Functional Peptide-Modified Liposomes for siRNA Delivery to Rat Retinal Pigment Epithelium Cells: Effect of Peptide Sequences.

Most retinal diseases involve the degeneration of choroidal retinal pigment epithelial (RPE) cells. Because of a blood-retina barrier (tight junction formation), RPE cells restrict the entry of hydrophilic macromolecules (e.g., small interfering RNA (siRNA)) through blood stream and eye drops. A cytoplasm-responsive stearylated (STR) peptide, STR-CH2R4H2C (CH2R4) enables stable siRNA complexation, cell permeation, and intracellular dynamics control. We previously demonstrated how CH2R4-modified liposomes promoted siRNA efficacy. We investigated the influence of amino acid sequences of functional peptides on cellular uptake pathways, siRNA transfection efficacy, and the permeation of peptide-modified liposomes in rat RPE-J cells. Four STR-peptides, consisting of arginine (R), cysteine (C), histidine (H), lysine (K) or serine (S), were designed based on CH2R4. We prepared siRNA-loaded, peptide-modified cationic liposomes (CH2R4-, CH2K4-, CH2S4-, SH2R4-, and SH2S4-lipoplexes). CH2R4-, CH2K4-, and SH2R4-lipoplexes induced cellular uptake by macropinocytosis by activating cytoskeletal F-actin, possibly due to cationic amino acids (arginine, lysine). SH2R4-lipoplexes were trapped in endosomes, whereas CH2R4- and CH2K4-lipoplexes enhanced endosomal siRNA release suggesting cysteine contributes to endosomal escape. Although cationic liposome-based, CH2S4- and SH2S4-lipoplexes (not including arginine and lysine) showed lower siRNA transfection efficiency. This difference may be because siRNAs were retained on both peptide moieties and cationic liposomes in CH2R4-, CH2K4- and SH2R4-lipoplexes, whereas in CH2S4- and SH2S4-lipoplexes, siRNAs were loaded to the cationic liposomes, but not on peptides. In three-dimensional spheroids, CH2R4- and CH2K4-modified liposomes promoted permeation through tight junctions. Thus, cationic amino acids and cysteine within peptide sequences of CH2R4 could be effective for siRNA delivery to the retina using functional peptide-modified liposomes.

A Multifunctional Hybrid Nanocarrier for Non-Invasive siRNA Delivery to the Retina.

Drug therapy for retinal diseases (e.g., age-related macular degeneration, the leading cause of blindness) is generally performed by invasive intravitreal injection because of poor drug delivery caused by the blood-retinal barrier (BRB). This study aimed to develop a nanocarrier for the non-invasive delivery of small interfering RNA (siRNA) to the posterior segment of the eye (i.e., the retina) by eyedrops. To this end, we prepared a hybrid nanocarrier based on a multifunctional peptide and liposomes, and the composition was optimized. A cytoplasm-responsive stearylated peptide (STR-CH2R4H2C) was used as the multifunctional peptide because of its superior ability to enhance the complexation, cell permeation, and intracellular dynamics of siRNA. By adding STR-CH2R4H2C to the surface of liposomes, intracellular uptake increased regardless of the liposome surface charge. The STR-CH2R4H2C-modified cationic nanocarrier demonstrated significant siRNA transfection efficiency with no cytotoxicity, enhanced siRNA release from endosomes, and effectively suppressed vascular endothelial growth factor expression in rat retinal pigment epithelium cells. The 2.0 mol% STR-CH2R4H2C-modified cationic nanocarrier enhanced intraocular migration into the retina after instillation into rat eyes.

Establishing a simple perfusion cell culture system for light-activated liposomes.

The off-target effects of light-activated or targeted liposomes are difficult to distinguish in traditional well plate experiments. Additionally, the absence of fluid flow in traditional cell models can lead to overestimation of nanoparticle uptake. In this paper, we established a perfusion cell culture platform to study light-activated liposomes and determined the effect of flow on the liposomal cell uptake. The optimal cell culturing parameters for the A549 cells under flow conditions were determined by monitoring cell viability. To determine optimal liposome treatment times, particle uptake was measured with flow cytometry. The suitability of commercial QuasiVivo flow-chambers for near-infrared light activation was assessed with a calcein release study. The chamber material did not hinder the light activation and subsequent calcein release from the liposomes. Furthermore, our results show that the standard cell culturing techniques are not directly translatable to flow cultures. For non-coated liposomes, the uptake was hindered by flow. Interestingly, hyaluronic acid coating diminished the uptake differences between the flow and static conditions. The study demonstrates that flow affects the liposomal uptake by lung cancer cell line A549. The flow also complicates the cell attachment of A549 cells. Moreover, we show that the QuasiVivo platform is suitable for light-activation studies.

Electroporation-Based ex Vivo Gene Delivery into Dendritic Cells by Anionic Polymer-Coated Versatile Nuclear Localization Signal/pDNA Complex.

In this study, we focused on a nuclear localization signal (NLS)-based versatile peptide vector, designed by us, combined with electroporation (EP) to establish an efficient gene delivery system to non-dividing or slow growing dendritic cells. We determined the intranuclear transport, gene expression, and cell viability in JAWS II mouse dendritic cells transfected with the green fluorescent protein (GFP) expression plasmid DNA alone (naked pEGFP); positive charged complex of NLS derivative STR-CH2SV40H2C, and pEGFP (binary complex); or negative charged complex of the binary complex with a biocompatible polyanion, γ-polyglutamic acid (ternary complex) combined with or without EP application. Although the binary complex showed higher nuclear transport and GFP expression in the absence of EP than those for naked pEGFP, the combination of EP significantly decreased the cell viability and did not improve the efficiency of compared gene expression. However, in the ternary complex, the intranuclear transport and GFP expression efficiency were significantly higher than those of naked pEGFP and the binary complex when combined with EP, and there was no decrease in cell viability. The results suggest that polyanion-coated ternary complex with EP is useful for non-viral gene delivery system into non-dividing cells for ex vivo gene/cell therapy.

Development of a Liquid Crystal Formulation that Can Penetrate the Stratum Corneum for Intradermal Delivery of Small Interfering RNA.

Topical delivery of small interfering RNA (siRNA) can be an attractive method for the treatment of skin diseases and improving the quality of life of patients. However, it is difficult for siRNA to pass through the two major barriers of the skin: the stratum corneum (SC) and tight junctions. We have previously reported that atopic dermatitis of skin without the SC can be efficiently treated by the intradermal administration of trans-activator of transcription (Tat) peptide and AT1002 (tight junction opening peptide). However, novel drug delivery systems are needed for effective SC penetration. Therefore, in the present study, we aimed to develop a lyotropic liquid crystalline (LC) system containing Tat and AT1002 for effective siRNA penetration through the SC. An LC formulation was prepared using selachyl alcohol and purified water, and its skin penetration ability was evaluated. No fluorescence was observed in mouse skin treated with a siRNA solution, as there was no intradermal localization of siRNA from naked siRNA. However, intradermal delivery of siRNA was remarkable and extensive with the LC formulation containing both Tat and AT1002. Semiquantitative analysis by brightness measurement revealed that the LC formulation containing both Tat and AT1002 had significantly enhanced intact skin permeability than other formulations. These results show that the functional peptides in the LC formulation increased SC penetration and intradermal delivery in the healthy skin. Therefore, this novel LC system may be useful in the treatment of various skin diseases.

In Vivo Fluorescence Imaging of Passive Inflammation Site Accumulation of Liposomes via Intravenous Administration Focused on Their Surface Charge and PEG Modification.

Nanocarriers such as liposomes have been attracting attention as novel therapeutic methods for inflammatory autoimmune diseases such as rheumatoid arthritis and ulcerative colitis. The physicochemical properties of intravenously administered nanomedicines enable them to target inflamed tissues passively. However, few studies have attempted to determine the influences of nanoparticle surface characteristics on inflammation site accumulation. Here, we aimed to study the effects of polyethylene glycol (PEG) modification and surface charge on liposome ability to accumulate in inflammatory sites and be uptake by macrophages. Four different liposome samples with different PEG modification and surface charge were prepared. Liposome accumulation in the inflammation sites of arthritis and ulcerative colitis model mice was evaluated by using in vivo imaging. There was greater PEG-modified than unmodified liposome accumulation at all inflammation sites. There was greater anionic than cationic liposome accumulation at all inflammation sites. The order in which inflammation site accumulation was confirmed was PEG-anionic > PEG-cationic > anionic > cationic. PEG-anionic liposomes had ~2.5× higher fluorescence intensity than PEG-cationic liposomes, and the PEG-liposomes had ~2× higher fluorescence intensity than non-PEG liposomes. All liposomes have not accumulated at the inflammation sites in healthy mice. Furthermore, cationic liposomes were taken up to ~10× greater extent by RAW264.7 murine macrophages. Thus, PEG-cationic liposomes that have the ability to accumulate in inflammatory sites via intravenous administration and to be taken up by macrophages could be useful.

Anti-Metastatic Effects on Melanoma via Intravenous Administration of Anti-NF-κB siRNA Complexed with Functional Peptide-Modified Nano-Micelles.

Controlling metastasis is an important strategy in cancer treatment. Nanotechnology and nucleic acids with novel modalities are promising regulators of cancer metastasis. We aimed to develop a small interfering RNA (siRNA) systemic delivery and anti-metastasis system using nanotechnology. We previously reported that polyethylene glycol-polycaprolactone (PEG-PCL) and functional peptide CH2R4H2C nano-micelle (MPEG-PCL-CH2R4H2C) has high siRNA silencing effects, indicated by increased drug accumulation in tumor-bearing mice, and has an anti-tumor effect on solid tumors upon systemic injection. In this study, we aimed to apply our micelles to inhibit metastasis and evaluated the inhibitory effect of anti-RelA siRNA (siRelA), which is a subunit of NF-κB conjugated with MPEG-PCL-CH2R4H2C, via systemic administration. We report that siRelA/MPEG-PCL-CH2R4H2C had a high cellular uptake and suppressed the migration/invasion of cells in B16F10 cells without toxicity. In addition, in a lung metastasis mouse model using intravenous administration of B16F10 cells treated with siRelA/MPEG-PCL-CH2R4H2C, the number of lung nodules in lung tissue significantly decreased compared to naked siRelA and siControl/MPEG-PCL-CH2R4H2C micelle treatments. Hence, we show that RelA expression can reduce cancer metastasis, and MPEG-PCL-CH2R4H2C is an effective siRNA carrier for anti-metastasis cancer therapies.

Correction: Therapeutic Effects in a Transient Middle Cerebral Artery Occlusion Rat Model by Nose-To-Brain Delivery of Anti-TNF-Alpha siRNA with Cell-Penetrating Peptide-Modified Polymer Micelles. Pharmaceutics, 2019, 11(9), 478.

The appropriate information for the reprint used with permission from [...].

Therapeutic Effects in a Transient Middle Cerebral Artery Occlusion Rat Model by Nose-To-Brain Delivery of Anti-TNF-Alpha siRNA with Cell-Penetrating Peptide-Modified Polymer Micelles.

We previously reported that siRNA delivery to the brain is improved by the nose-to-brain delivery route and by conjugation with polyethylene glycol-polycaprolactone (PEG-PCL) polymer micelles and the cell-penetrating peptide, Tat (PEG-PCL-Tat). In this study, we evaluated the nose-to-brain delivery of siRNA targeting TNF-α (siTNF-α) conjugated with PEG-PCL-Tat to investigate its therapeutic effects on a transient middle cerebral artery occlusion (t-MCAO) rat model of cerebral ischemia-reperfusion injury. Intranasal treatment was provided 30 min after infarction induced via suturing. Two hours after infarction induction, the suture was removed, and blood flow was released. At 22 h post-reperfusion, we assessed the infarcted area, TNF-α production, and neurological score to determine the therapeutic effects. The infarcted area was observed over a wide range in the untreated group, whereas shrinkage of the infarcted area was observed in rats subjected to intranasal administration of siTNF-α with PEG-PCL-Tat micelles. Moreover, TNF-α production and neurological score in rats treated by intranasal administration of siTNF-α with PEG-PCL-Tat micelles were significantly lower than those in untreated and naked siTNF-α-treated rats. These results indicate that nose-to-brain delivery of siTNF-α conjugated with PEG-PCL-Tat micelles alleviated the symptoms of cerebral ischemia-reperfusion injury.

Anti-RelA siRNA-Encapsulated Flexible Liposome with Tight Junction-Opening Peptide as a Non-invasive Topical Therapeutic for Atopic Dermatitis.

Small interfering RNA (siRNA) has been proposed as a novel treatment for atopic dermatitis (AD) because it suppresses sequence-specific mRNA expression. Indeed siRNA-based therapy achieves an almost complete cure with fewer side effects than currently available treatments. However, the tight junctions in the granular layer of the epidermis in the atopic skin are barriers to siRNA delivery. We previously reported the potential clinical utility of AT1002, a peptide that opens tight junctions. In the present study, we evaluated a topical siRNA-based therapy for AD using AT1002 in combination with a flexible liposome. The 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/cholesteryl hemisuccinate (CHEMS) liposome was chosen as a carrier for siRNA because of its highly flexible structure and permeability. We prepared siRNA-encapsulated DOPE/CHEMS liposomes and examined their physical properties, safety, uptake into RAW264.7 cells, and topical application in healthy and AD-affected skin. We then assessed the efficacy of anti-nuclear factor-kappa B (NF-κB) (RelA) siRNA (siRelA)-encapsulated DOPE/CHEMS liposomes with AT1002 in AD model mice. The siRNA-DOPE/CHEMS liposomes were absorbed significantly better than siRNA alone and they enhanced siRNA skin penetration without toxicity. Moreover, siRelA-DOPE/CHEMS liposomes with AT1002 alleviated AD symptoms and reduced the levels of inflammatory cytokines in AD model mice. Therefore, the combination of AT1002 and DOPE/CHEMS liposomes could be a dermally applied RNA interference therapeutic system for effective RNA delivery and AD treatment.

Transdermal anti-nuclear kappaB siRNA therapy for atopic dermatitis using a combination of two kinds of functional oligopeptide.

Nucleic acid-based targeting of nuclear factor kappaB (NF-κB) is gaining attention as a treatment option for skin diseases like atopic dermatitis (AD). Transdermal administration improves patient quality of life because of non-invasive; however, siRNA delivery into the skin can be challenging owing to the barrier of tight junctions in the granular layer. Therefore, we aimed to develop a delivery system of siRNA for topical skin application using functional peptides. We previously reported that combined treatment with a cytoplasm-responsive stearylated-arginine-rich peptide (STR-CH2R4H2C) and a tight junction opening peptide (AT1002) showed high siRNA permeability in the skin of AD-induced and normal mice. Here, we used murine macrophage RAW264.7 cells to examine siRNA permeation and the therapeutic effect of anti-NF-κB (RelA) siRNA (siRelA) complexed with STR-CH2R4H2C and AT1002 for AD-induced mice. We showed that significantly higher siRNA cellular uptake occurs after this treatment as well as decreased TNF-α and IL-6 expression. Additionally, we showed that effective siRNA transdermal delivery occurs with the suppression of the tight junction protein ZO-1. Moreover, topical skin application of siRelA with STR-CH2R4H2C and AT1002 improved AD-like symptoms in model mice. Thus, the combined treatment of STR-CH2R4H2C and AT1002 could serve as an effective transdermal siRNA therapeutic system for AD.

Intra-articular Retention and Anti-arthritic Effects in Collagen-Induced Arthritis Model Mice by Injectable Small Interfering RNA Containing Hydrogel.

Small interfering RNAs (siRNAs) are expected to offer a means of treating rheumatoid arthritis (RA) because they allow the specific silencing of genes related to RA pathogenesis. In our previous study, we reported that the siRNA targeted against RelA (anti-RelA siRNA), an important nuclear factor-kappaB (NF-κB) subdomain, was an effective therapeutic in atopic dermatitis and RA model animals. In this study, to develop an intra-articular injectable gel formulation against RA, we prepared a hydrogel that contains anti-RelA siRNA, and determined the in vitro release profile (%) and in vivo intra-articular retention of fluorescence-labeled model siRNA, and the anti-arthritic effects of the anti-RelA siRelA containing hydrogel in RA model mice. We selected the silk protein, sericin (SC), as an aqueous gel base, as it is a biocompatible and useful for forming hydrogels without a cross-linker. We showed that fluorescence-labeled model siRNA was continuously released from SC hydrogel in vitro, and retained in the knee joint of rats after injection of siRNA hydrogel. In addition, the knee joint thickness, clinical severity and incidence (%) in collagen-induced arthritis (CIA) mice as RA model treated with anti-RelA siRNA containing hydrogel were more improved than untreated, anti-RelA siRNA solution and negative control siRNA containing hydrogel group. Therefore, the intra-articular injectable sericin hydrogel formulation containing of anti-RelA siRNA could be a great potential therapeutic in rheumatoid arthritis.

Enhancement of nose-to-brain delivery of hydrophilic macromolecules with stearate- or polyethylene glycol-modified arginine-rich peptide.

Recently, nose-to-brain delivery is a highly versatile route, which, in combination with novel drugs being developed for treating intractable CNS diseases, is a promising approach for the treatment of disorders. Furthermore, nano-sized drug carriers may improve nose-to-brain drug delivery by their capability to increase the transmucosal penetration of the drugs across nasal mucosal tissue barrier. However, there is still not enough information regarding mechanism of absorption pathway from nasal cavity to brain using nanocarriers. In this study, to investigate the nose-to-brain transport pathway using nanocarriers, the distribution in whole brain, nasal mucosa, and trigeminal nerve after intranasal administration of two kinds of nanocarriers which have hydrophobic or hydrophilic moiety. We used CHHRRRRHHC peptide (CH2R4H2C) as basic peptide carriers, and modified with stearic acid (STR) as a hydrophobic moiety (STR-CH2R4H2C) or polyethylene glycol (PEG)-based block copolymer (PEG-PCL) as hydrophilic moiety (PEG-PCL-CH2R4H2C). The nose-to-brain drug delivery can be improved by using STR-CH2R4H2C and PEG-PCL-CH2R4H2C as carriers. Specifically, hydrophobic STR-CH2R4H2C is more suitable for the transport of drugs targeting the forebrain, while PEG-PCL-modified CH2R4H2C is more suitable for transporting drugs targeting the hindbrain or whole brain tissue. In conclusion, the results of this study support the possibility that drug delivery pathways can be controlled depending on the properties of different carrier complexes.

Systemic delivery of small interfering RNA targeting nuclear factor κB in mice with collagen-induced arthritis using arginine-histidine-cysteine based oligopeptide-modified polymer nanomicelles.

This study aimed to build an innovative system to deliver a systemic small interfering RNA (siRNA) treatment for rheumatoid arthritis. We combined arginine-histidine-cysteine based oligopeptide-modified polymer micelles with siRNA targeting the nuclear factor κB subunit, RelA (siRelA). This is a key molecule in the control of inflammation. We tested the cellular uptake of siRNA and its effects on inflammatory cytokine levels in vitro using synoviocytes, and siRNA distribution and therapeutic effects in vivo in mice with collagen-induced arthritis (CIA). These studies showed that arginine-histidine based oligopeptide modified micelles produced effective cellular siRNA uptake and suppressed inflammatory cytokine levels in synoviocytes. In vivo, these micelles produced marked accumulation of siRNAs in arthritic paws in CIA mice, with much less accumulation in healthy mice. The siRelA-polymer micelle complexes also produced more effective suppression of RelA mRNA expression and inflammatory cytokine levels in the arthritic paws of CIA mice and reduced their clinical symptom scores and paw thickness.

Development of an Innovative Intradermal siRNA Delivery System Using a Combination of a Functional Stearylated Cytoplasm-Responsive Peptide and a Tight Junction-Opening Peptide.

As a new category of therapeutics for skin diseases including atopic dermatitis (AD), nucleic acids are gaining importance in the clinical setting. Intradermal administration is noninvasive and improves patients' quality of life. However, intradermal small interfering RNA (siRNA) delivery is difficult because of two barriers encountered in the skin: intercellular lipids in the stratum corneum and tight junctions in the stratum granulosum. Tight junctions are the major barrier in AD; therefore, we focused on functional peptides to devise an intradermal siRNA delivery system for topical skin application. In this study, we examined intradermal siRNA permeability in the tape-stripped (20 times) back skin of mice or AD-like skin of auricles treated with 6-carboxyfluorescein-aminohexyl phosphoramidite (FAM)-labeled siRNA, the tight junction modulator AT1002, and the functional cytoplasm-responsive stearylated peptide STR-CH2R4H2C by using confocal laser microscopy. We found that strong fluorescence was observed deep and wide in the epidermis and dermis of back skin and AD-like ears after siRNA with STR-CH2R4H2C and AT1002 treatment. After 10 h from administration, brightness of FAM-siRNA was significantly higher for STR-CH2R4H2C + AT1002, compared to other groups. In addition, we confirmed the nontoxicity of STR-CH2R4H2C as a siRNA carrier using PAM212 cells. Thus, our results demonstrate the applicability of the combination of STR-CH2R4H2C and AT1002 for effective intradermal siRNA delivery.

Versatile nuclear localization signal-based oligopeptide as a gene vector.

To develop a versatile nuclear-targeted gene vector, nuclear localization signal (NLS) oligopeptides combining cysteine (C), histidine (H), and stearic acid (STR) were investigated in this study. The original SV40 sequence (SV40: Pro-Lys-Lys-Lys-Arg-Lys-Val) was selected as the NLS sequence, and physical characterizations of various NLS-based oligopeptides (CSV40C, STR-CSV40C, and STR-CH2SV40H2C), including mean diameter, zeta-potential, complex condensation, and decondensation, were evaluated. In addition, cellular and nuclear uptake of plasmid DNA (pDNA) and gene expression in COS7 and dendritic cells (JAWS II) were determined. As a result, C and STR enhanced formation of a smaller and more stable nano-complex with pDNA based on ionic interactions, the disulfide linkage and hydrophobic interactions. STR-CSV40C and STR-CH2SV40H2C had significantly higher cellular uptake ability and transfection efficiency than SV40 and CSV40C. In particular, STR-CH2SV40H2C had higher nuclear uptake and gene expression efficiency than STR-CSV40C. Furthermore, STR-CH2SV40H2C could deliver pDNA to the nuclei and had high gene expression efficiency in dendritic cells. Our results indicate that STR-CH2SV40H2C is a promising gene delivery system in non- or slow-dividing cells.

Prolongation of life in rats with malignant glioma by intranasal siRNA/drug codelivery to the brain with cell-penetrating peptide-modified micelles.

New therapeutic strategies are required to develop candidate drugs and ensure efficient delivery of these drugs to the brain and the central nervous system (CNS). Small interfering RNA (siRNA)-based therapies have been investigated as potential novel approaches for the treatment of brain disorders. Previously, we showed that Tat, a cell-penetrating peptide derived from HIV-Tat, and the modified block copolymers (MPEG-PCL-Tat) can form stable complexes with siRNA or can be loaded with an anticancer drug and efficiently deliver the drugs to the brain tissue via intranasal delivery. In this study, to develop a novel, efficient, and safe therapeutic strategy for managing brain disorders, we used MPEG-PCL-Tat micelles with a nose-to-brain delivery system to investigate its therapeutic effects on a rat model of malignant glioma using siRNA with a Raf-1 (siRaf-1)/camptothecin (CPT) codelivery system. MPEG-PCL-Tat and CPT-loaded MPEG-PCL-Tat can form a stable complex with siRNA with a particle size from 60 to 200 nm and a positive charge at N/P ratios up to 5. Additionally, MPEG-PCL-Tat/siRaf-1 and CPT-loaded MPEG-PCL-Tat/siRaf-1 have fostered cell death in rat glioma cells after the high cellular uptake of siRaf-1/drug by the MPEG-PCL-Tat carrier. Furthermore, compared to the unloaded MPEG-PCL-Tat/siRaf-1 complex, a CPT-loaded MPEG-PCL-Tat/siRaf-1 complex achieved the high therapeutic effect because of the additive effects of CPT and siRaf-1. These results indicate that drug/siRNA codelivery using MPEG-PCL-Tat nanomicelles with nose-to-brain delivery is an excellent therapeutic approach for brain and CNS diseases.

Cytoplasm-responsive nanocarriers conjugated with a functional cell-penetrating peptide for systemic siRNA delivery.

To develop a gene carrier for cancer therapy by systemic injection, we synthesized methoxypolyethylene glycol-polycaprolactone (MPEG-PCL) diblock copolymers conjugated with a cytoplasm-responsive cell-penetrating peptide (CPP), CH2R4H2C (C, Cys; H, His; R, Arg). The carrier/small interfering RNA (siRNA) complexes (N/P ratio of 20) had a particle size of approximately 50 nm and stabilized the siRNA against RNase. The cellular uptake ability of the carrier/FAM-siRNA complexes with fetal bovine serum was significantly higher than that of naked FAM-siRNA. In addition, the carrier/anti-vascular endothelial growth factor siRNA (siVEGF) complexes attained a significantly greater silencing effect than naked siVEGF with low cytotoxicity, resulting from higher uptake, early endosomal escape, and efficient release from the complexes in the cytoplasm. Furthermore, intravenous injection of MPEG-PCL-CH2R4H2C/siVEGF complexes had a significantly higher anti-tumor effect in S-180 tumor-bearing mice, which could be attributed to the rigid compaction of siRNA by ionic interactions and disulfide linkages in the CPP polymer micelles in the blood, as well as higher release following cleavage of the disulfide bonds in the reductive cytosol. Taken together, our data demonstrated that these cytoplasm-responsive polymer micelles conjugated with multi-functional CPP, could facilitate siVEGF delivery to tumor tissues after systemic injection and could exert an extremely strong anti-tumor effect.

Needle-free intravaginal DNA vaccination using a stearoyl oligopeptide carrier promotes local gene expression and immune responses.

The vaginal mucosa is the most common site of infection for viruses that are transmitted through heterosexual intercourse, including human immunodeficiency virus and papillomavirus. Thus, in order to prevent or respond to these infections, strong vaginal immunity is required as the first line of defense. We previously investigated the use of a needle-free injector as a mucosal vaccination tool in rabbits and demonstrated that this is a promising method for stimulating vaginal gene expression and immune responses. In order to improve gene expression, we have examined local vaginal gene transfection efficiency using a non-needle jet injector combined with an effective peptide carrier in rabbits. The carrier used was a stearoyl (STR) peptide with Cys (C), Arg (R) and His (H) residues that form disulfide cross linkages via Cys (STR-CH2R4H2C) which was developed in our previous study. As a result, vaginal gene expression using the needle-free injector combined with STR-CH2R4H2C carrier was significantly improved compared to that without STR-CH2R4H2C carrier. Moreover, intravaginal pDNA vaccination by the needle-free injector combined with STR-CH2R4H2C carrier and CpG-ODN promoted not only local vaginal IgA and IgG, but also serum IgG secretion, to a degree significantly higher than that of naked pDNA.

[Non-invasive ophthalmic liposomes for nucleic acid delivery to posterior segment of eye].

Nucleic acids like siRNA and pDNA are remarkable for treatment of ophthalmic diseases in posterior segment of eye such as age-related macular degeneration (AMD). However, hydrophilic and high molecule compounds are restricted in intraocular distribution through anterior segment of the eye. In addition, the ocular tissue has a blood-retinal barrier which restricts drug delivery thorough systemic administration. Therefore the invasive intravitreal injection has been generally applied for treatment of retinal diseases. The objective in this study is to prepare nucleic acid-loaded liposomes for effective gene delivery to posterior segment of eye by non-invasive ophthalmic administration such as eye-drops. The pDNA/PEI-complex loaded liposomes were prepared using detergent removal method. The obtained liposomes were lyophilized with optimal amount of a cryoprotectant to avoid changes in physical properties and, followed by adjustment of an appropriate volume and osmotic pressure as ophthalmic solution. The liposomes show high pDNA encapsulation efficiency and good cellular uptake ability in human retinal pigment epithelial cells (ARPE-19 cells). We further demonstrate that the modification of ligand which binds to specific receptor on the RPE cells to the liposomes may improve gene delivery efficacy to the posterior segment of eye by non-invasive ocular instillation.