Organ-on-a-chip platforms for studying drug delivery systems

Solange Massa

Novel microfluidic tools allow new ways to manufacture and test drug delivery systems. Organ-on-a-chip systems - microscale recapitulations of complex organ functions - promise to improve the drug development pipeline. This review highlights the importance of integrating microfluidic networks with 3D tissue engineered models to create organ-on-a-chip platforms, able to meet the demand of creating robust preclinical screening models. Specific examples are cited to demonstrate the use of these systems for studying the performance of drug delivery vectors and thereby reduce the discrepancies between their performance at preclinical and clinical trials. We also highlight the future directions that need to be pursued by the research community for these proof-of-concept studies to achieve the goal of accelerating clinical translation of drug delivery nanoparticles.

COREL-07177; No of Pages 13 Journal of Controlled Release xxx (2014) xxx–xxx Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel 4 5 6 7 8 9 a 1 0 a r t i c l e 11 21 12 22 13 14 Article history: Received 26 February 2014 Accepted 2 May 2014 Available online xxxx 15 16 17 18 19 20 Keywords: Organ-on-a-chip Drug delivery Nanoparticle Drug screening Toxicity R O O F 3 Nupura S. Bhise a,b, João Ribas a,b,c,d, Vijayan Manoharan a,b, Yu Shrike Zhang a,b, Alessandro Polini a,b, Solange Massa a,b, Mehmet R. Dokmeci a,b, Ali Khademhosseini a,b,e,f,⁎ Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, 02139, USA Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, 02139, USA Doctoral Programme in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology, Institute for Interdisciplinary Research, University of Coimbra, 3030-789 Coimbra, Portugal d Biocant — Biotechnology Innovation Center, 3060-197 Cantanhede, Portugal e Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston 02115, USA f Department of Physics, King Abdulaziz University, Jeddah 21569, Saudi Arabia b c i n f o a b s t r a c t P 2Q1 Organ-on-a-chip platforms for studying drug delivery systems Novel microﬂuidic tools allow new ways to manufacture and test drug delivery systems. Organ-on-a-chip systems — microscale recapitulations of complex organ functions — promise to improve the drug development pipeline. This review highlights the importance of integrating microﬂuidic networks with 3D tissue engineered models to create organ-on-a-chip platforms, able to meet the demand of creating robust preclinical screening models. Speciﬁc examples are cited to demonstrate the use of these systems for studying the performance of drug delivery vectors and thereby reduce the discrepancies between their performance at preclinical and clinical trials. We also highlight the future directions that need to be pursued by the research community for these proofof-concept studies to achieve the goal of accelerating clinical translation of drug delivery nanoparticles. © 2014 Published by Elsevier B.V. C T E D 1 33 31 E 32 1. Introduction 35 36 The rapidly developing ﬁeld of nanomedicine can signiﬁcantly impact human disease therapy [1,2]. The research progress accomplished in this ﬁeld, over the last few decades, has led to the development of nanomaterials, useful for designing carriers that deliver therapeutic payload to diseased cells. An ideal drug delivery system should be easy to manufacture and scale-up, low cost, biocompatible, biodegradable, possesses a high drug loading capacity and can be targeted to the site-of-interest in the body. Nanocarriers, also routinely referred to as nanoparticles, are a class of drug delivery systems that range in size from about 50 to 200 nm, allowing them to efﬁciently translocate across the cell membrane barrier. From a therapeutic standpoint, nanocarriers can prolong the systemic circulation time of the drug and signiﬁcantly reduce adverse side effects caused by off-target delivery at healthy tissue sites. This controlled release of drugs reduces the magnitude of overall drug exposure required for a therapeutic effect, thus avoiding higher drug doses and consequent adverse effects. A wide variety of drugs, including hydrophobic and hydrophilic small molecules, as well as biomacromolecules, 43 44 45 46 47 48 49 50 51 52 R O C 41 Q2 42 N 39 40 U 37 38 R 34 ⁎ Corresponding author at: Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge 02139, USA. E-mail address: alik@rics.bwh.harvard.edu (A. Khademhosseini). 23 24 25 26 27 28 29 30 can be encapsulated within nanoparticles by tailoring the chemistry of nanomaterials, polymeric or inorganic/metallic, to achieve the desired encapsulation capability and release kinetics. The ﬁrst use of nanoscale systems for drug delivery was reported in the 1970s, when liposomal Trojan horse nanoparticles were used for treating lysosomal storage disease [3,4]. Nanoparticles have also been developed as diagnostic agents to enhance the sensitivity for imaging techniques, including X-ray computed tomography (CT) and magnetic resonance imaging (MRI). An increase in available techniques to engineer more precise and sophisticated nanomaterials, and a deeper understanding of disease biology have catapulted a new generation of nanotherapeutics with improved properties. The above-mentioned advantages make nanoscale drug delivery systems appealing to the pharmaceutical companies and healthcare regulatory agencies. However, in spite of these rapid bench-side developments, the translation of therapeutic nanoparticles to the commercial pipeline has been less impressive [5]. Very few systems have been approved by the Food and Drug Administration (FDA), including Doxil, a liposomal formulation encapsulating the chemotherapeutic drug Doxorubicin, and Abraxane, based on the nanoparticle albuminbound (nab) technology to deliver Paclitaxel, a widely used drug for breast and pancreatic cancer [6]. This slow pace of bench-to-bedside translation can be attributed to several challenges, the most critical being the lack of robust preclinical tissue culture platforms that can mimic in vivo conditions and predict the performance of these nanoparticles within the human body. http://dx.doi.org/10.1016/j.jconrel.2014.05.004 0168-3659/© 2014 Published by Elsevier B.V. Please cite this article as: N.S. Bhise, et al., Organ-on-a-chip platforms for studying drug delivery systems, J. Control. Release (2014), http:// dx.doi.org/10.1016/j.jconrel.2014.05.004 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 N.S. Bhise et al. / Journal of Controlled Release xxx (2014) xxx–xxx 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 C 105 106 E 103 104 R 101 102 R F 94 95 O 92 93 C 90 91 U N 88 89 O Several parameters need to be studied for developing nanoparticles for clinical use. These include studying the fate of the nanoparticles inside the body and its toxicological effects, the mode of binding and internalization at the cellular level, the stability of the nanoparticles with respect to various physical and chemical conditions of the body, and, most importantly, the efﬁcacy when compared to free drugs [5]. Large-batch synthesis, toxicity assessment and efﬁcacy screening are the major levels at which clinical translation of nanotherapeutics faces set-back [16]. On the manufacturing front, scaling the small lab synthesis techniques to the large-scale production of nanoparticles has been challenging for the pharmaceutical companies [8]. Meanwhile, screening for the toxicity and efﬁcacy suffers from the paucity of preclinical models that would robustly predict the nanoparticles' behavior inside the human body [16]. For simultaneous evaluation of the above-mentioned parameters, predictive in vitro platforms are essential while developing drug delivery vectors [13,17]. The current gold standard for preclinical testing of nanotherapeutics is in vivo studies. These do not accurately predict human responses due to inter-species difference in genetic makeup, along with being extremely time-consuming, expensive, low-throughput and raising ethical concerns. The resolution for whole-animal imaging methods is limited, hindering visualization during transport of the theranostic agents in the target tissue. Being unable to reproduce its preclinical performance, many drug delivery systems which pass the preclinical phase fail to address the toxicity and efﬁcacy effects when compared to their free drug counterparts in human clinical trials [5]. Strikingly, the main reason cited for this effect is the use of animal models for optimization during drug carrier design [5], which brings back the obvious drawback of a certain degree of physiological irrelevance between human and animal models. Animal models need to be complemented with sophisticated in vitro platforms to ﬁll this gap. In current in vitro studies, drug delivery carriers are commonly tested in two-dimensional (2D) monolayer cell culture models. These 2D cultures involve growing on top of a ﬂat substrate (e.g., glass or polystyrene) a monolayer of single or multiple cell types that are either freshly isolated from human/animal tissues (primary cells) or are already established, immortalized cell lines. In these setups, drug delivery systems are usually mixed with culture media and directly applied on the cell monolayers, after which cellular responses are recorded. Among several published studies [18–21], the work of Xia and colleagues on the cellular uptake of gold nanoparticles (AuNPs) by SK-BR-3 breast tumor cells [22], stands out by devising a novel testing method. After culturing the cells on a piece of glass, the 86 87 R O 100 85 P 2. Limitations of current culture platforms used for developing drug delivery systems 83 84 substrate was carefully reversed and placed upside down before AuNPs, with different shapes and sizes, were added in the culture media. Such an approach successfully avoided the issues caused by rapid sedimentation of nanoparticles. Indeed, the amount of cellular uptake of nanoparticles in upright and inverted cultures was found to signiﬁcantly depend on the rate of diffusion/sedimentation of the nanoparticles. In spite of these novel approaches for 2D cell culture, it is gradually realized that there are many shortcomings with these “ﬂat” models to mimic the complex three-dimensional (3D) in vivo microenvironment, wherein the cells and extracellular matrix (ECM) exist in wellorganized architectures. Moreover, the nanoparticle delivery efﬁcacy differs considerably between 2D and 3D culture platforms [23]. Primary cells usually have a limited lifespan, undergo rapid phenotypic alterations, and show large variability over different batches of isolation; on the other hand, although established cell lines are more stable, many times they do not present genuine tissue-speciﬁc functions [24]. In this regard, efforts were shifted toward developing multiple 3D culture systems that can better recapitulate in vivo tissue functions. Multicellular spheroids are important 3D models for researchers [25–29]. These spheroids are formed by spontaneous aggregation of multiple cells held together by ECM secreted by residing cells. The apoptotic/ necrotic core of the spheroids contrasts with the proliferative cell layers on the periphery, providing a better mimic of in vivo tumor environment. Due to the importance and long-time usage of multicellular spheroids in both pharmaceutical studies and regenerative medicine, researchers have developed sophisticated methods that allow efﬁcient fabrication of uniform spheroids at relatively large scales, including the use of hanging drops, non-adhesive microwells, rotation cultures, or 3D porous scaffolds [30–37]. Multicellular tumor cylindroids have been used to study the effect of charge on the uptake of ﬂuorescein isothiocyanate (FITC) or doxorubicin (DOX)-conjugated AuNPs loaded with drugs, where diffusion is permitted only from the periphery to the center [38]. Kotov and co-workers directly utilized tumor spheroids for toxicity testing of CdTe quantum dots and AuNPs [39]. The toxic effects of these nanoparticles were compared with conventional 2D cultures, to reveal different responses of cells in terms of morphology, particle distribution, membrane integrity, mitochondrial activity, and apoptosis (Fig. 1). Besides multicellular spheroids, hydrogels and porous scaffolds have also been widely employed for constructing 3D tissue models at larger size scales [40–42]. There are a number of advantages associated with 3D cultures within a matrix. For example, the mechanical properties of the gels can be precisely modulated, which have been shown to determine the phenotypic behaviors of the cells [43–45]; the matrices can be fabricated to possess various hierarchical structures and any desired shape to accommodate speciﬁc target tissues. As an example, Huang and co-workers demonstrated that cancer cells became more tumorigenic when cultured in a ﬁbrin gel with a stiffness of approximately 90 Pa, as shown by in vivo tumor formation in mice even when only very few (10 or 100) tumor cells were injected, whereas the same number of tumor cells from stiff 2D substrates could not induce the formation of tumors [46]. Moreover, Mooney et al. cultured OSCC-3 oral squamous carcinoma cells within porous poly(lactide-co-glycolide) (PLG) scaffolds to create an in vitro tumor model [47]. They argued that tumor cells cultured in PLG scaffolds could better recapitulate their in vivo states than in 3D Matrigel or 2D substrates as shown by their morphological appearances, proliferation rates, distribution of oxygen concentrations, and secretion patterns of biomolecules. Although static culture systems based on multicellular spheroids or 3D matrices can recapitulate the in vivo functionality of tissues much better than 2D cultures, they fail to present dynamic ﬂow conditions that the cells usually experience in the body. The absence of homogenous perfusion results in improper gas and nutrient exchange through the core of the constructs. Additionally, the gravitational settling of nanoparticles in static conditions affects the outcome of dosage D 98 99 81 82 T 96 97 The development of microﬂuidic platforms for nanoparticle synthesis has shown to overcome several disadvantages of the traditional bulk synthesis methods such as scalability and batch-to-batch variability [7–9]. Microﬂuidic approaches have also been used as a tool for more sophisticated, faster and highly efﬁcient characterization of the biophysical properties of nanoparticles [10,11]. Additionally, the application of microfabrication techniques to tissue engineering aided in the creation of physiologically relevant disease models. Establishment of these techniques has paved the way for robust advances in tissue culture systems integrated with microﬂuidic networks [12]. More recently, the demand for high-throughput drug screening platforms with better preclinical predictability has translated into major developments in the organon-a-chip systems [13–15]. This review presents recent advances in in vitro tissue culture models by primarily emphasizing on organ-ona-chip platforms useful for studying the performance of drug delivery nanotherapeutics. The current challenges in the development of drug delivery systems are highlighted and the use of organ-on-chips as a potential solution is discussed by presenting speciﬁc examples of relevant proof-of-concept studies. 79 80 E 2 Please cite this article as: N.S. Bhise, et al., Organ-on-a-chip platforms for studying drug delivery systems, J. Control. Release (2014), http:// dx.doi.org/10.1016/j.jconrel.2014.05.004 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 N.S. Bhise et al. / Journal of Controlled Release xxx (2014) xxx–xxx Recent innovations in microﬂuidic technologies can provide novel synthesis and screening methods to address issues of scalability, batch-to-batch variability, and poor predictability that limit the pace of clinical translation of nanotherapeutics [16]. The integration of 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 239 240 3.1. Vascular platforms R B C E F U D N C O R A 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 Nanoparticles introduced intravenously have to traverse through 272 the vascular endothelial barrier to reach the target tissue. It is thus im- 273 portant to study the interactions of these carriers with the vasculature 274 E 241 T 218 219 C 216 217 R O O F 238 215 P 3. Organ-on-a-chip platforms as a potential solution 213 214 D 237 211 212 advanced 3D tissue engineered constructs with microﬂuidic network systems, termed ‘organ-on-a-chip’, provides a novel platform for better preclinical testing of drug delivery systems. Easy manipulation of micro-liter volume of liquids has made these models a platform where scaling and dynamic crosstalk between cells can be achieved [15]. The system geometry and structures recreate physiological length scales, concentration gradients, and the ﬂuid ﬂow generates mechanical forces that recapitulate the in vivo microenvironment experienced by cells [13,55]. Thus, these highly biomimetic platforms overcome the drawbacks with conventional tissue culture models as highlighted in the previous section [16]. By better mimicking the physiological conditions and more accurately predicting the effect of drug carriers, these sophisticated in vitro screening models can ﬁll the gap between the outcomes of animal studies and human clinical trials [16]. Since lung-on-a-chip [13,56], the ﬁeld of biomimetic organs-on-achip has expanded rapidly to encompass several organs including liver, kidney, heart, gut, breast, and blood vessels [55,57–59]. These studies have already shown that organ-on-a-chip platforms can generate responses similar to those observed in vivo toward nanoparticles [13,17]. Furthermore, multiple organ modules can be interconnected in a physiologically relevant scale and organized to form a human-ona-chip platform [60]. These systems can be used to study the fate of nanoparticles. The information obtained from such studies would allow rapid testing of new therapeutic designs. Thus, the use of physiologically relevant organ-on-a-chip models as a studying tool for drug delivery systems can accelerate the clinical translation of nanoparticles. The following sections of this review, ordered by organ-type, will discuss the progress, advantages and challenges of existing organ-ona-chip platforms, in the context of testing of drug delivery systems. E 236 optimization studies. Evaluating the drug carrier in static in vitro assays without considering mechanical forces and ﬂuid ﬂow has shown instances of false positives [13,17,22]. Hence, perfusion-based dynamic culture systems in bioreactors have become increasingly popular. At the same time, owing to the rapid development of microﬂuidics, the size of these bioreactors is being constantly reduced while the functionality is improved, enabling the development of improved systems for drug toxicity and efﬁcacy studies [48–52]. For example, Lee and coworkers constructed microﬂuidic chambers containing arrays of hydrodynamic traps, which was used to immobilize around ten tumor cells per trap (Fig. 2A). The entrapped tumor cells aggregated in the traps to form small spheroids (b50 μm) within several hours (Fig. 2B, C) [53]. Similarly, Cheung and co-workers developed a type of cell culture chip with arrays of microsieves for efﬁcient trapping of larger cell aggregates [54]. Treating the multicellular spheroids of LCC6/Her2 breast tumor cells in the microsieves with increasing concentrations of DOX, resulted in a reduction of the size of spheroid and viability of the cells. In addition a microﬂuidic platform to study the interaction of nanoparticle–aptamer conjugates with prostate cancer cells that expressed (LNCaP) or did not express (PC3) prostate speciﬁc surface antigen (PSMA) was developed [11]. For LNCaP cells, the binding of nanoparticles was higher in the presence of aptamer and decreased with increasing ﬂow rate, whereas in the case of PC3 the nanoparticles did not bind with or without aptamers (Fig. 3). Such advanced microﬂuidic culture models, where essentially any form of cell/tissue constructs (e.g., spheroids, hydrogels, and porous scaffolds) can be directly utilized on-chip for testing, can potentially be used for cytotoxicity and efﬁcacy assessment of drug delivery systems. 209 210 3 Q10 Fig. 1. The application of 3D scaffold-based multicellular tumor spheroids in drug testing. A–D) Confocal micrographs of live/dead staining showing (A) 2D culture and (C) 3D spheroid culture. B) 2D culture revealed signiﬁcant cell death after CdTe NP exposure. D) Spheroids showed much lower cell damage (especially in the central area) than the cells in 2D culture. E, F) SEM images of (E) 2D culture and (F) 3D spheroid culture at 24 h post CdTe treatment. E) In 2D culture most cells were dead with a large amount of cells detached. F) Cells in spheroids experienced much lower cellular damage than those in 2D culture. Reproduced with permission from Ref. [39]. Please cite this article as: N.S. Bhise, et al., Organ-on-a-chip platforms for studying drug delivery systems, J. Control. Release (2014), http:// dx.doi.org/10.1016/j.jconrel.2014.05.004 N.S. Bhise et al. / Journal of Controlled Release xxx (2014) xxx–xxx E C T E D P R O O F 4 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 R O 278 279 with regard to their vascular transport and toxicity. Some studies have already reproduced geometric features of vasculature, including straight channels [61–64], bifurcations [65,66], and a mixture of more complex features [67]. By mapping and replicating a microvascular network from a hamster muscle, a small microﬂuidic platform was created, reproducing several geometric features including bifurcations and tortuosities, as well as accounting for different shear stresses along the branches [67]. Another important parameter to be considered is particle size. A microﬂuidic vascular model was developed to study the differences in accumulation of micro- and nanosized spheres inside microchannels. It was reported that microspheres tend to locate more to the margins when compared to nanospheres [68]. This effect of size on accumulation, as well as hemodynamics and hemorheology, should be taken into account when designing drug delivery carriers targeted for vascular diseases and cancer. Additionally, these carriers also interact with blood components. A study using high concentrations of mesoporous silica nanoparticles showed that despite having no effects on platelet viability, it greatly increased platelet aggregation and adhesion to endothelium [62]. This emphasizes the evaluation of other parameters apart from viability when studying particle interaction with blood components. Another key aspect that vascular models are able to mimic is the inﬂuence of shear stress [63,65,69]. This is particularly important for nanoparticle delivery as it affects the endocytic uptake of nanoparticles by endothelial cells [69]. Narrowing in particular regions of the vascular system exhibits increased shear stress, which is an important factor in C 276 277 U N 275 R Fig. 2. The application of microﬂuidic bioreactors for drug testing. A) Schematic showing the tumor spheroid formation and culture in a microﬂuidic ﬂow chamber. B) Optical micrograph showing that MCF-7 cells could uniformly ﬁll all the traps. C) Optical micrographs showing the structure of the tumor spheroids: (left to right) phase contrast image and ﬂuorescence images showing cell membrane and nuclei. the development of thrombosis. A microﬂuidic model was developed to study the potential of shear responsive nanoparticles for targeting and treating embolic occlusions. The tissue plasminogen activator (tPA) loaded nanoparticles were able to dissolve preformed ﬁbrin clots inside this microﬂuidic channel. This in vitro observation was then validated in an ex-vivo mouse pulmonary embolism model, a shear-sensitive particle system allowed the use of a 100-fold lower dose of tPA to obtain the same lysing effect as achieved by free tPA (Fig. 4A) [63]. Such smart particles, which respond to biophysical cues, can target drugs more efﬁciently, reduce dosage and concomitantly lessen undesired secondary effects [63]. Vascular microﬂuidic models also take into account other aspects, such as particle functionalization [61,65]. Platforms can model the inﬂuence of particle functionalization combined with either shear stress [61] or geometric features as straight versus bifurcating channels [65]. Some studies have highlighted the effect of particle shape on adhesion to the channel walls (Fig. 4B) [66,70]. In case of an endothelialized channel, rod-shaped nanoparticles showed high speciﬁc targeting and lower non-speciﬁc accumulation as compared to sphere-shaped nanoparticles [70]. When engineering and designing novel carriers, one should take these shape effects into consideration. Microﬂuidic technologies have the potential for scalability and highthroughput analysis, and can characterize populations of nanoparticles in a quick and low cost manner [10], or even evaluate endothelial permeability [64]. Moreover, inherent characteristics of microﬂuidics, Please cite this article as: N.S. Bhise, et al., Organ-on-a-chip platforms for studying drug delivery systems, J. Control. Release (2014), http:// dx.doi.org/10.1016/j.jconrel.2014.05.004 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 5 P R O O F N.S. Bhise et al. / Journal of Controlled Release xxx (2014) xxx–xxx T C E R R O C 329 330 of action of drug carriers. A future challenge that still remains to be elucidated is the prioritization of parameters, and, perhaps, how to combine multiple features in a single, high-throughput capable, device. On-chip vasculature models are thus a relevant tool when considering the targeting of vascular diseases, tumor angiogenesis and usage of injectable drug delivery systems. N 327 328 such as laminar ﬂow, can be exploited to build superior models. For instance, hydrodynamic focusing [71] could be used for precisely targeting drug carriers to speciﬁc regions in microﬂuidic chips, and possibly study their subsequent transport to neighboring cells from a non-exposed region. Several of the above discussed models have the potential to leverage drug delivery by probing the efﬁcacy and mode U 325 326 E D Fig. 3. The application of microﬂuidic system for studying nanoparticle and cell interactions. A) Fluorescence images showing binding of rhodamine-labeled nanoparticles, without or with PSMA aptamer, to PSMA-positive LNCaP cell and PSMA-negative PC3 cells under static conditions. The left column shows phase contrast images of the cells and corresponding ﬂuorescence images of the nanoparticles are indicated in the right column. B) Fluorescence images showing binding of rhodamine-labeled nanoparticle–aptamer conjugates (red) to LNCaP cells under ﬂuid ﬂow conditions at ﬂow rates of 0.25, 1, and 4 μL/min. The cells were stained with Calcein AM (green) and DAPI (blue) to quantify cell viability. C) Quantiﬁcation of the number of nanoparticles bound to PC3 cells under static condition and to LNCaP cells under both static and ﬂuid ﬂow conditions. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) Reproduced with permission from Ref. [11]. Fig. 4. Microﬂuidic platforms provide insights on ﬂow dynamics and shape inﬂuence for carrier attachment. A) Stenotic regions have higher shear rate (top) and accumulate more nanoparticles (NP; bottom). B) Inﬂuence of particle shape (top) on attachment and accumulation in a 45° bifurcation, showing that disc-shaped particles attach more than spherical particles (bottom). Reproduced with permission from Ref. [63] and [66]. Please cite this article as: N.S. Bhise, et al., Organ-on-a-chip platforms for studying drug delivery systems, J. Control. Release (2014), http:// dx.doi.org/10.1016/j.jconrel.2014.05.004 331 332 333 334 335 336 6 N.S. Bhise et al. / Journal of Controlled Release xxx (2014) xxx–xxx 3.2. Cardiac platforms 3.3. Liver platforms 402 338 339 The need for targeting drugs for cardiovascular disease has become very critical [72]. Several drugs used for cardiovascular diseases have been subjected to FDA withdrawals [73]. Also a few commercial drugs used to treat other organ disorders have been identiﬁed to have adverse side effects on the cardiac tissue [74]. Inadequate screening models have become one of the major accountable factors for these failures [74,75]. With multitude of features to be researched for a drug and its carrier to successfully translate to clinics, testing them in the appropriate in vitro conditions has turned out to be the key aspect [16]. However, persisting difﬁculties in mimicking the physiology of the heart in vitro have hampered this process. Nevertheless, there are several reports on cardiac tissue engineering [76] and heart-on-a-chip [75] that promise to potentially emerge as physiologically relevant models for in vitro drug testing. Most of the developed platforms use primary cells derived from rats, however, more focus on integration of human cells with these models is required for mimicking human physiological response. Among the human cell sources, cardiomyocytes generated from human induced pluripotent stem cells have enormous potential to be pathophysiologically relevant [77]. They also facilitate the creation of platforms for mimicking genetic disorder in patient-speciﬁc cases, thereby paving the way for individualized therapy [77]. The culture of functional cardiomyocytes can beneﬁt from stimulation with electrical and mechanical forces [78], which cannot be reproduced in a simple cell culture setting. Additionally, to recreate a functional cardiac tissue, scaffolds can be used to recreate 3D tissue architecture [79]. The use of cardiomyocyte cell sheets has offered a possible platform for drug testing that exhibits an important physiological parameter of heart: a uniform contractile function. Stevens et al. [80] developed a scaffold free cardiac patch derived from human embryonic stem cells (hESCs) that showed electromechanical coupling with each beat. Shimizu et al. [81] fabricated these sheets with primary rat myocytes. When two of these sheets were overlaid, they exhibited a simultaneous and spontaneous beating after a week. Other attempts to record electrical stimulation have involved the use of cardiac tissue slices. Bussek et al. [82] implemented the use of guinea pig cardiac tissue slices to evaluate the performance of a potassium channel blocker. Even though different potentials were recorded when the slice was exposed to the drug, the experiment does not completely reproduce the effects in the human patient. Naturally, these reconstructions do not perform as the organ itself and fail to reproduce in vivo conditions but they can offer an advantage for further improvements by means of microﬂuidic platforms. Classically, most of the microﬂuidic platforms are fabricated using poly(dimethylsiloxane) (PDMS). To tune the stiffness of the PDMS surface for cardiomyocyte culture the microﬂuidic channels can be coated with a hydrogel of controllable matrix stiffness [14]. In a study by Annabi et al. two types of hydrogels, methacrylated gelatin (GelMA) and methacrylated tropoelastin (MeTro) were compared [14]. The cell attachment and alignment were dependent on the type of hydrogel used, whereas the beating was dependent on the stiffness of the matrix. In another report, cardiomyocytes were seeded on thin PDMS ﬁlms patterned with ﬁbronectin and cultured within a bioreactor [83]. The high-throughput format enabled testing of different geometric patterns of ﬁbronectin and monitoring up to forty thin ﬁlms in real-time in a single bioreactor. As the calcium dynamics of the cardiomyocytes are directly affected by disease conditions, Martewicz et al., developed a microﬂuidic platform where the calcium dynamics of the cardiomyocytes can be observed in real-time [84]. This platform could also control the oxygen concentration experienced by the cells, thus allowing induction of hypoxia. Concurrently, the differential response of calcium dynamics was monitored with varying oxygen concentration. These in vitro microﬂuidic platforms, with high-throughput capability, can accelerate the translation of a drug carrier to the clinic by recreating cardiovascular environment that better mimics human physiological conditions [75]. Drug hepatotoxicity is one of the main concerns in identifying new drugs [85], thus studying hepatoxicity of nanoparticles is very important for developing novel drug delivery platforms. Also with animal models failing to better predict outcomes of drug intake in humans [85], there is an extensive body of research focused toward developing in vitro models for evaluating hepatotoxity [86–89]. In vitro hepatic models aim at emulating functionality and mimicking the normal as well as pathophysiological architecture of liver. These models include 2D/3D mono- and co-cultures, hydrogel based engineered models, and organ-on-a-chip platforms of normal and diseased cells. Since most of the drug delivery and nanotoxicity studies are restricted to 2D cell culture plates [90–92], some important studies that describe the testing of nanoparticles in these platforms that more accurately predict in vivo behavior are highlighted. A recent study using lipid nanoparticles for siRNA delivery demonstrated the importance of using primary hepatocytes instead of immortalized cell lines for better translation of screening results to in vivo outcomes [93]. Assessment of nanoparticles should be evaluated within such clinically relevant models that can maintain the functionality of primary hepatocytes for long-term culture [86,94]. One such model used micropatterned collagen substrate for culturing primary hepatocytes in a high-throughput 24-well plate format [86]. In this model, primary hepatocytes were co-cultured with ﬁbroblasts to augment homotypic interactions with heterotypic interactions for maintaining the functionality of hepatocytes for a long period. This model represents a potentially useful platform for long-term analysis of liver nanotoxicity. In a study by Dragoni et al., the uptake and toxicity of AuNPs were investigated using precision cut rat liver slices [95]. The tissue slice model was used to evaluate the uptake of AuNPs by hepatocytes, Kupffer and endothelial cells present in the liver slice. However, tissue biopsies or slices cannot be used for high-throughput studies and show a rapid loss of functionality within days during in vitro culturing, thus limiting their application for long-term studies [96]. With extensive research on developing 3D hepatic spheroids for drug testing [87,88,97,98] and also with availability of highthroughput commercial platforms for formation of spheroids, liver spheroids represent a promising 3D construct for rapid clinically relevant evaluation of nanoparticles. Spheroids formed from primary human hepatocytes have been used for long-term drug studies [97]. In a work by Lee et al., the toxicity of gold nanoparticles and CdTe nanoparticles in 2D cell cultures and 3D spheroids were compared (Fig. 1) [39]. Results showed considerably reduced toxicity in spheroids when compared to 2D cultures and the reduction in toxicity was attributed to change in phenotype and complex cell–cell interaction that altered the transport of nanoparticles. Spheroids of cancerous hepatocytes have also been used as a model for mimicking in vivo tumor microenvironments as shown by England et al. [99]. They studied nanoparticle delivery to avascular regions in the spheroid core and used surface modiﬁcation strategy to enhance nanoparticle penetration within the tumor core. Nanoparticles exhibit differential behavior in static and ﬂow conditions [13,17], thus necessitating the development of dynamic microﬂuidic platforms for toxicity evaluation. Multiple biomimetic liver-on-a-chip platforms have been long established for drug toxicity testing [15,89,96,100]. Since liver is the major organ for drug metabolism, it is important to integrate a liver module with other organ modules to assess the cytotoxicity of prodrugs. In such models, the prodrugs are ﬁrst metabolized by the liver module before reaching the target organ of drug action. For example, Shuler and Sung developed a micro cell culture analog (μCCA) with 3D cultures of HepG2/C3A cells (as the liver module) and HCT-116 cells (as the tumor module), both in Matrigel, and Kasumi-1 myeloblasts (as the bone marrow module) in stiffer alginate gel (Fig. 5) [101]. The cytotoxic effect of Tegafur, an oral prodrug of 5-ﬂuorouracil (5-FU), on each organ analog was then 403 404 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 O R O P 359 360 D 357 358 E 355 356 T 353 354 C 351 352 E 349 350 R 347 348 R 345 346 O 343 344 C 342 U N 340 341 F 337 Please cite this article as: N.S. Bhise, et al., Organ-on-a-chip platforms for studying drug delivery systems, J. Control. Release (2014), http:// dx.doi.org/10.1016/j.jconrel.2014.05.004 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 7 C T E D P R O O F N.S. Bhise et al. / Journal of Controlled Release xxx (2014) xxx–xxx R E Fig. 5. The multi-chamber μCCA device. A) A PDMS μCCA device consisting of three chambers, which represented uterus, liver, and mammary tissues, respectively. B) Measurement of cell viability after treatment with 0.02% Triton. Triton treatment led to full mortality for all cell types. Reproduced with permission from Ref. [101]. 482 3.4. Lung platforms 483 484 The respiratory tract is one of the most signiﬁcant ports of entry in the human body. This can result in a number of infectious diseases as well as occupational pathology, but it also offers an appealing route of drug administration. The alveoli themselves offer a thin mucosal barrier (200–800 nm) with low enzymatic action, vast absorptive surface area (100 m2) and rapid access to the bloodstream while avoiding invasive procedures [104]. Aerosol nanocomplexes for gene therapy have proven to diffuse through the alveoli–capillary barrier in an efﬁcient manner compared to other particles due to their small size [105]. Other drug 476 477 478 479 485 486 Q3 487 488 489 490 491 O 474 475 C 472 473 N 470 471 U 468 469 R 480 481 tested using such a three organ-on-a-chip platform, where they found that the μCCA was capable of reproducing the metabolism of Tegafur to 5-FU in the liver module, whereas the metabolic conversion of Tegafur could not be accomplished with 2D cultures in 96-well plates. In a work by Wagner et al., a multi organ-on-a-chip model was developed for long-term culture by combining liver microtissues made of spheroids of HepaRG and hepatic stellate cells with skin biopsies [15]. Their microﬂuidic system integrated an inbuilt peristaltic pump with a total media volume of 300 μL, thus enabling cross-talk between skin and liver compartments proven by the dynamic level of albumin in mono- and co-culture systems. Such models could be channeled toward evaluating the toxicity of nanoparticles used in the cosmetic industry. Infectious disease models that recreate the hepatic stage of infections, such as hepatitis C viral infection, will also prove signiﬁcantly important for clinical translation of nanotherapeutics [102,86,103]. 467 delivery systems like zinc oxide nanoparticles (ZnONP) [106], silver nanoparticles (AgNP) [107] and doxorubicin micelles [108] have been used with 2D culture platforms to study the effect on cell function and apoptotic response. Multi-well plate cultures of MCF-7 and A549 lung cancer cell lines have been used to evaluate nanoparticles [109]. But these systems fail to reproduce a physiological environment for drug testing. To recreate the complexity of the human lung's structure, the use of 3D microﬂuidic systems that can introduce ﬂuid and solid mechanical forces [110] is essential to imitate the human alveoli environment. In an effort to develop biomimetic ‘lung-like’ microsystems, Huh et al. reproduced the alveolar–capillary interface in vitro by creating a layer of alveolar epithelial cells and endothelial cells both seeded on opposite sides of a porous ECM-coated membrane (Fig. 6A) [13]. The inﬂammatory and toxic effect of silica nanoparticles was evaluated by particle absorption through a vacuum strain model that mimics the physiological breathing motion. This cyclic mechanical force augmented the nanoparticle translocation across the alveolar–capillary barrier, enhancing the toxic and inﬂammatory response as indicated by increased reactive oxygen species (ROS) generation and intercellular adhesion molecule-1 (ICAM-1) expression. The nanoparticle translocation behavior was also evaluated in a whole mouse lung ventilation–perfusion model to demonstrate the signiﬁcance of using lung-on-a-chip systems to better predict physiological response. Yu et al. fabricated a ‘lung-likecompartment’ in one of the four multi-channel 3D microﬂuidic cell culture platform that also incorporated kidney, liver and adipose cells [111]. They evaluated gelatin microspheres as a controlled-release system for TGF-β1 encapsulated within the microspheres, and used A549 Please cite this article as: N.S. Bhise, et al., Organ-on-a-chip platforms for studying drug delivery systems, J. Control. Release (2014), http:// dx.doi.org/10.1016/j.jconrel.2014.05.004 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 8 N.S. Bhise et al. / Journal of Controlled Release xxx (2014) xxx–xxx A Alveolar epithelium C B Vacuum compartment Nanoparticles Cells Tubular cells Vacuum regulator Porous membrane Endothelium ECM-coated membrane Lower layer Interstitium Cell chamber Stretched F Unstretched Other organs that are important for studying nanocarrier mediated drug delivery are the kidney, spleen and gut [113–115]. Kidney and spleen play an important role in the clearance of systemically delivered nanoparticles. The kidney is one of the major excretion pathways in the human body, playing a key role in blood ﬁltration and water metabolism [116]. A study indicated that the glomerular ﬁltration barrier contributed toward the dissociation of siRNA from its cationic cyclodextrin carrier. Thus it is important to study the behavior of nanoparticles in the renal system especially when administered intravenously [113]. To replicate renal conditions, such as ﬂuid ﬂow and shear stress [117], Jang et al. created a microﬂuidic device that mimics the in vivo renal tubular environment [118]. The device consists of a PDMS platform designed to contain both ‘luminal’ and ‘tubular’ chambers separated by a porous membrane. Human primary kidney proximal tubular epithelial cells were grown on the ‘tubular’ side of the membrane while media was perfused into the ‘luminal’ side (Fig. 6B) [118]. The device demonstrated differential cell histologic arrangement and protein expression when using static and dynamic ﬂow conditions. The dynamic condition revealed an augmentation in the reabsorption function of the kidney. In the work by Shintu et al., a microﬂuidic device was used to measure the ‘metabolomic’ ﬁngerprint of the kidney using high-throughput screening approach [119]. This method provides for rapid screening of a compound target library that can be of further use in the drug delivery and discovery research. Spleen plays an important role in ﬁltering the bloodstream and is a vital part of the body's immunological response [120]. It forms a fundamental defense component against certain bacteria such as Streptococcus pneumoniae. Yung et al. [121] created a blood cleansing a platform that uses magnetic opsonins to target Candida albicans [121]. The pathogen 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 C E R 538 539 R 536 537 O 530 531 C 528 529 U N 526 527 3.6. Tumor platforms 579 The complex in vivo microenvironment of a tumor presents a unique challenge for the efﬁcient delivery of nanotherapeutics to the tumor site. The key features of this complex microenvironment include heterogenous vascularization, increased interstitial pressure and inadequate lymphatic drainage [122]. Signiﬁcant advances have been made in the chemical discovery, design and synthesis of chemotherapeutic drugs and diagnostic agents. However, their adverse side effects in healthy tissues greatly limit the maximum tolerated dose and thereby reduce their therapeutic efﬁcacy [123–125]. Researchers and pharmaceutical companies are pursuing the use of nanoparticles for encapsulating anti-cancer drugs and delivering them speciﬁcally to the tumor site as a potential solution to reduce the side effects [126]. Nanoparticles with surface targeting moieties have shown efﬁcacy in in vitro screening studies, but fail to show the same performance during in vivo trials. Several delivery barriers make nanotherapeutics ineffective within the human body and hinder successful clinical translation of these anticancer drug carriers. To solve this delivery problem, it is important to gain an understanding of the nanoparticle transport through the bloodstream, distribution at the target tissue and subsequent uptake by the cells. Preclinical models that mimic the in vivo 3D tumor architecture and dynamic ﬂow conditions are important for studying these delivery parameters and for toxicology assessment. The integration of microﬂuidic networks with 3D tissue engineered cultures offers a unique opportunity to probe nanoparticle transport barriers in a controlled manner. Additionally, these tumor-on-a-chip platforms can be used for studies to optimize nanoparticle dosage, to generate gradient drug concentrations and to develop personalized 580 581 P 534 535 524 525 563 D 3.5. Other organ platforms 522 523 is then removed by a micromagnetic–microﬂuidic separator and cleared from the blood. Such models are yet to be exploited for drug studies and can be of great use for optimizing drug delivery systems. Among other organs that could be used for drug testing is the guton-a-chip created by Kim et al. [55] This device recreates intestinal human environment and could be used to test oral drug absorption. The device consists of two chambers divided by a porous membrane (Fig. 6C). The membrane separates a columnar epithelium of Caco-2 cells from another chamber that lies below the porous structure. In addition, Lactobacillus rhamnosus GG bacteria was cultured together with the epithelial cells to enhance the barrier function of cells during this period. The inclusion of a Lactobacillus that resembles the intestinal ﬂora offers a step forward toward simulating the intestinal lumen. Future design strategies ought to consider the development of an organ-like platform that can mimic the in vivo complexity of the gut environment in a simple and efﬁcient way. E 533 520 521 T 532 cells to recreate a human lung microenvironment for drug testing. In the device, common media goes through this ﬁrst chamber of A549 cells and TGF-β1 encapsulated microspheres to continue to the other ‘organs’. The ‘lung’ compartment revealed an enhanced cellular function compared to the other uncompromised ‘organs’ showing a compartmental separation between the chambers and imitating the partial cross-talk between organs that occurs in the human body. Future endeavors must be oriented to combine the multiple cell types in the alveoli and the mechanical forces involved in ventilation. The reconstruction of a reliable alveolar–capillary barrier without a complex culturing process [112] as well as developing novel particles with effective transport properties are also upcoming challenges. A system that combines all of the above would be a reliable model that represents the inner organ conditions in the human body. 519 R O O Fig. 6. A) Schematic of the lung-on-a-chip platform developed to study nanoparticle transport. The transport of nanoparticles across the alveolar–capillary interface is augmented significantly when vacuum is applied to the side channels of the lung platform. Adapted from Ref. [13]. B) Schematic of the kidney-on-chip platform with two chambers divided by a porous membrane that separates the ‘luminal’ from the ‘tubular’ space recreating the in vivo conditions in the kidney. Adapted from Ref. [118]. C) The gut-on-a-chip platform includes two compartments divided by a porous membrane separating Caco-2 cells from the bottom chamber. A vacuum regulator is attached to the side chambers to assess the behavior of the cells under the effect of strain. Adapted from Ref. [55]. Please cite this article as: N.S. Bhise, et al., Organ-on-a-chip platforms for studying drug delivery systems, J. Control. Release (2014), http:// dx.doi.org/10.1016/j.jconrel.2014.05.004 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 N.S. Bhise et al. / Journal of Controlled Release xxx (2014) xxx–xxx R O O F 630 631 P 628 629 D 626 627 E 624 625 T 622 623 C 620 621 E 618 619 R 616 617 R 614 615 O 613 C 611 612 different concentrations and combinations of anti-cancer drugs on a parallel culture of PC3 prostate cancer cells. Their platform could capture the synergy between different sensitizer drugs (DOX or mitoxantrone in this case) and TRAIL (TNF-alpha Related Apoptosis Inducing Ligand). Such high-throughput systems can be used for screening and optimizing combinatorial delivery strategies using nanocarriers for cancer therapeutics before testing in humans [135,136]. Further, microﬂuidic systems that allow for testing anti-cancer drug sensitivity to a speciﬁc patient are promising for developing clinically relevant nanotherapeutics for personalized treatments [137]. The study by Zhao et al. addressed a key aspect of anti-cancer drug assessment, namely the integration of quantitative data acquisition methods with the on-chip platform for analyzing the cell apoptosis induced by the chemotherapeutic drug. This was achieved by using Annexin V conjugated quantum dots and Calcein AM as dual apoptotic probes [138]. Such real-time optical probing methods will be useful for monitoring the behavior of nanotherapeutics in the tumor-on-a-chip platforms. New delivery systems have been proposed in recent years to meet the demand for developing safer chemotherapeutic strategies for breast cancer, one of the commonest types of tumor in women [139]. These include liposomal nanoparticles used to deliver DOX [140] and albumin-bound Paclitaxel delivery system [141]. The anatomy of the mammary gland presents a number of challenges when it comes to clinical translation of these theranostics. Two of the major barriers are: the presence of a system of ducts in a branched manner with different sizes N 609 610 drug screening and treatment strategies [127]. For example, in a recent report by Albanese et al., a tumor-on-a-chip model was developed to investigate the transport behavior of AuNPs through 3D tumor-like spheroids of human melanoma cells immobilized in a PDMS chamber (Fig. 7A) [128]. Their microﬂuidic device allowed precise control of the media ﬂow conditions and the spheroids were coated with a laminin layer that acted as a barrier to AuNP transport, thus mimicking conditions found in vivo at the tumor site. The tissue penetration and accumulation of ﬂuorescent NPs under physiological ﬂow conditions were monitored in real-time. The nanoparticle diameter, surface functionalization and the ﬂow conditions in the microenvironment were shown to affect the accumulation of AuNPs near the tumor tissue (Fig. 7B, C). The nanoparticles functionalized with targeting groups accumulated in the periphery and failed to penetrate deep inside the core. Such studies provide critical insights to help design better nanoparticles for improved in vivo targeting. Pellegrino et al. have also designed a microﬂuidic platform to evaluate magnetic thermoresponsive polymers for controlled release of chemotherapeuitc drugs. This preliminary study was conducted without cells in PBS, but this system can be further developed for studying drug delivery to cells using thermoresponsive carriers [129]. Several other tumor-on-a-chip platforms have been reported to study the complex tumor microenvironment and to screen drugs [127,130–135]. In a recent study by Kim et al., a fully automated microﬂuidic system was developed for high-throughput screening of U 607 608 9 Fig. 7. Studying the nanoparticle (NP) tissue transport behavior using tumor-on-chip platform. A) The schematic of the microﬂuidic chip assembled on top of a microscope stage. B) The schematic (left), image (center) and graph (right) showing the effect of NP size on tissue accumulation. Four different sizes, 40 (red), 70 (blue), 110 (green) and 150 (orange) of PEGylated NPs were investigated. C–D) The effect of NP functionalization on tissue accumulation: C) PEGylated NPs and D) iron-transporting transferring (Tf) protein functionalized NP. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) Reproduced with permission from Ref. [128]. Please cite this article as: N.S. Bhise, et al., Organ-on-a-chip platforms for studying drug delivery systems, J. Control. Release (2014), http:// dx.doi.org/10.1016/j.jconrel.2014.05.004 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 N.S. Bhise et al. / Journal of Controlled Release xxx (2014) xxx–xxx 681 682 Pharmaceutical companies involved in drug discovery continuously aim to both reduce the cost of their research and speed up the development of new drugs. In this process, pre-clinical animal studies are the most expensive and time-consuming part, while not assuring human in vivo signiﬁcance. In vitro organ-on-a-chip platforms can revolutionize this process by making it less expensive, while representing a reliable way for drug discovery. However, further advances in the ﬁeld are needed to overcome engineering and biological challenges before entirely assessing their value as an efﬁcient and robust platform for screening nanotherapeutics. The ﬁnal goal is to develop a body-on-a-chip platform for systemic evaluation of drug delivery vectors. But current efforts are directed toward the development of individual organ platforms limiting their range of applications to a speciﬁc organ's functions. For example, liver being the most relevant organ for drug toxicity and metabolism, the development of a functional liver-on-a-chip system is the most important challenge currently faced by researchers. As mentioned in Section 3.3, several attempts have been developed in this direction, but the available platforms are yet unable to fully capture the complexities of in vivo drug metabolism in a robust manner. For developing a universal platform, the ﬂexibility and modularity of this technology, based on extremely versatile microfabrication techniques, should be exploited. The possibility of culturing different cell types employing the identical standard setups could lead to the development of several organ models on demand. The results obtained with such systems could be easily compared, and relevant drug-speciﬁc cell responses can be quickly noticed. In this context, the simplicity of the setup and the implementation of several parallel experiments will be a key aspect (e.g., for testing drug–dose responses as well as different target organs). Indeed, in many cases the platforms proposed so far have been optimized for one or two main parameters and the comparison between them is highly complicated. A crucial challenge is to build platforms that deliver comparable results, both to other systems and among different research groups. Furthermore, the inherent modularity of these systems allows for the development of several on-chip modules by only changing cell type. Interconnection of such modules would mimic the communication between different organs, making the search for potential new drugs a more effective and comprehensive process. However, highthroughput and automation, which are intrinsic capabilities of these 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 Acknowledgments 781 D E 683 684 T 676 677 C 674 675 E 672 673 R 670 671 R 668 669 O 666 667 C 664 665 U N 663 F 4. Future directions & conclusion 661 662 721 722 O 680 659 660 systems, yet remain a challenge. Developments and efforts on the ﬁeld of biosensors should be coupled to organs-on-a-chip, so that the evaluation of outcomes could be done in a quantitative and real-time manner. An ideal platform should include these sensors to monitor, in situ, various physical/chemical parameters, such as O2, pH or even proteins. A robust system has to show stable experimental conditions in order to correlate any signiﬁcant change in those parameters to the direct effect of the candidate drug. Commercially available sensors, and microfabricated ones, that allow a quick integration in a microﬂuidic system, will be combined to previously developed organ-on-a-chip platforms. Future platforms should integrate parenchymal and nonparenchymal cells, and allow long-term experiments without experiencing irreversible changes in cell function and behavior. Parameters such as cell proliferation and viability are quite immediate to assess, but changes in proteomic, genomic and epigenomic levels, that can dramatically affect drug metabolism, can be more difﬁcult to monitor. A stable and robust system to address these issues is a mandatory challenge that will be faced in the next generation organ-on-a-chip systems. New organ-on-a-chip platforms for drug discovery should also consider any problems arising from the interaction between a candidate drug vector and the microﬂuidic systems. Mathematical and computational ﬂuid dynamics (CFD) studies can help in this direction, providing a mechanistic understanding of drug carrier attachment in organ-on-achip systems. The known differential behavior of nanoparticle shape in terms of cell adhesion and distribution can be assessed by CFD, and potentially aid in the generation of better and more comprehensive models. For instance, in a computational study nanorods were shown to contact and adhere to the wall much better than spherical counterparts [147]. Moreover, a CFD model of a branched blood vessel showed that bifurcating sections had higher nanoparticle accumulation when compared to straight ones. In the same study, authors were able to model the trajectory and adhesive forces of nanoparticles under shear ﬂow [148]. Together, these and other works [149], set the stage for future CFD studies on particle–particle, particle–cell and particle– microﬂuidic device interaction (accumulation and adhesion). More complex CFD studies on these phenomena, especially within organon-a-chip, can help building next generation drug delivery systems, by better understanding their underlying biophysical aspects. From a material point of view, the devices developed in this ﬁeld show important differences that can determine different cell responses as well as affect drug concentration into the system, due to drug nonspeciﬁc adsorption to the channel walls. Though PDMS and polystyrene are the most popular materials due to several advantages [150], such as optical transparency and cell compatibility, other plastic materials have been used and a detailed comparison of their advantages/drawbacks in drug screening tests is missing. If an ideal material will be not found, research groups could decide to cover the microchannels with a cell layer for preventing drug adsorption, as successfully reported by Schimek et al. [151]. In the long run, organ-on-a-chip systems will be used also for personalized drug screening. As these systems will show strong standardization and reliability, the possibility to use patient-derived iPSCs, produced in vitro and then terminally differentiated, can solve one of the most important problems in disease treatment — patient-to-patient drug response variability — avoiding long and ineffective drug treatments and minimizing drug toxicity. In summary, organ-on-a-chip platforms for studying drug delivery systems hold great promise and some aspects of them have been investigated, but their full applicability and potential have yet to be realized. P 678 679 [142] and high pressure buildup during a diagnostic liquid procedure like the ﬂuid nipple aspiration or ductal lavage. Due to the increased pressure in the smaller ducts, the delivered theranostics fail to reach their target and in 20% of the patients these methods provide an inadequate number of cells to determine a diagnosis [143]. In their effort to reproduce the ductal system in a mammary gland and address the difﬁculties mentioned above, Grafton et al. developed a microﬂuidic breaston-a-chip system. This system consisted of laminin coated microﬂuidic channels with decreasing ductal size to culture a polarized monolayer of human mammary epithelial cells. In this model, they proposed the use of iron oxide superparamagnetic sub-micron particles (SMPs) with an external magnetic ﬁeld guidance for theranostic purposes of mammary gland neoplasias [144]. To further investigate breast neoplasia models, the same group attempted the reconstruction of tumor growth in the mammary gland. Knowing that most of the human mammary lesions ascend from the terminal lobular ductal units [145], they used a platform to reproduce the disease on a chip. Human breast epithelial cells and nodules (3–5 cells) of tumorigenic human mammary carcinoma cells were seeded in an acrylic hemichannel system leading to a model that mimics the tumor's organization in human biopsies [146]. Using patient biopsies in future studies, the tumor-on-a-chip models described above could serve as a personalized medicine platform to optimize an individualized chemotherapy regimen. 657 658 R O 10 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 The content is solely the responsibility of the authors and does not 782 necessarily represent the ofﬁcial views of the awarding agency. 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E 1124 1125 1126 Q9 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 13 Please cite this article as: N.S. Bhise, et al., Organ-on-a-chip platforms for studying drug delivery systems, J. Control. Release (2014), http:// dx.doi.org/10.1016/j.jconrel.2014.05.004 View publication stats 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221

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