Data set for "Development of Covalent Chitosan-Polyethylenimine Derivatives as Gene Delivery Vehicle: Synthesis, Characterization, and Evaluation" Int. J. Mol. Sci. 2021, 22, 3828. DOI: 10.3390/ijms22083828. Raw data for analytical techniques; description of experiments; metadata file.
The need for specific targeting strategies towards hepatocytes stems from the lack of
efficient therapeutic options to treat numerous serious liver diseases. Moreover, various
genetic disorders, such as α1-antitrypsin deficiency and hemophilia A and B, are depending
on an efficient gene delivery to defined cells, such as hepatocytes, preferentially avoiding
viral vectors. Since the asialoglycoprotein receptor is primarily expressed by liver
parenchymal cells, it offers a potential target for a cell specific delivery system.
First, the binding of various vectors was analyzed, using the human hepatocellular carcinoma
cell line HepG2 as an in vitro model. While the uptake of D-galactose as a monomer was
non-specific, the glycoprotein asialofetuin was analyzed as an alternative vector, which
represents the desialated derivative of fetuin, containing multi-antennary galactose-
terminating glycan residues. Next to a pronounced cellular accumulation, the uptake was
markedly inhibited in the presence of an excess of free asialofetuin, indicating specific
endocytosis through the asialoglycoprotein receptor. Therefore, asialofetuin was selected as
an ideal vector for the further development of a drug delivery system targeting liver
parenchymal cells.
Asialofetuin was covalently attached to pegylated liposomes, yielding a highly monodisperse
preparation with a particle size below 100 nm. A subsequently incubation with HepG2 cells
resulted in a specific endocytosis of the vesicles, providing an experimental proof of concept
for targeting hepatocytes in vitro. The delivery and intracellular accumulation in HepG2 cells
were investigated by incorporating various organic dyes and fluorescent semiconductor
nanocrystals, also known as quantum dots, into liposomes. The cellular uptake of
asialofetuin-conjugated liposomes, loaded with quantum dots, resulted in a bright fluorescent
signal, which was impaired by the need for a specific photoactivation prior to fluorescence
analysis. Despite their challenging optical properties, quantum dots are valuable
fluorochromes for further optimization of drug targeting strategies.
Finally, a proof of principle for a hepatocyte specific delivery was provided in vivo, by
intravenously injecting rats with asialofetuin-conjugated and pegylated liposomes, which
were taken up by the liver parenchymal cells. In contrast, accumulation in hepatocytes was
reduced by co-injecting free asialofetuin and conventional liposomes were uniquely engulfed
by Kupffer cells.
Summarized, asialofetuin-conjugated pegylated liposomes represent a novel approach,
combining desialated glycoproteins, which exhibit a high affinity towards the
asialoglycoprotein receptor, with long circulating vesicles, for a specific targeting of liver
parenchymal cells. This concept represents a most promising strategy for a hepatocyte
specific drug delivery system and gives the opportunity for further studies, such as the
isolated utilization of glycans only, to avoid immunogenic reactions.
These targeting strategies can be used to deliver drugs to diseased tissues or organs within
our body. This reflects our interests to modulate the pharmacokinetics of drugs using specific
formulation strategies. Two additional pharmacokinetic investigations of pharmaceutical
relevant substances were published in peer-reviewed journals. One study addresses the risk
of physical drug interactions of ceftriaxone with calcium in human plasma, and the second
one discusses the interaction potential of high doses of resveratrol with various cytochrome
P450 isoenzymes. These studies are presented in the section “Appendix”, to separate them
from the drug targeting approach of hepatocytes using liposomal formulations.
The organic anion transporting polypeptide (OATP) 2B1 is considered an emerging drug transporter that is found expressed in pharmacokinetically relevant organs such as the liver, small intestine, and kidney. Despite its interaction with various substrate drugs, the understanding of its in vivo relevance is still limited. In this study, we first validated the interaction of atorvastatin with rat OATP2B1 using transiently transfected HeLa cells. Moreover, we characterized our rSlco2b1-knockout and SLCO2B1-knockin rats for mRNA, protein expression, and localization of OATP2B1 in the liver, small intestine, and kidney. The transporter showed the highest expression in the liver followed by the small intestine. In humanized rats, human OATP2B1 is localized on the sinusoidal membrane of hepatocytes. In enterocytes of wildtype and humanized rats, the transporter was detected in the luminal membrane with the vast majority being localized subapical. Subsequently, we assessed atorvastatin pharmacokinetics in male wildtype, rSlco2b1-knockout, and SLCO2B1-knockin rats after a single-dose administration (orally and intravenously). Investigating the contribution of rat OATP2B1 or human OATP2B1 to oral atorvastatin pharmacokinetics revealed no differences in neither concentration-time profiles nor pharmacokinetic parameters. However, when comparing the pharmacokinetics of atorvastatin after intravenous administration in SLCO2B1-humanized rats and knockout animals, notable differences were observed. In particular, the systemic exposure (AUC) decreased by approximately 40% in humanized animals, while the clearance (CL) was 57% higher in animals expressing human OATP2B1. These findings indicate that human OATP2B1 influences pharmacokinetics of atorvastatin after intravenous administration, most likely by contributing to the hepatic uptake. Significance StatementWildtype, rSlco2b1-knockout and SLCO2B1-humanized Wistar rats were characterized for the expression of rat and human SLCO2B1/OATP2B1. Pharmacokinetic studies of atorvastatin over 24 hours were conducted in male wildtype, rSlco2b1-knockout, and SLCO2B1-humanized rats. After a single-dose i.v. administration, a lower systemic exposure and an increase in clearance were observed in SLCO2B1-humanized rats compared to knockout animals indicating a contribution of OATP2B1 to the hepatic clearance.
Superparamagnetic iron oxide nanoparticles (SPIONs) are clinically used as diagnostic agents, serving as a contrast media when exposed to a magnetic field. In article number 2000746, Jörg Huwyler and co-workers evaluate synchrotron radiation-based hard X-ray tomography (SRμCT) for the localization of SPIONs in soft tissues. In the cover, the tissue distribution of SPIONs is visualized after intravenous injection in a three-day old zebrafish embryo by SRμCT, with views within the blood vessel looking at the surrounding soft tissue and onto SPIONs engulfed by macrophages. Image: Courtesy of Jan Stephan Bolten.
Macrophage recognition of nanoparticles is highly influenced by particle size and surface modification. Due to the lack of appropriate in vivo screening models, it is still challenging and time-consuming to characterize and optimize nanomedicines regarding this undesired clearance mechanism. Therefore, we validate zebrafish embryos as an emerging vertebrate screening tool to assess the macrophage sequestration of surface modified particulate formulations with varying particle size under realistic biological conditions. Liposomes with different PEG molecular weights (PEG350-PEG5000) at different PEG densities (3.0-10.0 mol%) and particle sizes between 60 and 120 nm were used as a well-established reference system showing various degrees of macrophage uptake. The results of in vitro experiments, zebrafish embryos, and in vivo rodent biodistribution studies were consistent, highlighting the validity of the newly introduced zebrafish macrophage clearance model. We hereby present a strategy for efficient, systematic and rapid nanomedicine optimization in order to facilitate the preclinical development of nanotherapeutics.
Crystalline structures activate the NLRP3 inflammasome, leading to the production of IL-1β, however, the molecular interactions responsible for NLRP3 activation are not fully understood. Cathepsin B release from the ruptured phagolysosome and potassium ion efflux have been suggested to be critical for this activation. Here, we report that Cathepsin B redistribution was not a crucial event in crystal-induced IL-1β production. Silica and monosodium urate crystal-treated macrophages with undisturbed lysosomes demonstrated strong co-localization of ASC and Caspase-1, indicative of NLRP3 inflammasome activation. Importantly, we provided evidence to suggest that macrophage cell membrane binding to immobilized crystals was sufficient to induce IL-1β release, and this activation of the NLRP3 inflammasome was inhibited by blocking potassium efflux. Therefore, this work reveals additional complexity in crystalline structure-mediated NLRP3 inflammasome regulations.
Data set for "Development of Covalent Chitosan-Polyethylenimine Derivatives as Gene Delivery Vehicle: Synthesis, Characterization, and Evaluation" Int. J. Mol. Sci. 2021, 22, 3828. DOI: 10.3390/ijms22083828. Raw data for analytical techniques; description of experiments; metadata file.
Hydroxychloroquine (HCQ) is a quinoline derivate used for the treatment of malaria and rheumatoid disorders. During early phases of the SARS-CoV2 (COVID-19) pandemic, preliminary and later not substantiated reports suggested that HCQ might benefit COVID-19 patients. This had sparked a worldwide and rapidly rising demand for HCQ drug products. Consequently, patients with pre-existing rheumatic diseases in Switzerland were confronted with an acute drug shortage.<br><br>We have therefore designed, produced and characterized a generic HCQ drug formulation. The proposed HCQ formulation can be manufactured by using a minimal number of operation steps (mixing, wet granulation, sieving, blending, compression) and readily available pharmaceutical excipients.<br><br>HCQ tablets were manufactured by granulation of the active pharmaceutical ingredient (API), blending with the external phase and compaction using a non instrumented single punch tablet press. Analytics and identification of the API was performed by a combination of NMR, ESI-MS, FTIR and HPLC. HCQ tablets met the quality criteria for an immediate release HCQ dosage form.<br><br><div>We hope that free access to non-proprietary protocols covering analytical procedures, formulation design, and manufacturing instructions for HCQ tablets will help to bridge existing and future supply chain gaps.</div><div><br></div><div><br></div>
The mechanism governing pharmaceutical tablet disintegration is far from fully understood. Despite the importance of controlling a formulation’s disintegration process to maximize the active pharmaceutical ingredient's bioavailability and ensure predictable and consistent release profiles, the current understanding of the process is based on indirect or superficial measurements. Formulation design is, therefore, by and large, based on empirical knowledge and can be unpredictable and inefficient.We aim to help bridge the gap by generating a series of time-resolved X-ray micro-computed tomography (µCT) images capturing volumetric images of a broad range of mini-tablet formulations undergoing disintegration. Automated image segmentation was a prerequisite to overcoming the challenges of analyzing multiple time-series of heterogeneous tomographic images at high magnification. We devised and trained a convolutional neural network (CNN) based on the U-Net architecture for autonomous, rapid, and consistent image segmentation. We created our own µCT data reconstruction pipeline and parameterized it to deliver image quality optimal for our CNN-based segmentation.Our approach enabled us to visualize the internal microstructures of the tablets during disintegration and to extract parameters of disintegration kinetics from the time-resolved data. We determine by factor analysis the influence of the different formulation components on the disintegration process in terms of both qualitative and quantitative experimental responses. We relate our findings to known formulation component properties and established experimental results. Our direct imaging approach, enabled by deep learning-based image processing, delivers new insights into the disintegration mechanism of pharmaceutical tablets.
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