Translating innovative nanomaterials to medical products requires efficient manufacturing techniques that enable large-scale high-throughput synthesis and high reproducibility. Drug carriers in... Show moreTranslating innovative nanomaterials to medical products requires efficient manufacturing techniques that enable large-scale high-throughput synthesis and high reproducibility. Drug carriers in medicine embrace a complex subset of tasks calling for multi-functionality. Here, we report the synthesis of pro-drug-loaded core cross-linked polymeric micelles (CCPMs) in a continuous flow process, which combines the commonly separated steps of micelle formation, core cross-linking, functionalization, and purification into a single process. Redox-responsive CCPMs are formed from thiol-reactive polypept(o)ides of polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine) and functional cross-linkers based on dihydrolipoic acid hydrazide for pH-dependent release of paclitaxel. The precisely controlled microfluidic self-assembly and purification process allows the production of spherical micelles (Dh = 35 nm) with low polydispersity values (PDI<0.1) while avoiding toxic organic solvents and additives with unfavorable safety profiles. Self-assembly and cross-linking via slit interdigital micromixers produce 350-700 mg of CCPMs/h per single system, while purification by online tangential flow filtration successfully removes impurities (unimer ≤ 0.5%). The formed paclitaxel-loaded CCPMs possess the desired pH-responsive release profile, display stable drug encapsulation, an improved toxicity profile compared to Abraxane, and therapeutic efficiency in the B16F1-xenotransplanted zebrafish model. The combination of reactive polymers, functional cross-linkers, and microfluidics enables the continuous-flow synthesis of therapeutically active CCPMs in a single process. This article is protected by copyright. All rights reserved. Show less
Tuberculosis is the deadliest bacterial disease globally, threatening the lives of millions every year. New antibiotic therapies that can shorten the duration of treatment, improve cure rates, and... Show moreTuberculosis is the deadliest bacterial disease globally, threatening the lives of millions every year. New antibiotic therapies that can shorten the duration of treatment, improve cure rates, and impede the development of drug resistance are desperately needed. Here, we used polymeric micelles to encapsulate four second-generation derivatives of the antitubercular drug pretomanid that had previously displayed much better in vivo activity against Mycobacterium tuberculosis than pretomanid itself. Because these compounds were relatively hydrophobic and had limited bioavailability, we expected that their micellar formulations would overcome these limitations, reduce toxicities, and improve therapeutic outcomes. The polymeric micelles were based on polypept(o)ides (PeptoMicelles) and were stabilized in their hydrophobic core by π-π interactions, allowing the efficient encapsulation of aromatic pretomanid derivatives. The stability of these π-π-stabilized PeptoMicelles was demonstrated in water, blood plasma, and lung surfactant by fluorescence cross-correlation spectroscopy and was further supported by prolonged circulation times of several days in the vasculature of zebrafish larvae. The most efficacious PeptoMicelle formulation tested in the zebrafish larvae infection model almost completely eradicated the bacteria at non-toxic doses. This lead formulation was further assessed against Mycobacterium tuberculosis in the susceptible C3HeB/FeJ mouse model, which develops human-like necrotic granulomas. Following intravenous administration, the drug-loaded PeptoMicelles significantly reduced bacterial burden and inflammatory responses in the lungs and spleens of infected mice. Show less
Dal, N.K.; Speth, M.; Johann, K.; Barz, M.; Beauvineau, C.; Wohlmann, J.; ... ; Alonso-Rodriguez, N. 2021
With the increasing emergence of drug-resistant Mycobacterium tuberculosis strains, new and effective antibiotics against tuberculosis (TB) are urgently needed. However, the high frequency of... Show moreWith the increasing emergence of drug-resistant Mycobacterium tuberculosis strains, new and effective antibiotics against tuberculosis (TB) are urgently needed. However, the high frequency of poorly water-soluble compounds among hits in high-throughput drug screening (HTS) campaigns is a major obstacle in drug discovery. Moreover, in vivo testing using conventional animal TB models such as mice is time-consuming and costly, and represents a major bottleneck in lead compound discovery and development. Here, we report the use of the zebrafish embryo TB model, to evaluate the in vivo toxicity and efficacy of five poorly water-soluble nitronaphthofuran derivatives, which were recently identified to possess anti-tuberculosis activity in vitro. To aid solubilization compounds were formulated in biocompatible polymeric micelles (PM). Three of the five PM-formulated nitronaphthofuran derivatives showed low toxicity in vivo, significantly reduced bacterial burden and improved survival in infected zebrafish embryos. We propose the zebrafish embryo TB-model as a quick and sensitive tool for evaluating in vivo toxicity and efficacy of new anti-TB compounds during early stages of drug development. Thus, this model is well suited to pinpoint promising compounds for further development. Show less
The zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real-time... Show moreThe zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real-time observations of vascular flow of nanoparticles and their interactions with cells throughout the body. As a consequence, this system enables the acquisition of quantitative data that are difficult to obtain in rodents. Until now, a few studies using the zebrafish model have only described semiquantitative results on key nanoparticle parameters. Here, a MACRO dedicated to automated quantitative methods is described for analyzing important parameters of nanoparticle behavior, such as circulation time and interactions with key target cells, macrophages, and endothelial cells. Direct comparison of four nanoparticle (NP) formulations in zebrafish embryos and mice reveals that data obtained in zebrafish can be used to predict NPs' behavior in the mouse model. NPs having long or short blood circulation in rodents behave similarly in the zebrafish embryo, with low circulation times being a consequence of NP uptake into macrophages or endothelial cells. It is proposed that the zebrafish embryo has the potential to become an important intermediate screening system for nanoparticle research to bridge the gap between cell culture studies and preclinical rodent models such as the mouse. Show less
Fenaroli, F.; Repnik, U.; Xu, Y.T.; Johann, K.; Herck, S. van; Dey, P.; ... ; Griffiths, G. 2018
The enhanced permeability and retention (EPR) effect is the only described mechanism enabling nanoparticles (NPs) flowing in blood to reach tumors by a passive targeting mechanism. Here, using the... Show moreThe enhanced permeability and retention (EPR) effect is the only described mechanism enabling nanoparticles (NPs) flowing in blood to reach tumors by a passive targeting mechanism. Here, using the transparent zebrafish model infected with Mycobacterium marinum we show that an EPR-like process also occurs allowing different types of NPs to extravasate from the vasculature to reach granulomas that assemble during tuberculosis (TB) infection. PEGylated liposomes and other NP types cross endothelial barriers near infection sites within minutes after injection and accumulate close to granulomas. Although similar to 100 and 190 nm NPs concentrated most in granulomas, even similar to 700 nm liposomes reached these infection sites in significant numbers. We show by confocal microscopy that NPs can concentrate in small aggregates in foci on the luminal side of the endothelium adjacent to the granulomas. These spots are connected to larger foci of NPs on the ablumenal side of these blood vessels. EM analysis suggests that NPs cross the endothelium via the paracellular route. PEGylated NPs also accumulated efficiently in granulomas in a mouse model of TB infection with Mycobacterium tuberculosis, arguing that the zebrafish embryo model can be used to predict NP behavior in mammalian hosts. In earlier studies we and others showed that uptake of NPs by macrophages that are attracted to infection foci is one pathway for NPs to reach TB granulomas. This study reveals that when NPs are designed to avoid macrophage uptake, they can also efficiently target granulomas via an alternative mechanism that resembles EPR. Show less
Evensen, L.; Johansen, P.L.; Koster, G.; Zhu, K.; Hefindal, L.; Speth, M.; ... ; Griffiths, G. 2015
