Induction of immunological tolerance to myelinogenic glial-restricted progenitor allografts.

The immunological barrier currently precludes the clinical utilization of allogeneic stem cells. Although glial-restricted progenitors have become attractive candidates to treat a wide variety of neurological diseases, their survival in immunocompetent recipients is limited. In this study, we adopted a short-term, systemically applicable co-stimulation blockade-based strategy using CTLA4-Ig and anti-CD154 antibodies to modulate T-cell activation in the context of allogeneic glial-restricted progenitor transplantation. We found that co-stimulation blockade successfully prevented rejection of allogeneic glial-restricted progenitors from immunocompetent mouse brains. The long-term engrafted glial-restricted progenitors myelinated dysmyelinated adult mouse brains within one month. Furthermore, we identified a set of plasma miRNAs whose levels specifically correlated to the dynamic changes of immunoreactivity and as such could serve as biomarkers for graft rejection or tolerance. We put forward a successful strategy to induce alloantigen-specific hyporesponsiveness towards stem cells in the CNS, which will foster effective therapeutic application of allogeneic stem cells.

PET imaging of distinct brain uptake of a nanobody and similarly-sized PAMAM dendrimers after intra-arterial administration.

INTRODUCTION: We have recently shown that intracerebral delivery of an anti-VEGF monoclonal antibody bevacizumab using an intra-arterial (IA) infusion is more effective than intravenous administration. While antibodies are quickly emerging as therapeutics, their disadvantages such as large size, production logistics and immunogenicity motivate search for alternatives. Thus we have studied brain uptake of nanobodies and polyamidoamine (PAMAM) dendrimers. METHODS: Nanobodies were conjugated with deferoxamine (DFO) to generate NB(DFO)2. Generation-4 PAMAM dendrimers were conjugated with DFO, and subsequently primary amines were capped with butane-1,2-diol functionalities to generate G4(DFO)3(Bdiol)110. Resulting conjugates were radiolabeled with zirconium-89. Brain uptake of 89ZrNB(DFO)2 and 89ZrG4(DFO)3(Bdiol)110 upon carotid artery vs tail vein infusions with intact BBB or osmotic blood-brain barrier opening (OBBBO) with mannitol in mice was monitored by dynamic positron emission tomography (PET) over 30 min to assess brain uptake and clearance, followed by whole-body PET-CT (computed tomography) imaging at 1 h and 24 h post-infusion (pi). Imaging results were subsequently validated by ex-vivo biodistribution. RESULTS: Intravenous administration of 89ZrNB(DFO)2 and 89ZrG4(DFO)3(Bdiol)110 resulted in their negligible brain accumulation regardless of BBB status and timing of OBBBO. Intra-arterial (IA) administration of 89ZrNB(DFO)2 dramatically increased its brain uptake, which was further potentiated with prior OBBBO. Half of the initial brain uptake was retained after 24 h. In contrast, IA infusion of 89ZrG4(DFO)3(Bdiol)110 resulted in poor initial accumulation in the brain, with complete clearance within 1 h of administration. Ex-vivo biodistribution results reflected those on PET-CT. CONCLUSIONS: IA delivery of nanobodies might be an attractive therapeutic platform for CNS disorders where prolonged intracranial retention is necessary.

Proteolytic Rafts for Improving Intraparenchymal Migration of Minimally Invasively Administered Hydrogel-Embedded Stem Cells.

The physiological spaces (lateral ventricles, intrathecal space) or pathological cavities (stroke lesion, syringomyelia) may serve as an attractive gateway for minimally invasive deployment of stem cells. Embedding stem cells in injectable scaffolds is essential when transplanting into the body cavities as they secure favorable microenvironment and keep cells localized, thereby preventing sedimentation. However, the limited migration of transplanted cells from scaffold to the host tissue is still a major obstacle, which prevents this approach from wider implementation for the rapidly growing field of regenerative medicine. Hyaluronan, a naturally occurring polymer, is frequently used as a basis of injectable scaffolds. We hypothesized that supplementation of hyaluronan with activated proteolytic enzymes could be a viable approach for dissolving the connective tissue barrier on the interface between the scaffold and the host, such as pia mater or scar tissue, thus demarcating lesion cavity. In a proof-of-concept study, we have found that collagenase and trypsin immobilized in hyaluronan-based hydrogel retain 60% and 28% of their proteolytic activity compared to their non-immobilized forms, respectively. We have also shown that immobilized enzymes do not have a negative effect on the viability of stem cells (glial progenitors and mesenchymal stem cells) in vitro. In conclusion, proteolytic rafts composed of hyaluronan-based hydrogels and immobilized enzymes may be an attractive strategy to facilitate migration of stem cells from injectable scaffolds into the parenchyma of surrounding tissue.

Endoscopic treatment of intraductal pancreatic stent fragmentation.

BACKGROUND AND AIM: Complications of endoscopic treatment are reported more and more often as a result of popularization of pancreatic endotherapy. Our study presents the results of treatment in patients with intraductal pancreatic stent fragmentation diagnosed during endotherapy of chronic pancreatitis. METHODS: Retrospective analysis of 2496 endoscopic retrograde cholangiopancreatography procedures which were carried out in 607 patients at the Gastrointestinal Endoscopy Unit of the University Clinical Center in Gdansk. RESULTS: In the course of pancreatic endotherapy, intraductal pancreatic stent fragmentation was stated during 33 of 2496 (0.013%) procedures in 33 of 607 (5.44%) patients with chronic pancreatitis. In 33 patients, there were 46 intraductal fragments of broken stents. Most patients were asymptomatic. In 31/33 patients, fragments of broken stents were removed from the pancreatic duct endoscopically. In the case of two patients, endoscopic management was ineffective and they were treated surgically. Altogether, 44/46 stent fragments were removed endoscopically. Most fragments of pancreatic stents were removed during the first endoscopic procedure. One fragment of a broken stent was retrieved with polypectomy snare and four with Dormia basket. The remaining fragments of broken pancreatic stents were removed with rat-tooth forceps. CONCLUSIONS: Intraductal fragmentation of pancreatic stent is a rare complication of pancreatic endotherapy and it often has an asymptomatic course. Most fragments of broken pancreatic stents can be removed endoscopically from the pancreatic duct with an acceptable complication rate.

Imaging the DNA Alkylator Melphalan by CEST MRI: An Advanced Approach to Theranostics.

Brain tumors are among the most lethal types of tumors. Therapeutic response variability and failure in patients have been attributed to several factors, including inadequate drug delivery to tumors due to the blood-brain barrier (BBB). Consequently, drug delivery strategies are being developed for the local and targeted delivery of drugs to brain tumors. These drug delivery strategies could benefit from new approaches to monitor the delivery of drugs to tumors. Here, we evaluated the feasibility of imaging 4-[bis(2-chloroethyl)amino]-l-phenylalanine (melphalan), a clinically used DNA alkylating agent, using chemical exchange saturation transfer magnetic resonance imaging (CEST MRI), for theranostic applications. We evaluated the physicochemical parameters that affect melphalan's CEST contrast and demonstrated the feasibility of imaging the unmodified drug by saturating its exchangeable amine protons. Melphalan generated a CEST signal despite its reactivity in an aqueous milieu. The maximum CEST signal was observed at pH 6.2. This CEST contrast trend was then used to monitor therapeutic responses to melphalan in vitro. Upon cell death, the decrease in cellular pH from ∼7.4 to ∼6.4 caused an amplification of the melphalan CEST signal. This is contrary to what has been reported for other CEST contrast agents used for imaging cell death, where a decrease in the cellular pH following cell death results in a decrease in the CEST signal. Ultimately, this method could be used to noninvasively monitor melphalan delivery to brain tumors and also to validate therapeutic responses to melphalan clinically.

The role of endoscopic ultrasonography in endoscopic debridement of walled-off pancreatic necrosis--A single center experience.

BACKGROUND: In recent years the significance of endoscopic ultrasonography (EUS) has increased. In our center we have also performed EUS-guided cystostomy since 2011. Earlier we had performed this procedure without EUS. Introduction of EUS in our hospital has inspired us to perform a retrospective analysis of the influence of EUS use on the risk of complications and the effectiveness of endoscopic treatment of patients with WOPN. METHODS: Between years 2001 and 2013 176 patients with symptomatic WOPN underwent endoscopic treatment in the Department of Gastroenterology and Hepatology of Medical University of Gdansk. The results and complications of treatment in relation to the use of EUS during drainage/debridement were compared retrospectively. RESULTS: 64 patients underwent EUS-guided drainage/debridement of WOPN (group 1). In the case of 112 patients endoscopic drainage/debridement was performed without EUS control (group 2). In group 1 therapeutic success was achieved in 60/64 (93.75%) patients in comparison to 104/112 (92.9%) patients in group 2 (P = 0.870). The mean duration of drainage/debridement in both groups was 25 days - 4-173 days in group 1 and 4-112 days in group 2 (P = 0.519). The complications of endotherapy occurred in 9/64 (14.06%) patients from group 1 in comparison to 29/112 (25.9%) patients from group 2 (P = 0.047). CONCLUSIONS: In our study the use of EUS during endoscopic drainage/debridement of WOPN significantly reduced the number of procedure-related complications, mainly gastrointestinal bleeding. However, it had no influence on the duration of treatment or the effectiveness of therapy.

Asteroid hyalosis--current state of knowledge.

The search query into the Cochrane Library, Medline, Web of Science, Embase, Scopus and ScienceDirect enabled selection of research papers addressing the issue of asteroid hyalosis published in English between 1963 and January 2014. Asteroid hyalosis is a degenerative condition of the vitreous in which small, creamy or white, spherical particles (asteroid bodies) are randomly diffused within the vitreous. They consist mainly of calcium and phosphorus and have a structure of hydroxy lapatite. In 80.2-92.0% of cases the condition affects one eye only and it occurs in 0.36-1.96% of population, mostly in patients over 50 years of age and in males. Hypercholesterolemia and hypertension are systemic risk factors, but asteroid hyalosis is postulated to occur more often in retinitis pigmentosa and Leber amaurosis caused by mutations in lecithin retinol acyltransferase gene. Asteroid hyalosis also causes calcification of some intraocular lenses--mostly silicone ones. Vitreous of patients with asteroid hyalosis shows reduced gel liquefaction and anomalous vitreoretinal adhesion.

Adaptation and validation of the Columbia-Suicide Severity Rating Scale (C-SSRS) - screen version. / Polska adaptacja i walidacja wersji przesiewowej Skali Oceny Ryzyka Samobójstwa z Uniwersytetu Columbia (C-SSRS).

OBJECTIVES: To perform an adaptation and psychometric validation of the Polish version of the Columbia-Suicide Severity Rating Scale (C-SSRS) screen version in a clinical sample of patients admitted to the psychiatric hospital. METHODS: This was a single-center, observational and cross-sectional study. A total of 318 consecutive patients completed a set of questionnaires upon their admission to acute psychiatric units. The set comprised C-SSRS screener and the reference measures: the Suicidal Behaviors Questionnaire - Revised (SBQ-R), the Suicidal Ideation Attributes Scale (SIDAS), the Center of Epidemiological Studies Depression Scale - Revised (CESD-R), the Scale of Psychache, the Purpose in Life scale (PIL), and alcohol misuse screen test (CAGE). RESULTS: Cronbach's α of the C-SSRS was 0.89. Two latent components were identified in the factor analysis: (1) suicidal thoughts, intentions and plans, and (2) history of suicidal attempts. There were differences in the mean scores of all the utilized questionnaires (namely, SBQ-R, the Psychache scale, CAGE, SIDAS, PIL and CESD-R) between the C-SSRS risk groups (p=0.01). The C-SSRS risk group was associated with the category of the primary psychiatric diagnosis (p<0.001). CONCLUSIONS: The Polish version of the Columbia-Suicide Severity Rating Scale screener is a questionnaire with good psychometric features to assess the suicidal risk among psychiatric in-patients. It can be used for the purposes of a routine assessment of suicidal risk among hospitalized patients.

Multimodal, PSMA-Targeted, PAMAM Dendrimer-Drug Conjugates for Treatment of Prostate Cancer: Preclinical Evaluation.

Introduction: Prostate cancer (PC) is the second most common cancer and the fifth most frequent cause of cancer death among men. Prostate-specific membrane antigen (PSMA) expression is associated with aggressive PC, with expression in over 90% of patients with metastatic disease. Those characteristics have led to its use for PC diagnosis and therapies with radiopharmaceuticals, antibody-drug conjugates, and nanoparticles. Despite these advancements, none of the current therapeutics are curative and show some degree of toxicity. Here we present the synthesis and preclinical evaluation of a multimodal, PSMA-targeted dendrimer-drug conjugate (PT-DDC), synthesized using poly(amidoamine) (PAMAM) dendrimers. PT-DDC was designed to enable imaging of drug delivery, providing valuable insights to understand and enhance therapeutic response. Methods: The PT-DDC was synthesized through consecutive conjugation of generation-4 PAMAM dendrimers with maytansinoid-1 (DM1) a highly potent antimitotic agent, Cy5 infrared dye for optical imaging, 2,2',2"-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA) chelator for radiolabeling with copper-64 and positron emission tomography tomography/computed tomography (PET/CT), lysine-urea-glutamate (KEU) PSMA-targeting moiety and the remaining terminal primary amines were capped with butane-1,2-diol. Non-targeted control dendrimer-drug conjugate (Ctrl-DDC) was formulated without conjugation of KEU. PT-DDC and Ctrl-DDC were characterized using high-performance liquid chromatography, matrix assisted laser desorption ionization mass spectrometry and dynamic light scattering. In vitro and in vivo evaluation of PT-DDC and Ctrl-DDC were carried out in isogenic human prostate cancer PSMA+ PC3 PIP and PSMA- PC3 flu cell lines, and in mice bearing the corresponding xenografts. Results: PT-DDC was stable in 1×PBS and human blood plasma and required glutathione for DM1 release. Optical, PET/CT and biodistribution studies confirmed the in vivo PSMA-specificity of PT-DDC. PT-DDC demonstrated dose-dependent accumulation and cytotoxicity in PSMA+ PC3 PIP cells, and also showed growth inhibition of the corresponding tumors. PT-DDC did not accumulate in PSMA- PC3 flu tumors and did not inhibit their growth. Ctrl-DDC did not show PSMA specificity. Conclusion: In this study, we synthesized a multimodal theranostic agent capable of delivering DM1 and a radionuclide to PSMA+ tumors. This approach holds promise for enhancing image-guided treatment of aggressive, metastatic subtypes of prostate cancer.

Unlocking the Potential of Ultra-High Dose Fractionated Radiation for Effective Treatment of Glioblastoma in Mice.

Background: Current radiotherapy regimens for glioblastoma (GBM) have limited efficacy and fails to eradicate tumors. Regenerative medicine brings hope for repairing damaged tissue, opening opportunities for elevating the maximum acceptable radiation dose. In this study, we explored the effect of ultra-high dose fractionated radiation on tumor responses and brain injury in immunocompetent mice which can better mimic the tumor-host interactions observed in patients. We also evaluated the role of the hypoxia-inducible factor-1 alpha under radiation as potential target for combating radiation-induced brain injury. Methods: Naïve and Hif-1α+/- heterozygous mice received a fractionated daily dose of 20 Gy for three or five consecutive days. Magnetic resonance imaging (MRI) and histology were performed to assess brain injury post-radiation. The 2×105 human GBM1 luciferase-expressing cells were transplanted with tolerance induction protocol. Fractionated radiotherapy was performed during the exponential phase of tumor growth. Bioluminescence imaging, MRI, and immunohistochemistry staining were performed to evaluate tumor growth dynamics and radiotherapy responses. Additionally, animal lifespan was recorded. Results: Fractionated radiation of 5×20 Gy induced severe brain damage, starting 3 weeks after radiation. All animals from this group died within 12 weeks. In contrast, later onset and less severe brain injury were observed starting 12 weeks after radiation of 3×20 Gy. It resulted in complete GBM eradication and survival of all treated animals. Furthermore, Hif-1α+/- mice exhibited more severe vascular damage after fractionated radiation of 3×20 Gy. Conclusion: Ultra-high dose fractionated 3×20 Gy radiation has the potential to fully eradicate GBM cells at the cost of only mild brain injury. The Hif-1α gene is a promising target for ameliorating vascular impairment post-radiation, encouraging the implementation of neurorestorative strategies.

Ischemic brain injury: a consortium analysis of key factors involved in mesenchymal stem cell-mediated inflammatory reduction.

Increasing global birth rate, coupled with the aging population surviving into their eighth decade has lead to increased incidence diseases, hitherto designated as rare. Brain related ischemia, at birth, or later in life, during, for example stroke, is increasing in global prevalence. Reactive microglia can contribute to neuronal damage as well as compromising transplantion. One potential treatment strategy is cellular therapy, using mesenchymal stem cells (hMSCs), which possess immunomodulatory and cell repair properties. For effective clinical therapy, mechanisms of action must be understood better. Here multicentre international laboratories assessed this question together investigating application of hMSCs neural involvement, with interest in the role of reactive microglia. Modulation by hMSCs in our in vivo and in vitro study shows they decrease markers of microglial activation (lower ED1 and Iba) and astrogliosis (lower GFAP) following transplantation in an ouabain-induced brain ischemia rat model and in organotypic hippocampal cultures. The anti-inflammatory effect in vitro was demonstrated to be CD200 ligand dependent with ligand expression shown to be increased by IL-4 stimulation. hMSC transplant reduced rat microglial STAT3 gene expression and reduced activation of Y705 phosphorylated STAT3, but STAT3 in the hMSCs themselves was elevated upon grafting. Surprisingly, activity was dependent on heterodimerisation with STAT1 activated by IL-4 and Oncostatin M. Our study paves the way to preclinical stages of a clinical trial with hMSC, and suggests a non-canonical JAK-STAT signaling of unphosphorylated STAT3 in immunomodulatory effects of hMSCs.

3D printing-based frugal manufacturing of glass pipettes for minimally invasive delivery of therapeutics to the brain.

Objective: Intracerebral delivery of agents in liquid form is usually achieved through commercially available and durable metal needles. However, their size and texture may contribute to mechanical brain damage. Glass pipettes with a thin tip may significantly reduce injection-associated brain damage but require access to prohibitively expensive programmable pipette pullers. This study is to remove the economic barrier to the application of minimally invasive delivery of therapeutics to the brain, such as chemical compounds, viral vectors, and cells. Methods: We took advantage of the rapid development of free educational online resources and emerging low-cost 3D printers by designing an affordable pipette puller (APP) to remove the cost obstacle. Results: We showed that our APP could produce glass pipettes with a sharp tip opening down to 20 µm or less, which is sufficiently thin for the delivery of therapeutics into the brain. A pipeline from pipette pulling to brain injection using low-cost and open-source equipment was established to facilitate the application of the APP. Conclusion: In the spirit of frugal science, our device may democratize glass pipette-puling and substantially promote the application of minimally invasive and precisely controlled delivery of therapeutics to the brain for finding more effective therapies of brain diseases.

Unlocking the potential of ultra-high dose fractionated radiation for effective treatment of glioblastoma.

Background: Conventional radiation therapy for glioblastoma (GBM) has limited efficacy. Regenerative medicine brings hope for repairing damaged tissue, opening opportunities for elevating the maximum acceptable radiation dose. In this study, we explored the effect of ultra-high dose fractionated radiation on brain injury and tumor responses in immunocompetent mice. We also evaluated the role of the HIF-1α under radiation. Methods: Naïve and hypoxia-inducible factor-1 alpha (HIF-1α)+/- heterozygous mice received a fractionated daily dose of 20 Gy for three or five consecutive days. Magnetic resonance imaging (MRI) and histology were performed to assess brain injury post-radiation. The 2×105 human GBM1 luciferase-expressing cells were transplanted with tolerance induction protocol. Fractionated radiotherapy was performed during the exponential phase of tumor growth. BLI, MRI, and immunohistochemistry staining were performed to evaluate tumor growth dynamics and radiotherapy responses. Additionally, animal lifespan was recorded. Results: Fractionated radiation of 5×20 Gy induced severe brain damage, starting 3 weeks after radiation. All animals from this group died within 12 weeks. In contrast, later onset and less severe brain injury were observed starting 12 weeks after radiation of 3×20 Gy. It resulted in complete GBM eradication and survival of all treated animals. Furthermore, HIF-1α+/- mice exhibited more obvious vascular damage 63 weeks after fractionated radiation of 3×20 Gy. Conclusion: Ultra-high dose fractionated 3×20 Gy radiation can eradicate the GBM cells at the cost of only mild brain injury. The HIF-1α gene is a promising target for ameliorating vascular impairment post-radiation, encouraging the implementation of neurorestorative strategies.

Routine brush cytology and fluorescence in situ hybridization for assessment of pancreatobiliary strictures.

BACKGROUND: The specificity of brush cytology for detection of malignant pancreatobiliary strictures is high, but its sensitivity is moderate. Fluorescence in situ hybridization (FISH) can be used to detect chromosomal aneuploidy in biliary brushing specimens, and, according to some reports, it may improve the sensitivity of routine cytology. OBJECTIVE: To assess the role of routine cytology and FISH in detection of malignant pancreatobiliary strictures. DESIGN: Prospective study performed between September 2008 and August 2010. SETTING: University hospital. PATIENTS: This study involved 81 patients with bile duct or pancreatic duct strictures. INTERVENTION: Brush cytology obtained during ERCP from pancreatic duct or bile duct strictures and analysis of smears by routine cytology and FISH. MAIN OUTCOME MEASUREMENTS: Sensitivity, specificity, and positive and negative predictive values of routine cytology and FISH calculated with a 95% confidence interval. RESULTS: The sensitivity of routine cytology was 35.19%, and specificity was 100%. When atypia was identified as positive, the resultant sensitivity was 53.7%, and specificity was 100%. Sensitivity of FISH was 51.85%, and specificity was 88.89%. When either routine cytology was positive or atypia was observed or when the FISH result was positive, sensitivity was the highest (72.22%), and it was statistically significant in comparison with both routine cytology with atypia (P < .036) and FISH (P < .023), but specificity was lower than that of routine cytology (88.89% vs 100%). LIMITATIONS: Use of a DNA probe set that was designed for detection of urothelial carcinoma. Limited number of patients. CONCLUSION: FISH improved the sensitivity of routine cytology. Pancreatic duct brushings were a reliable material for detection of chromosomal abnormalities by FISH. The best diagnostic result was achieved by combining routine cytology with FISH.

Hyperosmolar blood-brain barrier opening using intra-arterial injection of hyperosmotic mannitol in mice under real-time MRI guidance.

The blood-brain barrier (BBB) is the main obstacle to the effective delivery of therapeutic agents to the brain, compromising treatment efficacy for a variety of neurological disorders. Intra-arterial (IA) injection of hyperosmotic mannitol has been used to permeabilize the BBB and improve parenchymal entry of therapeutic agents following IA delivery in preclinical and clinical studies. However, the reproducibility of IA BBB manipulation is low and therapeutic outcomes are variable. We demonstrated that this variability could be highly reduced or eliminated when the procedure of osmotic BBB opening is performed under the guidance of interventional MRI. Studies have reported the utility and applicability of this technique in several species. Here we describe a protocol to open the BBB by IA injection of hyperosmotic mannitol under the guidance of MRI in mice. The procedures (from preoperative preparation to postoperative care) can be completed within ~1.5 h, and the skill level required is on par with the induction of middle cerebral artery occlusion in small animals. This MRI-guided BBB opening technique in mice can be utilized to study the biology of the BBB and improve the delivery of various therapeutic agents to the brain.

The Role of Fecal Microbiota Transplantation in the Treatment of Inflammatory Bowel Disease.

The exact pathogenesis of inflammatory bowel disease (IBD) is still not completely understood. It is hypothesized that a genetic predisposition leads to an exaggerated immune response to an environmental trigger, leading to uncontrolled inflammation. As there is no known causative treatment, current management strategies for inflammatory bowel disease focus on correcting the excessive immune response to environmental (including microbial) triggers. In recent years, there has been growing interest in new avenues of treatment, including targeting the microbial environment itself. Fecal microbiota transplantation (FMT) is a novel treatment modality showing promising results in early studies. The article discusses the rationale for the use of FMT in inflammatory bowel disease and the yet-unresolved questions surrounding its optimal use in practice.

Mesenchymal Stem Cells Do Not Lose Direct Labels Including Iron Oxide Nanoparticles and DFO-89Zr Chelates through Secretion of Extracellular Vesicles.

Rapidly ageing populations are beset by tissue wear and damage. Stem cell-based regenerative medicine is considered a solution. Years of research point to two important aspects: (1) the use of cellular imaging to achieve sufficient precision of therapeutic intervention, and the fact that (2) many therapeutic actions are executed through extracellular vesicles (EV), released by stem cells. Therefore, there is an urgent need to interrogate cellular labels in the context of EV release. We studied clinically applicable cellular labels: superparamagnetic iron oxide nanoparticles (SPION), and radionuclide detectable by two main imaging modalities: MRI and PET. We have demonstrated effective stem cell labeling using both labels. Then, we obtained EVs from cell cultures and tested for the presence of cellular labels. We did not find either magnetic or radioactive labels in EVs. Therefore, we report that stem cells do not lose labels in released EVs, which indicates the reliability of stem cell magnetic and radioactive labeling, and that there is no interference of labels with EV content. In conclusion, we observed that direct cellular labeling seems to be an attractive approach to monitoring stem cell delivery, and that, importantly, labels neither locate in EVs nor affect their basic properties.

Imaging as a tool to accelerate the translation of extracellular vesicle-based therapies for central nervous system diseases.

Extracellular vesicles (EVs) are natural and diverse lipid bilayer-enclosed particles originating from various cellular components and containing an abundance of cargoes. Due to their unique properties, EVs have gained considerable interest as therapeutic agents for a variety of diseases, including central nervous system (CNS) disorders. Their therapeutic value depends on cell origin but can be further enhanced by enrichment of cargo when used as drug carriers. Therefore, there has been significant effort directed toward introducing them to clinical practice. However, it is essential to avoid the failures we have seen with whole-cell therapy, in particular for the treatment of the CNS. Successful launching of clinical studies is contingent upon the understanding of the biodistribution of EVs, including their uptake and clearance from organs and specific homing into the region of interest. A multitude of noninvasive imaging methods has been explored in vitro to investigate the spatio-temporal dynamics of EVs administered in vivo. However, only a few studies have been performed to track the delivery of EVs, especially delivery to the brain, which is the most therapeutically challenging organ. We focus here on the use of advanced imaging techniques as an essential tool to facilitate the acceleration of clinical translation of EV-based therapeutics, especially in the CNS arena. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Diagnostic Tools > in vivo Nanodiagnostics and Imaging.

Dual contrast agents for fluorescence and photoacoustic imaging: evaluation in a murine model of prostate cancer.

Prostate-specific membrane antigen (PSMA) is a promising diagnostic and therapeutic target for prostate cancer (PC). Poly(amidoamine) [PAMAM] dendrimers serve as versatile scaffolds for imaging agents and drug delivery that can be tailored to different sizes and compositions depending upon the application. We have developed PSMA-targeted PAMAM dendrimers for real-time detection of PC using fluorescence (FL) and photoacoustic (PA) imaging. A generation-4, ethylenediamine core, amine-terminated dendrimer was consecutively conjugated with on average 10 lysine-glutamate-urea PSMA targeting moieties and a different number of sulfo-cyanine7.5 (Cy7.5) near-infrared dyes (2, 4, 6 and 8 denoted as conjugates II, III, IV and V, respectively). The remaining terminal primary amines were capped with butane-1,2-diol functionalities. We also prepared a conjugate composed of Cy7.5-lysine-suberic acid-lysine glutamate-urea (I) and control dendrimer conjugate (VI). Among all conjugates, IV showed superior in vivo target specificity in male NOD-SCID mice bearing isogenic PSMA+ PC3 PIP and PSMA- PC3 flu xenografts and suitable physicochemical properties for FL and PA imaging. Such agents may prove useful in PC cancer detection and subsequent surgical guidance during excision of PSMA-expressing lesions.