microRNA Delivery and

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Theranostic Niosomes for Efficient siRNA/microRNA Delivery and Activatable Near-Infrared Fluorescent Tracking of Stem Cells Chuanxu Yang, Shan Gao, Ping Song, Frederik Dagnaes-Hansen, Maria Jakobsen, and Jørgen Kjems ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05513 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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ACS Applied Materials & Interfaces

Theranostic

Niosomes

for

Efficient

siRNA/microRNA Delivery and Activatable NearInfrared Fluorescent Tracking of Stem Cells Chuanxu Yang1,2*, Shan Gao1,2, Ping Song1, Frederik Dagnæs-Hansen3, Maria Jakobsen2, Jørgen Kjems1,2* 1. Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark 2. Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark 3. Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark

Correspondence Chuanxu Yang Interdisciplinary Nanoscience Center (iNANO) Department of Molecular Biology and Genetics Aarhus University Gustav Wieds Vej 14, DK-8000 Aarhus, Denmark E-mail: [email protected]

Jørgen Kjems Interdisciplinary Nanoscience Center (iNANO) Department of Molecular Biology and Genetics Aarhus University Gustav Wieds Vej 14, DK-8000 Aarhus, Denmark E-mail: [email protected]

KEYWORDS: Theranostics, siRNA, microRNA, Osteogenic differentiation, Indocyanine green, Stem cells tracking

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ABSTRACT RNA interference (RNAi) mediated gene regulation in stem cells offers great potential in regenerative medicine.

In this study, we developed a theranostic platform for efficient

delivery of small RNAs (siRNA/miRNA) to human mesenchymal stem cells (hMSCs) to promote differentiation, and meanwhile, to specifically label the transfected cells for in vivo tracking purpose. We encapsulated indocyanine green (ICG) in a nonionic surfactant vesicle, termed “niosome”, that is mainly composed of a nonionic surfactant sorbitan monooleate (Span 80) and a cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). This novel ICG containing niosome system (iSPN) demonstrated highly efficient siRNA and microRNA delivery in hMSCs. Specific inhibition of miR-138, a negative regulator of osteoblast differentiation, was achieved by iSPN/miR-138, which significantly promoted osteogenesis of hMSCs. Furthermore, iSPN exhibited OFF/ON activatable fluorescence upon cellular internalization, resulting in efficient NIR labeling and the capability to dynamically monitor stem cells in mice. In addition, iSPN/siRNA achieved simultaneous long-term cell tracking and in vivo gene silencing after implantation in mice. These results indicate that our theranostic niosomes could represent a promising platform for future development of stem cell-based therapy.


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1. INTRODUCTION Mesenchymal stem cells or marrow stromal cells (MSCs) hold great clinical potential for tissue engineering and cell therapy due to their ease of isolation, multipotent differentiation capability and low immunogenicity.1-2 However, the slow and uncontrolled differentiation process is the major issue to realize the therapeutic potential. Recently, small interfering RNAs (siRNAs) or microRNAs (miRNAs) have emerged as potent tools to control stem cell differentiation, by manipulating key factors involved in the differentiation process.3-4 Despite the great promise to use small RNA therapeutics to regulate stem cell fate, their large size, polyanionic properties and instability limit their bioavailablity. Thus, the design of efficient delivery systems is critical. Primary human cells, such as hMSCs are generally difficult to transfect compared to other cell lines.5-6 Although successful gene delivery has been achieved using viral vectors and electroporation, the practical and safety issues limit the clinical applications of these methods.7-8 Nonionic surfactant vesicles, termed “niosomes” have recently shown to be a promising drug delivery vectors. Niosomes have several advantages over traditional liposomes, including higher stability and loading capacity.9-10 More importantly, cationic niosomes composed of nonionic surfactants, such as sorbitan monooleate (Span 80) and glyceryl monooleate, were reported to mediate efficient siRNA delivery in several cancer cells with minimal toxicity.11-13 Therefore, cationic niosomes might be promising carriers of small RNAs to MSCs, which, however, has not been studied. The precise evaluation of the biodistribution, migration and proliferation of stem cells after transplantation poses another issue within stem cell therapy. Therefore, the development of efficient cell labeling and tracking system is urgently needed. To track stem cells, a variety of 3 ACS Paragon Plus Environment


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probes or contrast agents have been developed, including quantum dots14-15, iron oxide nanoparticles16, upconversion nanoparticles17 and semiconducting polymer dots18. Among different bioimaging techniques, optical imaging offers fast scanning, high sensitivity and non-invasiveness for diagnosis and cell tracking. Indocyanine green (ICG), an amphiphilic tricarbocyanine dye, has been approved by United Stated Food and Drug Administration (FDA) for clinical use and its near-infrared emission property makes it suitable for bioimaging applications. However, its tendency to form oligomers, instability and photobleaching limit its diagnostic applications.19 To overcome these problems, various encapsulation systems, such as silica nanoparticles, liposomes and polymeric nanoparticles have been developed.20-22 The combination of gene delivery and cell labeling capacity into a single unit to achieve theranostic properties is an attractive possibility. Recently, a few studies reported the possibility to combine plasmid DNA delivery and labeling of stem cells by use of polyethylenimine (PEI) coated magnetic nanoclusters23, cationic fluorescent mesoporous silica nanoparticles24 or PEI funcationalized quantum dots25. However, the combination of siRNA/miRNA delivery and tracking system for stem cells into one unit has been less studied. In this study, a theranostic platform (iSPN) was developed by encapsulation of ICG in a cationic niosomes composed of Span 80, DOTAP and TPGS. We investigated cellular uptake and gene silencing capacity of iSPN in hMSCs. The therapeutic application of iSPN was assessed by functional delivery of anti-miR-138 to promote osteogenic differentiation of hMSCs. Futhermore, the encapsulation of ICG in iSPN resulted in self-quenching and


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dequenched upon decomplexation. This OFF/ON activatable fluoresence from iSPN meidiated efficient NIR labeling and dynamically tracking of hMSCs in vivo. 2. MATERIALS AND METHODS 2.1 Materials D-α-tocopheryl polyethylene glycol-1000 succinate (TPGS), Span 80, and indocyanine green (ICG) were obtained from Sigma-Aldrich. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). siRNA against GFP (siGFP) with the sequene,

sense

5′-

GACGUAAACGGCCACAAGUTC-3′,

antisense

5′-

ACUUGUGGCCGUUUACGUCGC-3′ and Cy3-labeled siGFP were purchased from Ribotask (Odense, Denmark). Anti-miR-138, negative control anti-miR-NC and a scrambled negative

control

siRNA

(siNC)

with

the

sequence,

sense

5'-

UUCUCCGAACGUGUCACGUTT-3', antisense, 5'-ACGUGACACGUUCGGAGAATT-3' was provided by GenePharma (Shanghai, China). 2.2 Cell culture Human mesenchymal stem cells (hMSCs) and hMSCs stably expressing green fluorescent protein (GFP-hMSCs) were kindly provided by Dr. Moustapha Kassem (University of South Denmark, Odense, Denmark). The cells were maintained in minimum essential medium (Gibco,

Life

technologies)

with

10%

fetal

bovine

serum

(FBS)

and

1%

penicillin/streptomycin (P/S) at 37 °C, 5% CO2 and 100% humidity. 2.3 Preparation of SPN, iSPN and CL Empty niosome (SPN) and ICG loaded niosome (iSPN) were synthesized by ethanol 5 ACS Paragon Plus Environment


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injection method according to our previous report.11 In brief, DOTAP, Span 80, and TPGS were dissolved in absolute ethanol at concentrations of 50, 100, and 100 mg/ml, respectively. These components were combined at molar ratio of 50: 45: 5 (DOTAP/Span80/TPGS). The lipids mixture was then injected into ultrapure water followed by vigorous vortex mixing to assemble empty SPN. Cationic liposomes (CL) were prepared similarly by changing the components to DOTAP/DOPE/TPGS at 50:49:5 according to previous report.13 For synthesis of ICG encapsulated niosomes (iSPN), ICG was added to the lipids mixture in ethanol at weight ratio of 1: 10 (ICG/lipids) and then injected into water. The iSPN solution was loaded into a dialysis tube (MWCO, 12-14 kDa, Spectrumlabs) and dialyzed against ultrapure water to remove unencapsulated ICG. The encapsulation efficiency of ICG was quantified by measuring the fluorescent intensity of iSPN in lysis buffer (1% Triton X-100, 2% SDS in PBS) and normalized to that of feed ICG in lysis buffer using a Fluoremax 3 fluorometer (Horiba Jobin-Yvon). SPN and iSPN was characterized by Dynamic Light Scattering (DLS) at 25 °C using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) and their morphology was examined by transmission electron microscope (TEM, FEI Tecnai G2 Spirit). 2.4 iSPN/siRNA complex preparation and characterization To form iSPN/siRNA complexes, a stock solution of siRNA (20 µM) was mixed with iSPN at various weight ratios under vigorous vortexing. The solution was incubated at room temperature for 15 min before further analysis. The formation of iSPN/siRNA complex was examined by gel retardation assay. Samples were loaded into a 2% agarose gel containing SYBR Gold (Life Technologies) for nucleic


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acid staining and the electrophoresis was carried out at 120 mV for 45 min at room temperature. Image was acquired using GelDOC EZ System (Bio-Rad Laboratories Inc.). 2.5 Fluorescence quenching and de-quenching assay Fluorescence quenching and de-quenching of iSPN was analyzed by measuring the fluorescence spectrum before and after decomposition. First, the emission spectra of free ICG and iSPN solutions were measured using a fluorometer (Horiba Jobin-Yvon) at the excitation wavelength of 730 nm. Then, free ICG or iSPN stock solution was added to a lysis buffer (1% Triton X-100, 2% SDS in PBS) to trigger decomposition before recording emission spectrum. The maximum fluorescence of three independent experiments was used for statistical calculation. 2.6 Cellular uptake of iSPN/siRNA complex For visualization of siRNA uptake mediated by iSPN/siRNA complexes, hMSCs were seeded in 24-well plate (4×104 cells/well) in growth medium and incubated overnight. Cy3labeled siRNA were complexed with iSPN and added to the cells at final siRNA concentration of 50 nM. After incubation for 4 h, cells were washed three times with PBS and fixed with 4% paraformaldehyde. Then, cell membrane was stained with Wheat Germ Agglutinin, Alexa Fluor 488 (WGA-Alexa 488, Molecular Probes) and nucleus was stained with DAPI according to manufacturing protocols. Images were taken by using LSM 710 confocal microscope (Zeiss, Germany) and processed by ZEN program (Zeiss, Germany). Quantification of Cy3-siRNA uptake was conducted by measuring Cy3 intensity in cell lysate according to previous reports.26-27 In brief, transfected cells were lysed in 250 µL lysis buffer (1% Triton X-100, 2% SDS in PBS) on ice for 30 min. After removing cell debris by



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centrifugation, 100 µL of the supernatant was transferred to a black 96-well plate (NUNC) to measure the Cy3 fluorescent intensity using a FLUOstar OPTIMA (BMG, Labtechnologies). For intracellular tracking of siRNA, hMSCs were grown on an 8-well Tissue Culture Chamber (Sarstedt, Germany) and transfected as mentioned above. Thirty minutes before fixation, LysoTracker Green DND-26 (Molecular Probes) containing medium was added and incubated with cells at 37 °C. Afterwads, cells were fixed and the slides were mounted with ProLong Gold Antifade Reagent with DAPI (Molecular Probes) and then imaged by confocal microscope. 2.7 In vitro gene silencing GFP-hMSCs were plated on 24-well plate (4×104 cells/well) overnight. iSPN/siGFP complexes were added to the cells at final siRNA concentration of 50 nM and incubated overnight before replacing with fresh medium. Approximately 72 hours after transfection, the cells were imaged by Olympus IX71 microscope (Olympus, US) with the filter unit of excitation at 488 nm and emission at 515 nm. To quantify the silencing efficiency, cells were detached by 0.05% Trypsin-EDTA solution (Gibco, Invitrogen) and washed with PBS. The cells were analyzed by FACSCalibur flow cytometry (Becton Dickenson). A commercial transfection reagent lipofectamine 2000 (Invitrogen) was included as a control. 2.8 Cytotoxicity Cytotoxicity was evaluated using AlamarBlue assay (Molecular Probes, Life Technologies) according to manufacturer’s protocol. Cells were seeded in 96-well plate (1×104 cells/well) and transfected with iSPN/siRNA complex. After transfection for 48 h, the growth media was replaced with fresh media supplemented with 10% (v/v) of AlamarBlue reagent. After


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incubation for 2 h at 37 °C, the fluorescent intensity of the media was measured using a plate reader (FLUOstar OPTIMA, Moritex BioScience) at 540 nm excitation and 590 nm emission wavelength. 2.9 Osteogenic differentiation hMSCs were seeded in 24-well plates and incubated with iSPN/anti-miR-138 or iSPN/antimiR-NC (50 nM) overnight before osteogenic induction in differentiation media (growth medium supplemented with 10 nM dexamethasone, 0.2 mM l-ascorbic acid, 10 mM βglycerophosphate, and 10 nM 1,25-vitamin-D3). Matrix mineralization was visualized by Alizarin red S (ARS) staining. At day 14 of differentiation, cells were fixed and incubated in 2% ARS solution (pH = 4.2, Sigma– Aldrich) for 10 min at room temperature, followed by two washes with water to visualize the calcium deposits by microscope. Mineralization was further quantified by extracting the ARS stain with 10% cetylpyridinium chloride and measuring the absorption at 570 nm by plate reader. The expression level of miR-138 was quantified by Taqman small RNA assay (Applied Biosystems). Total RNA was isolated from cells by Trizol reagent (Invitrogen) and cDNA was synthesized by a TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems) and qRT-PCR was performed with a TaqMan® Universal PCR Master Mix and a specific TaqMan® MicroRNA assay (Applied Biosystems) according to manufacturer’s instructions. Osteogenic markers were quantified by real-time PCR. cDNA was synthesized by qScriptTM cDNA synthesis kit (Quanta) on a thermal cycler according to manufacturer’s instructions. Real-time PCR was performed using SYBR Green (Invitrogen) running on a LightCycler 480 Real-time PCR system (Roche) with the program: initial denaturation at 95 °C for 10 min, 9 ACS Paragon Plus Environment


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followed by 50 cycles of 95 °C for 15 s, 55 °C for 15 s, and 72 °C for 30 s. Primers used in reactions were obtained from Integrated DNA Technologies and the sequences were listed in Tab S1. 2.10 Near-infrared (NIR) imaging of hMSCs Cells were seeded in 24-well plates and incubated with either free ICG or iSPN/siRNA complexes for 1, 2, or 6 hours, respectively. Then, cells were washed three times with PBS and subsequently imaged with an IVIS®200 imaging system (Xenogen, Caliper Life Sciences, Hopkinton, MA, USA) using the ICG filter units of excitation of 710 - 760 nm and emission of 810 - 875 nm and scanned with auto-exposure. 2.11 In vivo tracking of hMSCs All animal experiments were performed in compliance with the guidelines established by the National Institutes of Health. hMSCs were incubated with iSPN/siRNA complexes overnight (～18 hrs) and harvested by Trypsin-EDTA solution (Gibco, Invitrogen) followed by three times washing with PBS. Female BALB/c nude mice (n=4) at 5-6 weeks of age (Janvier Lab, France) were injected intravenously through tail vein with labeled hMSCs (5 × 105/mouse) in PBS. Thereafter, mice were anesthetized by 2.5% isoflurane and scanned by IVIS®200 imaging system (excitation at 745 nm and emission at 840 nm) at 0.5, 2, 4, 6 and 24 hours post-injection. To examine biodistribution of labeled hMSCs, major organs including lung, liver, spleen, heart and kidneys were collected at 24 hours post-injection and imaged. Quantitative analysis of fluorescence intensity was perfomed using Living Image 4.2 software package (Caliper Life Science). The average radiant efficiency in region of interest (ROI) was measured (photons/sec/cm2/sr)/(µW/cm2), which presents radiance/illumination


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power density. 2.12 In vivo implantation of hMSCs hMSCs transfected with iSPN/siGFP or iSPN/siNC complexes overnight (～18 hrs) were harvested and washed three times with serum-free medium. One hundred microliter of hMSCs (1.5×106 cells/implant) were mixed with equal volume of Matrigel on ice (Corning, Catalog number: 356237) and injected subcutaneously into the back of 6-week-old nude mice (n=3). Non-transfected hMSCs were implanted similarly as controls. The mice were imaged by IVIS®200 imaging system as mentioned previously from Day 1 to Day 7 after implantation. One Day 7, the mice were sacrificed and the implants were collected, washed by saline twice and divided into two pieces, followed by fixation in 2% paraformaldehyde or RNA isolation by Trizol reagent, respectively. To evaluate the GFP silencing after implantation, fixed implants were further sectioned and stained with Hochest (Thermofisher Scientific) before visualized by confocal microscope. In addition, GFP expression level was further quantified by RT-qPCR as described previously. 2.13 Statistical analysis One-way ANOVA with Tukey test (OriginPro 8.1, OriginLab) was performed to determine statistical significance and P < 0.05 was considered as significant difference. 3. RESULTS AND DISCUSSION 3.1 Synthesis and Characterization of iSPN As illustrated in Scheme 1, indocyanine green encapsulated niosomes (iSPN) were synthesized by a simple ethanol injection method. The cationic niosome consists of a 11 ACS Paragon Plus Environment


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nonionic surfactant Span 80, a cationic lipid DOTA and a PEGylated lipid (TPGS). The encapsulation of amphiphilic ICG molecules in the niosomes resulted in dye self-quenching due to their close proximity. siRNA or miRNA could be further complexed through electrostatic interactions with the positive head group of DOTA on the surface of iSPN. From dynamic light scattering (DLS) measurement, iSPN exhibited a size of 164 nm and a zeta potential of +29.3 mV (Figure 1A, B). The encapsulation efficiency of ICG was ~ 95%. We further characterized the siRNA loading capability of iSPN. The size and zeta potential of iSPN/siRNA complexes formed at different iSPN/siRNA (N/P) ratios of 5, 10, 15, and 20 were measured. The size of iSPN/siRNA complexes appeared smallest at ratio 10 (~106 nm) and further increased at weight ratio of 15 and 20 (Figure 1A). Both iSPN and iSPN/siRNA complexes showed narrow size distribution from DLS measurement (Figure S1). Moreover, the zeta potential of iSPN/siRNA increased gradually with the increasing N/P ratio due to the negative charge of the RNA equilibrating the positive charge of DOTAP (Figure 1B). To further investigate the binding capability of siRNA, a gel retardation assay was performed (Figure 1C). The retardation of siRNA migration diminished gradually with the increasing of N/P ratio. No free siRNA molecules could be observed when the ratio reached 15, indicating that all siRNAs were fully complexed with iSPN. The morphology and size distribution was further analyzed by transmission electron microscopy (TEM, Figure 1D). Both SPN and iSPN showed spherical multilayered structure with narrow particle size distribution. Next, we investigated the optical properties of iSPN. The maximum fluorescence emission of free ICG solution was 810 nm (λmax), and following treatment with lysis buffer, λmax shifted to 832 nm (Figure 2A). ICG molecules are prone to assemble into oligomers in aqueous solution, leading to low fluorescence emission and blue-shifted spectrum.28 In 12 ACS Paragon Plus Environment

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contrast, the λmax of iSPN was the same as the lysed ICG solution but almost completely quenched. Upon treatment with lysis buffer, the ICG signals were activated. This OFF/ON fluorescence property is attributed to the distance change of ICG molecules from inside of iSPN state (self-quenching) to free state (dequenching). This quenching and dequenching phenomenon of iSPN was further confirmed by IVIS bioimaging (Figure 2C, D). We speculate that this OFF/ON fluorescence could be activated similarly by decomplexation of iSPN after cell internalization, thereby enabling efficient cell labeling (Scheme 1). In addition, the encapsulation of ICG in iSPN also resulted in enhanced aqueous stability comparing to free ICG (Figure S2). After storage for 4 weeks, the fluorescence intensity of iSPN remained above 90%, however, free ICG only remained ~ 40%. The size of 4-week-old iSPN is 154.5 ± 9.7 nm (PDI = 0.191), which is similar to freshly prepared iSPN (Figure S3). And the zeta potential of 4-week-old iSPN is + 44.5 ± 0.4 mV, which is slightly higher than the fresh iSPN. This long-term stability of iSPN is advantageous for further in vitro and in vivo applications. 3.2 Internalization of iSPN/siRNA by hMSCs Next, we examined the internalization of iSPN/siRNA complexes into hMSCs by confocal microscopy. Cy3-labeled siRNA was formulated with iSPN for visualization. Cell membrane was stained with WGA-Alexa 488 (green) and nucleus was stained with DAPI (blue). Confocal images clearly showed siRNA localized in the cytoplasm after transfection with iSPN/siRNA (Figure 3A) and the siRNA uptake increased proportionally with iSPN/siRNA weight ratio based on Cy3 fluorescence quantification in the cell lysate (Figure 3B). As cellular endocytotic pathways are known to influence transfection efficiency29-31, we next investigated the mechanism of iSPN/siRNA internalization by hMSCs. Following pre13 ACS Paragon Plus Environment


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treatment with various endocytosis inhibitors, including macropinocytosis inhibitors (EIPA and CCD), clathrin pathway inhibitor (CPZ) and caveolae inhibitor (GEN), significant reduction of siRNA internalization was observed (Figure 3C). Thus, macropinocytosis, clathrin, and caveolae pathways all appear to be involved in iSPN/siRNA internalization into hMSCs. Endosome escape is usually the most critical step for successful intracellular siRNA delivery.32 Therefore, we further studied the subcellular localization of siRNA after cellular uptake. Transfected hMSCs was stained with LysoTracker Green and nucleus with DAPI and imaged by confocal microscope (Figure 4). Although some colocalization (yellow) of siRNA and LysoTracker was observed in cells, the majority of siRNA (red) appeared separated from LysoTracker (green), indicating efficient endosome escaping. This process could be facilitated by the nonionic surfactant Span 80 that is known to promote cubic phase transition and destabilize endosomal membrane, causing subsequent release in the cytoplasm.13, 33 3.3 Transfection efficiency of iSPN/siRNA in hMSCs The silencing efficiency of iSPN/siRNA was evaluated by knockdown of GFP in GFP stably expressing hMSCs. From fluorescent microscopy, significant suppression of GFP expression was observed in iSPN/siGFP transfected cells when the N/P ratio was higher than 10 (Figure 5A). We further quantified silencing efficiency by flow cytometry, which showed that ～85% and ～88% silencing were achieved at N/P ratio of 15 and 20, respectively, which are significantly higher than lipofectamine 2000 (Figure 5B, C). No significant down-regulation of GFP was observed by iSPN/siNC, indicating the silencing effect was specific. In addition, we also evaluated the transfection of hMSCs by 4-week-old iSPN, which also achieved ～80%


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knockdown of GFP at N/P ratio of 20 (Figure S4), indicating a long shelf-life of the particles. We further compare the silencing activity of iSPN to traditional cationic liposome (CL) composed of DOTAP/DOPE/TPGS with size of 111.8 ± 2.1 nm and PDI of 0.21 (Figure S5). Both SPN/siGFP and iSPN/siGFP achieved significantly higher down-regulation of GFP than CL/siGFP (Figure S6). Similarly, Zhou, et al. also showed that cationic niosome achieved higher silencing effect in SK-HEP-1 cells compared to traditional cationic liposomes.13 We also examined the cytotoxicity of iSPN/siRNA in hMSCs by AlamarBlue assay. No significant cytotoxicity was observed for any of the formulations when siRNA concentration was below 100 nM (Figure S7). Further increasing the concentration of complexes to 200 nM some toxicity was detected, but still significantly lower than lipofectamine.

In fact,

iSPN/siRNA formulated at ratio 15 and concentration of 50 nM already achieved sufficient gene silencing in hMSCs, thus indicating the great biocompatibility of iSPN. Based on the in vitro transfection and toxicity results, we utilized iSPN/siRNA formulated at ratio 15 and concentration of 50 nM siRNA for further studies. 3.4 iSPN/anti-miR-138 Promotes Osteogenic Differentiation of hMSCs MicroRNAs are endogenous short non-coding RNAs that regulate multiple biologic processes through post-transcriptional gene silencing and in the past few years, microRNAs have emerged as important regulators for stem cells differentiation. Overexpression or suppression of microRNAs has been utilized to manipulate the state of stem cells through interfering with intracellular process.34 For example, miR-138 has been identified as a key suppressor of osteogenesis of hMSCs by targeting the focal adhesion kinase (FAK) signaling pathway and inhibition of miR-138 accelerated the osteognesis of hMSCs both in vitro and in



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vivo.35-36 To demonstrate the therapeutic potential of our iSPN system, we transfected hMSCs with anti-miR-138 loaded iSPN and evaluated the effect on osteogenic differentiation. We first examined the effect of introducing iSPN/anti-miR-138 on the expression level of miR-138 in hMSCs. From TaqMan small RNA analysis, iSPN/anti-miR-138 transfection resulted in significant suppression of miR-138 by ~90%, while iSPN/anti-miR-NC treated cells did not show any difference compared to untreated cells (Figure 6A). This transfection efficiency was significantly better than lipofectamine. We conclude that iSPN can deliver both double-stranded siRNA and single-stranded anti-miR to hMSCs, which could have broad application in stem cell therapy and regenerative medicine. Next, we evaluated the impact of iSPN/anti-miR-138 on the osteogenic differentiation of hMSCs. Inhibition of miR-138 strongly enhanced matrix mineralization and calcium deposition based on Alizarin red S staining (Figure 6 B, C). In addition, the expression of osteogenic markers, including alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) were also significantly increased upon iSPN/anti-miR-138 transfection, especially after differentiation for 14 days (Figure 6D). These results indicate that iSPN/anti-miR-138 significantly promotes the osteogenic differentiation of hMSCs, which could be applicable for bone regeneration. 3.5 hMSCs Labeling and In Vivo Tracking Monitoring the behaviour of stem cells in vivo after transplantation is important for both mechanism investigation and therapeutic evaluation. To examine the potential of iSPN for stem cell labeling, we incubated the hMSCs with iSPN/siRNA for different periods and performed the NIR imaging. As shown in Figure 7A, strong NIR signal were detected from iSPN/siRNA transfected hMSCs and the fluorescence increased along with the iSPN/siRNA 16 ACS Paragon Plus Environment

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incubation time. Quantitative measurement of the NIR intensity from IVIS imaging revealed that cells treated iSPN/siRNA showed significant higher fluorescence signals compared to cells treated by free ICG, especially after incubation for 2 and 6 hours (Figure 7B). Next, we explored the capability of our iSPN for tracking MSCs in vivo. iSPN labeled hMSCs were injected intravenously into nude mice and imaged by in vivo NIR imaging system. The fast scanning and high sensitivity of optical imaging allowed us to perform realtime monitoring of stem cell trafficking. NIR signals could be detected in both lung and liver at 30 min post-injection and the strongest accumulation of hMSCs in the mouse lung was observed at 2 hours post-injection (Figure 7C) and slowly decreased afterwards. The result was further confirmed by high resolution NIR imaging, which indicated slight translocation of hMSCs from lung to liver from 2 hours to 4 hours post-injection (Figure 7D). The NIR signals from lung and liver were further quantified from IVIS images (Figure 7E). At 24 h post-injection, major organs were collected and imaged to further characterize the biodistribution of iSPN labeled hMSCs. From ex vivo imaging (Figure 8), strong NIR signals were observed in lung and followed by liver whereas nearly no signals was observed in spleen, heart and kidneys, which is similar to the finding from previous reports.18, 37 Taken together, these results suggested that iSPN has great potential as NIR probes for stem cells tracking in vivo due to its high sensitivity and good temporal resolution. 3.6 hMSCs Transplantation In Vivo Gene modified mesenchymal stem cells have shown augmented therapeutic effect in several preclinical diseases models. Ex vivo modification of stem cells before transplantation is considered to be more applicable approach due to the good biosafety and effectiveness.38-40 However, the fate of modified MSCs after transplantation need to be carefully assessed. To 17 ACS Paragon Plus Environment


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further investigate translational potential of iSPN/siRNA system for stem cells therapy, we implanted the transfected hMSCs with Matrigel Matrix subcutaneously into the back of nude mice. Both the long-term cell tracking and in vivo gene silencing were evaluated. From the IVIS bioimaging, strong NIR signals were detected from Day 1 to Day 7 (Figure 9A) and only a slight decrease in NIR intensity at Day 5 (Figure 9B) was observed, implying that iSPN can serve as a long-term cell tracking probe. We further analyzed in vivo gene silencing in hMSCs 7 days after transplantation. From confocal microscopy analysis, strong inhibition of GFP expression was observed in the implant with iSPN/siGFP-treated cells (Figure 9C), while the implant with iSPN/siNC-treated cells showed strong GFP expression comparable to untreated cells. RT-qPCR analysis of implanted cells further confirmed that approximately 70% downregulation of GFP mRNA was achieved by iSPN/siGFP treatment (P < 0.05). From these results, we conclude that iSPN/siRNA has a great theranostic potential for simultaneous long-term cell tracking and gene silencing in vivo. Although several organic or inorganic NIR probes have been developed for stem cells tracking, such as quantum dots and semiconducting polymer dots,14, 18 the combination of siRNA/miRNAs delivery and cell labeling in one unit as a theranostic tool has been less reported. Recently, Li et al. reported peptide and cyclodextrin modified quantum dots for stem cells labeling and siRNA delivery.15 However, laborious work is involved for both particle synthesis and surface modification. Besides, our iSPN can efficiently deliver both double-stranded siRNA and single-stranded anti-miR to hMSCs, enabling broader applications for stem cell therapy. Furthermore, the induced fluorescent signal for bioimaging is advantageous for diagnosis by avoiding unspecific signals and generating superior targetto-background ratio, which has also been recently investigated in cancer or inflammatory 18 ACS Paragon Plus Environment

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diseases.41-42 In addition, iSPN showed a great theranostic potential for simultaneous longterm cell tracking and gene silencing after implantation in mice. Taken together, we believe that our iSPN/siRNA system represents a promising platform for stem cells research and regenerative medicine. 4. CONCLUSION In this study, we have developed a theranostic platform with the efficient siRNA/miRNA delivery and NIR labeling of stem cells. This novel niosome system (iSPN), consist of DOTAP, TPGS, a nonionic surfactant Span 80 and ICG. The nanosized iSPN with positive charge could complex with siRNA and mediated efficient intracellular delivery, resulting in specifically gene silencing in hMSCs. In addition, following inhibition of miR-138 by iSPN/anti-miR-138, enhanced osteogenic differentiation of hMSCs was achieved. Furthermore, iSPN exhibited OFF/ON activatable fluorescence upon decomposition or cellular internalization, resulting in efficient NIR labeling of stem cells and live tracking in animals. We conclude that the theranostic iSPN system represents a promising platform for stem cells research and regenerative medicine. ASSOCIATED CONTENT Supporting Information Primers for q-PCR reaction. Size distribution of iSPN and iSPN/siRNA by DLS. Fluorescent stability of free ICG and iSPN. Size characterization of 4-week-old iSPN by DLS. siRNA delivery in hMSCs by 4-week-old iSPN. Size characterization of cationic liposome. Comparison of gene silencing activity of cationic noisome and cationic liposome. Cytotoxicity of iSPN/siRNA.



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AUTHOR INFORMATION Corresponding Authors Chuanxu Yang, E-mail: [email protected] Jørgen Kjems, E-mail: [email protected] Notes The authors declare no conflict of interest. ACKNOWLEDGEMENTS This project was founded by LUNA Nanomedicine Center (the Lundbeck Foundation Nanomedicine Center for Individualized Management of Tissue Damage and Regeneration) and postdoctoral fellowship from the Lundbeck Foundation to C.Y. (Project No. 23750) and we also acknowledge the Ph.D. Scholarship from the China Scholarship Council (CSC) of the Ministry of Education of China for P.S..

Figures


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Scheme 1. Illustration showing the design of theranostic niosomes (iSPN) for intracellular delivery of siRNA/miRNA and activatable labeling of cells upon dequenching.

Figure 1. Characterization of iSPN. (A) Hydrodynamic size and (B) zeta potential of iSPN and iSPN/siRNA complexes measured by DLS. Data represent mean ± SD (n = 3). (C) Gel retardation assay for analysis of siRNA complexation by iSPN. Lane 1 and 7, free siRNA as control; lane 2 – 6, iSPN/siRNA complexes prepared at weight ratio (iSPN/siRNA, w/w) of 2.5, 5, 10, 15, 20, respectively. (D) Representative TEM images of SPN and iSPN. Scale bar, 100 nm.



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Figure 2. Optical properties and NIR imaging of iSPN. (A) Fluorescence spectrum (Excitation, 730 nm; emission 750 – 870 nm) of free ICG and iSPN in water or lysis buffer (LB). (B) Quantification of fluorescent intensity of free ICG and iSPN in water or LB at maximum emission wavelength. Results represent mean ± SD (n = 3, ***P < 0.001). (C) NIR imaging of free ICG and iSPN by an IVIS bioimaging system ) using the ICG filter units of excitation of 710 - 760 nm and emission of 810 - 875 nm. (D) Quantification of fluorescence intensity/radiant efficiency from NIR imaging.


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Figure 3. Characterization iSPN/siRNA uptake by hMSCs. (A) Representative confocal images of hMSCs transfected with iSPN/siRNA at N/P ratio of 5 and 20. hMSCs were incubated with iSPN/siRNA at siRNA concentration of 50 nM for 4 hours. Cell nucleus stained with DAPI (blue), membrane stained with WGA-Alexa 488 (green) and siRNA labeled with Cy3 (red). (B) Quantification of siRNA uptake by hMSCs by measuring Cy3 intensity in cell lysate. Untreated cells (Untr) and cells treated with naked siRNA were included as control. Results represent mean ± SD (n = 3). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. (C) Effect of endocytosis inhibitors on iSPN/siRNA uptake. hMSCs were 23 ACS Paragon Plus Environment


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transfected with iSPN/siRNA (N/P ratio 15, 50 nM) for 4 hours after 1 h pretreatment with EIPA (macropinocytosis, 100 µM), CPZ (clathrin pathway, 50 µM), GEN (caveolae pathway, 100 µM) or CCD (macropinocytosis, 2 µM). Uptake was quantified by flow cytometry. Results represent mean ± SD (n = 3). Significance: ***P < 0.001, vs non-inhibitor treated control.

Figure 4. Intracellular trafficking of siRNA. hMSCs transfected with iSPN/Cy3-siRNA (N/P ratio 15, 50 nM) for 4 hours and stained with Lysotracker Green and DAPI. Merged image shows significant endosome/lysosome escaping and partial co-localization of siRNA and Lysotracker (yellow). Scale bar, 20 µm.


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Figure 5. Gene silencing in hMSCs by iSPN/siRNA. GFP stably expressing hMSCs were transfected with iSPN/siGFP or iSPN/siNC at 50 nM for 72 hours. (A) Representative fluorescent images of (a) untreated cells; (b - e) cells transfected with iSPN/siGFP at N/P ratios 5, 10, 15 and 20; (f) cells transfected with iSPN/siNC at ratio 15. Scale bar, 200 µm. (B) Flow cytometry histogram of cells treated with indicated formulations. (C) Quantification of relative GFP expression from flow cytometry analysis. Results represent mean ± SD (n = 3). Significance: **P < 0.01, ***P < 0.001, vs cells transfected with Lipofect/siGFP.



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Figure 6. Enhanced osteogenic differentiation of hMSCs by iSPN/anti-miR-138. (A) Quantification of miR-138 level in hMSCs transfected with anti-miR-138 or anti-miR-NC by iSPN or lipofectamine (n = 3) ***P < 0.001, n.s., not significant vs untreated group. (B) Alizarin red S staining of transfected hMSCs under osteogenic differentiation for 14 days. (C) Quantification of mineralization / calcium deposition from by absorbance measurement of solubilized alizarin red from ARS staining (n=3). ***P < 0.001, n.s., not significant. (D) Q-PCR analysis of osteogenic markers expression including ALP, RUNX2 and osteocalcin (OCN) after osteogenic differentiation for day 7 (D7) and day 14 (D14). Results represent mean ± SD. Significance: *P < 0.05, vs non-transfected cells cultured in differentiation medium (DM only) group.


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Figure 7. Cell labeling and in vivo tracking. (A) NIR imaging of hMSCs incubated with iSPN/siRNA or free ICG at different time intervals (1, 2, and 6 hours; excitation at 710 - 760 nm, emission at 810 - 875 nm). (B) Quantification of fluorescent intensity from cells with indicated treatment (n = 4). Significance: *P < 0.05, ***P < 0.001. (C) hMSCs incubated with iSPN/siRNA overnight (～18 hrs) and injected into nude mice intravenously. Real-time tracking of iSPN/siRNA-labeled hMSCs in mice at different time intervals (M1-M4; 30 min,



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2 hrs, 4 hrs, 6 hrs and 24 hrs). A control mouse with buffer injection was included as control. (D) High resolution NIR imaging shows signal changes from 2 hrs to 6hrs post-injection. (E) Quantification of NIR signals from lung and liver (n = 4). Results represent mean ± SD.

Figure 8. Biodistribution of hMSCs in major organs. (A) Ex vivo NIR imaging of major organs, including lung, liver, spleen, heart, kidneys 24 hrs after i.v. injection of iSPN/siRNA complexes labeled hMSCs (excitation at 710 - 760 nm, emission at 810 - 875 nm). Mice 1-4, injection with labeled hMSCs; mice 5, injection with buffer. (B) Quantitative analysis of in vivo biodistribution of hMSCs using Living Image 4.3 software (Caliper Life Science). Each bar represents mean ± SD (n = 4). Significance: ***P < 0.001.


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Figure 9. In vivo implantation of hMSCs in nude mice. hMSCs were incubated with iSPN/siRNA

overnight ( ～ 18 hrs) and implanted subcutaneously in mice. (A)

Representative NIR imaging of hMSCs from Day1 to Day 7 after implantation (excitation at 710 - 760 nm, emission at 810 - 875 nm). (B) Quantification of NIR signals from implanted cells. Each bar represents mean ± SD (n = 3). (C) The hMSCs implants were harvested at Day 7. Sections were fixed and stained with Hoechst for confocal microscopy analysis of GFP silencing in vivo. Scar bar, 50 µm. (D) GFP expression of implanted cells was quantified by qPCR analysis. Each bar represents mean ± SD (n = 3). Significance: ** P < 0.01; n.s., not significant.



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