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Pharmaceutics, Volume 7, Issue 3 (September 2015) – 19 articles , Pages 90-362

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4522 KiB  
Article
Enhancement of Blood–Brain Barrier Permeability and Delivery of Antisense Oligonucleotides or Plasmid DNA to the Brain by the Combination of Bubble Liposomes and High-Intensity Focused Ultrasound
by Yoichi Negishi, Masaya Yamane, Naho Kurihara, Yoko Endo-Takahashi, Sanae Sashida, Norio Takagi, Ryo Suzuki and Kazuo Maruyama
Pharmaceutics 2015, 7(3), 344-362; https://doi.org/10.3390/pharmaceutics7030344 - 21 Sep 2015
Cited by 45 | Viewed by 9928
Abstract
The blood–brain barrier (BBB) is a major obstacle that prevents therapeutic drugs or genes from being delivered to the central nervous system. Therefore, it is important to develop methods to enhance the permeability of the BBB. We have developed echo-contrast gas (C3 [...] Read more.
The blood–brain barrier (BBB) is a major obstacle that prevents therapeutic drugs or genes from being delivered to the central nervous system. Therefore, it is important to develop methods to enhance the permeability of the BBB. We have developed echo-contrast gas (C3F8) entrapping liposomes (Bubble liposomes, BLs) that can work as a gene delivery tool in combination with ultrasound (US) exposure. Here, we studied whether the permeability of the BBB can be enhanced by the combination of BLs and high-intensity focused ultrasound (HIFU). Mice were intravenously injected with Evans blue (EB). BLs were subsequently injected, and the right hemispheres were exposed to HIFU. As a result, the accumulation of EB in the HIFU-exposed brain hemispheres was increased over that observed in the non-HIFU-exposed hemispheres, depending on the intensity and the duration of the HIFU. Similarly, the combination of BLs and HIFU allowed fluorescent-labeled antisense oligonucleotides to be delivered into the HIFU-exposed left hemispheres of the treated mice. Furthermore, a firefly luciferase-expressing plasmid DNA was delivered to the brain by the combination method of BLs and HIFU, which resulted in the increased gene expression in the brain at the focused-US exposure site. These results suggest that the method of combining BLs and HIFU together serves as a useful means for accelerating the permeability of BBB and thereby enabling antisense oligonucleotides or genes to be delivered to the focused brain site. Full article
(This article belongs to the Special Issue New Paradigm of Gene Therapy)
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2815 KiB  
Communication
Site-Specific Impact of a Regional Hydrodynamic Injection: Computed Tomography Study during Hydrodynamic Injection Targeting the Swine Liver
by Takeshi Yokoo, Tsutomu Kanefuji, Takeshi Suda, Kenya Kamimura, Dexi Liu and Shuji Terai
Pharmaceutics 2015, 7(3), 334-343; https://doi.org/10.3390/pharmaceutics7030334 - 16 Sep 2015
Cited by 8 | Viewed by 4413
Abstract
A hemodynamic study of hydrodynamic gene delivery (HGD) from the tail vein in rodents has inspired a mechanism and an approach to further improve the efficacy of this procedure. However, there is no report on the hemodynamics of a regional HGD, which is [...] Read more.
A hemodynamic study of hydrodynamic gene delivery (HGD) from the tail vein in rodents has inspired a mechanism and an approach to further improve the efficacy of this procedure. However, there is no report on the hemodynamics of a regional HGD, which is an inevitable approach in large animals. Here, we report the hemodynamics of a regional hydrodynamic injection in detail based on 3D volume data and the dynamism of tissue intensity over time by using computed tomography (CT) both during and after a regional hydrodynamic injection that targeted the liver of a pig weighing 15.6 kg. Contrast medium (CM) was injected at a steady speed of 20 mL/s for 7.5 s under the temporal balloon occlusion of the hepatic vein (HV). A retrograde flow formed a wedge-shaped strong enhancement area downstream of the corresponding HV within 2.5 s, which was followed by drainage into another HV beginning from the target area and the portal vein (PV) toward a non-target area of the liver. After the injection, the CM was readily eliminated from the PV outside the target area. These data suggest that an interventional radiology approach is effective in limiting the hydrodynamic impacts in large animals at a target area and that the burden overflowing into the PV is limited. A further investigation that simultaneously evaluates gene delivery efficiency and hemodynamics using CT is needed to establish feasible parameters for a regional HGD in large animals. Full article
(This article belongs to the Special Issue New Paradigm of Gene Therapy)
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1660 KiB  
Article
Optimization of a siRNA Carrier Modified with a pH-Sensitive Cationic Lipid and a Cyclic RGD Peptide for Efficiently Targeting Tumor Endothelial Cells
by Tomoya Hada, Yu Sakurai and Hideyoshi Harashima
Pharmaceutics 2015, 7(3), 320-333; https://doi.org/10.3390/pharmaceutics7030320 - 14 Sep 2015
Cited by 21 | Viewed by 5275
Abstract
In recent years, anti-angiogenic therapy has attracted much interest because it is a versatile approach to treating most types of tumors, and therefore would be expected to be applicable for various cancers. Severe adverse events in patients treated with currently available anti-angiogenic therapeutics [...] Read more.
In recent years, anti-angiogenic therapy has attracted much interest because it is a versatile approach to treating most types of tumors, and therefore would be expected to be applicable for various cancers. Severe adverse events in patients treated with currently available anti-angiogenic therapeutics have, however, been reported, and these are caused by their inhibitory effects in normal tissue. To achieve an efficient anti-angiogenic therapy with minimal toxicity, a drug delivery system (DDS) specific to tumor endothelial cells (TECs) is needed. Cyclic RGD (cRGD) is a well-known ligand against αVβ3 integrin that is expressed at high levels in the cell surface of TECs. To address this issue, we previously developed a cyclic RGD-equipped liposomal DDS (RGD-MEND) in which small interfering RNA (siRNA) was encapsulated. However, in the previous study, details of the preparation steps were not thoroughly examined. In this paper, to produce the most efficient delivery of therapeutic TECs, we explored optimum preparation conditions and components of the RGD-MEND. The cellular uptake and silencing ability of the RGD-MEND were investigated as a function of ligand density, poly(ethyleneglycol) linker length, and lipid composition. As a result, a knockdown efficiency that was five-fold higher than that of the previously reported one (ED50, from 4.0 to 0.75 mg/kg) was achieved. Full article
(This article belongs to the Special Issue New Paradigm of Gene Therapy)
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2711 KiB  
Article
Effect of Crosslinking Agent Concentration on the Properties of Unmedicated Hydrogels
by Rachel Shet Hui Wong, Mark Ashton and Kalliopi Dodou
Pharmaceutics 2015, 7(3), 305-319; https://doi.org/10.3390/pharmaceutics7030305 - 9 Sep 2015
Cited by 186 | Viewed by 10235
Abstract
Novel polyethylene oxide (PEO) hydrogel films were synthesized via UV crosslinking with varying concentrations of pentaerythritol tetra-acrylate (PETRA) as crosslinking agent. The aim was to study the effects of the crosslinking agent on the material properties of hydrogel films intended for dermatological applications. [...] Read more.
Novel polyethylene oxide (PEO) hydrogel films were synthesized via UV crosslinking with varying concentrations of pentaerythritol tetra-acrylate (PETRA) as crosslinking agent. The aim was to study the effects of the crosslinking agent on the material properties of hydrogel films intended for dermatological applications. Fabricated film samples were characterized using swelling studies, scanning electron microscopy, tensile testing and rheometry. Films showed rapid swelling and high elasticity. The increase of PETRA concentration resulted in significant increase in the gel fraction and crosslinking density (ρc), while causing a significant decrease in the equilibrium water content (EWC), average molecular weight between crosslinks (\({\overline{M}}_{c}\)), and mesh size (ζ) of films. From the scanning electron microscopy, cross-linked PEO hydrogel network appeared as cross-linked mesh-like structure with interconnected micropores. Rheological studies showed PEO films required a minimum of 2.5% w/w PETRA to form stable viscoelastic solid gels. Preliminary studies concluded that a minimum of 2.5% w/w PETRA is required to yield films with desirable properties for skin application. Full article
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6702 KiB  
Article
Topical Anti-Nuclear Factor-Kappa B Small Interfering RNA with Functional Peptides Containing Sericin-Based Hydrogel for Atopic Dermatitis
by Takanori Kanazawa, Yuki Shizawa, Mayu Takeuchi, Kuniko Tamano, Hisako Ibaraki, Yasuo Seta, Yuki Takashima and Hiroaki Okada
Pharmaceutics 2015, 7(3), 294-304; https://doi.org/10.3390/pharmaceutics7030294 - 7 Sep 2015
Cited by 27 | Viewed by 6183
Abstract
The small interfering RNA (siRNA) is suggested to offer a novel means of treating atopic dermatitis (AD) because it allows the specific silencing of genes related to AD pathogenesis. In our previous study, we found that siRNA targeted against RelA, an important nuclear [...] Read more.
The small interfering RNA (siRNA) is suggested to offer a novel means of treating atopic dermatitis (AD) because it allows the specific silencing of genes related to AD pathogenesis. In our previous study, we found that siRNA targeted against RelA, an important nuclear factor-kappa B (NF-κB) subdomain, with functional peptides, showed therapeutic effects in a mouse model of AD. In the present study, to develop a topical skin application against AD, we prepared a hydrogel containing anti-RelA siRNA and functional peptides and determined the intradermal permeation and the anti-AD effects in an AD mouse model. We selected the silk protein, sericin (SC), which is a versatile biocompatible biomaterial to prepare hydrogel as an aqueous gel base. We found that the siRNA was more widely delivered to the site of application in AD-induced ear skin of mice after topical application via the hydrogel containing functional peptides than via the preparation without functional peptides. In addition, the ear thickness and clinical skin severity of the AD-induced mice treated with hydrogel containing anti-RelA siRNA with functional peptides improved more than that of mice treated with the preparation formulated with negative siRNA. Full article
(This article belongs to the Special Issue New Paradigm of Gene Therapy)
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924 KiB  
Review
Facilitation of Drug Transport across the Blood–Brain Barrier with Ultrasound and Microbubbles
by Stephen Meairs
Pharmaceutics 2015, 7(3), 275-293; https://doi.org/10.3390/pharmaceutics7030275 - 31 Aug 2015
Cited by 57 | Viewed by 8882
Abstract
Medical treatment options for central nervous system (CNS) diseases are limited due to the inability of most therapeutic agents to penetrate the blood–brain barrier (BBB). Although a variety of approaches have been investigated to open the BBB for facilitation of drug delivery, none [...] Read more.
Medical treatment options for central nervous system (CNS) diseases are limited due to the inability of most therapeutic agents to penetrate the blood–brain barrier (BBB). Although a variety of approaches have been investigated to open the BBB for facilitation of drug delivery, none has achieved clinical applicability. Mounting evidence suggests that ultrasound in combination with microbubbles might be useful for delivery of drugs to the brain through transient opening of the BBB. This technique offers a unique non-invasive avenue to deliver a wide range of drugs to the brain and promises to provide treatments for CNS disorders with the advantage of being able to target specific brain regions without unnecessary drug exposure. If this method could be applied for a range of different drugs, new CNS therapeutic strategies could emerge at an accelerated pace that is not currently possible in the field of drug discovery and development. This article reviews both the merits and potential risks of this new approach. It assesses methods used to verify disruption of the BBB with MRI and examines the results of studies aimed at elucidating the mechanisms of opening the BBB with ultrasound and microbubbles. Possible interactions of this novel delivery method with brain disease, as well as safety aspects of BBB disruption with ultrasound and microbubbles are addressed. Initial translational research for treatment of brain tumors and Alzheimer’s disease is presented. Full article
(This article belongs to the Special Issue Drug Delivery to Brain)
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1592 KiB  
Review
Potential Use of Biological Proteins for Liver Failure Therapy
by Kazuaki Taguchi, Keishi Yamasaki, Hakaru Seo and Masaki Otagiri
Pharmaceutics 2015, 7(3), 255-274; https://doi.org/10.3390/pharmaceutics7030255 - 31 Aug 2015
Cited by 10 | Viewed by 6699
Abstract
Biological proteins have unlimited potential for use as pharmaceutical products due to their various biological activities, which include non-toxicity, biocompatibility, and biodegradability. Recent scientific advances allow for the development of novel innovative protein-based products that draw on the quality of their innate biological [...] Read more.
Biological proteins have unlimited potential for use as pharmaceutical products due to their various biological activities, which include non-toxicity, biocompatibility, and biodegradability. Recent scientific advances allow for the development of novel innovative protein-based products that draw on the quality of their innate biological activities. Some of them hold promising potential for novel therapeutic agents/devices for addressing hepatic diseases such as hepatitis, fibrosis, and hepatocarcinomas. This review attempts to provide an overview of the development of protein-based products that take advantage of their biological activity for medication, and discusses possibilities for the therapeutic potential of protein-based products produced through different approaches to specifically target the liver (or hepatic cells: hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells, and Kupffer cells) in the treatment of hepatic diseases. Full article
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3354 KiB  
Article
Development of Biodegradable Polycation-Based Inhalable Dry Gene Powders by Spray Freeze Drying
by Tomoyuki Okuda, Yumiko Suzuki, Yuko Kobayashi, Takehiko Ishii, Satoshi Uchida, Keiji Itaka, Kazunori Kataoka and Hirokazu Okamoto
Pharmaceutics 2015, 7(3), 233-254; https://doi.org/10.3390/pharmaceutics7030233 - 26 Aug 2015
Cited by 40 | Viewed by 8262
Abstract
In this study, two types of biodegradable polycation (PAsp(DET) homopolymer and PEG-PAsp(DET) copolymer) were applied as vectors for inhalable dry gene powders prepared by spray freeze drying (SFD). The prepared dry gene powders had spherical and porous structures with a 5~10-μm diameter, and [...] Read more.
In this study, two types of biodegradable polycation (PAsp(DET) homopolymer and PEG-PAsp(DET) copolymer) were applied as vectors for inhalable dry gene powders prepared by spray freeze drying (SFD). The prepared dry gene powders had spherical and porous structures with a 5~10-μm diameter, and the integrity of plasmid DNA could be maintained during powder production. Furthermore, it was clarified that PEG-PAsp(DET)-based dry gene powder could more sufficiently maintain both the physicochemical properties and in vitro gene transfection efficiencies of polyplexes reconstituted after powder production than PAsp(DET)-based dry gene powder. From an in vitro inhalation study using an Andersen cascade impactor, it was demonstrated that the addition of l-leucine could markedly improve the inhalation performance of dry powders prepared by SFD. Following pulmonary delivery to mice, both PAsp(DET)- and PEG-PAsp(DET)-based dry gene powders could achieve higher gene transfection efficiencies in the lungs compared with a chitosan-based dry gene powder previously reported by us. Full article
(This article belongs to the Special Issue New Paradigm of Gene Therapy)
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957 KiB  
Communication
Screening for Methylated Poly(⌊-histidine) with Various Dimethylimidazolium/Methylimidazole/Imidazole Contents as DNA Carrier
by Shoichiro Asayama, Takao Kumagai and Hiroyoshi Kawakami
Pharmaceutics 2015, 7(3), 224-232; https://doi.org/10.3390/pharmaceutics7030224 - 25 Aug 2015
Cited by 8 | Viewed by 5240
Abstract
Methylated poly(l-histidine) (PLH-Me), our original polypeptide, has controlled the contents of dimethylimidazolium, τ/π-methylimidazole and imidazole groups for efficient gene delivery. The screening for the PLH-Me as DNA carrier has been carried out by use of the PLH with 25 mol% (τ-methyl, 16 mol%; [...] Read more.
Methylated poly(l-histidine) (PLH-Me), our original polypeptide, has controlled the contents of dimethylimidazolium, τ/π-methylimidazole and imidazole groups for efficient gene delivery. The screening for the PLH-Me as DNA carrier has been carried out by use of the PLH with 25 mol% (τ-methyl, 16 mol%; π-methyl, 17 mol%; deprotonated imidazole, 41 mol%), 68 mol% (τ-methyl, 16 mol%; π-methyl, 8 mol%; deprotonated imidazole, 8 mol%) and 87 mol% (τ-methyl, 7 mol%; π-methyl, 4 mol%; deprotonated imidazole, 2 mol%) dimethylimidazolium groups, that is, PLH-Me(25), PLH-Me(68) and PLH-Me(87), respectively. The screening of the chemical structure of PLH-Me has been carried out for DNA carrier properties, which are the stability of its DNA polyion complexes and gene expression. The DNA complexes with the 25 mol% and 68 mol% dimethylated PLH-Me possessed almost same ability to retain DNA, as compared with the 87 mol% dimethylated PLH-Me, which was examined by competitive exchange with dextran sulfate. From the gene transfection experiment against HepG2 cells, human hepatoma cell line, the PLH-Me(25)/DNA complex was revealed to mediate highest gene expression. These results suggest that the dimethyl-imidazolium/methylimidazole/imidazole balance of the PLH-Me is important for DNA carrier design. Full article
(This article belongs to the Special Issue New Paradigm of Gene Therapy)
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1572 KiB  
Review
Image-Guided Hydrodynamic Gene Delivery: Current Status and Future Directions
by Kenya Kamimura, Takeshi Yokoo, Hiroyuki Abe, Yuji Kobayashi, Kohei Ogawa, Yoko Shinagawa, Ryosuke Inoue and Shuji Terai
Pharmaceutics 2015, 7(3), 213-223; https://doi.org/10.3390/pharmaceutics7030213 - 21 Aug 2015
Cited by 23 | Viewed by 8283
Abstract
Hydrodynamics-based delivery has been used as an experimental tool to express transgene in small animals. This in vivo gene transfer method is useful for functional analysis of genetic elements, therapeutic effect of oligonucleotides, and cancer cells to establish the metastatic cancer animal model [...] Read more.
Hydrodynamics-based delivery has been used as an experimental tool to express transgene in small animals. This in vivo gene transfer method is useful for functional analysis of genetic elements, therapeutic effect of oligonucleotides, and cancer cells to establish the metastatic cancer animal model for experimental research. Recent progress in the development of image-guided procedure for hydrodynamics-based gene delivery in large animals directly supports the clinical applicability of this technique. This review summarizes the current status and recent progress in the development of hydrodynamics-based gene delivery and discusses the future directions for its clinical application. Full article
(This article belongs to the Special Issue New Paradigm of Gene Therapy)
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976 KiB  
Article
Contribution of Epigenetic Modifications to the Decline in Transgene Expression from Plasmid DNA in Mouse Liver
by Lei Zang, Makiya Nishikawa, Mitsuru Ando, Yuki Takahashi and Yoshinobu Takakura
Pharmaceutics 2015, 7(3), 199-212; https://doi.org/10.3390/pharmaceutics7030199 - 7 Aug 2015
Cited by 7 | Viewed by 5092
Abstract
Short-term expression of transgenes is one of the problems frequently associated with non-viral in vivo gene transfer. To obtain experimental evidence for the design of sustainable transgene expression systems, the contribution of epigenetic modifications to the decline in transgene expression needs to be [...] Read more.
Short-term expression of transgenes is one of the problems frequently associated with non-viral in vivo gene transfer. To obtain experimental evidence for the design of sustainable transgene expression systems, the contribution of epigenetic modifications to the decline in transgene expression needs to be investigated. Bisulfite sequencing and reactivation by hydrodynamic injection of isotonic solution were employed to investigate methylation statues of CpG in transiently expressing plasmid, pCMV-Luc, in mouse liver after hydrodynamic delivery. The cytosines of CpGs in the promoter region of pCMV-Luc were methylated in mouse liver, but the methylation was much later than the decline in the expression. The expression from pre-methylated pCMV-Luc was insensitive to reactivation. Neither an inhibitor of DNA methylation nor an inhibitor of histone deacetylation had significant effects on transgene expression after hydrodynamic injection of pCMV-Luc. Partial hepatectomy, which reduces the transgene expression from the non-integrated vector into the genome, significantly reduced the transgene expression of human interferon γ from a long-term expressing plasmid pCpG-Huγ, suggesting that the CpG-reduced plasmid was not significantly integrated into the genomic DNA. These results indicate that the CpG-reduced plasmids achieve prolonged transgene expression without integration into the host genome, although the methylation status of CpG sequences in plasmids will not be associated with the prolonged expression. Full article
(This article belongs to the Special Issue New Paradigm of Gene Therapy)
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1330 KiB  
Article
Influence of Differing Analgesic Formulations of Aspirin on Pharmacokinetic Parameters
by Kunal Kanani, Sergio C. Gatoulis and Michael Voelker
Pharmaceutics 2015, 7(3), 188-198; https://doi.org/10.3390/pharmaceutics7030188 - 3 Aug 2015
Cited by 21 | Viewed by 7976
Abstract
Aspirin has been used therapeutically for over 100 years. As the originator and an important marketer of aspirin-containing products, Bayer’s clinical trial database contains numerous reports of the pharmacokinetics of various aspirin formulations. These include evaluations of plain tablets, effervescent tablets, granules, chewable [...] Read more.
Aspirin has been used therapeutically for over 100 years. As the originator and an important marketer of aspirin-containing products, Bayer’s clinical trial database contains numerous reports of the pharmacokinetics of various aspirin formulations. These include evaluations of plain tablets, effervescent tablets, granules, chewable tablets, and fast-release tablets. This publication seeks to expand upon the available pharmacokinetic information concerning aspirin formulations. In the pre-systemic circulation, acetylsalicylic acid (ASA) is rapidly converted into its main active metabolite, salicylic acid (SA). Therefore, both substances are measured in plasma and reported in the results. The 500 mg strength of each formulation was chosen for analysis as this is the most commonly used for analgesia. A total of 22 studies were included in the analysis. All formulations of 500 mg aspirin result in comparable plasma exposure to ASA and SA as evidenced by AUC. Tablets and dry granules provide a consistently lower Cmax compared to effervescent, granules in suspension and fast release tablets. Effervescent tablets, fast release tablets, and granules in suspension provide a consistently lower median Tmax compared to dry granules and tablets for both ASA and SA. This report reinforces the importance of formulation differences and their impact on pharmacokinetic parameters. Full article
(This article belongs to the Special Issue Drug Metabolism and Pharmacokinetics)
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1138 KiB  
Review
Neurosurgical Techniques for Disruption of the Blood–Brain Barrier for Glioblastoma Treatment
by Analiz Rodriguez, Stephen B. Tatter and Waldemar Debinski
Pharmaceutics 2015, 7(3), 175-187; https://doi.org/10.3390/pharmaceutics7030175 - 3 Aug 2015
Cited by 82 | Viewed by 9325
Abstract
The blood–brain barrier remains a main hurdle to drug delivery to the brain. The prognosis of glioblastoma remains grim despite current multimodal medical management. We review neurosurgical technologies that disrupt the blood–brain barrier (BBB). We will review superselective intra-arterial mannitol infusion, focused ultrasound, [...] Read more.
The blood–brain barrier remains a main hurdle to drug delivery to the brain. The prognosis of glioblastoma remains grim despite current multimodal medical management. We review neurosurgical technologies that disrupt the blood–brain barrier (BBB). We will review superselective intra-arterial mannitol infusion, focused ultrasound, laser interstitial thermotherapy, and non-thermal irreversible electroporation (NTIRE). These technologies can lead to transient BBB and blood–brain tumor barrier disruption and allow for the potential of more effective local drug delivery. Animal studies and preliminary clinical trials show promise for achieving this goal. Full article
(This article belongs to the Special Issue Drug Delivery to Brain)
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1847 KiB  
Communication
Novel Antitumor Strategy Utilizing a Plasmid Expressing a Mycobacterium tuberculosis Antigen as a “Danger Signal” to Block Immune Escape of Tumor Cells
by Yoshiyuki Koyama, Chieko Yoshihara and Tomoko Ito
Pharmaceutics 2015, 7(3), 165-174; https://doi.org/10.3390/pharmaceutics7030165 - 24 Jul 2015
Cited by 8 | Viewed by 5423
Abstract
Immune escape of tumor cells is one of the main obstacles hindering the effectiveness of cancer immunotherapy. We developed a novel strategy to block immune escape by transfecting tumor cells in vivo with genes of pathogenic antigens from Mycobacterium tuberculosis (TB). This induces [...] Read more.
Immune escape of tumor cells is one of the main obstacles hindering the effectiveness of cancer immunotherapy. We developed a novel strategy to block immune escape by transfecting tumor cells in vivo with genes of pathogenic antigens from Mycobacterium tuberculosis (TB). This induces presentation of the TB antigen on tumor cell surfaces, which can be recognized by antigen presenting cells (APCs) as a “danger signal” to stimulate antitumor immune response. This strategy is also expected to amplify the immune response against tumor-associated antigens, and block immune escape of the tumor. DNA/PEI/chondroitin sulfate ternary complex is a highly effective non-viral gene vector system for in vivo transfection. A therapeutic complex was prepared using a plasmid encoding the TB antigen, early secretory antigenic target-6 (ESAT-6). This was injected intratumorally into syngeneic tumor-bearing mice, and induced significant tumor growth suppression comparable to or higher than similar complexes expressing cytokines such as interleukin-2 (IL-2) and interleukin-12 (IL-12). Co-transfection of the cytokine-genes and the ESAT-6-gene enhanced the antitumor efficacy of either treatment alone. In addition, complete tumor regression was achieved with the combination of ESAT-6 and IL-2 genes. Full article
(This article belongs to the Special Issue New Paradigm of Gene Therapy)
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1687 KiB  
Article
Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine
by Yoshiyuki Koyama, Kikuya Sugiura, Chieko Yoshihara, Toshio Inaba and Tomoko Ito
Pharmaceutics 2015, 7(3), 152-164; https://doi.org/10.3390/pharmaceutics7030152 - 23 Jul 2015
Cited by 10 | Viewed by 5576
Abstract
We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI) and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of [...] Read more.
We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI) and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine “Max” (PEI “Max”), is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI “Max”/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI “Max”/polyanion small ternary complexes with high transfection efficiency. DNA/PEI “Max”/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice. Full article
(This article belongs to the Special Issue New Paradigm of Gene Therapy)
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875 KiB  
Article
Screening of mRNA Chemical Modification to Maximize Protein Expression with Reduced Immunogenicity
by Satoshi Uchida, Kazunori Kataoka and Keiji Itaka
Pharmaceutics 2015, 7(3), 137-151; https://doi.org/10.3390/pharmaceutics7030137 - 23 Jul 2015
Cited by 68 | Viewed by 9262
Abstract
Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, [...] Read more.
Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, RAW 264.7, modified mRNA tended to have reduced immunogenicity and increased protein expression compared to the unmodified mRNA. In contrast, in some cell types, such as hepatocellular carcinoma cells (HuH-7) and mouse embryonic fibroblasts (MEFs), protein expression was decreased by mRNA modification. Further analyses revealed that mRNA modifications decreased translation efficiency but increased nuclease stability. Thus, mRNA modification is likely to exert both positive and negative effects on the efficiency of protein expression in transfected cells and optimal mRNA formulation should be determined based on target cell types and transfection purposes. Full article
(This article belongs to the Special Issue New Paradigm of Gene Therapy)
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1271 KiB  
Article
Anti-Apoptotic Gene Delivery with cyclo-(d-Trp-Tyr) Peptide Nanotube via Eye Drop Following Corneal Epithelial Debridement
by Yu-Hsing Lee, Shwu-Fen Chang and Jiahorng Liaw
Pharmaceutics 2015, 7(3), 122-136; https://doi.org/10.3390/pharmaceutics7030122 - 17 Jul 2015
Cited by 13 | Viewed by 5772
Abstract
Corneal keratocyte apoptosis triggered by cornel debridement is one mechanism of corneal disorders. In this study, the feasibility of cyclo-(d-Trp-Tyr) peptide nanotubes (PNTs) as carriers of caspase 3 silence shRNA delivery was assessed. A model of epithelial injury by epithelial debridement was [...] Read more.
Corneal keratocyte apoptosis triggered by cornel debridement is one mechanism of corneal disorders. In this study, the feasibility of cyclo-(d-Trp-Tyr) peptide nanotubes (PNTs) as carriers of caspase 3 silence shRNA delivery was assessed. A model of epithelial injury by epithelial debridement was applied to investigate the feasibility of PNTs as gene delivery carriers on corneal injury. First, the PNTs were found within 2 μm in length and 300 nm in width by an atomic force microscope and confocal laser microscope system. Plasmid DNAs were observed to be associated with PNTs by atomic force microscope and confocal laser scanning microscope. The plasmids were associated with tyrosine of PNTs with a binding constant of 2.7 × 108 M−1. The stability of plasmid DNA with PNTs against the DNase was found at 60 min. Using thioflavin T pre-stained PNTs on the corneal eye drop delivery, the distribution of PNTs was in the epithelial and stroma regions. After corneal debridement, the rhodamine-labeled plasmid DNA and thioflavin T pre-stained PNTs were also delivered and could be observed in the stroma of cornea. PNTs complexed with anti-apoptotic plasmid caspase 3 silencing shRNA eye drop delivery decreased 41% of caspase 3 activity after the first dose by caspase 3 activity and Western blot analysis. Full article
(This article belongs to the Special Issue New Paradigm of Gene Therapy)
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761 KiB  
Article
Rapid Turbidimetric Assay to Determine the Potency of Daptomycin in Lyophilized Powder
by Eliane Gandolpho Tótoli and Hérida Regina Nunes Salgado
Pharmaceutics 2015, 7(3), 106-121; https://doi.org/10.3390/pharmaceutics7030106 - 9 Jul 2015
Cited by 22 | Viewed by 9552
Abstract
Daptomycin is an important antimicrobial for clinical practice, mainly because it remains very active against Gram-positive resistant strains, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. Development of microbiological methods for the analysis of antimicrobials is highly recommended, since they can provide important [...] Read more.
Daptomycin is an important antimicrobial for clinical practice, mainly because it remains very active against Gram-positive resistant strains, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. Development of microbiological methods for the analysis of antimicrobials is highly recommended, since they can provide important information about their biological activities, which physicochemical methods are not able to provide. Considering that there are no studies in the literature describing microbiological methods for the analysis of daptomycin, the aim of this work was to validate a microbiological method for the quantitation of daptomycin by the turbidimetric assay. Staphylococcus aureus was used as the test microorganism, and the brain heart infusion broth was used as the culture medium. The validation of the method was performed according to the ICH guidelines, and it was shown to be linear, precise, robust, accurate and selective, over a concentration range of 8.0 to 18.0 µg mL1. Student’s t-test showed the interchangeability of the proposed method with a previously-validated HPLC method. The developed turbidimetric method described in this paper is a convenient alternative for the routine quality control of daptomycin in its pharmaceutical dosage form. Full article
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Review
Transdermal Delivery of Drugs with Microneedles—Potential and Challenges
by Kevin Ita
Pharmaceutics 2015, 7(3), 90-105; https://doi.org/10.3390/pharmaceutics7030090 - 29 Jun 2015
Cited by 343 | Viewed by 21257
Abstract
Transdermal drug delivery offers a number of advantages including improved patient compliance, sustained release, avoidance of gastric irritation, as well as elimination of pre-systemic first-pass effect. However, only few medications can be delivered through the transdermal route in therapeutic amounts. Microneedles can be [...] Read more.
Transdermal drug delivery offers a number of advantages including improved patient compliance, sustained release, avoidance of gastric irritation, as well as elimination of pre-systemic first-pass effect. However, only few medications can be delivered through the transdermal route in therapeutic amounts. Microneedles can be used to enhance transdermal drug delivery. In this review, different types of microneedles are described and their methods of fabrication highlighted. Microneedles can be fabricated in different forms: hollow, solid, and dissolving. There are also hydrogel-forming microneedles. A special attention is paid to hydrogel-forming microneedles. These are innovative microneedles which do not contain drugs but imbibe interstitial fluid to form continuous conduits between dermal microcirculation and an attached patch-type reservoir. Several microneedles approved by regulatory authorities for clinical use are also examined. The last part of this review discusses concerns and challenges regarding microneedle use. Full article
(This article belongs to the Special Issue Microneedle Patches: Developing Strategies for Delivery)
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