No abstract

It has gradually become evident that nanomaterials, which are widely used in cosmetics, foods, and medicinal products, could induce substantial inflammation. However, the roles played by the physical characteristics of nanomaterials in inflammatory responses have not been elucidated. Here, we examined how particle size and surface modification influenced the inflammatory effects of nanosilica particles, and we investigated the mechanisms by which the particles induced inflammation. We compared the inflammatory effects of silica particles with diameters of 30-1,000 nm in vitro and in vivo. In macrophages in vitro, 30- and 70-nm nanosilica particles (nSP30 and nSP70) induced higher production of tumor necrosis factor-α (TNFα) than did larger particles. In addition, intraperitoneal injection of nSP30 and nSP70 induced stronger inflammatory responses involving cytokine production than did larger particles in mice. nSP70-induced TNFα production in macrophage depended on the production of reactive oxygen species and the activation of mitogen-activated protein kinases (MAPKs). Furthermore, nSP70-induced inflammatory responses were dramatically suppressed by surface modification of the particles with carboxyl groups in vitro and in vivo; the mechanism of the suppression involved reduction in MAPK activation. These results provide basic information that will be useful for the development of safe nanomaterials.

The use of gene transduction by viral vectors in human gene therapy is currently attracting a great deal of attention. In cancer therapy, gene therapy is expected to play a key role in next-generation treatments, as well as in complementing conventional treatment modalities, such as surgery, chemotherapy, and radiotherapy.1,2) Adenovirus vectors (Adv) have been very useful in clinical gene therapy because of their ability to propagate to a high titer and efficiently transduce cells and tissues regardless of the mitotic status of the cells. 3,4) Intratumoral injection of Adv has demonstrated therapeutic efficacy against primary tumors in several clinical trials. 5,6) However, the clinical application of Adv has been limited by several problems. The systemic administration of Adv can lead to the acute accumulation of Adv and transgene expression in the liver, which may cause marked hepatotoxicity and activate the acquired immune response. 4,7)Since systemic administration is required to treat distant metastases, it is currently difficult to use Adv to treat metastatic tumors, which are the major cause of mortality from cancer. Furthermore, because neutralizing antibodies (Abs) to Adv are highly prevalent in the human population, administration of high doses of Adv, which are needed to obtain sufficient therapeutic effects, causes negative side-effects. 8,9) In addition, neutralizing anti-Adv Abs are produced after administration of Adv in animal models.10,11) These preexisting or Adv-induced Abs can adversely affect the therapeutic effect of the Adv by attenuating the level of transgene expression.Covalent conjugation of polyethleneglycol (PEG) to the Adv surface, by a process called "PEGylation," is the one of the promising strategies to overcome these limitations. 4,12,13) Because of the steric hindrance of the PEG molecule, PEGylation can prolong the plasma half-life of molecules, prevent hepatic uptake, and alter the tissue distribution of the conjugates compared with the native form.14,15) The extended circulating lifetime in the blood induces the "enhanced permeability and retention" (EPR) effect, which is due to the leaky nature of tumor blood vessels. This effect results in increased delivery of the conjugates to tumor tissue. 16) We have examined the in vitro and in vivo characteristics of PEGylated Adv (PEG-Ad) manufactured using PEG of various molecular weights, and with various PEG modification rates. 17,18) With optimized modification conditions, PEG-Ad showed higher accumulation rates and transgene expression in tumors than unmodified Adv after systemic administration, due to the EPR effect.17,18) Furthermore, we showed that PEG-Ad encoding a therapeutic gene produced both stronger tumor-suppressive activity and fewer hepatotoxic side effects than unmodified Adv, suggesting that systemic administration of PEG-Ad has great potential for systemic cancer gene therapy. 17,18) In addition, we and others have reported that PEG-Ad can evade pre-existing neutralizing anti-Adv Abs, thereby making repeated vec...

Previously, we generated a cancer-specific gene therapy system using adenovirus vectors (Adv) conjugated to polyethylene glycol (Adv-PEG). Here, we developed a novel Adv that targets both tumor tissues and tumor vasculatures after systemic administration by conjugating CGKRK tumor vasculature homing peptide to the end of a 20-kDa PEG chain (Adv-PEG(CGKRK)). In a primary tumor model, systemic administration of Adv-PEG(CGKRK) resulted in ~500- and 100-fold higher transgene expression in tumor than that of unmodified Adv and Adv-PEG, respectively. In contrast, the transgene expression of Adv-PEG(CGKRK) in liver was about 400-fold lower than that of unmodified Adv, and was almost the same as that of Adv-PEG. We also demonstrated that transgene expression with Adv-PEG(CGKRK) was enhanced in tumor vessels. Systemic administration of Adv-PEG(CGKRK) expressing the herpes simplex virus thymidine kinase (HSVtk) gene (Adv-PEG(CGKRK)-HSVtk) showed superior antitumor effects against primary tumors and metastases with negligible side effects by both direct cytotoxic effects and inhibition of tumor angiogenesis. These results indicate that Adv-PEG(CGKRK) has potential as a prototype Adv with suitable efficacy and safety for systemic cancer gene therapy against both primary tumors and metastases.

Gene therapy is expected to play a key role in next-generation cancer therapy together with the conventional modalities for the treatment of cancer, such as surgery, chemotherapy, and radiotherapy. 1) Use of adenovirus vectors (Adv) in gene therapy has been developed in diverse animal models and clinical trials because Adv can be propagated to high yields and has the capacity to efficiently transduce a wide variety of cell types and tissues regardless of the mitotic status of the cell. 2,3) In several clinical trials, intratumoral injection of Adv has demonstrated therapeutic efficacy against primary tumors. 4,5) However, successful cancer gene therapy also requires treatment of unresectable primary cancers and distant metastases, which are the major cause of cancer mortality. To treat unresectable primary and metastatic cancers, Adv must be administered systemically, but its effects must be restricted to tumor cells. The major disadvantage in treating cancer with systemically administered conventional Adv is related to its accumulation in the liver immediately after systemic injection, which can cause severe liver toxicity. 6)Therefore, to enable the application of Adv to the treatment of both primary and metastatic cancers, it is necessary to develop Adv with the capacity for tumor-selective distribution and gene expression after systemic administration.In a previous study, we generated a dual cancer-specific targeting vector system by using Adv covalently conjugated to polyethylene glycol (PEG), for transductional targeting, and a cancer-specific promoter, for transcriptional targeting. 7)Covalent conjugation to PEG (PEGylation) can prolong the plasma half-life of Adv, prevent hepatic uptake, and alter the tissue distribution of Adv because of the steric hindrance of the PEG. 3,7,8) We showed that systemic administration of PEGylated Adv (PEG-Ad) with 20-kDa PEG at a 45% modification ratio resulted in higher tumor-selective transgene expression than unmodified Adv.7) In addition, we showed that the telomere reverse transcriptase (TERT) promoter-driven PEG-Ad (PEG-Ad-TERT), which contained the herpes simplex virus thymidine kinase (HSVtk) gene as the therapeutic gene, induced strong antitumor effects against metastasis without any side effects. By comparison, a conventional cytomegalovirus (CMV) promoter-driven PEG-Ad produced strong antitumor effects but induced unwanted side effects. 7)Therefore, PEG-Ad-TERT can be regarded as a prototype Adv with suitable efficacy and safety for systemic cancer gene therapy against metastasis, although PEG-Ad-CMV might not be suitable. However, it is not clear whether PEGAd-TERT can be used for the treatment of unresectable primary cancers.In this study, we examined the therapeutic effects of systemically administered PEG-Ad-TERT for the treatment of primary tumors. MATERIALS AND METHODS Mice and Cell LinesFemale 5-week-old C57BL6 mice were purchased from SLC Inc. (Hamamatsu, Japan). All procedures involving animal experimentation were performed in ; 7-6-8 Saito-Asagi, Ib...