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

Journal of Controlled Release 190 (2014) 82–93

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Journal of Controlled Release

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Review

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

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,⁎a Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, 02139, USAb Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, 02139, USAc Doctoral Programme in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology, Institute for Interdisciplinary Research, University of Coimbra, 3030-789 Coimbra, Portugald Biocant — Biotechnology Innovation Center, 3060-197 Cantanhede, Portugale Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston 02115, USAf Department of Physics, King Abdulaziz University, Jeddah 21569, Saudi Arabia

⁎ Corresponding author at: Division of BiomedicalMedicine, Brigham and Women's Hospital, Harvard MedUSA.

E-mail address: [email protected] (A. Khadem

http://dx.doi.org/10.1016/j.jconrel.2014.05.0040168-3659/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t
a r t i c l e i n f o
Article history:Received 26 February 2014Accepted 2 May 2014Available online 10 May 2014

Keywords:Organ-on-a-chipDrug deliveryNanoparticleDrug screeningToxicity

Novel microfluidic tools allow new ways to manufacture and test drug delivery systems. Organ-on-a-chipsystems — microscale recapitulations of complex organ functions — promise to improve the drug developmentpipeline. This review highlights the importance of integrating microfluidic networks with 3D tissue engineeredmodels to create organ-on-a-chip platforms, able to meet the demand of creating robust preclinical screeningmodels. Specific examples are cited to demonstrate the use of these systems for studying the performance ofdrug delivery vectors and thereby reduce the discrepancies between their performance at preclinical and clinicaltrials.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.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

The rapidly developing field of nanomedicine can significantly im-pact human disease therapy [1,2]. The research progress accomplishedin this field, over the last few decades, has led to the development ofnanomaterials, useful for designing carriers that deliver therapeuticpayload to diseased cells. An ideal drug delivery system should beeasy to manufacture and scale-up, low cost, biocompatible, biodegrad-able, possesses a high drug loading capacity and can be targeted to thesite-of-interest in the body. Nanocarriers, also routinely referred to asnanoparticles, are a class of drug delivery systems that range in sizefrom about 50 to 200 nm, allowing them to efficiently translocate acrossthe cell membrane barrier.

From a therapeutic standpoint, nanocarriers can prolong thesystemic circulation time of the drug and significantly reduce adverseside effects caused by off-target delivery at healthy tissue sites. This con-trolled release of drugs reduces themagnitude of overall drug exposurerequired for a therapeutic effect, thus avoiding higher drug doses andconsequent adverse effects. A wide variety of drugs, including hydro-phobic and hydrophilic small molecules, as well as biomacromolecules,

Engineering, Department ofical School, Cambridge 02139,

hosseini).

can be encapsulated within nanoparticles by tailoring the chemistry ofnanomaterials, polymeric or inorganic/metallic, to achieve the desiredencapsulation capability and release kinetics. The first use of nanoscalesystems for drug delivery was reported in the 1970s, when liposomalTrojan horse nanoparticles were used for treating lysosomal storagedisease [3,4]. Nanoparticles have also been developed as diagnosticagents to enhance the sensitivity for imaging techniques, includingX-ray computed tomography (CT) and magnetic resonance imaging(MRI). An increase in available techniques to engineer more preciseand sophisticated nanomaterials, and a deeper understanding of diseasebiology have catapulted a new generation of nanotherapeutics withimproved properties.

The above-mentioned advantages make nanoscale drug deliverysystems appealing to the pharmaceutical companies and healthcareregulatory agencies. However, in spite of these rapid bench-side devel-opments, the translation of therapeutic nanoparticles to the commercialpipeline has been less impressive [5]. Very few systems have beenapproved by the Food and Drug Administration (FDA), includingDoxil, a liposomal formulation encapsulating the chemotherapeuticdrug Doxorubicin, and Abraxane, based on the nanoparticle albumin-bound (nab) technology to deliver Paclitaxel, a widely used drug forbreast and pancreatic cancer [6]. This slow pace of bench-to-bedsidetranslation can be attributed to several challenges, the most criticalbeing the lack of robust preclinical tissue culture platforms that canmimic in vivo conditions and predict the performance of these nanopar-ticles within the human body.

83N.S. Bhise et al. / Journal of Controlled Release 190 (2014) 82–93

The development of microfluidic platforms for nanoparticle synthe-sis has shown to overcome several disadvantages of the traditional bulksynthesis methods such as scalability and batch-to-batch variability[7–9]. Microfluidic approaches have also been used as a tool for moresophisticated, faster andhighly efficient characterization of the biophys-ical properties of nanoparticles [10,11]. Additionally, the application ofmicrofabrication techniques to tissue engineering aided in the creationof physiologically relevant disease models. Establishment of these tech-niques has paved the way for robust advances in tissue culture systemsintegrated with microfluidic networks [12]. More recently, the demandfor high-throughput drug screening platforms with better preclinicalpredictability has translated into major developments in the organ-on-a-chip systems [13–15]. This review presents recent advances inin vitro tissue culture models by primarily emphasizing on organ-on-a-chip platforms useful for studying the performance of drug deliverynanotherapeutics. The current challenges in the development ofdrug delivery systems are highlighted and the use of organ-on-chipsas a potential solution is discussed by presenting specific examples ofrelevant proof-of-concept studies.

2. Limitations of current culture platforms used for developing drugdelivery systems

Several parameters need to be studied for developing nanoparticlesfor clinical use. These include studying the fate of the nanoparticlesinside the body and its toxicological effects, the mode of binding andinternalization at the cellular level, the stability of the nanoparticleswith 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 arethe major levels at which clinical translation of nanotherapeutics facesset-back [16]. On the manufacturing front, scaling the small lab synthe-sis techniques to the large-scale production of nanoparticles has beenchallenging for the pharmaceutical companies [8]. Meanwhile, screen-ing for the toxicity and efficacy suffers from the paucity of preclinicalmodels that would robustly predict the nanoparticles' behaviorinside the human body [16]. For simultaneous evaluation of theabove-mentioned parameters, predictive in vitro platforms are essentialwhile developing drug delivery vectors [13,17].

The current gold standard for preclinical testing of nanotherapeuticsis in vivo studies. These do not accurately predict human responsesdue to inter-species difference in genetic makeup, along with beingextremely time-consuming, expensive, low-throughput and raisingethical concerns. The resolution for whole-animal imaging methods islimited, hindering visualization during transport of the theranosticagents in the target tissue. Being unable to reproduce its preclinical per-formance, many drug delivery systemswhich pass the preclinical phasefail to address the toxicity and efficacy effects when compared to theirfree drug counterparts in human clinical trials [5]. Strikingly, the mainreason cited for this effect is the use of animal models for optimizationduring drug carrier design [5], which brings back the obvious drawbackof a certain degree of physiological irrelevance between human andanimal models.

Animal models need to be complementedwith sophisticated in vitroplatforms tofill this gap. In current in vitro studies, drug delivery carriersare commonly tested in two-dimensional (2D) monolayer cell culturemodels. These 2D cultures involve growing on top of a flat substrate(e.g., glass or polystyrene) a monolayer of single or multiple cell typesthat are either freshly isolated from human/animal tissues (primarycells) or are already established, immortalized cell lines. In these setups,drug delivery systems are usually mixed with culture media anddirectly applied on the cell monolayers, after which cellular responsesare recorded. Among several published studies [18–21], the work ofXia and colleagues on the cellular uptake of gold nanoparticles(AuNPs) by SK-BR-3 breast tumor cells [22], stands out by devising anovel testing method. After culturing the cells on a piece of glass, the

substrate was carefully reversed and placed upside down beforeAuNPs, with different shapes and sizes, were added in the culturemedia. Such an approach successfully avoided the issues caused byrapid sedimentation of nanoparticles. Indeed, the amount of cellularuptake of nanoparticles in upright and inverted cultures was foundto significantly depend on the rate of diffusion/sedimentation of thenanoparticles.

In spite of these novel approaches for 2D cell culture, it is graduallyrealized that there are many shortcomings with these “flat” models tomimic the complex three-dimensional (3D) in vivo microenvironment,wherein the cells and extracellular matrix (ECM) exist in well-organized architectures. Moreover, the nanoparticle delivery efficacydiffers considerably between 2D and 3D culture platforms [23]. Primarycells usually have a limited lifespan, undergo rapid phenotypic alter-ations, 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 cul-ture systems that can better recapitulate in vivo tissue functions. Multi-cellular spheroids are important 3D models for researchers [25–29].These spheroids are formed by spontaneous aggregation of multiplecells held together by ECM secreted by residing cells. The apoptotic/necrotic core of the spheroids contrasts with the proliferative cell layerson the periphery, providing a better mimic of in vivo tumor environ-ment. Due to the importance and long-time usage of multicellularspheroids in both pharmaceutical studies and regenerative medicine,researchers have developed sophisticated methods that allow efficientfabrication of uniform spheroids at relatively large scales, includingthe use of hanging drops, non-adhesive microwells, rotation cultures,or 3D porous scaffolds [30–37]. Multicellular tumor cylindroids havebeen used to study the effect of charge on the uptake of fluorescein iso-thiocyanate (FITC) or doxorubicin (DOX)-conjugated AuNPs loadedwith drugs, where diffusion is permitted only from the periphery tothe center [38]. Kotov and co-workers directly utilized tumor spheroidsfor toxicity testing of CdTe quantum dots and AuNPs [39]. The toxiceffects of these nanoparticles were compared with conventional 2Dcultures, to reveal different responses of cells in terms of morphology,particle distribution, membrane integrity, mitochondrial activity, andapoptosis (Fig. 1).

Besidesmulticellular spheroids, hydrogels and porous scaffolds havealso been widely employed for constructing 3D tissue models at largersize scales [40–42]. There are a number of advantages associated with3D cultures within a matrix. For example, the mechanical properties ofthe gels can be precisely modulated, which have been shown to deter-mine the phenotypic behaviors of the cells [43–45]; the matrices canbe fabricated to possess various hierarchical structures and any desiredshape to accommodate specific target tissues. As an example, Huangand co-workers demonstrated that cancer cells became more tumori-genic when cultured in a fibrin gel with a stiffness of approximately90 Pa, as shown by in vivo tumor formation in mice even when onlyvery few (10 or 100) tumor cells were injected, whereas the same num-ber of tumor cells from stiff 2D substrates could not induce the forma-tion of tumors [46]. Moreover, Mooney et al. cultured OSCC-3 oralsquamous carcinoma cells within porous poly(lactide-co-glycolide)(PLG) scaffolds to create an in vitro tumor model [47]. They arguedthat tumor cells cultured in PLG scaffolds could better recapitulatetheir in vivo states than in 3D Matrigel or 2D substrates as shown bytheir morphological appearances, proliferation rates, distribution ofoxygen concentrations, and secretion patterns of biomolecules.

Although static culture systems based on multicellular spheroids or3D matrices can recapitulate the in vivo functionality of tissues muchbetter than 2D cultures, they fail to present dynamic flow conditionsthat the cells usually experience in the body. The absence of homoge-nous perfusion results in improper gas and nutrient exchange throughthe core of the constructs. Additionally, the gravitational settling ofnanoparticles in static conditions affects the outcome of dosage

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optimization studies. Evaluating the drug carrier in static in vitro assayswithout considering mechanical forces and fluid flow has shown in-stances of false positives [13,17,22]. Hence, perfusion-based dynamicculture systems in bioreactors have become increasingly popular. Atthe same time, owing to the rapid development of microfluidics, thesize of these bioreactors is being constantly reducedwhile the function-ality is improved, enabling the development of improved systems fordrug toxicity and efficacy studies [48–52]. For example, Lee and co-workers constructedmicrofluidic chambers containing arrays of hydro-dynamic traps, which was used to immobilize around ten tumor cellsper trap (Fig. 2A). The entrapped tumor cells aggregated in the trapsto form small spheroids (b50 μm) within several hours (Fig. 2B, C)[53]. Similarly, Cheung and co-workers developed a type of cell culturechipwith arrays ofmicrosieves for efficient trapping of larger cell aggre-gates [54]. Treating the multicellular spheroids of LCC6/Her2 breasttumor cells in themicrosieves with increasing concentrations of DOX, re-sulted in a reduction of the size of spheroid and viability of the cells. In ad-dition 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 de-veloped [11]. For LNCaP cells, the binding of nanoparticles was higher inthe presence of aptamer and decreasedwith increasing flow rate, where-as in the case of PC3 the nanoparticles did not bind with or withoutaptamers (Fig. 3). Such advanced microfluidic culture models, where

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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) 3Dspheroid culture. B) 2D culture revealed significant cell death after CdTe NP exposure.D) Spheroids showed much lower cell damage (especially in the central area) thanthe cells in 2D culture. E, F) SEM images of (E) 2D culture and (F) 3D spheroid culture at24 h post CdTe treatment. E) In 2D culture most cells were dead with a large amount ofcells detached. F) Cells in spheroids experienced much lower cellular damage than thosein 2D culture.Reproduced with permission from Ref. [39].

essentially any form of cell/tissue constructs (e.g., spheroids, hydrogels,and porous scaffolds) can be directly utilized on-chip for testing, can po-tentially be used for cytotoxicity and efficacy assessment of drug deliverysystems.

3. Organ-on-a-chip platforms as a potential solution

Recent innovations in microfluidic technologies can provide novelsynthesis and screening methods to address issues of scalability,batch-to-batch variability, and poor predictability that limit the paceof clinical translation of nanotherapeutics [16]. The integration ofadvanced 3D tissue engineered constructs with microfluidic networksystems, termed ‘organ-on-a-chip’, provides a novel platform for betterpreclinical testing of drug delivery systems. Easy manipulation ofmicro-liter volume of liquids has made these models a platform wherescaling and dynamic crosstalk between cells can be achieved [15]. Thesystem geometry and structures recreate physiological length scales,concentration gradients, and the fluid flow generates mechanicalforces that recapitulate the in vivo microenvironment experienced bycells [13,55]. Thus, these highly biomimetic platforms overcome thedrawbacks with conventional tissue culture models as highlightedin the previous section [16]. By better mimicking the physiologicalconditions and more accurately predicting the effect of drug carriers,these sophisticated in vitro screening models can fill the gap betweenthe outcomes of animal studies and human clinical trials [16].

Since lung-on-a-chip [13,56], the field of biomimetic organs-on-a-chip has expanded rapidly to encompass several organs includingliver, kidney, heart, gut, breast, and blood vessels [55,57–59]. Thesestudies have already shown that organ-on-a-chip platforms can gener-ate responses similar to those observed in vivo toward nanoparticles[13,17]. Furthermore, multiple organ modules can be interconnectedin a physiologically relevant scale and organized to form a human-on-a-chip platform [60]. These systems can be used to study the fate ofnanoparticles. The information obtained from such studies wouldallow rapid testing of new therapeutic designs. Thus, the use of physio-logically relevant organ-on-a-chip models as a studying tool for drugdelivery systems can accelerate the clinical translation of nanoparticles.The following sections of this review, ordered by organ-type, willdiscuss the progress, advantages and challenges of existing organ-on-a-chip platforms, in the context of testing of drug delivery systems.

3.1. Vascular platforms

Nanoparticles introduced intravenously have to traverse throughthe vascular endothelial barrier to reach the target tissue. It is thus im-portant to study the interactions of these carriers with the vasculaturewith regard to their vascular transport and toxicity. Some studies havealready reproduced geometric features of vasculature, including straightchannels [61–64], bifurcations [65,66], and a mixture of more complexfeatures [67]. By mapping and replicating a microvascular networkfrom a hamster muscle, a small microfluidic platform was created,reproducing several geometric features including bifurcations andtortuosities, as well as accounting for different shear stresses alongthe branches [67]. Another important parameter to be considered is par-ticle size. Amicrofluidic vascularmodelwas developed to study the differ-ences in accumulation of micro- and nanosized spheres insidemicrochannels. It was reported that microspheres tend to locate moreto the margins when compared to nanospheres [68]. This effect of sizeon accumulation, as well as hemodynamics and hemorheology, shouldbe taken into account when designing drug delivery carriers targetedfor vascular diseases and cancer. Additionally, these carriers also interactwith blood components. A study using high concentrations of mesopo-rous silica nanoparticles showed that despite having no effects on plateletviability, it greatly increased platelet aggregation and adhesion to endo-thelium [62]. This emphasizes the evaluation of other parameters apartfrom viabilitywhen studying particle interactionwith blood components.

Fig. 2. The application of microfluidic bioreactors for drug testing. A) Schematic showingthe tumor spheroid formation and culture in a microfluidic flow chamber. B) Optical mi-crograph showing that MCF-7 cells could uniformly fill all the traps. C) Optical micro-graphs showing the structure of the tumor spheroids: (left to right) phase contrastimage and fluorescence images showing cell membrane and nuclei.


Another key aspect that vascular models are able to mimic is theinfluence of shear stress [63,65,69]. This is particularly important fornanoparticle delivery as it affects the endocytic uptake of nanoparticlesby endothelial cells [69]. Narrowing in particular regions of the vascularsystem exhibits increased shear stress, which is an important factor inthe development of thrombosis. A microfluidic model was developedto study the potential of shear responsive nanoparticles for targetingand treating embolic occlusions. The tissue plasminogen activator(tPA) loaded nanoparticles were able to dissolve preformed fibrinclots inside this microfluidic channel. This in vitro observation wasthen validated in an ex-vivo mouse pulmonary embolism model, ashear-sensitive particle system allowed the use of a 100-fold lowerdose 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 concomitantlylessen undesired secondary effects [63].

Vascular microfluidic models also take into account other aspects,such as particle functionalization [61,65]. Platforms canmodel the influ-ence of particle functionalization combinedwith either shear stress [61]or geometric features as straight versus bifurcating channels [65]. Somestudies have highlighted the effect of particle shape on adhesion to thechannel walls (Fig. 4B) [66,70]. In case of an endothelialized channel,rod-shaped nanoparticles showed high specific targeting and lowernon-specific accumulation as compared to sphere-shaped nanoparticles[70]. When engineering and designing novel carriers, one should takethese shape effects into consideration.

Microfluidic technologies have the potential for scalability and high-throughput analysis, and can characterize populations of nanoparticlesin a quick and low cost manner [10], or even evaluate endothelial per-meability [64]. Moreover, inherent characteristics of microfluidics,such as laminar flow, can be exploited to build superior models.For instance, hydrodynamic focusing [71] could be used for preciselytargeting drug carriers to specific regions in microfluidic chips, andpossibly study their subsequent transport to neighboring cells from anon-exposed region. Several of the above discussed models have the

potential to leverage drug delivery by probing the efficacy and modeof action of drug carriers. A future challenge that still remains to beelucidated is the prioritization of parameters, and, perhaps, how tocombine multiple features in a single, high-throughput capable, device.On-chip vasculature models are thus a relevant tool when consideringthe targeting of vascular diseases, tumor angiogenesis and usage ofinjectable drug delivery systems.

3.2. Cardiac platforms

The need for targeting drugs for cardiovascular disease has becomevery critical [72]. Several drugs used for cardiovascular diseases havebeen subjected to FDA withdrawals [73]. Also a few commercial drugsused to treat other organ disorders have been identified to have adverseside effects on the cardiac tissue [74]. Inadequate screeningmodels havebecome one of the major accountable factors for these failures [74,75].With multitude of features to be researched for a drug and its carrierto successfully translate to clinics, testing them in the appropriatein vitro conditions has turned out to be the key aspect [16]. However,persisting difficulties in mimicking the physiology of the heart in vitrohave hampered this process. Nevertheless, there are several reports oncardiac tissue engineering [76] and heart-on-a-chip [75] that promiseto potentially emerge as physiologically relevant models for in vitrodrug testing. Most of the developed platforms use primary cells derivedfrom rats, however, more focus on integration of human cells with thesemodels is required for mimicking human physiological response.Among the human cell sources, cardiomyocytes generated fromhuman induced pluripotent stem cells have enormous potential to bepathophysiologically relevant [77]. They also facilitate the creation ofplatforms for mimicking genetic disorder in patient-specific cases,thereby paving the way for individualized therapy [77].

The culture of functional cardiomyocytes can benefit from stimula-tion with electrical and mechanical forces [78], which cannot bereproduced in a simple cell culture setting. Additionally, to recreate afunctional cardiac tissue, scaffolds can be used to recreate 3D tissue ar-chitecture [79]. The use of cardiomyocyte cell sheets has offered a pos-sible platform for drug testing that exhibits an important physiologicalparameter of heart: a uniform contractile function. Stevens et al. [80]developed a scaffold free cardiac patch derived from human embryonicstem cells (hESCs) that showed electromechanical coupling witheach beat. Shimizu et al. [81] fabricated these sheets with primary ratmyocytes. When two of these sheets were overlaid, they exhibited asimultaneous and spontaneous beating after a week. Other attemptsto record electrical stimulation have involved the use of cardiac tissueslices. Bussek et al. [82] implemented the use of guinea pig cardiac tissueslices to evaluate the performance of a potassium channel blocker. Eventhough different potentials were recorded when the slice was exposedto the drug, the experiment does not completely reproduce the effectsin the human patient. Naturally, these reconstructions do not performas the organ itself and fail to reproduce in vivo conditions but they canoffer an advantage for further improvements by means of microfluidicplatforms.

Classically, most of the microfluidic platforms are fabricated usingpoly(dimethylsiloxane) (PDMS). To tune the stiffness of the PDMS sur-face for cardiomyocyte culture the microfluidic channels can be coatedwith a hydrogel of controllable matrix stiffness [14]. In a study byAnnabi et al. two types of hydrogels, methacrylated gelatin (GelMA)and methacrylated tropoelastin (MeTro) were compared [14]. The cellattachment and alignment were dependent on the type of hydrogelused, whereas the beatingwas dependent on the stiffness of thematrix.In another report, cardiomyocytes were seeded on thin PDMS filmspatterned with fibronectin and cultured within a bioreactor [83]. Thehigh-throughput format enabled testing of different geometric patternsof fibronectin and monitoring up to forty thin films in real-time ina single bioreactor. As the calcium dynamics of the cardiomyocytesare directly affected by disease conditions, Martewicz et al., developed a

PC3 LNCaP

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Fig. 3. The application of microfluidic system for studying nanoparticle and cell interac-tions. A) Fluorescence images showing binding of rhodamine-labeled nanoparticles, with-out or with PSMA aptamer, to PSMA-positive LNCaP cell and PSMA-negative PC3 cellsunder static conditions. The left column shows phase contrast images of the cells and cor-responding fluorescence images of the nanoparticles are indicated in the right column. B)Fluorescence images showing bindingof rhodamine-labeled nanoparticle–aptamer conju-gates (red) to LNCaP cells under fluid flowconditions atflow 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 conditionand to LNCaP cells under both static and fluid flow conditions.Reproduced with permission from Ref. [11].


microfluidic platformwhere the calciumdynamics of the cardiomyocytescan be observed in real-time [84]. This platform could also control theoxygen concentration experienced by the cells, thus allowing inductionof hypoxia. Concurrently, the differential response of calcium dynamicswas monitored with varying oxygen concentration. These in vitromicrofluidic platforms, with high-throughput capability, can accelerate

the translation of a drug carrier to the clinic by recreating cardiovascularenvironment that better mimics human physiological conditions [75].

3.3. Liver platforms

Drug hepatotoxicity is one of the main concerns in identifying newdrugs [85], thus studying hepatoxicity of nanoparticles is very impor-tant for developing novel drug delivery platforms. Also with animalmodels failing to better predict outcomes of drug intake in humans[85], there is an extensive body of research focused toward developingin vitro models for evaluating hepatotoxity [86–89]. In vitro hepaticmodels aim at emulating functionality and mimicking the normal aswell as pathophysiological architecture of liver. These models include2D/3D mono- and co-cultures, hydrogel based engineered models, andorgan-on-a-chip platforms of normal and diseased cells. Since most ofthe drug delivery and nanotoxicity studies are restricted to 2D cellculture plates [90–92], some important studies that describe the testingof nanoparticles in these platforms that more accurately predict in vivobehavior are highlighted.

A recent study using lipid nanoparticles for siRNA delivery demon-strated the importance of using primary hepatocytes instead of immor-talized cell lines for better translation of screening results to in vivooutcomes [93]. Assessment of nanoparticles should be evaluatedwithinsuch clinically relevant models that can maintain the functionality ofprimary hepatocytes for long-term culture [86,94]. One such modelused micropatterned collagen substrate for culturing primary hepato-cytes in a high-throughput 24-well plate format [86]. In this model,primary hepatocytes were co-cultured with fibroblasts to augmenthomotypic interactions with heterotypic interactions for maintainingthe functionality of hepatocytes for a long period. Thismodel representsa potentially useful platform for long-term analysis of liver nanotoxicity.In a study byDragoni et al., the uptake and toxicity of AuNPswere inves-tigated using precision cut rat liver slices [95]. The tissue slice modelwas used to evaluate the uptake of AuNPs by hepatocytes, Kupffer andendothelial cells present in the liver slice. However, tissue biopsies orslices cannot be used for high-throughput studies and show a rapidloss of functionality within days during in vitro culturing, thus limitingtheir application for long-term studies [96].

With extensive research on developing 3D hepatic spheroidsfor drug testing [87,88,97,98] and also with availability of high-throughput commercial platforms for formation of spheroids, liverspheroids represent a promising 3D construct for rapid clinically rele-vant evaluation of nanoparticles. Spheroids formed from primaryhuman hepatocytes have been used for long-term drug studies [97]. Inawork by Lee et al., the toxicity of gold nanoparticles and CdTe nanopar-ticles in 2D cell cultures and 3D spheroids were compared (Fig. 1) [39].Results showed considerably reduced toxicity in spheroids when com-pared to 2D cultures and the reduction in toxicity was attributed tochange in phenotype and complex cell–cell interaction that alteredthe transport of nanoparticles. Spheroids of cancerous hepatocyteshave also been used as a model for mimicking in vivo tumor microenvi-ronments as shown by England et al. [99]. They studied nanoparticledelivery to avascular regions in the spheroid core and used surfacemodification strategy to enhance nanoparticle penetration within thetumor core.

Nanoparticles exhibit differential behavior in static and flowconditions [13,17], thus necessitating the development of dynamicmicrofluidic platforms for toxicity evaluation. Multiple biomimeticliver-on-a-chip platforms have been long established for drug toxicitytesting [15,89,96,100]. Since liver is the major organ for drug metabo-lism, it is important to integrate a liver module with other organmodules to assess the cytotoxicity of prodrugs. In such models, theprodrugs are first metabolized by the liver module before reaching thetarget organ of drug action. For example, Shuler and Sung developed amicro cell culture analog (μCCA) with 3D cultures of HepG2/C3A cells(as the liver module) and HCT-116 cells (as the tumor module), both

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 morenanoparticles (NP; bottom). B) Influence of particle shape (top) on attachment and accumulation in a 45° bifurcation, showing that disc-shaped particles attach more than sphericalparticles (bottom).Reproduced with permission from Ref. [63] and [66].


in Matrigel, and Kasumi-1myeloblasts (as the bonemarrowmodule) instiffer alginate gel (Fig. 5) [101]. The cytotoxic effect of Tegafur, an oralprodrug of 5-fluorouracil (5-FU), on each organ analog was then testedusing such a three organ-on-a-chip platform,where they found that theμCCA was capable of reproducing the metabolism of Tegafur to 5-FU inthe liver module, whereas the metabolic conversion of Tegafur couldnot be accomplished with 2D cultures in 96-well plates.

In a work by Wagner et al., a multi organ-on-a-chip model was de-veloped for long-term culture by combining liver microtissues madeof spheroids of HepaRG and hepatic stellate cells with skin biopsies[15]. Their microfluidic system integrated an inbuilt peristaltic pumpwith a total media volume of 300 μL, thus enabling cross-talk betweenskin and liver compartments proven by the dynamic level of albuminin mono- and co-culture systems. Such models could be channeledtoward evaluating the toxicity of nanoparticles used in the cosmeticindustry. Infectious disease models that recreate the hepatic stage of in-fections, such as hepatitis C viral infection, will also prove significantlyimportant for clinical translation of nanotherapeutics [102,86,103].

3.4. Lung platforms

The respiratory tract is one of the most significant ports of entry inthe human body. This can result in a number of infectious diseases aswell as occupational pathology, but it also offers an appealing route ofdrug administration. The alveoli themselves offer a thinmucosal barrier(200–800 nm) with low enzymatic action, vast absorptive surface area(100 m2) and rapid access to the bloodstream while avoiding invasiveprocedures [104]. Aerosol nanocomplexes for gene therapy have provento diffuse through the alveoli–capillary barrier in an efficient mannercompared to other particles due to their small size [105]. Other drugdelivery systems like zinc oxide nanoparticles (ZnONP) [106], silvernanoparticles (AgNP) [107] and doxorubicin micelles [108] have beenused with 2D culture platforms to study the effect on cell function andapoptotic response. Multi-well plate cultures of MCF-7 and A549 lungcancer cell lines have been used to evaluate nanoparticles [109]. Butthese systems fail to reproduce aphysiological environment for drug test-ing. To recreate the complexity of the human lung's structure, the use of3D microfluidic systems that can introduce fluid and solid mechanicalforces [110] is essential to imitate the human alveoli environment.

In an effort to develop biomimetic ‘lung-like’ microsystems, Huhet al. reproduced the alveolar–capillary interface in vitro by creating alayer of alveolar epithelial cells and endothelial cells both seeded onopposite sides of a porous ECM-coated membrane (Fig. 6A) [13]. Theinflammatory and toxic effect of silica nanoparticles was evaluated byparticle absorption through a vacuum strain model that mimics thephysiological breathingmotion. This cyclicmechanical force augmentedthe nanoparticle translocation across the alveolar–capillary barrier, en-hancing the toxic and inflammatory response as indicated by increasedreactive oxygen species (ROS) generation and intercellular adhesionmolecule-1 (ICAM-1) expression. The nanoparticle translocation behav-ior was also evaluated in a whole mouse lung ventilation–perfusionmodel to demonstrate the significance of using lung-on-a-chip systemsto better predict physiological response. Yu et al. fabricated a ‘lung-like-compartment’ in one of the four multi-channel 3D microfluidic cellculture platform that also incorporated kidney, liver and adipose cells[111]. They evaluated gelatin microspheres as a controlled-release sys-tem for TGF-β1 encapsulated within the microspheres, and used A549cells to recreate a human lung microenvironment for drug testing. Inthe device, common media goes through this first chamber of A549cells and TGF-β1 encapsulated microspheres to continue to the other‘organs’. The ‘lung’ compartment revealed an enhanced cellular functioncompared to the other uncompromised ‘organs’ showing a compart-mental separation between the chambers and imitating the partialcross-talk between organs that occurs in the human body.

Future endeavors must be oriented to combine the multiple celltypes in the alveoli and the mechanical forces involved in ventilation.The reconstruction of a reliable alveolar–capillary barrier without acomplex culturing process [112] as well as developing novel particleswith effective transport properties are also upcoming challenges. Asystem that combines all of the above would be a reliable model thatrepresents the inner organ conditions in the human body.

3.5. Other organ platforms

Other organs that are important for studying nanocarrier mediateddrug delivery are the kidney, spleen and gut [113–115]. Kidney andspleen play an important role in the clearance of systemically deliverednanoparticles. The kidney is one of the major excretion pathways in

Fig. 5. Themulti-chamber μCCA device. A) A PDMS μCCA device consisting of three cham-bers, which represented uterus, liver, and mammary tissues, respectively. B) Measure-ment of cell viability after treatment with 0.02% Triton. Triton treatment led to fullmortality for all cell types.Reproduced with permission from Ref. [101].


the human body, playing a key role in blood filtration andwater metab-olism [116]. A study indicated that the glomerular filtration barrier con-tributed toward the dissociation of siRNA from its cationic cyclodextrincarrier. Thus it is important to study the behavior of nanoparticles in therenal system especially when administered intravenously [113]. To rep-licate renal conditions, such as fluid flow and shear stress [117], Janget al. created a microfluidic device that mimics the in vivo renal tubularenvironment [118]. The device consists of a PDMS platform designed tocontain both ‘luminal’ and ‘tubular’ chambers separated by a porousmembrane. Human primary kidney proximal tubular epithelial cellswere grown on the ‘tubular’ side of the membrane while media wasperfused into the ‘luminal’ side (Fig. 6B) [118]. The device demonstrateddifferential cell histologic arrangement and protein expression whenusing static and dynamic flow conditions. The dynamic conditionrevealed an augmentation in the reabsorption function of the kidney.In the work by Shintu et al., a microfluidic device was used to measurethe ‘metabolomic’ fingerprint of the kidney using high-throughputscreening approach [119]. This method provides for rapid screening ofa compound target library that can be of further use in the drug deliveryand discovery research.

Spleen plays an important role in filtering the bloodstream and is avital part of the body's immunological response [120]. It forms a funda-mental defense component against certain bacteria such as Streptococcuspneumoniae. Yung et al. [121] created a blood cleansing a platform thatuses magnetic opsonins to target Candida albicans [121]. The pathogenis then removed by a micromagnetic–microfluidic separator and clearedfrom the blood. Suchmodels are yet to be exploited for drug studies andcan be of great use for optimizing drug delivery systems.

Among other organs that could be used for drug testing is the gut-on-a-chip created by Kim et al. [55] This device recreates intestinalhuman 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-2cells from another chamber that lies below the porous structure. In ad-dition, Lactobacillus rhamnosus GG bacteria was cultured together withthe epithelial cells to enhance the barrier function of cells during thisperiod. The inclusion of a Lactobacillus that resembles the intestinalflora offers a step forward toward simulating the intestinal lumen.Future design strategies ought to consider the development of anorgan-like platform that can mimic the in vivo complexity of the gutenvironment in a simple and efficient way.

3.6. Tumor platforms

The complex in vivomicroenvironment of a tumor presents a uniquechallenge for the efficient delivery of nanotherapeutics to the tumorsite. The key features of this complex microenvironment include heter-ogenous vascularization, increased interstitial pressure and inadequatelymphatic drainage [122]. Significant advances have been made in thechemical discovery, design and synthesis of chemotherapeutic drugsand diagnostic agents. However, their adverse side effects in healthytissues greatly limit the maximum tolerated dose and thereby reducetheir therapeutic efficacy [123–125]. Researchers and pharmaceuticalcompanies are pursuing the use of nanoparticles for encapsulatinganti-cancer drugs and delivering them specifically to the tumor site asa potential solution to reduce the side effects [126]. Nanoparticleswith surface targetingmoieties have shown efficacy in in vitro screeningstudies, but fail to show the same performance during in vivo trials.Several delivery barriers make nanotherapeutics ineffective within thehuman body and hinder successful clinical translation of these anti-cancer drug carriers. To solve this delivery problem, it is important togain an understanding of the nanoparticle transport through the blood-stream, distribution at the target tissue and subsequent uptake by thecells. Preclinical models that mimic the in vivo 3D tumor architectureand dynamic flow conditions are important for studying these deliveryparameters and for toxicology assessment.

The integration of microfluidic networks with 3D tissue engineeredcultures offers a unique opportunity to probe nanoparticle transportbarriers in a controlled manner. Additionally, these tumor-on-a-chipplatforms can be used for studies to optimize nanoparticle dosage, togenerate gradient drug concentrations and to develop personalizeddrug screening and treatment strategies [127]. For example, in a recentreport by Albanese et al., a tumor-on-a-chip model was developed toinvestigate the transport behavior of AuNPs through 3D tumor-likespheroids of human melanoma cells immobilized in a PDMS chamber(Fig. 7A) [128]. Their microfluidic device allowed precise control ofthe media flow conditions and the spheroids were coated with a lami-nin layer that acted as a barrier to AuNP transport, thus mimickingconditions found in vivo at the tumor site. The tissue penetration andaccumulation of fluorescent NPs under physiological flow conditionswere monitored in real-time. The nanoparticle diameter, surfacefunctionalization and the flow conditions in the microenvironmentwere shown to affect the accumulation of AuNPs near the tumor tissue(Fig. 7B, C). The nanoparticles functionalized with targeting groupsaccumulated in the periphery and failed to penetrate deep inside thecore. Such studies provide critical insights to help design better nano-particles for improved in vivo targeting. Pellegrino et al. have also de-signed a microfluidic platform to evaluate magnetic thermoresponsivepolymers for controlled release of chemotherapeuitc drugs. Thispreliminary study was conducted without cells in PBS, but this systemcan be further developed for studying drug delivery to cells usingthermoresponsive carriers [129].

Several other tumor-on-a-chip platforms have been reportedto study the complex tumor microenvironment and to screen drugs[127,130–135]. In a recent study by Kim et al., a fully automatedmicrofluidic systemwas developed for high-throughput screening of dif-ferent concentrations and combinations of anti-cancer drugs on a paral-lel culture of PC3 prostate cancer cells. Their platform could capture the

Alveolar epithelium

EndotheliumPorous membrane

Nanoparticles

StretchedUnstretched

ECM-coated membrane

Tubular cells

Interstitium

Cells

Lower layer

Cell chamber

Vacuum compartment

Vacuum regulator

B

C

A

Fig. 6.A) Schematic of the lung-on-a-chip platformdeveloped to study nanoparticle trans-port. The transport of nanoparticles across the alveolar–capillary interface is augmentedsignificantly when vacuum is applied to the side channels of the lung platform. Adaptedfrom Ref. [13]. B) Schematic of the kidney-on-chip platform with two chambers dividedby a porous membrane that separates the ‘luminal’ from the ‘tubular’ space recreatingthe in vivo conditions in the kidney. Adapted from Ref. [118]. C) The gut-on-a-chip plat-form includes two compartments divided by a porous membrane separating Caco-2cells from the bottom chamber. A vacuumregulator is attached to the side chambers to as-sess the behavior of the cells under the effect of strain. Adapted from Ref. [55].


synergy between different sensitizer drugs (DOX ormitoxantrone in thiscase) and TRAIL (TNF-alpha Related Apoptosis Inducing Ligand). Suchhigh-throughput systems can be used for screening and optimizing com-binatorial delivery strategies using nanocarriers for cancer therapeuticsbefore testing in humans [135,136]. Further, microfluidic systems thatallow for testing anti-cancer drug sensitivity to a specific patient arepromising for developing clinically relevant nanotherapeutics for per-sonalized treatments [137]. The study by Zhao et al. addressed a key as-pect of anti-cancer drug assessment, namely the integration ofquantitative data acquisition methods with the on-chip platform for an-alyzing the cell apoptosis induced by the chemotherapeutic drug. Thiswas achieved by using Annexin V conjugated quantum dots and Calcein

AM as dual apoptotic probes [138]. Such real-time optical probingmethods will be useful for monitoring the behavior of nanotherapeuticsin the tumor-on-a-chip platforms.

New delivery systems have been proposed in recent years tomeet the demand for developing safer chemotherapeutic strategies forbreast cancer, one of the commonest types of tumor in women [139].These include liposomal nanoparticles used to deliver DOX [140] andalbumin-bound Paclitaxel delivery system [141]. The anatomy of themammary gland presents a number of challengeswhen it comes to clin-ical translation of these theranostics. Two of the major barriers are: thepresence of a system of ducts in a branchedmanner with different sizes[142] and high pressure buildup during a diagnostic liquid procedurelike the fluid nipple aspiration or ductal lavage. Due to the increasedpressure in the smaller ducts, the delivered theranostics fail to reachtheir target and in 20% of the patients these methods provide an inade-quate number of cells to determine a diagnosis [143]. In their effort toreproduce the ductal system in amammary gland and address the diffi-culties mentioned above, Grafton et al. developed a microfluidic breast-on-a-chip system. This system consisted of laminin coated microfluidicchannelswith decreasing ductal size to culture a polarizedmonolayer ofhuman mammary epithelial cells. In this model, they proposed the useof iron oxide superparamagnetic sub-micron particles (SMPs) with anexternal magnetic field guidance for theranostic purposes of mammarygland neoplasias [144]. To further investigate breast neoplasia models,the same group attempted the reconstruction of tumor growth in themammary gland. Knowing that most of the human mammary lesionsascend from the terminal lobular ductal units [145], they used a plat-form to reproduce the disease on a chip. Human breast epithelial cellsand nodules (3–5 cells) of tumorigenic human mammary carcinomacells were seeded in an acrylic hemichannel system leading to amodel that mimics the tumor's organization in human biopsies [146].Using patient biopsies in future studies, the tumor-on-a-chip modelsdescribed above could serve as a personalized medicine platform tooptimize an individualized chemotherapy regimen.

4. Future directions & conclusion

Pharmaceutical companies involved in drug discovery continuouslyaim to both reduce the cost of their research and speed up the develop-ment of new drugs. In this process, pre-clinical animal studies are ex-pensive and time-consuming, while not assuring human in vivosignificance. In vitro organ-on-a-chip platforms can revolutionize thisprocess by making it less expensive, while representing a reliable wayfor drug discovery. However, further advances in the field are neededto overcome engineering and biological challenges before entirelyassessing their value as an efficient and robust platform for screeningnanotherapeutics.

The final goal is to develop a body-on-a-chip platform for systemicevaluation of drug delivery vectors. But current efforts are directedtoward the development of individual organ platforms limiting theirrange of applications to a specific organ's functions. For example, liverbeing themost relevant organ for drug toxicity andmetabolism, the de-velopment of a functional liver-on-a-chip system is the most importantchallenge currently faced by researchers. As mentioned in Section 3.3,several attempts have been developed in this direction, but the availableplatforms are yet unable to fully capture the complexities of in vivo drugmetabolism in a robust manner. For developing a universal platform,the flexibility and modularity of this technology, based on extremelyversatilemicrofabrication techniques, should be exploited. The possibil-ity of culturing different cell types employing the identical standardsetups could lead to the development of several organ models ondemand. The results obtained with such systems could be easily com-pared, and relevant drug-specific cell responses can be quickly noticed.In this context, the simplicity of the setup and the implementation ofseveral parallel experiments will be a key aspect (e.g., for testingdrug–dose responses as well as different target organs). Indeed, in


many cases the platforms proposed so far have been optimized for one ortwo main parameters and the comparison between them is highly com-plicated. A crucial challenge is to build platforms that deliver comparableresults, both to other systems and among different research groups.

Furthermore, the inherent modularity of these systems allows forthe development of several on-chip modules by only changing celltype. Interconnection of such modules would mimic the communica-tion between different organs, making the search for potential newdrugs a more effective and comprehensive process. However, high-throughput and automation, which are intrinsic capabilities of thesesystems, yet remain a challenge. Developments and efforts on the fieldof biosensors should be coupled to organs-on-a-chip, so that the evalu-ation of outcomes could be done in a quantitative and real-timemanner.An ideal platform should include these sensors tomonitor, in situ, variousphysical/chemical parameters, such as O2, pH or even proteins. A robustsystem has to show stable experimental conditions in order to correlateany significant change in those parameters to the direct effect of thecandidate drug. Commercially available sensors, and microfabricatedones, that allow a quick integration in a microfluidic system, will becombined to previously developed organ-on-a-chip platforms.

Future platforms should integrate parenchymal and non-parenchymal cells, and allow long-term experiments withoutexperiencing irreversible changes in cell function and behavior. Parame-ters such as cell proliferation and viability are quite immediate to assess,but changes in proteomic, genomic and epigenomic levels, that candramatically affect drug metabolism, can be more difficult to monitor. Astable and robust system to address these issues is amandatory challengethat will be faced in the next generation organ-on-a-chip systems.

New organ-on-a-chip platforms for drug discovery should also con-sider any problems arising from the interaction between a candidatedrug vector and the microfluidic systems. Mathematical and computa-tional fluid dynamics (CFD) studies can help in this direction, providinga mechanistic understanding of drug carrier attachment in organ-on-a-chip systems. The known differential behavior of nanoparticle shape interms of cell adhesion and distribution can be assessed by CFD, andpotentially aid in the generation of better and more comprehensivemodels. For instance, in a computational study nanorods were shownto contact and adhere to the wall much better than spherical counter-parts [147]. Moreover, a CFD model of a branched blood vessel showedthat bifurcating sections had higher nanoparticle accumulation whencompared to straight ones. In the same study, authors were able tomodel the trajectory and adhesive forces of nanoparticles under shearflow [148]. Together, these and other works [149], set the stage for fu-ture CFD studies on particle–particle, particle–cell and particle–microfluidic device interaction (accumulation and adhesion). Morecomplex CFD studies on these phenomena, especially within organ-on-a-chip, can help building next generation drug delivery systems, bybetter understanding their underlying biophysical aspects.

From a material point of view, the devices developed in this fieldshow important differences that can determine different cell responsesas well as affect drug concentration into the system, due to drug non-specific adsorption to the channel walls. Though PDMS and polystyreneare the most popular materials due to several advantages [150], such asoptical transparency and cell compatibility, other plastic materials havebeen used and a detailed comparison of their advantages/drawbacks indrug screening tests is missing. If an ideal material will be not found,research groups could decide to cover the microchannels with a cell

Fig. 7. Studying the nanoparticle (NP) tissue transport behavior using tumor-on-chip plat-form. A) The schematic of themicrofluidic chip assembled on top of amicroscope stage. B)The schematic (left), image (center) and graph (right) showing the effect of NP size on tis-sue 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 ac-cumulation: C) PEGylatedNPs andD) iron-transporting transferring (Tf) protein function-alized NP.Reproduced with permission from Ref. [128].


layer for preventing drug adsorption, as successfully reported bySchimek et al. [151].

In the long run, organ-on-a-chip systems will be used also forpersonalized drug screening. As these systems will show strong stan-dardization and reliability, the possibility to use patient-derived iPSCs,produced in vitro and then terminally differentiated, can solve one ofthemost important problems in disease treatment— patient-to-patientdrug response variability — avoiding long and ineffective drug treat-ments and minimizing drug toxicity. In summary, organ-on-a-chipplatforms for studying drug delivery systems hold great promise andsome aspects of them have been investigated, but their full applicabilityand potential have yet to be realized.

Acknowledgments

The content is solely the responsibility of the authors and does notnecessarily represent the official views of the awarding agency. Theauthors acknowledge funding from the Office of Naval ResearchYoung National Investigator Award, the National Institutes of Health(HL092836, DE019024, EB012597, AR057837, DE021468, HL099073,EB008392), and the Presidential Early Career Award for Scientists andEngineers (PECASE). J.R. acknowledges the support from the PortugueseFoundation for Science and Technology (FCT; SFRH/BD/51679/2011).

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