Modeling iontophoretic drug delivery in a microfluidic device.

Iontophoresis employs low-intensity electrical voltage and continuous constant current to direct a charged drug into a tissue. Iontophoretic drug delivery has recently been used as a novel method for cancer treatment in vivo. There is an urgent need to precisely model the low-intensity electric fields in cell culture systems to optimize iontophoretic drug delivery to tumors. Here, we present an iontophoresis-on-chip (IOC) platform to precisely quantify carboplatin drug delivery and its corresponding anti-cancer efficacy under various voltages and currents. In this study, we use an in vitro heparin-based hydrogel microfluidic device to model the movement of a charged drug across an extracellular matrix (ECM) and in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Transport of the drug through the hydrogel was modeled based on diffusion and electrophoresis of charged drug molecules in the direction of an oppositely charged electrode. The drug concentration in the tumor extracellular matrix was computed using finite element modeling of transient drug transport in the heparin-based hydrogel. The model predictions were then validated using the IOC platform by comparing the predicted concentration of a fluorescent cationic dye (Alexa Fluor 594®) to the actual concentration in the microfluidic device. Alexa Fluor 594® was used because it has a molecular weight close to paclitaxel, the gold standard drug for treating TNBC, and carboplatin. Our results demonstrated that a 50 mV DC electric field and a 3 mA electrical current significantly increased drug delivery and tumor cell death by 48.12% ± 14.33 and 39.13% ± 12.86, respectively (n = 3, p-value <0.05). The IOC platform and mathematical drug delivery model of iontophoresis are promising tools for precise delivery of chemotherapeutic drugs into solid tumors. Further improvements to the IOC platform can be made by adding a layer of epidermal cells to model the skin.


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Intravenous chemotherapy is the traditional method for administering cytotoxic agents ions, which increases ion delivery out of the blood vessel; and 3) Electroosmotic flow 28 produces bulk motion of the solvent itself, which carries ions or neutral species within the 1 solvent 'stream' [19,22]. To date, most in vitro [21,[23][24][25][26] and in vivo [27,28] studies 2 focused on overcoming human epidermal membrane (HEM) drug resistance by using 3 iontophoresis with a short delivery duration of the electric field. There is a need, however, 4 to precisely control and predict the rate, direction, and distance of drug delivery in the 5 tumor extracellular matrix (ECM) when using iontophoresis techniques. After passing 6 through the HEM, iontophoretic drug delivery is still blocked by the ECM. Physiological 7 and biological barriers within the ECM not only decrease the efficacy of chemical 8 compounds, but also delay the compounds from reaching tumor cells in concentrations 9 sufficient enough to exert a therapeutic effect [14,17]. Barriers to iontophoretic drug 10 delivery created by the ECM is a critical issue that must be addressed. Using a diffusion-11 based model, our groups has previously [29] described that a 70% porosity, heparin-based 12 hydrogel was a biomimetic scaffolding for modeling the chemoresistance of MDA-MB-231 13 triple-negative breast cancer (TNBC) ECM [29]. Therefore, we chose to use a heparin-14 based hydrogel with 70% porosity to represent the ECM of an in vivo tumor [30,31]. 15 Previous microfluidic in vitro studies have been conducted to mimic the three-16 dimensional microenvironments of tumors using on-chip technologies including the tumor 17 vasculature network, which promotes drug resistance in the tumor microenvironment. In silico simulations are well-suited for testing combinations of multiple physical 5 laws (e.g., diffusion and electrophoresis) and are used for estimating drug concentration 6 profiles in the tumor [37][38][39]. However, the fundamental mathematical model that 7 incorporates the physics of electrophoresis transport is not well-defined. Based on the 8 work of Pascal et al. [40], it was demonstrated in their electrokinetics drug delivery model 9 [41] that the application of an electric field enhances drug delivery at both micro-and 10 macro-scales. However, a validated electrokinetics model [42] that can be used for the 11 prediction of the tumor response to chemotherapy in the presence of an applied low-12 intensity DC electric field has not been reported.

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To address these issues, we developed both iontophoresis-on-chip (IOC) platform  Our mathematical model enabled us to predict optimal parameters (electric field intensity, 22 direction), which were then validated in vitro. Our iontophoresis-on-chip (IOC) platform is 23 the first microfluidic system in the literature to offer the opportunity to investigate the effects The iontophoresis-on-chip (IOC) platform was designed to mimic tumor vasculature by 8 providing a top layer of arterial capillaries and two side channels of lymph capillaries in the 9 bottom layer ( Fig. 2A). The heparin-based hydrogel and cell culture medium (DMEM/F-10 12+10% FBS) is found between the top and the bottom layers and plays the role of a salt 11 bridge. Sterile stainless-steel acupuncture needles with a diameter of 0.12 mm (Kingli,12 China) were used as electrodes to construct the DC electric field circuit (Fig. 2B&C). The 13 negative electrode was placed in the top layer and the positive electrodes were placed in 14 the inlet and outlet of the bottom layer's hydrogel channel (Fig. 2A&B). The master mold 15 was patterned using two layers of photoresist: (i) The first layer (i.e., the top layer) consists 16 of a central well (1.5 mm wide and 3 mm thick) and has the role of supplying media to cells 17 that are encapsulated in hydrogel in the bottom layer; (ii) The second layer (i.e., the bottom 18 layer) consists of a cell culture chamber that has the dimensions of 3X3 mm 2 , two sink 19 channels with the dimensions of 0.5X3 mm 2 , 15 ports with the dimensions of 0.15X0.15 20 mm 2 between the cell culture chamber and each sink channel, and two side hydrogel 21 channels with the dimensions of 0.5X5 mm 2 ( Fig. 2A). The thickness of the bottom layer 22 was optimized to 400 µm using the SU8-2100 photoresist and was fabricated according to 23 the manufacturer's (MicroChem Corp.) instructions. Microfluidic devices were produced by 6 1 replica molding using Polydimethylsiloxane, (PDMS, Sylgard 184; Ellsworth Adhesives, 2 Wilmington, MA, USA) on the master wafer, and fabricated using standard microfabrication 3 techniques [52]. We aligned and bonded the two PDMS layers of the microfluidic platform 4 using a Nikon SMZ-1 stereo microscope and a Nordson MARCH (AP-300) oxygen plasma 5 bonder, respectively. After bonding the two layers, the inlets and outlets were punched 6 using a 0.75 mm biopsy puncher (Fig. 2D). Finally, a 6-well glass-bottom plate (MatTek,7 Ashland, MA, USA) was plasma-treated along with the PDMS IOC devices and the devices 8 were bonded to the plate using a hot plate (85°C for 10 min). Because of these dose response curves, we chose to use 2 nM of the drugs to use doses 2 less than the EC 90 for both paclitaxel and carboplatin in the microfluidic in vitro 3 environment. We tested the effect of electric field on the delivery of paclitaxel and 4 carboplatin separately to examine this effect on both charged (carboplatin) and neutral 5 (paclitaxel) drug delivery. 1 charge. We set the drug concentration to be less than the effective dose of 90% (ED 90 ), 2 which we measured in both standard well plates and in the microfluidic device (Fig. S1).

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Application of an external electric voltage resulted in faster delivery of anionic drugs 4 compared to cationic drugs [35]. We compared an anionic drug [53,54], carboplatin, to 5 the gold standard non-ionized drug, paclitaxel. We also studied TNBC cells cultured in 6 heparin-based hydrogel as a tumor ECM biomaterial [29,55]. In our device, we validated 7 the application of a 50 mV electric field to an ECM of 70% porosity to increase drug delivery 8 to a tumor's single cells. We also varied the drug type (charge). In this study, we controlled 9 low-intensity DC electric fields for electrophoretic drug delivery in the tumor's single cells. as the optimum electric field intensity to obtain a 79% cell death rate (Fig. 3B).

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Three non-dimensional numbers and the electric potential in the tumor region were 16 optimized using GRG (Generalized Reduced Gradient) nonlinear solver [56]. The optimal 17 value of (electric field intensity), 43.01 mV, was instrumental in eliminating multiple 2 18 microfluidic in vitro experiments for electric field intensity optimization. Table S1 and Table   19 S2 show the dependent and independent variables, their sensitivity ranges, and optimum 20 value to obtain the maximum fraction of cells killed. fraction of cells killed model [41] (Fig. 6C).

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The combined mathematical and experimental approach in our study included four 21 steps. First, we used a physics-based mathematical model [41], sensitivity analysis, and  second non-dimensional number is which is the ratio of electric potential in the drug , 10 source to the electric potential in the tumor. The fraction of cells killed decreases when 11 is close to 1, i.e., when there is no electric potential gradient. Therefore, when = 1 ( 1 = 12 the fraction of cells killed is minimized (0.44) (Fig. 3C). As increases , the 2 ) ( 1 > 2 ) 13 electrical potential gradient is in the opposite direction of the concentration gradient, so 14 the fraction of cells killed decreases. Note that means is 50 times more than = 1/50 2 15 , at which point the fraction of cells killed is maximized (0.79). The non-dimensional number is analyzed to examine the fraction of cells killed based 17 on drug uptake rate and diffusion. is the ratio of the drug uptake rate to drug diffusion.  High carboplatin diffusion can occur due to a decrease in the tumor drug uptake rate.

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Therefore, the number of cells killed at high carboplatin uptake leads to a ( = 14.2) 22 maximized fraction of cells killed (0.55) and a decrease in tumor drug resistance (Fig. 3D). where the fraction of tumor cells killed is 0.25. The fraction of cells killed reaches an 3 asymptote by increasing the value of , which depends on the properties of the drug. 4 Therefore, applying an electric field to enhance chemotherapy delivery of carboplatin is 5 essential to kill the maximum fraction of cells (0.79). The graph shows that the primary 6 sources of uncertainty in percent dead cells are the drug uptake rate and diffusivity, and  hydrogel microchannel (Fig. 4A, C&D). The finite element model of the concentration 9 profile in the microchannel was validated with an in vitro experiment of Alexa fluor 594® 10 cationic dye diffusion and electrophoretic delivery (Fig. 4C).

A low-intensity electric field increases carboplatin delivery in breast cancer
12 single cells 13 Based on model sensitivity analysis (section 3.2), we specified the low-intensity electric 14 field to be 50 mV. Drug concentration optimization (Fig. S1A) showed that 2 nM of 15 paclitaxel and carboplatin have around a 59% and 71% tumor cell death rate, respectively.

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Therefore, we specified operating at 2 nM drug concentrations, which is less than the EC 90

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(effective concentration for 90% of the cells being dead). A significant difference was 18 observed in the percent of dead cells by applying a 50 mV electric field and 3 mA electric 19 current for 3 h (Fig. 5A&C). The percent of dead cells increased by 22% (n=3, p-20 value<0.05) in the cell culture chamber (Fig. 5B) and 39% (n=3, p-value<0.05) in the 21 hydrogel channel (Fig. 5D).

Fraction of cells killed model validation with in vitro experiment 2
The "fraction of cells killed" model was developed to facilitate the prediction of the 3 iontophoresis outcome. The in vitro results of percent dead cells versus hydrogel depth 4 were used for model validation (Fig. 6A). A summary of results of the percent of dead cells 5 at different distances from the bottom of the chamber shows an increase in percent dead 6 cells when delivering carboplatin using electrophoresis (Fig. 6B). The validated fraction of 7 cells killed model shows an increase in the percent of dead cells when applying a 50 mV 8 electric field and 3 mA electric current for 3 h experimentally and mathematically (Fig. 6C). 9 Our results demonstrate that 50 mV of DC electric field and 3 mA of electric current 10 increases drug delivery by 48.12% and increases cell death by 39.13%. Our obtained 11 experimental results validated our recent drug transport model [41].

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The correlation between the fraction of cells killed model and the in vitro experiment 13 was measured using two different methods ( Overall, the statistical analysis indicated a strong correlation between the model and the 20 microfluidic experiment. View Article Online 1 mathematical model for the carboplatin concentration profile between two blood vessels 2 in a confined tumor volume was able to accurately predict our in vitro microfluidic results.

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In this study, we varied the drug type (charge) and cell density as the two main parameters 4 for the on-chip experiments. As expected, iontophoresis was only effective in increasing  and Computer Engineering for master mold fabrication. We would also like to thank Dr.

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Janet Webster for critically reading and editing the manuscript. There are no conflicts to declare. graph shows the variation in the fraction of tumor cells killed (f kill ) over a dimensionless kill 10 distance, y k , for different values of (which is the ratio of electric potential in the drug 11 source ( ) to the electric potential in the tumor ( )). When is 50 times more than , shows an accurate correlation between model and experiment ( Table 3).