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Detection of apoptosis in a rat model of focal cerebral ischemia using a homing peptide selected from in vivo phage display
Abstract
Focal cerebral ischemia, known as stroke, is caused by a sudden interruption in the blood supply to the brain. We attempted to identify peptides that can home to ischemic stroke tissue and detect the apoptosis of cells. A phage library displaying random peptides was screened for homing peptides to ischemic stroke tissue in a rat transient middle cerebral artery (MCA) occlusion model. After three rounds of in vivo screening, a phage clone displaying the most frequently occurring CLEVSRKNC sequence was selected. The CLEVSRKNC-phage preferentially homed to ischemic stroke tissue after intravenous administration into the MCA occlusion rats. The fluorescein-labeled synthetic CLEVSRKNC peptide, but not a scrambled control peptide, homed to ischemic stroke tissue with a lack of homing to non-ischemic brain tissue. The CLEVSRKNC peptide co-localized with a portion of neuronal cells, rather than with astrocytes, undergoing apoptosis at the penumbra region of stroke lesions. In autoradiographic studies, the uptake of the (131)I-labeled CLEVSRKNC peptide into an ischemic lesion increased at the first day and peaked at the third day after the injury. These results demonstrate that the CLEVSRKNC peptide can home to ischemic stroke tissue, while detecting apoptotic neuronal cells, and suggest it has applications as a targeting moiety for molecular imaging and selective drug delivery to stroke tissue.
... Annexin V and its derivatives have been labeled with radionuclides (such as [ 99m Tc] and [ 18 F]) and fluorescence dyes and have been used in several studies. Yagle et al. 11 radiolabeled recombinant human annexin V with N-succinimidyl-4-18 F-fluorobenzoic acid 18 F-SFB and evaluated in an animal model of apoptosis. They could quantify cell death in vivo. ...
... 14 In recent years, some peptides have been introduced through phage display technologies for the detection of apoptosis. [15][16][17][18][19] One of these peptides is LIKKPF (Leu-Ile-Lys-Lys-Pro-Phe) that has shown high affinity for PS in ELISA using phage display (K d = 2.5 nM), but has low sensitivity as an imaging agent. 16 In the authors' previous studies, they synthesized and radiolabeled LIKKPF with 18 F-FDG and [ 99m Tc]. ...
... [15][16][17][18][19] One of these peptides is LIKKPF (Leu-Ile-Lys-Lys-Pro-Phe) that has shown high affinity for PS in ELISA using phage display (K d = 2.5 nM), but has low sensitivity as an imaging agent. 16 In the authors' previous studies, they synthesized and radiolabeled LIKKPF with 18 F-FDG and [ 99m Tc]. The biologic properties of radiolabeled peptides were assessed in vitro and in vivo. ...
Background:
Early detection of apoptosis is very important for therapy and follow-up treatment in various pathologic conditions. Annexin V interacts strongly and specifically with phosphatidylserine, specific biomarkers of apoptosis with some limitations. Small peptides are suitable alternatives to annexin V. A reliable and noninvasive in vivo technique for the detection of apoptosis is in great demand. Based on our previous studies, three new peptide analogs of LIKKPF (Leu-Ile-Lys-Lys-Pro-Phe) as apoptosis imaging agents were developed.
Materials and methods:
Aoa-LIKKP-Cl-F, Aoe-LIKKP-Pyr-F, and Aoe-LIKKP-Nap-F were synthesized, functionalized with aminooxy, and radiolabeled with 18F-FDG. Their biologic properties were evaluated in vitro using apoptotic Jurkat cells. 18F-FDG-Aoe-LIKKP-Pyr-F peptide was injected into normal and apoptotic mice models for biodistribution and in vivo positron emission tomography/computed tomography imaging studies.
Results:
18F-FDG-Aoe-LIKKP-Pyr-F peptide showed higher affinity for apoptotic cells. The localization of peptide in apoptotic liver mice was confirmed in biodistribution and imaging studies.
Conclusion:
The results showed that Aoe-LIKKP-Pyr-F peptide is an auspicious agent for molecular imaging of apoptosis.
... For neural regeneration, the mostly used therapeutic stem cells are neural progenitor cells (NPCs), neural stem cells (NSCs), and embryonic stem cells (ESCs) [63][64][65][66][67][68][69][70][71]73,173]. The behaviors of the therapeutic stem cells can be controlled by functional peptides [174] or proteins to achieve their neuritogenesis and/or neural differentiation for the regeneration process [63][64][65][66][67][68][69][70][71]73,173]. ...
... For neural regeneration, the mostly used therapeutic stem cells are neural progenitor cells (NPCs), neural stem cells (NSCs), and embryonic stem cells (ESCs) [63][64][65][66][67][68][69][70][71]73,173]. The behaviors of the therapeutic stem cells can be controlled by functional peptides [174] or proteins to achieve their neuritogenesis and/or neural differentiation for the regeneration process [63][64][65][66][67][68][69][70][71]73,173]. Phage and phage display technique have been used for neural regeneration in a variety of ways. ...
... Phage biopanning has successfully been applied to identify NSC-, NPC-binding peptides, which have shown great potential for neural regenerative applications [63][64][65][66]73]. Arap et al. identified a murine NSCspecific peptide, CGLPYSSVC, through phage biopanning using the Ph. ...
Bacteriophage, also called phage, is a human-safe bacteria-specific virus. It is a monodisperse biological nanostructure made of proteins (forming the outside surface) and nucleic acids (encased in the protein capsid). Among different types of phages, filamentous phages have received great attention in tissue regeneration research due to their unique nanofiber-like morphology. They can be produced in an error-free format, self-assemble into ordered scaffolds, display multiple signaling peptides site-specifically, and serve as a platform for identifying novel signaling or homing peptides. They can direct stem cell differentiation into specific cell types when they are organized into proper patterns or display suitable peptides. These unusual features have allowed scientists to employ them to regenerate a variety of tissues, including bone, nerves, cartilage, skin, and heart. This review will summarize the progress in the field of phage-based tissue regeneration and the future directions in this field.
... Therefore, in the present study, we aimed to assess the PYC36D-TAT and JNKI-1D-TAT peptides in a permanent focal ischemia stroke model in a high and low dose trial. In addition, in one trial we also evaluated the PYC36L-D-TAT peptide fused to a cerebral ischemia homing peptide reported (Hong et al, 2008) to aid delivery into the brain. ...
... The D-retro-inverso form of the TAT peptide (D-TAT; H-GRRRQRRKKRG-NH2) and a scrambled version of the PYC36 peptide (PYC36Dscrambled-TAT; H-KRRGGILRYGQPQSQGRRRQRRKKRG-NH2) were also synthesized and used as controls. The HP-PYC36L-TAT peptide was synthesized in the L-form and consisted of the PYC36 and TAT peptides in addition to a brain homing peptide sequence (Hong et al, 2008); H-GRKKRRQRRRGLQGRRRQGYQSIKP CLEVSRKNC-NH2. All peptides were synthesized and high-performance liquid chromatography purified J Exp Stroke Transl Med (2012) Peptides were prepared in normal saline in 300 µl or 500 µl volumes for intravenous administration and stored at -80°C before use. ...
... In order to improve brain delivery due to a more compromised blood brain barrier and to better target the time of JNK/c-Jun activation, the peptides were administered 2 hours post-MCAO in trial 2, as compared to 1 hour post-occlusion used in trial 1. In addition, in trial 2 we used a modified PYC36D-TAT peptide (HP-PYC36D-TAT: IC50: 1.5µM in glutamate model) fused to a 9 amino acid sequence reported to "home" to ischemic brain tissue (Hong et al, 2008). Finally, we used Sprague Dawley rats, instead of Spontaneously Hypertensive rats used in our first study, as the later strain has an atypical patho-molecular response to glutamate receptor activation (Lecrux et al, 2007), which may have contributed to our previous negative findings. ...
... This transient modification approach would not pose long lasting influence on cells, and dissociated peptides from the membrane are biodegradable and even beneficial for ischemic tissue, thus paving the way for brain treatment study . Briefly, in the present study, the brain targeting CLEVSRKNC peptide (Hong et al., 2008) was selected and coated onto the cell surface via a lipid raft to induce the migration of MSCs to the ischemic lesion and to trigger a synergistic effect. This study investigated overall effect of targeting peptide coating onto MSCs, including whether or not membrane modification could result in cytotoxicity, cell behavior changes, and cell differentiation and the ability of peptides to induce cell homing. ...
... The CLEVSRKNC peptide can selectively home to ischemic brain tissue and detect apoptosis (Hong et al., 2008). The PA-peptide is supposed to be able to coat the cell because palmitate serves as an anchor to integrate into the cell membrane (Kean et al., 2012). ...
... The TTC-stained brain slices in Fig. 3c confirm the establishment of MCAO model animals. The CLEVSRKNC peptide can preferentially home to the ischemic hemisphere after circulation for either 15 min or 2 h in vivo (Hong et al., 2008). In the present study, we examined whether or not the PA-CLEVSRKNC peptide can successfully tract MSCs to ischemic brain tissue with the co-localization of peptide and MSCs. ...
... It was reported that a stroke-homing peptide (SHp, CLEVSRKNC) could be recruited to the ischemic site of the body. 16,17 For active drug delivery in the treatment of ischemic stroke, SHp was identified and optimized by in vivo phage display in a focal cerebral ischemia rat model. SHp selectively targets the ischemic site in the brain and colocalizes to a portion of neuronal cells undergoing apoptosis in the penumbra region of ischemic brain tissue. ...
... In 2008, a peptide CLEVSRKNC targeting ischemic brain tissue was first reported. 16 Synthetic CLEVSRKNC peptide, which resides in ischemic stroke tissue, can be used as a molecular probe for imaging and deliver drugs to ischemic tissues. 5 Therefore, we coupled CLEVSRKNC (SHp) with Fig. 8: Schematic illustration indicating that chemotherapy-induced NETs contribute to the development of CIPN by disturbing microcirculation. ...
Background:
Chemotherapy-induced peripheral neuropathy (CIPN) is a severe dose-limiting side effect of chemotherapy and remains a huge clinical challenge. Here, we explore the role of microcirculation hypoxia induced by neutrophil extracellular traps (NETs) in the development of CIPN and look for potential treatment.
Methods:
The expression of NETs in plasma and dorsal root ganglion (DRG) are examined by ELISA, IHC, IF and Western blotting. IVIS Spectrum imaging and Laser Doppler Flow Metry are applied to explore the microcirculation hypoxia induced by NETs in the development of CIPN. Stroke Homing peptide (SHp)-guided deoxyribonuclease 1 (DNase1) is used to degrade NETs.
Findings:
The level of NETs in patients received chemotherapy increases significantly. And NETs accumulate in the DRG and limbs in CIPN mice. It leads to disturbed microcirculation and ischemic status in limbs and sciatic nerves treated with oxaliplatin (L-OHP). Furthermore, targeting NETs with DNase1 significantly reduces the chemotherapy-induced mechanical hyperalgesia. The pharmacological or genetic inhibition on myeloperoxidase (MPO) or peptidyl arginine deiminase-4 (PAD4) dramatically improves microcirculation disturbance caused by L-OHP and prevents the development of CIPN in mice.
Interpretation:
In addition to uncovering the role of NETs as a key element in the development of CIPN, our finding provides a potential therapeutic strategy that targeted degradation of NETs by SHp-guided DNase1 could be an effective treatment for CIPN.
Funding:
This study was funded by the National Natural Science Foundation of China81870870, 81971047, 81773798, 82271252; Natural Science Foundation of Jiangsu ProvinceBK20191253; Major Project of "Science and Technology Innovation Fund" of Nanjing Medical University2017NJMUCX004; Key R&D Program (Social Development) Project of Jiangsu ProvinceBE2019732; Nanjing Special Fund for Health Science and Technology DevelopmentYKK19170.
... However, the nanoparticles did not exhibit specific distribution between the ischemic region and normal tissue [246]. On the contrary, a peptide (SHp, CLEVSRKNC sequence) selected from in vivo phage display could lead preferentially home to ischemic stroke tissue [247]. On this basis, Shp and T7 were modified on a liposome surface for targeting ischemic area and BBB penetration, respectively [142]. ...
... Interestingly, the cellular uptake of SHp-modified liposomes (SHp-P-LPs) in PC12 cells was involved with excitatory amino acids. Meanwhile, the SHp-P-LPs accumulated in the ischemic lesion rather than the contralateral hemisphere [247]. Consequently, it was speculated that the endow homing ability of SHp might be mediated by the overactivation of GR [142]. ...
Ischemic stroke is a vascular central nervous system (CNS) disease that leads a disability and death. Thrombolysis and neuroprotection are the primary treatment strategies of ischemic stroke. Recombinant tissue plasminogen activator (tPA) is currently the only therapeutic drug approved by the Food & Drug Administration (FDA) for the treatment of ischemic stroke, while the application of tPA is limited by narrow therapeutic window, rapid drug elimination, and risks of hemorrhagic transformation. Blood brain barrier (BBB) poses serious challenges to drug delivery into the brain, leading to many neuroprotective agents failing clinical trials. The drug delivery systems (DDS), including targeted nanocarriers and stimuli-responsive nanocarriers, could protect drugs from elimination, deliver drugs to desired sites such as thrombus and CNS, and specifically release drugs in preferred areas by responding to internal or external stimuli, thus increasing the therapeutic effects of drugs. This review summarizes some strategies employed in nanocarriers to achieve thrombus targeting or enhance the accumulation of neuroprotective agents in the brain and even ischemic regions and brain cells. Meanwhile, the stimuli responsive nanocarriers in response to different internal and external environments of thrombus or ischemic brain parenchyma are systematically discussed. This work also provides insights into the precise treatment of ischemic stroke.
... Reduced neuronal cell damage and suppressed brain damage [39] Simvastatin Pleiotropic effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition Improved biodistribution and brain accumulation of simvastatin [40] Citicoline Membrane repair and regeneration Alleviation of fatty acid-induced toxicity Suppressed ischemic brain damage and edema [41,43] To identify a novel targeting peptide for the region of ischemic stroke, Hong et al. performed an in vivo phage display using t-MCAO rats [47]. Through three cycles of in vivo screening, the authors identified a candidate peptide, namely CLEVSRKNC, termed strokehoming peptide (SHp). ...
... Transferrin receptor (TfR) (Expressed on cerebral endothelial cells) [44,45] Stroke-homing peptide (SHp; CLEVSRKNC) Unknown (Possible target: activated glutamate receptors) [42,47] AEPO EPO receptors (Upregulated on cerebral endothelial cells and neurons after ischemic stroke) [24] cRGD (Arg-Gly-Asp-D -Tyr-Lys) ...
Ischemic stroke is still one of the leading causes of high mortality and severe disability worldwide. Therapeutic options for ischemic stroke and subsequent cerebral ischemia/reperfusion injury remain limited due to challenges associated with drug permeability through the blood-brain barrier (BBB). Neuroprotectant delivery with nanoparticles, including liposomes, offers a promising solution to address this problem, as BBB disruption following ischemic stroke allows nanoparticles to pass through the intercellular gaps between endothelial cells. To ameliorate ischemic brain damage, a number of nanotherapeutics encapsulating neuroprotective agents, as well as surface-modified nanoparticles with specific ligands targeting the injured brain regions, have been developed. Combination therapy with nanoparticles encapsulating neuroprotectants and tissue plasminogen activator (t-PA), a globally approved thrombolytic agent, has been demonstrated to extend the narrow therapeutic time window of t-PA. In addition, the design of biomimetic drug delivery systems (DDS) employing circulating cells (e.g., leukocytes, platelets) with unique properties has recently been investigated to overcome the injured BBB, utilizing these cells’ inherent capability to penetrate the ischemic brain. Herein, we review recent findings on the application and utility of nanoparticle DDS, particularly liposomes, and various approaches to developing biomimetic DDS functionalized with cellular membranes/membrane proteins for the treatment of ischemic stroke.
... Apoptosis imaging is a non-invasive tool for understanding cellular processes contributing to a single-cell death. Apoptosis imaging probes provide opportunities to assess the following items: 1) prediction of disease/damage severity including ischemic or autoimmune disorders 2) acute organ transplant rejection [25] 3) distinguishing the irreversible brain and heart injuries from living tissues [26], and 4) response to therapy immediately after chemotherapy [27], radiotherapy [28], thermal therapy [29] and photodynamic therapy [30]. It is noteworthy that targeted apoptosis imaging has more advantages in comparison with canonical imaging techniques based on anatomical imaging modalities (magnetic resonance imaging (MRI), computed tomography (CT)) and functional 18 F-fluorodeoxyglucose ( 18 FDG)-PET, as mentioned below. ...
... The passage of such large peptide across the blood-brain barrier (BBB) was likely due to the disruption of BBB at 24 hours following cerebral ischemia. The authors suggested a new mechanism of uptake for 131 I-YCLEVSRKNC, which was dependent on the overexpression of calcium or potassium channel proteins or clusterin receptors in the stroke-related apoptotic regions [26]. ...
Apoptosis is a regulated cell death induced by extrinsic and intrinsic stimulants. Tracking of apoptosis provides an opportunity for assessment of cardiovascular and neurodegenerative diseases and also monitoring of cancer therapy at early stages. There are some key mediators in apoptosis cascade which could be considered as specific targets for delivering imaging or therapeutic agents. The targeted radioisotope-based imaging agents are able to sensitively detect the physiological signal pathways which make them suitable for apoptosis imaging at a single-cell level. Radiopeptides take advantage of both the high sensitivity of nuclear imaging modalities and favourable features of peptide scaffolds. Our aim is to review the characteristics of those radiopeptides targeting apoptosis with different mechanisms.
... These liposomal nanocarriers can gain additional features by incorporating functional molecules onto their membrane surfaces. Stroke-homing peptide (Hong et al. 2008) may be useful for selective drug delivery to ischemic brain tissue. ...
... Recently, the CLEVSRKNC (single letter sequence for Cys-Leu-Glu-Val-Ser-Arg-Lys-Asn-Cys) peptide has been demonstrated to be selective for ischemic brain tissue. (Hong et al. 2008)This peptide was screened from a phage peptide library based on a T7 415-1b phage vector displaying CX7C (C, cysteine; X, random peptides) (Pasqualini and Ruoslahti 1996). The CLEVSRKNC-phage preferentially targeted ischemic stroke tissue after intravenous administration in a rat MCA occlusion model. ...
... Focal cerebral ischemia is a common cause of death and serious long-term disabilities in many countries. It is caused by a sudden interruption of the blood supply to the brain (21). The mechanisms of ischemic neuronal injury include not only energy exhaustion, acidosis, Table 2. Effect of YGY-E on neuronal apoptosis in rat ischemic brain tissue TUNEL: TdT-mediated dUTP-biotin nick end labeling; Data are presented as mean ± S.E.M.; ** p < 0.01 vs. control group. ...
... Current treatments for ischemic strokes include intravenous thrombolytics, endovascular approaches, anticoagulation, neuroprotection, anti-platelet aggregation, etc. (21,22). Even if the efficacy of some treatment strategies has been proven, the number of acute stroke patients successfully treated remains disappointingly low because of the narrow therapeutic time window for each approach. ...
YGY-E is an active ingredient in traditional Chinese medical herbs which have anti-ischemic activity. The present work was designed to study its therapeutic time window in cerebral ischemic injury as well as its effect on neuronal apoptosis. Animals received an intravenous injection of YGY-E at 1, 3, and 6 h, respectively, after permanent focal cerebral ischemia induced by electrocoagulation of the middle cerebral artery. Infarct ratio and neurological function were employed to assess the effects of YGY-E on the therapeutic time window in this animal model. Furthermore, we evaluated effects of this compound on neuronal apoptosis and synthesis of Bcl-2 and Bax in ischemic brain tissue with in situ DNA end labeling (TUNEL), immunohistochemistry assay, and Western blot analysis. YGY-E (2-8 mg/kg) delivered at all the three time points dose-dependently decreased infarct ratio, neurological deficits, percentage of TUNEL-positive cells (p < 0.01) and Bax-positive cells (p < 0.01 or p < 0.05). In contrast, it increased the percentage of Bcl-2 positive cells (p < 0.01 or p < 0.05). These data demonstrated that YGY-E had protective effects against cerebral ischemia injuries in rats. But more importantly, they indicate that YGY-E has an unusually long (up to 6 h)therapeutic time window relative to classical drugs in treating cerebral ischemia. In addition, our results suggest that the anti-apoptotic effects of YGY-E are due to its regulation of the balance between Bcl-2 and Bax protein levels.
... In addition, Lv et al. modified the erythrocyte membrane with stroke homing peptide (SHp, CLEVSRKNC) [56]. Some studies have shown that CLEVSRKNC peptide can specifically target the ischemic site and co-locate with some apoptotic neurons in ischemic brain tissue [57]. In addition, SHp-modified liposomes can effectively be home to ischemic stroke sites [58]. ...
The efficacy and safety of treating ischemic stroke is still a challenging problem at this stage. Ischemic stroke has a special stroke microenvironment. In recent years, improving stroke microenvironment has become a new idea for treating ischemic stroke. At the same time, nanoparticles have unique physical and chemical properties and significant advantages in studying ischemic stroke. Therefore, in recent years, researchers have designed various types of nanoparticles in response to stroke microenvironment to treat ischemic stroke. In this review, we summarized and analyzed the ischemic areas targeted by nanoparticles in response to reactive oxygen species, pH, high expression of receptors in the blood–brain barrier, enrichment of molecules in the stroke microenvironment, light, magnetic harmony, and other stimuli. We analyze its advantages and disadvantages and look forward to the development prospect of this field. Hope to provide strategies for better treatment of ischemic stroke.
... Next, to address the delivery issue of RCMCNPs, the researchers chose to insert the stroke homing peptide (SHP) into the RBC membrane to enable RCMCNPs to target the ischemic area. The SHP has been experimentally shown to selectively target sites of cerebral ischemia [47,48]. Furthermore, to control the release of drugs, they synthesized a reactive oxygen species (ROS) bio-responsive polymer, PHB dextran, to prepare the drug-carrying NPs. ...
Neurological diseases (NDs) are a significant cause of disability and death in the global population. However, effective treatments still need to be improved for most NDs. In recent years, cell-membrane-coated nanoparticles (CMCNPs) as drug-targeting delivery systems have become a research hotspot. Such a membrane-derived, nano drug-delivery system not only contributes to avoiding immune clearance but also endows nanoparticles (NPs) with various cellular and functional mimicries. This review article first provides an overview of the function and mechanism of single/hybrid cell-membrane-derived NPs. Then, we highlight the application and safety of CMCNPs in NDs. Finally, we discuss the challenges and opportunities in the field.
... Unexplored impediments steam from the limited ability of drugs to penetrate the BBB and target the ischemic neuronal tissue, resulting in decreased efficient concentration of the neuroprotective agents (Saugstad, 2010;Ponnusamy and Yip, 2019). In this context, selective drug delivery systems such as stroke tissue-related homing peptides and nanoparticlesmediated agents are emerging (Hong et al., 2008;He et al., 2021). ...
Cerebral ischemia reperfusion injury is a debilitating medical condition, currently with only a limited amount of therapies aimed at protecting the cerebral parenchyma. Micro RNAs (miRNAs) are small, non-coding RNA molecules that via the RNA-induced silencing complex either degrade or prevent target messenger RNAs from being translated and thus, can modulate the synthesis of target proteins. In the neurological field, miRNAs have been evaluated as potential regulators in brain development processes and pathological events. Following ischemic hypoxic stress, the cellular and molecular events initiated dysregulate different miRNAs, responsible for long-terming progression and extension of neuronal damage. Because of their ability to regulate the synthesis of target proteins, miRNAs emerge as a possible therapeutic strategy in limiting the neuronal damage following a cerebral ischemic event. This review aims to summarize the recent literature evidence of the miRNAs involved in signaling and modulating cerebral ischemia-reperfusion injuries, thus pointing their potential in limiting neuronal damage and repair mechanisms. An in-depth overview of the molecular pathways involved in ischemia reperfusion injury and the involvement of specific miRNAs, could provide future perspectives in the development of neuroprotective agents targeting these specific miRNAs.
... GB was encapsulated into rHDL to form nanoparticles (NPs), after which PM was wrapped around rHDL-based NPs. With the aim to further increase the targetability of PM-cloaked NPs towards cerebral ischemic regions, PM was modified with a cerebral ischemic targeting peptide (CITP, its amino acid sequences: CLEVSRKNC) capable of homing to ischemic brain tissues probably due to the overactivation of glutamate receptors in ischemic neurons [30][31][32] . The in vitro targeting capability of CITP/PM/rHDL/GB NPs towards vascular injury sites and ischemic neurons was verified in rat adrenal pheochromocytoma PC12 cells exposed to oxygen and glucose deprivation/reoxygenation (OGD/R) by cellular uptake and collagen binding assays. ...
In this study, a dual-targeting biomimetic nanoplatform composed of reconstituted high-density lipoprotein (rHDL) that coated with platelet membrane (PM) and modified with cerebral ischemic targeting peptide (CITP) was developed to load ginkgolide B (GB) for anchoring vascular injury sites and ischemic neurons. The prepared CITP/PM/rHDL/GB nanoparticles (NPs) could selectively adhere to the exposed collagen, significantly elevate the cellular uptake and viability, and remarkably reduce the apoptosis ratio and the secretion level of proinflammatory cytokines in PC12 cells after oxygen and glucose deprivation/reoxygenation (OGD/R) stimulation. Ex vivo fluorescence imaging demonstrated that DiR-labeled CITP/PM/rHDL/GB NPs could efficiently distribute into brain tissues and mostly accumulate in the ischemic cerebral hemisphere of rats exposed to middle cerebral artery occlusion/reperfusion (MCAO/R). Besides, CITP/PM/rHDL/GB NPs could significantly strengthen the neuroprotective efficacy of GB against MCAO/R-induced cerebral ischemia/reperfusion (I/R) injury on rats, which was evidenced by reduced infarct rate, brain water content and neurological deficit scores as well as increased number of survival neurons owing to the inhibition on the release of proinflammatory cytokines and proapoptotic protein expression. Altogether, this platelet-like bioinspired nanoplatform could be utilized as an effective tool with enhanced targeting ability towards cerebral ischemic sites for ischemic stroke therapy.
... Based on the above background, a mitochondrial targeting molecule triphenylphosphine (TPP) was conjugated to melatonin (TPP-MLT) and increased the distribution of melatonin in intracellular mitochondria with the push of mitochondrial transmembrane potential [16], aiming to effectively scavenge and inhibit intracellular production of ROS under pathological conditions. Secondly, TPP-MLT was encapsulated in two-stage targeted micelles mediated by TGN peptide (TGNYKALHPHNG) with high affinity for BBB and the SHp peptide (CLEVSRKNC) with high affinity for the glutamate receptor of oxidative stress-damaged neural cells with up-regulated specificity during the occurrence of CIS [17,18]. TGN/SHp/TPP-MLT micelles increase the targeted transport of TPP-MLT to brain tissues, and further act on damaged cells by the mediation of SHp peptide to enhance the drug therapeutic effect, thus effectively protecting neural cells, and achieving the precise treatment of CIS. ...
Background
Effective amelioration of neuronal damages in the case of cerebral ischemic stroke (CIS) is essential for the protection of brain tissues and their functional recovery. However, most drugs can not penetrate the blood–brain barrier (BBB), resulting in the poor therapeutic outcomes.
Results
In this study, the derivatization and dual targeted delivery technologies were used to actively transport antioxidant melatonin (MLT) into the mitochondria of oxidative stress-damaged cells in brain tissues. A mitochondrial targeting molecule triphenylphosphine (TPP) was conjugated to melatonin (TPP-MLT) to increase the distribution of melatonin in intracellular mitochondria with the push of mitochondrial transmembrane potential. Then, TPP-MLT was encapsulated in dual targeted micelles mediated by TGN peptide (TGNYKALHPHNG) with high affinity for BBB and SHp peptide (CLEVSRKNG) for the glutamate receptor of oxidative stress-damaged neural cells.TGN/SHp/TPP-MLT micelles could effectively scavenge the overproduced ROS to protect neuronal cells from oxidative stress injury during CIS occurrence, as reflected by the improved infarct volume and neurological deficit in CIS model animals.
Conclusions
These promising results showed this stepwise-targeting drug-loaded micelles potentially represent a significant advancement in the precise treatment of CIS.
Graphical Abstract
... Based on the above background, a mitochondrial targeting molecule triphenylphosphine (TPP) was conjugated to melatonin (TPP-MLT) and increased the distribution of melatonin in intracellular mitochondria with the push of mitochondrial transmembrane potential [16], aiming to effectively scavenge and inhibit intracellular production of ROS under pathological conditions. Secondly, TPP-MLT was encapsulated in two-stage targeted micelles mediated by TGN peptide (TGNYKALHPHNG) with high a nity for BBB and the SHp peptide (CLEVSRKNC) with high a nity for the glutamate receptor of oxidative stress-damaged neural cells with up-regulated speci city during the occurrence of CIS [17,18]. ...
Background: Effective amelioration of neuronal damages in the case of cerebral ischemic stroke (CIS) is essential for the protection of brain tissues and their functional recovery. However, most drugs can not penetrate the blood-brain barrier (BBB), resulting in the poor therapeutic outcomes.
Results: In this study, the derivatization and dual targeted delivery technologies were used to actively transport antioxidant melatonin (MLT) into the mitochondria of oxidative stress-damaged cells in brain tissues. A mitochondrial targeting molecule triphenylphosphine (TPP) was conjugated to melatonin (TPP-MLT) to increase the distribution of melatonin in intracellular mitochondria with the push of mitochondrial transmembrane potential. Then, TPP-MLT was encapsulated in dual targeted micelles mediated by TGN peptide (TGNYKALHPHNG) with high affinity for BBB and SHp peptide (CLEVSRKNG) for the glutamate receptor of oxidative stress-damaged neural cells.TGN/SHp/TPP-MLT micelles could effectively scavenge the overproduced ROS to protect neuronal cells from oxidative stress injury during CIS occurrence, as reflected by the improved infarct volume and neurological deficit in CIS model animals.
Conclusions: These promising results showed this stepwise-targeting drug-loaded micelles potentially represent a significant advancement in the precise treatment of CIS.
... If NPs survive the hostility of the blood serum and cross the restricted BBB, they can be modified with certain ligands to target specific subsets or neural cells into the brain parenchyma. This is the case for the amino acid sequence "CLEVSRKNC", identified via the screening of a phage library, which might facilitate the mobilization of NPs towards ischemic penumbra areas due to the special affinity of this peptide sequence to binding neuronal cells at risk of being damaged [139,140]. ...
Ischemic stroke represents one of the most prevalent pathologies in humans and is a leading cause of death and disability. Anti-thrombolytic therapy with tissue plasminogen activator (t-PA) and surgical thrombectomy are the primary treatments to recanalize occluded vessels and normalize the blood flow in ischemic and peri-ischemic regions. A large majority of stroke patients are refractory to treatment or are not eligible due to the narrow time window of therapeutic efficacy. In recent decades, we have significantly increased our knowledge of the molecular and cellular mechanisms that inexorably lead to progressive damage in infarcted and peri-lesional brain areas. As a result, promising neuroprotective targets have been identified and exploited in several stroke models. However, these considerable advances have been unsuccessful in clinical contexts. This lack of clinical translatability and the emerging use of biomaterials in different biomedical disciplines have contributed to developing a new class of biomaterial-based systems for the better control of drug delivery in cerebral disorders. These systems are based on specific polymer formulations structured in nanoparticles and hydrogels that can be administered through different routes and, in general, bring the concentrations of drugs to therapeutic levels for prolonged times. In this review, we first provide the general context of the molecular and cellular mechanisms impaired by cerebral ischemia, highlighting the role of excitotoxicity, inflammation, oxidative stress, and depolarization waves as the main pathways and targets to promote neuroprotection avoiding neuronal dysfunction. In the second part, we discuss the versatile role played by distinct biomaterials and formats to support the sustained administration of particular compounds to neuroprotect the cerebral tissue at risk of damage.
... They found that the phage-borne rMSCbinding VTAMEPGQ-pVIII significantly increased the transfection efficiency up to 40% in rMSCs (Figure 3), confirming the important role of phage display technique in improving the gene delivery. An up-to-date example would be using primary cardiomyocytes (PCM) targeting peptide (WLSEAGPVVTVRALRGTGSW) identified by phage display as a specific targeting ligand for the delivery of apoptosis antigen 1 (Fas) small interfering RNA (siRNA) by a bioreducible polymer for the potential treatment of cardiovascular disease (Figure 4) (Jung et al., 2016;Lee et al., 2015) Human non-muscle-invasive bladder tumor cell line BIU-87 CSSPIGRHC (Jung et al., 2016;Yang et al., 2016) Hepatic stellate cells VHWDFRQWWQPS (Ma et al., 2015;Yang et al., 2016) Murine pancreas langerhan islet CVSNPRWKC, CHVLWSTRC (Ma et al., 2015;Yao et al., 2005) Rat transient middle cerebral artery occlusion model CLEVSRKNC Yao et al., 2005) HCC cell lines BEL-7402 AGKGTPSLETTP (Du et al., 2010;Hong, Choi, et al., 2008) Mice oral cancer xenografts SNPFSKPYGLTV, YPHYSLPGSSTL Du et al., 2010) Rat islet tumors CRGRRST, CKAAKNK, CRSRKG (Chang, Chiu, et al., 2009;Joyce et al., 2003) Rat glioblastoma NIPYNPY, YLGDTIEEL, VLASPLN, NLGLETS GNSLSFP, IRTPSTV, RRPVNCI, ELVKIFS, VAVTDSR, AISPRLSS, NISYNAY, DATRLSS, THVHMLS, HPTKWPL, SLPPKTT, AHEHTYA, VGNNNYP DHLHSSR, TCLQYLGRVV (Joyce et al., 2003;Schluesener et al., Schluesener & Xianglin, 2004) Abdominal skin of BALB/cA nude mice ACSSSPSKHCG (Schluesener et al., Schluesener & Xianglin, 2004;Chen et al., 2006) Gastrointestinal mucosal barrier YPRLLTP, CSKSSDYQC (Chen et al., 2006;Duerr, White, & Schluesener, 2004;Kang et al., 2008) Blood-cerebrospinal-fluid barrier (BCSFB) TPSYDTYAAELR (Duerr et al., 2004;Kang et al., 2008;Li, Feng, & Jiang, 2015) Atherosclerotic plaque-associated endothelium Atherosclerotic lesion surfaces CAPGPSKSC (Liu, Bhattacharjee, Boisvert, Dilley, & Edgington, 2003) under hypoxic conditions Fas siRNA/PCM-polymer polyplexes significantly silenced Fas gene in cardiomyocytes, resulting in the inhibition of cardiomyocyte apoptosis. Their finding confirmed that the targeting role of a PCM specific peptide for targeted gene delivery to the myocardium. ...
Display of a peptide or protein of interest on the filamentous phage (also known as bacteriophage), a biological nanofiber, has opened a new route for disease diagnosis and therapy as well as proteomics. Earlier phage display was widely used in protein–protein or antigen–antibody studies. In recent years, its application in nanomedicine is becoming increasingly popular and encouraging. We aim to review the current status in this research direction. For better understanding, we start with a brief introduction of basic biology and structure of the filamentous phage. We present the principle of phage display and library construction method on the basis of the filamentous phage. We summarize the use of the phage displayed peptide library for selecting peptides with high affinity against cells or tissues. We then review the recent applications of the selected cell or tissue targeting peptides in developing new targeting probes and therapeutics to advance the early diagnosis and targeted therapy of different diseases in nanomedicine. We also discuss the integration of antibody phage display and modern proteomics in discovering new biomarkers or target proteins for disease diagnosis and therapy. Finally, we propose an outlook for further advancing the potential impact of phage display on future nanomedicine.
This article is categorized under:
• Biology‐Inspired Nanomaterials > Protein and Virus‐Based Structures
... Stroke homing peptide (SHp) can home to ischemic brain tissue and co-localize with apoptotic neuronal cells after intravenous administration in the middle cerebral artery occlusion models [177]. In a conducted by Zhao's study et al., the researchers combined the stroke homing potential of SHp and the BBB targeting capacity of T7 peptide by conjugating both of the moieties to PEGylated liposomes [178]. ...
In recent years, nanomedicines have emerged as a promising method for central nervous system drug delivery, enabling the drugs to overcome the blood-brain barrier and accumulate preferentially in the brain. Despite the current success of brain-targeted nanomedicines, limitations still exist in terms of the targeting specificity. Based on the molecular mechanism, the exact cell populations and subcellular organelles where the injury occurs and the drugs take effect have been increasingly accepted as a more specific target for the next generation of nanomedicines. Dual and multi-targeted nanoparticles integrate different targeting functionalities and have provided a paradigm for precisely delivering the drug to the pathological site inside the brain. The targeting process often involves the sequential or synchronized navigation of the targeting moieties, which allows highly controlled drug delivery compared to conventional targeting strategies. Herein, we focus on the up-to-date design of pathological site-specific nanoparticles for brain drug delivery, highlighting the dual and multi-targeting strategies that were employed and their impact on improving targeting specificity and therapeutic effects. Furthermore, the background discussion of the basic properties of a brain-targeted nanoparticle and the common lesion features classified by neurological pathology are systematically summarized.
... From the same research group, Hong et al. reported on their results with dual-targeting liposomes modified with a peptide targeting TfR and strokehoming peptide (SHp; CLEVSRKNC), which was identified by another research group using an in vivo phage display in a rat model of focal cerebral ischemia. 47) The two peptidemodified ZL006-loaded dual-targeting liposomes showed significant accumulation in the ischemic region and inhibited neurological deficits and brain damage compared with each peptide-modified liposome alone. 48) Yemisci et al. investigated TfR-1-targeting antibody-modified chitosan nanoparticles carrying basis fibroblast growth factors, 49) and Han et al. constructed poly(lactic-co-glycolic acid) (PLGA) synthetic nanoparticles decorated with an MMP-2-targeting peptide, chlorotoxin. ...
Ischemic stroke is one of the leading causes of severe disability and death. In clinical settings, tissue plasminogen activator (t-PA) for thrombolytic therapy is the only globally approved drug for the treatment of ischemic stroke. However, the proportion of patients who receive t-PA therapy is extremely limited due to its narrow therapeutic time window (TTW) and the risk of cerebral hemorrhage. Cerebral ischemia–reperfusion (I/R) injury is also a serious problem for patients’ outcomes. Hence, the development of more effective therapies has been desired to prolong the TTW of t-PA and prevent cerebral I/R injury. For delivering drugs into the brain, the blood–brain barrier (BBB) must be overcome since it limits drug penetration into the brain, leading to insufficient therapeutic efficacy. As a distinctive pathology after an ischemic stroke, it was reported that the vascular permeability of the BBB is increased around the ischemic region. We found that nano-sized liposomes can pass through the disrupted BBB and accumulate in the I/R region, and that delivery of neuroprotective agents using a liposomal drug delivery system (DDS) is effective for the treatment of cerebral I/R injury. Moreover, we have recently demonstrated that combination therapy with liposomal drugs and t-PA can suppress the deleterious effects of t-PA and extend its TTW in a rat ischemic stroke model. These findings indicate that applications of nanoparticle DDS technology could be a hopeful approach to drug development for ischemic stroke therapy. In this review, we introduce our findings on ischemic stroke treatment using liposomal DDS and recent advances from other research groups.
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... Despite successful identification of unique ligands of disease and injury in AD and stroke respectively, utilization of phage display for TBI biomarkers has not been thoroughly conducted [101][102][103]. This slow adoption may be in part due to the difficulty of identifying biomarker candidates from the biopanning process. ...
Abstract Traumatic brain injury (TBI) affects 1.7 million people in the United States each year, causing lifelong functional deficits in cognition and behavior. The complex pathophysiology of neural injury is a primary barrier to developing sensitive and specific diagnostic tools, which consequentially has a detrimental effect on treatment regimens. Biomarkers of other diseases (e.g. cancer) have provided critical insight into disease emergence and progression that lend to developing powerful clinical tools for intervention. Therefore, the biomarker discovery field has recently focused on TBI and made substantial advancements to characterize markers with promise of transforming TBI patient diagnostics and care. This review focuses on these key advances in neural injury biomarkers discovery, including novel approaches spanning from omics-based approaches to imaging and machine learning as well as the evolution of established techniques.
... Similarly, following cerebral ischaemia, injured tissue can be targeted by the addition of a nine amino-acid sequence that specifically localises to the ischaemic region (Hong et al. 2008). ...
... Therapeutic techniques addressing strokes are in need of homing peptides that can target ischemic stroke tissue, identifying and detecting the apoptosis of cells. Hong et al. 104 discovered an ischemic stroke tissue-homing peptide sequence (CLEVSRKNC) and used fluorescein-labeled and 131 I-labeled peptides to radioimage ischemic stroke tissue and detect apoptotic neuronal cells. The fluorescent and autoradiographic images respectively demonstrated the viability of this peptide for targeting ischemic stroke tissue. ...
Novel affinity agents with high specificity are needed to make progress in disease diagnosis and therapy. Over the last several years, peptides have been considered to have fundamental benefits over other affinity agents, such as antibodies, due to their fast blood clearance, low immunogenicity, rapid tissue penetration, and reproducible chemical synthesis. These features make peptides ideal affinity agents for applications in disease diagnostics and therapeutics for a wide variety of afflictions. Virus-derived peptide techniques provide a rapid, robust, and high-throughput way to identify organism-targeting peptides with high affinity and selectivity. Here, we will review viral peptide display techniques, how these techniques have been utilized to select new organism-targeting peptides, and their numerous biomedical applications with an emphasis on targeted imaging, diagnosis, and therapeutic techniques. In the future, these virus-derived peptides may be used as common diagnosis and therapeutics tools in local clinics.
... In the current literature, there are limited numbers of ligands that have been characterized for targeted drug delivery to the ischemic microenvironment inside the brain. 35,36 In this study, we demonstrated CTX as a promising ligand for targeted drug delivery to the ischemic microenvironment inside the brain. CTX-modified NPs specifically located in the ischemic regions (Figure 3, F and G), whereas unmodified NPs did not have such a specificity. ...
Ischemic stroke is a leading cause of disability and death worldwide. Current drug treatment for stroke remains inadequate due to the existence of the blood-brain barrier. We proposed an innovative nanotechnology-based autocatalytic targeting approach, in which the blood-brain barrier modulator lexiscan is encapsulated in nanoparticles to enhance blood-brain barrier permeability and autocatalytically augment the brain stroke-targeting delivery efficiency of chlorotoxin-anchored nanoparticles. The nanoparticles efficiently and specifically accumulated in the brain ischemic microenvironment and the targeting efficiency autocatalytically increased with subsequent administrations. When Nogo-66 receptor antagonist peptide NEP1-40, a potential therapeutic agent for ischemic stroke, was loaded, nanoparticles significantly reduced infarct volumes and enhanced survival. Our findings suggest that the autocatalytic targeting approach is a promising strategy for drug delivery to the ischemic microenvironment inside the brain. Nanoparticles developed in this study may serve as a new approach for the clinical management of stroke.
... Research groups are developing peptides with high affinity for PS through phage display technology [12][13][14][15][16]. One of each refers to peptide LIKKPF which was conjugated with DTPA, labeled with Gadolinium, and examined in vitro and in vivo. ...
One of the early biochemical changes of apoptotic cells is exposure of phosphatidylserine on the external surface of the plasma membrane. The aim of current study is targeting Phosphatidyl serine (PS) using radiolabeled LIKKPF, which was functionalized with HYNIC and aminooxy, radiolabeled with 18FDG and assessed in vitro and in vivo. Results showed LIKKPF has less affinity to PS compared to original phage peptide, but high enough for specific binding to apoptotic cells. It is concluded the low affinity of radiolabeled LIKKPF might be attributed to hydrophobicity of peptide, therefor peptides used in future studies should be more hydrophobic compared to LIKKPF.
... Similarly, following cerebral ischaemia, injured tissue can be targeted by the addition of a nine amino-acid sequence that specifically localises to the ischaemic region (Hong et al. 2008). ...
The restricted ability of most proteins and peptides to cross the blood-brain barrier and/or plasma membrane limits their use as therapeutics following cerebral ischaemia. However, the discovery of cell-penetrating peptides has provided a means by which such molecules can be transported across the blood-brain barrier and plasma membrane. Many proteins/peptides have already been shown to have neuroprotective properties, and, due to their ability to block protein-protein interactions, provide a potentially rich source of new therapeutic compounds to prevent cell death following cerebral ischaemia. In this review, we give an overview of cell-penetrating peptides and their use experimentally to deliver neuroprotectant proteins/peptides into the brain following cerebral ischaemia.
Oxidative stress induced by ischemia‐reperfusion causes severe secondary injury in stroke patients. The blood‐brain barrier (BBB) and the challenges in targeting the stroke core hinder the therapeutic effect of drugs. This study introduces a precise biomimetic drug delivery system called SHp‐NM@Edv/RCD (SNM‐NPs), which possesses multiple stepwise targeting capabilities. SNM‐NPs are encapsulated by the neutrophil membranes (NMs) and exhibit a targeting effect (5.16‐fold) on the inflammatory microenvironment. The modification of stroke‐homing peptides (SHp) makes SNM‐NPs target damaged neurons faster, with a targeting efficiency 5.68 times higher than that of β‐cyclodextrins (RCD). Then, RCD encapsulated in SNM‐NPs responds to reactive oxygen species (ROS), leading to the release of edaravone (Edv), scavenges ROS, inhibits neuroinflammation, and reduces neuronal apoptosis by 90%. Mechanistically, SNM‐NPs deliver Edv precisely to the cerebral ischemia‐reperfusion injury (CIRI) site, resulting in the elimination of ROS, a decrease in the number of microglia, an improvement in tubulin expression in neurons, and the inhibition of neuronal apoptosis through Caspase 3 pathway. Preliminary experiments also show that SNM‐NPs exhibit a good safety profile both in intravenous therapy and in vitro cell experiments. As a result, SNM‐NPs hold promise for further development as effective and safe agents for target therapy of CIRI and other diseases.
Ischaemic stroke (IS) is the second most common cause of death worldwide. Traditional treatment strategies, including blood flow recanalization therapy and neuroprotective drugs, have reduced safety and effectiveness due to their lack of targeting and inability to penetrate the blood–brain barrier (BBB). Targeted nanoparticles (NPs) can improve BBB penetration and brain targeting by changing their composition and structure, which can integrate a variety of treatment components to match the complex pathological process of IS. In this review, we describe recent advances in IS therapy, specifically in the prevention, detection, and treatment of IS, by using targeted NPs, presenting basic knowledge and possible therapeutic targets to highlight their potential for early treatment in the clinic.
The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca ²⁺ ) uptake supports NVU function by buffering Ca ²⁺ and stimulating energy production. However, excessive mitochondrial Ca ²⁺ uptake causes toxic mitochondrial Ca ²⁺ overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca ²⁺ uptake and efflux in the brain are mediated by the mitochondrial Ca ²⁺ uniporter complex (MCU cx ) and sodium/Ca ²⁺ /lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCU cx inhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca ²⁺ overloading. These findings suggest that combining MCU cx inhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCU cx inhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCU cx , or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues.
High potency and safe therapies are still required for ischemic stroke, which is a leading cause of global death and disability. Herein, a reactive oxygen species (ROS)-responsive, transformable, and triple-targeting dl-3-n-butylphthalide (NBP) nanotherapy was developed for ischemic stroke. To this end, a ROS-responsive nanovehicle (OCN) was first constructed using a cyclodextrin-derived material, which showed considerably enhanced cellular uptake in brain endothelial cells due to notably reduced particle size, morphological transformation, and surface chemistry switching upon triggering via pathological signals. Compared to a nonresponsive nanovehicle, this ROS-responsive and transformable nanoplatform OCN exhibited a significantly higher brain accumulation in a mouse model of ischemic stroke, thereby affording notably potentiated therapeutic effects for the nanotherapy derived from NBP-containing OCN. For OCN decorated with a stroke-homing peptide (SHp), we found significantly increased transferrin receptor-mediated endocytosis, in addition to the previously recognized targeting capability to activated neurons. Consistently, the engineered transformable and triple-targeting nanoplatform, i.e., SHp-decorated OCN (SON), displayed a more efficient distribution in the injured brain in mice with ischemic stroke, showing considerable localization in endothelial cells and neurons. Furthermore, the finally formulated ROS-responsive transformable and triple-targeting nanotherapy (NBP-loaded SON) demonstrated highly potent neuroprotective activity in mice, which outperformed the SHp-deficient nanotherapy at a 5-fold higher dose. Mechanistically, our bioresponsive, transformable, and triple-targeting nanotherapy attenuated the ischemia/reperfusion-induced endothelial permeability and improved dendritic remodeling and synaptic plasticity of neurons in the injured brain tissue, thereby promoting much better functional recovery, which were achieved by efficiently enhancing NBP delivery to the ischemic brain tissue, targeting injured endothelial cells and activated neurons/microglial cells, and normalizing the pathological microenvironment. Moreover, preliminary studies indicated that the ROS-responsive NBP nanotherapy displayed a good safety profile. Consequently, the developed triple-targeting NBP nanotherapy with desirable targeting efficiency, spatiotemporally controlled drug release performance, and high translational potential holds great promise for precision therapy of ischemic stroke and other brain diseases.
Ischemic stroke (IS) is one of the most common causes of disability and death. Thrombolysis and neuroprotection are two current major therapeutic strategies to overcome the ischemic and reperfusion damages. Here, we have designed a novel peptide-templated MnO2 nanozyme (PNzyme/MnO2 ) that integrates the thrombolytic activity of the functional peptides with the ROS scavenging ability of the nanozyme. Through the self-assembled polypeptides that contains multiple functional motifs, the novel peptide-templated nanozyme is able to bind fibrin in the thrombus, cross blood brain barrier, and finally accumulate in the ischemic neuronal tissues, where the thrombolytic motif is "switch-on" by the action of thrombin. In mice and rat IS models, the PNzyme/MnO2 prolonged blood circulation time and exhibited strong thrombolytic action, and reduced the ischemic damages in brain tissues. Moreover, this peptide-templated nanozyme also effectively inhibited the activation of astrocytes and the secretion of pro-inflammatory cytokines. These data indicated that rationally designed PNzyme/MnO2 nanozyme exerts both thrombolytic and neuroprotective actions. Giving its long half-life in the blood and ability to target brain thrombi, the biocompatible nanozyme may serve as a novel therapeutic agent to improve the efficacy and prevent secondary thrombosis during the treatment of IS. This article is protected by copyright. All rights reserved.
Background:
Ischemic stroke is a condition in which an occluded blood vessel interrupts blood flow to the brain and causes irreversible neuronal cell death. Transplantation of regenerative stem cells has been proposed as a novel therapy to restore damaged neural circuitry after ischemic stroke attack. However, limitations such as low cell survival rates after transplantation remain significant challenges to stem cell-based therapy for ischemic stroke in the clinical setting. In order to enhance the therapeutic efficacy of transplanted stem cells, several biomaterials have been developed to provide a supportable cellular microenvironment or functional modification on the stem cells to optimize their reparative roles in injured tissues or organs.
Aim:
To discuss state-of-the-art functional biomaterials that could enhance the therapeutic potential of stem cell-based treatment for ischemic stroke and provide detailed insights into the mechanisms underlying these biomaterial approaches.
Methods:
The PubMed, Science Direct and Scopus literature databases were searched using the keywords of "biomaterial" and "ischemic stroke". All topically-relevant articles were then screened to identify those with focused relevance to in vivo, in vitro and clinical studies related to "stem cells" OR "progenitor cells" OR "undifferentiated cells" published in English during the years of 2011 to 2022. The systematic search was conducted up to September 30, 2022.
Results:
A total of 19 articles matched all the inclusion criteria. The data contained within this collection of papers comprehensively represented 19 types of biomaterials applied on seven different types of stem/progenitor cells, namely mesenchymal stem cells, neural stem cells, induced pluripotent stem cells, neural progenitor cells, endothelial progenitor cells, neuroepithelial progenitor cells, and neuroblasts. The potential major benefits gained from the application of biomaterials in stem cell-based therapy were noted as induction of structural and functional modifications, increased stem cell retention rate in the hostile ischemic microenvironment, and promoting the secretion of important cytokines for reparative mechanisms.
Conclusion:
Biomaterials have a relatively high potential for enhancing stem cell therapy. Nonetheless, there is a scarcity of evidence from human clinical studies for the efficacy of this bioengineered cell therapy, highlighting that it is still too early to draw a definitive conclusion on efficacy and safety for patient usage. Future in-depth clinical investigations are necessary to realize translation of this therapy into a more conscientious and judicious evidence-based therapy for clinical application.
The human brain is central, not only to normal biological function, but also to personal identity. Diseases and injuries to the brain can erase this sense of self. Delivering drugs to the brain is a critical challenge in addressing diseases and injuries that degrade human interaction. While delivering drugs to the brain can play a role in addressing a host of degenerative diseases of the central nervous system diseases like Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS), it is perhaps closer to being broadly clinically realized in the context of acute injuries or attacks. The subsequent review examines polymeric nanocarriers as a means to deliver drugs to the brain while using ischemic stroke as a specific example of an application. The explicit intravenous (IV) nature of ischemic stroke provides an opportunity to illustrate some of the opportunities and limits of nanocarrier drug delivery.KeywordsIschemic strokeTissue plasminogen activatorPoly(lactic-co-glycolic acid) nanocarriersMiddle cerebral artery occlusionBlood-brain barrier permeabilityFocused ultrasoundNear-infrared femtosecond-pulsed laser irradiationCarrier-mediated transcytosisTargeted deliveryEnzyme-responsive polymeric nanocarriersTriggered deliveryCore-shell nanocarriers
Central nervous system (CNS) diseases are among the most difficult to treat, mainly because the vast majority of the drugs fail to cross the blood-brain barrier (BBB) or to reach the brain at concentrations adequate to exert a pharmacological activity. The obstacle posed by the BBB has led to the in-depth study of strategies allowing the brain delivery of CNS-active drugs. Among the most promising strategies is the use of peptides addressed to the BBB. Peptides are versatile molecules that can be used to decorate nanoparticles or can be conjugated to drugs, with either a stable link or as pro-drugs. They have been used to deliver to the brain both small molecules and proteins, with applications in diverse therapeutic areas such as brain cancers, neurodegenerative diseases and imaging. Peptides can be generally classified as receptor-targeted, recognizing membrane proteins expressed by the BBB microvessels (e.g., Angiopep2, CDX, and iRGD), "cell-penetrating peptides" (CPPs; e.g. TAT47-57, SynB1/3, and Penetratin), undergoing transcytosis through unspecific mechanisms, or those exploiting a mixed approach. The advantages of peptides have been extensively pointed out, but so far few studies have focused on the potential negative aspects. Indeed, despite having a generally good safety profile, some peptide conjugates may display toxicological characteristics distinct from those of the peptide itself, causing for instance antigenicity, cardiovascular alterations or hemolysis. Other shortcomings are the often brief lifetime in vivo, caused by the presence of peptidases, the vulnerability to endosomal/lysosomal degradation, and the frequently still insufficient attainable increase of brain drug levels, which remain below the therapeutically useful concentrations. The aim of this review is to analyze not only the successful and promising aspects of the use of peptides in brain targeting but also the problems posed by this strategy for drug delivery.
There are limited neuroprotective strategies for various central nervous system conditions in which fast and sustained management is essential. Neuroprotection-based therapeutics have become an intensively researched topic in the neuroscience field, with multiple novel promising agents, from natural products to mesenchymal stem cells, homing peptides, and nanoparticles-mediated agents, all aiming to significantly provide neuroprotection in experimental and clinical studies. Dexmedetomidine (DEX), an α2 agonist commonly used as an anesthetic adjuvant for sedation and as an opioid-sparing medication, stands out in this context due to its well-established neuro-protective effects. Emerging evidence from preclinical and clinical studies suggested that DEX could be used to protect against cerebral ischemia, traumatic brain injury (TBI), spinal cord injury, neuro-degenerative diseases, and postoperative cognitive disorders. MicroRNAs (miRNAs) regulate gene expression at a post-transcriptional level, inhibiting the translation of mRNA into functional proteins. In vivo and in vitro studies deciphered brain-related miRNAs and dysregulated miRNA profiles after several brain disorders, including TBI, ischemic stroke, Alzheimer's disease, and multiple sclerosis, providing emerging new perspectives in neuroprotective therapy by modulating these miRNAs. Experimental studies revealed that some of the neuroprotective effects of DEX are mediated by various miRNAs, counteracting multiple mechanisms in several disease models, such as lipopolysaccharides induced neuroinflammation, β-amyloid induced dysfunction, brain ischemic-reperfusion injury, and anesthesia-induced neurotoxicity models. This review aims to outline the neuroprotective mechanisms of DEX in brain disorders by modulating miRNAs. We address the neuroprotective effects of DEX by targeting miRNAs in modulating ischemic brain injury, ameliorating the neurotoxicity of anesthetics, reducing postoperative cognitive dysfunction, and improving the effects of neurodegenerative diseases.
Phage display has been demonstrated as a powerful approach in the identification of lead compounds for the development of molecular imaging agents. It is an economical technique that covers a large area of diversity space and offers a high-throughput screening process with the availability of many types of phage clones and libraries, including peptides, cDNA, and antibodies. Isolated molecules identified from phage display determined to have optimal in vivo pharmacokinetics, specificity, and affinity can be labeled with radioisotopes, fluorophores, and nanoparticles for use in molecular imaging applications. The aim of this chapter is to provide a practical overview of the principles and applications of phage display in the context of identifying lead compounds for imaging agents. Key concepts on library constructions and selections, biopanning process, and lead compound and target identifications are covered to provide a state-of-the-art summary on molecular imaging agent development utilizing phage display technique.
The past decades have witnessed great progress in nanoparticle (NP)‐based brain‐targeting drug delivery systems, while their therapeutic potentials are yet to be fully exploited given that the majority of them are lost during the delivery process. Rational design of brain‐targeting drug delivery systems requires a deep understanding of the entire delivery process along with the issues that they may encounter. Herein, this review first analyzes the typical delivery process of a systemically administrated NPs‐based brain‐targeting drug delivery system and proposes a six‐step CRITID delivery cascade: circulation in systemic blood, recognizing receptor on blood‐brain barrier (BBB), intracellular transport, diseased cell targeting after entering into parenchyma, internalization by diseased cells, and finally intracellular drug release. By dissecting the entire delivery process into six steps, this review seeks to provide a deep understanding of the issues that may restrict the delivery efficiency of brain‐targeting drug delivery systems as well as the specific requirements that may guarantee minimal loss at each step. Currently developed strategies used for troubleshooting these issues are reviewed and some state‐of‐the‐art design features meeting these requirements are highlighted. The CRITID delivery cascade can serve as a guideline for designing more efficient and specific brain‐targeting drug delivery systems. This review first proposes a six‐step CRITID delivery cascade for dissecting the entire delivery process of brain‐targeting nanoparticle delivery systems. By reviewing the issues and requirements that limit delivery efficiency at each step, as well as corresponding strategies for troubleshooting, this review seeks to provide a guideline to design brain‐targeting drug delivery systems with improved delivery efficiency at each step.
Hypoxic-ischemic encephalopathy (HIE), initiated by the interruption of oxygenated blood supply to the brain, is a leading cause of death and lifelong disability in newborns. The pathogenesis of HIE involves a complex interplay of excitotoxicity, inflammation, and oxidative stress that results in acute to long term brain damage and functional impairments. Therapeutic hypothermia is the only approved treatment for HIE but has limited effectiveness for moderate to severe brain damage; thus, pharmacological intervention is explored as an adjunct therapy to hypothermia to further promote recovery. However, the limited bioavailability and the side-effects of systemic administration are factors that hinder the use of the candidate pharmacological agents. To overcome these barriers, therapeutic molecules may be packaged into nanoscale constructs to enable their delivery. Yet, the application of nanotechnology in infants is not well examined, and the neonatal brain presents unique challenges. Novel drug delivery platforms have the potential to magnify therapeutic effects in the damaged brain, mitigate side-effects associated with high systemic doses, and evade mechanisms that remove the drugs from circulation. Encouraging pre-clinical data demonstrates an attenuation of brain damage and increased structural and functional recovery. This review surveys the current progress in drug delivery for treating neonatal brain injury.
Effective treatments for brain diseases, including stroke, brain tumours, Alzheimer's disease and Parkinson's disease, are generally limited because of poor entry of therapeutic agents into the brain parenchyma. Most drugs cannot permeate into the brain because of the blood-brain barrier, enzymatic degradation, short circulation lifetimes, insufficient tissue penetration, and so on. Therefore, overcoming the above problems has become one of the most significant challenges in brain disease therapeutics. Nanotechnology offers intriguing potential for addressing these challenges via biological, physical or chemical modification strategies, which can be used for multiple functions such as genetic modification, imaging, and controlled drug release. This review presents the current research progress of nanoplatforms for brain disease diagnoses and intervention. The different routes for brain drug delivery, including intravenous, intranasal and local administrations, are discussed.
Neurodegenerative diseases and disorders seriously impact memory and cognition and can become life-threatening. Current medical techniques attempt to combat these detrimental effects mainly through the administration of neuromedicine. However, drug efficacy is limited by rapid dispersal of the drugs to off-target sites while the site of administration is prone to overdose. Many neuropathological conditions are accompanied by excessive reactive oxygen species (ROS) due to the inflammatory response. Accordingly, ROS-responsive drug delivery systems have emerged as a promising solution. To guide intelligent and comprehensive design of ROS-responsive drug delivery systems, this review article discusses the two following topics: (1) the biology of ROS in both healthy and diseased nervous systems and (2) recent developments in ROS-responsive, drug delivery system design. Overall, this review article would assist efforts to make better decisions about designing ROS-responsive, neural drug delivery systems, including the selection of ROS-responsive functional groups.
Ischemic stroke is a leading cause of death and disability worldwide and is in urgent need of new treatment options. The only approved treatment for stroke restores blood flow to the brain, but much of the tissue damage occurs during the subsequent reperfusion. Antioxidant therapies that directly address ischemia-reperfusion injury have shown promise in preclinical results. In this review, we discuss that reformulating antioxidant therapies as nanomedicine can potentially overcome the barriers that have kept these therapies from succeeding in the clinic. We begin by reviewing the pathophysiology of ischemic stroke with a focus on the effects of reperfusion injury. Next, we review nanotherapeutic systems designed to treat the disease with a focus on those addressing reperfusion injury. Mechanisms of passive and active transport required to traverse a blood-brain barrier are discussed. Finally, we conclude by outlining design parameters for potentially successful nanomedicines as front-line therapeutics for ischemic stroke.
Phage display is a powerful and widely used technique to find novel peptide ligands. A massive amount of peptide sequences have been identified for all kinds of materials, and peptides that may have targeting capabilities towards specific cells and tissues have received especial attention in biomedical sciences. As a result, it is increasingly harder to follow all the work that has been done, which sometimes leads to many promising ligands receiving little attention, together with the publication of false positives that have already been found. The aim of this review is to provide an updated and comprehensive list of phage-displayed peptides targeting different tissues and organs. The limitations of the technique are carefully analysed and the future perspectives envisaged.
Ischemic stroke is a leading cause of long-term disability and death worldwide. Current drug delivery vehicles for the treatment of ischemic stroke are less than satisfactory, in large part due to their short circulation lives, lack of specific targeting to the ischemic site, and poor controllability of drug release. In light of the upregulation of reactive oxygen species (ROS) in the ischemic neuron, we herein developed a bioengineered ROS-responsive nanocarrier for stroke-specific delivery of a neuroprotective agent, NR2B9C, against ischemic brain damage. The nanocarrier is composed of a dextran polymer core modified with ROS-responsive boronic ester and a red blood cell (RBC) membrane shell with stroke homing peptide (SHp) inserted. These targeted “core–shell” nanoparticles (designated as SHp-RBC-NP) could thus have controlled release of NR2B9C triggered by high intracellular ROS in ischemic neurons after homing to ischemic brain tissues. The potential of the SHp-RBC-NP for ischemic stroke therapy was systematically evaluated in vitro and in rat models of middle cerebral artery occlusion (MCAO). In vitro results showed that the SHp-RBC-NP had great protective effects on glutamate-induced cytotoxicity in PC-12 cells. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) testing further demonstrated that the bioengineered nanoparticles can drastically prolong the systemic circulation of NR2B9C, enhance the active targeting of the ischemic area in the MCAO rats, and reduce ischemic brain damage.
Molecular imaging of programmed cell death (apoptosis) in vivo is an innovative strategy for early assessment of treatment response and treatment efficacy in cancer patients. Externalization of phosphatidylserine (PS) to the cell membrane surface of dying cells makes this phospholipid an attractive molecular target for the development of apoptosis imaging probes. In this study, we have radiolabeled PS-binding 14-mer peptide FNFRLKAGAKIRFG (PSBP-6) with positron-emitter copper-64 (64Cu) for PET imaging of apoptosis. Peptide PSBP-6 was conjugated with radiometal chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) through an aminovaleric acid (Ava) linker for subsequent radiolabeling with 64Cu to prepare radiotracer 64Cu-NOTA-Ava-PSBP-6. PS-binding potencies of PSBP-6, NOTA-Ava-PSBP-6 and natCu-NOTA-Ava-PSBP-6 were determined in a competitive radiometric PS-binding assay. Radiotracer 64Cu-NOTA-Ava-PSBP-6 was studied in camptothecin-induced apoptotic EL4 mouse lymphoma cells and in a murine EL4 tumor model of apoptosis using dynamic PET imaging. Peptide PSBP-6 was also conjugated via an Ava linker with fluorescein isothiocyanate (FITC). FITC-Ava-PSBP-6 was evaluated in flow cytometry and fluorescence confocal microscopy experiments. Radiopeptide 64Cu-NOTA-Ava-PSBP-6 was synthesized in high radiochemical yields of >95%. The IC50 values for PS-binding potency of PSBP-6, NOTA-Ava-PSBP-6 and natCu-NOTA-PSBP-6 were 600 μM, 30 μM and 23 μM, respectively. A competitive radiometric cell binding assay confirmed binding of 64Cu-NOTA-Ava-PSBP-6 to camptothecin-induced apoptotic EL4 cells in a Ca2+-independent manner. PET imaging studies demonstrated significantly higher uptake of 64Cu-NOTA-Ava-PSBP-6 in apoptotic EL4 tumors (SUV5min 0.95 ± 0.04) compared to control tumors (SUV5min 0.74 ± 0.03). Flow cytometry studies showed significantly higher binding of FITC-Ava-PSBP-6 to EL4 cells treated with camptothecin compared to untreated cells. Fluorescence microscopy studies revealed that FITC-Ava-PSBP-6 was binding to cell membranes of early apoptotic cells, but was internalized in late apoptotic and necrotic cells. The present study showed that radiotracer 64Cu-NOTA-Ava-PSBP-6 holds promise as a first peptide-based PET imaging agent for molecular imaging of apoptosis. However, additional “fine-tuning” of 64Cu-NOTA-Ava-PSBP-6 is required to enhance PS-binding potency and in vivo stability to improve tumor uptake and retention.
A noninvasive method of detecting exposure of phosphatidylserine (PS) on the external surface of the plasma membrane such as nuclear imaging could assist the diagnosis and therapy of apoptosis related pathologies. The most studied imaging agent for apoptosis is Annexin V so far. Because of limitations of Annexin V other agents have been introduced such as small peptides and molecules. Radiopeptides that have affinity and bind to PS are good candidates for noninvasive imaging of apoptosis. The LIKKPF, introduced by Burtea et al, with nanomolar affinity for PS, was used as templete. The biological properties of LIKKPF radiolabeled with Tc-99 m was assessed in-vitro using apoptotic Jurkat cells and in-vivo using mouse model of liver apoptosis. The radiolabeled LIKKPF with 99mTc was stable in human serum at 37˚C for at least 2 h. Results showed that the radiolabeled LIKKPF has less affinity to PS compare to original phage peptide, but high enough for specific binding to apoptotic cells in-vitro and in-vivo. It is concluded that the less affinity of radiolabeled LIKKPF might be attributed to hydrophobicity of peptide. The future peptides should be more hydrophobic compare to LIKKPF.
Focal cerebral ischemia, known as stroke, causes serious long-term disabilities globally. Effective therapy for cerebral ischemia demands a carrier that can penetrate the blood-brain barrier (BBB) and subsequently target the ischemia area in brain. Here, we designed a novel neuroprotectant (ZL006) loaded dual targeted nanocarrier based on liposome (T7&SHp-P-LPs/ZL006) conjugated with T7 peptide (T7) and stroke homing peptide (SHp) for penetrating BBB and targeting ischemia area, respectively. Compared with non-targeting liposomes, T7&SHp-P-LPs/ZL006 could transport across BCEC cells and significantly enhance cellular uptake and reduce cells apoptosis of excitatory amino acid stimulated PC-12 cells. However, there was no significant difference in cellular uptake between SHp-modified and plain liposomes when PC-12 cells were incubated without excitatory amino acid. Besides, ex vivo fluorescent images indicated that DiR labeled T7&SHp-P-LPs could efficiently transport across BBB and mostly accumulated in ischemic region rather than normal cerebral hemisphere of MCAO rats. Furthermore, T7&SHp-P-LPs/ZL006 could enhance the ability of in vivo anti-ischemic stroke of MCAO rats. These results demonstrated that T7&SHp-P-LPs could be used as a safe and effective dual targeted nanocarrier for ischemic stroke treatment.
In this study we have assessed the ability of two TAT-fused peptides PYC36D-TAT and JNKI-1 D-TAT (JNKI-1 or XG-102), which respectively inhibit jun proto-oncogene (c-Jun) and c-Jun N-terminal kinase (JNK) activation, to reduce infarct volume and improve functional outcome (adhesive tape removal) following permanent focal cerebral ischemia/pMCAO in Sprague Dawley rats. In addition, PYC36L-TAT fused to an ischemic brain homing peptide (HP-PYC36L-TAT) was also assessed. Prior to animal experiments all PYC36D-TAT, JNKI-1 D-TAT and HP-PYC36L-TAT peptide batches were tested in vitro and protected cortical neurons against glutamate excitotoxicity. Rats were treated intravenously in two separate trials. Trial 1 used high peptide doses (PYC36D-TAT: 500, 1000 or 1500 nmol/kg; JNKI-1D-TAT: 500, 1000 or 1500 nmol/kg; PYC36Dscrambled-TAT: 1120 nmol/kg) administered 1 hour after MCAO. Trial 2 used lower doses (PYC36D-TAT: 50 or 250 nmol/kg; HP-PYC36L-TAT: 250 nmol/kg; JNKI-1 D-TAT: 250 nmol/kg; D-TAT: 250 nmol/kg) administered 2 hours after MCAO. Contrary to other stroke animal studies, but in line with our previous findings, no treatment significantly reduced infarct volume or improved functional score measurements compared to vehicle (saline) treated animals when assessed 24 hours post-MCAO.
In vivo selection of phage display libraries was used to isolate peptides that home specifically to tumor blood vessels. When
coupled to the anticancer drug doxorubicin, two of these peptides—one containing an αv integrin–binding Arg-Gly-Asp motif and the other an Asn-Gly-Arg motif—enhanced the efficacy of the drug against human breast
cancer xenografts in nude mice and also reduced its toxicity. These results indicate that it may be possible to develop targeted
chemotherapy strategies that are based on selective expression of receptors in tumor vasculature.
Firstly discovered in rete testis fluid, clusterin is a glycoprotein present in most of the other biological fluids. Several isoforms of clusterin are encoded from a single gene located on chromosome 8 in human species. Among the different isoforms, the secreted form of clusterin is expressed by a variety of tissues, including the nervous system under normal conditions. This form is presumed to play an anti-apoptotic role and seems to be a major determinant in cell survival and neuroplasticity after stroke. In animal models of this pathology, both neuronal and astroglial subpopulations express high levels of clusterin early after the ischemic damage. Recent lines of evidence point also to its possible involvement in neurodegenerative disorders. It is thought that in Alzheimer's disease the association between amyloidogenic peptides and clusterin contributes to limit A species misfolding and facilitates their clearance from the extracellular space. Thus, intercellular and intracellular factors that modulate local clusterin expression in the nervous system may represent potent targets for neurodegenerative disease therapies. In this review we provide a critical overview of the most recent data on the involvement of clusterin in neurodegenerative diseases with special reference to their putative clinical relevance.
In this study we wished to determine whether technetium-99m annexin V, an in vivo marker of cellular injury and death, could be used to noninvasively monitor neuronal injury following focal middle cerebral artery (MCA) occlusion/reperfusion injury. Sixteen adult male Sprague-Dawley rats (along with four controls) underwent left (unilateral) MCA intraluminal beaded thread occlusion for 2 h followed by reperfusion. One hour following tail vein injection of 5-10 mCi of (99m)Tc-annexin V, animals underwent either single-photon emission computerized tomography (SPECT) or autoradiography followed by immunohistochemical analyses. There was abnormal, bilateral, multifocal uptake of (99m)Tc-annexin V in each cerebral hemisphere as seen by both SPECT and autoradiography at 4 h and 1, 3, and 7 days after initiation of occlusion. The average maximal annexin V uptake at 4 h was 310%+/-85% and 365%+/-151% above control values (P<0.006) within the right and left hemispheres, respectively, peaking on day 3 with values of 925%+/-734% and 1,194%+/-643% (P<0.03) that decreased by day 7 to 489%+/-233% and 785%+/-225% (P<0.01). Total lesional volume of the left hemisphere was 226%, 261%, and 451% ( P<0.03) larger than the right at 4, 24, and 72 h after injury, respectively. Annexin V localized to the cytoplasm of injured neurons ipsilateral to the site of injury as well as to otherwise normal-appearing neurons of the contralateral hemisphere as confirmed by dual fluorescent microscopy. It is concluded that there is abnormal bilateral, multifocal annexin V uptake, greater on the left than on the right side, within 4 h of unilateral left MCA ischemic injury and that the uptake peaks at 3 days and decreases by 7 days after injury. This pattern suggests that neuronal stress may play a role in the response of the brain to focal injury and be responsible for annexin V uptake outside the region of ischemic insult.
Vascular endothelial growth factor (VEGF), a potent angiogenic molecule specific for vascular endothelial cells, is overexpressed in most tumors and closely associated with tumor growth and metastasis. It has been shown that a soluble fragment of VEGF receptor Flt-1 (sFlt-1) has anti-angiogenic properties by way of its antagonist activity against VEGF. In the present study, we demonstrated that the stable expression of sFlt-1 by endothelial cell targeted non-viral gene delivery inhibited the angiogenesis of endothelial cells. A targeted polymeric gene delivery system, PEI-g-PEG-RGD, was developed by incorporating the alphanubeta3/alphanubeta5 integrin-binding RGD peptide, ACDCRGDCFC (single-letter amino acid code), into the cationic polymer, polyethylenimine (PEI) via a hydrophilic polyethylene glycol (PEG) spacer. The functional analysis of therapeutic gene encoding sFlt-1/carrier complex was performed with an endothelial cell proliferation assay. The complex of sFlt-1 gene with PEI-g-PEG-RGD conjugate efficiently inhibited the proliferation of cultured endothelial cells, representing that expressed sFlt-1 predominantly bound to exogenous VEGF and blocked the binding of VEGF to the full-length Flt-1 receptor. These findings suggest that the combination of targeted gene carrier and sFlt-1 possesses the potential to be an efficient tool for the anti-angiogenic gene therapy to treat cancer.
Atherosclerosis, formerly considered a bland lipid storage disease, actually involves an ongoing inflammatory response. Recent advances in basic science have established a fundamental role for inflammation in mediating all stages of this disease from initiation through progression and, ultimately, the thrombotic complications of atherosclerosis. These new findings provide important links between risk factors and the mechanisms of atherogenesis. Clinical studies have shown that this emerging biology of inflammation in atherosclerosis applies directly to human patients. Elevation in markers of inflammation predicts outcomes of patients with acute coronary syndromes, independently of myocardial damage. In addition, low-grade chronic inflammation, as indicated by levels of the inflammatory marker C-reactive protein, prospectively defines risk of atherosclerotic complications, thus adding to prognostic information provided by traditional risk factors. Moreover, certain treatments that reduce coronary risk also limit inflammation. In the case of lipid lowering with statins, this anti-inflammatory effect does not appear to correlate with reduction in low-density lipoprotein levels. These new insights into inflammation in atherosclerosis not only increase our understanding of this disease, but also have practical clinical applications in risk stratification and targeting of therapy for this scourge of growing worldwide importance.
Imaging or drug delivery tools for atherosclerosis based on the plaque biology are still insufficient. Here, we attempted to identify peptides that selectively home to atherosclerotic plaques using phage display. A phage library containing random peptides was ex vivo screened for binding to human atheroma tissues. After three to four rounds of selection, the DNA inserts of phage clones were sequenced. A peptide sequence, CRKRLDRNC, was the most frequently occurring one. Intravenously injected phage displaying the CRKRLDRNC peptide was observed to home to atherosclerotic aortic tissues of low-density lipoprotein receptor-deficient (Ldlr-/-) mice at higher levels than to normal aortic tissues of wild-type mice. Moreover, a fluorescein- or radioisotope-conjugated synthetic CRKRLDRNC peptide, but not a control peptide, homed in vivo to atherosclerotic plaques in Ldlr((-/-)) mice, while homing of the peptide to other organs such as brain was minimal. The homing peptide co-localized with endothelial cells, macrophages, and smooth muscle cells at mouse and human atherosclerotic plaques. Homology search revealed that the CRKRLDRNC peptide shares a motif of IL-4 that is critical for binding to its receptor. The peptide indeed co-localized with IL-4 receptor (IL-4R) at atherosclerotic plaques. Moreover, the peptide bound to cultured cells expressing IL-4R on the cell surface and the binding was inhibited by the knockdown of IL-4R. These results show that the CRKRLDRNC peptide homes to atherosclerotic plaques through binding to IL-4R as its target and may be a useful tool for selective drug delivery and molecular imaging of atherosclerosis.
There is considerable evidence that complement activation occurs within the CNS in inflammatory and degenerative disorders, but little is known about its involvement in the pathophysiology of cerebral ischemia. Our study sought to characterize the glial response and the expression of complement factors after permanent focal cerebral ischemia in the mouse, using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. mRNA expression of glial fibrillary acidic protein (GFAP) increased at day 1 and peaked 3 days after middle cerebral artery (MCA) occlusion in the perifocal area. Immunohistochemical staining for GFAP indicated that astroglia were activated the day after MCA occlusion. Microglial activation, as assessed by lectin-binding experiments, increased by 1 day after MCA occlusion in the perifocal area and peaked at 3 days postocclusion. RT-PCR experiments demonstrated an increased expression of clusterin, C1qB, and C4 mRNA in the ischemic cortex, with a peak level at 3 days after MCA occlusion. Clusterin, C1qB, and C4 mRNA were located in the perifocal area, as assessed by in situ hybridization. Reactive astrocytes within the cortex medial to the ischemic lesion were found to be strongly immunoreactive for clusterin. In addition, we observed C1q-positive macrophage-like cells within the infarcted core at 3 days postocclusion. At 7 days after the onset of ischemia, increased C4 immunostaining was restricted to perifocal neurons. We conclude that local expression of complement components may contribute to the inflammation observed in this model, thereby representing an important process in secondary injury mechanisms after focal cerebral ischemia. GLIA 31:39–50, 2000. © 2000 Wiley-Liss, Inc.
Clusterin has been implicated in cell death both in peripheral tissues and in the central nervous system. In the present study, expression of clusterin in the cerebellar cortex was examined in two cases with hypoxic brain damage and in one case with cerebellar infarction. Intense staining of Purkinje cells was observed in each case, and these cells showed the shrunken and pyknotic appearance characteristic of irreversible ischaemic damage. In the cerebella of neurologically normal control cases, as well as in those of some other neurodegenerative diseases, no staining or only punctate staining of Purkinje cells was observed. The results provide additional evidence supporting an association of clusterin with dying neurons in human brain.
Preferential homing of tumour cells and leukocytes to specific organs indicates that tissues carry unique marker molecules accessible to circulating cells. Organ-selective address molecules on endothelial surfaces have been identified for lymphocyte homing to various lymphoid organs and to tissues undergoing inflammation, and an endothelial marker responsible for tumour homing to the lungs has also been identified. Here we report a new approach to studying organ-selective targeting based on in vivo screening of random peptide sequences. Peptides capable of mediating selective localization of phage to brain and kidney blood vessels were identified, and showed up to 13-fold selectivity for these organs. One of the peptides displayed by the brain-localizing phage was synthesized and shown to specifically inhibit the localization of the homologous phage into the brain. When coated onto glutaraldehyde-fixed red blood cells, the peptide caused selective localization of intravenously injected cells into the brain. These peptide sequences represent the first step towards identifying selective endothelial markers, which may be useful in targeting cells, drugs and genes into selected tissues.
Phage displaying an Arg-Gly-Asp (RGD)-containing peptide with a high affinity for alpha v integrins homed to tumors when injected intravenously into tumor-bearing mice. A substantially higher amount of alpha v-directed RGD phage than control phage was recovered from malignant melanomas and breast carcinoma. Antibodies detected the alpha v-directed RGD phage in tumor blood vessels, but not in several normal tissues. These results show that the alpha v integrins present in tumor blood vessels can bind circulating ligands and that RGD peptides selective for these integrins may be suitable tools in tumor targeting for diagnostic and therapeutic purposes.
Brain injury following transient or permanent focal cerebral ischaemia (stroke) develops from a complex series of pathophysiological events that evolve in time and space. In this article, the relevance of excitotoxicity, peri-infarct depolarizations, inflammation and apoptosis to delayed mechanisms of damage within the peri-infarct zone or ischaemic penumbra are discussed. While focusing on potentially new avenues of treatment, the issue of why many clinical stroke trials have so far proved disappointing is addressed. This article provides a framework that can be used to generate testable hypotheses and treatment strategies that are linked to the appearance of specific pathophysiological events within the ischaemic brain.
Contrary to previous dogmas, it is now well established that brain cells can produce cytokines and chemokines, and can express adhesion molecules that enable an in situ inflammatory reaction. The accumulation of neutrophils early after brain injury is believed to contribute to the degree of brain tissue loss. Support for this hypothesis has been drawn from many studies where neutrophil-depletion blockade of endothelial-leukocyte interactions has been achieved by various techniques. The inflammation reaction is an attractive pharmacologic opportunity, considering its rapid initiation and progression over many hours after stroke and its contribution to evolution of tissue injury. While the expression of inflammatory cytokines that may contribute to ischemic injury has been repeatedly demonstrated, cytokines may also provide "neuroprotection" in certain conditions by promoting growth, repair, and ultimately, enhanced functional recovery. Significant additional basic work is required to understand the dynamic, complex, and time-dependent destructive and protective processes associated with inflammation mediators produced after brain injury. The realization that brain ischemia and trauma elicit robust inflammation in the brain provides fertile ground for discovery of novel therapeutic agents for stroke and neurotrauma. Inhibition of the mitogen-activated protein kinase (MAPK) cascade via cytokine suppressive anti-inflammatory drugs, which block p38 MAPK and hence the production of interleukin-1 and tumor necrosis factor-alpha, are most promising new opportunities. However, spatial and temporal considerations need to be exercised to elucidate the best opportunities for selective inhibitors for specific inflammatory mediators.
There is considerable evidence that complement activation occurs within the CNS in inflammatory and degenerative disorders, but little is known about its involvement in the pathophysiology of cerebral ischemia. Our study sought to characterize the glial response and the expression of complement factors after permanent focal cerebral ischemia in the mouse, using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. mRNA expression of glial fibrillary acidic protein (GFAP) increased at day 1 and peaked 3 days after middle cerebral artery (MCA) occlusion in the perifocal area. Immunohistochemical staining for GFAP indicated that astroglia were activated the day after MCA occlusion. Microglial activation, as assessed by lectin-binding experiments, increased by 1 day after MCA occlusion in the perifocal area and peaked at 3 days postocclusion. RT-PCR experiments demonstrated an increased expression of clusterin, C1qB, and C4 mRNA in the ischemic cortex, with a peak level at 3 days after MCA occlusion. Clusterin, C1qB, and C4 mRNA were located in the perifocal area, as assessed by in situ hybridization. Reactive astrocytes within the cortex medial to the ischemic lesion were found to be strongly immunoreactive for clusterin. In addition, we observed C1q-positive macrophage-like cells within the infarcted core at 3 days postocclusion. At 7 days after the onset of ischemia, increased C4 immunostaining was restricted to perifocal neurons. We conclude that local expression of complement components may contribute to the inflammation observed in this model, thereby representing an important process in secondary injury mechanisms after focal cerebral ischemia.
With the advent of thrombolytic therapy for acute stroke, reperfusion-associated mechanisms of tissue injury have assumed greater importance. In this experimental study, we used several MRI techniques to monitor the dynamics of secondary ischemic damage, blood-brain barrier (BBB) disturbances, and the development of vasogenic edema during the reperfusion phase after focal cerebral ischemia in rats.
Nineteen Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion of 30 minutes, 60 minutes, or 2.5 hours with the suture occlusion model. MRI, including diffusion-weighted imaging (DWI), T2-weighted imaging, perfusion-weighted imaging, and T1-weighted imaging, was performed 5 to 15 minutes before reperfusion, as well as 0.5, 1.5, and 2.5 hours and 1, 2, and 7 days after withdrawal of the suture. Final infarct size was determined histologically at 7 days.
In the 30-minute ischemia group (and partially also after 60 minutes), DWI abnormalities reversed transiently during the early reperfusion period but recurred after 1 day, probably due to secondary ischemic damage. After 2.5 hours of ischemia, DWI abnormalities no longer reversed, and signal intensity on both DWI and T2-weighted images increased rapidly in the previously ischemic region due to BBB damage (enhancement on postcontrast T1-weighted images) and edema formation. Early BBB damage during reperfusion was found to be predictive of relatively pronounced edema at subacute time points and was probably related to the increased mortality rates in this experimental group (3 of 7).
Reperfusion after short periods of ischemia (30 to 60 minutes) appears to be mainly complicated by secondary ischemic damage as shown by the delayed recurrence of the DWI lesions, whereas BBB damage associated with vasogenic edema becomes a dominant factor with longer occlusion times (2.5 hours).
Active treatment of acute ischaemic stroke can only be successful as long as tissue in the area of ischaemic compromise is still viable. Therefore, the identification of the area of irreversible damage, and its distinction from the penumbral zone, may improve the estimation of the potential efficacy of various therapeutic strategies. Ten patients (seven male, three female, aged 52-75 years) with acute ischaemic stroke, in whom MRI delineated an infarct involving the cortex 3 weeks after the attack, were studied by [(11)C]flumazenil (FMZ) PET to assess their neuronal integrity, and regional cerebral blood flow (CBF) was measured by H(2)(15)O PET 2-12 h (median interval 6 h) after onset of symptoms. Cortical volumes of interest (3 mm radius) were placed on co-registered CBF, FMZ and on late MRI scans. Using initial CBF and FMZ binding data from volumes of interest finally located within or outside the cortical infarct, cumulative probability curves were computed to predict eventual infarction or non-infarction. Positive (at least 95% chance of infarct) and negative (at least 95% chance of non-infarct) prediction limits for CBF (4.8 and 14.1 ml/100 g/min, respectively) and for FMZ binding (3.4 and 5.5 times the mean of normal white matter, respectively) were determined to define the penumbral range. Using the lower FMZ binding threshold of 3.4 for irreversible tissue damage and the upper CBF value of 14.1 ml/ 100 g/min for the threshold of critical perfusion at or above which tissue will likely be preserved, various cortical subcompartments were identified: of the final cortical infarct (median size 25.7 cm(3)) a major portion comprising, on average, 55.1% showed FMZ binding critically decreased, thus predicting necrosis. In 20.5% of the final infarct, on average, CBF was in the penumbral range (<14.1 ml/100 g/min) and FMZ binding was above the critical threshold of irreversible damage. Only 12.9% of the final infarct exhibited neuronal integrity and CBF values above the penumbral range. Therefore, most of the final infarct is irreversibly damaged already at the time of the initial evaluation, when studied several hours after stroke onset. A much smaller portion is still viable but suffers from insufficient blood supply: this tissue may be salvaged by effective reperfusion. Only an even smaller compartment is viable and sufficiently perfused, but eventually becomes necrotic, mainly owing to delayed mechanisms, and may benefit from neuroprotective or other measures targeted at secondary damage. Therefore, early reperfusion is crucial in acute ischaemic stroke.
Clusterin is an enigmatic glycoprotein with a nearly ubiquitous tissue distribution and an apparent involvement in biological processes ranging from mammary gland involution to neurodegeneration in Alzheimer's disease. Its major form, a 75-80 kDa heterodimer, is secreted and present in physiological fluids, but truncated forms targeted to the nucleus have also been identified. Upregulation of clusterin mRNA and protein levels detected in diverse disease states and in in vitro systems have led to suggestions that it functions in membrane lipid recycling, in apoptotic cell death, and as a stress-induced secreted chaperone protein, amongst others. Recent studies of knockout mice have further complicated the picture by implicating clusterin in exacerbating neuronal death in hypoxia-ischemia. The question of whether clusterin is a multifunctional protein, or deploys a single primary function influenced by cellular context, remains a central issue continuing to stimulate interest in this unusual molecule.
Thrombolysis is the treatment of choice for acute stroke within 3 hours after symptom onset. Treatment beyond the 3-hour time window has not been shown to be effective in any single trial; however, meta-analyses suggest a somewhat lesser but still significant effect within 3 to 6 hours after stroke. It seems reasonable to apply improved selection criteria that allow differentiation between patients with and without a relevant indication for thrombolytic therapy.
The present literature on imaging in stroke has been thoroughly reviewed, covering Doppler ultrasound (DU), arteriography, CT, and MRI and including modern techniques such as perfusion CT, diffusion- and perfusion-weighted MRI (DWI, PWI), CT angiography and MR angiography (CTA, MRA), and CTA source image analysis (CTA-SI). The authors present their view of a comprehensive diagnostic approach to acute stroke, which challenges the concept of a rigid therapeutic time window.
Information about the presence or absence of a vessel occlusion, whether by means of DU, CTA, or MRA, is essential before recombinant tissue plasminogen activator is given in the 3- to 6-hour time window. Clear demarcation of the irreversibly damaged infarct core and the ischemic but still viable and thus salvageable tissue at risk of infarction as seen on DWI/PWI/MRA or alternatively CT/CTA/CTA-SI should be obtained before thrombolysis is initiated within 3 to 6 hours. Once these advanced techniques are used, the therapeutic time window can be extended with acceptable safety. However, comprehensive informed consent is mandatory, especially when thrombolytic therapy is considered beyond established time windows.
Tumour blood vessels express markers that are not present in resting blood vessels of normal tissues, but that can be shared by angiogenic vessels in non-malignant conditions. Many of these proteins are functionally important in the angiogenic process. Some tumours also contain lymphatic vessels, as well as channels that consist of cancer cells and their extracellular matrix. These special features of tumour vessels are good targets for cancer therapies.
Angiogenesis is a key process in the growth and metastasis of a tumor. Disrupting this process is considered a promising treatment strategy. Therefore, a drug delivery system specifically aiming at angiogenic tumor endothelial cells was developed. Alpha v beta 3-integrins are overexpressed on actively proliferating endothelium and represent a possible target. For this, RGD-peptides with affinity for this integrin were coupled to the distal end of poly(ethylene glycol)-coated long-circulating liposomes (LCL) to obtain a stable long-circulating drug delivery system functioning as a platform for multivalent interaction with alpha v beta 3-integrins. The results show that cyclic RGD-peptide-modified LCL exhibited increased binding to endothelial cells in vitro. Moreover, intravital microscopy demonstrated a specific interaction of these liposomes with tumor vasculature, a characteristic not observed for LCL. RGD-LCL encapsulating doxorubicin inhibited tumor growth in a doxorubicin-insensitive murine C26 colon carcinoma model, whereas doxorubicin in LCL failed to decelerate tumor growth. In conclusion, coupling of RGD to LCL redirected these liposomes to angiogenic endothelial cells in vitro and in vivo. RGD-LCL containing doxorubicin showed superior efficacy over non-targeted LCL in inhibiting C26 doxorubicin-insensitive tumor outgrowth. Likely, these RGD-LCL-doxorubicin antitumor effects are brought about through direct effects on tumor endothelial cells.
Historically, in vivo imaging methods have largely relied on imaging gross anatomy. More recently it has become possible to depict biological processes at the cellular and molecular level. These new research methods use magnetic resonance imaging (MRI), positron emission tomography (PET), near-infrared optical imaging, scintigraphy, and autoradiography in vivo and in vitro. Of primary interest is the development of methods using MRI and PET with which the progress of gene therapy in glioblastoma (herpes simplex virus-thymidine kinase) and Parkinson's disease can be monitored and graphically displayed. The distribution of serotonin receptors in the human brain and the duration of serotonin-receptor antagonist binding can be assessed by PET. With PET, it is possible to localize neurofibrillary tangles (NFTs) and beta-amyloid senile plaques (APs) in the brains of living Alzheimer disease (AD) patients. MR tracking of transplanted oligodendrocyte progenitors is feasible for determining the extent of remyelinization in myelin-deficient rats. Stroke therapy in adult rats with subventricular zone cells can be monitored by MRI. Transgene expression (beta-galactosidase, tyrosinase, engineered transferrin receptor) can also be visualized using MRI. Macrophages can be marked with certain iron-containing contrast agents which, through accumulation at the margins of glioblastomas, ameliorate the visual demarcation in MRI. The use of near-infrared optical imaging techniques to visualize matrix-metalloproteinases and cathepsin B can improve the assessment of tumor aggressiveness and angiogenesis-inhibitory therapy. Apoptosis could be detected using near-infrared optical imaging representation of caspase 3 activity and annexin B. This review demonstrates the need for neurohistological research if further progress is to be made in the emerging but burgeoning field of molecular imaging.
Peptide-based drugs are now viable alternatives to biopharmaceuticals, such as antibodies. Most of the past limitations of peptides have been removed by new technologies, so that peptides now face similar hurdles to antibodies. Phage-display technology provides novel peptides that bind protein targets with high affinity and specificity. Most marketed peptide-based drugs are receptor agonists derived from natural peptides. To address the need for antagonists, novel strategies have been developed for inhibiting receptor-ligand interactions. We review results from phage display in finding peptide drug candidates and conclude with some business benefits of developing peptides.
Diffusion-weighted MRI (DWI) in combination with perfusion-weighted MRI (PWI) has become a widely accepted modality for the selection of patients amenable for acute therapy, if a mismatch between these procedures suggests viable penumbral tissue. However, DWI as well as PWI yields semiquantitative measures limiting the definitions of irreversible damage and of potentially viable penumbral tissue. These limitations of PWI/DWI may be better understood if findings in individual patients are compared with the results from measurements of blood flow, oxygen metabolism, and benzodiazepine receptor binding obtained with positron emission tomography (PET). Comparative studies with PET and MRI were performed in 3 groups of patients: (1) In 12 acute stroke patients, results from DWI (median, 6.5 hours after symptom onset) and 11C-flumazenil (FMZ) PET (median, 85 minutes between DWI and PET) were compared with infarct extension 24 to 48 hours later on T2-weighted MRI. (2) In 11 acute stroke patients, results from PWI (median, 8 hours after symptom onset) were compared with cerebral blood flow measurements obtained with [15O]H2O PET (interval, 60 minutes between PWI and PET). (3) In 10 patients with acute (n=5) or chronic stroke (n=5), results from PWI/DWI were compared with PET of cerebral blood flow and oxygen consumption to detect mismatch or increased oxygen extraction fraction as surrogate markers of penumbra. Results were: (1) from regions with increased DWI intensity, decreased apparent diffusion coefficient (ADC) and decreased FMZ binding probability curves were computed for eventual infarction, and 95% prediction limits were determined. These limits predicted 83.5% (FMZ), 84.7% (DWI), and 70.9% (ADC) of the final infarct volume. However, the false-positive predictions were much higher for the DWI variables (5.1 and 3.6 cm3 for DWI and ADC versus a median of 0 for FMZ). (2) The comparison of volumes generated by different time to peak (TTP) thresholds (PWI) and hypoperfusion <20 mL/100 g per minute (PET) indicates that a TTP delay of 4 to 6 seconds yields a fair estimate of hypoperfusion. (3) The PWI/DWI mismatch with TTP >4 seconds did not reliably correspond to the penumbra as assessed by PET (oxygen extraction fraction >150%). Only 6 of 10 patients with a mismatch had areas of penumbra. In these cases, the penumbra volume was overestimated by MRI. DWI correlates with FMZ results and, with a few exceptions, yields a good estimate of acute tissue damage and final infarct volume. PWI measures seem to be less reliable; the TTP prolongation of >4 seconds assessed only 83% of the volume of hypoperfusion <20 mL/100 g per minute. The mismatch volume imprecisely depicts increased oxygen extraction fraction, and, despite its clinical role for selection of patients for eventual therapy, it does not to seem to be a reliable correlate of penumbra.
Identification of the ischemic penumbra in the acute stroke clinical setting is an important goal for stroke researchers and clinicians. Various models for imaging the penumbra with MRI have been proposed, including the pioneering diffusion-perfusion mismatch model and later multivariate approaches. A number of multicenter clinical trials are now under way to test these models and confirm the utility of MRI-based treatment decisions. Present knowledge about MRI visualization of the salvageable penumbra suggests a promising future in which MRI studies are performed routinely in the acute stroke setting and the data provided by these MRI studies assist in individualizing therapeutic decisions and identifying effective therapies that can be delivered at late time points.
Sites of neovascular angiogenesis are important chemotherapy targets. In this study, the synthesis, characterization, in-vivo imaging and biodistribution of a technetium-99m labeled, water-soluble, N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer carrying doubly cyclized Arg-Gly-Asp motifs (HPMA copolymer-RGD4C conjugate) are reported. In vitro endothelial cell adhesion assays indicated that HPMA copolymer-RGD4C conjugates inhibited alphaVbeta3-mediated endothelial cell adhesion while HPMA copolymer Arg-Gly-Glu control conjugates (HPMA copolymer-RGE4C conjugate) and hydrolyzed HPMA copolymer precursor (HPMA copolymer) showed no activity. The scintigraphic images of prostate tumor bearing SCID mice obtained 24 h post-i.v. injection indicated greater tumor localization of HPMA copolymer-RGD4C conjugate than the control, HPMA copolymer-RGE4C conjugate. The 24-h necropsy radioactivity data showed that HPMA copolymer-RGD4C conjugate had significantly higher (p<0.001) tumor localization compared to HPMA copolymer-RGE4C conjugate and HPMA copolymer. Also, HPMA copolymer-RGD4C conjugates had sustained tumor retention over 72 h and reasonably efficient clearance from the background organs. These results suggest that specific tumor angiogenesis targeting is possible with HPMA copolymer-RGD4C conjugates. This construct provides a foundation that should support targeted delivery of radionuclides and drugs to solid tumors for diagnostic and therapeutic applications.
Since the introduction of thrombolytic therapy as the foundation of acute stroke treatment, neuroimaging has rapidly advanced to empower therapeutic decision making. Diffusion-weighted imaging is the most sensitive and accurate method for stroke detection, and, allied with perfusion-weighted imaging, provides information on the functional status of the ischemic brain. It can also help to identify a response to thrombolytic and neuroprotective therapies. Additionally, multimodal magnetic resonance imaging, including magnetic resonance angiography, offers information on stroke mechanism and pathophysiology that can guide long-term medical management. Multimodal computed tomography is a comprehensive, cost-effective, and safe stroke imaging modality that can be easily implemented in the emergency ward and that offers fast and reliable information with respect to the arterial and functional status of the ischemic brain. Accessibility, contraindications, cost, speed, and individual patient-determined features influence which is the best imaging modality to guide acute stroke management.
We wished to determine the ability of radiolabeled annexin V to concentrate at sites of ischemic injury in patients with acute cerebral stroke. Secondly, we sought to correlate annexin V imaging in these patients with the degree of blood-brain barrier (BBB) breakdown.
Twelve patients with acute stroke had a complete neurological examination, including the National Institutes of Health (NIH) stroke scale and the Glasgow Coma Score (GCS). A non-contrast CT scan was performed on all patients. A SPECT of the brain was obtained 2 h after injection of annexin V. The integrity of the BBB was evaluated in seven patients using Tc-99m-DTPA brain SPECT.
All patients had an infarct in the MCA territory. Eight patients had abnormal increased annexin V activity, which was more common in patients with cortical strokes (P = 0.01). The concentration of annexin had no correlation to the volume of stroke, but it was significantly and inversely related to the GCS on admission (r = -0.7, P = 0.02). Foci of apoptosis were noted contralateral to the affected hemisphere as well. All seven patients who underwent DTPA SPECT showed breakdown of the BBB. DTPA uptake was significantly and positively associated with NIH score (r = 0.80, P = 0.01) and inversely associated with GCS (r = -0.89, P = -0.03).
This study shows that it is possible to identify in vivo regions of ischemic neuronal injury using radiolabeled annexin V in patients with acute stroke. Annexin imaging can play a major role in the selection of therapy in the initial period following stroke in adults.
Stroke, a disorder encompassing all cerebrovascular accidents, is a public health problem of immense proportions across the globe. Therapeutic efforts are directed at three aspects: prevention, acute treatment, and rehabilitation. Preventative measures, which in many instances mirror those for cardiovascular disease, can achieve the greatest public health impact. Measures that enhance the recovery of neurologic function and reduce neurologic disability after stroke can also affect a large population of handicapped stroke survivors. In the past 10 years, the greatest changes have occurred in the field of acute stroke treatment. Ultra-early-stage therapies with the potential to dramatically reverse severe neurologic deficits, or halt their progression, have caused a restructuring of the emergency care of neurologic patients. The parallels with the evolution of emergency treatment of acute coronary syndromes after 1970 are striking. This review focuses on aspects of stroke therapy that are either just entering, or soon to enter, current practice.
Bladder cancer is one of the most common tumors of the genitourinary tract. Here, we use phage display to identify a peptide that targets bladder tumor cells. A phage library containing random peptides was screened for binding to cells from human bladder tumor xenografts. Phage clones were further selected for binding to a bladder tumor cell line in culture. Six clones displaying the consensus sequence CXNXDXR(X)/(R)C showed selective binding to cells from primary human bladder cancer tissue. Of these, the CSNRDARRC sequence was selected for further study as a synthetic peptide. Fluorescein-conjugated CSNRDARRC peptide selectively bound to frozen sections of human bladder tumor tissue, whereas only negligible binding to normal bladder tissue was observed. When the fluorescent peptide was introduced into the bladder lumen, in a carcinogen-induced rat tumor model, it selectively bound to tumor epithelium. Moreover, when the peptide was intravenously injected into the tail vein, it homed to the bladder tumor but was not detectable in normal bladder and control organs. Next, we examined whether the peptide can detect tumor cells in urine. The fluorescent peptide bound to cultured bladder tumor cells but not to other types of tumor cell lines. Moreover, it bound to urinary cells of patients with bladder cancer, while showing little binding to urinary cells of patients with inflammation or healthy individuals. The CSNRDARRC peptide may be useful as a targeting moiety for selective delivery of therapeutics and as a diagnostic probe for the detection of bladder cancer.
Cell death is the basic neuropathological substrate in cerebral ischemia, and its non-invasive imaging may improve diagnosis and treatment for stroke patients. ApoSense is a novel family of low-molecular weight compounds for detection and imaging of cell death in vivo. We now report on imaging of cell death and monitoring of efficacy of neuroprotective treatment in vivo by intravenous administration of the ApoSense compound DDC (didansylcystine), in experimental stroke in rodents. Rats and mice were subjected to a short-term (2 h) or permanent occlusion of the middle cerebral artery (MCA) and injected with DDC or 3H-labeled DDC. Fluorescent and autoradiographic studies, respectively, were performed ex vivo, comprising assessment of DDC uptake in the infarct region, in correlation with tissue histopathology. Neuroprotection was induced by a caspase inhibitor (Q-VD-OPH), and its effect was monitored by DDC. Following its intravenous administration, DDC accumulated selectively in injured neurons within the region of infarct. Caspase inhibition exerted a 45% reduction in infarct volume, which was well reported by DDC. This is the first report on a small molecule probe for detection in vivo of cell death in cerebral stroke. DDC may potentially assist in addressing the current "neuroimaging/neurohistology gap", for molecular assessment of the extent of stroke-related cell death.
Ischemic stroke is a devastating disease with a complex pathophysiology. Animal modeling of ischemic stroke serves as an indispensable tool first to investigate mechanisms of ischemic cerebral injury, secondly to develop novel antiischemic regimens. Most of the stroke models are carried on rodents. Each model has its particular strengths and weaknesses. Mimicking all aspects of human stroke in one animal model is not possible since ischemic stroke is itself a very heterogeneous disorder. Experimental ischemic stroke models contribute to our understanding of the events occurring in ischemic and reperfused brain. Major approaches developed to treat acute ischemic stroke fall into two categories, thrombolysis and neuroprotection. Trials aimed to evaluate effectiveness of recombinant tissue-type plasminogen activator in longer time windows with finer selection of patients based on magnetic resonance imaging tools and trials of novel recanalization methods are ongoing. Despite the failure of most neuroprotective drugs during the last two decades, there are good chances to soon have effective neuroprotectives with the help of improved preclinical testing and clinical trial design. In this article, we focus on various rodent animal models, pathogenic mechanisms, and promising therapeutic approaches of ischemic stroke.
The objective of this article is to illustrate both the potential and the limitations of molecular imaging in stroke research. By molecular imaging we mean the visual representation of biological processes at the cellular and molecular level. The use of molecular imaging for stroke diagnosis is still at a very preliminary stage and many of these procedures have only been tested in animals. In rats, stroke therapy using stem cells can be monitored by magnetic resonance imaging (MRI), green fluorescent protein (GFP) or luciferase (LUC) imaging. The migration of macrophages, which take up intravenously administered iron-based contrast agents and then migrate to the area of infarction, can already be observed in stroke patients. With MRI, the new agent Gd-DTPA-sLexA that binds to E- and P-selectin can specifically visualize selectin-mediated early endothelial activation after transient focal ischemia "in vivo". Decreased glial fibrillary acidic protein (GFAP) gene expression can be imaged in vivo by scintigraphy 24 hours after cerebral ischemia using a peptide nucleic acid antisense conjugate labeled with 111In and that hybridizes to the rat GFAP mRNA. Technetium-99m hydrazine nicotinamide-labeled HYNIC-annexin V SPECT can not only detect sites of neuronal injury in stroke patients but also can monitor the effects of neuroprotective therapy with a monoclonal antibody raised against FasLigand (FasL) in rats. Finally, information about cell metabolism in the infarct region can be gained using certain intracellular tracers [e.g. 18F-fluoromisonidazole (FMISO)]. Imaging benzodiazepine receptors with 11C-flumazenil (FMZ) can distinguish between irreversibly damaged and viable penumbra tissue early after stroke.
Alpha v integrins as receptors for tumor targeting by circulating ligands
- Pasqualini
Inflammatory mediators and stroke: new opportunities for novel therapeutics
- Barone
Imaging-based decision making in thrombolytic therapy for ischemic stroke: present status
- Schellinger