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.

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.

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.

Bubble liposomes and ultrasound exposure improve localized morpholino oligomer delivery into the skeletal muscles of dystrophic mdx mice.

Duchenne muscular dystrophy (DMD) is a genetic disorder that is caused by mutations in the DMD gene that lead to an absence of functional protein. The mdx dystrophic mouse contains a nonsense mutation in exon 23 of the dystrophin gene; a phosphorodiamidate morpholino oligomer (PMO) designed to skip this mutated exon in the mRNA induces dystrophin expression. However, an efficient PMO delivery method is needed to improve treatment strategies for DMD. We previously developed polyethylene glycol (PEG)-modified liposomes (Bubble liposomes) that entrap ultrasound contrast gas and demonstrated that the combination of Bubble liposomes with ultrasound exposure is an effective gene delivery tool in vitro and in vivo. In this study, to evaluate the ability of Bubble liposomes as a PMO delivery tool, we tested the potency of the Bubble liposomes combined with ultrasound exposure to boost the delivery of PMO and increase the skipping of the mutated exon in the mdx mouse. The results indicated that the combination of Bubble liposomes and ultrasound exposure increased the uptake of the PMO targeting a nonsense mutation in exon 23 of the dystrophin gene and consequently increased the PMO-mediated exon-skipping efficiency compared with PMO injection alone, leading to significantly enhanced dystrophin expression. This increased efficiency indicated the potential of the combination of Bubble liposomes with ultrasound exposure to enhance PMO delivery for treating DMD. Thus, this ultrasound-mediated Bubble liposome technique may provide an effective, noninvasive, nonviral method for PMO therapy for DMD muscle as well as for other muscular dystrophies.

Combination of bubble liposomes and high-intensity focused ultrasound (HIFU) enhanced antitumor effect by tumor ablation.

Ultrasound (US) is used in the clinical setting not only for diagnosis but also for therapy. As a therapeutic US technique, high-intensity focused ultrasound (HIFU) can be applied to treat cancer in a clinical setting. Microbubbles increased temperature and improved the low therapeutic efficiency under HIFU; however, microbubbles have room for improvement in size, stability, and targeting ability. To solve these issues, we reported that "Bubble liposomes" (BLs) containing the US imaging gas (perfluoropropane gas) liposomes were suitable for ultrasound imaging and gene delivery. In this study, we examined whether BLs and HIFU could enhance the ablation area of the tumor and the antitumor effect. First, we histologically analyzed the tumor after BLs and HIFU. The ablation area of the treatment of BLs and HIFU was broader than that of HIFU alone. Next, we monitored the temperature of the tumor, and examined the antitumor effect. The temperature increase with BLs and HIFU treatment was faster and higher than that with HIFU alone. Moreover, treatment with BLs and HIFU enhanced the antitumor effect, which was better than with HIFU alone. Thus, the combination of BLs and HIFU could be efficacious for cancer therapy.

Diffusive delivery of plasmid DNA using zwitterionic carboxyalkyl poly(1-vinylimidazole) into skeletal muscle in vivo.

Zwitterionic carboxyalkyl poly(1-vinylimidazole) (CA-PVIm) polymers with imidazolium cations and carboxylate anions have been synthesized as a carrier for the in vivo delivery of plasmid DNA (pDNA) to skeletal muscle. From differential scanning calorimetry measurements, resulting CA-PVIm had intermediate water in hydration water as a biocompatible polymer. Notably, when the pDNA and resulting CA-PVIm were mixed, slight retarded bands of the pDNA were observed in agarose gel electrophoresis, suggesting the polyion complex (PIC) formation between the pDNA and CA-PVIm despite zwitterionic polymers. Resulting PICs maintained the higher-order structure of the pDNA. Using resulting pDNA PICs, the highest pDNA expression by intramuscular injection was achieved in the PIC with 7 mol% carboxymethylated PVIm, that is, CA1(7)-PVIm, observed in a widespread area by in vivo imaging system. These results suggest that the CA1(7)-PVIm/pDNA PIC is effective for the diffusive delivery of the pDNA into skeletal muscle for the treatment of serious muscle diseases.

Ultrasound-mediated gene delivery systems by AG73-modified Bubble liposomes.

Targeted gene delivery to neovascular vessels in tumors is considered a promising strategy for cancer therapy. We previously reported that "Bubble liposomes" (BLs), which are ultrasound (US) imaging gas-encapsulating liposomes, were suitable for US imaging and gene delivery. When BLs are exposed to US, the bubble is destroyed, creating a jet stream by cavitation, and resulting in the instantaneous ejection of extracellular plasmid DNA (pDNA) or other nucleic acids into the cytosol. We developed AG73 peptide-modified Bubble liposomes (AG73-BL) as a targeted US contrast agent, which was designed to attach to neovascular tumor vessels and to allow specific US detection of angiogenesis (Negishi et al., Biomaterials 2013, 34, 501-507). In this study, to evaluate the effectiveness of AG73-BL as a gene delivery tool for neovascular vessels, we examined the gene transfection efficiency of AG73-BL with US exposure in primary human endothelial cells (HUVEC). The transfection efficiency was significantly enhanced if the AG73-BL attached to the HUVEC was exposed to US compared to the BL-modified with no peptide or scrambled peptide. In addition, the cell viability was greater than 80% after transfection with AG73-BL. These results suggested that after the destruction of the AG73-BL with US exposure, a cavitation could be effectively induced by the US exposure against AG73-BL binding to the cell surface of the HUVEC, and the subsequent gene delivery into cells could be enhanced. Thus, AG73-BL may be useful for gene delivery as well as for US imaging of neovascular vessels.

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 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.

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.

Systemic delivery systems of angiogenic gene by novel bubble liposomes containing cationic lipid and ultrasound exposure.

Recently, we developed polyethyleneglycol (PEG)-modified liposomes (Bubble liposomes; BLs) entrapping ultrasound (US) gas and reported that the combination of BL and US exposure was an effective tool for the delivery of pDNA directly into skeletal muscles of an ischemic hindlimb model with local injection. To achieve gene delivery to deeper tissues, we attempted to prepare novel Bubble liposomes which were able to be loaded with pDNA and useful for systemic injection. We prepared BLs using cationic lipid and analyzed the interaction with the BLs and pDNA using flow cytometry. The solution of pDNA-loaded BLs (p-BLs) was further injected into the tail vein of hindlimb ischemia model mice, and transdermal US exposure was applied to ischemic hindlimb. The effects of transfection on angiogenic factors were investigated by real-time PCR. Blood flow was determined using a laser Doppler blood flow meter. The interaction with BLs and pDNA increased in the presence of DOTAP and short PEG chains and resulted in increased stability of pDNA in the serum. Transfection with pDNA encoding the bFGF gene using p-BLs and US induced various angiogenic factors and improved the blood flow. The gene delivery system into the ischemic hindlimb using the combination of p-BLs and US exposure could be an effective tool for angiogenic gene therapy via systemic injection.

Involvement of Ca²âº and ATP in enhanced gene delivery by bubble liposomes and ultrasound exposure.

Recently, we reported the accelerated gene transfection efficiency of laminin-derived AG73-peptide-labeled polyethylene glycol-modified liposomes (AG73-PEG liposomes) and cell penetrating TAT-peptide labeled PEG liposomes using PEG-modified liposomes, which trap echo-contrast gas, "Bubble liposomes" (BLs), and ultrasound (US) exposure. BLs and US exposure were reported to enhance the endosomal escape of AG73-PEG liposomes, thereby leading to increased gene expression. However, the mechanism behind the effect of BLs and US exposure on endosomes is not well understood. US exposure was reported to induce an influx of calcium ions (Ca²âº) by enhancing permeability of the cell membrane. Therefore, we examined the effect of Ca²âº on the endosomal escape and transfection efficiency of AG73-PEG liposomes, which were previously enhanced by BLs and US exposure. For cells treated with EGTA, the endosomal escape and gene expression of AG73-PEG liposomes were not enhanced by BLs and US exposure. Similarly, transfection efficiency of the AG73-PEG liposomes in ATP-depleted cells was not enhanced. Our results suggest that Ca²âº and ATP are necessary for the enhanced endosomal escape and gene expression of AG73-PEG liposomes by BLs and US exposure. These findings may contribute to the development of useful techniques to improve endosomal escape and achieve efficient gene transfection.

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.

Bubble liposomes and ultrasound promoted endosomal escape of TAT-PEG liposomes as gene delivery carriers.

We have previously developed laminin-derived AG73 peptide-labeled poly(ethylene glycol)-modified liposomes (AG73-PEG liposomes) for selective cancer gene therapy and reported that Bubble liposomes (BLs) and ultrasound (US) exposure could accelerate the endosomal escape of AG73-PEG liposomes, leading to the enhancement of transfection efficiency; however, it is still unclear whether BLs and US exposure can also enhance the transfection efficiency of other vectors. We therefore assessed the effect of BLs and US exposure on the gene transfection efficiency of trans-activating transcriptor (TAT) peptide modified PEG liposomes. Although TAT-PEG liposomes were efficiently internalized into cells, the efficacy of endosomal escape was insufficient. The transfection efficiencies of TAT-PEG liposomes were enhanced by about 30-fold when BLs and US exposure were used. We also confirmed that BLs and US exposure could not enhance the direct transportation of TAT-PEG liposomes into cells. Confocal microscopy showed that BLs and US exposure promoted endosomal escape of TAT-PEG liposomes. Our results suggested that BLs and US exposure could enhance transfection efficiency by promoting endosomal escape, which was independent of modified molecules of carriers. Thus, BLs and US exposure can be a useful tool to achieve efficient gene transfection by improving endosomal escape of various carriers.

Delivery of an angiogenic gene into ischemic muscle by novel bubble liposomes followed by ultrasound exposure.

PURPOSE: To develop a safe and efficient gene delivery system into skeletal muscle using the combination of Bubble liposomes (BL) and ultrasound (US) exposure, and to assess the feasibility and the effectiveness of BL for angiogenic gene delivery in clinical use. METHODS: A solution of luciferase-expressing plasmid DNA (pDNA) and BL was injected into the tibialis (TA) muscle, and US was immediately applied to the injection site. The transfection efficiency was estimated by a luciferase assay. The ischemic hindlimb was also treated with BL and US-mediated intramuscular gene transfer of bFGF-expressing plasmid DNA. Capillary vessels were assessed using immunostaining. The blood flow was determined using a laser Doppler blood flow meter. RESULTS: Highly efficient gene transfer could be achieved in the muscle transfected with BLs, and US mediated the gene transfer. Capillary vessels were enhanced in the treatment groups with this gene transfer method. The blood flow in the treated groups with this gene transfer method quickly recovered compared to other treatment groups (non-treated, bFGF alone, or bFGF+US). CONCLUSION: The gene transfer system into skeletal muscle using the combination of BL and US exposure could be an effective means for angiogenic gene therapy in limb ischemia.

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.

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.

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.

Preparation and characterization of laminin-derived peptide AG73-coated liposomes as a selective gene delivery tool.

Targeted gene delivery to cancer cells is considered as a promising strategy for cancer therapy. Since, several targeting ligands have been studied for cancer gene therapy, such as transferrin, folate, anisamide, RGD-peptide, and antibodies. We have focused on AG73 peptide, which is derived from the globular domain of the laminin α1 chain. AG73 peptide is known as a ligand for syndecans, one of the major heparin sulfate-containing transmembrane proteoglycans. Syndecan-2 is highly expressed in various cancer cells and plays a role in angiogenesis. In this study, we prepared AG73-labeled polyethyleneglycol-modified liposomes (AG73-PEG liposomes) for gene delivery tool to syndecan-2 overexpressing cancer cells, and assessed the characterization of AG73-PEG liposomes. We confirmed the conjugation of AG73 peptide to PEG liposomes by reverse-phase high-performance liquid chromatography analysis. Electron microscopy analysis showed that monodiseperse AG73-labeled lipsomes were prepared. We also assessed the gene transfection efficiency of AG73-PEG liposomes in syndecan-2 overexpressing cancer cells or syndecan-2 less expressing cancer cells. As a result, AG73-mediated liposomal gene transfection efficiency was increased by 100-fold in syndecan-2 overexpressing cancer cells compared to syndecan-2 less expressing cancer cells. These results suggested that AG73-PEG liposomes were successfully prepared from a point of view of the modification of AG73 peptide to PEG-liposomes and the particle size of liposomes, which presented nano size. Furthermore, our results suggest that AG73-PEG liposomes can be a useful targeted gene delivery vehicle for syndecan-2 overexpressing cancer cells.