[Show abstract][Hide abstract] ABSTRACT: Introduction
Current methods for early diagnosis of osteoarthritis (OA) are limited. We assessed whether in vivo detection of chondrocyte death by ApoPep-1 (CQRPPR), a peptide that binds to histone H1 of apoptotic and necrotic cells, could be used to detect the initiation of OA.
Apoptosis-induced ATDC5 cells were labeled with Annexin V and ApoPep-1. Surgical destabilization of the medial meniscus (DMM) was performed on both knees of 12-week-old male mice and severity of OA was determined by histological analysis according to the Osteoarthritis Research Society International (OARSI) guidelines. At 1, 2, 4, and 8 weeks post-surgery, mice were intravenously injected with fluorescence-labeled ApoPep-1 or control peptide and in vivo imaging was performed within 30 minutes of injection by near-infrared fluorescence (NIRF). Binding of ApoPep-1 to OA joints was demonstrated by ex vivo imaging and immunofluorescent staining using TUNEL and histone H1 and type II collagen antibodies.
Strong signals of ApoPep-1 were observed on the apoptotic ATDC5 cells. Knees corresponded to grade II, III, and V OA at 2, 4, and 8 weeks after DMM, respectively. Between 2 and 8 weeks after surgery, the in vivo NIRF signal at OA-ApoPep1-injected joints was consistently stronger than sham-operated or OA-control peptide-injected joints. ApoPep-1, TUNEL, and histone H1 signals were stronger in grade II OA cartilage than sham-operated cartilage when detected by immunofluorescent staining. Type II collagen expression was similar between grade II OA and sham group.
ApoPep-1 can be used to detect OA in vivo by binding to apoptotic chondrocytes. This is a novel, sensitive, and rapid method which can detect apoptotic cells in OA rodent models soon after its onset.
[Show abstract][Hide abstract] ABSTRACT: In this study, a simple, highly sensitive electrochemical biosensor for myoglobin was developed using a myoglobin-specific binding peptide as a sensing probe. A peptide (Myo-3R7, CPSTLGASC, 838 Da) identified by phage display and that specifically binds to myoglobin was covalently immobilized on a gold electrode functionalized via a dithiobis(succinimidyl propionate) (DSP) self-assembled monolayer (SAM). The peptide immobilization was confirmed with fluorescence microarray scanning and cyclic voltammetry (CV). The electrochemical performance of the biosensor with respect to myoglobin was characterized by CV and differential pulse voltammetry (DPV) using Fe(CN)6(3-)/Fe(CN)6(4-) as a redox probe. We successfully detected myoglobin in a broad working range of 17.8 to 1780 ng mL(-1) with a correlation coefficient (R(2)) of 0.998. The estimated limit of detection (LOD) was fairly low, 9.8 ng mL(-1) in 30 min. The electrochemical biosensor based on a myoglobin-specific binding peptide offers sensitivity, selectivity, and rapidity, making it an attractive tool for the early detection of cardiac infarction.
[Show abstract][Hide abstract] ABSTRACT: A number of peptide-based indicators have been identified and reported as potential apoptosis probes, offering great promise for early assessment of therapeutic efficacy in several types of cancer. Direct comparison of the newly developed probes with previously used ones would be an important step in assessing possible applications. Here, we compared the newly identified peptide-based phosphatidylserine (PS) indicator PSP1 (CLSYYPSYC) with annexin V, a common probe for molecular imaging of apoptotic cells, with respect to PS binding kinetics, apoptotic cell-targeting ability, and the efficacy of homing to apoptotic tumor cells in a mouse model after treatment with the anticancer agent camptothecin. Our results indicate that PSP1 efficiently targeted apoptotic cells and generated apoptosis/tumor-specific signals after cancer treatment in the animal model, whereas a similar dose of annexin V showed weak signals. The formation of a stable complex of PSP1 with PS might be one reason for the efficient in vivo targeting. We suggest that PSP1 has potential advantages for in vivo apoptotic cell imaging and could serve as a platform for the development of de novo peptide-based probes for apoptosis.
PLoS ONE 03/2015; 10(3):e0121171. DOI:10.1371/journal.pone.0121171 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We developed and tested a multicomponent peptide-woven siRNA nanocomplex (PwSN) comprising different peptides designed for efficient cellular targeting, endosomal escape, and release of siRNA. To enhance tumor-specific cellular uptake, we connected an interleukin-4 receptor-targeting peptide (I4R) to a nine-arginine peptide (9r), yielding I4R-9r. To facilitate endosomal escape, we blended endosomolytic peptides into the I4R-9r to form a multicomponent nanocomplex. Lastly, we modified 9r peptides by varying the number and positions of positive charges to obtain efficient release of siRNA from the nanocomplex in the cytosol. Using this step-wise approach for overcoming the biological challenges of siRNA delivery, we obtained an optimized PwSN with significant biological activity in vitro and in vivo. Interestingly, surface plasmon resonance analyses and three-dimensional peptide models demonstrated that our designed peptide adopted a unique structure that was correlated with faster complex disassembly and a better gene-silencing effect. These studies further elucidate the siRNA nanocomplex delivery pathway and demonstrate the applicability of our stepwise strategy to the design of siRNA carriers capable of overcoming multiple challenges and achieving efficient delivery.
PLoS ONE 02/2015; 10(2):e0118310. DOI:10.1371/journal.pone.0118310 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apoptosis has a role in many medical disorders and treatments; hence, its non-invasive evaluation is one of the most riveting research topics. Currently annexin V is used as gold standard for imaging apoptosis. However, several drawbacks, including high background, slow body clearance, make it a suboptimum marker for apoptosis imaging. In this study, we radiolabeled the recently identified histone H1 targeting peptide (ApoPep-1) and evaluated its potential as a new apoptosis imaging agent in various animal models. ApoPep-1 (CQRPPR) was synthesized, and an extra tyrosine residue was added to its N-terminal end for radiolabeling. This peptide was radiolabeled with (124)I and (131)I and was tested for its serum stability. Surgery- and drug-induced apoptotic rat models were prepared for apoptosis evaluation, and PET imaging was performed. Doxorubicin was used for xenograft tumor treatment in mice, and the induced apoptosis was studied. Tumor metabolism and proliferation were assessed by [(18)F]FDG and [(18)F]FLT PET imaging and compared with ApoPep-1 after doxorubicin treatment. The peptide was radiolabeled at high purity, and it showed reasonably good stability in serum. Cell death was easily imaged by radiolabeled ApoPep-1 in an ischemia surgery model. And, liver apoptosis was more clearly identified by ApoPep-1 rather than [(124)I]annexin V in cycloheximide-treated models. Three doxorubicin doses inhibited tumor growth, which was evaluated by 30-40 % decreases of [(18)F]FDG and [(18)F]FLT PET uptake in the tumor area. However, ApoPep-1 demonstrated more than 200 % increase in tumor uptake after chemotherapy, while annexin V did not show any meaningful uptake in the tumor compared with the background. Biodistribution data were also in good agreement with the microPET imaging results. All of the experimental data clearly demonstrated high potential of the radiolabeled ApoPep-1 for in vivo apoptosis imaging.
[Show abstract][Hide abstract] ABSTRACT: Adaptation to cellular stress is not a vital function of normal cells but is required of cancer cells, and as such might be a sensible target in cancer therapy. Piperlongumine is a naturally occurring small molecule selectively toxic to cancer cells. This study assesses the cytotoxicity of piperlongumine and its combination with cisplatin in head-and-neck cancer (HNC) cells in vitro and in vivo. The effect of piperlongumine, alone and in combination with cisplatin, was assessed in human HNC cells and normal cells by measuring growth, death, cell cycle progression, reactive oxygen species (ROS) production, and protein expression, and in tumor xenograft mouse models.
Piperlongumine killed HNC cells regardless of p53 mutational status but spared normal cells. It increased ROS accumulation in HNC cells, an effect that can be blocked by the antioxidant N-acetyl-L-cysteine. Piperlongumine induced selective cell death in HNC cells by targeting the stress response to ROS, leading to the induction of death pathways involving JNK and PARP. Piperlongumine increased cisplatin-induced cytotoxicity in HNC cells in a synergistic manner in vitro and in vivo. Piperlongumine might be a promising small molecule with which to selectively kill HNC cells and increase cisplatin antitumor activity by targeting the oxidative stress response.
[Show abstract][Hide abstract] ABSTRACT: Biopanning of phage displayed-peptide library was performed against myoglobin, a marker for the early assessment of acute myocardial infarction (AMI), to identify peptides that selectively bind to myoglobin. Using myoglobin-conjugated magnetic beads, phages that bound to myoglobin were collected and amplified for the next round of screening. A 148-fold enrichment of phage titer was observed after five rounds of screening relative to the first round. After phage binding ELISA, three phage clones were selected (3R1, 3R7 and 3R10) and the inserted peptides were chemically synthesized. The analysis of binding affinity showed that the 3R7 (CPSTLGASC) peptide had higher binding affinity (Kd=57nM) than did the 3R1 (CNLSSSWIC) and 3R10 (CVPRLSAPC) peptide (Kd=125nM and 293nM, respectively). Cross binding activity to other proteins, such as bovine serum albumin, troponin I, and creatine kinase-MB, was minimal. In a peptide-antibody sandwich ELISA, the selected peptides efficiently captured myoglobin. Moreover, the concentrations of myoglobin in serum samples measured by a peptide-peptide sandwich assay were comparable to those measured by a commercial antibody-based kit. These results indicate that the identified peptides can be used for the detection of myoglobin and may be a cost effective alternative to antibodies.
Journal of Biotechnology 07/2014; 187. DOI:10.1016/j.jbiotec.2014.07.435 · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Early decision on tumor response after anti-cancer treatment is still an unmet medical need. Here we investigated whether in vivo imaging of apoptosis using linear and cyclic (disulfide-bonded) form of ApoPep-1, a peptide that recognizes histone H1 exposed on apoptotic cells, at an early stage after treatment could predict tumor response to the treatment later. Treatment of stomach tumor cells with cistplatin or cetuximab alone induced apoptosis, while combination of cisplatin plus cetuximab more efficiently induced apoptosis, as detected by binding with linear and cyclic form of ApoPep-1. However, the differences between the single agent and combination treatment were more remarkable as detected with the cyclic form compared to the linear form. In tumor-bearing mice, apoptosis imaging was performed 1 week and 2 weeks after the initiation of treatment, while tumor volumes and weights were measured 3 weeks after the treatment. In vivo fluorescence imaging signals obtained by the uptake of ApoPep-1 to tumor was most remarkable in the group injected with cyclic form of ApoPep-1 at 1 week after combined treatment with cisplatin plus cetuximab. Correlation analysis revealed that imaging signals by cyclic ApoPep-1 at 1 week after treatment with cisplatin plus cetuximab in combination were most closely related with tumor volume changes (r2 = 0.934). These results demonstrate that in vivo apoptosis imaging using Apopep-1, especially cyclic ApoPep-1, is a sensitive and predictive tool for early decision on stomach tumor response after anti-cancer treatment.
PLoS ONE 06/2014; 9(6):e100341. DOI:10.1371/journal.pone.0100341 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Effective anticancer therapy can be achieved by designing a targeted drug-delivery system with high stability during circulation and efficient uptake by the target tumour cancer cells. We report here a novel nano-assembled drug-delivery system, formed by multivalent host-guest interactions between a polymer-cyclodextrin conjugate and a polymer-paclitaxel conjugate. The multivalent inclusion complexes confer high stability to the nano-assembly, which efficiently delivers paclitaxel into the targeted cancer cells via both passive and active targeting mechanisms. The ester linkages between paclitaxel and the polymer backbone permit efficient release of paclitaxel within the cell by degradation. This novel targeted nano-assembly exhibits significant antitumour activity in a mouse tumour model. The strategy established in this study also provides knowledge for the development of advanced anticancer drug delivery.
[Show abstract][Hide abstract] ABSTRACT: Various human solid tumors highly express IL-4 receptors which amplify the expression of some of anti-apoptotic proteins, preventing drug-induced cancer cell death. Thus, IL-4 receptor targeted drug delivery can possibly increase the therapeutic efficacy in cancer treatment. Macromolecular carriers with multivalent targeting moieties offered great advantages in cancer therapy as they not only increase the plasma half-life of the drug but also allow delivery of therapeutic drugs to the cancer cells with higher specificity, minimizing the deleterious effects of the drug on normal cells. In this study we designed a library of elastin like polypeptide (ELP) polymers containing tumor targeting AP1 peptide using recursive directional ligation method. AP1 was previously discovered as an atherosclerotic plaque and breast tumor tissue homing peptide using phage display screening method, and it can selectively bind to the interleukin 4 receptor (IL-4R). The fluorescently labeled [AP1-V12]6, an ELP polymer containing six AP1 enhanced tumor-specific targeting ability and uptake efficiency in H226 and MDA-MB-231 cancer cell lines in vitro. Surface plasmon resonance analysis showed that multivalent presentation of the targeting ligand in the ELP polymer increased the binding affinity towards IL-4 receptor compared to free peptide. The binding of [AP1-V12]6 to cancer cells was remarkably reduced when IL-4 receptors were blocked by antibody against IL-4 receptor further confirmed its binding. Importantly, the Cy5.5-labeled [AP1-V12]6 demonstrated excellent homing and longer retention in tumor tissues in MDA-MB-231 xenograft mouse model. Immunohistological studies of tumor tissues further validated the targeting efficiency of [AP1-V12]6 to tumor tissue. These results indicate that designed [AP1-V12]6 can serve as a novel carrier for selective delivery of therapeutic drugs to tumors.
PLoS ONE 12/2013; 8(12):e81891. DOI:10.1371/journal.pone.0081891 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During acute myocardial infarction (AMI), both apoptosis and necrosis of myocardial cells could occur and lead to left ventricular (LV) functional decline. Here we determined whether in vivo imaging signals of myocardial cell death by ApoPep-1 (CQRPPR), a peptide probe that binds to apoptotic and necrotic cells through histone H1, at an early stage after AMI showed correlation with the long-term heart function. AMI was induced using a rat model of ischemia and reperfusion (I/R) injury. Fluorescence-labeled ApoPep-1 was administered by intravenous injection into rats 2h after reperfusion. Ex vivo imaging of hearts isolated 2h after peptide injection showed higher levels of near-infrared fluorescence (NIRF) signals at hearts of I/R rats than those of sham-operated rats. The fluorescent peptide was rapidly cleared from the blood and did not bind to red and white blood cells. Localization of fluorescent ApoPep-1 at the area of cell death was demonstrated by co-staining of myocardial tissue with TUNEL. The intensity of in vivo NIRF imaging signals by homing of ApoPep-1 to injured myocardium of I/R rats obtained 2h after peptide injection (equivalent to 4h after injury) showed strong and moderate correlation with the change in the LV ejection fractions (r(2)=0.82) and the size of the fibrotic area (r(2)=0.64), respectively, observed at four weeks after injury. These results suggest that ApoPep-1-mediated in vivo imaging signals of myocardial cell death, including both apoptosis and necrosis, at an early stage of AMI could be a potential biomarker for assessment of long-term outcome of heart function.
[Show abstract][Hide abstract] ABSTRACT: Protein-cage nanoparticles are promising multifunctional platforms for targeted delivery of imaging and therapeutic agents owing to their biocompatibility, biodegradability, and low toxicity. The major advantage of protein-cage nanoparticles is the ability to decorate their surfaces with multiple functionalities through genetic and chemical modification to achieve desired properties for therapeutic and/or diagnostic purposes. Specific peptides identified by phage display can be genetically fused onto the surface of cage proteins to promote the association of nanoparticles with a particular cell type or tissue. Upon symmetrical assembly of the cage, peptides are clustered on the surface of the cage protein in bunches. The resulting PBNC (Peptide Bunches on NanoCage) offers the potential of synergistic increasing the avidity of the peptide ligands, thereby enhancing their blocking ability for therapeutic purposes. Here, we demonstrated proof-of-principle of PBNCs, fusing the interleukin-4 receptor (IL-4R)-targeting peptide, AP-1, identified previously by phage display, with ferritin-L-chain (FTL), which undergoes 24-subunit assembly to form highly stable AP-1-containing nanocage proteins (AP1-PBNC). AP1-PBNCs bound specifically to the IL-4R-expressing cell line, A549, and their binding and internalization were specifically blocked by anti-IL-4R antibody. AP1-PBNCs exhibited dramatically enhanced binding avidity to IL-4R compared with AP-1 peptide, measured by surface plasmon resonance spectroscopy. Furthermore, treatment with AP1-PBNCs in a murine model of experimental asthma diminished airway hyper-responsiveness and eosinophilic airway inflammation along with decreased mucus hyperproduction. These findings hold great promise that various PBNCs containing ligand specific peptides could be applied for therapeutics indifferent diseases, such as cancer.
[Show abstract][Hide abstract] ABSTRACT: In vitro phage display represents an emerging and innovative technology for the rapid isolation of high-affinity peptide ligands. Phage display technologies using phages comprising a vast library of peptides have become fundamental to the isolation of high-affinity binding ligands for diagnostic and therapeutic applications, e.g., ligand proteomics, discovery of novel protein-protein interactions, antibody engineering, targeted delivery of therapeutic agents, and development of imaging probes. This chapter describes the procedures for phage display selection of peptide ligands that selectively bind to aquaporin-2-expressing membrane fractions of rat kidney.