Biodistribution of Exosomes and Engineering Strategies for Targeted Delivery of Therapeutic Exosomes

Exosomes are cell-secreted nano-sized vesicles which deliver diverse biological molecules for intercellular communication. Due to their therapeutic potential, exosomes have been engineered in numerous ways for efficient delivery of active pharmaceutical ingredients to various target organs, tissues, and cells. In vivo administered exosomes are normally delivered to the liver, spleen, kidney, lung, and gastrointestinal tract and show rapid clearance from the blood circulation after systemic injection. The biodistribution and pharmacokinetics (PK) of exosomes can be modulated by engineering various factors such as cellular origin and membrane protein composition of exosomes. Recent advances accentuate the potential of targeted delivery of engineered exosomes even to the most challenging organs including the central nervous system. Major breakthroughs have been made related to various imaging techniques for monitoring in vivo biodistribution and PK of exosomes, as well as exosomal surface engineering technologies for inducing targetability. For inducing targeted delivery, therapeutic exosomes can be engineered to express various targeting moieties via direct modification methods such as chemically modifying exosomal surfaces with covalenton-covalent bonds, or via indirect modification methods by genetically engineering exosome-producing cells. In this review, we describe the current knowledge of biodistribution and PK of exosomes, factors determining the targetability and organotropism of exosomes, and imaging technologies to monitor in vivo administered exosomes. In addition, we highlight recent advances in strategies for inducing targeted delivery of exosomes to specific organs and cells.

Biodistribution of Exosomes and Engineering Strategies for Targeted Delivery of Therapeutic Exosomes

Exosomes are cell-secreted nano-sized vesicles which deliver diverse biological molecules for intercellular communication. Due to their therapeutic potential, exosomes have been engineered in numerous ways for efficient delivery of active pharmaceutical ingredients to various target organs, tissues, and cells. In vivo administered exosomes are normally delivered to the liver, spleen, kidney, lung, and gastrointestinal tract and show rapid clearance from the blood circulation after systemic injection. The biodistribution and pharmacokinetics (PK) of exosomes can be modulated by engineering various factors such as cellular origin and membrane protein composition of exosomes. Recent advances accentuate the potential of targeted delivery of engineered exosomes even to the most challenging organs including the central nervous system. Major breakthroughs have been made related to various imaging techniques for monitoring in vivo biodistribution and PK of exosomes, as well as exosomal surface engineering technologies for inducing targetability. For inducing targeted delivery, therapeutic exosomes can be engineered to express various targeting moieties via direct modification methods such as chemically modifying exosomal surfaces with covalenton-covalent bonds, or via indirect modification methods by genetically engineering exosome-producing cells. In this review, we describe the current knowledge of biodistribution and PK of exosomes, factors determining the targetability and organotropism of exosomes, and imaging technologies to monitor in vivo administered exosomes. In addition, we highlight recent advances in strategies for inducing targeted delivery of exosomes to specific organs and cells.

Extracellular vesicles in kidneys and their clinical potential in renal diseases

Extracellular vesicles (EVs), such as exosomes and microvesicles, are cell-derived lipid bilayer membrane particles, which deliver information from host cells to recipient cells. EVs are involved in various biological processes including the modulation of the immune response, cell-to-cell communications, thrombosis, and tissue regeneration. Different types of kidney cells are known to release EVs under physiologic as well as pathologic conditions, and recent studies have found that EVs have a pathophysiologic role in different renal diseases. Given the recent advancement in EV isolation and analysis techniques, many studies have shown the diagnostic and therapeutic potential of EVs in various renal diseases, such as acute kidney injury, polycystic kidney disease, chronic kidney disease, kidney transplantation, and renal cell carcinoma. This review updates recent clinical and experimental findings on the role of EVs in renal diseases and highlights the potential clinical applicability of EVs as novel diagnostics and therapeutics.

Extracellular vesicles in kidneys and their clinical potential in renal diseases

Extracellular vesicles (EVs), such as exosomes and microvesicles, are cell-derived lipid bilayer membrane particles, which deliver information from host cells to recipient cells. EVs are involved in various biological processes including the modulation of the immune response, cell-to-cell communications, thrombosis, and tissue regeneration. Different types of kidney cells are known to release EVs under physiologic as well as pathologic conditions, and recent studies have found that EVs have a pathophysiologic role in different renal diseases. Given the recent advancement in EV isolation and analysis techniques, many studies have shown the diagnostic and therapeutic potential of EVs in various renal diseases, such as acute kidney injury, polycystic kidney disease, chronic kidney disease, kidney transplantation, and renal cell carcinoma. This review updates recent clinical and experimental findings on the role of EVs in renal diseases and highlights the potential clinical applicability of EVs as novel diagnostics and therapeutics.

Erratum: A Method for Generating Mouse Model of Stroke: Evaluation of Parameters for Blood Flow, Behavior, and Survival

We correct a typo in the title.

A Method for Generate a Mouse Model of Stroke: Evaluation of Parameters for Blood Flow, Behavior, and Survival

Stroke is one of the common causes of death and disability. Despite extensive efforts in stroke research, therapeutic options for improving the functional recovery remain limited in clinical practice. Experimental stroke models using genetically modified mice could aid in unraveling the complex pathophysiology triggered by ischemic brain injury. Here, we optimized the procedure for generating mouse stroke model using an intraluminal suture in the middle cerebral artery and verified the blockage of blood flow using indocyanine green coupled with near infra-red radiation. The first week after the ischemic injury was critical for survivability. The survival rate of 11% in mice without any treatment but increased to 60% on administering prophylactic antibiotics. During this period, mice showed severe functional impairment but recovered spontaneously starting from the second week onward. Among the various behavioral tests, the pole tests and neurological severity score tests remained reliable up to 4 weeks after ischemia, whereas the rotarod and corner tests became less sensitive for assessing the severity of ischemic injury with time. Further, loss of body weight was also observed for up 4 weeks after ischemia induction. In conclusion, we have developed an improved approach which allows us to investigate the role of the cell death-related genes in the disease progression using genetically modified mice and to evaluate the modes of action of candidate drugs.

Constitutive Expression of MAP Kinase Phosphatase-1 Confers Multi-drug Resistance in Human Glioblastoma Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Current treatment of glioblastoma after surgery consists of a combination of fractionated radiotherapy and temozolomide. However, it is difficult to completely remove glioblastoma because it has uncertain boundaries with surrounding tissues. Moreover, combination therapy is not always successful because glioblastoma has diverse resistances. To overcome these limitations, we examined the combined effects of chemotherapy and knockdown of mitogen-activated protein kinase phosphatase-1 (MKP-1). MATERIALS AND METHODS: We used ten different anti-cancer drugs (cisplatin, cyclophosphoamide, doxorubicin, epirubicin, etoposide, 5-fluorouracil, gemcitabine, irinotecan, mitomycin C, and vincristine) to treat glioblastoma multiforme (GBM) cells. Knockdown of MKP-1 was performed using siRNA and lipofectamine. The basal level of MKP-1 in GBM was analyzed based on cDNA microarray data obtained from the Gene Expression Omnibus (GEO) databases. RESULTS: Anti-cancer drug-induced cell death was significantly enhanced by knockdown of MKP-1, and this effect was most prominent in cells treated with irinotecan and etoposide. Treatment with these two drugs led to significantly increased phosphorylation of c-Jun N-terminal kinase (JNK) in a time-dependent manner, while pharmacological inhibition of JNK partially inhibited drug-induced cell death. Knockdown of MKP-1 also enhanced drug-induced phosphorylation of JNK. CONCLUSION: Increased MKP-1 expression levels could be the cause of the high resistance to conventional chemotherapeutics in human GBM. Therefore, MKP-1 is an attractive target for overcoming drug resistance in this highly refractory malignancy.

Proapoptotic Ginsenosides Compound K and Rh2 Enhance Fas-induced Cell Death of Human Astrocytoma Cells Through Distinct Apoptotic Signaling Pathways / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Malignant astrocytomas are among the commonest primary brain tumors and they have a grave prognosis, and so there is an urgent need to develop effective treatment. In this study, we investigated the molecular mechanisms that are responsible for the anti-tumor effect of ginsenosides on human astrocytoma cells. MATERIALS AND METHODS: We tested 13 different ginsenosides for their anti-tumor effect on human malignant astrocytoma cells in conjunction with Fas stimulation. In addition, the cell signaling pathways were explored by using pharmacological inhibitors and performing immunoblot analysis. DCF-DA staining and antioxidant experiments were performed to investigate the role of reactive oxygen species as one of the apoptosis-inducing mechanisms. RESULTS: Among the 13 different ginsenoside metabolites, compound K and Rh2 induced apoptotic cell death of the astrocytoma cells in a caspase- and p38 MAPK-dependent manner, yet the same treatment had no cytotoxic effect on the primary cultured human astrocytes. Combined treatment with ginsenosides and Fas ligand showed a synergistic cytotoxic effect, which was mediated by the reduction of intracellular reactive oxygen species. CONCLUSION: These results suggest that ginsenoside metabolites in combination with Fas ligand may provide a new strategy to treat malignant astrocytomas, which are tumors that are quite resistant to conventional anti-cancer treatment.

Multiplex Analysis of Cytokines in the Serum and Cerebrospinal Fluid of Patients With Alzheimer's Disease by Color-Coded Bead Technology

Background and purpose: The availability and promise of effective treatments for neurodegenerative disorders are increasing the importance of early diagnosis. Having molecular and biochemical markers of Alzheimer's disease (AD) would complement clinical approaches, and further the goals of early and accurate diagnosis. Combining multiple biomarkers in evaluations significantly increases the sensitivity and specificity of the biochemical tests. Methods: In this study, we used color-coded bead-based Luminex technology to test the potential of using chemokines and cytokines as biochemical markers of AD. We measured the levels of 22 chemokines and cytokines in the serum and cerebrospinal fluid (CSF) of 32 de novo patients (13 controls, 11 AD, and 8 Parkinson's disease [PD]). Results: MCP-1 was the only cytokine detectable in CSF, and its levels did not differ between control and disease groups. However, the serum concentration of eotaxin was significantly higher in AD patients than in the control group. Conclusions: The analysis of multiple inflammatory mediators revealed marginal differences in their CSF and serum concentrations for the differential diagnosis of AD and PD. These results provide evidence that immunological responses are not major contributors to the pathogenesis of AD and PD.

The Role of the Ubiquitin-Proteasome Pathway in Neurodegenerative Disorders

Impaired function of the Ubiquitin (Ub)/proteasome pathway is one of the molecular mechanisms underlying aging process and neurodegenerative disorders such as Parkinson's Disease and Alzheimer's Disease (AD). Among many vital cellular functions, the Ub/proteasome pathway regulates immune responses via mediating activation of NF-kappa B by pro-inflammatory signals. Dysfunction of this pathway may aberrantly affect the signaling of pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), which are abundantly present in AD brains. To address this, chemokine expression was measured as a readout for IL-1beta and TNF-alpha signaling in human astrocytes. Proteasome inhibitors, MG-132 and lactacystin, suppressed IL-1beta and TNF-alpha-induced expression of MCP-1, RANTES and IP-10, but not that of IL-8. In addition, human astrocytes underwent apoptotic cell death upon treatment with IL-1beta and TNF-alpha only in the presence of the proteasome inhibitors. These results suggest that inhibition of the Ub/proteasome pathway dysregulates pro-inflammatory cytokine signaling in human astrocytes, leading to divergent chemokine expression and enhanced cell death. Therefore, we propose that the immuno-pathologic role of astrocytes in AD brains should be re-evaluated under the circumstances of impaired function of the Ub/proteasome pathway.

Application of Proteomics and Protein Chip Analysis in the Diagnosis of Neurodegenerative Disorders

As we face an increase of the adult population suffering from dementia, a typical senile disorder, it is imperative to develop appropriate tools for early detection and differential diagnosis of dementia. Recently, proteomics techniques have been proposed to be used for diagnosis of neurodegenerative disorders by identifying numerous biological markers that are known to increase or decrease in the cerebrospinal fluid or serum of dementic patients. Protein chip analysis, one of the most important techniques of proteomics, is suggested to be useful for examining various modifications of proteins as the high throughput screening method using small volumes of precious samples in a short period. We describe here a list of biological markers, such as A beta, APP, tau, ubiquitin, S100B, soluble IL-6 receptor, beta 2 micoglobulin and prostaglandin E2, proposing that these molecules can be used as biochemical markers of dementia. Therefore, we suggest that the proteomic approaches to analyze the amount and modifications of these proteins might be powerful tools for early detection and differential diagnosis of various neurodegenerative disorders as well as assessment of disease progress.

Diffusion-weighted image and MR spectroscopic analysis of a case of MELAS with repeated attacks

We report the clinical and MR manifestations of an 18 year-old girl with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. Recurrent status epilepticus caused reversible cytotoxic edema on diffusion-weighted images (DWI). Initial and one month follow-up MR spectroscopy, after seizure control, showed some discrepancies in the ratio of metabolites. N-acetylaspartate (NAA) partially recovered (NAA/creatine (Cr) ratio: 1.27-->1.84). This was because of a normalization of decreased NAA due to cellular dysfunction as a result of status epilepticus. A low ratio of NAA/Cr due to abnormal mitochondria remained in the decreased state. Reversible NAA/Cr ratios in the acute lesion suggested that NAA reflects the neuronal function as well as the level of neuronal structural damage. The altered NAA/Cr ratio better correlated with the abnormal signal intensity area of T2-weighted images (T2WI) and DWI than the lactate (Lac)/Cr ratio. With conservative treatment with anti-epileptics not accompanied by coenzyme Q or sodium dichloroacetate, lactate persistently increased (Lac/Cr ratio: 1.01-->1.21) because of the continued production of lactate in cells with respiratory deficiency, which is the main pathology of MELAS.

Transient improvement of pyruvate metabolism after coenzyme Q therapy in Kearns-Sayre syndrome: MRS study

Coenzyme Q therapy has been used to support metabolic derangements in patients with mitochondrial encephalomyopathies. Biochemical analysis of the living human brain can be performed by magnetic resonance spectroscopy (MRS). We report upon a KSS patient who was serially imaged with localized proton MRS to monitor the efficacy of CoQ treatment. A 17-year-old girl with KSS was serially imaged with localized proton MRS performed on a GE 1.5 T SIGNA MRI/MRS system. The elevated lactate contents of lesions decreased after one month of CoQ therapy but were re-elevated 10 months after treatment. We conclude that MRS presents us with a powerful tool for monitoring the effects of therapeutic trials in mitochondrial encephalomyopathies.

Effects of Cycloheximide and Dexamethasone on Fas - Mediated Apopthsis in Primary Human Astrocytes / 대한면역학회지

Astrocytes are major glial cells in central nervous system (CNS) and are known to express death receptors or ligands that can induce apoptosis of astrocytes or other brain cells. We have previously confirmed that cultured human astrocytes express fas and fas ligand and their expression may be regulated by various cytokines found in CNS. Because fas can rnediate cell death known as apoptosis, we investigated fas-mediated cell death in cultured human astrocytes and evaluated factors that may influence the fas-mediated apoptosis in astrocytes. Pretreatment of interferon-r and TNF-a increased cell death in astrocytes. Cell death induced by fas ligation was confirmed as apoptosis by phosphatidylserine translocation in cell membrane. Cycloheximide, protein synthesis inhibitor, potentiated fas-mediated cell death. However, buthionine sulfoxine did not potentiate fas-mediated apoptosis. Dexamethasone blocked cell death in dose-dependent and time-dependent manners. These findings collectively show that fas expressed on cultured human fetal astrocytes can induce apoptotic cell death after pretreatment of interferon-r and/or TNF-a. Therefore, the fas-fas ligand system in CNS may regulate the glial degeneration and may participate the neuronal loss in certain conditions. Furthermore, fas-mediated apoptosis of astrocytes can be potentiated by protein synthesis inhibitors and can be blocked by dexamethasone.

Analysis of the T Lymphocyte Subsets in the Peripheral Blood of Patients with Myasthenia Gravis

It is known that the activated peripheral T lymphocytes are increased in patients with autoimmune disorders such as systemic lupus erythematosis, rheumatoid arthritis, multiple sclerosis and autoimmune thyroiditis, but not in the patients with myasthenia gravis on previous studies. We investigated the subsets of T cells in peripheral blood of the patients with myasthenia gravis using flow cytometric analysis. Forty-three patients of myasthenia gravis who were not on steroid or other immunosuppressants were chosen, and thirty-six age-matched healthy persons were evaluated as controls. The peripheral blood mononuclear cells from patients and controls were stained with FITC or PE-conjugated monoclonal antibodies to several surface molecules expressed on T cells within 6 hours after collection. The samples were analysed by flow cytometry within 24 hours. In patients with myasthenia gravis, the expressions of DR and CD25 molecules on the T lymphocytes were increased significantly compared to those of the control group. The expression of CD25 was increased on CD4+ T cells, but not on CD8+ T cells . The expression of DR molecule was increased on CD8+ T cells, but not on CD4+ T cells. Therefore we suggest that the activated T cells are increased in myasthenia gravis and CD25 surface markers on CD4+ T cells may be a more sensitive indicator of immune status.

Expression of interleukin-2 receptor in primary cultured adult human astrocytes

BACKGROUND: Astrocytes are the major glial cells involved with the chemical homeostasis and mechanical supports of central nervous system. Recently, astrocytes were found to actively synthesize and secrete many immunologically active cytokines and express receptors for these mediators, which proposed their autocrine and paracrine roles in the pathogenesis of many neurodegenerative or autoimmune diseases, I.e., multiple sclerosis, Alzheimer's disease, etc. The identification of various chemical mediators secreted by astrocytes and receptors expressed on astrocytes seems to be crucial for understanding their pathogenetic roles in these diseases. Our investigation was conducted to test the expression of interleukin-2 receptor alpha Subunit (IL-2Ralpha) on primary cultured astrocytes, which has not been studied yet. METHODS: Astrocytes were obtained from the surgical specimen of a patient with intractable temporal lobe epilepsy. Neuropathological examination of the specimen revealed hippocampal sclerosis only with normal lateral temporal neocortex from which cultured astrocytes were obtained. All experiments were performed within 2 months after starting primary culture. Cultured astrocytes were incubated with IL-1, IL-2, IL-6, TGF-beta And TNF-alpha For 24 hours. RT-PCR was performed to investigate the transcription of IL-2R. RESULTS: RT-PCRs for the IL-2Ralpha Showed constitutional expression on the adult cultured astrocytes, which was increased by IL-1, IL-2, IL-6 and TNF-alpha But decreased by treatment of TGF-beta. CONCLUSIONS: Adult astrocytes expressed IL-2Ralpha Constitutively, which were upregulated by IL-1, IL-2, IL-6 and TNF-alpha. These findings suggest the immunocompetence of astrocytes, which may be important in the pathogenesis of many neurological diseases.

MRI follow-up study and 1H-MR spectroscopic finding in a patient with Kearns-Sayre syndrome

Serial Brain MRI was performed on a seventeen-year-old girl with Kearns-Sayre syndrome. At the age of 11, she complained bilateral ptosis. Two years later, bilateral blepharoplasty was done and brain MRI was taken. T2-weighted MRI sequence showed high signal intensity areas in the brainstem, thalamus and white matter of the cerebrum and cerebellum bilaterally. Four years later, chronic progressive external ophthalmoplegia developed and serial MRI and proton MRS were taken. Follow-up MRI showed similar but slightly progressed findings compared with previous films. The proton MR spectroscopic imaging demonstrated focal localization of abnormally increased lactate content in the involved area of the brain.