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Therapeutic strategies based on antisense oligonucleotides and therapeutic genes are being extensively investigated for the treatment of hereditary muscle diseases and hold great promise. However, the cellular uptake of these polyanions to the muscle cells is inefficient. Therefore, it is necessary to develop more effective methods of gene delivery into the muscle tissue. The A2G80 peptide (VQLRNGFPYFSY) from the laminin α2 chain has high affinity for α-dystroglycan (α-DG) which is expressed on the membrane of muscle cells. In this study, we designed a peptide-modified A2G80 with oligoarginine and oligohistidine (A2G80-R9-H8), and prepared peptide/plasmid DNA (pDNA) complex, to develop an efficient gene delivery system for the muscle tissue. The peptide/pDNA complex showed α-DG-dependent cellular uptake of the A2G80 sequence and significantly improved gene transfection efficiency mediated by the oligohistidine sequence in C2C12 myoblast cells. Further, the peptide/pDNA complex promoted efficient and sustained gene expression in the Duchenne muscular dystrophy mouse models. The A2G80-R9-H8 peptide has the potential for use as a specific carrier for targeting muscle in gene therapy in muscular dystrophy.

The polyion complexes (PICs) between plasmid DNA (pDNA) and succinylated branched polyethylenimine (bPEI-Et-COOH) were formed for in vivo pDNA delivery by muscular injection. Transmission electron ...

Autoimmune diseases present a significant clinical problem, highlighting the need for the development of novel or improved therapeutic methods. One of the factors that causes autoimmune diseases is a defect in the clearance of apoptotic cells by phagocytes. Thus, improved apoptotic cell processing has been considered as a strategy to treat autoimmune diseases. However, therapeutic strategies focusing on apoptotic cell clearance have not been approved till date. We have reported that liposomes composed of phosphatidylserine (PS liposomes) exhibit anti-inflammatory or immunosuppressive effects in macrophages. A PS liposome display PS on its surface, which plays a crucial role in the phagocytosis of apoptotic cells by marginal zone macrophages (MZMs), a key player in the clearance of apoptotic cells, by recognizing PS exposed on the surface of apoptotic cells. Therefore, we hypothesized that PS liposomes could be used as "antigen delivery vesicles" to act as a substitute for apoptotic cells in the treatment of autoimmune diseases. In this study, we showed that systemically administered PS liposomes accumulated in the marginal zone of the spleen due to recognition of surface-displayed PS by MZMs because it was observed that liposomes without PS did not accumulate in the marginal zone. In conclusion, PS liposomes may be useful vehicles to function as active agents and/or antigens against autoimmune diseases.

Conversion of CD4+CD25+FOXP3+ T regulatory cells (Tregs) from the immature (CD45RA+) to mature (CD45RO+) phenotype has been shown during development and allergic reactions. The relative frequencies of these Treg phenotypes and their responses to oxidative stress during development and allergic inflammation were analysed in samples from paediatric and adult subjects. The FOXP3lowCD45RA+ population was dominant in early childhood, while the percentage of FOXP3highCD45RO+ cells began increasing in the first year of life. These phenotypic changes were observed in subjects with and without asthma. Further, there was a significant increase in phosphorylated ERK1/2 (pERK1/2) protein in hydrogen peroxide (H2O2)-treated CD4+CD25high cells in adults with asthma compared with those without asthma. Increased pERK1/2 levels corresponded with increased Ca2+ response to T cell receptor stimulation. mRNA expression of peroxiredoxins declined in Tregs from adults with asthma. Finally, CD4+CD25high cells from paediatric subjects were more sensitive to oxidative stress than those from adults in vitro. The differential Treg sensitivity to oxidative stress observed in children and adults was likely dependent on phenotypic CD45 isoform switching. Increased sensitivity of Treg cells from adults with asthma to H2O2 resulted from a reduction of peroxiredoxin-2, -3, -4 and increased pERK1/2 via impaired Ca2+ response in these cells.

Recently, the development of drug delivery systems (DDSs) for clinical application of anticancer drugs and gene therapy has rapidly progressed. In particular, DDS carriers used for chemotherapy and gene therapy are required to selectively deliver drugs and genes to cancer cells. Both the carrier and the molecule must in combination be highly selective in most cases. Possible candidate targeting molecules are the laminins, major basement membrane proteins that interact with various cells through their multiple constituent active peptide sequences. Laminin-derived peptides bind to various cellular receptors and have been used for DDSs as a targeting moiety. Here, we review the progress in laminin-derived peptide-conjugated DDSs. Drug and gene carriers as well as ultrasound diagnostic contrast agents utilizing laminin-derived peptides for selective targeting are useful components of DDSs and play important roles in cancer and in the neovasculature.

To cleave biocompatible poly(ethylene glycol) (PEG) from the mono-ion complex (MIC) for sustainable cellular uptake in vivo, ω-amide-pentylimidazolium end-modified PEG with an ester bond, that is, APe-Im-E-PEG, has been synthesized. The hydrolysis of the resulting APe-Im-E-PEG proceeded during the incubation for 2 weeks under physiological conditions, which was confirmed by gel filtration chromatography. APe-Im-E-PEG formed the MIC with plasmid DNA (pDNA), assessed by agarose gel retardation assay. Furthermore, dynamic light scattering measurement and transmission electron microscopy observations have estimated that the particle size of the resulting MIC was approximately 30 nm, with a rather flexible structure. The APe-Im-E-PEG/pDNA MIC incubated for 2 weeks exhibited hemolytic activity at endosomal pH, presumably because the pH-sensitive carboxyl groups revealed after the hydrolysis of an ester bond of APe-Im-E-PEG. The APe-Im-E-PEG/pDNA MIC enhanced the gene expression 2 weeks after transfection in vivo by intramuscular administration in mice. Consequently, in vivo sustainable gene expression has been achieved by the molecular design of APe-Im-E-PEG for cellular uptake and endosomal escape proceeded by temporal hydrolysis of the ester bond.

In recent years, the use of stimuli-responsive carriers and physical energies, such as ultrasound, magnetic force, electric force, and light, in combination therapy has attracted attention as useful gene and oligonucleotide delivery systems. These systems allow target-specific delivery to be achieved relatively easily at the application site of physical energy. Ultrasound-mediated delivery has attracted particular interest because of its noninvasive nature. Microbubbles are ultrasound contrast agents that can act as echo enhancers. Under appropriate conditions, microbubbles or nanosized bubbles can also enhance the efficiency of drug, gene, and oligonucleotide delivery by ultrasound exposure. Therefore, the combination of ultrasound technology and bubbles is expected to be a fusion diagnostic and therapeutic system known as the theranostic system. In this review, we summarize the use of micro- and nanobubbles in ultrasound-mediated gene and oligonucleotide delivery systems, and discuss their potential as therapeutic tools.

An advantage of mucosal vaccines over conventional parenteral vaccines is that they can induce protective immune responses not only at mucosal surfaces but also in systemic compartments. Despite this advantage, few live attenuated or inactivated mucosal vaccines have been developed and applied clinically. We recently showed that the intranasal immunization of ovalbumin (OVA) with class B synthetic oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motif (CpG ODN)-loaded cationic liposomes synergistically exerted both antigen-specific mucosal immunoglobulin A (IgA) and systemic immunoglobulin G (IgG) responses in mice. However, the mechanism underlying the mucosal adjuvant activity of CpG ODN-loaded liposomes remains unknown. In the present study, we showed that the intranasal administration of CpG ODN-loaded cationic liposomes elicited interleukin (IL)-6 release in nasal tissues. Additionally, pre-treatment with an anti-IL-6 receptor (IL-6R) antibody attenuated antigen-specific nasal IgA production but not serum IgG responses. Furthermore, the intranasal administration of OVA and CpG ODN-loaded cationic liposomes increased the number of IgA+/CD138+ plasma cells and IgA+/B220+ B cells in the nasal passages. This increase was markedly suppressed by pre-treatment with anti-IL-6R blocking antibody. In conclusion, IL-6 released by CpG ODN-loaded cationic liposomes at the site of administration may play a role in the induction of antigen-specific IgA responses by promoting differentiation into IgA+ plasma cells for IgA secretion from B cells.

The structure-activity relationship of mono-ion complexes (MICs) for plasmid DNA (pDNA) delivery by muscular injection is demonstrated. MICs were formed between pDNA and monocationic poly(ethylene glycol) (PEG) macromolecules. As monocationic PEGs, the &omega, amide-pentylimidazolium (APe-Im) end-modified PEGs with a stable amide (Am) and hydrolytic ester (Es) bond, that is, APe-Im-Am-PEG and APe-Im-Es-PEG, respectively, are synthesized. The difference between the APe-Im-Am-PEG and APe-Im-Es-PEG was only a spacer structure between a terminal cation and a PEG chain. The resulting pDNA MICs with APe-Im-Am-PEG at a charge ratio (+/&minus, ) of 32 or 64 were more stable than those with APe-Im-Es-PEG in the presence of serum proteins. The highest gene expression by muscular injection was achieved using the APe-Im-Am-PEG/pDNA MIC at a charge ratio (+/&minus, ) of 32 with a smaller particle diameter of approximately 50 nm, as compared to that charge ratio of 64. Consequently, the pDNA MIC with the monocationic PEG with a stable amide spacer, as compared to a hydrolytic ester spacer, is considered to be suitable for the highest gene expression by muscular injection.

The polyion complexes (PICs) between plasmid DNA (pDNA) and succinylated branched polyethylenimine (bPEI-Et-COOH) were formed for

Background: RNA interference (RNAi)-based therapy has gained attention because of its potent genesilencing effect and high specificity. However, the efficient delivery of nucleic acids to the target site is a major challenge to the clinical implementation. Recently, ultrasound-mediated gene delivery systems have been developed and attracted interest due to its safety and site-specificity. By the combination with contrast agents, called microbubbles, not only the delivery effects but also the imaging effects are significantly enhanced. We developed lipid bubbles (LBs) entrapping an ultrasound contrast gas to enhance the efficacy of ultrasound-mediated delivery and imaging. In this review, we summarize ultrasound-mediated nucleic acid delivery systems and discuss the possibility of combining LBs and ultrasound for RNAi-based therapies. Methods: We prepared polyethylene glycol-modified liposomes and entrapped an echo-contrast gas within the liposomes. Small interfering RNA (siRNA) were transfected into cells and muscles using LBs and ultrasound. Moreover, we also developed nucleic acid-loaded LBs using cholesterol-conjugated siRNA or positively-charged lipid for an efficient systemic delivery of siRNA and microRNA. The usability of LBs for RNA delivery system was evaluated by the silencing effects of target genes and the therapeutic effects on ischemia hind limb. Results: A combination of LBs and therapeutic ultrasound was able to enhance the gene silencing effects by siRNA. Nucleic acid-loaded LBs were able to efficiently deliver siRNA or microRNA by systemic administration. A combination of LBs and diagnostic ultrasound also enhanced the imaging efficiency. Using a hindlimb ischemia mouse model, microRNA-loaded LBs could lead to increased angiogenic factors and improved blood flow. Conclusion: Ultrasound technology is widely used in clinical settings not only for diagnosis but also for therapy. Ultrasonic devices are being actively developed. Computer-controlled ultrasound systems can provide precise exposure to the target site. The combination of precise ultrasound exposure and LBs might be useful for target site-specific nucleic acids delivery, and holds potential to be developed into a beneficial therapeutic and diagnostic system for various diseases.

Infectious diseases are the second leading cause of death worldwide, suggesting that there is still a need for the development of new and improved strategies for combating pathogens effectively. Streptococcus pneumoniae is the most virulent bacteria causing pneumonia with high mortality, especially in children and the elderly. Because of the emergence of antibiotic resistance in S. pneumoniae, employing a serotype-independent mucosal vaccine would be the best approach to prevent and treat the diseases caused by S. pneumoniae. In this study, we have developed a pneumococcal nasal vaccine, consisting of pneumococcal surface protein A (PspA) and cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and cholesteryl 3β-N-(dimethylaminoethyl)-carbamate (DC-chol) (DOTAP/DC-chol liposome). The efficiency of this cationic liposome-based PspA nasal vaccine was examined in a murine model of S. pneumoniae infection. Intranasal vaccination with PspA and DOTAP/DC-chol liposomes conferred protective immunity against lethal inhalation of S. pneumoniae, improving the survival rate of infected mice. Moreover, intranasal immunization with PspA and DOTAP/DC-chol liposomes not only induced the production of PspA-specific IgA and IgG by both mucosal and systemic compartments but also elicited PspA-specific Th17 responses, which play a pivotal role in controlling S. pneumoniae infection by host innate immune response. We further demonstrated that DOTAP/DC-chol liposomes enhanced PspA uptake by nasal dendritic cells (DCs), which might be a mechanism for the induction of protective immune responses to S. pneumoniae infection. These results show that DOTAP/DC-chol liposome would be an efficient mucosal vaccine system for a serotype-independent universal nasal vaccine against pneumococcal infection.

Liposomes have been used as targeting carriers for drug delivery systems (DDSs), and the carriers are able to be modified with targeting ligands, such as antibodies and peptides. To evaluate the targetability of DDS carriers modified with a targeting ligand, culture cells expressing the targeting molecules as well as small animals are used. Furthermore, in vitro and in vivo screening analyses must be repeatedly performed. Therefore, it is important to establish an easy and high-precision screening system for targeting carriers. With this aim, we focused that whether this ex vivo system could easily support assessment of interaction between targeting ligand and its receptor under physiological environment and further screen the DDS carrier-modified with targeting moiety. We examined targeting ability via in vitro, ex vivo, and in vivo analyses using integrin αvβ3-targeting C16Y-L. For the in vitro analysis, the cellular uptake of C16Y-L was higher than that of control liposomes in colon26 cells. For the ex vivo analysis, we performed an immunohistochemical analysis using colon26 tumor sections. C16Y-L was specifically attached to the tumor sections, as found in the in vitro analysis. Moreover, to evaluate the ex vivo-in vivo correlation, we examined the intratumoral localization of C16Y-L. This result showed that C16Y-L was accumulated not only in the tumor tissue but also in the tumor vasculature after the intravenous injection of C16Y-L, suggesting that the ex vivo peptide-modified liposomal analysis was correlated with the in vivo analysis. Thus, the ex vivo peptide-modified liposomal analysis may be an easy and rapid screening system with high-precision and for consideration in in vivo conditions.

The COVID-19 pandemic, caused by a highly virulent and transmissible pathogen, has proven to be devastating to society. Mucosal vaccines that can induce antigen-specific immune responses in both the systemic and mucosal compartments are considered an effective measure to overcome infectious diseases caused by pathogenic microbes. We have recently developed a nasal vaccine system using cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane and cholesteryl 3β-N-(dimethylaminoethyl)carbamate in mice. However, the comprehensive molecular mechanism(s), especially the host soluble mediator involved in this process, by which cationic liposomes promote antigen-specific mucosal immune responses, remain to be elucidated. Herein, we show that intranasal administration of cationic liposomes elicited interleukin-6 (IL-6) expression at the site of administration. Additionally, both nasal passages and splenocytes from mice nasally immunized with cationic liposomes plus ovalbumin (OVA) were polarized to produce IL-6 when re-stimulated with OVA in vitro. Furthermore, pretreatment with anti-IL-6R antibody, which blocks the biological activities of IL-6, attenuated the production of OVA-specific nasal immunoglobulin A (IgA) but not OVA-specific serum immunoglobulin G (IgG) responses. In this study, we demonstrated that IL-6, exerted by nasally administered cationic liposomes, plays a crucial role in antigen-specific IgA induction.

There is an increased demand for vaccines to prevent and/or treat illness and mortality caused by the infectious diseases. We have recently established that liposomes composed of cationic lipids act as adjuvant for nasal vaccine formulation. However, the molecular mechanism(s) behind the adjuvant effect remain unrevealed. To this end, we have studied the enhancement of antigen uptake by murine dendritic cell line, DC2.4 cells, by the cationic liposomes and the specific pathways involved in the process. We have observed that the uptake of ovalbumin (OVA) into DC2.4 cells is greatly increased when co-cultured with the cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] (DC-chol). However, this enhancement was blocked by pretreatment of DC2.4 cells with chlorpromazine and methyl-β-cyclodextrin, indicating the involvement of clathrin- and caveolin-independent lipid raft-dependent endocytic pathways in the process. Our results implied, at least in part, that enhanced uptake of antigens induced by the cationic liposomes could be a possible mechanism for the induction of immune responses. Although further studies are needed to understand the precise mechanisms behind the adjuvant effects of DOTAP/DC-chol liposome, this approach is quite useful for the development of vaccine system to combat various diseases

[Image: see text] Inhibition of myostatin is a promising strategy for treatment of muscle atrophic disorders. We had already identified a 23-mer peptide (1) as a synthetic myostatin inhibitor, and structure–activity relationship studies with 1 afforded a potent 22-mer peptide derivative (3). Herein, we report the shortest myostatin inhibitory peptide so far. Among chain-shortened 16-mer peptidic inhibitors derived from the C-terminal region of 3, peptide inhibitor 8a with β-sheet propensity was twice as potent as 22-mer inhibitor 3 and significantly increased not only muscle mass but also hind limb grip strength in Duchenne muscular dystrophic model mice. These results suggest that 8a is a promising platform for drug development treating muscle atrophic disorders.

We previously developed novel liposomal nanobubbles (Bubble liposomes [BL]) that oscillate and collapse in an ultrasound field, generating heat and shock waves. We aimed to investigate the feasibility of cancer therapy using the combination of BL and ultrasound. In addition, we investigated the anti-tumor mechanism of this cancer therapy. Colon-26 cells were inoculated into the flank of BALB/c mice to induce tumors. After 8 days, BL or saline was intratumorally injected, followed by transdermal ultrasound exposure of tumor tissue (1 MHz, 0–4 W/cm[2], 2 min). The anti-tumor effects were evaluated by histology (necrosis) and tumor growth. In vivo cell depletion assays were performed to identify the immune cells responsible for anti-tumor effects. Tumor temperatures were significantly higher when treated with BL + ultrasound than ultrasound alone. Intratumoral BL caused extensive tissue necrosis at 3–4 W/cm[2] of ultrasound exposure. In addition, BL + ultrasound significantly suppressed tumor growth at 2–4 W/cm[2]. In vivo depletion of CD8[+] T cells (not NK or CD4[+] T cells) completely blocked the effect of BL + ultrasound on tumor growth. These data suggest that CD8[+] T cells play a critical role in tumor growth suppression. Finally, we concluded that BL + ultrasound, which can prime the anti-tumor cellular immune system, may be an effective hyperthermia strategy for cancer treatment.

Gene therapy is promising for the treatment of many diseases including cancers and genetic diseases. From the viewpoint of safety, ultrasound (US)-mediated gene delivery with nano/ microbubbles was recently developed as a novel non-viral vector system. US-mediated gene delivery using nano/microbubbles are able to produce transient changes in the permeability of the cell membrane after US-induced cavitation while reducing cellular damage and enables the tissue-specific or the site-specific intracellular delivery of gene both in vitro and in vivo. We have recently developed novel lipid nanobubbles (Lipid Bubbles). These nanobubbles can also be used to enhance the efficacy of the US-mediated genes (plasmid DNA, siRNA, and miRNA etc.) delivery. In this review, we describe US-mediated delivery systems combined with nano/microbubbles and discuss their feasibility as non-viral vector systems.

Abnormal splicing of the chloride channel 1 (CLCN1) gene causes myotonic dystrophy type 1 (DM1). Therefore, controlling the alternative splicing process of this gene by antisense oligonucleotides can be a promising treatment for DM1. In this study, we describe an efficient phosphorodiamidate morpholino oligomer (PMO) delivery method by ultrasound-mediated bubble liposomes, which is a known gene delivery tool with ultrasound exposure, to treat skeletal muscles in a DM1 mouse model, HSA

Infectious disease remains a substantial cause of death. To overcome this issue, mucosal vaccine systems are considered to be a promising strategy. Yet, none are approved for clinical use, except for live-attenuated mucosal vaccines, mainly owing to the lack of effective and safe systems to induce antigen-specific immune responses in the mucosal compartment. We have reported that intranasal vaccination of an antigenic protein, with cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane and 3&beta, [N-(N&prime, N&prime, dimethylaminoethane)-carbamoyl], induced antigen-specific mucosal and systemic antibody responses in mice. However, precise molecular mechanism(s) underlying the mucosal adjuvant effects of cationic liposomes remain to be uncovered. Here, we show that a host double-stranded DNA (dsDNA), released at the site of cationic liposome injection, plays an essential role for the mucosal adjuvanticity of the cationic liposome. Namely, we found that nasal administration of the cationic liposomes induced localized cell death, at the site of injection, resulting in extracellular leakage of host dsDNA. Additionally, in vivo DNase I treatment markedly impaired OVA-specific mucosal and systemic antibody production exerted by cationic liposomes. Our report reveals that host dsDNA, released from local dying cells, acts as a damage-associated molecular pattern that mediates the mucosal adjuvant activity of cationic liposomes.

Abnormal splicing of the chloride channel 1 (CLCN1) gene causes myotonic dystrophy type 1 (DM1). Therefore, controlling the alternative splicing process of this gene by antisense oligonucleotides can be a promising treatment for DM1. In this study, we describe an efficient phosphorodiamidate morpholino oligomer (PMO) delivery method by ultrasound-mediated bubble liposomes, which is a known gene delivery tool with ultrasound exposure, to treat skeletal muscles in a DM1 mouse model, HSALR. Effective delivery of PMO using this technique can help control the alternative splicing of the Clcn1 gene via exon skipping and enhance the expression of Clcn1 protein in skeletal muscles and the amelioration of myotonia. Thus, exon skipping by PMO delivery with ultrasound-mediated BLs may be feasible in myotonic dystrophy model mice.

Insulin is a key peptide hormone used for the treatment of both type I and type II diabetes. To maximize the effect of the treatment of these diseases, the addition of poly(ethylene glycol) (PEGylation) methods for the insulin are widely developed. Here, to make these PEGylation methods the simplest, we report the byproduct-free intact modification of insulin by cholesterol end-modified poly(ethylene glycol) with urethane, propyl, and methoxy groups (that is, Chol-U-Pr-mPEG). The noncovalent PEGylation by the Chol-U-Pr-mPEG has been achieved by the simple mixing of insulin with the Chol-U-Pr-mPEG in aqueous solution, followed by freeze-drying. The formation of the Chol-U-Pr-mPEG/insulin complex has proceeded without byproducts, such as N-hydroxysuccinimide, formed by the conventional covalent PEGylation using an active ester group. The byproduct-free PEGylation has preserved insulin conformation as well as primary structure. The intact PEGylation has protected insulin from hydrolysis by protease. The resulting insulin modified by the Chol-U-Pr-mPEG has sustainably suppressed the level of blood glucose, as compared to naked insulin, in mice. Consequently, the Chol-U-Pr-mPEG/insulin complex formation offers the byproduct-free intact PEGylation of insulin for in vivo protein delivery.

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration, caused by nonsense or frameshift mutations in the dystrophin (DMD) gene. Antisense oligonucleotides can be used to induce specific exon skipping; recently, a phosphorodiamidate morpholino oligomer (PMO) has been approved for clinical use in DMD. However, an efficient PMO delivery strategy is required to improve the therapeutic efficacy in DMD patients. We previously developed polyethylene glycol (PEG)-modified liposomes containing ultrasound contrast gas, "Bubble liposomes" (BLs), and found that the combination of BLs with ultrasound exposure is a useful gene delivery tool. Here, we describe an efficient PMO delivery strategy using the combination of BLs and ultrasound exposure to treat muscles in a DMD mouse model (mdx). This ultrasound-mediated BL technique can increase the PMO-mediated exon-skipping efficiency, leading to significantly increased dystrophin expression. Thus, the combination of BLs and ultrasound exposure may be a feasible PMO delivery method to improve therapeutic efficacy and reduce the PMO dosage for DMD treatment.

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration, caused by nonsense or frameshift mutations in the dystrophin (DMD) gene. Antisense oligonucleotides can be used to induce specific exon skipping; recently, a phosphorodiamidate morpholino oligomer (PMO) has been approved for clinical use in DMD. However, an efficient PMO delivery strategy is required to improve the therapeutic efficacy in DMD patients. We previously developed polyethylene glycol (PEG)-modified liposomes containing ultrasound contrast gas, "Bubble liposomes" (BLs), and found that the combination of BLs with ultrasound exposure is a useful gene delivery tool. Here, we describe an efficient PMO delivery strategy using the combination of BLs and ultrasound exposure to treat muscles in a DMD mouse model (mdx). This ultrasound-mediated BL technique can increase the PMO-mediated exon-skipping efficiency, leading to significantly increased dystrophin expression. Thus, the combination of BLs and ultrasound exposure may be a feasible PMO delivery method to improve therapeutic efficacy and reduce the PMO dosage for DMD treatment.

Sonoporation is a promising method to intracellularly deliver synthetic gene carriers that have lower endocytotic uptake than viral carriers. Here, we applied sonoporation to deliver genes via polyethylene glycol (PEG)-grafted polymeric carriers that specifically respond to hyperactivated protein kinase A (PKA). PEG-grafted polymeric carrier/DNA polyplexes were not efficiently delivered into cells via the endocytotic pathway because of the hydrophilic PEG layer surrounding the polyplexes. However, the delivery of polyplexes into cells was significantly increased by sonoporation. The delivered polyplexes exhibited PKA-responsive transgene expression in PKA-overexpressing cells, but not in cells with low PKA activation. These results show that the sonoporation-mediated delivery of PEG-modified PKA-responsive polyplexes is a promising approach for safely applying gene therapy to abnormal cells with hyperactivated PKA.