D-T7 Peptide-Modified PEGylated Bilirubin Nanoparticles Loaded with Cediranib and Paclitaxel for Antiangiogenesis and Chemotherapy of Glioma.

The blood-brain tumor barrier (BTB) and blood-brain barrier (BBB) have always been the major barriers in glioma therapy. In this report, we proposed D-T7 peptide-modified nanoparticles actively targeted glioma by overcoming the BBB and BTB to improve the antiglioma efficacy. Glioma-targeting experiments showed that the penetration effect of the D-T7 peptide-modified nanoparticles was 7.89-fold higher than that of unmodified nanoparticles. Furthermore, cediranib (CD) and paclitaxel (PTX) were used for the combination of the antiangiogenesis and chemotherapy for glioma. PEGylated bilirubin nanoparticles (BRNPs) were selected as a suitable drug delivery system (CD&PTX@TBRBPs) owing to the antioxidant, anti-inflammatory, and reactive oxygen species-responsive ability. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and apoptosis assays showed that CD&PTX@TBRBPs had the highest cytotoxicity and the median survival time of the CD&PTX@TBRNP group was 3.31-fold and 1.23-fold longer than that of the saline and CD&PTX@BRNP groups, respectively. All the results showed that we constructed a novel and accessible peptide-modified dual drug carrier with an enhanced antiglioma effect.

Computational modeling of synergistic interaction between αVß3 integrin and VEGFR2 in endothelial cells: Implications for the mechanism of action of angiogenesis-modulating integrin-binding peptides.

Cooperation between VEGFR2 and integrin αVß3 is critical for neovascularization in wound healing, cardiovascular ischemic diseases, ocular diseases, and tumor angiogenesis. In the present study, we developed a rule-based computational model to investigate the potential mechanism by which the Src-induced integrin association with VEGFR2 enhances VEGFR2 activation. Simulations demonstrated that the main function of integrin is to reduce the degradation of VEGFR2 and hence stabilize the activation signal. In addition, receptor synthesis rate and recruitment from internal compartment were found to be sensitive determinants of the activation state of VEGFR2. The model was then applied to simulate the effect of integrin-binding peptides such as tumstatin and cilengitide on VEGFR2 signaling. Further, computational modeling proposed potential molecular mechanisms for the angiogenesis-modulating activity of other integrin-binding peptides. The model highlights the complexity of the crosstalk between αVß3 integrin and VEGFR2 and the necessity of utilizing models to elucidate potential mechanisms in angiogenesis-modulating peptide therapy.

Systemic delivery of siRNA by T7 peptide modified core-shell nanoparticles for targeted therapy of breast cancer.

Systemic delivery of siRNA is the most challenging step to transfer RNAi to clinical application for breast cancer therapy. In this study, the tumor targeted, T7 peptide modified core-shell nanoparticles (named as T7-LPC/siRNA NPs) were constructed to achieve effective systemic delivery of siRNA. The core-shell structure of T7-LPC/siRNA NPs enables them to encapsulate siRNA in the core and protect it from RNase degradation during circulation. In vitro cellular uptake and gene silencing experiments demonstrated that T7-LPC/siEGFR NPs could deliver EGFR siRNA into breast cancer cells through receptor mediated endocytosis and effectively down-regulate the EGFR expression. In vivo distribution study proved the T7-LPC/siRNA NPs could deliver fluorescence labeled siRNA to the tumor site more efficiently than the non-targeted PEG-LPC/siRNA NPs after intravenous administration. Furthermore, the experiments of in vivo tumor therapy confirmed that intravenous administration of T7-LPC/siEGFR NPs led to an effective EGFR down-regulation and an obvious inhibition of breast tumor growth, with little activation of immune responses and negligible body weight loss. These results suggested that T7-LPC/siRNA NPs could be an effective and safe systemic siRNA delivery system for RNAi-based breast cancer therapy.

uPARAP/endo180 directs lysosomal delivery and degradation of collagen IV.

Collagen turnover is crucial for tissue homeostasis and remodeling and pathological processes such as cancer invasion, but the underlying molecular mechanisms are poorly understood. A major pathway appears to be internalization and degradation by fibroblasts. We now show that the endocytic transmembrane glycoprotein urokinase plasminogen activator receptor-associated protein (uPARAP/endo180) directs collagen IV for lysosomal delivery and degradation. In wild-type fibroblasts, fluorescently labeled collagen IV was first internalized into vesicular structures with diffuse fluorescence eventually appearing uniformly within the wild-type cells after longer incubation times. In these cells, some collagen-containing vesicles were identified as lysosomes by staining for LAMP-1. In contrast, collagen IV remained extracellular and associated with fiber-like structures on uPARAP/endo180-deficient fibroblasts. Blocking lysosomal cysteine proteases with the inhibitor E64d resulted in strong accumulation of collagen IV in lysosomes in wild-type cells, but only very weak intracellular fluorescence accumulation in uPARAP/endo180-deficient fibroblasts. We conclude that uPARAP/endo180 is critical for targeted delivery of collagen IV to lysosomes for degradation implicating the receptor in normal and malignant extracellular matrix degradation. A similar localization pattern was observed for collagen V, suggesting that uPARAP/endo180 might be generally involved in collagen degradation.

Localization of type IV collagen a 1 to a 6 chains in basement membrane during mouse molar germ development.

The dental basement membrane (BM) putatively mediates epithelial-mesenchymal interactions during tooth morphogenesis and cytodifferentiation. Type IV collagen alpha chains, a major network-forming protein of the dental BM, was studied and results disclosed distinct expression patterns at different stages of mouse molar germ development. At the dental placode and bud stage, the BM of the oral epithelium expressed alpha 1, alpha 2, alpha 5 and alpha 6 chains while the gubernaculum dentis, in addition to the above four chains, also expressed a 4 chain. An asymmetrical expression for alpha 4, alpha 5 and alpha 6 chains was observed at the bud stage. At the early bell stage, the BM associated with the inner enamel epithelium (IEE) of molar germ expressed alpha 1, alpha 2 and alpha 4 chains while the BM of the outer enamel epithelium (OEE) expressed only alpha 1 and a 2 chains. With the onset of dentinogenesis, the collagen a chain profile of the IEE BM gradually disappeared. Howeverfrom the early to late bell stage, the gubernaculum dentis consistently expressed alpha 1, alpha 2, alpha 5 and a 6 chains resembling fetal oral mucosa. These findings suggest that stage- and position-specific distribution of type IV collagen alpha subunits occur during molar germ development and that these changes are essential for molar morphogenesis and cytodifferentiation.

Evaluation of radiolabeled type IV collagen fragments as potential tumor imaging agents.

The objective of this study was to examine radiopharmaceuticals that target the alpha3beta1 integrin to determine if these agents target tumors for diagnostic imaging and/or targeted radiotherapy of cancer. Prior studies had shown that residues 531-542 from the alpha1 chain of type IV collagen bind a variety of tumor cell alpha3beta1 integrins. A peptide mimic of this sequence containing all D-amino acids (designated D-Hep-III) was synthesized by solid-phase methods. The tetraazamacrocyclic chelator, TETA, was conjugated to the peptide while it was resin-bound. TETA-D-Hep-III and D-Hep-III were radiolabeled with 64Cu and 125I, respectively, in high specific activity and radiochemical purity. Heterologous competitive binding assays between D-Hep-III and either 125I-D-Hep-III or 64Cu-TETA-D-Hep-III indicated low micromolar affinity of D-Hep-III. The biodistribution of each radiolabeled analogue of D-Hep-III was carried out in rats and tumor-bearing mice. Both analogues were rapidly cleared from the blood in normal rats, with the kidneys receiving the highest accumulation of each. SKOV3 human ovarian tumor cells, known to strongly express alpha3beta1, were xenografted in SCID mice. Localization of 125I-D-Hep III and 64Cu-TETA-D-Hep III in the xenografts were low (<2% ID/g), and in the case of 125I-D-Hep III, not inhibited by a competitive dose of D-Hep III. The low tumor accumulation is likely not due to receptor down-regulation, but rather due to the weak affinity of the radioligands for the alpha3beta1 integrin.

Adhesion and proliferation of rat vascular smooth muscle cells (VSMC) on polyethylene implanted with O+ and C+ ions.

Polyethylene was implanted with 30-keV oxygen (PE/O+) or 23-keV carbon ions (PE/C+) at 10(13) to 5 x 10(15) ions cm(-2) doses in order to improve the adhesion of vascular smooth muscle cell (VSMC) to the polymer surface in vitro because of its oxidation and carbon-enrichment. The concentration of -CO- groups in the PE/O+ and PE/C+ samples increased only up to doses of 3 x 10(14) and 10(15) ions cm(-2), respectively, and then declined. At the same time, the concentration of these groups, measured at a dose of 3 x 10(14) ions cm(-2), was higher in PE/O+ than in PE/C+ samples. Similarly, the number of initially-adhering rat VSMC (24 h after seeding) increased only up to a dose of 3 x 10(13) and 10(15) ions cm(-2) on PE/O+ and PE/C+ samples, respectively. In addition, between doses of 10(13) and 10(14) ions cm(-2), this number was about two to three times higher on PE/O+ samples. On the other hand, the surface wettability increased proportionally to the implanted ion dose, especially above a dose of 10(14) ions cm(-2). Thus, the number of initially-adhered cells appeared to be positively correlated with the amount of the oxygen group present at the polymer surface rather than with the surface wettability. The higher cell adhesion was accompanied by adsorption of fluorescent dye-conjugated collagen IV in larger amounts. The highest numbers of initially-adhered cells were usually associated with the lowest rates of subsequent proliferation (measured by the doubling time, BrdU labelling and M