RESUMO
The current treatments for wounds often fail to induce adequate healing, leaving wounds vulnerable to persistent infections and development of drug-resistant microbial biofilms. New natural-derived nanoparticles were studied to impair bacteria colonization and hinder the formation of biofilms in wounds. The nanoparticles were fabricated through polyelectrolyte complexation of chitosan (CS, polycation) and hyaluronic acid (HA, polyanion). UV-induced photo-crosslinking was used to enhance the stability of the nanoparticles. To achieve this, HA was methacrylated (HAMA, degree of modification of 20 %). Photo-crosslinked nanoparticles obtained from HAMA and CS had a diameter of 478 nm and a more homogeneous size distribution than nanoparticles assembled solely through complexation (742 nm). The nanoparticles were loaded with the antimicrobial agent bacitracin (BC), resulting in nanoparticles with a diameter of 332 nm. The encapsulation of BC was highly efficient (97 %). The BC-loaded nanoparticles showed significant antibacterial activity against gram-positive bacteria Staphylococcus aureus, Methicillin-resistant S. aureus and S. epidermidis. Photo-crosslinked HAMA/CS nanoparticles loaded with BC demonstrated inhibition of biofilm formation and a positive effect on the proliferation of mammalian cells (L929). These crosslinked nanoparticles have potential for the long-term treatment of wounds and controlled antibiotic delivery at the location of a lesion.
RESUMO
The cholecystokinin type 2 receptor (CCK2-R) represents an ideal target for cancer therapy since it is overexpressed in several tumors and is associated with poor prognosis. Nastorazepide (Z-360), a selective CCK2-R antagonist, has been widely investigated as a CCK2-R ligand for targeted therapy; however, its high hydrophobicity may represent a limit to cell selectivity and optimal in vivo biodistribution. Here, we present three new fluorescent Z-360 derivatives (IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho) in which nastorazepide was linked, through spacers bearing different saccharides (glucose (G), lactose (L), and maltotriose (M)), to sulforhodamine B. A fourth compound (IP-002H-Rho) with no pendant sugar was also synthesized as a control. Through two-dimensional (2D) and three-dimensional (3D) in vitro studies, we evaluated the compound association with and selectivity for CCK2-R-overexpressing cells (A431-CCK2-R+) vs CCK2-R-underexpressing cells (A431 WT). 2D in vitro studies highlighted a progressive increase of IP-002x-Rho association with A431-CCK2-R+ cells according to the linker hydrophilicity, that is, maltotriose > lactose > glucose > hydrogen, with IP-002M-Rho showing a 2.4- and a 1.36-fold higher uptake than IP-002G-Rho and IP-002L-Rho, respectively. Unexpectedly, IP-002H-Rho showed a similar cell association to that of IP-002L-Rho but with no difference between the two tested cell lines. On the contrary, association with A431-CCK2-R+ cells as compared to the A431 WT was found to be 1.08-, 1.14-, and 1.37-fold higher for IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho, respectively, proving IP-002M-Rho to be the best-performing compound, as also confirmed by competition studies. Trafficking studies on A431-CCK2-R+ cells incubated with IP-002M-Rho suggested the coexistence of receptor-mediated endocytosis and simple diffusion. On the contrary, a high and selective uptake of IP-002M-Rho by A431-CCK2-R+ cells only was observed on 3D scaffolds embedded with cells, underlining the importance of 3D models in in vitro preliminary evaluation.
Assuntos
Receptor de Colecistocinina B , Humanos , Receptor de Colecistocinina B/antagonistas & inibidores , Receptor de Colecistocinina B/metabolismo , Linhagem Celular Tumoral , Trissacarídeos/química , Lactose/análogos & derivados , Lactose/química , Glucose/metabolismoRESUMO
The treatment of bone defects is a clinical challenge. Bone tissue engineering is gaining interest as an alternative to current treatments, with the development of 3D porous structures (scaffolds) helpful in promoting bone regeneration by ensuring temporary functional support. In this work, methacrylated silk fibroin (SilMA) sponges were investigated as scaffolds for bone tissue engineering by exploiting the combination of physical (induced by NaCl salt during particulate leaching) and chemical crosslinking (induced by UV-light exposure) techniques. A biomimetic approach was adopted to better simulate the extracellular matrix of the bone by introducing either natural (mussel shell-derived) or synthetic-origin hydroxyapatite nanoparticles into the SilMA sponges. The obtained materials were characterized in terms of pore size, water absorption capability and mechanical properties to understand both the effect of the inclusion of the two different types of nanoparticles and the effect of the photocrosslinking. Moreover, the SilMA sponges were tested for their bioactivity and suitability for bone tissue engineering purposes by using osteosarcoma cells, studying their metabolism by an AlamarBlue assay and their morphology by scanning electron microscopy. Results indicate that photocrosslinking helps in obtaining more regular structures with bimodal pore size distributions and in enhancing the stability of the constructs in water. Moreover, the addition of naturally derived hydroxyapatite was observed to be more effective at activating osteosarcoma cell metabolism than synthetic hydroxyapatite, showing a statistically significant difference in the AlamarBlue measurement on day 7 after seeding. The methacrylated silk fibroin/hydroxyapatite nanocomposite sponges developed in this work were found to be promising tools for targeting bone regeneration with a sustainable approach.