Poly(methyl methacrylate) with Oleic Acid as an Efficient Candida albicans Biofilm Repellent.

Poly(methyl methacrylate) (PMMA), widely used in dentistry, is unfortunately a suitable substrate for Candida (C.) albicans colonization and biofilm formation. The key step for biofilm formation is C. albicans ability to transit from yeast to hypha (filamentation). Since oleic acid (OA), a natural compound, prevents filamentation, we modified PMMA with OA aiming the antifungal PMMA_OA materials. Physico-chemical properties of the novel PMMA_OA composites obtained by incorporation of 3%, 6%, 9%, and 12% OA into PMMA were characterized by Fourier-transform infrared spectroscopy and water contact angle measurement. To test antifungal activity, PMMA_OA composites were incubated with C. albicans and the metabolic activity of both biofilm and planktonic cells was measured with a XTT test, 0 and 6 days after composites preparation. The effect of OA on C. albicans morphology was observed after 24 h and 48 h incubation in agar loaded with 0.0125% and 0.4% OA. The results show that increase of OA significantly decreased water contact angle. Metabolic activity of both biofilm and planktonic cells were significantly decreased in the both time points. Therefore, modification of PMMA with OA is a promising strategy to reduce C. albicans biofilm formation on denture.

Hexosomes with Undecylenic Acid Efficient against Candida albicans.

Due to the growing issues with fungal infections, especially with Candida, there is still a need to develop novel anti-Candida materials. One of the known antifungal agents is undecylenic acid (UA), which still cannot be efficiently used due to its oily nature, and thus limited solubility. By taking advantage of the properties of UA, we developed an emulsion with hexagonal phase, i.e., hexosomes, whose structure and morphology was studied by small-angle X-ray scattering and cryo-electron microscopy, respectively. The presence of UA in the hexosome was confirmed by spectroscopy. Moreover, we studied the anti-Candida effect of hexosomes and their cytotoxicity toward human cells. The minimal inhibitory concentration for the 50% and 90% Candida-growth reduction was found at 0.01 and 0.16 wt % hexosomes, respectively (i.e., 2 and 32 pghex/C.a.cell, respectively). The percentage of metabolically active Candida was reduced by 72-96% at hexosome concentrations of 1.0-8.2 pghex/C.a.cell as compared to untreated Candida. Furthermore, at the same concentration range the embedded filamentation test after 24 and 48 h showed the inhibition of both the filamentation and growth of Candida, while the preliminary toxicity test showed that hexosomes were nontoxic for human cells. All these render the here-developed hexosomes with UA efficient and promising anti-Candida agents.

Fungicidal PMMA-Undecylenic Acid Composites.

Undecylenic acid (UA), known as antifungal agent, still cannot be used to efficiently modify commercial dental materials in such a way that this affects Candida. Actually, issues with Candida infections and fungal resistance compromise the use of Poly(methyl-methacrylate) (PMMA) as dental material. The challenge remains to turn PMMA into an antifugal material, which can ideally affect both sessile (attached) and planktonic (free-floating) Candida cells. We aimed to tackle this challenge by designing PMMA-UA composites with different UA concentrations (3-12%). We studied their physico-chemical properties, the antifungal effect on Candida and the cytotoxicity toward human cells. We found that UA changes the PMMA surface into a more hydrophilic one. Mainly, as-preparation composites with ≥6% UA reduced sessile Candida for >90%. After six days, the composites were still efficiently reducing the sessile Candida cells (for ~70% for composites with ≥6% UA). Similar results were recorded for planktonic Candida. Moreover, the inhibition zone increased along with the UA concentration. The antifungal effect of UA was also examined at the surface of an UA-loaded agar and the minimal inhibitory concentration (MIC90) was below the lowest-studied 0.0125% UA. Furthermore, the embedded filamentation test after 24 h and 48 h showed complete inhibition of the Candida growth at 0.4% UA.

Folic acid on iron oxide nanoparticles: platform with high potential for simultaneous targeting, MRI detection and hyperthermia treatment of lymph node metastases of prostate cancer.

The overexpression of the folate receptor in most cancers has been widely exploited to specifically deliver folic acid (FA) coupled nanomedicines to tumors. However, complex coupling chemistry is often used to bind FA to the nanoparticles. Furthermore, very little has been reported for the targeting of nanomedicines to lymph node metastases (LNMs) of prostate cancer. We here report the simple and aqueous coating of iron oxide nanoparticles (IONPs) with FA for theranostics of LNMs of prostate cancer. FA was directly bound to the IONPs' surface without the use of any linker, simultaneously playing the role of the coating molecule and targeting agent. We measured for FA-IONPs a hydrodynamic diameter around 100 nm and a negative surface charge, what is needed to access and to be retained in the lymphatic system for the LNMs targeting. We also show that FA-IONPs are specifically uptaken by prostate cancer cells expressing the prostate specific membrane antigen, including LNMs cells. FA-IONPs also displayed both high relaxivity for MRI detection and high specific absorption rate needed for hyperthermia treatment of tumors. Our study provides a theranostic platform for targeting LNMs of prostate cancer with high potential for their detection by MRI and treatment by hyperthermia.

Tuning Properties of Iron Oxide Nanoparticles in Aqueous Synthesis without Ligands to Improve MRI Relaxivity and SAR.

Aqueous synthesis without ligands of iron oxide nanoparticles (IONPs) with exceptional properties still remains an open issue, because of the challenge to control simultaneously numerous properties of the IONPs in these rigorous settings. To solve this, it is necessary to correlate the synthesis process with their properties, but this correlation is until now not well understood. Here, we study and correlate the structure, crystallinity, morphology, as well as magnetic, relaxometric and heating properties of IONPs obtained for different durations of the hydrothermal treatment that correspond to the different growth stages of IONPs upon initial co-precipitation in aqueous environment without ligands. We find that their properties were different for IONPs with comparable diameters. Specifically, by controlling the growth of IONPs from primary to secondary particles firstly by colloidal and then also by magnetic interactions, we control their crystallinity from monocrystalline to polycrystalline IONPs, respectively. Surface energy minimization in the aqueous environment along with low temperature treatment is used to favor nearly defect-free IONPs featuring superior properties, such as high saturation magnetization, magnetic volume, surface crystallinity, the transversal magnetic resonance imaging (MRI) relaxivity (up to r2 = 1189 mM-1·s-1 and r2/r1 = 195) and specific absorption rate, SAR (up to 1225.1 W·gFe-1).

Versatility of Pyridoxal Phosphate as a Coating of Iron Oxide Nanoparticles.

Pyridoxal 5'-phosphate (PLP) is the most important cofactor of vitamin B6-dependent enzymes, which catalyses a wide range of essential body functions (e.g., metabolism) that could be exploited to specifically target highly metabolic cells, such as tumour metastatic cells. However, the use of PLP as a simultaneous coating and targeting molecule, which at once provides colloidal stability and specific biological effects has not been exploited so far. Therefore, in this work iron oxide nanoparticles (IONPs) were coated by PLP at two different pH values to tune PLP bonding (e.g., orientation) at the IONP surface. The surface study, as well as calculations, confirmed different PLP bonding to the IONP surface at these two pH values. Moreover, the obtained PLP-IONPs showed different zeta potential, hydrodynamic radius and agglomeration state, and consequently different uptake by two metastatic-prostate-cancer cell lines (LnCaP and PC3). In LnCaP cells, PLP modified the morphology of IONP-containing intracellular vesicles, while in PC3 cells PLP impacted the amount of IONPs taken up by cells. Moreover, PLP-IONPs displayed high magnetic resonance imaging (MRI) r2 relaxivity and were not toxic for the two studied cell lines, rendering PLP promising for biomedical applications. We here report the use of PLP simultaneously as a coating and targeting molecule, directly bound to the IONP surface, with the additional high potential for MRI detection.

Methods of protein corona isolation for magnetic nanoparticles.

Nanoparticles (NPs) in contact with a biological environment get covered by proteins and some are loosely bound and some are tightly bound. The latter form a hard protein corona (HPC) which is known to determine their biological behavior. Therefore, in order to study the biological behaviour of NPs one needs to start from the HPC. However, established methods and standards of HPC isolation are still not known. This is especially a challenge in the case of magnetic NPs which form a major branch of nanomedicine. Therefore, we developed a novel HPC isolation method, a multi-step centrifugation method (MSCM), for single-domain magnetic NPs. The MSCM was applied to iron oxide NPs in interaction with human blood and lymph serum with different dilutions in triplicate. The analysis of the composition of the obtained HPCs showed the reproducibility of the MSCM. This new method was also compared with the existing magnetic separation method (MagSep) and a study of the obtained HPC allowed us to establish the validity limits of MagSep and MSCM on only superparamagnetic NPs and on any single-domain magnetic NPs, respectively. Surprisingly, the HPCs obtained by these two isolation methods were quite different, up to 50%, suggesting that only these proteins, which are found in the HPCs of both isolation methods, are in fact real HPCs.

Assessment of nanoparticles' safety: corrected absorbance-based toxicity test.

We developed a method to correct absorbance-based toxicity tests to remove the influence of the deposited dose of nanoparticles. As a model of absorbance-based toxicity tests, we chose the frequently used MTS test. The corrected cell viabilities obtained with the MTS test matched the results of the fluorescence-based PrestoBlue® test.

Protein Corona: Impact of Lymph Versus Blood in a Complex In Vitro Environment.

In biological environments, the surface of nanoparticles (NPs) are modified by protein corona (PC) that determines their biological behavior. Unfortunately, in vitro tests still give different PC than in vivo tests causing in vitro-in vivo discrepancy; hence, in vitro studies are not indicative for the NPs' behavior in vivo. Here is demonstrated that PC in vitro is strongly influenced by the type of extracellular fluid (ECF), blood or lymph, by their high and low flow conditions and transitions between ECFs, and a combination of these parameters. As a result, this in vitro study approaches fluidic and dynamic variations to which NPs are exposed in vivo: different ECF that NPs encounter first in different injection routes, different transitions in-between ECFs during circulation, and simultaneous change in the exposed flow in these transitions. The most-abundant proteins in PCs are found to be not the most abundant in ECFs, but those having high affinity for binding to the surface of NPs. Moreover, some proteins are differently abundant in PCs at different flows, which indicate force-promoted binding, catch bonds. These results suggest that future in vitro studies should consider more complex incubation conditions to improve the in vitro-in vivo consistency necessary for translational research.