BioNanoSci. (2014) 4:59–70 DOI 10.1007/s12668-013-0117-2
Magnetic Resonance Imaging for Monitoring of Magnetic Polyelectrolyte Capsule In Vivo Delivery Qiangying Yi & Danyang Li & Bingbing Lin & Anton M. Pavlov & Dong Luo & Qiyong Gong & Bin Song & Hua Ai & Gleb B. Sukhorukov
Published online: 10 December 2013 # Springer Science+Business Media New York 2013
Abstract Layer-by-layer (LbL) assembled polyelectrolyte capsules have been widely studied as promising delivery systems due to their well-controlled architectures. Although their potential applications in vitro have been widely investigated, at present, it is still a challenging task to track their realtime delivery in vivo, where and how they would be located following their administration. In this work, the noninvasive magnetic resonance imaging (MRI) technique was applied to monitor the delivery of polyelectrolyte capsules in vivo, incorporating magnetite nanoparticles as imaging components. First, MRI scan was performed over 6 h after sample administration at the magnetic field of 3.0 T; magnetic capsules, both poly(allylamine hydrochloride)/poly(styrenesulfonate sodium salt)-based and poly-L -arginine hydrochloride/dextran sulfate (Parg/DS)-based, were detected mostly in the liver region, where the transverse relaxation time (T2) was shortened and hypointense images were visualized, demonstrating a contrast-enhanced MRI effect between liver and adjacent tissue. A continuous MRI scan found that the contrastenhanced MRI effect can last up to 30 h; in the mean time, The authors Q. Yi and D. Li contributed equally to this work. Q. Yi : A. M. Pavlov : G. B. Sukhorukov (*) School of Engineering and Materials Science, Queen Mary University of London, Mile End Road, London E1 4NS, UK e-mail:
[email protected] D. Li : B. Lin : D. Luo : H. Ai (*) National Engineering Research Centre for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, People’s Republic of China e-mail:
[email protected] Q. Gong : B. Song : H. Ai Department of Radiology, West China Hospital, Sichuan University, 37 Guoxuexiang Road, Chengdu 610041, People’s Republic of China
the Parg/DS-based capsules with smaller diameter were found to have a pronounced clearance effect, which resulted in a weakened MRI effect in the liver. No obvious toxicity was found in animal studies, and all mice survived after MRI scans. Histology study provided evidences to support the MRI results, and also revealed the destination of these magnetic capsules over 30 h after administration. Keywords Magnetite . Capsule . MRI . Liver . Spleen
1 Introduction Layer-by-layer (LbL) self-assembled polyelectrolyte capsules have been widely studied over the past few decades and have been developed as potential delivery systems for various applications [1, 2]. Driven by electrostatic interactions of desired building blocks, LbL capsules with precise controlled multilayer architecture and properties can be easily obtained [3, 4]. Promisingly, these fabricated capsules could engineer numerous solutions to meet the diverse requirements in the field of medical and pharmaceutical applications. These shelllike formations serve as steady and efficient carriers for loading of cargo substances with varied molecular weights, shapes, and types [5]. Moreover, the stepwise polymer deposition procedure facilitates the modification and functionalization of the capsule formations, which further allow the release of the encapsulated substances at targeted sites in a controlled manner [6, 7]. Prior to practical applications in vivo, such as drug delivery, tracking, and diagnosis, basic considerations of fabricated capsules with biosafety and efficacy in organ or tissue need to be well investigated. In vitro biological evaluations helped researchers narrow the choices of biocompatible subjects for further in vivo use. Recent contributions of the in vitro
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research on polyelectrolyte capsules for delivery uses, comprising of important features such as biostability, biocompatibility, intracellular fate, etc., have been well studied and summarized [3, 8, 9]. However, the most essential work here should be emphasized on the investigation of the delivery, how and where these capsules would be transported in real physiological and pathological condition in vivo. Unfortunately, there are very few research works concerning the real-time detection of these capsules in vivo, due to the difficulty to realize continuous monitoring of them in organs or tissues. As a powerful and reliable tool developed in (bio-)medical imaging use, magnetic resonance imaging (MRI) allows the real-time visualization of living organisms and related interactions at molecular or cellular level [10, 11]. Specially, MRI offers the available technique here to monitor the capsule delivery in vivo, with the help of possible imaging components. Considering the well-controlled and unique structures of the capsule shells, iron oxide-based magnetic (e.g., magnetite) nanoparticles should be one series of optimal imaging components. Moreover, the magnetite nanoparticles are easy to be introduced into capsule shells or cavities as charged components after a simple modification process [12]. These magnetite nanoparticles are able to dramatically shorten the transverse relaxation time (T2) in a mononuclear phagocyte system (e.g., liver, spleen) and thus provide decreased signal intensity in a T2-wieghted image [13]. For example, superparamagnetic iron oxide (SPIO) nanoparticle possessing extremely high T2 relaxivity has made essential contribution to liver MRI imaging and drug delivery tracking [14], benefiting from its selective uptake by Kupffer cells in liver, spleen, and bone marrow [15]. Furthermore, these magnetite nanoparticles behave quite biosafe for in vivo application, because of the low administration amount as well as their degradation products would participate in the iron metabolism of the human body [16, 17]. Design of delivery vesicles for potential use in vivo requires considerations of many parameters, such as size and stability. Besides, the featured surface with functional sites would also affect the in vivo delivery as well as potential functionalities of fabricated delivery vesicles. For example, biocompatible building blocks could be used to eliminate or reduce possible biotoxicity to minimum level [8]; hydrophilic segment, for instance the polyethylene glycol (PEG), could be used to repel protein absorption and to ensure a longer circulation time in bloodstream [18]; and unique functional groups/ components could be used to achieve potential detection, controlled delivery, as well as site-specific manipulation [19]. Consequently, these parameters should be addressed as important considerations for the fabrication and delivery of LbL polyelectrolyte capsule systems. In this work, we cope with the challenges and report on the monitoring of the magnetic polyelectrolyte capsules in vivo. Generally, incorporating the magnetite nanoparticles as
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imaging probes, typical organs or tissues with deposited capsules could be visualized as hypointense signal under a 3-T clinical magnetic field. Comparing the continuous MRI scans, in vivo capsule delivery path and preservation duration could be estimated qualitatively. Practically, related information could provide useful data to estimate working time window of the capsule systems for potential applications. Generally, five types of capsule systems with different shell structures were studied as typical examples here. Besides the specific MRI signal intensity that was influenced by possible capsule architecture as well as accumulation, the distribution of these magnetic capsules over a certain period after administration was verified by histology studies.
2 Materials and Methods 2.1 Materials Poly(L -lysine)20 kDa-graft -poly(ethylene glycol)2 kDa (PLLPEG) was purchased from SuSoS AG. Poly(allylamine hydrochloride) (PAH, 70 kDa), poly(styrenesulfonate) (PSS) sodium salt (70 kDa), poly-L -arginine (Parg) hydrochloride (15–17 kDa), dextran sulfate (DS) sodium salt (∼100 kDa), iron(II) chloride (FeCl2), iron(III) chloride (FeCl3), ammonium hydroxide solution (NH4OH, 28 wt%), citric acid, ethylenediaminetetraacetic acid (EDTA), and other chemicals were purchased from Sigma-Aldrich. 2.2 Methods 2.2.1 Magnetite Nanoparticle Preparation Superparamagnetic (Fe3O4) nanoparticles were synthesized according to the previous well-known Massart coprecipitation method and stabilized by modification of the particle surface with citric acid, as described by Minko and coworkers [12]. After dialysis against water, magnetic nanoparticles with adsorbed citric acid layers were stabilized in water. The freshly prepared nanoparticles were characterized by using a transmission electron microscope (TEM, JEOL 2010) operating at 200 kV and dynamic light scattering (DLS) technique with Malvern Zetasizer Nano ZS (Malvern Instruments Ltd.). 2.2.2 Magnetic Capsule Fabrication For capsule preparation, polyelectrolytes were alternatively deposited on prepared CaCO3 microparticles (∼3 μm, the same batch) by using LbL assembly technique [20]. Particularly, formed superparamagnetic nanoparticles were introduced as the negatively charged layers for fabrication of magnetic capsules [21]. Adsorption of the magnetite nanoparticles occurred by immersion of the prepared CaCO 3
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microparticles in diluted water suspension of the nanoparticles in a ratio of 1:50. In the meantime, capsules without magnetic nanoparticles were also prepared as controls. Basically, two different multilayer systems, PAH/PSS-based and Parg/DSbased, were adsorbed on the templates. After core removal with treatment of 0.2 M EDTA, hollow capsules with different shell structures were obtained. Prior to introducing capsules to in vivo tests, the capsules were autoclaved at 121 °C for 30 min to sterilize and shrink them [22]. Diluted microcapsules were coated with gold and observed under a scanning electron microscope (SEM, FEI Inspect-F) with an accelerating voltage of 10 kVand spot size of 3.5 at a working distance of approximately 10 mm. Capsule size distribution was expressed as mean ± SD of at least 50 capsules per sample of random measurement of SEM images by using the software Image-Pro Plus v 6.0. 2.2.3 In Vivo MRI Studies Before an MRI effect study, the capsule samples were concentrated, resulting in an Fe concentration of 1 mg/ml of all the magnetite-containing capsule suspensions, which was determined by atomic absorption spectroscopy (AAS) (AA800, PerkinElmer, USA). All the tests involving animals were carried out in the National Engineering Research Centre for Biomaterials and West China Hospital (Sichuan University, China). All animal works were performed under guidelines determined by the institutional committees for animal welfare and use of human subjects. In vivo MRI effect of all the capsule samples was studied by using a 3-T MRI imaging system (Philips Medical System) incorporating a mouse coil (Philips) for transmission and reception of the signal. Commercial Philips clinical sequences T2-weighted spin echo (T2W SE) was used to collect corresponding information with the following parameters: repetition time (TR)=762 ms, TS=100 ms, FOV=40 mm× 40 mm, and slice thickness=1 mm. BALB/c mice (20∼25 g) were anesthetized with 150 μl of 1 % pentobarbital sodium through intraperitoneal injection. Through intravenous injection in the tail, 50 μl of capsule suspensions with/without magnetic nanoparticles was administrated. Continuous MRI scans were performed 6 and 30 h after sample injection. In the meantime, a healthy BALB/c mouse without sample injection was studied as the negative control. 2.2.4 Histology Analysis After the second MRI scan, mice were sacrificed, and liver and spleen tissue were removed and fixed with 4 % paraformaldehyde before histology analysis. Then, the fixed tissues were embedded in paraffin and cut into slides of 5 μm. Adjacent slides were prepared for histological analysis using Prussian blue staining (iron staining).
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3 Results and Discussion 3.1 Magnetic Capsules Coprecipitating ferric (Fe3+) and ferrous (Fe2+) ions in aqueous solution at high pH allows the simplest approach to generate superparamagnetic nanoparticles [12]. Figure 1 shows the characterization of the prepared magnetite nanoparticles. These nanoparticles were polydispersed as presented in the TEM image; most of them were found to be less than 20 nm in diameter (Fig. 1a) [23]. The DLS data illustrated that these particles has an average size of 48.5 nm in water, with distribution ranging from 32.7 to 91.3 nm (Fig. 1b). This difference obtained from two measurements could be explained as the aggregates of magnetite nanoparticles in water. However, comparing with the microscaled capsule formations, such magnetic particle aggregates would have negligible influence on capsule fabrication. On the other hand, such aggregates of magnetite naonoparticles have been found to have abilities to greatly improve T2 relaxivity over single ones [24]. These magnetite nanoparticles were negatively charged due to the existence of citric acid coating on the surfaces. Their zeta potential was measured to be −25.6 mV in water (Fig. 1c), which made them good building blocks as the negatively charged layers for LbL capsule fabrication. After LbL assembly and template removal process, hollow capsules with different shell structures were obtained, as shown in Table 1. Samples #1 to #5 referred to these capsules with magnetite nanoparticles in their shells, while the samples C1 and C2 represented the PAH/PSS and Parg/DS capsules without magnetite, respectively. Mainly, these capsules could be classified into two types: PAH/PSS-based and Parg/DSbased capsules. The former is the most commonly studied synthetic capsules in many research works [25, 26]; the latter has been investigated as a biocompatible system in many cases [27–29]. The capsule samples size was about 3 μm in diameter after fabrication, as shown in Table 1 and Fig. 2. For all the magnetic capsules, much rougher surfaces were observed due to the existence of magnetite nanoparticles in their shells. Size control of the candidate delivery systems/vehicles is necessary for their in vivo studies; a longer circulation time in bloodstream usually requires smaller vehicle size [30]. Typical examples have been demonstrated in related research works, where nanoscaled (
