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Zebrafish as a visual and dynamic model to study the transport of nanosized drug delivery systems across the biological barriers

https://doi.org/10.1016/j.colsurfb.2017.05.022Get rights and content

Highlights

  • Transport of C6-NCs across biological barriers has been studied in zebrafish model.

  • Higher transport efficiency was observed with small sized NCs.

  • Accumulation and excretion of C6-NCs in different organs have been investigated.

  • Lipid raft active pathways participated the transport of NCs.

  • Intact C6-NCs together with the free dissolved form of C6 were observed in vivo.

Abstract

With the wide application of nanotechnology to drug delivery systems, a simple, dynamic and visual in vivo model for high-throughput screening of novel formulations with fluorescence markers across biological barriers is desperately needed. In vitro cell culture models have been widely used, although they are far from a complimentary in vivo system. Mammalian animal models are common predictive models to study transport, but they are costly and time consuming. Zebrafish (Danio rerio), a small vertebrate model, have the potential to be developed as an “intermediate” model for quick evaluations. Based on our previously established coumarin 6 nanocrystals (C6-NCs), which have two different sizes, the present study investigates the transportation of C6-NCs across four biological barriers, including the chorion, blood brain barrier (BBB), blood retinal barrier (BRB) and gastrointestinal (GI) barrier, using zebrafish embryos and larvae as in vivo models. The biodistribution and elimination of C6 from different organs were quantified in adult zebrafish. The results showed that compared to 200 nm C6-NCs, 70 nm C6-NCs showed better permeability across these biological barriers. A FRET study suggested that intact C6-NCs together with the free dissolved form of C6 were absorbed into the larval zebrafish. More C6 was accumulated in different organs after incubation with small sized NCs via lipid raft-mediated endocytosis in adult zebrafish, which is consistent with the findings from in vitro cell monolayers and the zebrafish larvae model. C6-NCs could be gradually eliminated in each organ over time. This study demonstrated the successful application of zebrafish as a simple and dynamic model to simultaneously assess the transport of nanosized drug delivery systems across several biological barriers and biodistribution in different organs, especially in the brain, which could be used for central nervous system (CNS) drug and delivery system screening.

Introduction

In recent decades, the applications of nanotechnology for medicine and more specifically for drug delivery systems (DDSs) have increased rapidly [1]. Different nanosized DDSs are being developed for various drug delivery applications, such as enhancing the solubility or bioavailability of therapeutic compounds [2]. After administration and before entering the circulation system or the targeted tissues, several biological barriers need to be overcome for these DDSs, such as blood brain barrier (BBB), gastrointestinal (GI) barrier and blood retinal barrier (BRB), among others [3], [4], [5]. There are many factors affecting the transport of DDSs across these barriers, such as particle size and surface properties [6]. To facilitate the rapid development of DDSs, an ideal model that is suitable for high-throughput screening (HTS) of the novel formulation or excipient across several biological barriers was desperately needed. Many models have been developed to mimic these barriers at various levels, ranging from in silico to several in vitro and in vivo models [7], [8], [9]. Cell culture models have also been used successfully. For example, the Caco-2 cell line has been recognized as a GI model for many years [10]. This cellular model, however, lacks the complex metabolism and physiology of biological barrier in vivo [11]. Similarly, although several cellular systems have been established as in vitro BBB models, they are still far from being ideal models to mimic BBB properties due to the severe down-regulation of the transporter function [12]. In addition, cellular models also face challenges due to poor batch-to-batch consistency. For instance, it has been reported that the BBB function in primary cultured brain capillary endothelial cells (BCEC), often used as an in vitro BBB model, varies from batch to batch [13]. In the same sense, BRB models based on primary cells reportedly lack reliability and reproducibility [14]. Currently, mice and rats are the most widely used animal models to evaluate whether DDSs can permeate across those in vivo barriers. These models are costly and often time consuming, however, which renders them unsuitable for permeability screening of DDS [15]. Therefore, the lack of a simple and dynamic in vivo model to assess the capability of various drug delivery systems across the biological barriers has slowed down the development of novel formulations and excipients.

In recent years, the zebrafish (Danio rerio) has emerged as a useful vertebrate model for studying the development and maintenance of biological barriers, due to the structure and function of the barriers being very similar to that of mammals at certain levels [16]. Zebrafish BBB function is developed by 3 days post fertilization (dpf), and it has restricted permeability to many compounds, which is similar to that of humans. The expression of tight junctions and efflux transport systems are also found in the cerebral microvessels [16], [17]. Similarly, the hyaloid vasculature in the zebrafish retina develops a barrier functions at 3 dpf [18]. Moreover, zebrafish also have a functional GI barrier with the intestinal epithelium maturing gradually until 120 h post-fertilization (hpf) [19], [20]. Tight junction proteins, such as claudin, are specifically expressed in the intestine at 56 hpf [21]. Transcripts of multidrug resistance proteins were detected in the intestine of developing embryos and in adult tissues [22]. Different from larval or adult zebrafish, embryos have another barrier-chorion that plays an essential role in protecting embryos from external influences [23]. More importantly, due to the transparency of the whole body, embryos and larval zebrafish can be used for tracking DDSs in vivo directly and non-invasively. The pharmacokinetic profiling and biodistribution of DDSs are often affected by the transport across different barriers [24], [25]. Therefore, we expect that zebrafish could be used to explore the in vivo fate of nanosized DDSs including absorption, distribution and elimination.

Nanocrystals (NCs), a carrier nearly free colloidal delivery system in the nano-sized range, show emerging applications in both clinical practice and commercial products. NCs provide an option for the delivery of poorly soluble drugs and receive more attention. The in vivo behavior of NCs, however, is not well understood [2]. In our previous study, coumarin 6 nanocrystals (C6-NCs) have been used to study the transport mechanism of NCs through the MDCKII monolayer and larval zebrafish. Compared to coarse C6, NCs could dramatically increase the uptake of C6 in MDCKII epithelial cells and larval zebrafish. Lipid rafts, ER/Golgi, and Golgi/plasma membrane pathways were involved in the transport process [26].

In this study, C6-NCs were employed as the model nano DDS to investigate the transport features of biological barriers in zebrafish. Coumarin 6, a highly fluorescent and lipophilic compound (log P = 6.9), is selected as a good hydrophobic model compound, as it is nearly insoluble in water. Due to its autofluorescence, the in vivo behavior of the NCs could be imaged by monitoring fluorescence. Two different sized C6-NCs (70 and 200 nm) were prepared as per our previously published paper [26]. Then, the transport of C6-NCs across biological barriers was traced in real time in different developmental stages of zebrafish, including embryos and larvae from 3 dpf to 7 dpf. To monitor the in vivo fate of NCs, larval zebrafish of 7 dpf with fully developed biological barriers were chosen. Hybrid NCs technology and fluorescence resonance energy transfer (FRET) technology were used. With the aim of extending the application of zebrafish in development of DDS, the biodistribution and elimination of C6-NCs in different organs were compared using adult zebrafish. In addition, MβCD, a transport inhibitor that could disrupt membrane lipid rafts by extracting membrane cholesterol, was used to study the transport mechanism of NCs across the intestine in adult zebrafish.

Section snippets

Materials

Coumarin-6 (purity >98%), 1-phenyl-2-thiourea (PTU), low gelling temperature agarose, ethyl 3-aminobenzoate methanesulfonate (MS-222), methyl-β-cyclodextran (MβCD) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI) were obtained from Sigma Aldrich (St. Louis, MO, USA). Hydroxy Propyl Methyl Cellulose (HPMC) was purchased from Shanghai Colorcon Coating Technology Limited (Shanghai, China). 4′,6-Diamidino-2-Phenylindole (DAPI) was supplied by Thermo Fisher Scientific (USA). Methanol

Preparation and characterization of C6-NCs with two mean particle size

C6-NCs at 400 ng/ml of 70 nm (mean particle size was 70.74 ± 2.85 nm with the polydistribution index of 0.21 ± 0.015) and 200 nm (mean particle size was 211.9 ± 9.8 nm with the polydistribution index of 0.19 ± 0.045) were prepared by the anti-solvent precipitation method reported on previously by our group [26].

Transport of C6-NCs in embryonic zebrafish in vivo

In the study of permeability of NCs across chorion, embryos (3 hpf) were incubated with C6-NCs (400 ng/ml) for different time periods and subsequently observed under a fluorescence microscope. The

Conclusions

In the present study, zebrafish at different developmental stages were used to assess the transport of NCs with particle sizes of 70 nm and 200 nm across several biological barriers, including chorion, GI barrier, BRB and BBB. Higher uptake and transport efficiency were observed after incubation with the small sized C6-NCs. Along with the maturation of these biological barriers from 3 hpf to 7 dpf, the transport and biodistribution of C6-NCs were also changed. Hybrid nanocrystal technology and FRET

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81528021) and University of Macau (Research Grant MYRG112 (Y2-L3)-ICMS13-ZY and MYRG2014-00040-ICMS-QRCM).

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