Hyaluronan‐Based Nanohydrogels for Targeting Intracellular S. Aureus in Human Keratinocytes

Abstract

Staphylococcus aureus is one of the most significant human pathogens that is frequently isolated in a wide range of superficial and systemic infections. The ability of S. aureus to invade and survive within host cells such as keratinocytes and host immune cells has been increasingly recognized as a potential factor in persistent infections and treatment failures. The incorporation of antibiotics into hyaluronan-cholesterol nanohydrogels represents a novel paradigm in the delivery of therapeutic agents against intracellular bacteria. The work presented herein shows that NHs quickly enter human keratinocytes and accumulate into lysosomes. When used for targeting intracellular S. aureus the antimicrobial activity of loaded levofloxacin is enhanced, possibly changing the antibiotic intracellular fate from cytosol to lysosome. Indeed, gentamicin, an antibiotic that predominantly accumulates in lysosomes, shows significant and equal antibacterial activity when entrapped into NHs. These results strongly suggest that lysosomal formulations may display preferential activity toward intracellular S. aureus, opening new avenues for the use of HA-based NHs for treatment of such skin infections.

Pursuing Intracellular Pathogens with Hyaluronan. From a ‘Pro-Infection’ Polymer to a Biomaterial for ‘Trojan Horse’ Systems

“…HA is already extensively used for topical administration in cosmetics or ophthalmology. The incorporation of antimicrobials within HA-based nanogels may represent an interesting approach for enhancing the intracellular delivery of some drugs, thus targeting to pathogens that are a common cause of chronic topical infections (e.g., S. aureus ) [ 113 ]. Moreover, the local HA administration could reduce the drawbacks due to the in vivo u ptake and catabolism of HA into cells that express LYVE-1 or HARE (e.g., liver and kidney).…”

“…Therefore, among all the drug delivery systems for antimicrobial activity, the HA-based nano-carriers may represent a novel paradigm for targeting to cells where pathogens persist, enhancing the intracellular drug concentration in the specific sub-cellular compartments. Indeed, such nano-carriers can be customised in order to target lysosomes [ 113 ] or to escape the endosome, being released into the cytoplasm [ 114 ].…”

“…Even if a number of works have shown HA-based nano-carriers, are internalised by cells especially by CD44 receptors [ 115 , 116 , 117 ], the subsequent intracellular trafficking of these nano-systems is often not clear or is not investigated. It has been demonstrated self-assembled HA-cholesterol nanohydrogels (HA-CH NHs) (HA M w = 2.2 × 10 5 , CH degree of functionalisation = 15% (mol/mol, %), mean diameter~180 nm) quickly accumulate (within 1 h) in acidic endosomal and lysosomal compartments of human keratinocytes (HaCaT cells), reaching the highest co-localisation with those vesicles in 4 h [ 113 ]. Intracellularly, ApoTome analysis showed HA-CH NHs located into vesicle-like structures, those with a diameter of approximately 0.3 µm close to the plasma membrane and those in larger vesicles, with diameter up to 1.5 µm close to the nucleus.…”

“…The reported results showed entrapped LVF was able to eradicate both intracellular microorganisms after only 2 h of incubation, whilst free LVF was ineffective intracellularly. A similar study was conducted on human keratinocytes (HaCaT cells) infected with S. aureus [ 113 ]; infected cells were incubated with LVF or gentamicin (GM)-loaded HA-CH NHs or their controls (free LVF, GM, NHs). These two antibiotics were selected as they have different intracellular pathways: LVF is a cytosolic drug, whilst GM is a lysosomal one.…”

“…Though it is still not clear where the sub-cellular sites in which the loaded antibiotic was acting, the experiment was designed to remove any extracellular pathogen, so only intracellular microorganisms were counted after treatment. Moreover, as the free NHs was not effective against both intracellular and extracellular pathogens, it can be stated the antibacterial activity was only due to the intracellular LVF [ 113 , 133 ].…”