Non-invasive Tracking and Regenerative Capabilities of Transplanted

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Biological and Medical Applications of Materials and Interfaces

Non-invasive Tracking and Regenerative Capabilities of Transplanted Human Umbilical-cord Derived Mesenchymal Stem Cells Labeled with I-III-IV Semiconducting Nanocrystals in Liver-Injured Living Mice Shashank Shankar Chetty, Selvarasu Praneetha, Kavitha Govarthanan, Rama Shanker Verma, and Arumugam Vadivel Murugan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19953 • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 11, 2019

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ACS Applied Materials & Interfaces

Non-invasive Tracking and Regenerative Capabilities of Transplanted Human Umbilicalcord Derived Mesenchymal Stem Cells Labeled with I-III-IV Semiconducting Nanocrystals in Liver-Injured Living Mice Shashank Shankar Chettya, Selvarasu Praneethaa, Kavitha Govarthananb, Rama Shanker Vermab*, Arumugam Vadivel Murugana*

aAdvanced

Functional Nanostructured Materials Laboratory, Centre for Nanoscience

and Technology, Madanjeet School of Green Energy Technologies, Pondicherry University (A Central University), Puducherry-605 014, India.

bBhupat

and Jyoti Mehta School of Biosciences, Department of Biotechnology, Indian

Institute of Technology-Madras (IIT-M), Chennai-600 036, India.

ACS Paragon Plus Environment

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*Corresponding Author: E-mail: [email protected], [email protected] & [email protected]

KEYWORDS: mesenchymal stem cell labeling, in vivo fluorescence tracking, liver regenerative therapy, functionalized I-III-VI CuInS2-ZnS nanocrystals, microwave refluxing

ABSTRACT: Acute liver injury is a critical syndrome ascribed to prevalent death of hepatocyte and imperatively requires a liver transplantation. Such methodology is certainly hampered due to deficit of healthy donors. In this regard, stem cells-based regenerative therapies are attractive in repairing injured tissues and organs for medical applications. However, it is crucial to understand the migration, engraftment and regeneration capabilities of transplanted stem cells in the living animal models. For the first time, we demonstrate rapid labeling of umbilical cord-derived mesenchymal stem cells (MSCs) with near-infrared (NIR) fluorescent CuInS2-ZnS nanocrystals (CIZS-NCs) to develop an innovative nano-bioconjugates (MSCs-CIZS-NBCs) that exhibit 98% labeling efficiency. Prior to nano-bioconjugates synthesis, the pristine CIZS-NCs were


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prepared via two-step, hot-injection, rapid and low-cost domestic-microwave-refluxing (MWR) method within 6 min. As-synthesied CIZS-NCs display high photoluminescence quantum yield (~88%) and long-lived lifetime (23.4 μS). In contrast to unlabeled MSCs, the MSCs-CIZS nano-bioconjugates shows an excellent biocompatibility without affecting the stemness, as confirmed by cell viability, immuno-phenotyping (CD44+, CD105+, CD90+), multi-lineage-specific gene expressions and differentiation into adipocytes, osteocytes and chondrocytes. The in vivo fluorescence tracking analyses revealed that the MSCs-CIZS-NBCs after tail-vein injection were initially trapped in lungs and gradually engrafted in the injured liver within 2 h. The regeneration potential of MSCs-CIZS-NBCs were confirmed via renewal of portal tract comprising of portal veins, bile ducts and hepatic arteries around the hepatocytes. Consequently, no apparent inflammations, necrosis or apoptosis were observed in acetaminophen (APAP)-induced liver-injured BALB/c mice model over 3 days after transplantation, as corroborated

using

laser-scanning

confocal

microscopy,

histopathological

and

hematological analyses. Hence, our innovative NIR-fluorescent MSCs-CIZS-NBCs offers


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an off-the-self technology for non-invasive tracking of transplanted MSCs in acute-liver injured animal model and vitalize for future image-guided cell-therapies.

1. Introduction Mesenchymal stem cells (MSCs) therapy has engrossed enormous potential in several clinical applications specifically for the therapeutics of lung injury, liver failure, bone regeneration and cardiac repair.1 Under definite in vitro and in vivo environments, MSCs exhibit multi-lineage differentiation potential into numerous tissue-specific cells namely chondrocytes, hepatocytes, osteoblasts, adipocytes, cardiomyocytes, islet cells and endothelial cells.2 The multi-potency, low immunogenicity and self-renewal capability of adult MSCs have been the promising aspects for repairing impaired tissues and regulating the immune responses. However, in vivo migration, engraftment and differentiation ability of transplanted MSCs are not thoroughly understood.3 In this regard, it is necessary to develop high-resolution imaging systems and nanoprobes for better understanding of therapeutic mechanisms in regenerative medicine.4 An ideal tracking technique should possess certain requisites such as higher sensitivity, non-invasiveness and should be nondegradable to visualise the transplanted MSCs in vivo at any anticipated time and anatomic positions.5 The current techniques mainly include magnetic resonance imaging (MRI),6,7 positron emission tomography (PET),8,9 fluorescence imaging,10,11 ultrasound12 and photoacoustic imaging.12,13 Zhou et al reported MRI for imaging superparamagnetic iron oxide (SPIO)-labeled bone marrow-derived mesenchymal stem cells (BMSCs) in rats with liver fibrosis.6 However, the results revealed difference in liver tissue arrangement in rats with lower signal-to-noise (SNR) ratio of ~15 and concluded SPIO-based MRI might not be appropriate for long-term monitoring of administered BMSCs. Schönitzer et al. reported tracking of MSCs using Dopamine Type 2


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Receptor and 18F-Fallypride for PET imaging.8 However, the location of MSC-D2R80A were not visualized post-transplantation owing to low tracer uptake and exhibited low SNR of 2.2. Jokers et al. reported silica-coated gold nanorods (Si-GNRs) as contrast probe for labeling MSCs for PA imaging in 3 h.12 However, the work concluded certain challenges in PA imaging such as signal/frequency distortion due to volume change, inaccuracy in remodeling, tissue background, light scatter, excitation source attenuation and inefficient for deep-tissue imaging. In particular, fluorescence imaging offers high resolution at single-cells and sub-cellular levels using green fluorescent proteins/luciferase expressions accomplished using gene transfection for long-term tracking.14,15 However, gene transfection requires selection and clonal expansion that demands prolonged culturing of MSCs, which are considered unsuitable due to restricted proliferative ability and impaired homing potential due to loss of certain surface biomarkers.16 Alternatively, the use of fluorescent proteins suffers from narrow penetration depth, greater interference and auto-fluorescence.17 An ensemble of these challenges could be prudently overcome by nearinfrared (NIR) imaging agents such as DiR, indocyanine green dye etc. that are employed for stem cell tracking.18,19 Nevertheless, limited brightness, smaller Stroke’s shift and poor photostability of the organic dyes still impart practical difficulties for high sensitivity and long-term tracking of MSCs.20 Several reports focuses on conjugation of nanocrystals (NCs) with peptides or small molecules for the internalization into stem cells. For example, Yukawa and co-workers mixed QD 655 with cell-penetrating peptides (CCPs) octa-arginine peptide (R8) for transduction to adipose tissue-derived stem cells (ASCs) and studied the differentiation into both osteogenic and adipogenic cells. However, chondrogenic


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lineage differentiation that are crucial cells found in healthy cartilages, hematological, histopathological evaluation to confirm the liver functions during the regeneration, immno-fluorescence studies to ensure engraftment of peptide conjugated QD655 were not examined to understand the theranostics mechanism of acute liver injury.21 Few works reported conjugation with CCPs like CGGGRGD, APWHLSSQYSRT etc. that increases the transduction efficiency and cost of therapy.22 Lin and co-workers used Qtrackers to label murine embryonic stem cells (ESCs). However, the results suggested that QD-labelling appears to diminish transplanted stem cell behavior and leads to false signals during in vivo tracking.23 Recently, Chen et al. and Zhang et al. reported preparation of semiconductor polymer dots in conjugation with octa-arginine peptide (R8) for labeling of MSCs and cancer cells.24,25 However, they exhibited low photoluminescence quantum yield (PLQY) of ~21% and low SNR of ∼1.5. Over the past years, several fluorescent inorganic semiconductor NCs such as II-VI, I-III-VI and III-V has been proposed as versatile fluorescent nano-probes for bioimaging and molecular diagnostics.26-31 Particularly, highly fluorescent II-VI based NCs mostly consist of toxic heavy-metal ions such as Pb2+, Cd2+, Hg2+ etc. thus limiting their practical applicability in biological systems.32 Several groups have demonstrated that these toxic element adversely influence the biocompatibility due to the eventual release of heavy-metal ions to cellular environment.32,33 Alternatively, visible-to-near-infrared fluorescent CuInS2 (CIS), I-III-


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VI2 based semiconductor with optical bandgap of ~1.45 eV has gained increased attraction as a promising nanoprobe for in vivo imaging applications. Numerous synthetic methods such as hydrothermal, hot-injection, solvothermal and thermolysis have been adopted to develop bright fluorescent and photo-stable CuInS2-ZnS (CIZS) NCs.34-40 However, these methodologies requires either longer refluxing time (˃ 3 h), intricate experimental system and multiple solvents at higher temperature of 200-230 °C to facilitate nucleation and utilizes ancillary solvents in the inert gas (N2/Ar) or vacuum environment. These conditions could be ameliorated via sustainable chemical method to prepare state-of-the-art nanoprobe for bio-photonic applications. Therefore, it is essential to explore an innovative, biocompatible and fluorescent NCs to synthesize via energy-efficient, novel, low-cost synthetic approach for further advances in the development of nanoprobes.41 In this regard, microwave-assisted synthesis offer an in situ volume heating in few minutes with homogenous formation of NCs.42-45 Hong et al. demonstrated microwavesolvothermal synthesis of CIZS-NCs in 40 min for silicon solar cell in air-exposing atmosphere and exhibited PLQY of 56% with no PL lifetime analysis.44 Mange et al. presented microwave-hydrothermal synthesis of CIZS-NCs using thioglycolic acid as stabilizing agent with lower PLQY of 1% and no PL lifetime studies.45 Recently, our group has demonstrated the NIR-fluorescent CIZS-NCs via one-step, non-injection, microwavesolvothermal reaction condition (200 - 230 oC, 900 W) in 5 min with PLQY of 77% and lower PL lifetime of 310 ns for facile labeling of normal cells, cancer cells and primitive animal model (zebrafish embryo).42 Subsequently, we also investigated the imaging capability of CIZS-NCs with bio-molecule, for example folic acid as a tumor targeting agent to prepare folic acid-CIZS-


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NCs conjugates for active targeting to tumor site in B16F10 induced tumor-bearing C57BL/6 mice model for cancer diagnosis application.46 In spite of the aforementioned progress of CIZS-NCs in energy and biotechnological applications, the utilization of CIZS-NCs especially in labeling of stem cells for tracking migration, engraftment and regenerative-assisted therapy remains to be absolutely unexplored. Hence, it has motivated us to develop umbilical cord-derived mesenchymal stem cell with CIZSNCs as an innovative nano-bioconjugates for non-invasive tracking and regenerative therapy in advance animal model. In the present study, we demonstrate for the first time a two-step, hotinjection and low-cost domestic microwave-refluxing (MW-R) method to synthesize highly NIR-fluorescent CIZS-NCs within 6 min at 800 W in argon gas atmosphere using 1-dodecanetiol (DDT) as sulfur sourcing solvent. The DDT-CIZS-NCs presented state-of-the-art PLQY of 87.83% in organic phase with long-lived PL lifetime of 23.4 μS.34-40 The DDT-CIZS-NCs were subsequently phase-transferred using 11-mercaptoundecanoic acid (MUA) via ligand exchange into aqueous phase for simplistic labelling of MSCs to develop an innovative MSCs-CIZSNBCs with 98% labeling efficiency in 2 h, without requiring any transfection agents (such as R8, Tat peptide etc.) or electroporation processes. For the first time, the MSCs-CIZS-NBCs have been used for non-invasive tracking the migration, engraftment and regeneration capabilities in acetaminophen-induced acute liver-injured BALB/c mice model.

2. Experimental Section 2.1 Materials


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Zinc stearate (Zn(St)2), Indium (III) acetate (In(Ac)3, 99.9%), Copper (I) iodide (CuI, 99.9%), 1dodecanethiol (DDT, 98%), 11-mercaptoundecanoic acid (MUA, 95%), bovine serum albumin (BSA), acetaminophen (APAP, ≥99.0%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dimethyl sulfoxide (DMSO, ≥99.9%), Dichloro-dihydro-fluorescein diacetate (DCFH-DA, ≥97%) were all purchased from Sigma-Aldrich. Tetramethylammonium hydroxide pentahydrate (TMAH, 97%) was purchased from HiMedia were used without further purification. 2.2 Microwave-refluxing (MW-R) synthesis of DDT-CuInS2-ZnS NCs The CIZS-NCs with different In/Zn ratios (1:0-1:6) were synthesized by fixing In concentration and increasing Zn concentration. Briefly, CuI (0.05 mM, 9.9 mg) and In(Ac)3 (0.1 mM, 29.19 mg) were mixed in 10 mL of 1-DDT for 30 sec in 50 ml three-neck round bottom flask. Further, the flask was transferred into domestic microwave oven and attached tightly with the condenser bottom. The reaction mixture was degassed and backfilled with argon followed by microwave irradiation at 800 W for 4 min to form dark red colored CIS solution (Figure 1a). For the ZnS alloying, increasing proportion of Zn(St)2 (0-0.6 mM) in 2 ml of 1-DDT were prepared in prior by solubilizing the mixture on a hot plate at 60 ºC. This mixture was hot-injected into presynthesized CIS solution and microwave irradiated at 800 W for 2 min. The irradiation time was optimized depending on the optical performance of CIZS-NCs. The CIZS-NCs solution was precipitated using chloroform-methanol mixture with surplus of acetone and centrifuged (10 min 10,000 rpm). The red precipitate was dispersed in chloroform (5 mL) for phase transformation from oil-soluble to water-soluble phase via modified ligand exchange procedure47 also provided in supporting information. 2.3 Analytical characterization


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X-ray diffraction (XRD) analysis of synthesized CIZS-NCs was carried out by Rigaku Ultima IV with graphite monochromated Cu Kα radiation (λ = 1.54 Å) source. High-resolution transmission electron microscopy (HR-TEM) was analyzed using JEOL JEM-7100 electron microscope operated at 200 kV accelerating voltage. The Zeta (ζ) potential and hydrodynamic diameter (HD) were analyzed using Zeta sizer (Nano ZS, Malvern). X-ray photoelectron spectroscopy (XPS) was analyzed using PHOBIOS HSA3500 DLSEGD analyzer with 1486.74 eV excitation energy. Fourier-transform infrared (FT-IR) spectroscopy was analyzed by Thermo Nicolet Model 6700. UV-visible spectra were recorded using Varian Model 5000. The steady-state PL spectra were analyzed by Fluorolog - FL3-11 (JobinYvon) at 470 nm excitation wavelength. The PLQY of CIZS-NCs were measured with rhodamine-6G dye (QY-95%, in ethanol) as a reference using the equation 1.48 QY = QYR x (I/IR) x (AR/A) x (n2/nR2)

(1)

Where, I and n denotes integral PL intensity and refractive index (n = 1.33 for water, n = 1.44 for chloroform), respectively. A and R denotes optical density and reference, respectively. Timeresolved single photon counting (TRSPC) were analyzed by Fluorolog - FL3-11 (JobinYvon) using femtosecond laser at 488 nm. The PL lifetime of CIZS-NCs were analyzed using equation 2.42 τav = (A1τ12 + A2τ 22 + A3τ 32)/( A1τ 1 + A2τ 2 + A3τ 3)

(2)

Where, τav represent the average fluorescence lifetime, A refers to amplitude and τ is PL lifetime of time-resolved decay curve. 2.4 Isolation and Expansion of MSCs from umbilical cord The experiments using umbilical cord-derived MSCs were approved by Institute stem cells ethical committee of Indian Institute of Technology Madras (IIT-M), India. Briefly, fresh


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umbilical cords were collected from the hospital after parental consent and transported in Dulbecco’s phosphate buffered saline (DPBSA) solution with 1x antibiotics and anti-mycotic solution (Gibco). Briefly, MSCs from umbilical cord connective tissues (Wharton’s jelly) were isolated by mechanically separating the blood vessels and digesting enzymatically by using Type 1 Collagenase (Worthington biochemical corporation, New Jersey) at 37 ºC for 12 h.49,50 The cells suspension was filtered via 100 µm cell strainer (BD biosciences) to remove clumps and constituted to 50 ml using complete media containing α-MEM (Lonza) supplemented with 1x non-essential amino acids (NEAA) (Gibco), 10% fetal bovine serum (FBS) (Gibco) and 2 mM L-Glutamine (Gibco). Filtered cells suspension was centrifuged and washed twice with complete media. The pellet was finally seeded at a density of 1x107 cells/ml in 25 cm2 T-flask with complete medium in 5% CO2 humidified incubator at 37 ºC. The cultured MSCs were then passaged using accutase (Gibco) at 80% confluency prior to in vitro and in vivo studies. 2.5 Cell viability and ROS production The biocompatibility of water-soluble MUA-CIZS-NCs with MSCs was evaluated by standard MTT assay. Briefly, MSCs (passage 2) were seeded with 5000 cells/well in a 96-well plate (Coster, US) and cultured for 12 h prior to addition of CIZS-NCs. Following 24, 48, and 72 h of incubation with increasing concentrations of MUA-functionalized CIZS-NCs (0, 6.25, 12.5, 25, 50, 100, 250, 500, 1000 μg/mL), the MSCs were gently washed thrice with PBS. Then, 20 μL MTT in culture medium (5 mg/mL concentration) was added in each well and incubated for 3 h. The culture medium was aspirated and DMSO solution (100 uL) was added in each well for dissolution of formazan under gentle shaking for 10 min. The absorbance values were measured at 570 nm by microplate reader (GENios, Tecan). Cell viability (%) was analyzed by absorbance ratio of MSCs incubated with increasing concentration of MUA-CIZS-NCs to control MSCs in


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culture medium. To evaluate reactive oxygen species (ROS) production, increasing concentration of MUA-CIZS-NCs (0, 6.25, 12.5, 25, 50, 100, 250, 500, 1000 μg/mL) incubated with MSCs for 24, 48 and 72 h were rinsed thrice with PBS. Then, the treated MSCs were incubated with 25 μM DCFH-DA working solution (in DMSO: PBS at 1: 400 v/v) for 45 min at 37 ºC in dark. The fluorescence intensity (excitation: 495 nm; emission: 529 nm) was measured by infinite F200 microplate reader (Tecan group Ltd., Switzerland) and intensity from MSCs incubated with increasing concentration of MUA-functionalized CIZS-NCs in culture medium were normalized with cells incubated only with culture medium. 2.6 Labeling of MSCs, flow cytometry quantification and in vitro imaging The MSCs were seeded onto cover slip placed in 6-well plate (1x105 cells/well) and incubated with MUA-functionalized CIZS-NCs (10 ug/ml concentration) in 2 mL complete media for 2 h to prepare MSCs-CIZS-NBCs. At different time interval, the MSCs incubated with MUACIZS-NCs were rinsed thrice with PBS and trypsinized for flow cytometry analysis using FACS Conto flow cytometer (BD, USA) or fixed with paraformaldehyde (PFA) for fluorescence imaging using inverted fluorescent microscope (Carl Zeiss, Germany). Additionally, the actin filaments were stained using 100 μM of Alexa Fluor 488-phalloidin (Invitrogen) for 30 min and nuclei were stained with 1 μM of Hoechst (Invitrogen) for 5 min in PBS. 2.7 Flow cytometry phenotyping and gene expression For phenotype analysis, the MSCs-CIZS-NBCs were incubated with FITC-conjugated antibodies at 4 ºC for 30 min in dark. After incubation, 10,000 events were examined using FACS Conto flow cytometer (BD, USA). All the antibodies were purchased from BD biosciences: CD90-FITC (555595), CD105-FITC (561443) and CD44-FITC (553133). For


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mRNA expression analysis of differentiated MSCs, the total RNA was extracted from MSCsCIZS-NBCs using TRI reagent (Sigma Aldrich, USA). Approximately, 2 µg of total extracted RNA was transformed into cDNA in 20 µl reaction volume by Murine Leukemia Virus Reverse Transcriptase (MLV-RT, Thermo scientific, USA) and oligo-dT primers (NEB, USA). Semiquantitative real time-PCR was performed using primers for specific gene sequence (Supporting information Table S2) of differentiated MSCs and supplemented SYBR master mix in the Applied biosystems 7500 Real-Time PCR Systems (Life Technologies, USA). A two-step cycling process was executed involving 10 min cycle at 95 °C, followed by 40 cycles of denaturation (95 °C for 10 sec), annealing (60 °C for 30 sec) and primer extension (72 ºC for 30 s) with extra elongation cycle at 72 ºC for 10 min. After amplification, the melting curve evaluation was carried out to corroborate the specificity of the obtained product. Relative expression of mRNA was quantified by normalizing with β-actin expression as reference gene and relative fold change was determined by 2-ΔΔct method. 2.8 Long-term fluorescence intensity and migration assay The MSCs-CIZS-NBCs were harvested at days 1, 3 and 7 day (routine passage was performed), rinsed and suspended in PBS for flow cytometry measurements. The MSCs migration assay was examined in CytoSelect Transwell 24-well microplate with 8.0 µm pore size (Cell Biolabs). 105 MSCs-CIZS-NBCs were seeded in the lower compartment and incubated at 37 ºC for 2 h to promote surface attachment. Further, 105 unlabeled MSCs were seeded in insert well (upper compartment) and incubated for 12, 24 and 36 h. The medium was removed and non-migratory MSCs were detached by swabbing the inside of insert well through a wet cotton swab. The insert


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well was incubated in a cell staining solution (crystal violet 0.09%) for 10 min and rinsed with deionized water. The migrated MSCs-CIZS-NBCs were visualized under bright-field microscope. For NCs leakage assay, the lower and upper compartment were labeled with Hoechst (1 μM) for 5 min and imaged in fluorescent microscope at 12, 24 and 36 h.51 2.9 Induced differentiation and histological staining The MSCs-CIZS-NBCs (10,000 cells/cm2) in 12-well plate were rinsed with PBS. The adipogenic differentiation was induced by supplementing the base medium with 1 μM dexamethasone, 10 μg/mL insulin, 200 μM indomethacin, 0.5 mM isobutylmethylxanthine and changed every 2-3 days for 14 days. The adipogenic differentiation was observed by staining the lipid deposits using Oil Red O and picrosirius red. The osteogenic differentiation was induced by supplementing base medium with 10 mM β-glycerol phosphate, 0.2 mM L-ascorbic acid 2phosphate, 10 nM dexamethasone and changed every 2-3 days for 14 days. The osteogenic differentiation was imaged by alizarin red stain for mineral deposition. The chondrogenic differentiation was induced by supplementing base medium with 6.25 μg/mL insulin, 5.33 mg/mL linolenic acid, 6.25 g/mL selenous acid, 6.25 g/mL transferrin,1.25 mg/mL BSA, 0.35 mM proline, 1mM sodium pyruvate, 100 nM dexamethasome, 10 ng/mL transforming growth factor, 0.1 mM L-ascorbic acid and changed every 2-3 days for 28 days. The chondrogenic differentiation was visualized using toluidine blue stain. Finally, the images of differentiated MSCs-CIZS-NBCs were taken using phase contrast microscope (Carl Zeiss, Germany). The optical density (OD) of differentiation staining was analyzed by Image J software. 2.10 In vitro and in vivo detection sensitivity


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Different dilutions of MSCs-CIZS-NBCs (1.2×103, 3.0×103, 5.5×103, 7.3×103, 9.1×103) in 1 mL PBS were seeded in 6-well plate and imaged under IVIS Lumia III imaging system (excitation: 470 nm, emission: 570 nm) and radiant efficiency in each well was determined using region of intensity (ROI). Similarly, different dilutions of MSCs-CIZS-NBCs (1.2×103, 3.0×103, 5.5×103, 7.3×103, 9.1×103) in 20 μL PBS were injected subcutaneously into the back of BALB/c mice. The detection sensitivity of MSCs-CIZS-NBCs fluorescence was visualized under IVIS Lumia III imaging system (excitation: 470 nm, emission: 570 nm) and the radiant efficiency in region of intensity (ROI) was compared quantitatively. 2.11 In vivo tacking, engraftment and regeneration of injured liver All the animal studies conform with the approved guidelines from Institutional Animal Ethics Committee (IAEC) of Indian Institute of Technology, Madras (IIT-M), India. Acetaminophen (APAP) was dissolved in sterile normal saline (NS) just prior to use. To develop acute liver injury model, male BALB/c mice (18-20 g) was injected with 500 mg/kg of acetaminophen interperitoneally.52 The intoxication of acetaminophen was confirmed by histopathological and hematological analyses. After 4 h, MSCs-CIZS-NBCs (5×106) were administered intravenously via tail vein in the acute liver injured BALB/c mice. To evaluate the distribution of MSCs-free MUA-CIZS-NCs, 5 μg/μL concentration of 50 μl MUA-CIZS-NCs in PBS was administered in acute liver injured BALB/c mice as control group. For in vivo imaging, acute liver-injured and healthy mice were imaged at several time intervals under IVIS Lumia III imaging system (excitation: 470 nm, emission: 590 nm). For ex vivo studies, major organs (spleen, kidney, heart, liver, lungs) were excised for imaging under IVIS Lumia III imaging system in the same


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conditions. The region of intensity (ROI) was measured by Living Image Software version 4.3.1. The in situ engraftment of the transplanted MSCs-CIZS-NBCs in liver of acetaminopheninduced liver injured mice was confirmed using laser scanning confocal microscope (LSM 710, Carl Zeiss, Germany). 2.12 Histopathological, hematological, immunofluorescence and ICP-MS analysis All major organs (spleen, kidney, heart, liver, lungs) from healthy, acetaminophen-induced liverinjured and MSCs-CIZS-NBCs transplanted BALB/c mice were harvested and embedded in paraffin to prepare tissue sections. The sections were stained with hematoxylin and eosin for histopathological investigation. The proliferating cell nuclear antigen (PCNA)-positive cells were characterized by specific antibody (AVIVA system biology, China) in the liver section via immunohistochemical (IHC) staining. The microscopic images of all the tissue sections were captured using phase contrast microscope (Carl Zeiss, Germany). For hematological analysis, blood sample from all the three mice groups were collected via orbital bleeding and stored at -20 ºC after isolating the serum. The complete blood count, biochemical examination, liver function test and lipid profile were analyzed using automated analyzer (Mindray, China). For immunofluorescence assay, the liver tissues were harvested from liver-injured BALB/c mice after transplantation of unlabeled MSCs and MSCs-CIZS-NBCs at day 3. The tissues were fixed with 4% PFA to cut into 10 μm thick sections and blocked with 3% BSA for 1 h. The sections were incubated with mouse anti-human nuclei (1:100, HuNu; Millipore, US) for overnight at 4 ºC. Then, the sections were incubated with fluorescent Cy3 conjugated goat antimouse IgG (1:500, Invitrogen, US) second antibody for 2 h in dark. The sections were stained with 1 μM of Hoechst for 5 min and imaged under fluorescent microscope.26 For ICP-MS


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analysis, the major organs (spleen, kidney, lungs, liver, heart) and blood were heated in 0.5 mL of nitric acid (trace-metal grade) for 2 h. The digested solution was used to quantify copper and zinc concentration using inductively coupled plasma-mass spectroscopy (ICP-MS) (Teledyne Leeman, Prodigy XP) to understand the bio-distribution of MSCs-CIZS-NBCs in acute liver injured BALB/c mice. 2.13 Statistical test The experimental data are shown as mean ± standard deviation (SD). The statistical significance was analyzed at Probability, P 70% cell viablity, thus confirming the enhanced biocompatibility of MUACIZS-NCs (Figure 2b). The ROS production in MSCs with increasing concentrations of MUACIZS NCs were analyzed. We observed slight increase in ROS levels (~13.29%) with increasing concentration of MUA-CIZS-NCs till 1 mg/mL (Figure 2c). While, at the working concentration of MUA-CIZS-NCs (10 μg/mL), the MSCs exhibited ~96.5% cell viability and minimal ROS production. The MUA-CIZS-NCs (10 μg/mL) were incubated MSCs to prepare an innovative MSCs-CIZS-NBCs that demonstrated labeling efficiency of ~98% after 2 h as analyzed using flow cytometry (Figure 2d), which was much significant than > 3 h co-incubation reported.10-13 The MSCs-CIZS-NBCs were observed by laser scanning confocal microscope (LSCM) in low-


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exposure-low-gain conditions (Figure 2g). The MSCs nuclei were labeled with Hoechst (Figure 2e) and actin filaments with Alexa Fluor 488-phalloidin (Figure 2f) to visualise the internalization of MUA-CIZS-NCs. The overlay image confirmed efficient labeling of MSCs using MUA-CIZS-NCs in 2 h (Figure 2h). The MSCs did not showed auto-fluorescence in the absence of MUA-CIZS-NCs. It is noteworthy that the MSCs-CIZS-NBCs exhibited bright NIRfluorescence in cytoplasm near the cell membrane with substantial signal-to-noise ratio (SNR) of 17.6 (Supporting information Figure S5). The carboxyl terminated CIZS-NCs are favorable to MSCs internalization via endocytosis pathway.58 Thus, our work demonstrate convenient and economical labeling protocol for direct labeling MSCs without requiring any transfection agent. 3.4 Immunophenotyping and mRNA expression studies Phenotypic expressions and multi-potent differentiation capabilities are the two vital factors to characterize stem cells. The immuno-profiles were examined using flow cytometry in accordance with the principle descriptions of MSCs by International Society for Cellular Therapy (ISCT).59 Presently, no single specific marker has been recognized for identification of multi-potent MSCs. These cells could serve as positives for adhesion markers for example CD44 (hyaluronan receptor), mesenchymal markers such as CD90 (thy-1), CD105 (endoglin) etc.59 The flow cytometry analysis of surface biomarkers in MSCs-CIZS-NBCs revealed no significant changes in the expressions of CD90 (Figure 2j), CD105 (Figure 2l), CD44 (Figure 2n) with respect to control MSCs (Figure 2i, k, m). This confirmed that the labeling of MSCs using MUA-CIZSNCs does not affect proliferation and multi-potency properties. To investigate the differentiation abilities of MSCs-CIZS-NBCs, mRNA gene expressions for specific lineages were examined


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using RT-PCR protocol. These biomarkers are involved in transcriptional activation, regulation of glucose metabolism and fatty acid storage.

Figure 2. (a) Morphology of umbilical cord-derived MSCs. (b) in vitro cell viability (%) and (c) ROS production in MSCs incubated with increasing concentration of MUA-CIZS-NCs for 24, 48 and 72 h. (d) Flow cytometry analysis of MSCs-CIZS-NBCs. In vitro fluorescence imaging of MSCs (e) Hoechst, (f) Alexa Fluor 488-phalloidin staining, (g) MUA-CIZS-NCs labelling, (h) overlay image. Immunophenotyping of MSCs markers by flow cytometry: (i, j) CD90, (k, l) CD105, (m, n) CD44 for control and MSCs-CIZS-NBCs, respectively. Gene expression profile of MSCs-CIZS-NBCs after (o) adipogenic differentiation (CEBP A, CEBP B, PPAR G, FABP 4), (p) osteogenic differentiation


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(Collagen I, Runx2, Opn), (q) Chondrogenic differentiation (Collagen I, Collagen II, Sox9). Data are presented as mean ± SD, n = 3/group.

It was observed that the expression of CEBP-α and CEBP-β were down-regulated due to terminal differentiation of MSCs-CIZS-NBCs into adipocytes. The gene expressions of PPAR-γ and FABP 4 were sustained due to the presence of exogenous PPAR-γ agonist (indomethacin) in the induction media that enabled the accumulation of fat droplets in adipocytes. For osteogenic differentiation, Runx2 (Runt-associated transcription factor 2), Collagen type I, Opn (Osteopontin) were investigated (Figure. 2p).50,59,60 These genes are involved in transcriptional activation of MSCs to osteocytes and play a crucial role in bio-mineralization during bone formation. The expressions of osteogenic-associated genes were similar in comparison to control MSCs, which suggested that osteogenic differentiation potential of MSCs were retained after labeling with MUA-CIZS-NCs. For chondrogenic differentiation, Collagen type I & II and sox9 (SRY-Box 9) were evaluated (Figure 2q). 50,59,60 These genes are cartilage-specific transcription factors, which play a significant function in chondrogenesis and cartilage formation through the accumulation of sulfated proteoglycans. We observed that the expressions of chondrogenicassociated genes resembled with control MSCs. The high level of Collagen type II to Collagen type I expression ratio revealed the characteristic features of hyaline-like cartilage tissues formation. The multi-lineage differentiation profiling suggests significant potential of MUACIZS-NCs as amicable for facile labeling of umbilical cord-derived MSCs. 3.5 Long-term fluorescence intensity and migration potential Direct labeling approach are facile, convenient and economical as they do involve any genetic modification of MSCs. Considering the fact that every cells internalize certain concentration of


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NCs passed on to their daughter cells during proliferation, it is important to monitor intensity of fluorescence in MSCs for long-term tracking studies. Flow cytometry analysis was used to measure the fluorescence intensity of MSCs-CIZS-NBCs for 1, 3 and 7 day (Figure 3a, b).


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Figure 3. (a) Flow cytogram of MUA-CIZS-NCs fluorescence vs forward scatter of MSCs and (b) longterm tracking of MSCs-CIZS-NBCs for 1, 3 and 7 day. (c) Transwell experiment setup with MSCs-

CIZS-NBCs (upper) and unlabeled MSCs (lower) compartment, respectively separated by a porous membrane (0.8 µm). (d) Migration potential of MSCs and MSCs-CIZS-NBCs at 12, 24 and 36 h. Fluorescence images of (e) MSCs-CIZS-NBCs cultured in upper compartment and (f) MSCs in lower compartment.

We observed that fluorescence intensity of MSCs-CIZS-NBCs were 1000-folds greater than the unlabeled MSCs. The intensity of fluorescence progressively reduced as cells proliferate at 3 and 7 day. However, the MSCs-CIZS-NBCs were significantly detected with 10-fold higher fluorescence intensity at 7 day. The decrease in time-lapse fluorescence could be possibly due to subsequent MSCs proliferation. One of a major challenge in labeling and tracking of stem cell involve leakage of NCs during proliferation and their effect on the migratory behavior of MSCs.26 A transwell experiments was performed by culturing the MSCs-CIZS-NBCs in upper compartment and unlabeled MSCs in lower compartment separated by a porous membrane (0.8 µm) to validate the migration of MSCs and diffusion of free MUA-CIZS-NCs into lower compartment (Figure 3c). We observed that the migration of MSCs were unaffected even after labeling with MUA-CIZS-NCs and migratory MSCs on under the lower compartment increased with time (12

Non-invasive Tracking and Regenerative Capabilities of Transplanted

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