Multichannel-optical imaging for in vivo evaluating the safety and therapeutic efficacy of stem cells in tumor model in terms of cell tropism, proliferation and NF-κB activity.

Stem cell-based cancer treatment has garnered significant attention, yet its safety and efficacy remain incompletely understood. The nuclear factor-kappa B (NF-κB) pathway, a critical signaling mechanism involved in tumor growth, angiogenesis, and invasion, serves as an essential metric for evaluating the behavior of stem cells in tumor models. Herein, we report the development of a triple-channel imaging system capable of simultaneously monitoring the tropism of stem cells towards tumors, assessing tumor proliferation, and quantifying tumor NF-κB activity. In this system, we generated a CRISPR-Cas9 gene-edited human glioblastoma cell line, GE-U87-MG, which provided a reliable readout of the proliferation and NF-κB activity of tumors by EF1α-RFLuc- and NF-κB-GLuc-based bioluminescent imaging, respectively. Additionally, near infrared-II emitting Tat-PEG-AgAuSe quantum dots were developed for tracking of stem cell tropism towards tumor. In a representative case involving human mesenchymal stem cells (hMSCs), multichannel imaging revealed no discernible effect of hMSCs on the proliferation and NF-κB activity of GE-U87-MG tumors. Moreover, hMSCs engineered to overexpress the necrosis factor-related apoptosis-inducing ligand were able to inhibit NF-κB activity and growth of GE-U87-MG in vivo. Taken together, our imaging system represents a powerful and feasible approach to evaluating the safety and therapeutic efficacy of stem cells in tumor models.

Recent advances of engineered and artificial drug delivery system towards solid tumor based on immune cells.

Precise drug delivery in cancer treatment is a long-standing concern of modern medicine. Compared with traditional molecular medicines and nano-medicines, emerging cell-based biomimetic delivery strategies display numerous merits, including successive biological functions, innate biocompatibility and superior security since they originate from living organisms, providing a very promising approach. Among them, immune cells receive increasing attention because of their inherent ability in tumor resistance, pathogen elimination, and other significant physiological functions. Herein, we investigated the recent advances on immune cell-based high efficient delivery and therapeutic strategies in solid tumor treatment, mainly focus on T cells, natural killer cells and macrophages, which have been used as drug cargos directly or provided membrane/exosomes as nanoscale drug delivery systems. We also discuss the further potential applications and perspective of this innovative strategy, as well as the predictable challenges in forward exploration in this emerging area.

Linear-branched poly(ß-amino esters)/DNA nano-polyplexes for effective gene transfection and neural stem cell differentiation.

Controllable regulation of stem cell differentiation is a critical concern in stem cell-based regenerative medicine. In particular, there are still great challenges in controlling the directional differentiation of neural stem cells (NSCs) into neurons. Herein, we developed a novel linear-branched poly(ß-amino esters) (S4-TMPTA-BDA-DT, STBD) through a two-step reaction. The synthesized linear-branched polymers possess multiple positively charged amine terminus and degradable intermolecular ester bonds, thus endowing them with excellent properties such as high gene load, efficient gene delivery, and effective gene release and transcription in cells. In the mCherry transfection test, a high transfection efficiency of approximately 70% was achieved in primary NSCs after a single transfection. Moreover, STBD also showed high biocompatibility to NSCs without disturbing their viability and neural differentiation. With the high gene delivery property, STBD is capable of delivering siRNA (shSOX9) expression plasmid into NSCs to significantly interfere with the expression of SOX9, thus enhancing the neuronal differentiation and maturation of NSCs. The STBD/DNA nano-polyplex represents a powerful non-viral approach of gene delivery for manipulating the differentiation of stem cells, showing broad application prospects in NSC-based regenerative therapy for treating neurodegenerative diseases.

Whole-Body Fluorescence Imaging in the Near-Infrared Window.

Fluorescence imaging is one of the most widely used in vivo imaging methods for both fundamental research and clinical practice. Due to the reduced photon scattering, absorption, and autofluorescence in tissues, the emerging near-infrared (NIR) imaging (650-1700 nm) can afford deep tissue imaging with high spatiotemporal resolution and in vivo report the anatomical structures as well as the physiological activities in a whole-body level. Here, we give a brief introduction to fluorescence imaging in the first NIR (NIR-I, 650-950 nm) and second NIR (NIR-II, 1000-1700 nm) windows, summarize the recently developed NIR fluorophores and their applications in whole-body vascular system imaging, precision cancer theranostics, and regenerative medicine. Finally, the clinical applications and future prospects of in vivo NIR fluorescence imaging are also discussed.

A Nanoformulation-Mediated Multifunctional Stem Cell Therapy with Improved Beta-Amyloid Clearance and Neural Regeneration for Alzheimer's Disease.

Alzheimer's disease (AD) is a common dementia that is currently incurable. The existing treatments can only moderately relieve the symptoms of AD to slow down its progress. How to achieve effective neural regeneration to ameliorate cognitive impairments is a major challenge for current AD treatment. Here, the therapeutic potential of a nanoformulation-mediated neural stem cell (NSC) therapy capable of simultaneous Aß clearance and neural regeneration is investigated in a murine model. Genetically engineered NSCs capable of stably and continuously expressing neprilysin (NEP) are developed to enhance Aß degradation and NSC survival in the brain. A PBAE-PLGA-Ag2 S-RA-siSOX9 (PPAR-siSOX9) nanoformulation with high gene/drug deliverability is synthesized to overcome AD microenvironment-associated adverse effects and to promote neuronal differentiation of the NEP-expressing NSCs. For achieving accurate stereotactic transplantation, Ag2 S quantum-dot-based fluorescence imaging is used to guide NSC transplantation in real time. This strategy shows numerous benefits, including efficient and long-lasting Aß degradation, improved neural regeneration, and accurate cell transplantation. It is shown that a single administration of this therapy achieves long-term efficacy (6 months) with respect to memory reversal and improvement of learning deficits.

Rapid Unperturbed-Tissue Analysis for Intraoperative Cancer Diagnosis Using an Enzyme-Activated NIR-II Nanoprobe.

Accurate intraoperative tissue identification is critical to tumor surgery. However, conventional methods are labor- and time-intensive, which greatly delay the intraoperative decision-making. Herein, a matrix metalloproteinase (MMP)14-activated NIR-II nanoprobe (A&MMP@Ag2 S-AF7P) is presented for rapid unperturbed-tissue analysis for ex vivo and in vivo neuroblastoma diagnosis. A&MMP@Ag2 S-AF7P displays negligible fluorescence in normal tissues but is activated quickly by inhibiting the fluorescence resonance energy transfer (FRET) between Ag2 S QDs and A1094 mediated by MMP14 overexpressed in neuroblastoma; meanwhile, the exposure of the membrane penetrating peptide R9 (TAT-peptide) results in efficient internalization of nanoprobes in the cancer cells, providing superior tumor-to-normal (T/N) tissue ratio. Instant illumination of the lesion and well-defined tumor margins make the nanoprobes a suitable rapid diagnostic reagent for cancer surgical or tissue biopsy procedures.

Advanced Near-Infrared Light for Monitoring and Modulating the Spatiotemporal Dynamics of Cell Functions in Living Systems.

Light-based technique, including optical imaging and photoregulation, has become one of the most important tools for both fundamental research and clinical practice, such as cell signal sensing, cancer diagnosis, tissue engineering, drug delivery, visual regulation, neuromodulation, and disease treatment. In particular, low energy near-infrared (NIR, 700-1700 nm) light possesses lower phototoxicity and higher tissue penetration depth in living systems as compared with ultraviolet/visible light, making it a promising tool for in vivo applications. Currently, the NIR light-based imaging and photoregulation strategies have offered a possibility to real-time sense and/or modulate specific cellular events in deep tissues with subcellular accuracy. Herein, the recent progress with respect to NIR light for monitoring and modulating the spatiotemporal dynamics of cell functions in living systems are summarized. In particular, the applications of NIR light-based techniques in cancer theranostics, regenerative medicine, and neuroscience research are systematically introduced and discussed. In addition, the challenges and prospects for NIR light-based cell sensing and regulating techniques are comprehensively discussed.

Tumor Microenvironment-Activated NIR-II Nanotheranostic System for Precise Diagnosis and Treatment of Peritoneal Metastasis.

Activatable theranostic systems show potential for improved tumor diagnosis and therapy owing to high detection specificities, effective ablation, and minimal side-effects. Herein, a tumor microenvironment (TME)-activated NIR-II nanotheranostic system (FEAD1) for precise diagnosis and treatment of peritoneal metastases is presented. FEAD1 was fabricated by self-assembling the peptide Fmoc-His, mercaptopropionic-functionalized Ag2 S quantum dots (MPA-Ag2 S QDs), the chemodrug doxorubicin (DOX), and NIR absorber A1094 into nanoparticles. We show that in healthy tissue, FEAD1 exists in an NIR-II fluorescence "off" state, because of Ag2 S QDs-A1094 interactions, while DOX remains in stealth mode. Upon delivery of FEAD1 to the tumor, the acidic TME triggers its disassembly through breakage of the Fmoc-His metal coordination and DOX hydrophobic interactions. Release of A1094 switches on Ag2 S fluorescence, illuminating the tumor, accompanied by burst release of DOX within the tumor tissue, thereby achieving precise tumor theranostics. This TME-activated theranostic strategy holds great promise for future clinical applications.

NIR-II Fluorescent Self-Assembled Peptide Nanochain for Ultrasensitive Detection of Peritoneal Metastasis.

Fluorescence-guided cytoreductive surgery is one of the most promising approaches for facile elimination of tumors in situ, thereby improving prognosis. Reported herein is a simple strategy to construct a novel chainlike NIR-II nanoprobe (APP-Ag2 S-RGD) by self-assembly of an amphiphilic peptide (APP) into a nanochain with subsequent chemical crosslinking of NIR-II Ag2 S QDs and the tumor-targeting RGD peptide. This probe exhibits higher capability for cancer cell detection compared with that of RGD-functionalized Ag2 S QDs (Ag2 S-RGD) at the same concentration. Upon intraperitoneal injection, superior tumor-to-normal tissue signal ratio is achieved and non-vascularized tiny tumor metastatic foci as small as about 0.2 mm in diameter could be facilely eliminated under NIR-II fluorescent imaging guidance. These results clearly indicate the potential of this probe for fluorescence-guided tumor staging, preoperative diagnosis, and intraoperative navigation.

Programmable Chemotherapy and Immunotherapy against Breast Cancer Guided by Multiplexed Fluorescence Imaging in the Second Near-Infrared Window.

Combined chemotherapy and immunotherapy have demonstrated great potential in cancer treatment. However, it is difficult to provide clear information of the pharmacokinetics and pharmacodynamics of chemodrugs and transplanted immune cells in vivo by traditional approaches, resulting in inadequate therapy. Here, a multiplexed intravital imaging strategy by using fluorescence in the second near-infrared window (NIR-II) is first developed to visualize the two events of chemotherapy and immunotherapy in vivo, so that a combinational administration is programed to improve the therapeutical effects against a mouse model of human breast cancer. In detail, Ag2 Se quantum dots (QDs) (λEm = 1350 nm) loaded with stromal-cell-derived factor-1α (SDF-1α) and chemodrug doxorubicin (DOX) are first administrated to deliver the SDF-1α and DOX to the tumor site. After their arrival, monitored by Ag2 Se QD fluorescence, natural killer (NK)-92 cells labeled with Ag2 S QDs (λEm = 1050 nm) are intravenously injected so that the cells are recruited to the tumor by the chemotaxis of SDF-1α, which is visualized by Ag2 S QD fluorescence. Such an imaging approach allows simultaneous evaluation of the behaviors of individual injections in vivo, and facilitates optimized administration regimens, resulting in enhanced tumor inhibition.

Recent Advances in Tracking the Transplanted Stem Cells Using Near-Infrared Fluorescent Nanoprobes: Turning from the First to the Second Near-Infrared Window.

Stem cell-based regenerative medicine has attracted tremendous attention for its great potential to treat numerous incurable diseases. Tracking and understanding the fate and regenerative capabilities of transplanted stem cells is vital for improving the safety and therapeutic efficacy of stem cell-based therapy, therefore accelerating the clinical application of stem cells. Fluorescent nanoparticles (NPs) have been widely used for in vivo tracking of the transplanted stem cells. Among these fluorescent NPs, near-infrared (NIR) NPs have greatly improved the sensitivity, tissue penetration depth, spatial and temporal resolutions of the fluorescence imaging-based stem cell tracking technologies due to the reduced absorption, scattering, and autofluorescence of NIR fluorescence in tissues. Here, this review summarizes the recent studies regarding the tracking of transplanted stem cells using NIR NPs and emphasizes the recent advances of fluorescence imaging in the second NIR window (NIR-II, 1000-1700 nm). Furthermore, the challenges and future prospects of the NIR NP-based technologies are also discussed.

An Ultra-Robust and Crystalline Redeemable Hydrogen-Bonded Organic Framework for Synergistic Chemo-Photodynamic Therapy.

The low structural stability of hydrogen-bonded organic frameworks (HOFs) is a thorny issue retarding the development of HOFs. A rational design approach is now proposed for construction of a stable HOF. The resultant HOF (PFC-1) exhibits high surface area of 2122 m2 g-1 and excellent chemical stability (intact in concentrated HCl for at least 117 days). A new method of acid-assisted crystalline redemption is used to readily cure the thermal damage to PFC-1. With periodic integration of photoactive pyrene in the robust framework, PFC-1 can efficiently encapsulate Doxorubicin (Doxo) for synergistic chemo-photodynamic therapy, showing comparable therapeutic efficacy with the commercial Doxo yet considerably lower cytotoxicity. This work demonstrates the notorious stability issue of HOFs can be properly addressed through rational design, paving a way to develop robust HOFs and offering promising application perspectives.

Tumor-induced disorder of iron metabolism in major organs: a new insight from chemical speciation of iron.

Objective To investigate the evolution of iron speciation in major organs of tumor-bearing mice and its role in cancer formation and cancer-associated complications. Methods The concentration and chemical speciation of iron in the spleen, liver, lung, kidney, heart, blood, muscle, and tumor tissue of healthy mice and tumor-bearing mice were studied by synchrotron radiation-based total reflection X-ray fluorescence spectrometry (SR-TXRF) coupled with X-ray absorption spectroscopy (XAS). Results The TXRF and XAS results showed that the iron content, especially the ferritin content, significantly decreased in the blood and spleen but significantly increased in the liver, lung, and muscle of mice after tumor implantation. The chemical speciation of iron in the tumor mainly comprised ferrous-sulfide-like iron and ferritin. Conclusion The tumors disturbed the iron metabolism in major organs, and the evolution of iron may be involved in iron deficiency anemia, cancer growth, and immunity. Additionally, iron speciation-based markers may be further developed as clinical indicators for cancer and cancer-associated complications.

Revealing the Fate of Transplanted Stem Cells In Vivo with a Novel Optical Imaging Strategy.

Stem-cell-based regenerative medicine holds great promise in clinical practices. However, the fate of stem cells after transplantation, including the distribution, viability, and the cell clearance, is not fully understood, which is critical to understand the process and the underlying mechanism of regeneration for better therapeutic effects. Herein, we develop a dual-labeling strategy to in situ visualize the fate of transplanted stem cells in vivo by combining the exogenous near-infrared fluorescence imaging in the second window (NIR-II) and endogenous red bioluminescence imaging (BLI). The NIR-II fluorescence of Ag2 S quantum dots is employed to dynamically monitor the trafficking and distribution of all transplanted stem cells in vivo due to its deep tissue penetration and high spatiotemporal resolution, while BLI of red-emitting firefly luciferase (RfLuc) identifies the living stem cells after transplantation in vivo because only the living stem cells express RfLuc. This facile strategy allows for in situ visualization of the dynamic trafficking of stem cells in vivo and the quantitative evaluation of cell translocation and viability with high temporal and spatial resolution, and thus reports the fate of transplanted stem cells and how the living stem cells help, regeneration, for an instance, of a mouse with acute liver failure.

Engineered Multifunctional Nanomedicine for Simultaneous Stereotactic Chemotherapy and Inhibited Osteolysis in an Orthotopic Model of Bone Metastasis.

A novel multifunctional Ag2 S quantum dot (QD)-based nanomedicine of alendronate (Ald)/doxorubicin (DOX)@Ag2 S is developed for highly effective bone tumor therapy in an orthotopic model. The bone-targeting and osteolysis inhibition of Ald and the chemotherapeutic effect of DOX on the bone tumor are in situ visualized by Ag2 S QDs with emission in the second near-infrared window.

Improved osteogenesis and upregulated immunogenicity in human placenta-derived mesenchymal stem cells primed with osteogenic induction medium.

BACKGROUND: Mesenchymal stem cells (MSCs) are widely used in cell-based therapy owing to their multilineage potential and low immunogenicity. However, low differentiation efficiency and unpredictable immunogenicity of allogeneic MSCs in vivo limit their success in therapeutic treatment. Herein, we evaluated the differentiation potential and immunogenicity of human placenta-derived MSCs manipulated with osteogenic priming and dedifferentiation process. METHODS: MSCs from human placentas were subjected to osteogenic induction and then cultivated in osteogenic factor-free media; the obtained cell population was termed dedifferentiated mesenchymal stem cells (De-MSCs). De-MSCs were induced into osteo-, chondro- and adipo-differentiation in vitro. Cell proliferation was quantified by a Cell-Counting Kit-8 or tritiated thymidine ([(3)H]-TdR) incorporation. Meanwhile, the osteogenesis of De-MSCs in vivo was assayed by real-time PCR and histological staining. The expressions of stem cell markers and co-stimulatory molecules on De-MSCs and lymphocytes from primed BALB/c mouse with De-MSCs were determined by flow cytometry. RESULTS: De-MSCs exhibited some properties similar to MSCs including multiple differentiation potential and hypoimmunogenicity. Upon re-osteogenic induction, De-MSCs exhibited higher differentiation capability than MSCs both in vitro and in vivo. Of note, De-MSCs had upregulated immunogenicity in association with their osteogenesis, reflected by the alternated expressions of co-stimulatory molecules on the surface and decreased suppression on T cell activation. Functionally, De-MSC-derived osteoblasts could prime lymphocytes of peripheral blood and spleen in BALB/c mice in vivo. CONCLUSIONS: These data are of great significance for the potential application of De-MSCs as an alternative resource for regenerative medicine and tissue engineering. In order to avoid being rejected by the host during allogeneic De-MSC therapy, we suggest that immune intervention should be considered to boost the immune acceptance and integration because of the upregulated immunogenicity of De-MSCs with redifferentiation in clinical applications.

In vivo real-time visualization of mesenchymal stem cells tropism for cutaneous regeneration using NIR-II fluorescence imaging.

Mesenchymal stem cells (MSCs) have shown great potential for cutaneous wound regeneration in clinical practice. However, the in vivo homing behavior of intravenously transplanted MSCs to the wounds is still poorly understood. In this work, fluorescence imaging with Ag2S quantum dots (QDs) in the second near-infrared (NIR-II) window was performed to visualize the dynamic homing behavior of transplanted human mesenchymal stem cells (hMSCs) to a cutaneous wound in mice. Benefiting from the desirable spatial and temporal resolution of Ag2S QDs-based NIR-II imaging, for the first time, the migration of hMSCs to the wound was dynamically visualized in vivo. By transplanting a blank collagen scaffold in the wound to help the healing, it was found that hMSCs were slowly recruited at the wound after intravenous injection and were predominantly accumulated around the edge of wound. This resulted in poor healing effects in terms of slow wound closure and thin thickness of the regenerated skin. In contrast, for the wound treated by the collagen scaffold loaded with stromal cell derived factor-1α (SDF-1α), more hMSCs were recruited at the wound within a much shorter time and were homogenously distributed across the whole wound area, which enhances the re-epithelialization, the neovascularization, and accelerates the wound healing.

Double-walled Au nanocage/SiO2 nanorattles: integrating SERS imaging, drug delivery and photothermal therapy.

In this work, a novel type of nanomedical platform, the double-walled Au nanocage/SiO(2) nanorattle, is successfully fabricated by combining two "hollow-excavated strategies"--galvanic replacement and "surface-protected etching". The rational design of double-walled nanostructure based on gold nanocages (AuNCs) and hollow SiO(2) shells functionalized respectively with p-aminothiophenol (pATP) and Tat peptide simultaneously renders the nanoplatforms three functionalities: 1) the whole nanorattle serves as a high efficient drug carrier thanks to the structural characteristics of AuNC and SiO(2) shell with hollow interiors and porous walls; 2) the AuNC with large electromagnetic enhancement acts as a sensitive surface-enhanced Raman scattering (SERS) substrate to track the internalization process of the nanorattles by human MCF-7 breast cancer cells, as well as an efficient photothermal transducer for localized hyperthermia cancer therapy due to the strong near-infrared absorption; 3) Tat-functionalized SiO(2) shell not only improves biocompatibility and cell uptake efficiency resulting in enhanced anticancer efficacy but also prevents the AuNCs from aggregation and provides the stability of AuNCs so that the SERS signals can be used for cell tracking in high fidelity. The reported chemistry and the designed nanostructures should inspire more interesting nanostructures and applications.

Fates of Fe3O4 and Fe3O4@SiO2 nanoparticles in human mesenchymal stem cells assessed by synchrotron radiation-based techniques.

Superparamagnetic iron oxide nanoparticles (SPIOs) have been widely used as the magnetic resonance imaging (MRI) contrast agent in biomedical studies and clinical applications, with special interest recently in in vivo stem cell tracking. However, a full understanding of the fate of SPIOs in cells has not been achieved yet, which is particularly important for stem cells since any change of the microenvironment may disturb their propagation and differentiation behaviors. Herein, synchrotron radiation-based X-ray fluorescence (XRF) in combination with X-ray absorption spectroscopy (XAS) were used to in situ reveal the fate of Fe3O4 and Fe3O4@SiO2 NPs in human mesenchymal stem cells (hMSCs), in which the dynamic changes of their distribution and chemical speciation were precisely determined. The XAS analysis evidences that Fe3O4 NPs cultured with hMSCs are quite stable and almost keep their initial chemical form up to 14 days, which is contradictory to the previous report that Fe3O4 NPs were unstable in cell labeling assessed by using a simplified lysosomal model system. Coating with a SiO2 shell, Fe3O4@SiO2 NPs present higher stability in hMSCs without detectable changes of their chemical form. In addition, XRF analysis demonstrates that Fe3O4@SiO2 NPs can label hMSCs in a high efficiency manner and are solely distributed in cytoplasm during cell proliferation, making it an ideal probe for in vivo stem cell tracking. These findings with the help of synchrotron radiation-based XAS and XRF improve our understanding of the fate of SPIOs administered to hMSCs and will help the future design of SPIOs for safe and efficient stem cells tracking.

In vivo real-time visualization of tissue blood flow and angiogenesis using Ag2S quantum dots in the NIR-II window.

Improving the tissue penetration depth and spatial resolution of fluorescence-based optical nanoprobes remains a grand challenge for their practical applications in in vivo imaging, due to the scattering and absorption and endogenous autofluorescence of living tissues. Here, we present that Ag2S quantum dots (QDs), containing no toxic ions, exhibiting long circulation time and high stability, act as a new kind of fluorescent probes in the second near-infrared window (NIR-II, 1000-1350 nm) which enable in vivo monitoring of lymphatic drainage and vascular networks with deep tissue penetration and high spatial and temporal resolution. In addition, NIR-II fluorescence imaging with Ag2S QDs provide ultrahigh spatial resolution (~40 µm) that permits us to track angiogenesis mediated by a tiny tumor (2-3 mm in diameter) in vivo. Our results indicate that Ag2S QDs are promising NIR-II fluorescent nanoprobes that could be useful in surgical treatments such as sentinel lymph node (SLN) dissection as well in assessment of blood supply in tissues and organs and screening of anti-angiogenic drugs.