Dual-modal magnetic resonance and photoacoustic tracking of tendon stem cell labeled with PLGA/IO MPs

Objective To prepare PLGA microparticles loaded with Iron oxide (PLGA/IO MPs) and explore their feasibility of the rat tendon stem cells (TSCs) labeled with the particles and the multimodal imaging of Ultrasonic (US)/Photoacoustic (PA)/Magnetic resonance (MR) in vitro. Methods The PLGA/IO MPs were prepared using double emulsification, and physical and chemical properties were tested and US/PA/MRI imaging was performed. The TSCs were labeled with PLGA/IO MPs, and transmission electron microscopy (TEM) and prussian blue staining were performed to test labeling effects, then the US, PA and MRI imaging of labeled TSCs were performed. Results The diameter and Zeta potential of prepared PLGA/IO MPs were (801.5±165.6)nm and (6.36±3.36)mV [the Zeta potential of microparticles which including poly-L-Lysine(PLL) was about (3.16±3.69)mV], respectively. PLGA/IO MPs could be imaged by US/PA/MRI multimodal imaging. After labeling, the PLGA/IO MPs were distributed in cytoplasm of labeled TSCs which could be imaged by US, PA, MRI simultaneously. Conclusions The TSCs can be labeled with PLGA/IO MPs effectively, and imaged by using multimodal US/PA/MRI imaging in vitro, which will lay foundation for noninvasive and multimodal tracking of transplanted TSCs in vivo. Key words: Ultrasonography; Iron oxide/PLGA microparticles; Tendon stem cells; Multimodal imaging; Photoacoustic imaging; Magnetic resonance imaging

Stem cells have been used to promote the repair of rotator cuff injury, but their fate after transplantation is not clear. Therefore, contrast agents with good biocompatibility for labeling cell and a reliable technique to track cell are necessary. Here, we developed a micron-sized PLGA/IO MPs to label tendon stem cells (TSCs) and demonstrated that PLGA/IO MPs were safe and efficient for long-term tracking of TSCs by using dual-modal MR and Photoacoustic (PA) imaging both in vitro and in rat rotator cuff injury. Moreover, TSCs improved the repair of injury and the therapeutic effect was not affected by PLGA/IO MPs labeling. We concluded that PLGA/IO particle was a promising dual-modal MR/PA contrast for noninvasive long-term stem cell tracking.

Objective. To evaluate the feasibility of visualizing bone marrow-derived human mesenchymal stem cells (MSCs) labeled with a gold-coated magnetic resonance (MR)-active multifunctional nanoparticle and injected via the carotid artery for assessing the extent of MSC homing in glioma-bearing mice. Materials and methods. Nanoparticles containing superparamagnetic iron oxide coated with gold (SPIO@Au) with a diameter of ∼82 nm and maximum absorbance in the near infrared region were synthesized. Bone marrow-derived MSCs conjugated with green fluorescent protein (GFP) were successfully labeled with SPIO@Au at 4 μg ml−1 and injected via the internal carotid artery in six mice bearing orthotopic U87 tumors. Unlabeled MSCs were used as a control. The ability of SPIO@Au-loaded MSCs to be imaged using MR and photoacoustic (PA) imaging at t = 0 h, 2 h, 24 h, and 72 h was assessed using a 7 T Bruker Biospec experimental MR scanner and a Vevo LAZR PA imaging system with a 5 ns laser as the excitation source. Histological analysis of the brain tissue was performed 72 h after MSC injection using GFP fluorescence, Prussian blue staining, and hematoxylin-and-eosin staining. Results. MSCs labeled with SPIO@Au at 4 μg ml−1 did not exhibit cell death or any adverse effects on differentiation or migration. The PA signal in tumors injected with SPIO@Au-loaded MSCs was clearly more enhanced post-injection, as compared with the tumors injected with unlabeled MSCs at t = 72 h. Using the same mice, T2-weighted MR imaging results taken before injection and at t = 2 h, 24 h, and 72 h were consistent with the PA imaging results, showing significant hypointensity of the tumor in the presence of SPIO@Au-loaded MSCs. Histological analysis also showed co-localization of GFP fluorescence and iron, thereby confirming that SPIO@Au-labeled MSCs continue to carry their nanoparticle payloads even at 72 h after injection. Conclusions. Our results demonstrated the feasibility of tracking carotid artery-injected SPIO@Au-labeled MSCs in vivo via MR and PA imaging.

Merging metal organic framework with hollow organosilica nanoparticles as a versatile nanoplatform for cancer theranostics

Reliable cell tracking is essential to understand the fate of stem cells following implantation, and thus promote the clinical application of stem cell therapy. Dual or multiple modal imaging modalities mediated by different types of multifunctional contrast agent are generally needed for efficient cell tracking. Here, we created a new contrast agent—PLGA/iron oxide microparticles (PLGA/IO MPs) and characterized the morphology, structure and function of enhancing both photoacoustic (PA) and magnetic resonance imaging (MRI). Both PA and MRI signal increased with increased Fe concentration of PLGA/IO MPs. Fluorescent staining, Prussian blue staining and transmission electron microscope (TEM) certified that PLGA/IO MPs were successfully encapsulated in the labeled TSCs. The established PLGA/IO MPs demonstrated superior ability of dual-modal PA/MRI tracking of TSCs without cytotoxicity at relatively lower Fe concentrations (50, 100 and 200 μg/mL). The optimal Fe concentration of PLGA/IO MPs was determined to be 100 μg/mL, thus laying a foundation for the further study of dual-modal PA/MRI tracking of TSCs in vivo and promoting the repair of injured tendon.

Reporter genes are useful scientific tools for analyzing promoter activity, transfection efficiency, and cell migration. The current study has validated the use of tyrosinase (involved in melanin production) as a dual reporter gene for magnetic resonance and photoacoustic imaging. MCF-7 cells expressing tyrosinase appear brown due to melanin. Magnetic resonance imaging of tyrosinase-expressing MCF-7 cells in 300 μL plastic tubes displayed a 34 to 40% reduction in T1 compared to normal MCF-7 cells when cells were incubated with 250 μM ferric citrate. Photoacoustic imaging of tyrosinase-expressing MCF-7 cells in 700 μm plastic tubes displayed a 20 to 57-fold increase in photoacoustic signal compared to normal MCF-7 cells. The photoacoustic signal from tyrosinase-expressing MCF-7 cells was significantly greater than blood at 650 nm, suggesting that tyrosinase-expressing cells can be differentiated from the vasculature with in vivo photoacoustic imaging. The imaging results suggest that tyrosinase is a useful reporter gene for both magnetic resonance and photoacoustic imaging.

PurposePhotoacoustic (PA) imaging is a recently developed breast cancer imaging technique. In order to enhance successful clinical implementation, we quantified the potential clinical value of different scenarios incorporating PA imaging by means of multi-criteria analysis. From this analysis, the most promising area of application for PA imaging in breast cancer diagnosis is determined, and recommendations are provided to optimize the design of PA imaging.MethodsThe added value of PA imaging was assessed in two areas of application in the diagnostic track. These areas include PA imaging as an alternative to x-ray mammography and ultrasonography in early stage diagnosis, and PA imaging as an alternative to Magnetic Resonance Imaging (MRI) in later stage diagnosis. The added value of PA imaging was assessed with respect to four main criteria (costs, diagnostic performance, patient comfort and risks). An expert panel composed of medical, technical and management experts was asked to assess the relative importance of the criteria in comparing the alternative diagnostic devices. The judgments of the experts were quantified based on the validated pairwise comparison technique of the Analytic Hierarchy Process, a technique for multi-criteria analysis. Sensitivity analysis was applied to account for the uncertainty of the outcomes.ResultsAmong the considered alternatives, PA imaging is the preferred technique due to its non-invasiveness, low cost and low risks. However, the experts do not expect large differences in diagnostic performance. The outcomes suggest that design changes to improve the diagnostic performance of PA imaging should focus on the quality of the reconstruction algorithm, detector sensitivity, detector bandwidth and the number of wavelengths used.ConclusionThe AHP method was useful in recommending the most promising area of application in the diagnostic track for which PA imaging can be implemented, this being early diagnosis, as a substitute for the combined use of x-ray mammography and ultrasonography.

Ultrasound imaging, one of the common diagnosis techniques, is frequently used since it is safe, cost-efficient technique and real-time imaging can be conducted. However, various organs and tissues reflect ultrasonic waves, which leads to difficulty in imaging small biomolecules and to a low spatial resolution for deep-tissue images. As such, there have been significant advances in photonics and optical molecular probes in recent years, and photoacoustic (PA) tomography (PAT) has emerged as a promising modality that can overcome the limitations of ultrasound. PAT relies on the photoacoustic effect, which is the conversion of absorbed optical energy into acoustic energy. Since fewer biomolecules exhibit the photoacoustic effect compared to the scattering or reflection of ultrasound, PAT can be employed to generate high-resolution images. PAT also has a number of other advantages when compared to conventional biomedical imaging modalities such as optical tomography, ultrasound imaging, computed tomography, positron emission tomography and magnetic resonance imaging. This review provides a general overview of the contrast agents used for PAT, including organic, inorganic and hybrid contrast agents, and describes their application. This review also identifies limitations of current PAT contrast agents and suggests future research directions for their development.

The efficacy of tendon-derived stem cells (TDSCs) for the promotion of tendon and tendon-bone junction repair has been reported in animal studies. Modulation of the tendon stem cell niche in vivo has also been reported to influence tendon structure. There is a need to have specific and reliable markers that can define TDSCs in vitro and tendon stem cells in situ for several reasons: to understand the basic biology of TDSCs and their subpopulations in vitro; to understand the identity, niches and functions of tendon/progenitor stem cells in vivo; to meet the governmental regulatory requirements for quality of TDSCs when translating the exciting preclinical findings into clinical trial/practice; and to develop new treatment strategies for mobilizing endogenous stem/progenitor cells in tendon. TDSCs were reported to express the common mesenchymal stem cell (MSC) markers and some embryonic stem cell (ESC) markers, and there were attempts to use these markers to label tendon stem cells in situ. Are these stem cell markers useful for the identification of TDSCs in vitro and tracking of tendon stem cells in situ? This review aims to discuss the values of the panel of MSC, ESC and tendon-related markers for the identification of TDSCs in vitro. Important factors influencing marker expression by TDSCs are discussed. The usefulness and limitations of the panel of MSC, ESC and tendon-related markers for tracking stem cells in tendon, especially tendon stem cells, in situ are then reviewed. Future research directions are proposed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0097-y) contains supplementary material, which is available to authorized users.

Magnetic resonance (MR) and photoacoustic (PA) imaging are currently being investigated as complementing strategies for applications requiring sensitive detection of cells in vivo. While combined MR/PAI detection of cells requires biocompatible cell labeling probes, water-based synthesis of dual-modality MR/PAI probes presents significant technical challenges. Here we describe facile synthesis and characterization of hybrid modular dextran-stabilized gold/iron oxide (Au-IO) multimetallic nanoparticles (NP) enabling multimodal imaging of cells. The stable association between the IO and gold NP was achieved by priming the surface of dextran-coated IO with silver NP resulting from silver(I) reduction by aldehyde groups, which are naturally present within the dextran coating of IO at the level of 19-23 groups/particle. The Au-IO NP formed in the presence of silver-primed Au-IO were stabilized by using partially thiolated MPEG5-gPLL graft copolymer carrying residual amino groups. This stabilizer served as a carrier of near-infrared fluorophores (e.g., IRDye 800RS) for multispectral PA imaging. Dual modality imaging experiments performed in capillary phantoms of purified Au-IO-800RS NPs showed that these NPs were detectible using 3T MRI at a concentration of 25 μM iron. PA imaging achieved approximately 2.5-times higher detection sensitivity due to strong PA signal emissions at 530 and 770 nm, corresponding to gold plasmons and IRDye integrated into the coating of the hybrid NPs, respectively, with no "bleaching" of PA signal. MDA-MB-231 cells prelabeled with Au-IO-800RS retained plasma membrane integrity and were detectable by using both MR and dual-wavelength PA at 49 ± 3 cells/imaging voxel. We believe that modular assembly of multimetallic NPs shows promise for imaging analysis of engineered cells and tissues with high resolution and sensitivity.

To date, imaging-guided multimodality therapy is important to improve the accuracy of the diagnosis of renal fibrosis, and nanoplatforms for imaging-guided multimodality diagnosis are gaining more and more attention. There are many limitations and deficiencies in clinical use for early-stage diagnosis of renal fibrosis, and multimodal imaging can contribute more thoroughly and provide in-detail information for effective clinical diagnosis. Melanin is an endogenous biomaterial, and we developed an ultrasmall particle size melanin nanoprobe (MNP-PEG-Mn) based on photoacoustic (PA) and magnetic resonance (MR) dual-modal imaging. MNP-PEG-Mn nanoprobe, with the average diameter about 2.7 nm, can be passively targeted for accumulation in the kidney, and it has excellent free radical scavenging and antioxidant abilities without further exacerbating renal fibrosis. Using the normal group signal as a control, the dual-modal imaging results showed that the MR imaging (MAI) and PA imaging (PAI) signals reached the strongest at 6 h when MNP-PEG-Mn entered the 7 day renal fibrosis group via the left vein of the tail end of the mice; however, the strength of the dual-modal imaging signal and the gradient of signal change were significantly weaker in the 28 day renal fibrosis group than in the 7 day renal fibrosis group and normal group. The phenomenon preliminarily indicates that as a PAI/MRI dual-modality contrast medium candidate, MNP-PEG-Mn has outstanding ability in clinical application potential.

Rationale: Photothermal therapy (PTT) alone is easy to cause cancer recurrence and fail to completely resist metastasis, yet recurrence and metastasis are two major difficulties in cancer treatment. Titanium disulfide (TiS2) nanosheet anchored iron oxide nanoparticles (IO NPs) with strong absorption in the second near-infrared (NIR-II) window and excellent magnetic properties is developed as therapeutic agent for NIR-II photoacoustic (PA) imaging and magnetic resonance (MR) imaging guided NIR-II PTT triggered immunotherapy.Methods: The TiS2 nanosheets were prepared through a modified colloidal chemistry approach, and TSIO nanoagents were prepared by using a one pot self-assembly technique. The magnetic targeting capability of TSIO nanoagents were monitored by NIR-II PA, MR and thermal imaging in vivo. The NIR-II PTT combined with immunotherapy effect was investigated in mouse breast cancer tumor-bearing mice.Results: The TSIO nanoplatform showed enhanced tumor accumulation when a magnetic field was applied and had the ability to real time monitor the treatment process via dual NIR-II PA and MR imaging. In addition, the magnetic targeted NIR-II PA/MR imaging guided PTT provides an effective way to reverse the immunosuppression inside a tumor and to cooperate with immunotherapy to improve therapeutic outcome of the primary, distal and metastatic tumors.

BackgroundTendon injury is a significant clinical problem due to poor healing and a high reinjury rate; successful treatment is limited by our poor understanding of endogenous tendon stem cells. Recent evidence suggests that adult stem cells are phenotypically diverse, even when comparing stem cells isolated from the same tissue from the same individual, and may in fact exist on a spectrum of proliferation and differentiation capacities. Additionally, the relationships between and clinical relevance of this phenotypic variation are poorly understood. In particular, tenogenic capacity has not been studied in comparison to tenogenic differentiation and cell proliferation. Toward this end, we performed a comprehensive assessment of cell proliferation and differentiation capacity toward four connective tissue lineages (tendon, cartilage, bone, and adipose) using tendon stem cell lines derived from single cells released directly from tendon tissue to (1) evaluate the differences, if any, in tenogenic potential, and (2) identify the relationships between differentiation phenotypes and proliferation capacity.MethodsTendon stem cells were derived from the endotenon of superficial digital flexor tendon from 3 horses. The cell suspension from each horse was separately plated simultaneously (1) at moderate density to generate a heterogenous population of cells—parent tendon cell line—and (2) at low density to separate single cells from each other to allow isolation of colonies that derive from single mother cells—clonal tendon stem cell lines.Thirty clonal tendon stem cell lines—10 from each horse—and each parent tendon cell line were assessed for tenogenesis, tri-lineage differentiation, and cell proliferation. Differentiation was confirmed by lineage-specific cell staining and quantified by the relative gene expression of lineage-specific markers. Statistical significance was determined using analysis of variance and post hoc Tukey’s tests.ResultsThree distinct differentiation phenotypes—differentiation potency toward all 4 tissue lineages and two tri-lineage differentiation potencies—were identified in tendon clonal stem cell lines. These phenotypes were differentiation toward (1) tendon, cartilage, bone, and adipose (TCOA); (2) tendon, cartilage, and bone (TCO); and (3) tendon, cartilage, and adipose (TCA). Further, clonal cell lines that differentiated toward all four lineages had the highest expression of scleraxis and mohawk upon tenogenesis. Moreover, cell proliferation was significantly different between phenotypic groups, as evidenced by increased numbers of cumulative cell population doublings in clonal cell lines that did not differentiate toward adipose.ConclusionsOur study provides evidence of the heterogenous character of adult stem cells and identifies key differences in tendon stem cell differentiation and proliferative potentials from the same individual and from the same tendon. Isolation of tendon stem cell lines with the capacity to differentiate into all four connective tissue lineages may yield improved therapeutic benefits in clinical models of repair and promote a native, regenerative phenotype in engineered tendons. Future studies may be targeted to understanding the functional contributions of each tendon stem cell phenotype in vivo and identifying additional cell phenotypes.

<h3>Introduction</h3> Ligament and tendon are prone to degeneration through ageing and injury and current therapies are largely ineffective. The identification of a cell population within tendon with stem cell-like characteristics (Bi, 2007) holds potential for regeneration of tendon and ligament. Tendon stem cells differentiate into tenocytes (Zhang, 2010); the predominant cell type within tendon, responsible for producing extracellular matrix (ECM). The local stem cell environment (niche) is vital for stem cell maintenance and function in many tissues, and tenascin C in particular has been shown to play an important role within stem cell niches (Garcion, 2004). Tendon and ligament are composed of fascicles and interfascicular matrix (IFM) which vary considerably in composition providing definitive niches within the tissue. This study aims to characterise ECM components of the stem cell niche in equine tendon and canine ligament, which are prone to age-related degeneration. The goal of this research is to produce an <i>in vitro</i> environment for stem cells which mimics the stem cell niche, for treatment of tendon and ligament disease. <h3>Methods</h3> Putative stem cells were isolated from equine superficial digital flexor tendon (SDFT) and canine anterior cruciate ligament (ACL) by low-density plating and differential adhesion to plastic and fibronectin substrates. Cells were analysed by flow cytometry using antibodies to mesenchymal stem cell markers CD90, CD73 and CD105, as well as qRT-PCR for stem cell and tenogenic markers. ECM components of the fibroblast and stem cell niche were analysed using radioisotope labelling. Cells were labelled with ¹⁴C-labelled amino acids to specifically label newly synthesised collagenous (proline) and non-collagenous (lysine/arginine) ECM, prior to extraction of ECM. Immuno-histochemistry and histology were conducted to analyse the structure and composition of SDFT. <h3>Results</h3> Tendon and ligament cells formed colonies after low-density plating, however only ligament cells formed colonies after differential adhesion to fibronectin. A subpopulation of tendon cells expressed CD90 in both freshly isolated cells and putative stem cells, but were CD105 and CD73 negative. Putative tendon stem cells, isolated by differential fibronectin adhesion did not exhibit increased expression of stem cell markers when compared with tenocytes. However there was a significant increase in expression of stem cell markers in putative ligament stem cells compared with ligamentocytes. Tenocytes and putative tendon stem cells (isolated by low-density plating) labelled with ¹⁴C-labelled amino acids both displayed similar labelling profiles. Histological analysis of SDFT tissue highlighted the varied structure and composition of tendon, with tenascin C expression confined to IFM (see Figure.1). <h3>Conclusion</h3> The absence of stem cell marker expression in putative stem cell populations indicates that further testing of stem cell isolation procedures is required. Published techniques for tendon stem cell isolation in humans and other mammals do not appear to be effective for isolation of equine tendon stem cells. Alternatively it is possible that the equine tendon cell population consists of a heterogenous mixture of cells at different stages of differentiation. We are currently optimising stem cell isolation techniques and conducting tri-lineage differentiation assays. Tendon stem cells in other species show tri-lineage differentiation, however it is possible that equine tendon stem cells may be restricted to tenogenic differentiation. Future experiments aim to identify ECM components of the stem cell niche by mass spectrometry and microarray comparison of tendon and ligament tissue, stem cells and fibroblasts. <h3>References</h3> Bi <i>et al</i>. Nat Med. 2007;13: 1219–1227 Garcion <i>et al</i>. Development. 131:3423–3432 Zhang <i>et al</i>. Am J Sport Med. 38:2477–2486

Stem cell imaging in vivo is critical to elucidate the homing, distribution, survival, and repair mechanisms and to evaluate the therapeutic effects of engrafted stem cells. Unfortunately, unimodal imaging of stem cells does not simultaneously satisfy all current requirements owing to their intrinsic limitations. Obviously, bimodal or multimodal imaging of stem cells is a promising strategy for circumventing this issue. This study aimed to design and synthesize a novel dual-modal polyethylene glycol-modified magnetic nanoparticle (Fe3+-PEG-MNP) based on natural biomaterials including melanin and Fe ions for photoacoustic (PA) and magnetic resonance (MR) imaging of stem cells in vivo. The Fe3+-PEG-MNPs were characterized and their PA/MR imaging capability and cytotoxicity were evaluated. Bone marrow mesenchymal stem cells (BM-MSCs) labeled with Fe3+-PEG-MNPs were subjected to PA and MR imaging in vitro and in vivo. Consequently, Fe3+-PEG-MNPs displayed many superior properties, including ultra-small particle size, higher stability, water solubility, easy labeling of cells, lower cytotoxicity, high biosafety, excellent capability of PA/MR imaging, high sensitivity and long-term monitoring in vitro and in vivo. In particular, PA and MR signals of labeled BM-MSCs were maintained for at least 35 and 28 d, respectively, in vivo. Therefore, Fe3+-PEG-MNPs are ideal dual-modal PA/MR nanoparticles for non-invasive and effective monitoring of engrafted stem cells in vivo.