they become repellent to the adsorption of opsonic proteins and resistant to unspecific phagocytosis. The currently most considered strategy relies on the immobilisation of a poly(ethylene glycol) (PEG) corona onto the microparticles' surface. In the first chapter of this review, we discuss the unique physicochemical properties of PEG, which make it the polymer of choice to render the surfaces of microparticles repellent to the adsorption of proteins and resistant to cellular recognition. Furthermore, we present various technologies for the preparation of microparticles with PEGylated surfaces. Another aspect is the decoration of the PEGylated surfaces with suitable ligands for cell specific recognition and targeting. Finally, we review miscellaneous applications of PEGylated microparticles, mainly focusing on the fields of drug delivery, targeting and vaccination. Although still in its infancy, the PEGylation of microparticles holds promise towards future biomedical applications.

Stable stealth function for hollow polyelectrolyte microcapsules through a poly(ethylene glycol) grafted polyelectrolyte adlayer.

Prospective biomedical applications of hollow polyelectrolyte microcapsules, for example, as drug delivery systems, require surface modifications that help to escape clearance by the mononuclear phagocytic system (MPS). Layer-by-layer assembled microcapsules that were alternatingly composed of polystyrene sulfonate (PSS) and polyallylamine hydrochloride (PAH) were coated with adlayers of poly(ethylene glycol) (PEG)-grafted poly-L-lysine (PLL-g-PEG) and poly-L-glutamic acid (PGA-g-PEG). Their effects on MPS recognition were studied in primary cell cultures of human monocyte derived dendritic cells and macrophages. PGA-g-PEG coatings had no significant effect on cellular recognition, which may be explained by insufficient PEG density of the adlayer. Contrary, PLL-g-PEG effectively blocked phagocytosis of coated microcapsules. In addition, PLL-g-PEG coatings showed efficient adlayer stability for at least 3 weeks, and PAH/PSS microcapsules did not impair phagocyte viability. Our results demonstrate that layer-by-layer assembled polyelectrolyte microcapsules coated with a PEG-grafted polyelectrolyte, PLL-g-PEG, represent a promising platform for a drug delivery system that escapes fast clearance by the MPS.

Phagocytosis of poly(L-lysine)-graft-poly(ethylene glycol) coated microspheres by antigen presenting cells: Impact of grafting ratio and poly(ethylene glycol) chain length on cellular recognition.

Microparticulate carrier systems have significant potential for antigen delivery. The authors studied how microspheres coated with the polycationic copolymer poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) can be protected against unspecific phagocytosis by antigen presenting cells, a prerequisite for selective targeting of phagocytic receptors. For this aim the authors explored the influence of PLL-g-PEG architecture on recognition of coated microspheres by antigen presenting cells with regard to both grafting ratio and molecular weight of the grafted PEG chains. Carboxylated polystyrene microspheres (5 microm) were coated with a small library of PLL-g-PEG polymers with PLL backbones of 20 kDa, grafting ratios from 2 to 20, and PEG side chains of 1-5 kDa. The coated microspheres were characterized by their zeta-potential and resistance to IgG adsorption. Phagocytosis of these microspheres by human monocyte derived dendritic cells (DCs) and macrophages (MPhi) was quantified by phase contrast microscopy and by analysis of the cells' side scattering in a flow cytometer. Generally, increasing grafting ratios impaired the protein resistance of coated microspheres, leading to higher phagocytosis rates. For DC, long PEG chains of 5 kDa decreased the phagocytosis of coated microspheres even in the case of considerable IgG adsorption. In addition, preferential adsorption of dysopsonins is discussed as another factor for decreased phagocytosis rates. For comparison, the authors studied the cellular adhesion of DC and MPhi to PLL-g-PEG coated microscopy slides. Remarkably, DC and MPhi were found to adhere to relatively protein-resistant PLL-g-PEG adlayers, whereas phagocytosis of microspheres coated with the same copolymers was inefficient. Overall, PLL(20)-[3.5]-PEG(2) was identified as the optimal copolymer to ensure resistance to both phagocytosis and cell adhesion. Finally, the authors studied coatings made from binary mixtures of PLL-g-PEG type copolymers that led to microspheres with combined properties. This enables future studies on cell targeting with ligand modified copolymers.