Development of a concise and reliable method for quantifying the antibody loaded onto lipid nanoparticles modified with Herceptin.

Antibodies are essential components of the immune system with a wide range of molecular targets. They have been recognized as modalities for treating several diseases and more than 130 approved antibody-based therapeutics are available for clinical use. However, limitations remain associated with its efficacy, tissue permeability, and safety, especially in cancer treatment. Nanoparticles, particularly those responsive to external stimuli, have shown promise in improving the efficacy of antibody-based therapeutics and tissue-selective delivery. In this study, we developed a reliable and accurate method for quantifying the amount of antibody loaded onto lipid nanoparticles modified with Herceptin® (Trastuzumab), an antibody-based therapeutic used to treat HER2-positive cancers, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. This method proved to be a suitable alternative to commonly used protein quantification techniques, which are limited by lipid interference present in the samples. Furthermore, the amount of Herceptin modified on the liposomes, measured by this method, was confirmed by Herceptin's antibody-dependent cell-mediated cytotoxicity activity. Our results demonstrate the potential of this method as a critical tool for developing tissue-selective antibody delivery systems, leading to improved efficacy and reduced side effects of antibody-based therapeutics.

Organic Phase-Soluble Nanomagnetically Cationic Phospholipid: Synthesis, Characterization, and In Vitro Transfection Activity.

The presented work describes the synthesis and characterization of a novel magnetic cationic phospholipid (MCP) system with a stable dopamine anchor as well as its transfection activity study. The synthesized architectural system increases the biocompatibility of iron oxide and promises applications of magnetic nanoparticles in living cells. The MCP system is soluble in organic solvents and can be easily adapted to prepare magnetic liposomes. We created complexes with liposomes containing MCP and other functional cationic lipids and pDNA as gene delivery tools, which possessed the ability to enhance the efficiency of transfection, particularly the process of interaction with cells by inducing a magnetic field. The MCP is able to create iron oxide nanoparticles and has the potential for the materials to prepare the system for site-specific gene delivery with the application of an external magnetic field.

Development of a Gene and Nucleic Acid Delivery System for Skeletal Muscle Administration via Limb Perfusion Using Nanobubbles and Ultrasound.

Strategies for gene and nucleic acid delivery to skeletal muscles have been extensively explored to treat Duchenne muscular dystrophy (DMD) and other neuromuscular diseases. Of these, effective intravascular delivery of naked plasmid DNA (pDNA) and nucleic acids into muscles is an attractive approach, given the high capillary density in close contact with myofibers. We developed lipid-based nanobubbles (NBs) using polyethylene-glycol-modified liposomes and an echo-contrast gas and found that these NBs could improve tissue permeability by ultrasound (US)-induced cavitation. Herein, we delivered naked pDNA or antisense phosphorodiamidate morpholino oligomers (PMOs) into the regional hindlimb muscle via limb perfusion using NBs and US exposure. pDNA encoding the luciferase gene was injected with NBs via limb perfusion into normal mice with application of US. High luciferase activity was achieved in a wide area of the limb muscle. DMD model mice were administered PMOs, designed to skip the mutated exon 23 of the dystrophin gene, with NBs via intravenous limb perfusion, followed by US exposure. The number of dystrophin-positive fibers increased in the muscles of mdx mice. Combining NBs and US exposure, which can be widely delivered to the hind limb muscles via the limb vein, could be an effective therapeutic approach for DMD and other neuromuscular disorders.

Gene and oligonucleotide delivery via micro- and nanobubbles by ultrasound exposure.

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.

Development of an Antibody Delivery Method for Cancer Treatment by Combining Ultrasound with Therapeutic Antibody-Modified Nanobubbles Using Fc-Binding Polypeptide.

A key challenge in treating solid tumors is that the tumor microenvironment often inhibits the penetration of therapeutic antibodies into the tumor, leading to reduced therapeutic efficiency. It has been reported that the combination of ultrasound-responsive micro/nanobubble and therapeutic ultrasound (TUS) enhances the tissue permeability and increases the efficiency of delivery of macromolecular drugs to target tissues. In this study, to facilitate efficient therapeutic antibody delivery to tumors using this combination system, we developed therapeutic antibody-modified nanobubble (NBs) using an Fc-binding polypeptide that can quickly load antibodies to nanocarriers; since the polypeptide was derived from Protein G. TUS exposure to this Herceptin®-modified NBs (Her-NBs) was followed by evaluation of the antibody's own ADCC activity, resulting the retained activity. Moreover, the utility of combining therapeutic antibody-modified NBs and TUS exposure as an antibody delivery system for cancer therapy was assessed in vivo. The Her-NBs + TUS group had a higher inhibitory effect than the Herceptin and Her-NBs groups. Overall, these results suggest that the combination of therapeutic antibody-modified NBs and TUS exposure can enable efficient antibody drug delivery to tumors, while retaining the original antibody activity. Hence, this system has the potential to maximize the therapeutic effects in antibody therapy for solid cancers.

Ternary Complexes of pDNA, Neuron-Binding Peptide, and PEGylated Polyethyleneimine for Brain Delivery with Nano-Bubbles and Ultrasound.

In brain-targeted delivery, the transport of drugs or genes across the blood-brain barrier (BBB) is a major obstacle. Recent reports found that focused ultrasound (FUS) with microbubbles enables transient BBB opening and improvement of drug or gene delivery. We previously developed nano-sized bubbles (NBs), which were prepared based on polyethylene glycol (PEG)-modified liposomes containing echo-contrast gas, and showed that our NBs with FUS could also induce BBB opening. The aim of this study was to enhance the efficiency of delivery of pDNA into neuronal cells following transportation across the BBB using neuron-binding peptides. This study used the RVG-R9 peptide, which is a chimeric peptide synthesized by peptides derived from rabies virus glycoprotein and nonamer arginine residues. The RVG peptide is known to interact specifically with the nicotinic acetylcholine receptor in neuronal cells. To enhance the stability of the RVG-R9/pDNA complex in vivo, PEGylated polyethyleneimine (PEG-PEI) was also used. The ternary complexes composed of RVG-R9, PEG-PEI, and pDNA could interact with mouse neuroblastoma cells and deliver pDNA into the cells. Furthermore, for the in vivo experiments using NBs and FUS, gene expression was observed in the FUS-exposed brain hemispheres. These results suggest that this systemic gene delivery system could be useful for gene delivery across the BBB.

Development of A2G80 peptide-gene complex for targeted delivery to muscle cells.

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.

Alpha-dystroglycan binding peptide A2G80-modified stealth liposomes as a muscle-targeting carrier for Duchenne muscular dystrophy.

Safe and efficient gene therapy for the treatment of Duchenne muscular dystrophy (DMD), a genetic disorder, is required. For this, the muscle-targeting delivery system of genes and nucleic acids is ideal. In this study, we focused on the A2G80 peptide, which has an affinity for α-dystroglycan expressed on muscle cell membranes, as a muscle targeted nanocarrier for DMD and developed A2G80-modified liposomes. We also prepared A2G80-modified liposomes coated with long- and short-chain PEG, called A2G80-LSP-Lip, to improve the blood circulation of liposomes using microfluidics. The liposomes had a particle size of approximately 80 nm. A2G80-LSP-Lip showed an affinity for the muscle tissue section of mice by overlay assay. When the liposomes were administered to DMD model mice (mdx mice) via the tail vein, A2G80-LSP-Lip accumulated efficiently in muscle tissue compared to control liposomes. These results suggest that A2G80-LSP-Lip can function as a muscle-targeting liposome for DMD via systemic administration, and may be a useful tool for DMD treatment.

Microbubbles and Nanobubbles with Ultrasound for Systemic Gene Delivery.

The regulation of gene expression is a promising therapeutic approach for many intractable diseases. However, its use in clinical applications requires the efficient delivery of nucleic acids to target tissues, which is a major challenge. Recently, various delivery systems employing physical energy, such as ultrasound, magnetic force, electric force, and light, have been developed. Ultrasound-mediated delivery has particularly attracted interest due to its safety and low costs. Its delivery effects are also enhanced when combined with microbubbles or nanobubbles that entrap an ultrasound contrast gas. Furthermore, ultrasound-mediated nucleic acid delivery could be performed only in ultrasound exposed areas. In this review, we summarize the ultrasound-mediated nucleic acid systemic delivery system, using microbubbles or nanobubbles, and discuss its possibilities as a therapeutic tool.

Development of Antibody-Modified Nanobubbles Using Fc-Region-Binding Polypeptides for Ultrasound Imaging.

Ultrasound (US) imaging is a widely used imaging technique. The use of US contrast agents such as microbubbles, which consist of phospholipids and are filled with perfluorocarbon gases, has become an indispensable component of clinical US imaging, while molecular US imaging has recently attracted significant attention in combination with efficient diagnostics. The avidin-biotin interaction method is frequently used to tether antibodies to microbubbles, leading to the development of a molecular targeting US imaging agent. However, avidin still has limitations such as immunogenicity. We previously reported that lipid-based nanobubbles (NBs) containing perfluorocarbon gas are suitable for US imaging and gene delivery. In this paper, we report on the development of a novel antibody modification method for NBs using Fc-region-binding polypeptides derived from protein A/G. First, we prepared anti-CD146 antibody-modified NBs using this polypeptide, resulting in high levels of attachment to human umbilical vein endothelial cells expressing CD146. To examine their targeting ability and US imaging capability, the NBs were administered to tumor-bearing mice. The contrast imaging of antibody-modified NBs was shown to be prolonged compared with that of non-labeled NBs. Thus, this antibody modification method using an Fc-binding polypeptide may be a feasible tool for developing a next-generation antibody-modified US imaging agent.

Exon Skipping by Ultrasound-Enhanced Delivery of Morpholino with Bubble Liposomes for Myotonic Dystrophy Model Mice.

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.

Nucleic Acid Delivery System by the Combination of Lipid bubbles and Ultrasound.

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.

Development of a Screening System for Targeting Carriers Using Peptide-Modified Liposomes and Tissue Sections.

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 αvß3-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.

PMO Delivery System Using Bubble Liposomes and Ultrasound Exposure for Duchenne Muscular Dystrophy 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.

Gene delivery systems by the combination of lipid bubbles and ultrasound.

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.

Preparation of Angiopep-2 Peptide-Modified Bubble Liposomes for Delivery to the Brain.

In the development of therapeutic approaches for central nervous system diseases, a significant obstacle is efficient drug delivery across the blood-brain barrier owing to its low permeability. Various nanocarriers have been developed for brain-targeted drug delivery by modification with specific ligands. We have previously developed polyethylene glycol-modified liposomes (Bubble liposomes [BLs]) that entrap ultrasound (US) contrast gas and can serve as both plasmid DNA or small interfering RNA carriers and US contrast agents. In this study, we attempted to prepare brain-targeting BLs modified with Angiopep-2 (Ang2) peptide (Ang2-BLs). Ang2 is expected to be a useful ligand for the efficient delivery of nanocarriers to the brain. We showed that Ang2-BLs interacted specifically with brain endothelial cells via low-density lipoprotein receptor-related protein-1. We also confirmed that Ang2-BLs could entrap US contrast gas and had US imaging ability as well as unmodified BLs. Furthermore, we demonstrated that Ang2-BLs accumulated in brain tissue after intravascular injection. These results suggested that Ang2-BLs may be a useful tool for brain-targeted delivery and US imaging via systemic administration.

MicroRNA Imaging in Combination with Diagnostic Ultrasound and Bubble Liposomes for MicroRNA Delivery.

MicroRNA (miRNA) is expected to play an important role in the diagnosis and therapy of various diseases. In miRNA therapy, the development of delivery tools to the target site is considered to be essential. By using a delivery tool possessing imaging ability, miRNA colocalized with the carrier could be visualized after administration. We prepared polyethylene glycol (PEG)-modified liposomes containing echo-contrast gas, "Bubble liposomes" (BLs), and confirmed that BLs containing cationic lipid were capable of loading miRNA. Furthermore, we also achieved the imaging and delivery of systemically injected miRNA to target site in combination with ultrasound exposure. MiRNA-loaded BLs could be a useful tool for imaging and therapy.

Potential effect of cationic liposomes on interactions with oral bacterial cells and biofilms.

CONTEXT: Although oral infectious diseases have been attributed to bacteria, drug treatments remain ineffective because bacteria and their products exist as biofilms. Cationic liposomes have been suggested to electrostatically interact with the negative charge on the bacterial surface, thereby improving the effects of conventional drug therapies. However, the electrostatic interaction between oral bacteria and cationic liposomes has not yet been examined in detail. OBJECTIVE: The aim of the present study was to examine the behavior of cationic liposomes and Streptococcus mutans in planktonic cells and biofilms. MATERIALS AND METHODS: Liposomes with or without cationic lipid were prepared using a reverse-phase evaporation method. The zeta potentials of conventional liposomes (without cationic lipid) and cationic liposomes were -13 and 8 mV, respectively, and both had a mean particle size of approximately 180 nm. We first assessed the interaction between liposomes and planktonic bacterial cells with a flow cytometer. We then used a surface plasmon resonance method to examine the binding of liposomes to biofilms. We confirmed the binding behavior of liposomes with biofilms using confocal laser scanning microscopy. RESULTS: The interactions between cationic liposomes and S. mutans cells and biofilms were stronger than those of conventional liposomes. Microscopic observations revealed that many cationic liposomes interacted with the bacterial mass and penetrated the deep layers of biofilms. DISCUSSION AND CONCLUSION: In this study, we demonstrated that cationic liposomes had higher affinity not only to oral bacterial cells, but also biofilms than conventional liposomes. This electrostatic interaction may be useful as a potential drug delivery system to biofilms.

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.

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.

Nonviral gene delivery systems by the combination of bubble liposomes and ultrasound.

The combination of therapeutic ultrasound (US) and nano/microbubbles is an important system for establishing a novel and noninvasive gene delivery system. Genes are delivered more efficiently using this system compared with a conventional nonviral vector system such as the lipofection method, resulting in higher gene expression. This higher efficiency is due to the gene being delivered into the cytosol and bypassing the endocytosis pathway. Many in vivo studies have demonstrated US-mediated gene delivery with nano/microbubbles, and several gene therapy feasibility studies for various diseases have been reported. In addition, nano/microbubbles can deliver genes site specifically by the control of US exposure site. In the present review, we summarize the gene delivery systems by the combination of nano/microbubbles and US, describe their properties, and assess applications and challenges of US theranostics.