Antibody Delivery into the Brain by Radiosensitizer Nanoparticles for Targeted Glioblastoma Therapy.

Background: Glioblastoma is the most lethal primary brain malignancy in adults. Standard of care treatment, consisting of temozolomide (TMZ) and adjuvant radiotherapy (RT), mostly does not prevent local recurrence. The inability of drugs to enter the brain, in particular antibody-based drugs and radiosensitizers, is a crucial limitation to effective glioblastoma therapy. Methods: Here, we developed a combined strategy using radiosensitizer gold nanoparticles coated with insulin to cross the blood-brain barrier and shuttle tumor-targeting antibodies (cetuximab) into the brain. Results: Following intravenous injection to an orthotopic glioblastoma mouse model, the nanoparticles specifically accumulated within the tumor. Combining targeted nanoparticle injection with TMZ and RT standard of care significantly inhibited tumor growth and extended survival, as compared to standard of care alone. Histological analysis of tumors showed that the combined treatment eradicated tumor cells, and decreased tumor vascularization, proliferation, and repair. Conclusions: Our findings demonstrate radiosensitizer nanoparticles that effectively deliver antibodies into the brain, target the tumor, and effectively improve standard of care treatment outcome in glioblastoma.

Multifunctional nanoprobe for real-time in vivo monitoring of T cell activation.

Genetically engineered T cells are a powerful new modality for cancer immunotherapy. However, their clinical application for solid tumors is challenging, and crucial knowledge on cell functionality in vivo is lacking. Here, we fabricated a nanoprobe composed of dendrimers incorporating a calcium sensor and gold nanoparticles, for dual-modal monitoring of engineered T cells within a solid tumor. T cells engineered to express a melanoma-specific T-cell receptor and loaded with the nanoprobe were longitudinally monitored within melanoma xenografts in mice. Fluorescent imaging of the nanoprobe's calcium sensor revealed increased intra-tumoral activation of the T cells over time, up to 24 h. Computed tomography imaging of the nanoprobe's gold nanoparticles revealed the cells' intra-tumoral distribution pattern. Quantitative analysis revealed the intra-tumoral T cell quantities. Thus, this nanoprobe reveals intra-tumoral persistence, penetration and functional status of genetically engineered T cells, which can advance T cell-based immunotherapy and promote next-generation live cell imaging.

'Golden' exosomes as delivery vehicles to target tumors and overcome intratumoral barriers: in vivo tracking in a model for head and neck cancer.

Exosomes are promising vectors for anti-tumor therapy, due to their biocompatibility, low immunogenicity, and innate ability to interact with target cells. However, promoting clinical application of exosome-based therapeutics requires elucidation of key issues, including exosome biodistribution, tumor targeting and accumulation, and the ability to overcome tumor barriers that limit the penetration of various nano-carriers and drugs. Here, we examined these parameters in exosomes derived from mesenchymal stem cells (MSC-exo) and from the A431 squamous cell carcinoma line (A431-exo), which both have potential use in cancer therapy. Using our novel technique combining gold nanoparticle (GNP) labeling of exosomes and non-invasive computed tomography imaging (CT), we longitudinally and quantitatively tracked the two intravenously-injected exosome types in A431 tumor-bearing mice. CT imaging up to 48 h and subsequent ex vivo analysis revealed tumor homing abilities of both exosome types, yet there was significantly higher tumor accumulation of MSC-exo as compared to A431-exo. Moreover, MSC-exo demonstrated the ability to penetrate the tumor and distribute throughout its bulk, while non-encapsulated GNPs remained concentrated at the tumor periphery. Histological analysis showed penetration of MSC-exo not only into the tumor tissue, but also into tumor cell cytoplasm. While the proportion of biodistribution between organs at 48 h was similar for both exosome types, more rapid clearance was indicated for A431-exo. Thus, our findings demonstrate an effect of exosome type on tumor targeting abilities and biodistribution, and suggest that MSC-exo may have superior abilities for tumor-targeted therapy.

Glucose-Functionalized Liposomes for Reducing False Positives in Cancer Diagnosis.

Fluorodeoxyglucose-positron emission tomography (18F-FDG-PET) is a powerful tool for cancer detection, staging, and follow-up. However, 18F-FDG-PET imaging has high rates of false positives, as it cannot distinguish between tumor and inflammation regions that both feature increased glucose metabolic activity. In the present study, we engineered liposomes coated with glucose and the chelator dodecane tetraacetic acid (DOTA) complexed with copper, to serve as a diagnostic technology for differentiating between cancer and inflammation. This liposome technology is based on FDA-approved materials and enables complexation with metal cations and radionuclides. We found that these liposomes were preferentially uptaken by cancer cell lines with high metabolic activity, mediated via glucose transporter-1. In vivo, these liposomes were avidly uptaken by tumors, as compared to liposomes without glucose coating. Moreover, in a combined tumor-inflammation mouse model, these liposomes accumulated in the tumor tissue and not in the inflammation region. Thus, this technology shows high specificity for tumors while evading inflammation and has potential for rapid translation to the clinic and integration with existing PET imaging systems, for effective reduction of false positives in cancer diagnosis.

Nanoparticle contrast agents for X-ray imaging applications.

X-ray imaging is the most widely used diagnostic imaging method in modern medicine and several advanced forms of this technology have recently emerged. Iodinated molecules and barium sulfate suspensions are clinically approved X-ray contrast agents and are widely used. However, these existing contrast agents provide limited information, are suboptimal for new X-ray imaging techniques and are developing safety concerns. Thus, over the past 15 years, there has been a rapid growth in the development of nanoparticles as X-ray contrast agents. Nanoparticles have several desirable features such as high contrast payloads, the potential for long circulation times, and tunable physicochemical properties. Nanoparticles have also been used in a range of biomedical applications such as disease treatment, targeted imaging, and cell tracking. In this review, we discuss the principles behind X-ray contrast generation and introduce new types of X-ray imaging modalities, as well as potential elements and chemical compositions that are suitable for novel contrast agent development. We focus on the progress in nanoparticle X-ray contrast agents developed to be renally clearable, long circulating, theranostic, targeted, or for cell tracking. We feature agents that are used in conjunction with the newly developed multi-energy computed tomography and mammographic imaging technologies. Finally, we offer perspectives on current limitations and emerging research topics as well as expectations for the future development of the field. This article is categorized under: Diagnostic Tools > in vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Multifunctional Dendrimer-Entrapped Gold Nanoparticles for Labeling and Tracking T Cells Via Dual-Modal Computed Tomography and Fluorescence Imaging.

Nanosystems for monitoring and tracking T cells provide an important basis for evaluating the functionality and efficacy of T cell-based immunotherapy. To this end, we designed herein an efficient nanoprobe for T cell monitoring and tracking using poly(amidoamine) (PAMAM) dendrimer-entrapped gold nanoparticles (Au DENPs) conjugated with Fluo-4 for dual-mode computed tomography (CT) and fluorescence imaging. In this study, PAMAM dendrimers of generation 5 (G5) were modified with hydroxyl-terminated polyethylene glycol (PEG) and then used to entrap 2.0 nm Au NPs followed by acetylation of the excess amine groups on the dendrimer surface. Subsequently, the calcium ion probe was covalently attached to the dendrimer nanohybrids through the PEG hydroxyl end groups to gain the functional {(Au0)25-G5.NHAc-(PEG)14-(Fluo-4)2} nanoprobe. This nanoprobe had excellent water solubility, high X-ray attenuation coefficient, and good cytocompatibility in the given concentration range, as well as a high T cell labeling efficiency. Confocal microscopy and flow cytometry results demonstrated that the nanoprobe was able to fluorescently sense activated T cells. Moreover, the nanoprobe was able to realize both CT and fluorescence imaging of subcutaneously injected T cells in vivo. Thus, the developed novel dendrimer-based nanosystem may hold great promise for advancing and improving the clinical application of T cell-based immunotherapy.

Intranasal Delivery of Mesenchymal Stem Cell Derived Exosomes Loaded with Phosphatase and Tensin Homolog siRNA Repairs Complete Spinal Cord Injury.

Individuals with spinal cord injury (SCI) usually suffer from permanent neurological deficits, while spontaneous recovery and therapeutic efficacy are limited. Here, we demonstrate that when given intranasally, exosomes derived from mesenchymal stem cells (MSC-Exo) could pass the blood brain barrier and migrate to the injured spinal cord area. Furthermore, MSC-Exo loaded with phosphatase and tensin homolog small interfering RNA (ExoPTEN) could attenuate the expression of PTEN in the injured spinal cord region following intranasal administrations. In addition, the loaded MSC-Exo considerably enhanced axonal growth and neovascularization, while reducing microgliosis and astrogliosis. The intranasal ExoPTEN therapy could also partly improve structural and electrophysiological function and, most importantly, significantly elicited functional recovery in rats with complete SCI. The results imply that intranasal ExoPTEN may be used clinically to promote recovery for SCI individuals.

Ultrasmall Oxygen-Deficient Bimetallic Oxide MnWOX Nanoparticles for Depletion of Endogenous GSH and Enhanced Sonodynamic Cancer Therapy.

Sonodynamic therapy (SDT) triggered by ultrasound (US) has attracted increasing attention owing to its abilities to overcome critical limitations including low tissue-penetration depth and phototoxicity in photodynamic therapy. Herein, the design of a new type of sonosensitizer is revealed, namely, ultrasmall oxygen-deficient bimetallic oxide MnWOX nanoparticles, for multimodal imaging-guided enhanced SDT against cancer. As-made MnWOX nanoparticles with poly(ethylene glycol) (PEG) modification show high physiological stability and biocompatibility. Interestingly, such MnWOX -PEG nanoparticles exhibit highly efficient US-triggered production of 1 O2 and •OH, higher than that of previously reported sonosensitizers (e.g., protoporphyrin IX and titanium dioxide), because the oxygen-deficient structure of MnWOX serves as an electron trap site to prevent electron-hole recombination. The glutathione depletion capability of MnWOX -PEG can also further favor SDT-triggered cancer cell killing. With efficient tumor homing as illustrated by computer tomography and magnetic resonance imaging, MnWOX -PEG enables effective destruction of mouse tumors under US stimulation. After accomplishing its therapeutic functions, MnWOX -PEG can be metabolized by the mouse body without any long-term toxicity. Herein, a new type of sono-sensitizing agent with high SDT efficacy, multimodal imaging functions, and rapid clearance is presented, an agent which is promising for noninvasive SDT cancer treatment.

Golden Exosomes Selectively Target Brain Pathologies in Neurodegenerative and Neurodevelopmental Disorders.

Exosomes, nanovesicles that are secreted by different cell types, enable intercellular communication at local or distant sites. Alhough they have been found to cross the blood brain barrier, their migration and homing abilities within the brain remain unstudied. We have recently developed a method for longitudinal and quantitative in vivo neuroimaging of exosomes based on the superior visualization abilities of classical X-ray computed tomography (CT), combined with gold nanoparticles as labeling agents. Here, we used this technique to track the migration and homing patterns of intranasally administrated exosomes derived from bone marrow mesenchymal stem cells (MSC-exo) in different brain pathologies, including stroke, autism, Parkinson's disease, and Alzheimer's disease. We found that MSC-exo specifically targeted and accumulated in pathologically relevant murine models brains regions up to 96 h post administration, while in healthy controls they showed a diffuse migration pattern and clearance by 24 h. The neuro-inflammatory signal in pathological brains was highly correlated with MSC-exo accumulation, suggesting that the homing mechanism is inflammatory-driven. In addition, MSC-exo were selectively uptaken by neuronal cells, but not glial cells, in the pathological regions. Taken together, these findings can significantly promote the application of exosomes for therapy and targeted drug delivery in various brain pathologies.

Can molecular profiling enhance radiotherapy? Impact of personalized targeted gold nanoparticles on radiosensitivity and imaging of adenoid cystic carcinoma.

Personalized molecular profiling has an established role in selection of treatment for metastatic disease; however, its role in improving radiosensitivity and functional imaging has not been evaluated. In the current study, we examined molecular profiling as a tool for designing personalized targeted gold nanoparticles (GNP) to serve as dual-modal tumor radiosensitizers and functional imaging enhancers. To this end, molecular profiling of a patient's salivary gland adenoid cystic carcinoma (ACC) was performed, and anaplastic lymphoma kinase (ALK) mutation was detected. The extracted tumor was subcutaneously injected into mice, which were then treated either with radiation, the specific ALK inhibitor crizotinib, or a combination of therapies. One of these combinations, namely, ALK-targeted GNP (via crizotinib coating), was found to enhance radiation treatment, as demonstrated by a significant decrease in tumor volume over 24 days. In parallel, ALK-targeted GNP substantially augmented tumor visualization via computed tomography. The mechanism of radiosensitivity enhancement was mostly related to a diminished cell repair mechanism in tumors, as demonstrated by proliferating cell nuclear antigen staining. These findings indicate that personalized molecular profiling is an effective technique for enhancing cancer theranostics.

Therapeutic Effect of Astroglia-like Mesenchymal Stem Cells Expressing Glutamate Transporter in a Genetic Rat Model of Depression.

Recent studies have proposed that abnormal glutamatergic neurotransmission and glial pathology play an important role in the etiology and manifestation of depression. It was postulated that restoration of normal glutamatergic transmission, by enhancing glutamate uptake, may have a beneficial effect on depression. We examined this hypothesis using unique human glial-like mesenchymal stem cells (MSCs), which in addition to inherent properties of migration to regions of injury and secretion of neurotrophic factors, were differentiated to express high levels of functional glutamate transporters (excitatory amino acid transporters; EAAT). Additionally, gold nanoparticles (GNPs), which serve as contrast agents for CT imaging, were loaded into the cells for non-invasive, real-time imaging and tracking of MSC migration and final location within the brain. MSC-EAAT (2×105; 10 µl) were administered (i.c.v.) to Flinder Sensitive Line rats (FSLs), a genetic model for depression, and longitudinal behavioral and molecular changes were monitored. FSL rats treated with MSC-EAAT showed attenuated depressive-like behaviors (measured by the forced swim test, novelty exploration test and sucrose self-administration paradigm), as compared to controls. CT imaging, Flame Atomic Absorption Spectroscopy analysis and immunohistochemistry showed that the majority of MSCs homed specifically to the dentate gyrus of the hippocampus, a region showing structural brain changes in depression, including loss of glial cells. mRNA and protein levels of EAAT1 and BDNF were significantly elevated in the hippocampus of MSC-EAAT-treated FSLs. Our findings indicate that MSC-EAATs effectively improve depressive-like manifestations, possibly in part by increasing both glutamate uptake and neurotropic factor secretion in the hippocampus.

Differentiating Between Cancer and Inflammation: A Metabolic-Based Method for Functional Computed Tomography Imaging.

One of the main limitations of the highly used cancer imaging technique, PET-CT, is its inability to distinguish between cancerous lesions and post treatment inflammatory conditions. The reason for this lack of specificity is that [(18)F]FDG-PET is based on increased glucose metabolic activity, which characterizes both cancerous tissues and inflammatory cells. To overcome this limitation, we developed a nanoparticle-based approach, utilizing glucose-functionalized gold nanoparticles (GF-GNPs) as a metabolically targeted CT contrast agent. Our approach demonstrates specific tumor targeting and has successfully distinguished between cancer and inflammatory processes in a combined tumor-inflammation mouse model, due to dissimilarities in angiogenesis occurring under different pathologic conditions. This study provides a set of capabilities in cancer detection, staging and follow-up, and can be applicable to a wide range of cancers that exhibit high metabolic activity.

In-vitro Optimization of Nanoparticle-Cell Labeling Protocols for In-vivo Cell Tracking Applications.

Recent advances in theranostic nanomedicine can promote stem cell and immune cell-based therapy. Gold nanoparticles (GNPs) have been shown to be promising agents for in-vivo cell-tracking in cell-based therapy applications. Yet a crucial challenge is to develop a reliable protocol for cell upload with, on the one hand, sufficient nanoparticles to achieve maximum visibility of cells, while on the other hand, assuring minimal effect of particles on cell function and viability. Previous studies have demonstrated that the physicochemical parameters of GNPs have a critical impact on their efficient uptake by cells. In the current study we have examined possible variations in GNP uptake, resulting from different incubation period and concentrations in different cell-lines. We have found that GNPs effectively labeled three different cell-lines - stem, immune and cancer cells, with minimal impairment to cell viability and functionality. We further found that uptake efficiency of GNPs into cells stabilized after a short period of time, while GNP concentration had a significant impact on cellular uptake, revealing cell-dependent differences. Our results suggest that while heeding the slight variations within cell lines, modifying the loading time and concentration of GNPs, can promote cell visibility in various nanoparticle-dependent in-vivo cell tracking and imaging applications.

A challenge for theranostics: is the optimal particle for therapy also optimal for diagnostics?

Theranostics is defined as the combination of therapeutic and diagnostic capabilities in the same agent. Nanotechnology is emerging as an efficient platform for theranostics, since nanoparticle-based contrast agents are powerful tools for enhancing in vivo imaging, while therapeutic nanoparticles may overcome several limitations of conventional drug delivery systems. Theranostic nanoparticles have drawn particular interest in cancer treatment, as they offer significant advantages over both common imaging contrast agents and chemotherapeutic drugs. However, the development of platforms for theranostic applications raises critical questions; is the optimal particle for therapy also the optimal particle for diagnostics? Are the specific characteristics needed to optimize diagnostic imaging parallel to those required for treatment applications? This issue is examined in the present study, by investigating the effect of the gold nanoparticle (GNP) size on tumor uptake and tumor imaging. A series of anti-epidermal growth factor receptor conjugated GNPs of different sizes (diameter range: 20-120 nm) was synthesized, and then their uptake by human squamous cell carcinoma head and neck cancer cells, in vitro and in vivo, as well as their tumor visualization capabilities were evaluated using CT. The results showed that the size of the nanoparticle plays an instrumental role in determining its potential activity in vivo. Interestingly, we found that although the highest tumor uptake was obtained with 20 nm C225-GNPs, the highest contrast enhancement in the tumor was obtained with 50 nm C225-GNPs, thus leading to the conclusion that the optimal particle size for drug delivery is not necessarily optimal for imaging. These findings stress the importance of the investigation and design of optimal nanoparticles for theranostic applications.

Nanomedicine for Cancer Immunotherapy: Tracking Cancer-Specific T-Cells in Vivo with Gold Nanoparticles and CT Imaging.

Application of immune cell-based therapy in routine clinical practice is challenging due to the poorly understood mechanisms underlying success or failure of treatment. Development of accurate and quantitative imaging techniques for noninvasive cell tracking can provide essential knowledge for elucidating these mechanisms. We designed a novel method for longitudinal and quantitative in vivo cell tracking, based on the superior visualization abilities of classical X-ray computed tomography (CT), combined with state-of-the-art nanotechnology. Herein, T-cells were transduced to express a melanoma-specific T-cell receptor and then labeled with gold nanoparticles (GNPs) as a CT contrast agent. The GNP-labeled T-cells were injected intravenously to mice bearing human melanoma xenografts, and whole-body CT imaging allowed examination of the distribution, migration, and kinetics of T-cells. Using CT, we found that transduced T-cells accumulated at the tumor site, as opposed to nontransduced cells. Labeling with gold nanoparticles did not affect T-cell function, as demonstrated both in vitro, by cytokine release and proliferation assays, and in vivo, as tumor regression was observed. Moreover, to validate the accuracy and reliability of the proposed cell tracking technique, T-cells were labeled both with green fluorescent protein for fluorescence imaging, and with GNPs for CT imaging. A remarkable correlation in signal intensity at the tumor site was observed between the two imaging modalities, at all time points examined, providing evidence for the accuracy of our CT cell tracking abilities. This new method for cell tracking with CT offers a valuable tool for research, and more importantly for clinical applications, to study the fate of immune cells in cancer immunotherapy.

Nanoparticle-based CT imaging technique for longitudinal and quantitative stem cell tracking within the brain: application in neuropsychiatric disorders.

A critical problem in the development and implementation of stem cell-based therapy is the lack of reliable, noninvasive means to image and trace the cells post-transplantation and evaluate their biodistribution, final fate, and functionality. In this study, we developed a gold nanoparticle-based CT imaging technique for longitudinal mesenchymal stem cell (MSC) tracking within the brain. We applied this technique for noninvasive monitoring of MSCs transplanted in a rat model for depression. Our research reveals that cell therapy is a potential approach for treating neuropsychiatric disorders. Our results, which demonstrate that cell migration could be detected as early as 24 h and up to one month post-transplantation, revealed that MSCs specifically navigated and homed to distinct depression-related brain regions. We further developed a noninvasive quantitative CT ruler, which can be used to determine the number of cells residing in a specific brain region, without tissue destruction or animal scarification. This technique may have a transformative effect on cellular therapy, both for basic research and clinical applications.