Higher Loading of Gold Nanoparticles in PAD Mesenchymal-like Stromal Cells Leads to a Decreased Exocytosis.

Cell therapy is an important new method in medicine and is being used for the treatment of an increasing number of diseases. The challenge here is the precise tracking of cells in the body and their visualization. One method to visualize cells more easily with current methods is their labeling with nanoparticles before injection. However, for a safe and sufficient cell labeling, the nanoparticles need to remain in the cell and not be exocytosed. Here, we test a glucose-PEG-coated gold nanoparticle for the use of such a cell labeling. To this end, we investigated the nanoparticle exocytosis behavior from PLX-PAD cells, a cell type currently in clinical trials as a potential therapeutic agent. We showed that the amount of exocytosed gold from the cells was influenced by the uptake time and loading amount. This observation will facilitate the safe labeling of cells with nanoparticles in the future and contribute to stem cell therapy research.

High-Throughput Method for the Simultaneous Determination of Doxorubicin Metabolites in Rat Urine after Treatment with Different Drug Nanoformulations.

Doxorubicin (DOX) is one of the most effective cytotoxic agents against malignant diseases. However, the clinical application of DOX is limited, due to dose-related toxicity. The development of DOX nanoformulations that significantly reduce its toxicity and affect the metabolic pathway of the drug requires improved methods for the quantitative determination of DOX metabolites with high specificity and sensitivity. This study aimed to develop a high-throughput method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for the quantification of DOX and its metabolites in the urine of laboratory animals after treatment with different DOX nanoformulations. The developed method was validated by examining its specificity and selectivity, linearity, accuracy, precision, limit of detection, and limit of quantification. The DOX and its metabolites, doxorubicinol (DOXol) and doxorubicinone (DOXon), were successfully separated and quantified using idarubicin (IDA) as an internal standard (IS). The linearity was obtained over a concentration range of 0.05-1.6 µg/mL. The lowest limit of detection and limit of quantitation were obtained for DOXon at 5.0 ng/mL and 15.0 ng/mL, respectively. For each level of quality control (QC) samples, the inter- and intra-assay precision was less than 5%. The accuracy was in the range of 95.08-104.69%, indicating acceptable accuracy and precision of the developed method. The method was applied to the quantitative determination of DOX and its metabolites in the urine of rats treated by novel nanoformulated poly(lactic-co-glycolic acid) (DOX-PLGA), and compared with a commercially available DOX solution for injection (DOX-IN) and liposomal-DOX (DOX-MY).

Testing of aerosolized ciprofloxacin nanocarriers on cystic fibrosis airway cells infected with P. aeruginosa biofilms.

The major pathogen found in the lungs of adult cystic fibrosis (CF) patients is Pseudomonas aeruginosa, which builds antibiotic-resistant biofilms. Pulmonary delivery of antibiotics by inhalation has already been proved advantageous in the clinic, but the development of novel anti-infective aerosol medicines is complex and could benefit from adequate in vitro test systems. This work describes the first in vitro model of human bronchial epithelial cells cultivated at the air-liquid interface (ALI) and infected with P. aeruginosa biofilm and its application to demonstrate the safety and efficacy of aerosolized anti-infective nanocarriers. Such a model may facilitate the translation of novel therapeutic modalities into the clinic, reducing animal experiments and the associated problems of species differences. A preformed biofilm of P. aeruginosa PAO1 was transferred to filter-grown monolayers of the human CF cell line (CFBE41o-) at ALI and additionally supplemented with human tracheobronchial mucus. This experimental protocol provides an appropriate time window to deposit aerosolized ciprofloxacin-loaded nanocarriers at the ALI. When applied 1 h post-infection, the nanocarriers eradicated all planktonic bacteria and reduced the biofilm fraction of the pathogen by log 6, while CFBE41o- viability and barrier properties were maintained. The here described complex in vitro model approach may open new avenues for preclinical safety and efficacy testing of aerosol medicines against P. aeruginosa lung infection.

Microparticle formulations alter the toxicity of fenofibrate to the zebrafish Danio rerio embryo.

A wide variety of active pharmaceutical ingredients are released into the environment and pose a threat to aquatic organisms. Drug products using micro- and nanoparticle technology can lower these emissions into the environment by their increased bioavailability to the human patients. However, due to this enhanced efficacy, micro- and nanoscale drug delivery systems can potentially display an even higher toxicity, and thus also pose a risk to non-target organisms. Fenofibrate is a lipid-regulating agent and exhibits species-related hazards in fish. The ecotoxic effects of a fenofibrate formulation embedded into a hydroxypropyl methylcellulose microparticle matrix, as well as those of the excipients used in the formulation process, were evaluated. To compare the effects of fenofibrate without a formulation, fenofibrate was dispersed in diluted ISO water alone or dissolved in the solvent DMF and then added to diluted ISO water. The effects of these various treatments were assessed using the fish embryo toxicity test, acridine orange staining and gene expression analysis assessed by quantitative RT polymerase chain reaction. Exposure concentrations were assessed by chemical analysis. The effect threshold concentrations of fenofibrate microparticle precipitates were higher compared to the formulation. Fenofibrate dispersed in 20%-ISO-water displayed the lowest toxicity. For the fenofibrate formulation as well as for fenofibrate added as a DMF solution, greater ecotoxic effects were observed in the zebrafish embryos. The chemical analysis of the solutions revealed that more fenofibrate was present in the samples with the fenofibrate formulation as well as fenofibrate added as a DMF solution compared to fenofibrate dispersed in diluted ISO water. This could explain the higher ecotoxicity. The toxic effects on the zebrafish embryo thus suggested that the formulation as well as the solvent increased the bioavailability of fenofibrate.

Ciprofloxacin-loaded polymeric nanoparticles incorporated electrospun fibers for drug delivery in tissue engineering applications.

Presented work focuses on the development of biodegradable polymer nanoparticles loaded with antibiotics as drug delivery systems deposited on electrospun scaffolds for tissue engineering. The innovative ciprofloxacin-loaded poly(DL-lactide-co-glycolide) NPs ensure a continuous slow release and high local concentration at the site of action for an optimal therapy. The local delivery of antibiotics as an integrated part of electrospun scaffolds offers an effective, safe, and smart enhancement supporting tissue regeneration. Presented data provides solid scientific evidence for fulfilling the requirements of local nano antibiotic delivery systems with biodegradability and biocompatibility for a wide range of tissue engineering applications, including middle ear tissues (e.g., tympanic membranes) which are subject to bacterial infections. Further characterization of such systems, including in vivo studies, is required to ensure successful transfer from lab to clinical applications. Graphical abstract .

Evaluation of potential environmental toxicity of polymeric nanomaterials and surfactants.

Nanomaterials have gained huge importance in various fields including nanomedicine. Nanoformulations of drugs and nanocarriers are used to increase pharmaceutical potency. However, it was seen that polymeric nanomaterials can cause negative effects. Thus, it is essential to identify nanomaterials with the least adverse effects on aquatic organisms. To determine the toxicity of polymeric nanomaterials, we investigated the effects of poly(lactic-co-glycolid) acid (PLGA), Eudragit® E 100 and hydroxylpropyl methylcellulose phthalate (HPMCP) on zebrafish embryos using the fish embryo toxicity test (FET). Furthermore, we studied Cremophor® RH40, Cremophor® A25, Pluronic® F127 and Pluronic® F68 applied in the generation of nanoformulations to identify the surfactant with minimal toxic impact. The order of ecotoxicty was HPMCP < PLGA < Eudragit® E100 and Pluronic® F68 < Pluronic® F127 < Cremophor® RH40 < Cremophor® A25. In summary, HPMCP and Pluronic® F68 displayed the least toxic impact, thus suggesting adequate environmental compatibility for the generation of nanomedicines.

Ciprofloxacin-loaded PLGA nanoparticles against cystic fibrosis P. aeruginosa lung infections.

Current pulmonary treatments against Pseudomonas aeruginosa infections in cystic fibrosis (CF) lung suffer from deactivation of the drug and immobilization in thick and viscous biofilm/mucus blend, along with the general antibiotic resistance. Administration of nanoparticles (NPs) with high antibiotic load capable of penetrating the tight mesh of biofilm/mucus can be an advent to overcome the treatment bottlenecks. Biodegradable and biocompatible polymer nanoparticles efficiently loaded with ciprofloxacin complex offer a solution for emerging treatment strategies. NPs were prepared under controlled conditions by utilizing MicroJet Reactor (MJR) to yield a particle size of 190.4±28.6nm with 0.089 PDI. Encapsulation efficiency of the drug was 79% resulting in a loading of 14%. Release was determined to be controlled and medium-independent in PBS, PBS+0.2% Tween 80 and simulated lung fluid. Cytotoxicity assays with Calu-3 cells and CF bronchial epithelial cells (CFBE41o-) indicated that complex-loaded PLGA NPs were non-toxic at concentrations ≫ MICcipro against lab strains of the bacteria. Antibacterial activity tests revealed enhanced activity when applied as nanoparticles. NPs' colloidal stability in mucus was proven. Notably, a decrease in mucus turbidity was observed upon incubation with NPs. Herewith, ciprofloxacin complex-loaded PLGA NPs are introduced as promising pulmonary nano drug delivery systems against P.aeruginosa infections in CF lung.

Optimization of ciprofloxacin complex loaded PLGA nanoparticles for pulmonary treatment of cystic fibrosis infections: Design of experiments approach.

Design of Experiments (DoE) is a powerful tool for systematic evaluation of process parameters' effect on nanoparticle (NP) quality with minimum number of experiments. DoE was employed for optimization of ciprofloxacin loaded PLGA NPs for pulmonary delivery against Pseudomonas aeruginosa infections in cystic fibrosis (CF) lungs. Since the biofilm produced by bacteria was shown to be a complicated 3D barrier with heterogeneous meshes ranging from 100nm to 500nm, nanoformulations small enough to travel through those channels were assigned as target quality. Nanoprecipitation was realized utilizing MicroJet Reactor (MJR) technology based on impinging jets principle. Effect of MJR parameters flow rate, temperature and gas pressure on particle size and PDI was investigated using Box-Behnken design. The relationship between process parameters and particle quality was demonstrated by constructed fit functions (R2=0.9934 p<0.0001 and R2=0.9983 p<0.0001, for particle size and PDI, respectively). Prepared nanoformulations varied between 145.2 and 979.8nm with PDI ranging from 0.050 to 1.00 and showed encapsulation efficiencies >65%. Response surface plots provided experimental data-based understanding of MJR parameters' effect, thus NP quality. Presented work enables ciprofloxacin loaded PLGA nanoparticle preparations with pre-defined quality to fulfill the requirements of local drug delivery under CF disease conditions.

Eco-friendly fabrication of 4% efficient organic solar cells from surfactant-free P3HT:ICBA nanoparticle dispersions.

Photo-active layers from non-stabilized P3HT:ICBA nanoparticles enable the fabrication of inverted organic solar cells from eco-friendly, alcoholic dispersions. Exhibiting power conversion efficiencies (PCEs) ≈4%, the devices are competitive to state-of-the-art P3HT:ICBA solar cells from chlorinated solvents. Upon thermal annealing, the short circuit current density and consequently the PCE of the inverted solar cells improve radically due to a more intimate contact of the nanoparticles and hence an enhanced charge carrier extraction.

In vitro susceptibilities of Candida spp. to Panomycocin, a novel exo-beta-1,3-glucanase isolated from Pichia anomala NCYC 434.

Panomycocin, the killer toxin of Pichia anomala NCYC 434 (K5), is a 49 kDa monomeric glycoprotein with exo-beta-1,3-glucanase activity (patent pending). In this study we evaluated the in vitro activity of panomycocin against a panel of 109 human isolates of seven different pathogenic Candida spp. using microdilution and time-kill methods. Panomycocin was most active against C. tropicalis, C. pseudotropicalis and C. glabrata with MIC(90) values of 1 microg/ml. It displayed significant activity against C. albicans and C. parapsilosis with MIC(90) values of 4 and 2 microg/ml, respectively. For C. krusei, the MIC(90) value was 8 microg/ml. Panomycocin was fungicidal against all the tested Candida spp. The MFC values were only one or 2 dilutions higher than the MICs with the exception of C. krusei isolates with MFCs greater than or equal to 4xMIC. Results of this study indicated that panomycocin could be considered as a natural antifungal agent against Candida infections and has significant potential for further investigation.

In vitro activity of panomycocin, a novel exo-beta-1,3-glucanase isolated from Pichia anomala NCYC 434, against dermatophytes.

Killer proteins that are produced and secreted into the environment by certain yeast strains are considered as promising antifungal agents. In this study, in vitro activity of Pichia anomala NCYC 434 (K5) killer protein, panomycocin, which is a 49 kDa glycoprotein with an exo-beta-1,3-glucanase activity was tested against 41 isolates of dermatophytes. Minimum inhibitory concentrations (MICs) were determined by a broth microdilution method based on the reference document M38-A of Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS). For panomycocin MIC determinations two end point criteria MIC-2 (prominent growth inhibition) and MIC-0 (complete growth inhibition) were recorded. All the tested isolates (Microsporum spp. and Trichophyton spp.) were found susceptible to panomycocin. The MIC-2 values ranged from 0.25 to 2 microg ml(-1) and MIC-0 values ranged from 1 to 8 microg ml(-1). These results showed that panomycocin is active in vitro against fungal strains that cause superficial infections and highlighted its probable use as a topical antifungal agent.