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.

Nose-to-brain/spinal cord delivery kinetics of liposomes with different surface properties.

The administration of liposomes via nose-to-brain delivery is expected to become a strategy for efficient drug delivery to the central nervous system. Efficient nose-to-brain delivery and the kinetics of drugs administered in this manner depend on the properties of liposomes. However, there is a lack of basic knowledge of which liposomes are suitable for this purpose. Here, a qualitative study of intranasally administered liposomes (positively charged, neutral, and negatively charged, with or without polyethylene glycol [PEG] modification; particle size <100 nm) was performed to elucidate their dynamics in the brain and spinal cord. Additionally, a quantitative investigation was performed to ascertain their distribution in each part of the brain and spinal cord. The effects of liposome surface charge and PEG modification on the kinetics and distribution post intranasal administration were investigated via two experiments. Qualitative evaluation was performed via ex vivo observation after intranasal administration of fluorescently labeled liposomes. Neutral PEG-modified liposomes were distributed throughout the brain and spinal cord 60 min after administration, and the fluorescence intensity increased with time. By contrast, non-PEG-modified neutral liposomes showed particularly strong fluorescence in the olfactory bulb, and the fluorescence was localized in the anterior part of the brain. Positively charged liposomes showed low fluorescence around the lateral part of the brain and lumbar spinal cord 60 min after administration. Low fluorescence was observed in the whole brain and spinal cord, with strong fluorescence being observed in the olfactory bulb after 120 min of administration. Negatively charged liposomes showed no fluorescence at 60 min after administration, but low fluorescence was observed throughout the brain and spinal cord 120 min after administration. We quantified the radioactivity in the brain and spinal cord after intranasal administration of radioisotope-labeled liposomes. Neutral liposomes showed the highest distribution by area under the drug concentration-time curve (AUC60-120) in the brain and spinal cord compared to other liposomes. Compared with negatively charged liposomes, positively charged liposomes had a higher distribution in the olfactory bulb and forebrain, while negatively charged liposomes had a higher distribution in the hindbrain and bulbospinal tract cord. In addition, the distribution of PEG-modified neutral liposomes in the brain and spinal cord was significantly enhanced compared to that of non-PEG-modified neutral liposomes after 90 min of intranasal administration. These results indicate that surface charge and PEG modification strongly affect the efficiency of nose-to-brain delivery kinetics, and that PEG-modified neutral liposomes are excellent carriers for drug delivery to a wide area of the brain and spinal cord.

Systemic delivery of siRNA to the colon using peptide modified PEG-PCL polymer micelles for the treatment of ulcerative colitis.

Ulcerative colitis (UC) is a refractory inflammatory bowel disease that causes inflammation and ulcers in the digestive tract, and significantly reduces the patient's quality of life. While existing UC treatments have many challenges, nanotechnology, and small interfering RNA (siRNA) based formulations are novel and promising for UC treatment. We previously reported that intravenous administration of MPEG-PCL-CH2R4H2C nanomicelles had high inflammatory site accumulation and remarkable therapeutic effects on rheumatoid arthritis by a phenomenon similar to enhanced permeability and retention effect. In this study, we investigated the effects of siRNA delivered using MPEG-PCL-CH2R4H2C nanomicelles through intravenous administration to the inflammation site of dextran sulfate sodium-induced colitis mice. The MPEG-PCL-CH2R4H2C micelles had optimum physical properties and high siRNA compaction ability. Moreover, model-siRNA delivered through MPEG-PCL-CH2R4H2C showed higher accumulation in the inflammatory site than that of the naked siRNA. Furthermore, intravenous administration of MPEG-PCL-CH2R4H2C/siRelA micelles, targeting siRelA, a subunit of NF-κB, significantly decreased the shortening of large intestine, clinical score, and production of inflammatory cytokines compared the 5-ASA and naked siRelA. These results suggest that MPEG-PCL-CH2R4H2C is a useful carrier for the systemic delivery and accumulation of siRNA, thus improving its therapeutic effect.

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.

Topical Anti-Nuclear Factor-Kappa B Small Interfering RNA with Functional Peptides Containing Sericin-Based Hydrogel for Atopic Dermatitis.

The small interfering RNA (siRNA) is suggested to offer a novel means of treating atopic dermatitis (AD) because it allows the specific silencing of genes related to AD pathogenesis. In our previous study, we found that siRNA targeted against RelA, an important nuclear factor-kappa B (NF-κB) subdomain, with functional peptides, showed therapeutic effects in a mouse model of AD. In the present study, to develop a topical skin application against AD, we prepared a hydrogel containing anti-RelA siRNA and functional peptides and determined the intradermal permeation and the anti-AD effects in an AD mouse model. We selected the silk protein, sericin (SC), which is a versatile biocompatible biomaterial to prepare hydrogel as an aqueous gel base. We found that the siRNA was more widely delivered to the site of application in AD-induced ear skin of mice after topical application via the hydrogel containing functional peptides than via the preparation without functional peptides. In addition, the ear thickness and clinical skin severity of the AD-induced mice treated with hydrogel containing anti-RelA siRNA with functional peptides improved more than that of mice treated with the preparation formulated with negative siRNA.