Globally, peptide-based anticancer therapies have drawn much attention. Marine organisms are a reservoir of anticancer peptides that await discovery. In this study, we aimed to identify cytotoxic oligopeptides from Sarcophyton glaucum. Peptides were purified from among the S. glaucum hydrolysates produced by alcalase, chymotrypsin, papain, and trypsin, guided by a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay on the human cervical cancer (HeLa) cell line for cytotoxicity evaluation. Purification techniques adopted were membrane ultrafiltration, gel filtration chromatography, solid phase extraction (SPE), and reversed-phase high-performance liquid chromatography (RP-HPLC). Purified peptides were identified by de novo peptide sequencing. From papain hydrolysate, three peptide sequences were identified: AGAPGG, AERQ, and RDTQ (428.45, 502.53, and 518.53 Da, respectively). Peptides synthesized from these sequences exhibited cytotoxicity on HeLa cells with median effect concentration (EC₅₀) values of 8.6, 4.9, and 5.6 mmol/L, respectively, up to 5.8-fold stronger than the anticancer drug 5-fluorouracil. When tested at their respective EC₅₀, AGAPGG, AERQ, and RDTQ showed only 16%, 25%, and 11% cytotoxicity to non-cancerous Hek293 cells, respectively. In conclusion, AERQ, AGAPGG, and RDTQ are promising candidates for future development as peptide-based anticancer drugs.
Amino Acid Sequence ; Animals ; Anthozoa/chemistry* ; Antineoplastic Agents/pharmacology* ; Chromatography, Gel ; Chromatography, High Pressure Liquid ; Chromatography, Reverse-Phase ; Cytotoxins/pharmacology* ; Drug Discovery ; HEK293 Cells ; HeLa Cells ; Humans ; Hydrolysis ; Marine Toxins/pharmacology* ; Oligopeptides/pharmacology* ; Solid Phase Extraction ; Tandem Mass Spectrometry

Objective: To analysis the biomechanical and biocompatible properties of calcium phosphate cement (CPC) enhanced by chitosan short nanofibers(CSNF) and Arg-Gly-Asp (RGD). Methods: Chitosan nanofibers were prepared by electrospinning, and cut into short fibers by high speed dispersion. CPC with calcium phosphorus ratio of 1.5:1 was prepared by Biocement D method. The composition and structure of CPC, CSNF, RGD modified CSNF (CSNF-RGD), CSNF enhanced CPC (CPC-CSNF), RGD modified CPC-CSNF (CPC-CSNF-RGD) were observed by infrared spectrum, X-ray diffraction (XRD) and scan electron microscopy (SEM). The mechanical properties were measured by universal mechanical testing instrument. The adhesion and proliferation of MC3T3 cells were assessed using immunofluorescence staining and MTT method. Results: The distribution of CSNF in the scaffold was homogeneous, and the porous structure between the nanofibers was observed by SEM. The infrared spectrum showed the characteristic peaks at 1633 nm and 1585 nm, indicating that RGD was successfully grafted on chitosan nanofibers. The XRD pattern showed that the bone cement had a certain curability. The stain-stress test showed that break strengths were (17.74±0.54) MPa for CPC-CSNF and (16.67±0.56) MPa for CPCP-CSNF-RGD, both were higher than that of CPC(all P<0.05). The immunofluorescence staining and MTT results indicated that MC3T3 cells grew better on CPC-CSNF-RGD after 240 min of culture(all P<0.05). Conclusion: CSNF-RGD can improve the biomechanical property and biocompatibility of CPC, indicating its potential application in bone tissue repair.
3T3 Cells ; Animals ; Biocompatible Materials ; Bone Cements ; chemistry ; metabolism ; pharmacology ; Calcium Phosphates ; metabolism ; Cell Proliferation ; drug effects ; Chitosan ; chemistry ; pharmacology ; Mice ; Nanofibers ; chemistry ; Oligopeptides ; chemistry

Faldaprevir analogue molecule (FAM) has been reported to effectively inhibit the catalytic activity of HCV NS3/4A protease, making it a potential lead compound against HCV. A series of HCV NS3/4A protease crystal structures were analyzed by bioinformatics methods, and the FAM-HCV NS3/4A protease crystal structure was chosen for this study. A 20.4 ns molecular dynamics simulation of the complex consists of HCV NS3/4A protease and FAM was conducted. The key amino acid residues for interaction and the binding driving force for the molecular recognition between the protease and FAM were identified from the hydrogen bonds and binding free energy analyses. With the driving force of hydrogen bonds and van der Waals, FAM specifically bind to the active pocket of HCV NS3/4A protease, including V130-S137, F152-D166, D77-D79 and V55, which agreed with the experimental data. The effect of R155K, D168E/V and V170T site-directed mutagenesis on FAM molecular recognition was analyzed for their effect on drug resistance, which provided the possible molecular explanation of FAM resistance. Finally, the system conformational change was explored by using free energy landscape and conformational cluster. The result showed four kinds of dominant conformation, which provides theoretical basis for subsequent design of Faldaprevir analogue inhibitors based on the structure of HCV NS3/4A protease.
Antiviral Agents ; chemistry ; Carrier Proteins ; chemistry ; Drug Resistance, Viral ; Endopeptidases ; Hepacivirus ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; Oligopeptides ; chemistry ; Protease Inhibitors ; chemistry ; Serine Proteases ; Thiazoles ; chemistry ; Viral Nonstructural Proteins ; chemistry

Gabexate mesilate (GM) is a trypsin inhibitor, and mainly used for treatment of various acute pancreatitis, including traumatic pancreatitis (TP), edematous pancreatitis, and acute necrotizing pancreatitis. However, due to the characteristics of pharmacokinetics, the clinical application of GM still needs frequently intravenous administration to keep the blood drug concentration, which is difficult to manage. Specially, when the blood supply of pancreas is directly damaged, intravenous administration is difficult to exert the optimum therapy effect. To address it, a novel thermosensitive in-situ gel of gabexate mesilate (GMTI) was developed, and the optimum formulation of GMTI containing 20.6% (w/w) P-407 and 5.79% (w/w) P188 with different concentrations of GM was used as a gelling solvent. The effective drug concentration on trypsin inhibition was examined after treatment with different concentrations of GMTI in vitro, and GM served as a positive control. The security of GMTI was evaluated by hematoxylin-eosin (HE) staining, and its curative effect on grade II pancreas injury was also evaluated by testing amylase (AMS), C-reactive protein (CRP) and trypsinogen activation peptide (TAP), and pathological analysis of the pancreas. The trypsin activity was slightly inhibited at 1.0 and 5.0 mg/mL in GM group and GMTI group, respectively (P<0.05 vs. P-407), and completely inhibited at 10.0 and 20.0 mg/mL (P<0.01 vs. P-407). After local injection of 10 mg/mL GMTI to rat leg muscular tissue, muscle fiber texture was normal, and there were no obvious red blood cells and infiltration of inflammatory cells. Furthermore, the expression of AMS, CRP and TAP was significantly increased in TP group as compared with control group (P<0.01), and significantly decreased in GM group as compared with TP group (P<0.01), and also slightly inhibited after 1.0 and 5.0 mg/mL GMTI treatment as compared with TP group (P<0.05), and significantly inhibited after 10.0 and 20.0 mg/mL GMTI treatment as compared with TP group (P<0.01). HE staining results demonstrated that pancreas cells were uniformly distributed in control group, and they were loosely arranged, partially dissolved, with deeply stained nuclei in TP group. Expectedly, after gradient GMTI treatment, pancreas cells were gradually restored to tight distribution, with slightly stained nuclei. This preliminary study indicated that GMTI could effectively inhibit pancreatic enzymes, and alleviate the severity of trauma-induced pancreatitis, and had a potential drug developing and clinic application value.
Amylases ; metabolism ; Animals ; C-Reactive Protein ; metabolism ; Delayed-Action Preparations ; chemical synthesis ; pharmacokinetics ; pharmacology ; Gabexate ; chemistry ; pharmacokinetics ; pharmacology ; Gels ; Male ; Muscle, Skeletal ; drug effects ; enzymology ; Oligopeptides ; metabolism ; Pancreas ; drug effects ; enzymology ; pathology ; Pancreatitis ; drug therapy ; enzymology ; etiology ; pathology ; Poloxamer ; chemistry ; Rats ; Rats, Sprague-Dawley ; Serine Proteinase Inhibitors ; chemistry ; pharmacokinetics ; pharmacology ; Temperature ; Wounds, Penetrating ; complications ; drug therapy ; enzymology ; pathology

The purpose of the study is to construct R8 peptide (RRRRRRRR) and pH sensitive polyethylene glycols (PEG) co-modified liposomes (Cl-Lip) and utilize them in breast cancer treatment. The co-modified liposomes were prepared with soybean phospholipid, cholesterol, DSPE-PEG2K-R8 and PEG5K-Hz-PE (pH sensitive PEG). The size and zeta potential of Cl-Lip were also characterized. The in vitro experiment demonstrated that the Cl-Lip had high serum stability in 50% fetal bovine serum. The cellular uptake of Cl-Lip under different pre-incubated conditions was evaluated on 4T1 cells. And the endocytosis pathway, lysosome escape ability and tumor spheroid penetration ability were also evaluated. The results showed the particle size of the Cl-Lip was (110.4 ± 5.2) nm, PDI of the Cl-Lip was 0.207 ± 0.039 and zeta potential of the Cl-Lip was (-3.46 ± 0.05) mV. The cellular uptake of Cl-Lip on 4T1 cells was pH sensitive, as the cellular uptake of Cl-Lip pre-incubated in pH 6.0 was higher than that of pH 7.4 under each time point. The main endocytosis pathways of Cl-Lip under pH 6.0 were micropinocytosis and energy-dependent pathway. At the same time, the Cl-Lip with pre-incubation in pH 6.0 had high lysosome escape ability and high tumor spheroid penetration ability. All the above results demonstrated that the Cl-Lip we constructed had high pH sensitivity and is a promising drug delivery system.
Animals ; Cell Line, Tumor ; Cell-Penetrating Peptides ; chemical synthesis ; chemistry ; Cholesterol ; chemistry ; Drug Delivery Systems ; Liposomes ; Mice ; Oligopeptides ; chemical synthesis ; chemistry ; Particle Size ; Phospholipids ; chemistry ; Polyethylene Glycols

Since the release rate of protein in hydrogels is directly dependent upon the size of the protein and the hydrogel, how to deliver low molecular weight protein for prolonged periods has always been a problem. In this article, we present a usage of self-assembling peptide (P3) with the RGD epitope on its N terminus. The concentration of the released insulin-like growth factor 1 (IGF-1) was determined by UV-vis spectroscopy and the release kinetics suggested a notable reduction of the IGF-1 release rate. Cell entrapment experiments revealed that IGF-1 delivery by biotinylated nanofibers could promote the proliferation of the mouse chondrogenic ATDC5 cells when compared with cells embedded within nanofibers with untethered IGF-1.
Animals ; Biotin ; Cell Line ; Delayed-Action Preparations ; Drug Carriers ; chemistry ; Hydrogels ; chemistry ; Insulin-Like Growth Factor I ; pharmacology ; Mice ; Nanofibers ; Oligopeptides ; chemistry

OBJECTIVE: To prepare Arg-Gly-Asp (RGD) and cell penetrating peptide TAT co-modified paclitaxel loaded liposome (RGD/TAT-LP-PTX) for MCF-7 cell inhibition. METHODS: The co-modified liposome was prepared by film-ultrasonic method. The appearance, particle size and zeta potential were evaluated. The cellular uptake by MCF-7 cells in vitro was used to evaluate the targeting efficiency. The anti-proliferation efficiency of RGD/TAT-LP-PTX was evaluated by MTT assay. Tumor spheroids were used to evaluate anti-tumor ability of RGD/TAT-LP-PTX in vitro. RESULTS: The particle diameter of the co-modified liposome was (138.8 ± 12.4) nm with the Zeta potential of (25.85 ± 2.75) mV. The entrapment efficiency of PTX was 88.3%. The RGD/TAT-LP uptaken by MCF-7 cells at 4 h was 1.79 times higher than that at 2 h. The co-modified liposome uptaken by MCF-7 cells was 2.25 and 2.72 times higher than that of TAT-LP and RGD-LP, respectively. The anti-proliferation rate of RGD/TAT-LP-PTX increased with time. The inhibition rate of RGD/TAT-LP-PTX for MCF-7 cells at 48 h was 1.78 times higher than that at 24 h. The MTT assay demonstrated the cell viability of RGD/TAT-LP-PTX was 1.65, 1.82 and 2.55 times higher than that of TAT-LP-PTX, RGD-LP-PTX and LP-PTX, respectively. CONCLUSION Co-modified liposome may serve as a promising breast cancer delivery system for antitumor drugs.
Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; Cell Survival ; Humans ; Liposomes ; MCF-7 Cells ; drug effects ; Oligopeptides ; chemistry ; Paclitaxel ; pharmacology ; Particle Size ; Peptide Fragments ; chemistry ; Spheroids, Cellular ; drug effects

iRGD-modified sterically stabilized liposomes loaded doxorubicin (iRGD-SSL-DOX) were prepared and their cellular toxicity and anti-tumor efficacy were evaluated, comparing to doxorubixin loaded sterically stabilized liposomes (SSL-DOX) and RGD modified doxorubixin loaded sterically stabilized liposomes (RGD-SSL-DOX). The iRGD peptide, with both tumor targeting and cell penetrating functions, was conjugated to DSPE-PEG-NHS and DSPE-PEG-iRGD was obtained. DSPE-PEG-RGD was gained in the same way. iRGD-SSL-DOX, RGD-SSL-DOX and SSL-DOX were prepared by ammonium sulfate gradient method. The size and zeta potential of the liposomes were characterized by dynamic laser light scattering. The cellular toxicity study was done on B16 melanoma cell line and the anti-tumor efficacy study was carried on B16 cell line bearing C57BL/6 mice. The results showed that the particle sizes of liposomes were all around 90-100 nm. DOX entrapment efficiency was above 95%. The formulations were with good preparation reproducibility. iRGD-SSL-DOX showed no significant difference in B16 cellular toxicity with SSL-DOX and RGD-SSL-DOX, but the anti-tumor efficacy on B16 melanoma bearing C57BL/6 mice was significantly better than that of SSL-DOX, similar as that of RGD-SSL-DOX. Therefore, iRGD modified liposomes loaded DOX would be a promising drug delivery system for tumor therapy.
Animals ; Antibiotics, Antineoplastic ; administration & dosage ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Doxorubicin ; administration & dosage ; pharmacology ; Drug Carriers ; Drug Delivery Systems ; Liposomes ; Male ; Melanoma, Experimental ; pathology ; Mice ; Mice, Inbred C57BL ; Molecular Weight ; Neoplasm Transplantation ; Oligopeptides ; chemistry ; pharmacology ; Particle Size ; Phosphatidylethanolamines ; chemistry ; Polyethylene Glycols ; chemistry ; Tumor Burden ; drug effects

To investigate the rat intestinal absorption of stearic acid-octaarginine (SA-R8) modified solid lipid nanoparticles containing paclitaxel (SA-R8-PTX-SLN), compared with the commercially available preparation of PTX (Taxol) and PTX-loaded solid lipid nanoparticles (PTX-SLN), the in situ intestinal absorption of SA-R8-PTX-SLN was investigated by means of single-pass rat intestinal perfusion technique. The absorptions of the preparations were investigated at different intestinal segments, different drug concentrations and in the presence of P-glycoprotein inhibitor (verapamil). The results showed that PTX could be absorbed at each intestinal segment and the three preparations all showed maximum absorptions at the duodenum. The cumulative absorptions of three preparations at each intestinal segment appeared SA-R8-PTX-SLN > PTX-SLN > Taxol (P < 0.05). SA-R8-PTX-SLN showed a liner absorption manner at the duodenum in the examined drug concentration range. The cumulative absorptions of Taxol and PTX-SLN were significantly promoted after the addition of P-glycoprotein inhibitor (verapamil) into the preparation (P < 0.05), but absorption of SA-R8-PTX-SLN existed no significantly difference compared with the preparation without verapamil (P > 0.05). SA-R8 and SLN might both effectively improve the oral absorption of PTX in the intestinal tract.
ATP-Binding Cassette, Sub-Family B, Member 1 ; antagonists & inhibitors ; Animals ; Antineoplastic Agents, Phytogenic ; administration & dosage ; chemistry ; pharmacokinetics ; Cell-Penetrating Peptides ; chemistry ; Drug Carriers ; Intestinal Absorption ; drug effects ; Lipids ; chemistry ; Male ; Nanoparticles ; Oligopeptides ; chemistry ; Paclitaxel ; administration & dosage ; chemistry ; pharmacokinetics ; Perfusion ; Rats ; Rats, Sprague-Dawley ; Stearic Acids ; chemistry ; Verapamil ; pharmacology

Chemotherapy remains one of the major tools, along with surgery, radiotherapy, and more recently targeted therapy, in the war against cancer. There have appeared a plethora of highly potent cytotoxic drugs but the poor discriminability between cancerous and healthy cells of these agents limits their broader application in clinical settings. Therapeutic antibodies have emerged as an important class of biological anticancer agents, thanks to their ability in specific binding to tumor-associated antigens. While this important class of biologics can be used as single agents for the treatment of cancer through antibody-dependent cell cytotoxicity (ADCC), their therapeutical efficacy is often limited. Antitumor antibody drug conjugates (ADCs) combine the target-specificity of monoclonal antibody (mAb) and the highly active cell-killing drugs, taking advantages of the best characteristics out of both components. Thus, insufficiency of most naked mAbs in cancer therapy has been circumvented by arming the immunoglobulin with cytotoxic drugs. Here mAbs are used as vehicles to transport potent payloads to tumor cells. ADCs contain three main components: antibody, linker and cytotoxics (also frequently referred as payload). Antibodies can recognize and specifically bind to the tumor-specific antigens, leading to an antibody-assisted internalization, and payload release. While ADC has demonstrated tremendous success, a number of practical challenges limit the broader applications of this new class of anticancer therapy, including inefficient cellular uptake, low cytotoxicity, and off-target effects. This review article aims to cover recent advances in optimizing linkers with increased stability in circulation while allowing efficient payload release within tumor cells. We also attempt to provide some practical strategies in resolving the current challenges in this attractive research area, particularly to those new to the field.
Aminobenzoates ; pharmacology ; therapeutic use ; Animals ; Antibodies, Monoclonal ; pharmacology ; therapeutic use ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Cell Survival ; drug effects ; Cytotoxins ; pharmacology ; therapeutic use ; Drug Design ; Humans ; Immunoconjugates ; chemistry ; pharmacology ; therapeutic use ; Maytansine ; pharmacology ; therapeutic use ; Neoplasms ; drug therapy ; pathology ; Oligopeptides ; pharmacology ; therapeutic use