Graphene oxide cellular patches for mesenchymal stem cell-based cancer therapy

Accepted Manuscript Graphene oxide cellular patches for mesenchymal stem cell-based cancer therapy Smruthi Suryaprakash, Mingqiang Li, Yeh-Hsing Lao, Hong-Xia Wang, Kam W. Leong PII:

S0008-6223(17)31264-2

DOI:

10.1016/j.carbon.2017.12.031

Reference:

CARBON 12661

To appear in:

Carbon

Received Date: 31 May 2017 Revised Date:

4 December 2017

Accepted Date: 8 December 2017

Please cite this article as: S. Suryaprakash, M. Li, Y.-H. Lao, H.-X. Wang, K.W. Leong, Graphene oxide cellular patches for mesenchymal stem cell-based cancer therapy, Carbon (2018), doi: 10.1016/ j.carbon.2017.12.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Chemodrugs

MSC-Go

RI PT

Graphene oxide

TE D

M AN U

SC

+

Mesenchymal Stem Cell – GO/drug conjugate

AC C

EP

drugs tumor 500cells mm

Tumor

tumor-homing & on-site drug release

ACCEPTED MANUSCRIPT

Graphene oxide cellular patches for mesenchymal stem cell-based cancer therapy Smruthi Suryaprakash1, Mingqiang Li1, Yeh-Hsing Lao1, Hong-Xia Wang1, Kam W Leong*1,2 1

Department of Biomedical Engineering, Columbia University, New York, NY, 10027, USA Department of Systems Biology, Columbia University Medical Center, New York, NY, 10032, USA

RI PT

2

Abstract

SC

Graphene oxide (GO) is a versatile platform for drug delivery, but poor accumulation at the tumor site limits the translation of this technology. Mesenchymal stem cells (MSC) on the other

M AN U

hand is tumor trophic but is limited to delivering only protein-based drugs. Our solution is to combine the individual attributes of GO and MSC by loading GO on the surface of MSC to create a unique drug delivery platform wherein, GO can be used to load a range of therapeutics and MSC can carry the drug effectively to the site of the tumor. Here, we demonstrated that GO can be loaded with two types of chemo drugs, doxorubicin and mitoxantrone, with similar efficiency (>30%). The drug-GO complex was loaded on MSC without affecting the MSC

TE D

viability significantly. On exposure to GO-mitoxantrone, 87% of MSC survived while only 28% of the cancer cells (LN18) survived, demonstrating the selective killing of the cancer cells but not MSC. Furthermore, drug-GO loading did not affect the tumor trophic property of MSC, and coculture of the drug-GO loaded MSC with LN18 showed a dose-dependent killing of LN18.

AC C

for cancer therapy.

EP

Together, these results show the feasibility of using drug-GO loaded MSC as an effective carrier

1. Introduction

An effective cancer therapy would discern cancer cells from normal cells and allow for robust killing of the cancer cells only. However most anti-tumor drugs are not specific and * Corresponding Author Email Address: [email protected]

ACCEPTED MANUSCRIPT

indiscriminately kill the cells around them. Targeted delivery of these drugs using nanoparticles have also not been successful because of masking of the targeting ligands by the biological the injected nanoparticles penetrate the tumor[1].

RI PT

components. Survey of the literature for the past 10 years has shown that only 0.7% (median) of

Thus, there is a need to develop a more effective drug delivery system that will allow for targeted

SC

killing of cancer cells.

Modified graphene oxide (GO), a 2D graphene sheet functionalized with carboxylic acid,

M AN U

epoxide and hydroxyl group has been successfully used in various biomedical applications including bioimaging, gene delivery, drug delivery and tissue engineering[2]. The high specific surface area allows for high drug loading though - stacking, electrostatic or hydrophobic interaction with small molecules, proteins, and nucleic acid. Sun et al have shown high loading of DOX on graphene via adsorption and pH dependent-release[3]. Since the tumor microenvironment is acidic this allows for site-specific release of the drug. While graphene oxide

TE D

is a versatile platform to deliver a wide range of therapeutics with controlled release, the low accumulation of graphene oxide at the tumor site is of serious concern for effective translation of this technology[4], [5].

EP

Mesenchymal stem cells (MSC) on the other hand have inherent tumor trophic property making it an attractive candidate for cancer therapy[6]. The site-specific delivery of chemotherapeutics ensures minimal off-target effects, while immune compatibility allows for allogeneic

AC C

transplantation. It has additional advantages: it is deemed to be safe as reported by more than 350 clinical trials, it is easily isolated from a wide range of sources and it allows for facile genetic manipulation. Collectively these factors make MSC an ideal carrier for cancer therapy[7].

Therapies using MSC, however, have been limited to genetically engineering the cells to secrete anti-tumor peptidic drugs. Various strategies have been used for targeted treatment of different cancer types such as delivery of (1) interleukins to regulate the inflammatory and immune response[8], (2) interferons to inhibit tumor growth[9], (3) enzymes to activate prodrug[10], and

ACCEPTED MANUSCRIPT

(4) pro- apoptotic proteins such as TRAIL to inhibit cancer cell proliferation[11]. This however, precludes the delivery of non-peptidic drugs and therapeutic nucleic acids that can combat tumor progression via different mechanisms. Cancer is highly heterogeneous in nature and is constantly evolving. It is highly unlikely that any one anti-tumor drug would be successful.

RI PT

Hence, being able to deliver a wide range of therapeutics is highly desired for cancer therapy[12].

Drug-loaded NP are usually coupled with MSC therapy through either cellular [16]

or surface anchorage[17]. In case of cellular internalization, the

SC

internalization[13]-[15]

loading efficiency depends on a plethora of particle characteristics such as charge, hydrophobicity, size and shape. The stability and availability of the drug internalized by the MSC

M AN U

depends on the drug’s ability to escape the late endosome, and the effects of the drug within MSC are of concern. In case of surface anchorage, the interaction might be short-lived depending on the specific receptor to which they bind. The cell also has the potential to turn immunogenic after engineering due to the alteration in membrane composition[18]. These methods release the drugs through either exocytosis or simple diffusion making it harder to control and predict the release. There is also a high possibility that the cells would excrete the

TE D

drugs before reaching the target site. Hence, there is a need to develop a system that is capable of controlled delivery of a wide variety of drugs while taking advantage of MSC’s tumor trophic properties for targeted delivery.

EP

We propose to combine the drug delivery capability and the cellular interaction of GO to load drugs onto the surface of MSC. On one hand, the graphene oxide flakes allow for cellular interaction mediated by the hydrophobic interactions, electrostatic forces and hydrogen bonding

AC C

between the GO and proteins from the cell culture media[19], versatility for adsorbing a wide range of therapeutics, and minimal internalization into MSC. On the other hand, the tumortrophic MSC can carry the drug-loaded GO adsorbed on the cell surface to the tumor site and ideally the tumor cell, thereby circumventing the issue of low penetration and poor cell specificity. To test the hypothesis, we used doxorubicin hydrochloride (DOX) and mitoxantrone (MTX) as model drugs adsorbed on GO through physical mixing. The GO-loaded MSC was then tested against cancer cells to evaluate the tumor trophic nature and killing efficacy of the system.

ACCEPTED MANUSCRIPT

2. Experimental 2.1. Loading and release of graphene oxide

RI PT

GO used in all the experiments were bought from Graphene Supermarket. The GO used was 5mg/mL and the size of the flakes ranged from 1-6 µm. To prepare GO-Drug, GO was sonicated and mixed with different ratio of the drugs: DOX, and MTX (Sigma). The complex was left in a shaker overnight in the dark. The complex was then washed thrice to remove the unbound drug.

SC

The absorbance of DOX at 478nm and MTX at 610nm in the supernatant was used to measure the amount of drug loaded onto GO. Following this, GO-DOX and GO-MTX at the highest loading level was prepared and added to the dialysis bag with a molecular weight cut off 1000

M AN U

Da. Half the dialysis bags were suspended in 5 mL of PBS at pH7.4 and the rest at PBS at pH5.5. Samples were collected at regular interval and the amount of drug released was measured using absorbance plate reader. 2.2. Culture of MSC

TE D

MSC that was used in the experiments was obtained from Texas A&M Health Science Center, Institute for regenerative medicine. For all the experiments the cells between passage 3-6 was used. MSC was cultured using alpha Minimum Essential Medium Eagle (Thermo Fisher Scientific) supplemented by 20% FBS and 1% Penicillin Streptomycin (Pen Strep, Thermo

EP

Fisher Scientific). The cancer cells, LN18, and MDA-MB-231 were cultured using Dulbecco’s Modified Eagle’s Medium supplemented (Invitrogen) by 10% FBS (Atlanta Biologics), and 1%

AC C

Pen Strep. GBM8 was cultured using Neurobasal medium (Thermo Fisher Scientific) supplemented by L-Glutamine (Thermo Fisher Scientific), B27 (Thermo Fisher Scientific), N2 (Thermo Fisher Scientific), Heparin , Fibroblast growth factor, Epidermal growth factor and Pen strep. All the cells used in the study were cultured at 37oC, 5% CO2 and 95% relative humidity.

2.3. Formation and characterization of MSC-GO complex

ACCEPTED MANUSCRIPT

The MSC and GO were incubated for 4 hours at 37oC, 5% CO2 and 95% relative humidity to allow the GO to adhere to MSC. Following this the cells were washed twice, trypsinized and replated in a new plate and cultured for further experiments. To visualize the interaction of MSC and GO, GO was labeled with Dil (1µg/mL, Thermoscientific) overnight at 37oC[19]. The

RI PT

complex was washed thrice with PBS to remove the unbound dye. This complex at concentration of 10ug/mL of GO was used to bind to MSC. After binding the cells were trypsinized and analyzed using fluorescence microscopy (Nikon 10X) to visualize the binding, and flow cytometry to measure the binding efficiency. MSC-GO complex was also analyzed using a Zeiss

SC

SIGMA VP scanning electron microscope (SEM) operated at 5.00 Kv. For this the cells were first fixed with 4% paraformaldehyde. Dehydration of the fixed cells was carried out sequentially using ethanol of concentration 30%, 50%, 70%, 90% and 100%. The sample was then washed

M AN U

thrice with Hexamethyldisilazane (Sigma) and allowed to dry at room temperature and coated with gold in sputter coater before analysis using SEM. 2.4. Migration

Migration assay was performed using the two-chamber cell culture insert (ibidi). The cancer cell

TE D

LN18 expressing mcherry and MSC-GO-Drug (0, 2.5 µg/mL, 5 µg/mL, and 10 µg/mL) were plated in adjacent chamber (0.5mm separation) of the cell culture insert at a density of 7500 cells/well which is 70% confluency. The cells were left overnight to attach and the next day the cell culture insert was removed to create a well-defined gap of 500 µm. The movement of the

EP

cells were then captured every hour for 48 hours using a live cell imaging system. ImageJ software was used to track the individual cells for migratory capabilities using the plugin

AC C

manual tracking and chemotaxis. 2.5. Viability Test

To understand the effect of GO and GO-Drug on the cells, MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay was performed. Briefly, the cells were plated in a 96 well and exposed to a rage of concentrations of either GO, GO-DOX, or GO-MTX. After 24 hours, the MTT reagent was added and the cells were incubated at 37oC for 2 hours before measuring

ACCEPTED MANUSCRIPT

the absorbance at 570nm using a plate reader. The cell viability was calculated by comparing to untreated cells which was assumed to be 100% viable.

RI PT

2.6. Characterization of toxicity of MSC-GO-Drug The cell killing efficacy of the system was studied using three different models. In the first coculture model LN19 and MSC-GO-Drug were cultured together for 48 hours. The cancer cell was transduced with luciferase and the viability of the cancer cells was measured using Steady

SC

Glo (Promega). In the second model, LN18 and MSC-GO-Drug were plated at a ratio of 1:2 using the previously described migration protocol. After 48hrs the viability of the cancer cells was measured using the Steady Glo. Finally, in the third model, a transwell insert was used to

M AN U

plate LN18 and MSC-GO-Drug in the plate and insert respectively at a ratio of 1:2. After 48 hours the cancer cells were lysed and the amount of luciferase was measured using Steady Glo. 2.7. Statistical Analysis

The data in the figures are represented by mean and standard error of mean. One-way analysis

TE D

of variance (ANOVA) with the Tukey significant difference post hoc test was performed when more than two means were compared. T-test was performed when comparing two means. A value of p < 0.05 was considered to denote statistical significance.

EP

3. Results and Discussion

AC C

3.1. Loading and Release

The large surface area of GO makes it an excellent candidate to load a wide range of therapeutics. The drug loading level increased with the ratio of drug:GO in the solution. At 0.5 mg/mL of drug the DOX loading capacity was 0.34 mg of DOX per mg of GO, and the MTX loading capacity was 0.35 mg of MTX/mg of GO (Figure 1A). The interaction between GO and the drug is mostly governed by the - stacking as well as hydrogen bonding between the –OH and –COOH on GO and –OH and –NH2 on DOX [20]. In case of the release (Figures 1B and C) for both DOX and MTX we can see a burst release within the first 20 hours. However, since most of the drugs are released within the first three days this could potentially limit the ability to

ACCEPTED MANUSCRIPT

target invasive glioblastoma cells that have spread to the contra-lateral hemisphere. Additionally, in case of both drugs double the amount of drug was released at the lower pH of 5.5 (29% DOX, 40% MTX) compared with the normal physiological pH of 7.4 (15% DOX, 20%MTX). This could be due to the partial dissociation of hydrogen bonding at low pH leading

RI PT

to the release of the drugs. This would be useful because the pH surrounding the cancer cells would be lower than the physiological pH [21], which could trigger the release of the drug from GO. This could possibly be a secondary level of targeting to release the drugs close to the site of

(B)

(C)

M AN U

(A)

SC

the tumor.

TE D

Figure 1. Characterization of GO-DOX and GO-MTX complex (A) Loading of DOX and MTX on GO at different initial concentrations (B) Cumulative release of DOX at pH 5.5 and pH 7.4 (C) Cumulative release of MTX at pH 5.5 and pH 7.4. Data are presented as average ± standard

EP

error of mean (n = 3; significance: *: p < 0.05; ***: p< 0.001; determined by Student’s T-test).

AC C

3.2. MSC-GO loading and characterization To understand the long-term toxicity of GO on MSC, the cells were incubated with increasing concentrations of GO ranging from 0.2µg/mL to 100µg/mL for 1,3 and 5 days. At the end of each time point MTT assay showed that the cell viability was above 80% for GO concentrations up to 100µg/mL (Figure 2C).

Although GO quenches fluorescence signal, we used previously

published protocol of using high concentrations of Di-l to overcome this issue. The fluorescent image of the MSC-GO shows that the GO is localized on the surface of MSC (Figure 2A). The size of the particle is known to affect the cellular uptake efficiency. While most studies have

ACCEPTED MANUSCRIPT

shown uptake of GO using nanographene oxide (20-300nm), we used larger flakes (1-6 µm) which are not easily internalized by MSC. The interaction between the cells and GO is probably aided by the extracellular matrix (ECM) adsorption on the surface of GO via hydrophobic, electrostatic, and hydrogen bonding. GO is known to effectively absorb proteins though

RI PT

hydrophobic interactions, electrostatic forces, and hydrogen bonding. The absorption of ECM graphene oxide can facilitate the cell adhesion on GO. Flow cytometry analysis showed that more than 95% of the cells were associated with GO (Figure 2B), further demonstrating the efficient interaction between GO and MSC. SEM analysis further confirmed that the GO

EP

TE D

M AN U

SC

remained attached to the surface of the cells after coating and trypsinization (Figure 2D).

Figure 2. Characterization of MSC-GO complex (A) Fluorescent image of MSC (green) and GO

AC C

(red) (B) Flow cytometry analysis of MSC and MSC-GO (C) Cell viability of MSC across a range of GO concentrations for Day 1, 3 and 5 (D) SEM image of MSC-GO complex. Data are presented as average ± standard error of mean (n = 3).

3.3. GO-Drug toxicity

ACCEPTED MANUSCRIPT

The ability of MSC to carry GO-Drug hinges on its ability to survive at concentrations that are toxic to the cancer cells. Avoiding cellular damages is crucial for effective delivery. GO loaded with DOX and MTX were added to MSC as well as a range of cancer cells (LN18, GBM8, MDAMB-231) to evaluate the toxicity. LN18 is a human glioblastoma cell line, GBM8 is a patient

RI PT

derived human glioblastoma cell line, and MDA-MB-231 is a human breast adenocarcinoma cell line. At concentrations of 5µg/mL of GO-MTX, 87% of MSC survived while only 28% of LN18, 21% of GBM8, and 37% of MDA-MB-231 survived (Figure 3). This wide gap in toxicity has been reported previously and attributed to possible drug-resistant ability of MSC[16], [17]. This

SC

indicates that we can load GO-Drug on to MSC at concentrations that are not toxic to MSC but can still effectively kill the cancer cells. The largest difference was seen with LN18 and this cell type was used for all future experiments.

TE D AC C

EP

(B)

M AN U

(A)

Figure 3. (A) Cell viability of cancer cells across a range of GO-DOX concentrations (B) Cell viability of cancer cells across a range of GO-MTX concentrations. Data are presented as average ± standard error of mean (n = 3).

ACCEPTED MANUSCRIPT

3.4. Migration To understand the effect of GO on the migration potential of MSC, different concentrations of GO (0, 2.5 µg/mL, 5 µg/mL, and 10 µg/mL) was loaded on to MSC and its migration towards

RI PT

cancer cells were analyzed. Using manual tracking and chemotaxis we analyzed various parameters such as distance travelled, velocity, directionality. MSC without cancer cells moved significantly slower and had lower directionality (Supplementary Figure 6). The Rayleigh test for MSC without and with cancer cells had p-value of 0.277 and 2.16*10^-7 further

SC

demonstrating that the cancer cells are required for directional movement of MSC. The migration of MSC characterized by the velocity and directionality was not affected by the presence of GO. Irrespective of the concentration of GO the MSC-GO complex can migrate

M AN U

towards the tumor cells with a high degree of directionality. While the mechanism for the migration of MSC towards cancer cells is still under investigation the most popular hypothesis is the interaction of the C-X-C chemokine receptor type 4 (CXCR-4) from MSC towards the SDF-1 gradient created by the cancer cells[22]. On analyzing the CXCR4 levels of MSC coated with different concentrations of GO we found a two-fold increase (Supporting Information), confirming the results that the migration is not affected by the presence of GO.

Cancer Cell (LN18)

(C)

T=48 hrs

AC C

EP

Cancer Cell (LN18) T=0 hrs

(B)

MSC Graphene Oxide

MSC Graphene Oxide

TE D

(A)

Figure 4. Effect of GO on migration of MSC (A) Live cell image of MSC-GO (GO is green) moving towards LN18 (Red) at time 0 hours and 48 hours (B) Velocity of the cells under different GO concentrations (C) Directionality is the ratio of the euclidian distance and the accumulated distance as measured by MSC under different GO concentrations. 3.5. Toxicity

ACCEPTED MANUSCRIPT

LN18 was cultured with MSC-GO-Drug and in case of both DOX and MTX the cancer cells were killed in a dose-dependent manner. At the ratio of 2:1 (MSC: cancer cells), the amount of LN18 that survived was 46%. For the same ratio the amount of LN18 that survived with MTX and DOX

RI PT

loaded GO are 40.7% and 106.23% (Supplementary Figure 4). This demonstrates the need to have a platform that can deliver a wide range of therapeutics because not all cells are sensitive to the same drug. To test our hypothesis that the MSC-GO-Drug needs to carry the drug and migrate towards the cancer cells to kill it, we used three different models: Co-culture, Migration

SC

and Transwell. In case of the transwell model, the MSC and cancer cells have a barrier in between hence only the drug that has been released from GO can diffuse and kill the cancer cells. In the migration model the MSC needs to carry the GO-Drug to the cancer cells 500um

M AN U

apart to kill them. Finally, in the third co-culture model the cells and the cancer cells are cocultured so the killing is done through cell-cell contact. The co-culture model had the highest cell death (54%) followed by the migration model (53%) and finally the transwell model (27%). Furthermore, there was a delay in the killing observed in the migration model (Supplementary Figure 3). Collectively they show that the drug does not easily detach from the complex, but rather needs to be carried by MSC towards the cancer cells for effective killing. This again

AC C

EP

TE D

would raise the cancer-cell specificity of this delivery system.

ACCEPTED MANUSCRIPT

(A)

(B) Co-culture model

M AN U

500 500 µµm m

SC

Migration Model Cell-Cell contact between MSC and cancer cells on (C) migration

RI PT

Cell-Cell contact between MSC and cancer cells

Transwell Model

Only diffusion of molecules between MSC and cancer cells

Cancer cell

TE D

MSC-GO

Figure 5. Functional testing of MSC-GO-Drug (A) The three different toxicity models that were studied (B) Cell viability of LN 18 under different amounts of MSC-GO-Drug (C) Toxicity of

EP

LN18 under the different models tested. Data are presented as average ± standard error of mean (n = 3; significance: **: p < 0.01; n.s.: not significant; determined by One-way ANOVA).

AC C

4. Conclusion

This study shows that the MSC-GO is an effective vehicle to carry the drugs to the tumor cells. MSC-GO forms a strong bond which was visualized by fluorescence imaging, flow cytometry and SEM. The migration of this complex is not affected by the concentration of GO. MSC-GO-Drug is also capable of killing the cancer cells in a dose-dependent manner. Finally, we could show that MSC-GO-Drug needs to be in cell-cell contact to be an effective system. This versatile system can be used to load a wide range of therapeutics and can be used as an effective cancer therapeutic to deliver the drugs locally and effectively.

ACCEPTED MANUSCRIPT

Acknowledgement

RI PT

This work is supported by NIH (HL109442, AI096305, UH3TR000505, and GM110494); Guangdong Innovative and Entrepreneurial Research Team Program, China (2013S086) and Global Research Laboratory Program, Korea (2015032163). S.S. acknowledges the fellowship,

AC C

EP

TE D

M AN U

SC

Faculty for the Future, from Schlumberger Foundation.

Reference

[1]

S. Wilhelm, A. J. Tavares, Q. Dai, S. Ohta, J. Audet, H. F. Dvorak, and W. C. W. Chan,

ACCEPTED MANUSCRIPT

“Analysis of nanoparticle delivery to tumours,” Nature Reviews Materials, vol. 1, p. 16014, Apr. 2016. [2]

S. Goenka, V. Sant, and S. Sant, “Graphene-based nanomaterials for drug delivery and tissue engineering,” Journal of Controlled Release, vol. 173, pp. 75–88, Jan. 2014. X. Sun, Z. Liu, K. Welsher, J. T. Robinson, A. Goodwin, S. Zaric, and H. Dai, “Nano-

RI PT

[3]

Graphene Oxide for Cellular Imaging and Drug Delivery.,” Nano Res, vol. 1, no. 3, pp. 203–212, 2008. [4]

T. Zhou, X. Zhou, and D. Xing, “Controlled release of doxorubicin from graphene oxide

SC

based charge-reversal nanocarrier,” Biomaterials, vol. 35, no. 13, pp. 4185–4194, Apr. 2014. [5]

K. Yang, J. Wan, S. Zhang, B. Tian, Y. Zhang, and Z. Liu, “The influence of surface

M AN U

chemistry and size of nanoscale graphene oxide on photothermal therapy of cancer using ultra-low laser power.,” Biomaterials, vol. 33, no. 7, pp. 2206–2214, Mar. 2012. [6]

K. Shah, “Mesenchymal stem cells engineered for cancer therapy,” vol. 64, no. 8, pp. 739–748, Jun. 2012.

[7]

J. S. Park, S. Suryaprakash, Y.-H. Lao, and K. W. Leong, “Engineering mesenchymal Aug. 2015.

[8]

TE D

stem cells for regenerative medicine and drug delivery,” Methods, vol. 84, pp. 3–16, C. H. Ryu, S.-H. Park, S. A. Park, S. M. Kim, J. Y. Lim, C. H. Jeong, W.-S. Yoon, W.-I. Oh, Y.-C. Sung, and S.-S. Jeun, “Gene therapy of intracranial glioma using interleukin 12-secreting human umbilical cord blood-derived mesenchymal stem cells.,” Human [9]

EP

Gene Therapy, vol. 22, no. 6, pp. 733–743, Jun. 2011. C. Ren, S. Kumar, D. Chanda, L. Kallman, J. Chen, J. D. Mountz, and S. Ponnazhagan, “Cancer gene therapy using mesenchymal stem cells expressing interferon- in a mouse

AC C

prostate cancer lung metastasis model,” Gene Ther, vol. 15, no. 21, pp. 1446–1453, Jul. 2008.

[10]

C. Altaner, V. Altanerova, M. Cihova, K. Ondicova, B. Rychly, L. Baciak, and B. Mravec, “Complete regression of glioblastoma by mesenchymal stem cells mediated prodrug gene therapy simulating clinical therapeutic scenario.,” Int. J. Cancer, vol. 134, no. 6, pp. 1458–1465, Mar. 2014.

[11]

L. S. Sasportas, R. Kasmieh, H. Wakimoto, S. Hingtgen, J. A. J. M. van de Water, G. Mohapatra, J. L. Figueiredo, R. L. Martuza, R. Weissleder, and K. Shah, “Assessment of therapeutic efficacy and fate of engineered human mesenchymal stem cells for cancer

ACCEPTED MANUSCRIPT

therapy.,” Proc. Natl. Acad. Sci. U.S.A., vol. 106, no. 12, pp. 4822–4827, Mar. 2009. [12]

M. E. Godsey, S. Suryaprakash, and K. W. Leong, “Materials innovation for co-delivery of diverse therapeutic cargos.,” RSC Adv., vol. 3, no. 47, pp. 24794–24811, Dec. 2013.

[13]

X. Zhang, S. Yao, C. Liu, and Y. Jiang, “Tumor tropic delivery of doxorubicin-polymer

RI PT

conjugates using mesenchymal stem cells for glioma therapy,” Biomaterials, vol. 39, pp. 269–281, Jan. 2015. [14]

S. Kang, S. H. Bhang, S. Hwang, J.-K. Yoon, J. Song, H.-K. Jang, S. Kim, and B.-S. Kim, “Mesenchymal Stem Cells Aggregate and Deliver Gold Nanoparticles to Tumors for

[15]

SC

Photothermal Therapy.,” ACS Nano, vol. 9, no. 10, pp. 9678–9690, Sep. 2015.

X. Huang, F. Zhang, H. Wang, G. Niu, K. Y. Choi, M. Swierczewska, G. Zhang, H. Gao, Z. Wang, L. Zhu, H. S. Choi, S. Lee, and X. Chen, “Mesenchymal stem cell-based cell

M AN U

engineering with multifunctional mesoporous silica nanoparticles for tumor delivery,” Biomaterials, vol. 34, no. 7, pp. 1772–1780, Feb. 2013. [16]

L. Pascucci, V. Coccè, A. Bonomi, D. Ami, P. Ceccarelli, E. Ciusani, L. Viganò, A. Locatelli, F. Sisto, S. M. Doglia, E. Parati, M. E. Bernardo, M. Muraca, G. Alessandri, G. Bondiolotti, and A. Pessina, “Paclitaxel is incorporated by mesenchymal stromal cells and released in exosomes that inhibit in vitro tumor growth: A new approach for

[17]

TE D

drug delivery.,” J Control Release, vol. 192, pp. 262–270, Jul. 2014. L. Li, Y. Guan, H. Liu, N. Hao, T. Liu, X. Meng, C. Fu, Y. Li, Q. Qu, Y. Zhang, S. Ji, L. Chen, D. Chen, and F. Tang, “Silica Nanorattle–Doxorubicin-Anchored Mesenchymal Stem Cells for Tumor-Tropic Therapy,” ACS …, vol. 5, no. 9, pp. 7462–7470, Aug. [18]

EP

2011.

Q. Wang, H. Cheng, H. Peng, H. Zhou, P. Y. Li, and R. Langer, “Non-genetic engineering of cells for drug delivery and cell-based therapy.,” Advanced drug delivery

[19]

AC C

…, Dec. 2014.

J. Park, B. Kim, J. Han, J. Oh, S. Park, S. Ryu, S. Jung, J.-Y. Shin, B. S. Lee, B. H. Hong,

D. Choi, and B.-S. Kim, “Graphene Oxide Flakes as a Cellular Adhesive: Prevention of Reactive Oxygen Species Mediated Death of Implanted Cells for Cardiac Repair.,” ACS

Nano, May 2015. [20]

X. Yang, X. Zhang, Z. Liu, Y. Ma, Y. Huang, and Y. Chen, “High-Efficiency Loading and Controlled Release of Doxorubicin Hydrochloride on Graphene Oxide,” J. Phys. Chem. C, vol. 112, no. 45, pp. 17554–17558, Oct. 2008.

[21]

J. L. Wike-Hooley, J. Haveman, and H. S. Reinhold, “The relevance of tumour pH to the

ACCEPTED MANUSCRIPT

treatment of malignant disease,” Radiotherapy and Oncology, vol. 2, no. 4, pp. 343– 366, Dec. 1984. [22]

Z. Cheng, L. Ou, X. Zhou, F. Li, X. Jia, Y. Zhang, X. Liu, Y. Li, C. A. Ward, L. G. Melo, and D. Kong, “Targeted migration of mesenchymal stem cells modified with CXCR4

RI PT

gene to infarcted myocardium improves cardiac performance.,” Mol Ther, vol. 16, no. 3,

EP

TE D

M AN U

SC

pp. 571–579, Mar. 2008.

AC C

Supporting Information

Figure 1. Characterization of Graphene Oxide (A) Fourier-transform infrared spectroscopy profile (B) Ultraviolent-visible spectroscopy spectrum (C) Scanning electron microscope image (D) Size distribution of graphene oxide analyzed from SEM images.

ACCEPTED MANUSCRIPT

50

90

80

70

B

40 30 20 10 0 220

4000 3500 3000 2500 2000 1500 1000 500

320

420

520

620

720

820

Wavelength (nm) 5

C Size (µm)

4 3 2

M AN U

1

D

SC

Wave number (cm-1)

RI PT

A

Relative Intensity (a.u.)

% Transmittance

100

0

Graphene Oxide

Figure 2. Cell viability of cancer cells across a range of DOX concentrations. Data are presented as average ± standard error of mean (n = 3).

TE D

100

MSC

80

MDA-MB-231

60 40

EP

Cell viability (%)

120

LN18

20

AC C

0 0.1

1

GBM8

10

100

[DOX] (µg/mL)

Figure 3. Cell death of LN18 under different time points in a Migration Model. Data are presented as average ± standard error of mean (n = 3).

ACCEPTED MANUSCRIPT

80

Cell death (%)

** 60

RI PT

40 20 0

24 hrs

48 hrs

SC

MSC-GO-MTX

M AN U

Figure 4. Cell viability of GBM8 under different amounts of MSC-GO-Drug. Data are presented as average ± standard error of mean (n = 3). DOX MTX

Cell viability (%)

120 100 80 60 40

TE D

20 0 0.1

1

2

3

4

MSC-GO/cancer cell (GBM8)

EP

Figure 5. Fold change of CXCR4 for different concentrations of GO coated MSC.

3

10

0

2. 5

1

5

2

AC C

CXCR4 Fold Change

4

GO (µg/mL)

Figure 6. Live cell image of MSC-GO (GO is green) moving without cancer cells at time 0 hours and 48 hours

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

Figure 7. Tracking of MSC migration (Left) MSC with cancer cells (Right) MSC without cancer cells