Anna Salvati

Nanoparticle rigidity influences cellular uptake and in vivo behavior, yet the underlying mechanisms remain unclear. Here we used lipids with identical head groups but different acyl chains to prepare liposomes with comparable surface composition, size and zeta potential, but distinct mechanical properties. Rigid liposomes showed much higher uptake than soft ones, mediated by distinct endocytic mechanisms. Proteomic analysis revealed that rigid and soft liposomes adsorbed very different coronas upon dispersion in serum. RNA interference and competition studies indicated that the enhanced uptake of rigid liposomes is likely driven by the lower abundance of multiple apolipoproteins in their corona, and possibly the higher abundance of apolipoprotein H and of a pattern of low-abundant corona proteins. Importantly, a strong difference in uptake was observed only when liposomes were added to cells in the presence of serum. Hence, the different uptake efficiency is not solely due to the different mechanical properties, but results from the effect of liposome mechanical properties on corona formation.

Positive nanoparticles have often a higher uptake than neutral and negative nanoparticles. This is usually attributed to electrostatic interactions with negatively charged proteoglycans on the cell membrane. However, upon contact with serum, nanoparticles adsorb a biomolecule corona and tend toward neutrality, suggesting that electrostatic interactions alone cannot explain the different uptake. Here, we used oppositely charged liposomes as an example to explore at a fundamental level why positive nanoparticles usually show a higher uptake than the negative ones. Two proteomic-based approaches were combined to compare their protein coronas and the cell surface proteins involved in their internalization. The results showed that the higher uptake of the positive liposomes used for this study could not be simply explained by the involvement of specific corona proteins and dominating cell surface proteins. Instead, small differences in the abundances of a large number of corona proteins and cell surface proteins were observed, including multiple low-abundance proteins. Importantly, the positive liposomes had higher uptake than the negative liposomes only when added to cells in the presence of serum, suggesting that the higher uptake likely resulted from the observed subtle differences in their corona and the collective contribution and interactions with multiple cell surface proteins.

Staphylococcus aureus is a Gram-positive bacterium capable of infecting multiple types of cells, organs, and tissues in the human body. Treatment can become highly challenging, especially in the case of intracellular infections and upon biofilm formation. Additionally, this pathogen has developed several antimicrobial resistance mechanisms, and resistant strains such as methicillin-resistant S. aureus (MRSA) are among the most difficult to treat. Within this context, nanomedicine can offer novel and more efficient treatments against S. aureus. Here, we first introduce the challenges in the treatment of S. aureus infections, focusing on intracellular infections and biofilms, and challenges associated with the development of resistance. We then provide an overview of the multiple applications of nanomedicine against S. aureus infection and discuss how nanomedicine may overcome the challenges in reaching this pathogen and eliminating it, including potential solutions less prone to generating resistance. Finally, we discuss the current clinical development of antimicrobial nanomedicines, where only one out of 35 completed trials has so far targeted MRSA, indicating that most research is still at the preclinical stage. Challenges in the clinical translation of antimicrobial nanomedicines are discussed, together with strategies to support the development of these promising therapeutic agents.

Monitoring nanoparticle uptake and intracellular trafficking by cells is required to understand how nanomedicines are processed by cells. Many methods currently applied to achieve this test the involvement of known mechanisms and pathways after blocking them, or by measuring colocalization of nanoparticles with known intracellular compartments. However, unknown and non‐canonical mechanisms are often involved in nanomedicine uptake and the organelles in which they are trafficked are not fully identified. Therefore novel methods to characterize how and where nanomedicines are internalized and trafficked in unperturbed cells are highly sought. Here a novel platform is presented to characterize uptake and intracellular trafficking of nanoparticles without requiring prior knowledge on the involved mechanisms and their intracellular location. After uptake and cell lysis, organelle sorting by fluorescence assisted cell sorting is used to isolate and purify the organelles in which nanoparticles are internalized and trafficked. Next, the composition of the recovered organelles with nanoparticles is determined by organelle proteomics. Using polystyrene nanoparticles to validate the method, it is shown that this workflow allows to discover novel receptors involved in nanoparticle uptake and proteins mediating nanoparticle trafficking toward the lysosomes. The knowledge gained is essential to test and improve novel formulations for nanomedicine applications.

Small amphiphilic gold nanoparticles with mixed ligand surfaces were loaded with antibiotics. The nanoparticles showed antimicrobial activity against Staphylococcus aureus and were able to penetrate biofilms and eradicate the bacteria.

In acute myeloid leukemia (AML), disease relapse is often observed because of therapy‐resistant leukemic stem cells that re‐initiate the disease. Leukemic stem cells can tightly associate with mesenchymal stromal cells inside the bone marrow, which is considered to further drive drug resistance. Here, the cell membrane of bone marrow stromal cells is used to prepare cell membrane nanoparticles and study their interactions with AML cells. Cell membrane liposomes (CM‐Liposomes) of different charge are prepared and either used directly, or after deposition on silica cores to modulate nanoparticle mechanical properties. Nanoparticle size, zeta potential and coating efficiency are analyzed by dynamic light scattering (DLS) and cryo electron microscopy (Cryo‐EM) imaging. Atomic force microscopy (AFM) is used to characterize the mechanical properties of CM‐Liposomes and confirm bilayer deposition on silica cores. Finally, uptake by leukemic cells is determined. No difference in uptake is found between soft CM‐Liposomes and liposomes of the same composition without membrane components. Instead, after deposition on a rigid core, uptake is higher for the cell membrane particles. Preliminary results on primary cells from leukemia patients confirm this observation. These results show that nanoparticle rigidity strongly affects the interaction between cell membrane nanoparticles and the targeted cells.

Trehalose has sparked considerable interest in a variety of pharmaceutical applications as well as in cryopreservation. Recently, there have been growing efforts in the development of trehalose delivery nanocarriers to address the issue of the poor bioavailability of trehalose. The majority of the strategies comprise physical entrapment of trehalose, since its covalent, yet biolabile, conjugation is challenging. Here, we present research on trehalose-releasing nanogels, in which covalent, yet biolabile, conjugation of trehalose was achieved through the co-incorporation of trehalose (meth)acrylate(s) together with hydrophilic primary/secondary acrylamides in one polymeric network. In this case, the primary and secondary amide groups participated in ester hydrolysis in the (meth)acrylate units, making the hydrolysis feasible under physiologically relevant conditions. A set of nanogels with precisely selected compositions were synthesized, characterized, and then studied to evaluate the influence of various structural and environmental factors on the release rate of trehalose. The study also provides insights into some other aspects that are important in view of potential biomedical applications, including specific interactions of nanogels through their terminal α-d-glucopyranosyl moieties from pendant trehalose, protein corona formation, and cellular uptake.