Targeted Delivery of Glypican 3 (GPC3) Antibody-Modified MicroRNA (miR let-7b-5p) Polymer Nanoparticles to Sorafenib-Resistant Hepatsocellular Carcinoma Cells.

The miR let-7b-5p (a kind of microRNAs) has many pathophysiological regulation effects, including human hepatocellular carcinoma (HCC) pathogenesis. This study investigated whether nanoparticle-mediated miR let-7b-5p could jointly enhance the therapeutic effect of sorafenib on HCC by inhibiting the proliferation of HCC cells, inducing apoptosis, and reversing drug resistance. We evaluated the level of miR let-7b-5p in sorafenib-resistant HepG2 cells (HepG2R) and HepG2 HCC cells by qRT-PCR and analyzed the biological effects of hepatocellular carcinoma treated with sorafenib with miR let-7b-5p, and further studied the toxicity of nanoparticles (Ab-miR-NPs) that deliver miR let-7b-5p mimics and target GPC3 on the surface of hepatocellular carcinoma cells. Results showed that, in HepG2 cells, the expression level of miR let-7b-5p was significantly higher than that in HepG2R cells. Targeted nanoparticle Ab-miR-NPs mediated the delivery of miR let-7b-5p to the HCC cytoplasm and released miRNA after being broken down, down-regulating the expression of IGF1R and inhibiting AKT/mTOR and Ras/Raf signal transmission. Ab-miR-NPs not only enhanced the proliferation of sorafenib in cultured HepG2R cells and induced cell apoptosis efficiency, but they also improved the anti-tumor activity in the mouse models. These results indicated that GPC3 antibody-modified PLGA-PLL (polylactic acid-glycolic acetic copolymer grafted hyper-branched polylysine) loaded miR let-7b-5p polymer nanoparticles combined with sorafenib may be a new treatment strategy for HCC resistant to sorafenib.

Titania nanotube-based protein delivery system to inhibit cranial bone regeneration in Crouzon model of craniosynostosis.

BACKGROUND: Craniosynostosis is a developmental disorder characterized by the premature fusion of skull sutures, necessitating repetitive, high-risk neurosurgical interventions throughout infancy. This study used protein-releasing Titania nanotubular implant (TNT/Ti) loaded with glypican 3 (GPC3) in the cranial critical-sized defects (CSDs) in Crouzon murine model (Fgfr2c342y/+ knock-in mutation) to address a key challenge of delaying post-operative bone regeneration in craniosynostosis. MATERIALS AND METHODS: A 3 mm wide circular CSD was created in two murine models of Crouzon syndrome: (i) surgical control (CSDs without TNT/Ti or any protein, n=6) and (ii) experimental groups with TNT/Ti loaded with GPC3, further subdivided into the presence or absence of chitosan coating (on nanotubes) (n=12 in each group). The bone volume percentage in CSDs was assessed 90 days post-implantation using micro-computed tomography (micro-CT) and histological analysis. RESULTS: Nano-implants retrieved after 90 days post-operatively depicted well-adhered, hexagonally arranged, and densely packed nanotubes with average diameter of 120±10 nm. The nanotubular architecture was generally well-preserved. Compared with the control bone volume percentage data (without GPC3), GPC3-loaded TNT/Ti without chitosan coating displayed a significantly lower volume percent in cranial CSDs (P<0.001). Histological assessment showed relatively less bone regeneration (healing) in GPC3-loaded CSDs than control CSDs. CONCLUSION: The finding of inhibition of cranial bone regeneration by GPC3-loaded TNT/Ti in vivo is an important advance in the novel field of minimally-invasive craniosynostosis therapy and holds the prospect of altering the whole paradigm of treatment for affected children. Future animal studies on a larger sample are indicated to refine the dosage and duration of drug delivery across different ages and both sexes with the view to undertake human clinical trials.

Glypican-based drug releasing titania implants to regulate BMP2 bioactivity as a potential approach for craniosynostosis therapy.

Advances in molecular biology and nanomedicine based therapies hold promise to obviate the need of multiple surgical interventions (associated with current management) in craniosynostosis by preventing bone re-ossification. One such adjunctive therapy involves application of glypicans 1 and 3 (GPC1 and GPC3) that are BMP inhibitors implicated in downregulating the BMP2 activity in prematurely fusing sutures. Electrochemically anodized Titania nanotube (TNT) arrays have been recognized as a promising localized, long-term drug delivery platform for bone-related therapies. This study presents the application of nanoengineered TNT/Ti implants loaded with recombinant glypicans for craniosynostosis therapy. By using Dual luciferase Reporter assay, we tested the biofunctionality of eluted glypicans from the TNT/Ti implants for BMP2 bioactivity regulation in C2C12 murine myoblast cell line. BMP2 activity was inhibited significantly for up to 15days by the glypicans released from polymer-coated TNT/Ti implants, indicating their potential application in adjunctive craniosynostosis treatment.

Plasmodium parasite as an effective hepatocellular carcinoma antigen glypican-3 delivery vector.

We have previously demonstrated that malaria parasite infection has an anti-tumor effect in a mouse model. This research aimed to investigate the possibility of using Plasmodium parasite as a novel vaccine vector for hepatocellular carcinoma (HCC) immunotherapy. We constructed a Plasmodium yoelii 17XNL strain (P.y) expressing murine glypican-3 (GPC3) protein (P.y-GPC3), and examined its therapeutic potency in a murine Hepa1-6-induced hepatoma model that highly expressed GPC3 protein. The prerequisites for invoking a CD8+ T cell response were assessed after P.y-based immunization, which included obviously increased concentrations of T helper cell type 1 (Th1)-associated cytokines, such as IL-2, IFN-γ and TNF-α, in serum and preferential expansion of the CD8α+ dendritic cell (DC) subset with higher expression of CD80 and CD86 molecules. Compared with uninfected and wild-type P.y-infected mice, a significant GPC3-specific cytotoxic T lymphocyte (CTL) response was detected in P.y-GPC3 vaccinated mice. Furthermore, P.y-GPC3-based vaccination dramatically inhibited Hepa1-6-induced tumor growth in the implanted HCC and prolonged the survival of tumor-bearing mice. We concluded that a Plasmodium-based vector is highly efficient in inducing tumor antigen-specific T cell-mediated immunity and protection against tumor cells. More broadly, this strategy supported our hypothesis that Plasmodium parasites, as novel therapeutic antigen vectors, may be applicable to tumor immunotherapy for patients with HCC.

Analysis of cytotoxic T lymphocytes from a patient with hepatocellular carcinoma who showed a clinical response to vaccination with a glypican­3­derived peptide.

Glypican-3 (GPC3), which is a carcinoembryonic antigen, is overexpressed in human hepatocellular carcinoma (HCC). Previously, we performed a phase I clinical trial of GPC3­derived peptide vaccination in patients with advanced HCC, and reported that GPC3 peptide vaccination is safe and has clinical efficacy. Moreover, we proposed that a peptide­specific CTL response is a predictive marker of overall survival in patients with HCC who receive peptide vaccination. In this study, we established GPC3­derived peptide-specific CTL clones from the PBMCs of an HLA-A*02:07-positive patient with HCC who was vaccinated with an HLA-A2-restricted GPC3 peptide vaccine and showed a clinical response in the phase I clinical trial. Established CTL clones were analyzed using the IFN-γ ELISPOT assay and a cytotoxicity assay. GPC3 peptide-specific CTL clones were established successfully from the PBMCs of the patient. One CTL clone showed cytotoxicity against cancer cell lines that expressed endogenously the GPC3 peptide. The results suggest that CTLs have high avidity, and that natural antigen-specific killing activity against tumor cells can be induced in a patient with HCC who shows a clinical response to vaccination with the GPC3144-152 peptide.

Remarkable tumor lysis in a hepatocellular carcinoma patient immediately following glypican-3-derived peptide vaccination: an autopsy case.

We recently reported the safety, immunological and clinical responses to a GPC3-derived peptide vaccine in a phase I clinical trial of patients with advanced hepatocellular carcinoma (HCC). We conducted a subsequent trial in advanced HCC to assess the histopathological findings before and after vaccination with the GPC3 peptide. Here, we present the clinical course and the pathological study including the autopsy of a patient with advanced HCC in the ongoing clinical trial. A 62-year old patient suffering from HCC refractory to sorafenib therapy received the GPC3 peptide vaccine. The patient had fever and remarkably impaired liver function twice after vaccination. Contrast-enhanced CT after the second vaccination showed multiple low-density areas in the liver tumor, indicating tumor necrosis. In contrast, the tumor thrombus in the right atrium increased. The patient discontinued protocol treatment due to disease progression and died 30 days after the second vaccination. An autopsy was performed to determine the main cause of death and to evaluate the antitumor effect of the vaccination. A histological examination showed central necrosis in most of the intrahepatic tumor. The main cause of death was circulatory failure due to tumor thrombus, which occupied most of the right atrium. An immunohistochemical analysis revealed infiltration of CD8-positive T cells in the residual carcinoma, but not within the cirrhotic area. Ex vivo IFN-γ enzyme-linked immunospot analysis revealed vaccine-induced immune-reactivity against the GPC3 peptide. A histopathological examination at the estimated time of a strong immunological response demonstrated a GPC3 peptide vaccination-induced cytotoxic T-lymphocyte response with an anti-tumor effect.

Intracerebral chondroitinase ABC and heparan sulfate proteoglycan glypican improve outcome from chronic stroke in rats.

Physical and chemical constraints imposed by the periinfarct glial scar may contribute to the limited clinical improvement often observed after ischemic brain injury. To investigate the role of some of these mediators in outcome from cerebral ischemia, we treated rats with the growth-inhibitory chondroitin sulfate proteoglycan neurocan, the growth-stimulating heparan sulfate proteoglycan glypican, or the chondroitin sulfate proteoglycan-degrading enzyme chondroitinase ABC. Neurocan, glypican, or chondroitinase ABC was infused directly into the infarct cavity for 7 d, beginning 7 d after middle cerebral artery occlusion. Glypican and chondroitinase ABC reduced glial fibrillary acidic protein immunoreactivity and increased microtubule-associated protein-2 immunoreactivity in the periinfarct region, and glypican- and chondroitinase ABC-treated rats showed behavioral improvement compared with neurocan- or saline-treated rats. Glypican and chondroitinase ABC also increased neurite extension in cortical neuron cultures. Glypican increased fibroblast growth factor-2 expression and chondroitinase ABC increased brain-derived neurotrophic factor expression in these cultures, whereas no such effects were seen following neurocan treatment. Thus, treatment with glypican or enzymatic disruption of neurocan with chondroitinase ABC improves gross anatomical, histological, and functional outcome in the chronic phase of experimental stroke in rats. Changes in growth factor expression and neuritogenesis may help to mediate these effects.

HLA-A2 and -A24-restricted glypican-3-derived peptide vaccine induces specific CTLs: preclinical study using mice.

We previously reported that glypican-3 (GPC3) is uniquely overexpressed in human hepatocellular carcinoma and melanoma and that it is an ideal tumor antigen for immunotherapy in mouse models. We recently identified both HLA-A24 (A*2402) and H-2Kd-restricted GPC3298-306 (EYILSLEEL) and HLA-A2 (A*0201)-restricted GPC3144-152 (FVGEFFTDV), both of which can induce GPC3-reactive cytotoxic T cells (CTLs). The present study was a preclinical study in a mouse model that was conducted in order to design an optimal schedule for clinical trial of GPC3-derived peptide vaccine. When BALB/c mice were intradermally vaccinated at the base of the tail with Kd-restricted GPC3298-306 peptide mixed with incomplete Freund's adjuvant (IFA), the peptide-specific CTLs were induced. But the peptide alone could not induce peptide-specific CD8+ T cells. Furthermore, proteomic analyses showed that IFA protected the peptide against degradation in the human serum. Peptide-reactive CTLs were induced by peptide vaccine in a dose-dependent manner. In addition, at least two vaccinations with a single dose >10 microg were needed for the induction of GPC3298-306-specific CTLs. But repeated vaccination with a lower dose of GPC3298-306 did not induce peptide-specific CTLs. Similarly, induction of an Ag-specific immune response by HLA-A2 GPC3144-152 depended on the dose administered. The results of this study suggested that IFA is one of the indispensable adjuvants for peptide-based immunotherapy, and that the immunological effect of peptide vaccines depends on the dose of peptide injected.