ngr-peptide and Fibrosarcoma

A new drug-targeting system for CD13(+) tumors has been developed, based on ultrasound-sensitive nanobubbles (NBs) and cell-permeable peptides (CPPs). Here, the CPP-doxorubicin conjugate (CPP-DOX) was entrapped in the asparagine-glycine-arginine (NGR) peptide modified NB (CPP-DOX/NGR-NB) and the penetration of CPP-DOX was temporally masked; local ultrasound stimulation could trigger the CPP-DOX release from NB and activate its penetration. The CPP-DOX/NGR-NBs had particle sizes of about 200 nm and drug entrapment efficiency larger than 90%. In vitro release results showed that over 85% of the encapsulated DOX or CPP-DOX would release from NBs in the presence of ultrasound, while less than 1.5% of that (30 min) without ultrasound. Cell experiments showed the higher cellular CPP-DOX uptake of CPP-DOX/NGR-NB among the various NB formulations in Human fibrosarcoma cells (HT-1080, CD13(+)). The CPP-DOX/NGR-NB with ultrasound treatment exhibited an increased cytotoxic activity than the one without ultrasound. In nude mice xenograft of HT-1080 cells, CPP-DOX/NGR-NB with ultrasound showed a higher tumor inhibition effect (3.1% of T/C%, day 24), longer median survival time (50 days) and excellent body safety compared with the normal DOX injection group. These results indicate that the constructed vesicle would be a promising drug delivery system for specific cancer treatment.

Topics: Animals; Cell Line, Tumor; Cell-Penetrating Peptides; Doxorubicin; Drug Delivery Systems; Female; Fibrosarcoma; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Oligopeptides; Ultrasonics

Asparagine-glycine-arginine (NGR)-containing peptides targeting aminopeptidase N (APN)/CD13 can be an excellent candidate for targeting ligands in molecular tumor imaging. In this study, we developed two NGR-containing hexapeptides, and evaluated the diagnostic performance of Tc-99m labeled hexapeptides as molecular imaging agents in an HT-1080 fibrosarcoma-bearing murine model. Peptides were synthesized using Fmoc solid-phase peptide synthesis. Radiochemical purity of Tc-99m was evaluated using instant thin-layer chromatography. The uptake of two NGR-containing hexapeptides within HT-1080 cells was evaluated in vitro. In HT-1080 fibrosarcoma tumor-bearing mice, gamma images were acquired. A biodistribution study was performed to calculate percentage of the injected dose per gram of tissue (%ID/g). Two hexapeptides, glutamic acid-cysteine-glycine (ECG)-NGR and NGR-ECG were successfully synthesized. After radiolabeling procedures with Tc-99m, the complexes Tc-99m hexapeptides were prepared in high yield. The uptake of Tc-99m ECG-NGR within the tumor cells had been assured by in vitro studies. The gamma camera imaging in the murine model showed that Tc-99m ECG-NGR was accumulated substantially in the subcutaneously engrafted tumor. However, Tc-99m NGR-ECG was accumulated minimally in the tumor. Two NGR-containing hexapeptides, ECG-NGR and NGR-ECG were developed as molecular imaging agents to target APN/CD13 in HT-1080 fibrosarcoma. Tc-99m ECG-NGR showed a significant uptake in the tumor, and it is a good candidate for tumor imaging.

Topics: Animals; Cell Line, Tumor; Female; Fibrosarcoma; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Oligopeptides; Radionuclide Imaging; Radiopharmaceuticals; Technetium

Some specific types of tumor cells and tumor endothelial cells represented CD13 proteins and act as receptors for Asn-Gly-Arg (NGR) motifs containing peptide. These CD13 receptors can be specifically recognized and bind through the specific sequence of cyclic NGR (cNGR) peptide and presented more affinity and specificity toward them. The cNGR peptide was conjugated to the poly(ethylene glycol) (PEG) terminal end in the poly(lactic-co-glycolic) acid PLGA-PEG block copolymer. Then, the ligand conjugated nanoparticles (cNGR-DNB-NPs) encapsulating docetaxel (DTX) were synthesized from preformed block copolymer by the emulsion/solvent evaporation method and characterized for different parameters. The various studies such as in vitro cytotoxicity, cell apoptosis, and cell cycle analysis presented the enhanced therapeutic potential of cNGR-DNB-NPs. The higher cellular uptake was also found in cNGR peptide anchored NPs into HUVEC and HT-1080 cells. However, free cNGR could inhibit receptor mediated intracellular uptake of NPs into both types of cells at 37 and 4 °C temperatures, revealing the involvement of receptor-mediated endocytosis. The in vivo biodistribution and antitumor efficacy studies indicated that targeted NPs have a higher therapeutic efficacy through targeting the tumor-specific site. Therefore, the study exhibited that cNGR-functionalized PEG-PLGA-NPs could be a promising approach for therapeutic applications to efficient antitumor drug delivery.

Topics: Animals; Antineoplastic Agents; Apoptosis; CD13 Antigens; Cell Cycle; Cell Proliferation; Cells, Cultured; Docetaxel; Drug Carriers; Drug Delivery Systems; Fibrosarcoma; Flow Cytometry; Hemolysis; Human Umbilical Vein Endothelial Cells; Humans; Lactic Acid; Macrophages; Mice; Mice, Inbred BALB C; Nanoparticles; Oligopeptides; Phagocytosis; Polyethylene Glycols; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Taxoids; Tissue Distribution

Peptides containing asparagine-glycine-arginine (NGR) and arginine-glycine-aspartic acid (RGD) sequence are being developed for tumor angiogenesis-targeted imaging and therapy. The aim of this study was to compare the efficacy of NGR- and RGD-based probes for imaging tumor angiogenesis in HT-1080 tumor xenografts. Two PET probes, (68)Ga-NOTA-G₃-NGR2 and ⁶⁸Ga-NOTA-G₃-RGD2, were successfully prepared. In vitro stability, partition coefficient, tumor cell binding, as well as in vivo biodistribution properties were also analyzed for both PET probes. The results revealed that the two probes were both hydrophilic and stable in vitro and in vivo, and they were excreted predominately and rapidly through the kidneys. For both probes, the higher tumor uptake and lower accumulation in vital organs were determined. No significant difference between two probes was observed in terms of tumor uptake and the in vivo biodistribution properties. We concluded that these two probes are promising in tumor angiogenesis imaging. ⁶⁸Ga-NOTA-G₃-NGR2 has the potential as an alternative for PET imaging in patients with fibrosarcoma, and it may offer an opportunity to noninvasively monitor CD13-targeted therapy.

Topics: Adenocarcinoma; Animals; Biological Transport; Cell Line, Tumor; Drug Stability; Female; Fibrosarcoma; Gallium Radioisotopes; Humans; Hydrophobic and Hydrophilic Interactions; Mice, Nude; Molecular Imaging; Neovascularization, Pathologic; Oligopeptides; Positron-Emission Tomography; Radiopharmaceuticals; Renal Elimination; Tissue Distribution; Xenograft Model Antitumor Assays

tTF-NGR consists of the extracellular domain of tissue factor and the peptide GNGRAHA, a ligand of the surface protein aminopeptidase N and of integrin αvβ3. Both surface proteins are upregulated on endothelial cells of tumor vessels. tTF-NGR shows antitumor activity in xenografts and inhibition of tumor blood flow in cancer patients. We performed random TMS(PEG)12 PEGylation of tTF-NGR to improve the antitumor profile of the molecule. PEGylation resulted in an approximately 2-log step decreased procoagulatory activity of the molecule. Pharmacokinetic studies in mice showed a more than 1-log step higher mean area under the curve. Comparison of the LD10 values for both compounds and their lowest effective antitumor dose against human tumor xenografts showed an improved therapeutic range (active/toxic dose in mg/kg body weight) of 1/5 mg/kg for tTF-NGR and 3/>160 mg/kg for TMS(PEG)12 tTF-NGR. Results demonstrate that PEGylation can significantly improve the therapeutic range of tTF-NGR.

Topics: Animals; Antineoplastic Agents; Blood Vessels; Cell Line, Tumor; Female; Fibrosarcoma; Humans; Mice; Models, Molecular; Oligopeptides; Polyethylene Glycols; Protein Conformation; Thromboplastin; Xenograft Model Antitumor Assays

In the present study, we prepared NGR-modified sterically stabilized liposomes containing paclitaxel (NGR-SSL-PTX) in order to evaluate their potential targeting to aminopeptidase N receptors expressed on tumor endothelial cells and the tumor cell surface and its anti-angiogenic activity following metronomic administration. NGR-SSL-PTX was prepared by a thin-film hydration method. The in vitro targeting characteristics of NGR-modified liposomes on HUVEC (human umbilical vein endothelial cells), HT1080 (human fibrosarcoma cells) and MCF-7 (human breast adenocarcinoma cells) were then investigated. The effect of NGR-SSL-PTX on HUVEC proliferation and migration was also tested. The pharmacokinetics of NGR-SSL-PTX was studied in rats. The in vivo targeting activity of NGR-modified liposomes was investigated in HT1080 tumor-bearing mice. The anti-tumor activity of NGR-SSL-PTX following metronomic administration was evaluated in HT1080 tumor-bearing mice in vivo. The targeting activity of the NGR-modified liposomes was demonstrated by in vitro flow cytometry and confocal microscopy as well as in vivo confocal immunofluorescence microscopy and bio-distribution experiments. The results of endothelial cell proliferation and migration and microvessel density (MVD) confirmed the anti-angiogenic activity of NGR-SSL-PTX in vitro and in vivo. The sustained circulation of NGR-SSL-PTX was shown in the pharmacokinetic study. NGR-SSL-PTX is able to improve treatment efficacy producing the most significant anti-tumor activity and anti-angiogenic following metronomic administration.

Topics: Administration, Metronomic; Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Diffusion; Female; Fibrosarcoma; Humans; Liposomes; Metabolic Clearance Rate; Mice; Mice, Inbred BALB C; Mice, Nude; Nanocapsules; Oligopeptides; Organ Specificity; Paclitaxel; Rats; Rats, Sprague-Dawley; Tissue Distribution; Treatment Outcome

Targeting and inhibiting angiogenesis is a promising strategy for treatment of cancer. NGR peptide motif is a tumor-homing peptide, which could bind with CD13 expressed on tumor blood vessels. Lidamycin is a highly potent antitumor antibiotic, which is composed of an apoprotein (LDP) and an active enediyne chromophore (AE). Here, an NGR-integrated and enediyne-energized apoprotein composed of cyclic NGR peptide and lidamycin was developed by a two-step procedure. Firstly, we prepared the fusion protein composed of NGR peptide and LDP by recombinant DNA technology. Then, AE was reloaded to the fusion protein to get NGR-LDP-AE. Our experiments showed that NGR-LDP could bind to CD13-expressing HT-1080 cells, whereas the recombinant LDP (rLDP) showed weak binding. NGR-LDP-AE exerted highly potent cytotoxicity to cultured tumor cells in vitro. In vivo antitumor activity was evaluated in murine hepatoma 22 (H22) model and human fibrosarcoma HT-1080 model. At the tolerable dose, NGR-LDP-AE and lidamycin inhibited H22 tumor growth by 94.8 and 66.9%, and the median survival time of the mice was 62 and 37 days, respectively. In the HT-1080 model, NGR-LDP-AE inhibited tumor growth by 88.6%, which was statistically different from that of lidamycin (74.5%). Immunohistochemical study showed that NGR-LDP could bind to tumor blood vessels. Conclusively, these results demonstrate that fusion of LDP with CNGRC peptide delivers AE to tumor blood vessels and improves its antitumor activity.

Topics: Aminoglycosides; Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Apoproteins; CD13 Antigens; Cell Line, Tumor; Enediynes; Female; Fibrosarcoma; Hep G2 Cells; Humans; Liver Neoplasms, Experimental; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Oligopeptides; Protein Binding; Recombinant Fusion Proteins