Supplementary Data

Tumortropic Adipose-Derived Stem Cells Carrying Smart

Nanotherapeutics for Targeted Delivery and Dual-Modality Therapy of

Orthotopic Glioblastoma

Wen-Chia Huang,‡a I.-Lin Lu,‡a,b Wen-Hsuan Chiang,a Yi-Wen Lin,a Yuan-Chung Tsai,a

Hsin-Hung Chen,c Chien-Wen Chang,a Chi-Shiun Chianga and Hsin-Cheng Chiua*

aDepartment of Biomedical Engineering and Environmental Sciences,

National Tsing Hua University, Hsinchu 300, Taiwan

bDepartment of Surgery, Hsinchu Mackay Memorial Hospital, Hsinchu

30071, Taiwan

cDepartment of Chemical Engineering, National Chung Hsing

University, Taichung 402, Taiwan

*To whom correspondence should be addressed. Fax: 886-35718649. Tel: 886-

35750829. E-mail: hscchiu@mx.nthu.edu.tw (H.-C. Chiu)

‡The authors contributed equally to this work.

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Synthesis of oleic acid-coated SPIONs.

Oleic acid-coated SPIONs were prepared through the co-precipitation of FeCl2 and

FeCl3 via addition of NH4OH [1]. In brief, FeCl2 and FeCl3 (1:2 molar ratio) were mixed

in deionized water and heated to 80 oC under nitrogen. NH4OH was added dropwise by

syringe under vigorous stirring while the high temperature being further maintained for

30 min. This was followed by the addition of oleic acid and the reaction was allowed to

proceed at 95 oC for 90 min. The oleic acid-coated SPIONs were washed repeatedly with

ethanol and collected by centrifugation. The resultant pellets were resuspended in

chloroform. The superparamagnetic and magnetic properties of oleic acid-coated SPIONs

were characterized on an MPMS XL-7 Quantum Design SQUID magnetometer at 300 K.

The applied magnetic field varied in the range 1×104~−1×104 Oe in order to attain the

hysteresis loops.

Synthesis of poly(-glutamic acid-co-distearyl -glutamate) (poly(-GA-co-DSGA))

The esterification reaction of poly(-GA) (Mw 15.6 kDa) with N-

hydroxysuccinimide (NHS; equivalent to the molar concentration of the -GA residues)

was performed in dimethyl sulfoxide (DMSO)/pyridine (3/1 (v/v)) solution at 4 oC for 48

h under stirring, using N,N'-dicyclohexylcarbodiimide (DCC; equivalent to the molar

concentration of the -GA residues) as the coupling reagent [2]. The resulting mixture

was then filtered repeatedly to remove the undesired byproduct, N,N-dicyclohexylurea.

The solution was added into a DMSO solution containing distearin (15 mol% with

respect to the original -GA residues in poly(-GA)) and 4-dimethylaminopyridine

(DMAP) as the catalyst. The transesterification reaction was carried out at the ambient

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temperature for 7 days. The hydrolysis of the unreacted active N-succinimidyl esters of

-GA residues was carried out by addition of Tris buffer (pH8.0) under stirring at 4 oC for

5 days. After thorough dialysis (Cellu Sep MWCO 12000~14000) against deionized

water, the purified copolymer was then collected by lyophilization. The synthetic route

and chemical structure of poly(-GA-co-DSGA) are illustrated in Fig. S1. The DSGA

content was determined by 1H-NMR in DMSO-d6 at ambient temperature. Based on the

relative integral ratio of the feature signals of glutamic α-CH proton at δ 4.1 ppm in the

poly(-GA) backbone and methyl (-CH3) protons at δ 0.86 ppm from distearyl moieties

(Fig. S1), the DSGA content was estimated to be ca 9.24 mol%. Poly(-GA-co-DSGA)

(MW 2.18×104 g/mol) used in this work was thus obtained. The polydispersity was

estimated to be ca 1.52 by gel permeation chromatography [2].

Characterizations of SPNPs

The mean particle size and size distribution of SPNPs were determined by dynamic

light scattering (DLS) on a Malvern ZetaSizer Nano Series instrument with He-Ne laser 4

mW, λ = 633 nm. The structural morphology of NPs was examined by transmission

electron microscopy (TEM) on a JEOL JEM-1200 CXII operating at an accelerating

voltage of 120 kV. The sample was prepared by placing a few drops of the suspension on

a 300-mesh carbon-covered copper grid and dried at ambient temperature for 2 days prior

to measurements. To determine the SPION content, the SPNP suspension was first freeze-

dried and the powders were dissolved into a concentrated hydrochloric acid solution

(36~38%) at 80 oC until complete dissolution of SPIONs. The iron concentration was

determined by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7500ce,

Japan). The loading content of SPIONs thus obtained was expressed as the weight

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percentage of the embedded SPIONs in the lyophilized SPNPs. To estimate the loading

content of PTX, the lyophilized sample was dissolved in DMSO and subsequently

analyzed with high performance liquid chromatography (HPLC, Ascentis C18 column,

Supelco analytical; Agilent 1100 HPLC system, USA) equipped with UV detection at

227 nm. The isocratic elution comprising an acetonitrile-water-methanol (1/1/1 by

volume) co-solvent at a flow rate of 1 mL/min was used. Drug loading efficiency (DLE)

and content (DLC) were calculated by the following formulas, respectively:

DLE (%)=Weight of loaded PTXWeight of PTX ∈feed

× 100 %

DLC (% )= Weight of loaded PTXWeight of lyophilized NPs

× 100 %

Similarly, the amounts of PTX released from nanoparticles in vitro under different

conditions were determined using HPLC. The aqueous samples were lyophilized and re-

dissolved in acetonitrile. The resulting solution was filtered (0.22 m filter). The sample

was then analyzed by HPLC under the conditions identical to that for the drug loading

measurement described above. The magnetic hyperthermia characterization of SPNPs in

aqueous solution was performed under the high frequency magnetic field (HFMF)

treatment. The HFMF generator consisted of a 7-loop magnetic coil (35 mm in diameter),

a functional generator, an amplifier and a cooling water circulation system to produce

alternative magnetic field. The frequency and strength of the magnetic field were set at 37

kHz and 2.5 kA/m, respectively. The temperature of the NP suspensions after HFMF

treatment was monitored by a thermocouple meter. Another experiment to characterize

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the superparamagnetic property of SPNPs was carried out simply by comparison of the

aggregation of the SPNPs in aqueous solution in the absence and presence of external

magnetic field. Photographs were taken before and after the magnetic alignment. The X-

ray powder diffraction (XRD) measurement of lyophilized SPNP (or oleic acid-coated

SPION) powders was conducted on a powder diffractometer (Mac Science MXP-18)

with a Cu target at 30 kV and 20 mA.

Isolation of mesenchymal stem cells from adipose tissues

The ADSCs were harvested from adipose tissues in the lateral epididymis region of

male mice in culture medium (-MEM) [3]. The tissue was mechanically separated and

processed to eliminate the adipose tissue subjacent. The dermal adipose sheets were

collected and immersed in phosphate buffered saline (PBS) containing 5%

penicillin/streptomycin mixture (P/S). The collected tissue was minced and placed into a

PBS solution containing 0.075% collagenase Type I and 2% P/S for tissue digestion.

After 0.5 h reaction, 10 mL of -MEM medium containing 20% fetal bovine serum

(FBS, Hyclone) and 1% P/S were added into the above solution for neutralizing the

collagenase Type I activity.  The sample was then centrifuged at 2000 rpm for 5 min and

re-suspended in an ice-cold red blood cell lysis buffer (1.0 mL; Sigma–Aldrich, USA) for

10 min. The cell lysis reaction was neutralized with 20 mL of -MEM medium

containing 20% FBS and 1% P/S. The resulting mixture was centrifuged and the pellet

was re-dispersed in 3 mL culture medium, followed by filtration through a 70-μm pore

size filter. The cells were collected and incubated at 37 oC for 72 h on 60×15 mm cell

culture dish. The ADSCs were identified by the levels of CD73, CD90 and CD105

expression prior to in vitro and in vivo studies.

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Fig. S1 Synthetic route, chemical structure and 1H-NMR spectrum of poly(-glutamic

acid-co-distearyl -glutamate) (poly(-GA-co-DSGA)) in DMSO-d6 at ambient

temperature.

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Fig. S2. (a) X-ray diffraction profiles of the oleic acid-coated SPIONs (black) and SPNPs

(red). (b) Photographs of the SPNP suspension in the absence (left) and presence (right)

of external magnetic field. (c) Magnetization curve of the oleic acid-coated SPIONs and

SPNPs at 300 K.

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Fig. S3. (a) Cellular uptake of DiO-labeled SPNPs by ADSCs with different incubation

times evaluated by flow cytometry. (b) Fluorescence microscopic images of ADSCs

incubated with DiO-labeled SPNPs for 4 h. The NP concentration of 0.3 mg/mL was

used. The cell nuclei and endosome/lysosome compartments were stained with Hoechst

33258 and LysoTracker Red DND-99, respectively.

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Fig. S4. (a) In vivo NIR fluorescence images attained at preset time intervals from

subcutaneous tumor-bearing mice receiving PBS, SPNPs and SPNP-loaded ADSCs,

respectively. A single dose of therapeutics was given intravenously into the mice when

the tumors reached a volume of ca 120 mm3. (b) Ex vivo fluorescence images of major

organs and tumors harvested from the subcutaneous tumor-bearing mice 72 h post

intravenous administration. (c) Quantification of the Cy5.5 fluorescence intensity from

whole major organs and tumors 72 h post injection. Symbols and error bars are mean ±

S.D. *P < 0.05.

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Fig. S5. (a) Tumor growth curves of the subcutaneous ALTS1C1 tumors implanted on

the C57BL/6J mice receiving different therapeutic formulations. Two doses were given

via tail vein at day 0 and 1, respectively, when the tumors reached a volume of ca 120

mm3. HFMF was applied 48 h post each therapeutic injection, if applicable. (b) Mass of

the tumors harvested from the subcutaneous tumor-bearing mice at day 14. Values are

means ± SD. *P < 0.05, **P < 0.01. (n = 5 per group)

Biodistribution and antitumor efficacy on subcutaneous tumor model

The subcutaneous ALTS1C1 tumor model established on C57BL/6J mice was

employed to assess the efficiency of ADSC-based targeted delivery and therapy in vivo.

Tumor-bearing mice (C57BL/6J) were treated with SPNP-loaded ADSCs via tail vein

injection, followed by examination of therapeutic accumulation at tumor sites by IVIS.

To facilitate NIR imaging, SPNPs were labeled with an NIR fluorophore, Cy5.5, prior to

the cellular internalization. As shown in Fig. S4a, at 24, 48 and 72 h post tail vein

injection with SPNPs alone or SPNP-carrying ADSCs, considerable fluorescence signals

of Cy5.5 were observed in tumor. Although the tumors from the mice treated with SPNPs

alone show a slightly higher fluorescence signal than that with SPNP-loaded ADSCs 24 h

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post injection, a more prominent signal in tumor was found by the cell-based delivery as

compared to the NP-based counterpart 48 h post administration. This result demonstrates

the effective tumor targeting of the ADSC-based delivery system, though a prolonged

time is required for the cellular Trojan infiltrating to tumor via extravasation compared to

the NP-based delivery via blood circulation alongside tumor EPR effect. To further

examine the biodistribution of SPNPs in tumor-bearing mice, the ex vivo NIR imaging of

solid tumors and major organs harvested 72 h post administration was performed. Fig.

S4b confirms a significantly enhanced fluorescence signal of Cy5.5 in tumors with only a

slight signal observed in liver from the mice receiving the cellular therapy, indicating the

superior performance of the ADSC-based approach on selective accumulation in tumor.

In contrast, by the NP-based formulation, the signal was mostly found in liver rather than

in tumor due to the existence of plentiful Kupffer cells highly capable to remove foreign

substances by phagocytosis from blood circulation. The quantitative data from the

fluorescence intensities of different organs and tumors are summarized in Fig. S4c. The

result confirms the prominent reduction of therapeutic accumulation in liver, spleen and

other major organs except the tumor with the nanotherapeutics being transported by

ADSCs. It can therefore be concluded that in spite of a slow accumulation as compared to

SPNPs alone, the prominent ability to selectively accumulate in tumor encourages

ADSCs to serve as a promising targeted delivery system of tumor therapy.

Along with the selective tumor accumulation of ADSC-based delivery being

demonstrated, the subcutaneous tumor model was also employed for examining the

efficacy of the ADSC-based approach involving the combined treatments of HFMF-

mediated hyperthermia and released PTX. The in vivo antitumor efficacy of NP- and cell-

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based formulations in terms of tumor volume and mass was evaluated for up to 14 days

from the therapeutic injection via tail vein. As shown in Fig. S5, with HFMF treatment,

the ADSC-based group shows the best antitumor efficacy with the remnant tumor mass of

1.20 ± 0.46 g at day 14, as compared to 3.85 g of the PBS group. Owing to a poor

accumulation in tumor (Fig. S4), the effect of NP-based approach is rather limited, yet

better than those without the HFMF activation as evidenced by a relatively smaller tumor

mass (2.12 ± 0.61 g) shown in Fig. S5b. A statistically significant difference (*P < 0.05)

between these two major approaches was observed, clearly demonstrating the enhanced

therapeutic efficacy by improving selective tumor accumulation with the cell-based

delivery. On the other hand, the antitumor effect of both cell- and NP-based approaches

in the absence of HFMF stimulus was rather limited due to the slow release of PTX from

SPNPs along with PLGA degradation, thus rendering the tumor of relatively large mass

(ca 2.6 ~ 3.1 g). No apparent change in body weight was observed for the subcutaneous

tumor-bearing mice receiving different treatments (data not shown). The cell-based

approach adopted in this study undoubtedly represents an advance in effective anticancer

treatment by virtue of the enhanced active targeting and combined

hyperthermia/chemotherapies.

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Fig. S6. H&E-stained images of major organs. The brain tumor (ALTS1C1)-bearing mice

receiving different therapeutic formulations were sacrificed 2-day post the final HFMF

treatment. Four daily doses of therapeutics were given intravenously for four consecutive

days into the mice 14-day post intracranial injection of ALTS1C1 glioma cells into the

right cerebral hemisphere, followed by the repeated HFMF treatments once per day 48 h

post each therapeutic administration. Scale bars are 50 m.

Reference

[1] M. Bloemen, W. Brullot, T.T. Luong, N. Geukens, A. Gils, T. Verbiest, Improved functionalization of oleic acid-coated iron oxide nanoparticles for biomedical applications, J. Nanopart. Res., 14 (2012) 1100.

[2] W.H. Chiang, W. C. Huang, M.Y. Shen, C.H. Wang, Y.F. Huang, S.C. Lin, C.S. Chern, H.C. Chiu, Dual-Layered Nanogel-Coated Hollow Lipid/Polypeptide Conjugate Assemblies for Potential pH-Triggered Intracellular Drug Release, Plos One, 9 (2014) e92268.

[3] B.A. Bunnell, M. Flaat, C. Gagliardi, B. Patel, C. Ripoll, Adipose-derived stem cells: Isolation, expansion and differentiation, Methods, 45 (2008) 115-120.

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