**1. Introduction**

The past years have witnessed a boost in the importance and growth of novel biopharmaceuticals which include peptides, proteins and antibodies [1]. When we consider the USFDA approvals from the year 2017 to 2019, more than 30% of the approved drugs were biologics (2017: 12 of 34; 2018: 17 of 42; 2019: 10 of 48) [2]. Compared to the traditional small drug molecules, biologics provide high target specificity and pharmacokinetics resulting in minimal off-target effects, making them more favorable [3,4]. Even though they exhibit increased effectiveness for a wide range of diseases including cancer and metabolic disorders, they face major challenges. One of the biggest challenges is the delivery of biologics to achieve their maximum therapeutic potential. This is attributed to their structural complexity, decreased stability, distribution, and permeability across biological barriers [5]. Nanoparticulate systems constitute an effective strategy to improve the delivery of biologics. The biologics can be either encapsulated inside the nanoparticles (NPs) or loaded onto their surface by covalent conjugation or by physical adsorption [6].

Silica NPs (SiNP) are an attractive platform especially for protein delivery due to their excellent biocompatibility, potential for surface modification, safety, tunability, and stability. They have been studied previously for intra- and extracellular protein delivery and enzyme mobilization [7–9]. The functionalization of SiNPs offers diverse prospects, especially in the field of protein delivery. Hollow-type mesoporous SiNPs with surface amino functionalization were used for the delivery of bovine viral diarrhea virus protein, and show promising potential for the development of nanoparticle-based recombinant subunit vaccines [10]. Wolley et al. successfully demonstrated the conjugation of platelet activationspecific antibodies to polyamido-amine dendrimer-functionalized SiNPs, facilitating the development of a diagnostic tool in cardiovascular disease [11]. Furthermore, carboxylic acid functionalization of SiNPs has been reported to improve protein-loading efficacy and sustained release, and to improve the particles' thermal stability and adaptability [12,13]. However, all these benefits remain of limited value if the functionalized nanomaterial exerts an enhanced or altered immunological effect, specifically when directed against the cargo (adaptive immunity). Immunological inertness is thus, for many applications, desired when considering functionalized nanoparticles for drug delivery. Ideally, they should remain undetectable by the innate immune system, display a low propensity to activate the adaptive immune system, and serve the purpose of delivering their cargo effectively to the target site. In this regard, surface functionalization of particulate systems should be scrutinized, as the functional groups or molecules associated with the surface of NPs can cause immunomodulation or induce an unexpected immune recognition resulting in an unwanted response. For example, polyethylene glycol (PEG) can induce an anti-PEG immune response and immunological memory, thereby resulting in reduced clinical efficacy and increased adverse effects [14]. Furthermore, even if the free molecule in solution exhibits weak immunogenicity, it can become immunogenic when associated with a carrier [15,16]. For instance, while PEG by itself is considered as immunologically inert and safe, PEGylation of nanoparticles results in the induction of anti-PEG antibodies, leading to the enhanced clearance of NPs from the blood and hypersensitivity reactions in patients [16]. Moreover, the physicochemical properties of nanomaterials, including their coatings, have been reported to determine the outcome of an immune response [17]. Thus, it is essential to elucidate the immune effects of functionalized nanoparticles at an early stage during development of nanobiopharmaceutics.

Dendritic cells are the most potent antigen-presenting cells (APCs) that can prompt the initiation of a primary immune response. Hence, they are highly relevant cells to investigate the immune effects of functionalized nanomaterials. Several reports have revealed the influence of the physicochemical properties of nanoparticles on the molecular mechanisms leading to an immune response. This includes the recognition by immune cells, efficiency of uptake, APC maturation, antigen processing, presentation, and T cell differentiation [18–20]. For example, nanoparticles with an optimum size of 50 nm were found to be taken up more effectively compared smaller or larger particles [21].

In this study, we compared the immune effects of aminopropyltriethoxysilane- (SiNP\_Aconferring amino (NH2) group) and Meldrum's acid- (SiNP\_M-conferring a carboxy (COOH) group) functionalized silica particles with uncoated, i.e., SiNPs, silica NPs in APCs. SiNP\_M was used as an alternative to previously investigated undecanoic acid-functionalized particles due to increased stability in suspension and, thus better performance in immunological assays involving APCs. Dendritic cells derived from human peripheral blood monocytes, termed monocyte-derived dendritic cells (moDCs) were used as APCs to study immune effects. We have previously observed alterations in the antigen processing patterns when Bet v 1 was conjugated to SiNPs due to the structural alteration of the protein at the nanomaterial interface [22]. Thus, the immune effects of the particles with and without conjugation to Bet v 1 (the model protein) were also explored here. Bet v 1, the major birch pollen allergen is a well-characterized, well-studied protein; its properties as an allergen facilitate specific immunological assessments in follow-up studies to determine its suitability as a candidate for allergen-specific immunotherapy. Here, we investigated the two major molecular mechanisms contributing to the initiation or modulation of an immune response, including the uptake of antigen and the maturation of the APCs.
