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Standalone Lab-On-a-Chip Systems Towards the Evaluation of Therapeutic Biomaterials in Individualized Disease Treatment Danny Jian Hang Tng, Peiyi Song, Rui Hu, Chengbin Yang, Cher Heng Tan, and Ken-Tye Yong ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00369 • Publication Date (Web): 07 Sep 2015 Downloaded from http://pubs.acs.org on September 22, 2015

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ACS Biomaterials Science & Engineering

Standalone Lab-On-a-Chip Systems towards the Evaluation of Therapeutic Biomaterials in Individualized Disease Treatment Danny Jian Hang Tng1†, Peiyi Song1†, Rui Hu1, Chengbin Yang1, Cher Heng Tan2 and Ken-Tye Yong1* 1 School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 2 Department of Diagnostic Radiology, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore 308433

†

These authors contributed equally to this work. *Corresponding author: Ken-Tye Yong, PhD, School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Tel: +65-6790-5444, Email: [email protected]

Keywords: Biocompatible, Electrochemical, Individualized, Lab-on-a-Chip, Standalone Abstract As each tumor is unique, treatments should be individualized in terms of their drug formulation and time dependent dosing. In vitro Lab-On-a-Chip (LOC) drug testing is a viable avenue to individualize treatments. A drug testing platform in the form of a customizable Standalone LOC system is proposed for treatment individualization in vitro. The platform was used to individualize the treatment of pancreatic cancer by using PANC-1 and MIA PaCa-2 cell lines cultured on-chip. Using on-chip drug uptake, growth and migration inhibition assays, the therapeutic effect of various treatment combinations was analyzed. Thereafter, optimized treatments were devised for each cell line. The individualized dosage for MIA PaCa-2 cell line was found to be between 0.05 – 0.1 µg/µl of doxorubicin (DOX), where the greatest growth and migration inhibition effects were observed. As the PANC-1 cell line showed resistance to DOX only formulations, a multidrug approach was used for individualized treatment. Compared to the DOX only formulations, the individualized treatment produced the same degree of migration inhibition but with 5 – 10 times lower concentration of DOX, potentially minimizing the side-effects of the treatment. Furthermore, the individualized treatment had an average of 672.4% higher rate of growth inhibition. Finally, a preliminary study showed how a tested formulation from the LOC system can be translated for use by employing a nanoparticle system for controlled delivery, producing similar therapeutic effects. The use of such systems in clinical practice could potentially revolutionize treatment formulation by maximizing the therapeutic effects of existing treatments while minimizing their potential side effects through individualization of treatment. 1 ACS Paragon Plus Environment


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1. Introduction Therapy individualization involves the customization of treatment by customizing the treatment dosing, formulation, concentration, etc., in order to maximize the therapeutic outcomes of the treatment, whilst keeping the potential side effects to the minimum.1, 2 This approach to treatment has been receiving greater attention in areas such as cancer treatment, due to the severity of the disease as well as the harsh side effects caused by anti-cancer medications. Customizable treatment approaches such as multifunctional nanoparticles,3-5 cancer genomics6 and implantable drug delivery devices1 which have site specific targeting and controlled drug release capabilities are some of the well pursued avenues of treatment individualization. In practice, due to the great number of permutations customizations that these treatments can have, individualization of treatment per patient is an uphill task, as each treatment combination is evaluated for its effectiveness. Automated bulk testing approaches such as Lab-On-a-Chip (LOC) drug screening devices are attractive solutions as they require microliter amounts of samples and reagents, conserving the limited cell samples.7 These LOC systems feature miniaturized but still functionally identical versions of traditional testing systems, as the miniaturization process does not alter the interactions between the cells and reagents.8 The basic methodology of these systems is to utilize various on-chip assays to profile the therapeutic outcome of a treatment candidate using cancer cell samples. To adapt current LOC systems for treatment individualization applications, they must be highly customizable rather than the typical configuration of identical parallel tests. The key issue is that there are significant variances in each tumor,1, 9, 10 therefore a customized approach to testing is also required to find the individualized treatment instead of bulk testing conditions. Customized testing in high volume is a limitation in present LOC systems as they have large offchip dependencies such as fluid pumps for fluid manipulation.11 Due to the large combinations of assays for every tumor sample, customized high throughput testing for individualized treatment is a great challenge for LOC testing systems as an unrealistically large number of off-chip dependencies would be required.12 In this respect, rather than conventional LOC systems, standalone LOC systems which can be configured to function without external dependencies through the integration of all the required components for operation, are more suitable for individualized testing applications. Standalone LOC systems integrate the required functional components on-chip, such as on-chip cell imaging13-16 and on-chip fluid mixers,17 thereby removing their reliance on external dependencies. Within 2 ACS Paragon Plus Environment

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these LOC systems, on-chip fluid actuation have emerged as one of the vital areas in need of greater development for the realization of standalone LOC systems. On-chip microactuators are essential components of the core functional modules within the LOC testing systems as they provide the actuation force required for the delivery of the test reagents to the cell culture chambers. Currently, there is a lack of on-chip actuation methods which are compatible with miniaturized on-chip in vitro applications. Popular methods such as electrowetting,18 have relatively low flow rates, while thermal19 and microthrusters (physical20 and chemical21, 22) methods produce significant heating, which can influence the test results. Current on-chip actuation methods result in LOC systems which are unable to be easily sterilized as high temperatures would cause the chemical propellant to be prematurely discharged or damage membrane structures. Furthermore, since many of the current on-chip microactuators utilize exothermic chemical reactions for actuation that can reach up to 70 - 100 °C,22 high density integration of these microactuators would be very challenging. Temperatures of more than 65 °C have been shown to cause cellular damage,23 limiting the use of these actuators for in vitro applications. The increased temperature also has some implications, as the drug formulations used can be negatively affected, causing structural change or unintended release from their delivery vehicles.24, 25 This would significantly reduce the available candidates for drug testing as only temperature insensitive drugs would give consistent results. Due to these limitations, standalone LOCs have only been applied to a small range of applications such as disposable assays19 and Point of Care (POC) diagnostics.26 Therefore, for the realization of a standalone LOC system for customized drug testing to develop individualized treatments, a platform which allows ease of customization as well as testing without temperature induced influences is greatly needed. In this work, we demonstrate this platform, which focuses on overcoming the limitations based on on-chip fluid manipulation. A standalone LOC system was prototyped for the in vitro testing of multiple drug formulations to find an individualized treatment for two pancreatic carcinoma cell lines. For fluid manipulation, on-chip electrochemical microactuators are integrated into the LOC. These microactuators do not cause significant temperature increase during operation as they generate actuation force from gases produced in an endothermic electrolysis reaction,27 making the LOC system suitable for in vitro drug testing. PANC-1 and MIA PaCa-2 pancreatic cancer cell lines were cultured on-chip for the drug testing. Using on-chip drug uptake, growth and migration inhibition assays, the 3 ACS Paragon Plus Environment


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therapeutic effects of different administered drug formulations could be studied. With the presented LOC device, different concentrations of Doxorubicin (DOX), a known anti-cancer drug, was administered to the cell cultures. Between the PANC-1 and MIA PaCa-2 cell lines, a significant difference in uptake of as high as 26.6% was observed. This difference in response to the same treatment highlights the need for treatment individualization. These tests also reveal that an individualized dosage range for MIA PaCa-2 was found to be between 0.05 – 0.1 µg/µl of DOX, which had the highest observed growth and migration inhibition effects. As the PANC-1 cell line showed some resistance to DOX only formulations, multidrug formulations were tested and individualized for treatment. The two drugs used were DOX and Anti-Insulin Growth Factor (αIGF), another anti-cancer reagent.28,

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Compared to the DOX only formulations, the individualized

treatment produced the same degree of migration inhibition utilize 5 – 10 times lower concentration of DOX, potentially minimizing the side-effects of treatment. Furthermore the individualized treatment had an average of 672.4% higher rate of growth inhibition. Finally, a preliminary study showed how tested formulations from the LOC system can be translated for use by employing a nanoparticle system for controlled delivery. In vitro tests revealed these nanoparticle systems had similar drug release profiles as the LOC systems and caused similar growth inhibition effects. These results strongly suggest that with the customization of treatment, it is possible to maximize treatment outcomes whilst minimizing the risk of complications due to treatment side-effects. The reduction in the concentration of DOX used is an important improvement, since many anti-cancer reagents such as DOX have potentially serious side effects.30 The implementation of such customizable LOC systems at the clinical level could potentially revolutionize treatment with fully on-chip in vitro individualized testing solutions31, on-chip on-demand drug production.32

2 Materials and Methods 2.1 Fabrication of On-chip Actuator AZ5214E image reversal photoresist (Merck Performance Materials, Darmstadt, Germany) was spin coated onto polished glass substrates (Latech Scientific Supply, Singapore) using a spin coater set to spin at 4000 rpm for 30 s. After which the glass substrate was baked at 105 ˚C for 2 min. The electrode structures were then pattered onto the photoresist with a mask aligner (Karl Suss MA-6, Suss MicroTec, Germany). The exposure was performed at 420 W of power for 2.5 s under hard contact settings. The exposed samples were 4 ACS Paragon Plus Environment

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then baked at 110 ˚C for 7.5 min, followed by a flood exposure at 420 W of power for 30 s, as part of an image reversal process. The final pattern was obtained by developing the image reversed samples by immersing them into photoresist developer as per vendor instructions (AZ developer, Branchburg, NJ). Using physical vapor deposition (Electron bean evaporation, Denton Vacuum DV502A), 5 metal layers of titanium (Ti) and platinum (Pt) were deposited alternately by electron beam evaporation. A 5-layer nanosandwich structure was created.31 As part of a lift off process, the samples were then soaked in acetone to release the metal structures by removing the unwanted photoresist.

2.2 Fabrication of LOC Module and Arduino Shield LOC Control Module The microfluidic pattern for the LOC was made using a soft lithography process, where Polydimethylsiloxane (Sylgard 184, 10 parts silicone to 1 part fixing agent, Dow Corning, MI, USA) was poured and cured in a mold made of patterned SU-8 2000 negative epoxy photoresist (Microchem, Westborough, MA, USA). To create the mold, SU-8 photoresist was coated onto a silicon wafer substrate using a spin coater set to spin at 800 rpm for 60 s. The silicon wafer was purchased from Latech Scientific Supply (Singapore). The coated silicon wafer was then baked for 4 h at 110 ˚C to evaporate the excess solvent from the photoresist. The mold was then patterned using a mask aligner (SUSS MicroTec MJB4) and a mask under hard contact settings with an exposure of 90 s at 420 W (I-line). The exposed photoresist was then baked for 2 h at 95 ˚C. After which, the unexposed photoresist is removed by soaking the sample in SU8 developer (Gersteltec Engineering Solutions, Pully, Switzerland). The cured PDMS microfluidic structure was then attached onto the on-chip actuators fabricated in 2.1. Uncured PDMS was used as an adhesive and the assembly was cured under vacuum. Next, wire bonding using conductive silver paint (RS Components, Singapore) was performed to attach pins to the bond pads of the on-chip actuators. The assembly was then fitted into a standard IC socket using the bonded pins thus forming the LOC module’s connection pins. To prepare the LOC module for cell applications, it was sterilized through a steam autoclave process at 125 °C at 15 PSI for 10 mins. For programmed control of the LOC module, an Arudino UNO microcontroller (Arduino, Italy) was used. A custom made shield was used to interface the LOC module to the microcontroller which was built using 1.5KΩ resistors, PN2222ABU Bipolar Transistors and LEDs (Red,

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Yellow, Green). The desired operation program which was used to trigger the on-chip actuators sequentially for the different on-chip applications was flashed onto the microcontroller memory using USB connection.

2.3 On-Chip Actuator Characterization The on-chip electrochemical actuators were characterized for their flow rate using different constant bias voltages. The actuators were left running for 20 s at each test voltage and the water displaced from the actuators was measured using a micropipette. The flow rate was computed as the average volume of water displaced per second. For reach test voltage, 4 identical test chips were used. The power consumption at each test voltage of the actuator was measured using the internal source measure unit of the voltage supply. To monitor the temperature fluctuations of the actuator, a laser infrared digital temperature gun was used to probe the actuators at each test voltage after 20 s of operation.

2.4. Cell Culture PANC-1 pancreatic cancer cell line cells (CRL-1469, American Type Culture Collection) were cultivated in nutrient solution consisting of Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Life Technologies), 10% Fetal Bovine Serum (FBS, Gibco, Life Technologies), 5% penicillin and streptomycin antibiotic solution (0.1 mg/ml). MIA PaCa-2 pancreatic cancer cells (CRL-1420, American Type Culture Collection) were cultivated in nutrient solution consisting of DMEM, 10% FBS, 2.5% Horse Serum (American Type Culture Collection), 5% penicillin and streptomycin antibiotic solution (0.1 mg/ml). After culturing to 90% confluence, trypsin solution was used to dissociate the cells from their cell culture plates and cell suspensions of 2 x 103 cells/ml concentration were made. The cell reservoirs of the LOC chip were then filled with the cell solution using a syringe and then kept in a humidified incubator at 37 °C with 5% CO2.

2.5 Preparation of Drug Test Formulations The drug test formulations were prepared by mixing different amounts of Doxorubicin (DOX, SigmaAldrich, Singapore), anti-human Insulin-like Growth Factor-1 Receptor (αIGF, Biomed Diagnostics, Singapore) with DMEM medium to form the required concentrations.


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2.6 Cancer Cell Growth Inhibition Studies The test solutions with the required drug concentrations were loaded into the drug delivery reservoirs using a syringe and a 30 gauge needle. Each LOC has 3 drug delivery chambers. For the experiments using only DOX formulations, 2 chambers were loaded with DOX solutions and 1 chamber was loaded with culture medium. For the experiments using DOX and αIGF formulations, 2 chambers were loaded DOX solutions and 1 chamber was loaded with αIGF solution. For the reservoirs containing the test solutions, the microactuator was programmed to activate the electrochemical actuator with a 7 V bias for 2 s at 10 min intervals until one chamber was empty and then the next chamber with the test solution was used to continue the delivery. For the reservoir containing the culture medium/αIGF solution, the electrochemical actuator was activated with a 7 V bias for 2 s at 20 min intervals. Every 24 h, the cells in each culture chamber were imaged (Nikon eclipse Ti-U) and counted to measure the degree of growth inhibition against a control where all 3 drug delivery chambers contained only culture medium.

2.7 Cancer Cell Migration Studies PANC-1 and MIA PaCa-2 pancreatic cancer cell lines were cultured on-chip until the cell culture chambers reached 80% confluence. For the migration test, 1 of the delivery chambers was loaded with the test solution and the other 2 delivery chambers were loaded with culture medium. The microcontroller was then programmed to activate the electrochemical actuator at 7 V bias for 500 s to deliver the test solution to the cell chamber. After 1 h of incubation, the microcontroller activated another actuator containing culture medium with 7 V bias for 500 s to flush the test solution from the cell chamber. After which, using a sterile 30 gauge needle, a vertical streak at the center of each cell culture chamber was made for a wound healing assay to measure the cells migration capabilities after drug treatment. After 24 h, the last delivery chamber with culture medium was activated with a 7 V bias for 2 s at 20 minute intervals. Every 24 h thereafter, the cells in each culture chamber were imaged (Nikon eclipse Ti-U) and the wound area was measured using ImageJ image processing software.



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2.8 Drug Uptake Studies PANC-1 and MIA PaCa-2 pancreatic cancer cell lines were cultured on-chip until the cell culture chambers reached 80% confluence. For the migration test, 1 of the delivery chambers was loaded with the test solution and the other 2 delivery chambers were loaded with culture medium. The microcontroller was then programmed to activate the electrochemical actuator at 7 V bias for 500 s to deliver the test solution to the cell chamber. After 1 h of incubation, the microcontroller activated another actuator containing culture medium with 7 V bias for 500 s to flush the test solution from the cell chamber. The cells in each culture chamber were then imaged (Nikon eclipse Ti-U) using an excitation of 480 nm to measure the fluorescent intensity of the cells corresponding the uptake of DOX.

2.9 Nanoparticle Synthesis Poly(dl-lactide/glycolide) (PLGA, 50:50, MW 150 000, Polysciences, Taipei, Taiwan) was dissolved in chloroform to make a solution of 80 mg/ml concentration. Doxorubicin (DOX, Sigma Aldrich, Singapore) and Anti-human Insulin-like Growth Factor-1 Receptor (αIGF, Biomed Diagnostics, Singapore) were added drop wise to the PLGA solution under vigorous agitation in an ice bath. This resulting mixture was then added drop wise to a solution of 1 mg/ml Methoxy-Poly (Ethylene Glycol) - 1, 2-Distearoyl-sn-Glycero-3Phosphoethanolamine-N, (mPEG-DSPE, MW 2000, Laysan Bio, Alabama, United States) MW 2,000. The drop wise addition was performed under sonication with a tip sonicator (Sonic Dismembrator, ultrasonic Processor FB-505, Fisher Scientific) in an ice bath. The drop wise addition was stopped when the PLGA/DSPE ratio of 0.05 was reached, forming the drug nanoparticles. To remove the excess choloroform, the nanoparticle solution was put into a vacuum desiccator for 24 h. For purification, the nanoparticle solution was dialyzed against Phosphate Buffer Solution (PBS, pH 7.4) using a MWCO 12000 dialysis tubing (Snake skin, Sigma Aldrich, Singapore) for 4 cycles throughout a 72 h period. DOX loading was then verified using Ultraviolet Visible (UV-VIS) spectroscopy at 480 nm absorption wavelength (UV-VIS Spectrometer UV-2450, Shimadzu Instruments). The concentration was then calculated using the BeerLambert Law.


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2.10 Particle Size and Zeta Potential Characterization The hydrodynamic diameter of the synthesized nanoparticles in 2.9 was characterized using Dynamic Light Scattering (Nanobrook 90 Plus, Brookhaven Instruments, NY, United States). Measurements were all performed in triplicate and the solutions were 10X diluted using Deionized (DI) water.

2.11 In vitro Drug Release from Nanoparticles 1ml of each nanoparticle solution (1 mg/ml) was sealed within porous tubing (snakeskin dialysis tubing, MWCO 12000, Sigma Aldrich, Singapore). The sealed tube was then put into a glass vial with 19 ml of PBS solution (pH 7.4) and kept under constant, gentle agitation. At predefined timings, PBS solution was analysed using UV-VIS. The absorption at 480 nm was then used to compute the amount of DOX released from the nanoparticles using Beer-Lambert Law. The PBS in the glass vials was replaced with fresh solution after each UV-VIS measurement.

2.12 Nanoparticle Treatment Studies The nanoparticle drug formulations were loaded into 3 of the delivery reservoirs and the cell culture reservoir using a syringe and needle. The microcontroller was then programmed to activate the one of the electrochemical actuators 24 h later, with a 7 V bias lasting 500 s. This replaced the nanoparticle solution within the cell culture reservoir and it was repeated every 24 h. Also, at each 24 h interval, the cells in the cell culture reservoir were imaged using optical microscopy and the cells were counted to determine the degree of inhibition as compared to control cell cultures which were delivered with culture solution instead of nanoparticles.

3. Results and discussion 3.1 Standalone LOC System Design As previously discussed in section 1, the current limitation impeding the realization of standalone LOC systems for in vitro applications such as treatment individualization testing systems, is compatible fully on9 ACS Paragon Plus Environment


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chip fluid manipulation. We illustrate how this limitation can be overcome through the use of an LOC system with on-chip electrochemical microactuators, which have been used extensively in many drug delivery applications.1,33-35 The LOC was controlled by an open-source programmable Arduino UNO microcontroller (Figure 1A) and the custom fabricated LOC Module (Figure 1E). The two components communicate with each other via the custom built Arduino shield (Figure 1C) to form the complete LOC system in Figure 1B and were powered by a single 9 V battery. The Arduino shield had a socket to plug in the LOC module and headers on the back to connect with the microcontroller. LED indicators were used to indicate which electrochemical actuator was in activation. The Arduino shield circuitry is simplistic and can be miniaturized into a single chip for practical application. The LOC module consisted of a layer made of PDMS with the drug delivery and cell culture chambers interconnected by microchannels mounted on top of on-chip electrochemical actuators on glass below. The LOC module is transparent, allowing cell imaging with optical microscopes. Additionally, PDMS is highly biocompatible (Figure 1H) and has a self-sealing ability, allowing the drug delivery chambers to be refilled with new drugs for long term testing.36 The LOC module consisted of 4 identical units in a 2 X 2 array and more units can be added if required. Each unit had 3 drug delivery chambers with on-chip actuators at the bottom (Figure 1F), connected via microchannel to a cell culture reservoir (Figure 1D). The actuators featured a 5-layer nanosandwiched metal structure, ensuring high reliability.31 More information on the on-chip electrochemical actuators performance and characterization is included in the supporting information, Figure S1. Of all the evaluated current on-chip actuation methods (Table 1), electrochemical actuation is the highly suitable for fully on-chip in vitro applications. Firstly, other actuators such as microthrusters are heat activated, relying on internal heaters to release pressurized gases from chambers20 or produce gases from chemical propellants.21 Thus, the high temperatures during conventional sterilization such as autoclaving could cause unintended actuation. In contrast, the electrochemical actuators are unaffected by sterilization and remain structurally uncompromised after autoclave (Figure 1G). Secondly, these actuators produce significant heating of up to 70 - 100 °C,22 which can negatively affect the test reagents and cell cultures. This issue becomes greater with increasing chip miniaturization. In contrast, the presented LOC system operated at constant temperature as shown in the supporting information Figure S1C. This is a key property as even small temperature fluctuations have been known to influence therapeutic outcome,37, 38 drug toxicity39, 40 and 10 ACS Paragon Plus Environment

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drug release from their associated thermo-sensitive nanoparticle carriers.41, 42 As the development of such thermo-sensitive drugs intensifies, so will the demand for such testing systems such as the one presented in this paper. Thirdly, many of the present on-chip actuators have non-linear input energy to flow rate controllability, 21 making customized dosage profiles challenging. As a demonstration of its ability in treatment individualization, the LOC device was specifically customized for the treatment of two cancer cell lines, PANC-1 and MIA PaCa-2, which are both pancreatic carcinoma, but originate from different patients. The two cell lines were cultured on-chip and treatment was formulated in 3 stages (Figure 1I). At each stage, the effect of the tested treatment was evaluated based drug uptake, growth inhibition and migration inhibition assays performed with the help of the LOC. Once a treatment was found which was effective at stopping both cell migration and growth with minimal side-effect inducing reagents, no additional individualization would be necessary. First, both cell lines response to a generalized treatment was tested. In clinical settings, this treatment would be one which was broadly applied to treat a particular disease. For simplicity, Doxorubicin (DOX), which is a well-known anti-cancer reagent,43 administered with 0.01 µg/µl concentration was assumed as the generalized treatment. Second, the generalized treatment was individualized for each cell line by altering the concentration of the treatment. Finally, the treatment was further individualized by changing the administered drug formulation. As an example, the standalone LOC was used to individualize treatment for the PANC-1 cell line and can be repeated with any other cell line.



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Figure 1: Prototype Standalone Lab-On-a-Chip (LOC) system for treatment individualization. (A) Programmable Arduino Microcontroller. (B) Fully Assembled Standalone LOC. (C) Custom made Arduino Shield which interfaces the microcontroller and the LOC module. (D) Schematic illustration of the LOC module, with a 2 X 2 array of identical units. Each unit consists of 1 cell culture chamber and 3 delivery chambers with integrated on-chip electrochemical actuators. (E) LOC module where the drug testing is performed. (F) Top view of the LOC module. (G) Magnified view of the electrochemical actuator (4 mm diameter) after autoclave at 125 °C at 15 PSI for 10 mins showing no delamination. (H) Brightfield image of cultured cells in cell culture chamber. (I) Example approach for using the proposed LOC platform for treatment individualization. 3.2 Response to Generalized Treatment and Individualization by Dosage Adjustment In most cases, there is already a generalized treatment being used, however it is unlikely to produce optimal therapeutic results as it is not optimized for the individual. Both cell lines were first treated with the generalized treatment of 0.01 µg/µl of DOX and then subsequently tested with other treatments consisting of different concentrations of DOX. Using drug uptake, growth inhibition and migration inhibition assays were performed with the help of the standalone LOC and the therapeutic effects of each treatment were analyzed. 12 ACS Paragon Plus Environment

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As previously mentioned, a treatment is individualized if it has the optimal migration and growth inhibition characteristics, but with the least potential side effect. Otherwise, further individualization would be necessary by tweaking the treatment formulation which will be discussed in section 3.3.

3.2.1 Drug Uptake Studies Using the LOC device, programmed administration of different formulations of DOX to the cancer cells were examined for their uptake of DOX. Being a fluorescent chemotherapeutic drug, the fluorescent intensity of DOX observed in cells corresponds closely to its cellular uptake.44 The fluorescence signal from both cell lines confirmed their uptake of DOX (Figure 2A). The relative fluorescent intensities were measured and illustrated in Figure 2B. Both cell lines showed an increase in cell fluorescent intensity when treated with higher concentrations of DOX. In general, PANC-1 cells showed a higher intensity, suggesting a higher uptake of DOX than MIA PaCa-2. Particularly for 0.05 µg/µl and 0.1 µg/µl concentrations of DOX, PANC-1 cells (528 and 1026, respectively) had significantly higher fluorescent intensity than MIA PaCa-2 cells (244 and 747, respectively). This variance in DOX uptake may be due to the differences of responses of both cell lines to the same treatment. As previously mentioned, the LOC system operated at constant temperature, thus any fluctuation in fluorescent intensity due to temperature change278, 279 would have been unlikely. Additionally, although in this case DOX is fluorescent, this same approach can be used on nonfluorescent drugs by labeling them with a fluorescent dye first.45



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Figure 2: Measurement of drug uptake using fluorescent intensity and growth inhibition after administration of different concentrations of DOX to both cell lines. (A) For each cell line, left column represents bright field images and right column represents the fluorescent image. (B) Fluorescent intensities of the cells after 1 h of incubation with the different concentrations of DOX. (C) Growth inhibition effect on the MIA PaCa-2 cell line after treatment with different concentrations of DOX. (D) Growth inhibition effect on the PANC-1 cell line after treatment with different concentrations of DOX. (Vertical lines represent standard deviation, n = 4 and scale bar represents 50 µm for all cell images). 3.2.2 Growth Inhibition Studies Cancer cell growth is one of the foremost concerns during treatment. The challenge is to find drug treatments which are able to inhibit or kill cancer cells specific to different tumors.1,

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The LOC device was

programmed to administer various concentrations of DOX (0.01 µg/µl, 0.02 µg/µl, 0.05 µg/µl and 0.06 µg/µl) using a pulsatile dosage over 72 h. The corresponding changes in the cell counts of the treated PANC-1 and 14 ACS Paragon Plus Environment

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MIA PaCa-2 cultures are presented in Figure 2C and 2D, respectively. The cultures treated with 0.00 µg/µl concentration DOX solution (control) showed a significant increase in cell count after 72 h (PANC-1 533% and MIA PaCa-2 273%). This suggests that the operation of the on-chip actuators had minimal impact on the cell growth. Other on-chip actuation methods may be unsuitable as they produce heat upon operation (Table 1). Temperatures above 42 – 45 °C have been shown to cause tissue disorganization and nuclear pyknosis.46 Additionally, unlike traditional LOC devices, the presented device sustains the cell cultures on-chip without the use of any off-chip pumps. For the MIA PaCa-2 cultures, formulations with higher DOX concentrations were observed to have enhanced inhibition effects (Figure 2D). Interestingly, in the first 48 h, cell count reduction was 12% more when treated with the 0.06 µg/µl formulation as compared to the 0.05 µg/µl formulation. However at 72 h, cultures treated by both 0.05 µg/µl and 0.06 µg/µl formulations had comparable cell counts (27% and 31%, respectively). This suggests that in the long term, dosages above 0.05 µg/µl may not produce additional inhibitory effects which would outweigh the added risk of overdose. Thus the suitable dosage for the growth inhibition of MIA PaCa-2 would be 0.01 – 0.05 µg/µl. In contrast, when the same formulations were administered to PANC-1 cells, the response was vastly different (Figure 2C). Firstly, although an inhibitory effect was observed from the treatment with the 0.01 µg/µl formulation in MIA PaCa-2 cultures, in PANC-1 cell cultures, there was an 8% initial increase in cell count for the first 24 h. Secondly, treatment with the 0.02 µg/µl, 0.05 µg/µl and 0.06 µg/µl formulations resulted in a significantly lower inhibitory effect. Especially after 24 h, the inhibitory effect was much lower. This suggests that PANC-1 cancer cells may have gained resistance to DOX after 24 h. It was also observed that the 0.06 µg/µl formulation did not show an increase in inhibitory effect as compared to the 0.05 µg/µl formulation and cell counts were almost identical (83% at 24 Hrs, 80% at 48 Hrs and 77% at 72 Hrs). From these results, DOX alone may not be an ideal drug for the treatment of PANC-1 as much higher concentrations might be needed to produce the desired inhibitory effect. Thus the treatment had to be further individualized in order to increase the therapeutic effects while avoiding the side-effects.



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3.2.3 Migration Inhibition Studies Chemotactic migration is a component of cancer cell migration an indicator of cancer metastasis.47 Thus it is of great interest to find treatments which can inhibit cancer cell migration. Using the presented LOC, formulations with different DOX concentrations (0.005 µg/µl, 0.01 µg/µl, 0.05 µg/µl and 0.10 µg/µl) were studied for their migration inhibitory effects using a wound healing assay. When treated with the 0.00 µg/µl formulations (control), the wound size decreased with time, fully closing at 48 h for both cell lines (Figure 3A and 3C). This again demonstrated the suitability of the presented LOC system for in vitro applications, as the results are consistent with other microfluidic wound healing assays with off-chip pumps.48 When tested with the other formulations containing various concentrations of DOX, it was observed that the formulations with higher DOX concentrations produced a larger migration inhibitory effect. For the MIA PaCa-2 cell line, only the 0.1 µg/µl formulation produced a sufficient migration inhibitory effect, maintaining the wound size at 88.9% after 48 h (Figure 3D). Formulations with lower DOX concentrations of 0.01 µg/µl and 0.05 µg/µl only had a mild migration inhibitory effect where the wound size decreased to 40.0% and 26.1%, respectively after 48 h. Additionally, compared to the control, the culture treated with the 0.005 µg/µl formulation had a 16.4% larger wound size after 24 h, however at 48 h, its difference in wound size was only 1.5%. This suggests that dosages below 0.005 µg/µl would not be effective for migration inhibition in the long term. In contrast, all of the tested DOX formulations had higher migration inhibitory effects on PANC-1 colonies as seen from the larger wound sizes (Figure 3B). After 48 h of treatment with the 0.005 µg/µl and 0.01 µg/µl formulations, the wound size was maintained at 60.9% and 64.6%, respectively. For the 0.05 µg/µl and 0.1 µg/µl formulations, migration was inhibited almost completely with the wound size 84.2% and 96.8%, respectively. However, with the 0.1 µg/µl formulation, cell adhesion was compromised causing the cells to break away from one another after 48 h (Figure 3A). This suggests that although formulations with higher concentrations of DOX would be more effective, drug toxicity might be an issue.


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Figure 3: Wound healing assay performed on the LOC module measuring the migration inhibition after treatment with treatments consisting of different concentrations of DOX. (A) Insets of bright field images of the wound healing assay showing the cell migration of PANC-1 cells into the wound area (yellow outline) 24 and 48 h after treatment. (B) Measured changes in wound area for PANC-1 cells treated with different concentrations of DOX. (C) Insets of bright field images of the wound healing assay showing the cell migration of MIA PaCa-2 cells into the wound area (yellow outline) 24 and 48 h after treatment. (D) Measured changes in wound area for MIA PaCa-2 cells treated with different concentrations of DOX. (Scale bar of 200 µm applies globally to all the insets, vertical bars represent standard deviation of each measurement and the number of samples, n = 4.) 3.3 Further Individualization of Treatment for PANC-1 Cell Line Considering both the growth and migration inhibition studies, it is possible that an individualized dosage for treating the MIA PaCa-2 cell line with a DOX dosage of somewhere between 0.05 µg/µl (highest growth inhibition) and 0.1 µg/µl (almost complete cell migration). However, for the PANC-1 cell line, no clear dosage range could be established. Greater growth inhibition is required and high dosages might not be the answer as dosages above 0.05 µg/µl did not produce increased inhibitory effects. Therefore, further 17 ACS Paragon Plus Environment


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individualization of treatment for PANC-1 was necessary. It was apparent that DOX alone may be insufficient in treating PANC-1. Therefore, for further individualization, the LOC system was used to test a new formulation, the combination of DOX and Anti Insulin-like Growth Factors (αIGF) using the same assays. αIGF is another reagent with known anti-cancer properties.29 Insulin-like Growth Factors (IGF) have a protective function against Reactive Oxidative Species (ROS),49 whereas DOX induces cellular damage via ROS,50 thus αIGF could potentially inhibit the antagonistic action of IGF, promoting enhanced damage to cancer cells when administered with DOX.51

3.3.1 Growth Inhibition Studies for PANC-1 Cells The LOC system was used to administer different concentrations of DOX (0.01 µg/µl, 0.02 µg/µl, 0.05 µg/µl, 0.06 µg/µl), each with αIGF at a fixed concentration of 0.01 µg/µl. When compared to the treatments with DOX alone, the combination treatment had higher growth inhibition effects as observed from the lower percentage of cell counts at all the time points (Figure 4A). On average, the combination formulations achieved lower cell counts of 12.8% at 24 h, 27.5% at 48 h and 38.8% at 72 h of treatment. It was observed that the combination formulation with 0.06 µg/µl DOX had the greatest reduction in cell count at 48 h (44.0%). However at 72 h, the difference in cell count with combination formulation with 0.05 µg/µl DOX was only 4.0%. This again suggests that treatments with more than 0.05 µg/µl dosage of DOX may not produce higher growth inhibition effects which would outweigh the increased risk of overdose. Drug resistance after 24 h was an issue with the treatment of PANC-1 with DOX only formulations. In the analysis of the rate of change of cell counts during treatment (Figure 4B), DOX only formulations had a rate of change of -0.458 to -0.726 Au/h at 24 h. However at 48 and 72 h, it declined to an average of -0.124 Au/h. In comparison, the combination formulations had more consistent performance, with an average rate of change of -0.714 Au/h at 48 Hrs and -0.664 Au/h at 72 Hrs. Furthermore, at low concentrations, the combination formulation with 0.01 µg/µl DOX achieved a rate of change of -0.540 Au/h at 24 h, this was in contrast to the positive rate of change of +0.330 Au/h when using the DOX only formulations. Furthermore, the combination drug formulation at low concentrations of 0.01 µg/µl DOX and αIGF had a cell count of 62% after 72 h and was almost as effective as the DOX only formulations using 0.05 and 0.06 µg/µl DOX concentrations, which had a comparable cell count of 77%. Overall, the DOX with αIGF formulations had an 18 ACS Paragon Plus Environment

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average 672.4% higher rate of growth inhibition than the formulations with DOX only. This highlights the potential of such testing platforms to find individualized drug formulations where combinations of low concentration drugs can have comparable therapeutic effects to highly concentrated single drug treatments. Thus maximizes treatment effects whilst minimizing potential side effects.

Figure 4: Comparison of the growth inhibition of PANC-1 cultures using combination drug therapy with DOX and αIGF formulations against the DOX only formulations. (A) Comparison of the changes in cell counts after treatment with different concentrations of DOX alone against the combination treatment with different concentrations of DOX co-delivered with a fixed concentration of αIGF at 0.01 µg/µl. (B) Comparison on the rate of decrease in cell counts using treatment with different concentrations of DOX alone against combination treatment with different concentrations of DOX co-delivered with a fixed concentration of αIGF at 0.01 µg/µl. (*Au represents arbitrary units calculated using the rate of change of the cell counts between observation time points, Vertical lines indicate the standard deviation and the number of samples used in each measurement, n = 4.) 3.3.2 Migration Inhibition Studies for PANC-1 Cells In the earlier migration studies, it was observed that larger DOX concentrations of 0.05 or 0.1 µg/µl could achieve almost complete migration inhibition. However, the use of high concentrations increases the risk of severe side effects. Another treatment strategy is required to minimize these risks. Using the presented LOC module, the combination treatments (DOX and αIGF) were tested to evaluate their migration inhibition abilities with a wound healing assay and the results are presented in Figure 5A and 5B. As IGF are important promoters of cell migration,52-54 the combination treatment with αIGF was expected to inhibit IGF, thus 19 ACS Paragon Plus Environment


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reducing cell migration. Indeed, treatment using only αIGF maintained the wound size at 54% after 48 h (Figure 5B), supporting the notion of αIGF inhibiting cell migration. The combination treatment showed great migration inhibition capabilities where αIGF, together with 0.005 or 0.01 µg/µl DOX maintained the wound area at 92% and 100% at 48 h of treatment, respectively. In contrast, the same treatments without αIGF reported an average wound area of 63%. The combination treatment thus had relatively the same inhibition effects as DOX only formulations which had 5 – 10 times more DOX chemotherapeutic agent, reducing the risk of complications from high concentrations of DOX. These results show that there is significant potential such LOC testing systems to individualize treatments, finding formulations with the required therapeutic effect whilst minimizing the potential risks of side effects. Lastly, these results show the presented LOC system has the potential to test many types of drug candidates. αIGF is a protein based reagent, of which are demanding in terms of handling, as fluctuations in testing conditions can cause changes in surface configuration or agglomeration. This is a serious limitation in current standalone LOCs as the integrated on-chip actuators can reach temperatures up to 220 °C, causing significant heating which would denature protein based drugs. Furthermore, current studies show some advanced drugs such as nanoparticles are highly sensitive to their environment, where temperatures of 32 °C to 41.5 °C are already high enough to cause premature drug release from their carriers.24, 55

Figure 5: Effect of the combination treatment on cell migration compared against the DOX only treatments. (A) Insets of bright field images of the wound healing assay showing the cell migration of PANC-1 cells into the wound area (yellow outline) after treatment with different concentrations of DOX with and without 0.01 µg/µl αIGF (B) Measured average changes in wound size for PANC-1 cells after treatment with different concentrations of DOX with and without 0.01 µg/µl αIGF (Scale bars of 200 µm apply globally to all the insets, vertical bars represent standard deviation of each measurement and the number of samples, n = 4.) 20 ACS Paragon Plus Environment

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3.4 Preliminary Study on Transition to Nanoparticle Delivery Systems In order to implement the treatment as tested by the presented LOC device, controlled delivery systems such as an implantable device or a nanoparticle vehicle would be required. As a preliminary demonstration, a nanoparticle system using a PLGA core and mPEG-DSPE outer shell was used as a controlled drug release system to deliver DOX and αIGF to PANC-1 cell cultures. Both PLGA and mPEG-DSPE are highly biocompatible biomaterials56,

57

and are FDA approved pharmaceutical materials56,

58

which have been

extensively used for their sustained drug release characteristics.59-62 The synthesized nanoparticles were loaded with 0.05 µg/µl DOX only or 0.05 µg/µl DOX and αIGF. The respective hydrodynamic diameters of these particles were 61.5 nm and 66.7 nm respectively. Additional information on the particle size distributions used can be found in the supporting information Figure S3. As a demonstration, the nanoparticles were designed to have the same 24 h drug release profile as the LOC biased at 7 V, continuously releasing 2 s pulses of 0.05 µg/µl DOX solution every 10 minutes (Figure 6A). In order to keep the nanoparticle release profile as close to the LOC system as possible, the nanoparticle solutions were replaced every 24 h. Comparing the growth inhibition effect of the nanoparticle solutions with the LOC system (Figure 6B), the data suggests that the nanoparticle system was able to mimic the results from LOC system to some degree. From Figure 6B, the cell counts of the cultures treated with the DOX loaded nanoparticles were similar to that of the cultures treated with the LOC, for both DOX only (solid line) and DOX with αIGF (dashed line). These results illustrate the potential of such LOC systems to aid in the design of advanced nanoparticle drugs, leading to more individualized treatments.



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Figure 6: Evaluation of therapeutic Nanoparticles with reference to treatment performed on the LOC system. (A) Drug release from loaded nanoparticles (DOX release) and drug release by the presented LOC system (LOC Release) when biased at 7V delivering 0.05 µg/µl of DOX for 2 s every 10 minutes into PBS. (B) Growth inhibitory effect of DOX and DOX with αIGF (DOX + αIGF) using nanoparticles and using the presented LOC system on the PANC-1 cell line. (Vertical lines indicate the standard deviation and the number of samples used in each measurement, n = 4.) *Represents significant differences between the DOX and DOX + αIGF treatments at the 0.95% significance level using two tailed t-test. 4. Conclusion Standalone LOC systems are able to integrate many functionalities on-chip, allowing them to be highly customizable for individualized applications. Such applications would be challenging to implement with traditional LOC systems as they would require an unrealistic number of off-chip components. As a demonstration, a standalone LOC drug testing system was prototyped to perform treatment individualization for PANC-1 and MIA PaCa-2 pancreatic cancer cell lines. The presented system was specifically engineered for high compatibility for cell cultures and a wide variety of test reagents, overcoming the challenges faced by current standalone LOC systems. The presented system was used to test a variety of formulations consisting of different concentrations of DOX and αIGF, which have known anti-cancer properties. To analyze the effect of each formulation on PANC-1 and MIA PaCa-2 cell cultures, drug uptake, growth inhibition and migration inhibition assays were performed with the help of the presented LOC. It was observed that both cell lines responded differently when tested with the same formulations, highlighting the need for such systems to individualize treatment for patients. Treatment was individualized by first adjusting the dosage of DOX and a treatment window of between 0.05 – 0.1 µg/µl was found for the MIA PaCa-2 cell line which has the highest growth inhibition and almost complete cell migration. Further individualization was performed for the PANC-1 cell line using a combination of DOX with αIGF, as PANC-1 showed some resistance to DOX only formulations. Compared to the DOX only formulations, the DOX with αIGF formulations had an average 672.4% higher rate of growth inhibition and depending on the formulation, the same migration inhibition effects could be achieved using 5 – 10 times lower DOX concentration, reducing the risk of side effects due to DOX. Finally, a preliminary study was presented illustrating how a tested formulation from the LOC can be translated for use by employing a nanoparticle system for controlled delivery. In vitro tests reveal that the nanoparticle system loaded with the same tested formulations had similar growth inhibition effects as those tested on the LOC system using programmed delivery. These 22 ACS Paragon Plus Environment

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studies show the great potential of such LOC systems in the formation of individualized medicine and it is predicted that such systems would be in great demand in order to increase the therapeutic effects of treatments for individual patients.

5. Supporting Information On-chip actuator performance data, drug uptake studies using PANC-1 cell lines, and nanoparticle size distributions.

6. Acknowledgements This work was supported by the Singapore Ministry of Education (Grants Tier 2 MOE2010-T2-2-010 (M4020020.040 ARC2/11) and NEWRI Seed Funding (M4061180.A91; M06080003), NTU-NHG Innovation Collaboration Grant (No. M4061202.040), A*STAR Science and Engineering Research Council (No. M4070176.040) and School of Electrical and Electronic Engineering at NTU.



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Method

Max Flow Rate (µl/s)

Power (mW)

Heat generated at actuator

Autoclave Compatibility (Sterility)

Controllability

Other Factors

Application

Compatible. No thermally affected components.

Linear controllability, flexible dosing

High consistency and reliablity (>500s continuous actuation). Refillable.

Self Actuated On-Chip LOC

Discrete dosing, requires active actuation to prevent backflow

Requires continuous active actuation or passive one way values to prevent backflow

Disposable LOC for biological enzyme analysis

Non-linear controllability, flexible dosing (Current vs pressure)

NA

On-Chip pressure generator

Linear Controllability

Voltage control results in very accurate droplet handling

Portable digital microfluidic platform

Discrete dosing

Fabrication process to incorporate pressurized gas is challenging

Disposable LOC, for POC Diagnostics

Flexible dosing

Energetic material deposition is non uniform resulting in differing flow rates

Portable and disposable LOC

0.025 – Electrochemica l31 (This Work)

0.065 (This work),

1.5 5.5

No Heating in linear region

0.631

Thermal expansion19

Solid Chemical microthruster 21

Electrowetting on diaelectric

7 x 10-6*

220 800

< 80 °C

Incompatible. Expandable spheres will rupture at 125 °C.

0.283*

236 321*

70 °C 100 °C

Incompatible. Solid propellant decomposes at 70 - 100 °C.

0.0021*

18, 63, 64

Physical microthruster 20,

1.56*

0.01 0.1 64

40

26

NR

Compatible. No thermally affected components.

220 °C

Possibly Compatible. Gas must be filled during fabrication and heat may weaken the chambers

100 °C

Compatible. Energetic material decomposes at 225 °C

26

Liquid chemical microthruster22

37.5*

90

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Table 1: Comparison of various standalone on-chip LOC Systems with on-chip actuators. *Indicates the values were calculated based on the reported specifications of that paper. Electrochemical Method refers to the method used in this study.


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Standalone Lab-On-a-Chip Systems towards the Evaluation of Therapeutic Biomaterials in Individualized Disease Treatment Danny Jian Hang Tng1†, Peiyi Song1†, Rui Hu1, Chengbin Yang1, Cher Heng Tan2 and Ken-Tye Yong1* 1 School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 2 Department of Diagnostic Radiology, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore 308433

†

These authors contributed equally to this work. *Corresponding author: Ken-Tye Yong, PhD, School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Tel: +65-6790-5444, Email: [email protected]

Synopsis Standalone Lab-On-a-Chip drug testing system with customizable on-chip assays was fabricated for the individualization of cancer treatment. The drug treatments were prepared by using nanoparticle formulations that had effective therapeutic effects on the pancreatic cancer cell lines.

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