Although conventional vaccines have generated major successes in the control of infectious diseases, several obstacles remain in their development against chronic diseases (HIV, tuberculosis), against which no current candidate vaccines yet ensure protection. The transcutaneous route of vaccine administration appears to be a promising approach of targeting vaccines toward antigen-presenting cells (APCs) and thus improving immune responses. We investigated the suitability of nanoparticles in this approach. We found a high density of Langerhans cells (LCs) around hair follicles that, when sorted, readily internalized all size particles. However, flow cytometry after transcutaneous application of 40, 750, or 1,500 nm nanoparticles on human skin samples revealed that only 40 nm particles entered epidermal LC. Fluorescence and laser scan microscopies, which were carried out to identify the penetration pathway of transcutaneously applied nanoparticles, revealed that only 40 nm particles deeply penetrate into vellus hair openings and through the follicular epithelium. We conclude that 40 nm nanoparticles, but not 750 or 1,500 nm nanoparticles, may be efficiently used to transcutaneously deliver vaccine compounds via the hair follicle into cutaneous APCs.

The influence of the surface functionalization of silica particles on their colloidal stability in physiological media is studied and correlated with their uptake in cells. The surface of 55 ± 2 nm diameter silica particles is functionalized by amino acids or amino- or poly(ethylene glycol) (PEG)-terminated alkoxysilanes to adjust the zeta potential from highly negative to positive values in ethanol. A transfer of the particles into water, physiological buffers, and cell culture media reduces the absolute value of the zeta potential and changes the colloidal stability. Particles stabilized by L-arginine, L-lysine, and amino silanes with short alkyl chains are only moderately stable in water and partially in PBS or TRIS buffer, but aggregate in cell culture media. Nonfunctionalized, N-(6-aminohexyl)-3-aminopropyltrimethoxy silane (AHAPS), and PEG-functionalized particles are stable in all media under study. The high colloidal stability of positively charged AHAPS-functionalized particles scales with the ionic strength of the media, indicating a mainly electrostatical stabilization. PEG-functionalized particles show, independently from the ionic strength, no or only minor aggregation due to additional steric stabilization. AHAPS stabilized particles are readily taken up by HeLa cells, likely as the positive zeta potential enhances the association with the negatively charged cell membrane. Positively charged particles stabilized by short alkyl chain aminosilanes adsorb on the cell membrane, but are weakly taken up, since aggregation inhibits their transport. Nonfunctionalized particles are barely taken up and PEG-stabilized particles are not taken up at all into HeLa cells, despite their high colloidal stability. The results indicate that a high colloidal stability of nanoparticles combined with an initial charge-driven adsorption on the cell membrane is essential for efficient cellular uptake.

In this study, the skin penetration and cellular uptake of amorphous silica particles with positive and negative surface charge and sizes ranging from 291 ± 9 to 42 ± 3 nm were investigated. Dynamic light scattering measurements and statistical analyses of transmission electron microscopy images were used to estimate the degree of particle aggregation, which was a key aspect to understanding the results of the in vitro cellular uptake experiments. Despite partial particle aggregation occurring after transfer in physiological media, particles were taken up by skin cells in a size-dependent manner. Functionalization of the particle surface with positively charged groups enhanced the in vitro cellular uptake. However, this positive effect was contrasted by the tendency of particles to form aggregates, leading to lower internalization ratios especially by primary skin cells. After topical application of nanoparticles on human skin explants with partially disrupted stratum corneum, only the 42 ± 3 nm particles were found to be associated with epidermal cells and especially dendritic cells, independent of their surface functionalization. Considering the wide use of nanomaterials in industries and the increasing interest for applications in pharmaceutics and cosmetics versus the large number of individuals with local or spread impairment of the skin barrier, e.g., patients with atopic dermatitis and chronic eczema, a careful dissection of nanoparticle-skin surface interactions is of high relevance to assess possible risks and potentials of intended and unintended particle exposure.

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