ABSTRACT
Cancer-specific diagnosis is challenging. Phage display is an approach that could contribute to finding new specific biomarkers. In this study, we developed a new peptide probe specific for gastric cancer and validated it for gastric cancer-specific theranostics. We isolated linear peptides by screening a combinatorial phage library for a cancer stem cell marker, LGR5 protein. Among these, peptides with high selectivity against gastric cancer cells were selected and examined for therapeutic poteintial in vitro as well as in vivo. Through leucine-rich G protein-coupled receptor 5 (LGR5) protein-based phage display, we obtained a hydrophilic 7-mer peptide sequence (STCTRSR, named STC). Both the STC-peptide-conjugated fluorescent dye and chlorin e6 (Ce6) displayed a significantly higher intensity in gastric cancer cells compared to that in healthy cells. In mice with gastric cancer, the fluorescence in the tumors was 3.4× more intense when treated with the Ce6-STC conjugate compared to that with free Ce6 and conferred higher phototoxicity after single laser irradiation. Repeated photodynamic therapy could further reduce the tumor volume after treating these mice with the Ce6-STC conjugate. The treatment with the Ce6-STC conjugate exhibited a significantly lower fluorescence in the liver than that with free Ce6. In conclusion, we confirmed that the STC peptide is a gastric cancer-specific probe that could be useful in gastric cancer theranostics. In conclusion, considering its targeting ability and hydrophilicity, various hydrophobic chemotherapeutic agents could be revisited for gastric cancer treatment in combination with the probe described in this study.
Subject(s)
Photochemotherapy , Porphyrins , Stomach Neoplasms , Mice , Animals , Stomach Neoplasms/drug therapy , Peptides , Drug Delivery Systems , Hydrophobic and Hydrophilic Interactions , Porphyrins/chemistry , Photosensitizing Agents/chemistry , Cell Line, TumorABSTRACT
BACKGROUND: The duodenum has emerged as a key player in metabolic diseases. The objective was to evaluate the safety and efficacy of intra-duodenal PDT using methylene blue in managing glycemic control and weight reduction. METHODS: Optimal concentration of methylene blue and conditions for intra-duodenal PDT were determined through in vitro experiments. After injecting methylene blue into the duodenum, we performed intra-duodenal PDT. High-fat diet rats were used to assess the efficacy of intra-duodenal PDT through measures of oral glucose tolerance, insulin sensitivity, and weight change. Immunohistochemical staining was also conducted to examine GLP-1 and GIP-producing cells in the ileum and duodenum, respectively. RESULTS: Introduodenal PDT reduced villous height of duodenum at 48 h, which was fully recovered at 30 days without complications. Rats treated with PDT showed significantly lower blood glucose levels with glucose loading and improved insulin sensitivity than rats in the sham-treatment group. The PDT group also had a significant reduction in body weight compared to the sham-treatment group at 30 days after intervention, although food intake was not significantly different between the two groups. Numbers of GLP-1 and GIP producing cells in the ileum and irradiated area were significantly higher in the PDT group than in the sham-treatment group. CONCLUSIONS: Intra-duodenal PDT using methylene blue showed a feasible therapeutic modality in improving metabolic parameters. However, large animal experiments and mechanism studies are needed to determine the clinical relevance. The possibility of repeating this treatment every 30 days and its accompanying complications should be further studied.
Subject(s)
Insulin Resistance , Photochemotherapy , Mice , Rats , Animals , Methylene Blue/therapeutic use , Photochemotherapy/methods , Photosensitizing Agents/pharmacology , Photosensitizing Agents/therapeutic use , Duodenum , Disease Models, Animal , Glucagon-Like Peptide 1ABSTRACT
Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a stem cell marker in gastric cancer. In this study, we aimed to produce the LGR5-targeting peptide probe for the use of molecular imaging for gastric cancer. We used phage display libraries to produce a LGR5-specific peptide probe. This peptide was validated for targeting gastric cancer with in vitro and in vivo studies. This peptide was tagged with fluorescein isothiocyanate (FITC) and cyanine 5.5 (Cy5.5). We used two normal and three gastric cancer cell lines. Immunocytochemistry (ICC) and fluorescence-activated cell sorting (FACS) analysis were used to validate the target specificity of the peptide. After three rounds of bio-panning, we found a novel 7-mer peptides, IPQILSI (IPQ∗). FITC-conjugated IPQ∗ showed 2 to 10 times higher fluorescence in gastric cancer cells vs. control cells in ICC. This discrimination was consistently observed using Cy5.5-conjugated IPQ∗ in ICC. FACS analysis showed right shift of peak point in gastric cancers compared to the control cells. In the peritoneal metastasis animal model, we could find Cy5.5-conjugated IPQ∗ accumulated specifically to gastric tumors. In conclusion, IPQ∗ peptide showed a specific probe for gastric cancer diagnosis. This probe can be applied to theragnosis for gastric cancer diagnosis including peritoneal metastasis.
Subject(s)
Molecular Imaging/methods , Peptides/chemistry , Receptors, G-Protein-Coupled/analysis , Stomach Neoplasms/diagnostic imaging , Animals , Carbocyanines/chemistry , Cell Line, Tumor , Fluorescein-5-isothiocyanate/chemistry , Fluorescent Dyes/chemistry , Humans , Mice, Nude , Optical Imaging/methodsABSTRACT
AIM: Colon cancer is one of the leading causes of cancer-related mortality. However, specific biomarkers for its diagnosis or treatment are not established well. METHODS: We developed a colon-cancer specific peptide probe using phage display libraries. We validated the specificity of this probe to colon cancer cells with immunohistochemical staining and FACS analysis using one normal cell and five colon cancer cell lines. RESULTS: This peptide probe maintained binding affinity even after serum incubation. For therapeutic applications, this peptide probe was conjugated to hematoporphyrin, a photosensitizer, which showed a significantly enhanced cellular uptake and high photodynamic effect to kill tumor cells. As another application, we made a nanoparticle modified from the peptide probe. It efficiently delivered SN-38, an anticancer drug, into tumor cells, and its tumor-targeting ability was observed in vivo after intravenous injection to the same xenograft model. CONCLUSION: The noble dodecapeptide probe can be a promising candidate for both colon tumor diagnosis and targeted drug delivery.
ABSTRACT
BACKGROUND/AIMS: Pancreatic cancer has a very poor prognosis, and early diagnosis is a way to increase the survival rate of patients. The purpose of this study was to develop pancreatic cancer-specific peptides for imaging studies. METHODS: Three pancreatic cancer cell lines, MIA PaCa-2, UACC-462, and BxPC-3, and a control cell line, CCD841, were used. Biopannings were performed on MIA PaCa-2 using a phage display library. After this, the peptides were synthesized and labeled with fluorescein isothiocyanate (FITC). Immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), and fluorescence- activated cell sorter (FACS) were performed to examine the specific binding. To examine its therapeutic applications, a photosensitizer, chlorin e6 (Ce6), was conjugated on the peptide and photodynamic therapy was performed. Cell survival was investigated using a [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay. RESULTS: After three biopannings, the phages were amplified from 1.4×104 to 3.2×105 plaque-forming units. The most strongly binding phage was selected from the ELISA and ICC results. FITC-labeled peptide, M5, in the three pancreatic cancer cell lines showed significantly higher immunofluorescence in the ICC experiments than that of CCD841. The higher binding ability to MIA PaCa-2 cells was confirmed from FACS analysis, which showed a right shift compared to CCD841. M5 bound to Ce6 showed a significantly lower cell survival rate than that of Ce6 alone in photodynamic therapy, which was observed consistently as a change in the tumor size and fluorescence intensity in MIA PaCa-2 cell-implanted animal models. CONCLUSIONS: This study showed that the noble peptide, M5, binds specifically to the pancreatic cancer cell line, MIA PaCa-2. The M5 peptide has potential use in future optical diagnostic and therapeutic purposes.
Subject(s)
Cell Surface Display Techniques/methods , Peptides/metabolism , Animals , Cell Line, Tumor , Cell Survival/drug effects , Chlorophyllides , Fluorescein-5-isothiocyanate/chemistry , Humans , Light , Mice , Mice, Nude , Optical Imaging , Pancreatic Neoplasms/diagnosis , Pancreatic Neoplasms/drug therapy , Peptides/chemistry , Photochemotherapy , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacology , Photosensitizing Agents/therapeutic use , Porphyrins/chemistryABSTRACT
BACKGROUND: Improving the water solubility of hydrophobic photosensitizer and increasing its accumulation in tumor tissue are essential for in vivo photodynamic therapy (PDT). Considering commercialization or clinical application in future, it will be promising to achieve these purposes by developing new agents with simple and non-toxic structure. RESULTS: We conjugated multiple chlorin e6 (Ce6) molecules to gelatin polymer, synthesizing two types of gelatin-Ce6 conjugates with different amounts of Ce6: gelatin-Ce6-2 and gelatin-Ce6-8. The resulting conjugates remained soluble in aqueous solutions for a longer time than hydrophobic Ce6. The conjugates could generate singlet oxygen and kill tumor cells upon laser irradiation. After intravenous injection into SCC-7 tumor-bearing mice, gelatin-Ce6-2 showed prolonged blood circulation and highly increased accumulation in tumor tissue as observed in real-time imaging in vivo. After laser irradiation, gelatin-Ce6-2 suppressed tumor growth completely and enabled improved PDT compared to free Ce6 and gelatin-Ce6-8. CONCLUSIONS: This work demonstrates that a simple structure based on photosensitizer and gelatin can highly improve water solubility and stability. Superior tumor tissue accumulation and increased therapeutic efficacy of gelatin-Ce6 during in vivo PDT showed its high potential for clinical application.
Subject(s)
Gelatin/chemistry , Photosensitizing Agents/pharmacology , Porphyrins/pharmacology , Animals , Cell Line, Tumor , Cell Survival , Chlorophyllides , Drug Carriers , Humans , Mice , Neoplasm Transplantation , Phototherapy , Polymers/chemistry , Porphyrins/chemistry , Singlet Oxygen/metabolism , Solubility , Tissue DistributionABSTRACT
Recently, increased attention has been focused on endoscopic disinfection after outbreaks of drug-resistant infections associated with gastrointestinal endoscopy. The aims of this study were to investigate the bactericidal efficacy of methylene blue (MB)-based photodynamic therapy (PDT) on Pseudomonas aeruginosa (P. aeruginosa), which is the major cause of drug-resistant postendoscopy outbreak, and to assess the synergistic effects of hydrogen peroxide addition to MB-based PDT on biofilms. In planktonic state of P. aeruginosa, the maximum decrease was 3 log10 and 5.5 log10 at 20 and 30 J cm-2 , respectively, following MB-based PDT. However, the maximum reduction of colony forming unit (CFU) was decreased by 2.5 log10 and 3 log10 irradiation on biofilms. The biofilm formation was significantly inhibited upon irradiation with MB-based PDT. When the biofilm state of P. aeruginosa was treated with MB-based PDT with hydrogen peroxide, the CFU was significantly decreased by 6 log10 after 20 J cm-2 , by 7 log10 after 30 J cm-2 irradiation, suggesting significantly higher efficacy than MB-based PDT alone. The implementation of the combination of hydrogen peroxide with MB-based PDT through working channels might be appropriate for preventing early colonization and biofilm formation in the endoscope and postendoscopy outbreak.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Hydrogen Peroxide/pharmacology , Methylene Blue/chemistry , Photochemotherapy , Colony Count, Microbial , Drug Synergism , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/growth & developmentABSTRACT
Targeted drug delivery has been an important issue for tumor therapy including photodynamic therapy (PDT). The purpose of our study is to increase the targeting efficiency of photosensitizer (PS) using folate-modified nanoparticles (NPs) to tumor site in vivo. Folate receptor is over-expressed on the surface of many human cancer cells. We prepared poly (lactic-co-glycolic acid) (PLGA) NPs containing pheophorbide a (Pba), a PS that is used in PDT and generates free radical for killing cancer cells. The surface of NPs was composed of phospholipids modified with polyethylene glycol (PEG) and folate (FA). The size of the resulting FA-PLGA-Pba NPs was about 200â¯nm in PBS at pH 7.4 and they were stable for long time. They showed faster cellular uptake to MKN28 human gastric cancer cell line than control PLGA-Pba NPs by high-affinity binding with folate receptors on cell surface. In MTT assay, FA-PLGA-Pba NPs also showed enhanced tumor cell killing compared to control PLGA-Pba NPs. In vivo and ex vivo imaging showed high accumulation of FA-PLGA-Pba NPs in tumor site during 24â¯h after intravenous injection to MKN28 tumor-bearing mice model. These results demonstrate that our FA-PLGA-Pba NPs are useful for tumor-targeted delivery of PS for cancer treatment by PDT.