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)

Nano-in-micro-particles consisting of PLGA nanoparticles embedded in chitosan microparticles via spray-drying enhances their uptake in the olfactory mucosa

Intranasal delivery has gained prominence since 1990, when the olfactory mucosa was recognized as the window to the brain and the central nervous system (CNS); this has enabled the direct site specific targeting of neurological diseases for the first time. Intranasal delivery is a promising route because general limitations, such as the blood-brain barrier (BBB) are circumvented. In the treatment of multiple sclerosis (MS) or Alzheimer’s disease, for example, future treatment prospects include specialized particles as delivery vehicles. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are well known as promising delivery systems, especially in the area of nose-to-brain (N2B) delivery. Chitosan is also broadly known as a functional additive due to its ability to open tight junctions. In this study, we produced PLGA nanoparticles of different sizes and revealed for the first time their size-time-dependent uptake mechanism into the lamina propria of porcine olfactory mucosa. The intracellular uptake was observed for 80 and 175 nm within only 5 min after application to the epithelium. After 15 min, even 520 nm particles were detected, associated with nuclei. Especially the presence of only 520 nm particles in neuronal fibers is remarkable, implying transcellular and intracellular transport via the olfactory or the trigeminal nerve to the brain and the CNS. Additionally, we developed successfully specialized Nano-in-Micro particles (NiMPs) for the first time via spray drying, consisting of PLGA nanoparticles embedded into chitosan microparticles, characterized by high encapsulation efficiencies up to 51%, reproducible and uniform size distribution, as well as smooth surface. Application of NiMPs accelerated the uptake compared to purely applied PLGA nanoparticles. NiMPs were spread over the whole transverse section of the olfactory mucosa within 15 min. Faster uptake is attributed to additional paracellular transport, which was examined via tight-junction-opening. Furthermore, a separate chitosan penetration gradient of ∼150 µm caused by dissociation from PLGA nanoparticles was observed within 15 min in the lamina propria, which was demonstrated to be proportional to an immunoreactivity gradient of CD14. Due to the beneficial properties of the utilized chitosan-derivative, regarding molecular weight (150-300 kDa), degree of deacetylation (80%), and particle size (0.1-10 µm) we concluded that M2-macrophages herein initiated an anti-inflammatory reaction, which seems to already take place within 15 min following chitosan particle application. In conclusion, we demonstrated the possibility for PLGA nanoparticles, as well as for chitosan NiMPs, to take all three prominent intranasal delivery pathways to the brain and the CNS; namely transcellular, intracellular via neuronal cells, and paracellular transport.European Union’s Horizon 2020 research and innovation programm

Nanoparticle preparation process using novel microjet reactor technology for enhancing dissolution rates of poorly water soluble drugs

In this study a novel method MicroJet reactor technology was developed to enable the custom preparation of nanoparticles. rnDanazol/HPMCP HP50 and Gliclazide/Eudragit S100 nanoparticles were used as model systems for the investigation of effects of process parameters and microjet reactor setup on the nanoparticle properties during the microjet reactor construction. rnFollowing the feasibility study of the microjet reactor system, three different nanoparticle formulations were prepared using fenofibrate as model drug. Fenofibrate nanoparticles stabilized with poloxamer 407 (FN), fenofibrate nanoparticles in hydroxypropyl methyl cellulose phthalate (HPMCP) matrix (FHN) and fenofibrate nanoparticles in HPMCP and chitosan matrix (FHCN) were prepared under controlled precipitation using MicroJet reactor technology. Particle sizes of all the nanoparticle formulations were adjusted to 200-250 nm. rnThe changes in the experimental parameters altered the system thermodynamics resulting in the production of nanoparticles between 20-1000 nm (PDI<0.2) with high drug loading efficiencies (96.5% in 20:1 polymer:drug ratio).rnDrug releases from all nanoparticle formulations were fast and complete after 15 minutes both in FaSSIF and FeSSIF medium whereas in mucodhesiveness tests, only FHCN formulation was found to be mucoadhesive. Results of the Caco-2 studies revealed that % dose absorbed values were significantly higher (p<0.01) for FHCN in both cases where FaSSIF and FeSSIF were used as transport buffer.rnIn der vorliegenden Arbeit wurde eine neuartige Methode entwickelt - die Mikrojetreaktor-Technologie - um die spezifische Herstellung von Nanopartikeln zu ermöglichen. rnDanzol/HPMCP HP50 und Gliclazid/Eudragit S100 Nanopartikel wurden als Modellsysteme für die Untersuchung des Einflusses der verschiedenen Prozess- und konstruktionsbedingten Parameter am Mikrojetreaktor auf die Eigenschaften der Nanopartikel genutzt. rnNach Abschluss einer initialen Machbarkeitsstudie am Mikrojetreaktorsystem wurden drei verschiedene Nanopartikelformulierungen unter Verwendung des Modellwirkstoffes Fenofibrat durch kontrollierte Fällung im Mikrojetreaktor hergestellt. Dies waren Fenofibrat Nanopartikel, stabilisiert mit Poloxamer 407 (FN), Fenofibrat Nanopartikel in einer Hydroxypropylmethylcellulosephthalat (HPMCP) Matrix (FHN) und Fenofibrat Nanopartikel in einer HPMCP/Chitosan Matrix (FHCN). Bei allen Formulierungen konnte die Partikelgröße auf 200-250 nm eingestellt werden. rnVariationen der experimentellen Parameter resultierten in thermodynamischen Änderungen des Systems, welche die Herstellung von Nanopartikeln zwischen 20-1000nm (PDI <0,2) mit hohen Wirkstoffbeladungen (96,5% bei einem Polymer:Wirkstoff Verhältnis von 20:1).rnFenofibrat Formulierungen zeichneten sich durch eine schnelle und vollständige Wirkstofffreisetzung in FaSSIF und FeSSIF Medien innerhalb von 15 Minuten aus. Die Mucoadhäsionstests ergaben nur für die FHCN Formulierung eine Mucoadhäsion. Die Caco-2 Studien zeigten sowohl bei der Nutzung von FaSSIF als auch FeSSIF Medien als Transportpuffersystem für FHCN signifikant höhere (p<0,01) prozentuale Absorptionswerte. r

K5 tipi öldürücü maya toksik proteinin yüksek miktarlarda üretilmesi ve saflaştırılması, insan ve bitki patojenik mantarları üzerindeki inhibasyon etkisinin tayini ve endüstriyel amaçlı kullanım için formülasyonunun geliştirilmesi

TÜBİTAK TBAG01.12.200

Screening of Surfactants for Improved Delivery of Antimicrobials and Poly-Lactic-co-Glycolic Acid Particles in Wound Tissue

Topical wound management is often a challenge due to the poor penetration of antimicrobials in wound tissue and across the biofilm matrix where bacteria are embedded. Surfactants have been used for decades to improve the stability of formulations, increase drug solubility, and enhance penetration. In this study, we screened different detergents with respect to their cytotoxicity and their ability to improve the penetration of poly-lactic-co-glycolic acid (PLGA) particles in wound tissue. Among the tested surfactants, Kolliphor SLS and Tween 80 increased the penetration of PLGA particles and had a limited cytotoxicity. Then, these surfactants were used to formulate PLGA particles loaded with the poorly water-soluble antibiotic ciprofloxacin. The antimicrobial efficacy of the formulations was tested in a wound infection model based on human ex vivo skin. We found that even though PLGA particles had the same antimicrobial efficiency than the particle-free drug formulation, thanks to their solubilizing and anti-biofilm properties, the surfactants remarkably improved the antimicrobial activity of ciprofloxacin with respect to the drug formulation in water. We conclude that the use of Tween 80 in antimicrobial formulations might be a safe and efficient option to improve the topical antimicrobial management of chronic wound infections

Development of a Hot-Melt-Extrusion-Based Spinning Process to Produce Pharmaceutical Fibers and Yarns

Fibers and yarns are part of everyday life. So far, fibers that are also used pharmaceutically have mainly been produced by electrospinning. The common use of spinning oils and the excipients they contain, in connection with production by melt extrusion, poses a regulatory challenge for pharmaceutically usable fibers. In this publication, a newly developed small-scale direct-spinning melt extrusion system is described, and the pharmaceutically useful polyvinyl filaments produced with it are characterized. The major parts of the system were newly developed or extensively modified and manufactured cost-effectively within a short time using rapid prototyping (3D printing) from various materials. For example, a stainless-steel spinneret was developed in a splice design for a table-top melt extrusion system that can be used in the pharmaceutical industry. The direct processing of the extruded fibers was made possible by a spinning system developed called Spinning-Rosi, which operates continuously and directly in the extrusion process and eliminates the need for spinning oils. In order to prevent instabilities in the product, further modifications were also made to the process, such as a the moisture encapsulation of the melt extrusion line at certain points, which resulted in a bubble-free extrudate with high tensile strength, even in a melt extrusion line without built-in venting

Ciprofloxacin-loaded PLGA nanoparticles against Cystic Fibrosis P. aeruginosa Lung Infections.

Current pulmonary treatments against Pseudomonasaeruginosa 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±28.6 nm 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 Calu3 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