Manipulation of Macrophage Uptake by Controlling the Aspect Ratio of Graft Copolymer Micelles.

Nanostructures of drug carriers play a crucial role in nanomedicine due to their ability to influence drug delivery. There is yet no clear consensus regarding the optimal size and shape (e.g., aspect ratio) of nanoparticles for minimizing macrophage uptake, given the difficulties in controlling the shape and size of nanoparticles while maintaining identical surface properties. Here, we employed graft copolymer self-assembly to prepare polymer micelles with aspect ratios ranging from 1.0 (spherical) to 10.8 (cylindrical) and closely matched interfacial properties. Notably, our findings emphasize that cylindrical micelles with an aspect ratio of 2.4 are the least susceptible to macrophage uptake compared with both their longer counterparts and spherical micelles. This reduced uptake of the short cylindrical micelles results in a 3.3-fold increase in blood circulation time compared with their spherical counterparts. Controlling the aspect ratio of nanoparticles is crucial for improving drug delivery efficacy through better nanoparticle design.

A Simple Preparation Method of Gelatin Hydrogels Incorporating Cisplatin for Sustained Release.

The objective of this study was to develop a new preparation method for cisplatin (CDDP)-incorporated gelatin hydrogels without using chemical crosslinking nor a vacuum heating instrument for dehydrothermal crosslinking. By simply mixing CDDP and gelatin, CDDP-crosslinked gelatin hydrogels (CCGH) were prepared. CDDP functions as a crosslinking agent of gelatin to form the gelatin hydrogel. Simultaneously, CDDP is incorporated into the gelatin hydrogel as a controlled release carrier. CDDP's in vitro and in vivo anticancer efficacy after incorporation into CCGH was evaluated. In the in vitro system, the CDDP was released gradually due to CCGH degradation with an initial burst release of approximately 16%. CDDP metal-coordinated with the degraded fragment of gelatin was released from CCGH with maintaining the anticancer activity. After intraperitoneal administration of CCGH, CDDP was detected in the blood circulation while its toxicity was low. Following intraperitoneal administration of CCGH in a murine peritoneal dissemination model of human gastric cancer MKN45-Luc cell line, the survival time was significantly prolonged compared with free CDDP solution. It is concluded that CCGH prepared by the CDDP-based crosslinking of gelatin is an excellent sustained release system of CDDP to achieve superior anticancer effects with minimal side effects compared with free CDDP solution.

Single-component nanodiscs via the thermal folding of amphiphilic graft copolymers with the adjusted flexibility of the main chain.

Nanodiscs have attracted considerable attention as structural scaffolds for membrane-protein research and as biomaterials in e.g. drug-delivery systems. However, conventional disc-fabrication methods are usually laborious, and disc fabrication via the self-assembly of amphiphiles is difficult. Herein, we report the formation of polymer nanodiscs based on the self-assembly of amphiphilic graft copolymers by adjusting the persistence length of the main chain. Amphiphilic graft copolymers with a series of different main-chain persistence lengths were prepared and these formed, depending on the persistence length, either rods, discs, or vesicles. Notably, polymer nanodiscs were formed upon heating a chilled polymer solution without the need for any additives, and the thus obtained nanodiscs were used to solubilize a membrane protein during cell-free protein synthesis. Given the simplicity of this disc-fabrication method and the ability of these discs to solubilize membrane proteins, this study considerably expands the fundamental and practical scope of graft-copolymer nanodiscs and demonstrates their utility as tools for studying the structure and function of membrane proteins.

Preparation of cationic proteoliposomes using cell-free membrane protein synthesis: the chaperoning effect of cationic liposomes.

Membrane protein reconstituted cationic liposomes are constructed using cell-free membrane protein synthesis in the presence of cationic liposomes. The chaperon effect of cationic liposomal membrane assists in folding the functional conformation of membrane protein. This preparation method enables the provision of the usage of proteoliposomes for drug delivery.

Proteoliposome Engineering with Cell-Free Membrane Protein Synthesis: Control of Membrane Protein Sorting into Liposomes by Chaperoning Systems.

Integral membrane proteins (IMPs) modulate key cellular processes; their dysfunctions are closely related to disease. However, production of IMPs in active conformations for further study is hindered by aggregation and toxicity in living expression systems. IMPs are therefore produced in cell-free systems employing liposome chaperoning, but membrane integration of the nascent IMPs is suboptimal and orientation of the integrated proteins remains uncontrollable. Thus, an artificial membrane protein sorting system is developed, based on polyhistidine/nickel-chelate affinity, combined with cell-free membrane protein synthesis. Its proof of concept is demonstrated with a N-terminal hexahistadine-fused conexin-43 (NHis-Cx43) model IMP. Nickel-chelating liposomes efficiently incorporate twofold newly synthesized NHis-Cx43 compared with Cx43. NHis-Cx43, when synthesized in this system, forms dye-permeable hemichannels, similar to plasma membrane pores formed by Cx43 in cells. The topology of incorporated NHis-Cx43 indicates two orientations in the liposomal membranes. However, NHis-Cx43 orientation is controlled, resulting in single topology, by combination of the natural molecular chaperone DnaKJE. Successful synthesis and at least 4.5-fold increase lipid incorporation are also achieved with three other NHis-fused IMPs, including α-helix and ß-barrel IMPs. Overall, this simple membrane protein sorting system is usable combined with molecular chaperones to prepare proteoliposomes for many applications.

Targeted Delivery of Interferon Gamma Using a Recombinant Fusion Protein of a Fibrin Clot-Binding Peptide With Interferon Gamma for Cancer Gene Therapy.

Accelerated formation of fibrin clots in a tumor microenvironment can be used for targeted delivery of interferon gamma (IFNγ) to tumor cells. Here, we selected cysteine-arginine-glutamic acid-lysine-alanine (CREKA) as the fibrin clot-binding peptide and designed 2 types of fusion proteins for tumor targeting. The CREKA peptide was fused to IFNγ's C-terminus, with or without a matrix metalloproteinase-2 (MMP2)-cleavable linker (IFNγ-mmp-CREKA or IFNγ-CREKA, respectively). The former was designed to release IFNγ from IFNγ-mmp-CREKA bound to fibrin clots, to ensure IFNγ's function in the tumor milieu. IFNγ-activated sequence-dependent reporter gene expression in B16-BL6 cells revealed that the biological activities of IFNγ-CREKA and IFNγ were comparable, whereas that of IFNγ-mmp-CREKA was approximately 60% that of IFNγ. Plasma concentrations of IFNγ-CREKA and IFNγ-mmp-CREKA remained at effective levels for at least 4 weeks after gene transfer into mice. After gene transfer to tumor-bearing mice, intratumoral concentration of IFNγ in pCpG-IFNγ-mmp-CREKA group was tended to be higher than those of the other groups. Inhibition of colon-26 tumor growth was significantly more with gene transfer of IFNγ-mmp-CREKA than with IFNγ or IFNγ-CREKA. These results indicate that targeted delivery of IFNγ to fibrin clots through IFNγ-mmp-CREKA fusion can enhance the therapeutic efficacy of IFNγ in cancer gene therapy.

Comprehensive study of liposome-assisted synthesis of membrane proteins using a reconstituted cell-free translation system.

Membrane proteins play pivotal roles in cellular processes and are key targets for drug discovery. However, the reliable synthesis and folding of membrane proteins are significant problems that need to be addressed owing to their extremely high hydrophobic properties, which promote irreversible aggregation in hydrophilic conditions. Previous reports have suggested that protein aggregation could be prevented by including exogenous liposomes in cell-free translation processes. Systematic studies that identify which membrane proteins can be rescued from irreversible aggregation during translation by liposomes would be valuable in terms of understanding the effects of liposomes and developing applications for membrane protein engineering in the context of pharmaceutical science and nanodevice development. Therefore, we performed a comprehensive study to evaluate the effects of liposomes on 85 aggregation-prone membrane proteins from Escherichia coli by using a reconstituted, chemically defined cell-free translation system. Statistical analyses revealed that the presence of liposomes increased the solubility of >90% of the studied membrane proteins, and ultimately improved the yields of the synthesized proteins. Bioinformatics analyses revealed significant correlations between the liposome effect and the physicochemical properties of the membrane proteins.

Contribution of Epigenetic Modifications to the Decline in Transgene Expression from Plasmid DNA in Mouse Liver.

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.

Enhancement of anticancer effect of interferon-γ gene transfer against interferon-γ-resistant tumor by depletion of tumor-associated macrophages.

Tumor-associated macrophages (TAMs) negatively affect the therapeutic effects of anticancer agents. To examine the role of TAMs in interferon (IFN)-γ gene therapy, we selected two types of solid tumors, which varied in the number of TAMs, and investigated the effects of IFN-γ gene transfer on tumor growth. Many TAMs were detected in the solid tumors of murine adenocarcinoma colon-26 cells, whereas few TAMs were detected in murine melanoma B16-BL6 cells. IFN-γ gene transfer hardly suppressed the growth of colon-26 tumors, whereas it was effective in suppressing the growth of B16-BL6 tumors. The antiproliferative effects of IFN-γ on cultured colon-26 cells were similar to those on cultured B16-BL6 cells. To evaluate the role of TAMs, we injected clodronate liposomes (CLs) modified with poly(ethylene glycol) (PEG) to functionally deplete TAMs in tumor-bearing mice. Repeated injections of PEG-CLs significantly retarded the growth of colon-26 tumors and combination with IFN-γ gene transfer further inhibited the growth. In contrast, PEG-CLs hardly retarded the growth of B16-BL6 tumors. These results clearly indicate that TAM depletion is effective in enhancing the therapeutic effect of IFN-γ in TAM-repleted and IFN-γ-resistant tumors.

Long-term elimination of hepatitis C virus from human hepatocyte chimeric mice after interferon-γ gene transfer.

Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis, liver failure, and hepatocellular carcinoma. Although the combination therapy employing pegylated interferon (IFN)-α and ribavirin is effective, this treatment is effective in only approximately 50% patients with genotype 1 HCV infection. IFN-γ is a potent anti-HCV agent that exhibits its antiviral action through a receptor distinct from that for IFN-α. Therefore, IFN-γ application might provide an alternative approach to IFN-α-based therapies. However, recombinant IFN-γ protein exhibits a poor pharmacokinetic property, that is, a very short half-life. It is our hypothesis that sustained IFN-γ serum concentrations produced by gene transfer could effectively eliminate HCV in vivo. We examined the in vivo antiviral activity in human hepatocyte chimeric mice infected with genotype 1b HCV at high HCV RNA titers (10(5)-10(7) copies/ml). The human IFN-γ-expressing plasmid vector pCpG-huIFNγ exhibited prolonged transgene expression in mice compared with the plasmid vector pCMV-huIFNγ. Moreover, the gene transfer of pCpG-huIFNγ eliminated HCV from the liver of the chimeric mice for a sustained period. On the contrary, administration of pCMV-huIFNγ could not eliminate HCV. In conclusion, we found that a single pCpG-huIFNγ injection resulted in long-term elimination of HCV RNA in chimeric mice, providing, for the first time, direct evidence that chronic infection with high titer HCV in vivo can be treated by sustained IFN-γ treatment.

Prevention of adverse events of interferon γ gene therapy by gene delivery of interferon γ-heparin-binding domain fusion protein in mice.

Sustained gene delivery of interferon (IFN) γ can be an effective treatment, but our previous study showed high levels of IFNγ-induced adverse events, including the loss of body weight. These unwanted events could be reduced by target-specific delivery of IFNγ after in vivo gene transfer. To achieve this, we selected the heparin-binding domain (HBD) of extracellular superoxide dismutase as a molecule to anchor IFNγ to the cell surface. We designed three IFNγ derivatives, IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, each of which had 1, 2, or 3 HBDs, respectively. Each plasmid-encoding fusion proteins was delivered to the liver, a model target in this study, by hydrodynamic tail vein injection. The serum concentration of IFNγ-HBD2 and IFNγ-HBD3 after gene delivery was lower than that of IFNγ or IFNγ-HBD1. Gene delivery of IFNγ-HBD2, but not of IFNγ-HBD3, effectively increased the mRNA expression of IFNγ-inducible genes in the liver, suggesting liver-specific distribution of IFNγ-HBD2. Gene delivery of IFNγ-HBD2-suppressed tumor growth in the liver as efficiently as that of IFNγ with much less symptoms of adverse effects. These results indicate that the adverse events of IFNγ gene transfer can be prevented by gene delivery of IFNγ-HBD2, a fusion protein with high cell surface affinity.

Expression profile-dependent improvement of insulin sensitivity by gene delivery of interleukin-6 in a mouse model of type II diabetes.

Type II diabetes is one of the most problematic metabolic disorders and is associated with secondary conditions such as heart disease and eye complications. Interleukin-6 (IL-6), a multifunctional cytokine, could influence conditions of altered glucose metabolism such as insulin resistance in diabetic patients. However, a consensus about the role of IL-6 on glucose metabolism has not been reached. The aim of the present study is to investigate whether the expression of IL-6 affects glucose metabolism in diet-induced obesity (DIO) mice, a model of type II diabetes and obesity, using gene delivery of IL-6. DIO mice received hydrodynamic or intramuscular injection of IL-6-expressing plasmid to investigate the importance of the site of IL-6 expression. DIO mice that received a sustained IL-6 gene transfer showed similar glucose levels to lean mice in a glucose tolerance test. DIO mice exhibited reduced food intake and low body and epididymal fat weights after IL-6 gene transfer. IL-6 gene delivery reduced the mRNA expression of metabolism-related genes in the liver, skeletal muscle, and adipose tissue of DIO mice. The metabolic status of DIO mice receiving intramuscular injections was moderately better than that of DIO mice receiving hydrodynamic injections. The infiltration of inflammatory cells into the sites where the IL-6-expressing plasmid DNA was delivered was observed. Transient expression of IL-6 had limited effects on all parameters examined. These results indicate that the expression of IL-6 has an effect on obesity and the metabolism of glucose and lipid in diabetic mice and that the expression site of IL-6 is not an important factor.

Gene delivery of albumin binding peptide-interferon-gamma fusion protein with improved pharmacokinetic properties and sustained biological activity.

We have demonstrated that gene delivery of a fusion protein of mouse interferon (IFN) γ with mouse serum albumin (IFNγ-MSA) was effective in prolonging the circulation half-life of IFNγ in mice. However, the fusion to MSA greatly reduced the biological activity of IFNγ to less than 1%. In this study, we designed IFNγ fusion proteins with a 20 amino-acid long albumin-binding peptide (ABP) to prolong the in vivo half-life of IFNγ without reducing its biological activity. IFNγ-ABP and ABP-IFNγ, two fusion proteins with the ABP being fused to the C- or N-terminal of IFNγ, retained 40%-50% biological activities determined using a gamma-activated sequence-dependent luciferase assay. These fusion proteins exhibited the ability to bind to MSA. Gene delivery of IFNγ-ABP or ABP-IFNγ to mice using the hydrodynamic injection method resulted in a sustained concentration of IFNγ in the serum compared with gene delivery of IFNγ. In addition, the growth of mouse colon carcinoma CT-26 cells in the lung was efficiently inhibited by gene delivery of the IFNγ fusion proteins. These results indicate that the fusion of ABP is a useful approach to achieving prolonged retention in the blood circulation through binding to serum albumin and retaining biological activity.

[Control of spatiotemporal distribution of interferon γ by genetically fusing functional peptides].

Type II interferon (IFNγ) is a representative Th1 cytokine and it possesses a variety of functions, including immune regulation, antiviral and antitumor activity. Because of its multifunctional nature, IFNγ is expected to be applied to the treatment of autoimmune diseases, cancer and viral infection. Although IFNγ has therapeutic potential for such diseases, the clinical use of IFNγ has been limited due to its short in vivo half-life and serious adverse effects. In contrast, gene delivery of IFNγ is an alternative approach to increasing the retention time of IFNγ. To extend transgene expression after plasmid DNA (pDNA) gene transfer, we designed and developed pDNA with varying numbers of CpG motifs. CpG-reduced pDNA resulted in more durable transgene expression than its CpG replete counterpart in mice. Comparison of the effect of promoter/enhancer elements on transgene expression showed that ROSA26 promoter-mediated IFNγ expression was safe because of the lack of an initial surge after hydrodynamic gene transfer. We also designed an IFNγ-mouse serum albumin (MSA) fusion protein, IFNγ-MSA. Gene transfer of this fusion protein resulted in a sustained concentration of IFNγ fusion protein in mouse serum, and inhibited tumor metastasis in mice. These results provide experimental evidence that IFNγ gene therapy can be a useful treatment for a variety of diseases.

Constant and steady transgene expression of interferon-γ by optimization of plasmid construct for safe and effective interferon-γ gene therapy.

BACKGROUND: Hydrodynamic injection of pmCMV(enh) -hEF-1(prom) -muIFN-γ, a plasmid DNA (pDNA) expressing murine interferon (IFN)-γ with a murine cytomegalovirus (mCMV) enhancer and a human elongation factor (EF)-1 promoter, has been proven effective for the treatment of cancer and atopic dermatitis in mice. However, the initial peak of IFN-γ soon after injection was quite high compared to the steady level for subsequent periods, which could cause unwanted adverse effects. Therefore, in the present study, aiming to optimize the efficacy/side-effect ratio of IFN-γ gene transfer, we have developed plasmid vectors encoding murine IFN-γ under the control of different combinations of promoter and enhancer sequences. METHODS: The promoter and enhancer sequence of pmCMV(enh) -hEF-1(prom) -huIFN-γ, a prototype plasmid expressing human IFN-γ, was replaced or deleted to obtain various pDNAs. To assess the transgene expression profile, each pDNA was delivered to mice by hydrodynamic injection and the serum IFN-γ concentration was measured periodically. On the basis of the results obtained, murine IFN-γ expressing pDNAs were constructed and the body weight change was monitored as an indicator of adverse effects. RESULTS: The prototype pmCMV(enh) -hEF-1(prom) -huIFN-γ showed a high but declining concentration of IFN-γ. Those containing hROSA26 promoter expressed the transgene in a more constant manner with no initial high concentrations and scarcely reduced the body weight. CONCLUSIONS: These results indicate that hROSA26 promoter, irrespective of the presence and type of enhancers, is suitable for achieving constant and steady level of transgene expression and effective in avoiding the body weight loss caused by high concentrations of IFN-γ.

Prolonged circulation half-life of interferon γ activity by gene delivery of interferon γ-serum albumin fusion protein in mice.

Gene delivery of mouse interferon (IFN) γ has been shown to inhibit metastatic tumor growth and onset of atopic dermatitis in mouse models. In this study, we tried to increase the circulation half-life of IFNγ after its gene delivery by designing a novel fusion protein of IFNγ with mouse serum albumin (MSA). Western blot analysis confirmed that IFNγ-MSA was expressed as a fusion protein, but hardly formed dimer as IFNγ did. The biological activity of IFNγ-MSA, which was examined using a plasmid expressing luciferase under the control of gamma-activated sequence elements, was about 200-fold lower than the activity of IFNγ. Intravenous injection of the proteins into mice confirmed that the circulation half-life of IFNγ was significantly prolonged by the modification. A hydrodynamic injection of a plasmid expressing IFNγ-MSA resulted in a sustained concentration in mouse serum; it resulted in about sixfold greater area under the concentration-time curve and about threefold longer mean residence time of IFNγ activity than those of IFNγ. Gene delivery of IFNγ-MSA inhibited tumor metastasis to a similar level to that of IFNγ despite the reduced activity of IFNγ-MSA. These results indicate that gene delivery of IFNγ-MSA is a promising approach to prolong the circulation half-life of IFNγ activity.

Persistent interferon transgene expression by RNA interference-mediated silencing of interferon receptors.

BACKGROUND: The in vivo half-life of interferons (IFNs) is very short, and its extension would produce a better therapeutic outcome in IFN-based therapy. Delivery of IFN genes is one solution for providing a sustained supply. IFNs have a variety of functions, including the suppression of transgene expression, through interaction with IFN receptors (IFNRs). This suppression could prevent IFNs from being expressed from vectors delivered. Silencing the expression of IFNAR and IFNGR, the receptors for type I and II IFNs, respectively, in cells expressing IFNs may prolong transgene expression of IFNs. METHODS: Mouse melanoma B16-BL6 cells or mouse liver were selected as a site expressing IFNs (not a target for IFN gene therapy) and IFN-expressing plasmid DNA was delivered with or without small interfering RNA (siRNA) targeting IFNRs. RESULTS: Transfection of B16-BL6 cells with siRNA targeting IFNAR1 subunit (IFNAR1) resulted in the reduced expression of IFNAR on the cell surface. This silencing significantly increased the IFN-beta production in cells that were transfected with IFN-beta-expressing plasmid DNA. Similar results were obtained with the combination of IFN-gamma and IFNGR. Co-injection of IFN-beta-expressing plasmid DNA with siRNA targeting IFNAR1 into mice resulted in sustained plasma concentration of IFN-beta. CONCLUSIONS: These results provide experimental evidence that the RNAi-mediated silencing of IFNRs in cells expressing IFN, such as hepatocytes, is an effective approach for improving transgene expression of IFNs when their therapeutic target comprises cells other than those expressing IFNs.

Effect of the content of unmethylated CpG dinucleotides in plasmid DNA on the sustainability of transgene expression.

BACKGROUND: Nonviral gene transfer generally suffers from short-term expression of transgenes. We have previously demonstrated that plasmids with reduced CpG content exhibited a more prolonged expression of murine interferon (IFN)-beta or IFN-gamma, which was effective in inhibiting metastatic tumor growth. A further extension of the duration of transgene expression could be achieved by controlling the number and location of CpG motifs in plasmid DNA. METHODS: Luciferase-expressing plasmids with differing CpG content were injected into the tail vein of mice by the hydrodynamic injection method. The effects of CpG content on the duration of transgene expression were examined, focusing on cytosine methylation and pro-inflammatory cytokines. Based on the findings, IFN-gamma-expressing plasmids were constructed and their transgene expression and inhibitory effect on pulmonary metastasis were evaluated. RESULTS: Plasmids with a few CpG motifs showed a prolonged luciferase activity in the liver. Methylation of CpG motifs in plasmids reduced the expression and the extent of this reduction was greater for plasmids with a high CpG content. Pro-inflammatory cytokines hardly affected the expression. pCpG-Mu gamma, the IFN-gamma-expressing plasmid, which contains 20 CpG motifs only in the cDNA region, exhibited a sustained IFN-gamma concentration at therapeutic levels, and had a great inhibitory effect on the pulmonary metastasis of tumor cells. CONCLUSIONS: The duration of transgene expression of IFN-gamma was successfully increased by reducing the CpG content of IFN-expressing plasmid vector, which resulted in an increased anticancer activity of IFN gene transfer.