An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake.

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations.

The molecular role of connexin 43 in human trophoblast cell fusion.

Connexin expression and gap junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43)/gap junction A1 (GJA1) are required for cytotrophoblast fusion into the syncytium, the outer functional layer of the human placenta. Cx43 also impacts intracellular signaling through protein-protein interactions. The transcription factor GCM1 and its downstream target ERVW-1/SYNCYTIN-1 are key players in trophoblast fusion and exert their actions through the ERVW-1 receptor SLC1A5/ASCT-2/RDR/ATB(0). To investigate the molecular role of the Cx43 protein and its interaction with this fusogenic pathway, we utilized stable Cx43-transfected cell lines established from the choriocarcinoma cell line Jeg3: wild-type Jeg3, alphahCG/Cx43 (constitutive Cx43 expression), JpUHD/Cx43 (doxycyclin-inducible Cx43 expression), or JpUHD/trCx43 (doxycyclin-inducible Cx43 carboxyterminal deleted). We hypothesized that truncation of Cx43 at its C-terminus would inhibit trophoblast fusion and protein interaction with either ERVW-1 or SLC1A5. In the alphahCG/Cx43 and JpUHD/Cx43 lines, stimulation with cAMP caused 1) increase in GJA1 mRNA levels, 2) increase in percentage of fused cells, and 3) downregulation of SLC1A5 expression. Cell fusion was inhibited by GJIC blockade using carbenoxylone. Neither Jeg3, which express low levels of Cx43, nor the JpUHD/trCx43 cell line demonstrated cell fusion or downregulation of SLC1A5. However, GCM1 and ERVW-1 mRNAs were upregulated by cAMP treatment in both Jeg3 and all Cx43 cell lines. Silencing of GCM1 prevented the induction of GJA1 mRNA by forskolin in BeWo choriocarcinoma cells, demonstrating that GCM1 is upstream of Cx43. All cell lines and first-trimester villous explants also demonstrated coimmunoprecipitation of SLC1A5 and phosphorylated Cx43. Importantly, SLC1A5 and Cx43 gap junction plaques colocalized in situ to areas of fusing cytotrophoblast, as demonstrated by the loss of E-cadherin staining in the plasma membrane in first-trimester placenta. We conclude that Cx43-mediated GJIC and SLC1A5 interaction play important functional roles in trophoblast cell fusion.

Site-specific PEGylation of HR2 peptides: effects of PEG conjugation position and chain length on HIV-1 membrane fusion inhibition and proteolytic degradation.

Peptides derived from the HR1 or HR2 regions of the HIV-1 envelope glycoprotein gp41 have been shown to be effective inhibitors to prevent virus-host cell membrane fusion. These peptide drugs, however, suffer from relatively short plasma half-lives and are susceptible to enzymatic degradation. Modification of peptides/proteins with poly(ethylene glycol) (PEG) is a well-established strategy to overcome these limitations. This manuscript presents the results of a systematic study on the influence of the site of PEGylation of HR2-derived peptides, as well as of PEG molecular weight on the biological activity and proteolytic stability of these conjugates. Investigation of the fusion inhibitory efficacy of the conjugates in a model cell-cell based assay revealed a loss in activity for the PEGylated peptides as compared to the wild-type HR2-derived peptide. The loss of activity, however, can be minimized by controlling the site of PEGylation, more specifically, by introducing the PEG chain at one of the more central positions along the non-interacting α-helical surface of the peptides. The proteolytic stability of the PEG-peptide conjugates was assessed in a trypsin-based model assay, which revealed an up to 3.4-fold increase in degradation half-life that may help to compensate for the lower inhibitory efficacy of the PEG-peptide conjugates as compared to the wild-type peptide. The results of this study emphasize the power of site-specific PEGylation to improve the stability of peptide/protein drugs while minimizing adverse effects on biological activity.

Erythropoietin mimetic compound AGEM400(HES) binds to the same receptor as erythropoietin but displays a different spectrum of activities.

EPO mimetic peptides (EMPs) have a completely different structure than erythropoietin (EPO) or new generation recombinant erythropoiesis stimulating agents (ESAs) like Darbepoietin alfa (Aranesp) and continuous erythropoiesis stimulating agent (CERA). This study intended to compare the effects of a novel compound called AGEM400(HES), consisting of a dimeric EMP conjugated to hydroxyethyl starch (HES), to those of recombinant EPO. AGEM400(HES) efficiently stimulated erythropoiesis in vitro and efficiently stimulated survival of EPO-dependent cell line UT7/EPO. It also efficiently induced phosphorylation of signaling proteins in these models. However, AGEM400(HES) was shown to have weak or absent effects on survival of, and signaling in, three different EPO-responsive hematopoietic cell lines. In the latter models, when added in excess to moderate concentrations of EPO, AGEM400(HES) inhibited the activity of EPO in a fashion indicating receptor binding competition between EPO and AGEM400(HES). It was furthermore shown, using stably transfected BA/F3 cells, that the degree of responsiveness of a cell to AGEM400(HES) relative to its responsiveness to EPO, correlated with the level of EPO receptor surface expression. The findings presented raise intriguing possibilities because they imply that not all side-effects said to be associated with EPO must necessarily be elicited by AGEM400(HES) too.

Evaluation of fusogenic trophoblast surface epitopes as targets for immune contraception.

Syncytial trophoblast fusion is an essential step in the process of implantation. This project is aimed at the immunological inhibition of syncytial trophoblast fusion as a novel approach to contraception. Fusion-inhibiting recombinant antibodies were generated and used together with autoantibodies from patients with repetitive in vitro fertilization (IVF) failure that were shown to inhibit syncytial fusion and are expected to inhibit implantation, to generate anti-idiotypic peptides. These peptides mimic trophoblast epitopes essential for syncytial fusion and are, therefore, considered specific immunogens for the generation of antibodies that will inhibit implantation. To verify their physiological role in humans, 300 anti-idiotypic peptides were tested for their binding capacity to patient autoantibodies associated with repetitive IVF failure, habitual abortion and preeclampsia. Of these, only three peptides were found to selectively bind to autoantibodies of patients with repetitive IVF failure and were considered safe and efficient enough for evaluation in preclinical and clinical studies required for the development of immune contraceptives. When used as immunogens, these peptides are expected to elicit an antibody response inhibiting syncytial fusion and thus implantation. Furthermore, the action of these antibodies needs to be restricted to the stage of syncytium formation at the time of implantation so as not to cause complications of pregnancy in those cases where they fail to have a contraceptive effect. To exclude potential side effects on other systems, toxicological experiments in animals are in progress.

Detection of Peptide-based nanoparticles in blood plasma by ELISA.

AIMS: The aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma. MATERIALS & METHODS: A convenient enzyme linked immunosorbent assay (ELISA) was developed for the detection of peptides functionalized with biotin and fluorescein groups. As a proof of principle, polymerized pentafluorophenyl methacrylate nanoparticles linked to biotin-carboxyfluorescein labeled peptides were intravenously injected in Wistar rats. Serial blood plasma samples were analyzed by ELISA and by liquid chromatography mass spectrometry (LC/MS) technology. RESULTS: The ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS. CONCLUSIONS: We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions.

Monoclonal antibody CD133-2 (AC141) against hematopoietic stem cell antigen CD133 shows crossreactivity with cytokeratin 18.

CD133 is an antigen expressed on hematopoietic progenitor cells and on some epithelial cells. We previously reported that a commercially available antibody against CD133, CD133-2/AC141, also reacted with an intracellular protein in placental trophoblasts. Here we show by 2D electrophoresis and mass spectroscopy that this reactivity is with cytokeratin 18, a cytokeratin present in most simple epithelia. Immunohistochemistry (IHC) with CD133-2/AC141 on a trophoblast cell line displayed a staining pattern typical for the cytoskeleton. Cryostat sections of stratified epithelia lacking cytokeratin 18 did not react with CD133-2/AC141. In conclusion, care must be taken not to misinterpret staining patterns using CD133-2/AC141 in IHC.

C-Terminal truncations of syncytin-1 (ERVWE1 envelope) that increase its fusogenicity.

Syncytin-1, the envelope protein of ERVWE1, an endogenous retrovirus of the HERV-W family, plays an important role in regulating fusion of the placental trophoblast. At least one of its receptors is expressed on a variety of human cell types. Its ability to fuse cells makes it an attractive candidate molecule in gene therapy against cancer. We studied the relevance of sequences in the cytoplasmic tail of syncytin-1 for inducing cell-cell fusion. We generated a series of C-terminally truncated syncytin-1 variants. Sequences immediately adjacent to the transmembrane region of syncytin-1 were necessary for inducing optimal fusion, whereas the extreme C-terminus of syncytin-1 partially inhibited its fusogenicity. Two variants of syncytin-1, truncated after residues 483 and 515, were significantly hyperfusogenic compared to wild-type syncytin-1. Cellular and cell-surface expression levels of these two variant proteins were similar to those of wild-type syncytin-1. In testing the latter we found that only a very minor portion of recombinantly expressed cellular syncytin-1 was fully mature and expressed on the cell surface. Our results contribute to the understanding of the structure-function relationship of syncytin-1, and might have implications for the use of this molecule in gene therapy.