Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs.

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.

NFC-Based Wearable Optoelectronics Working with Smartphone Application for Untact Healthcare.

With growing interest in healthcare, wearable healthcare devices have been developed and researched. In particular, near-field communication (NFC) based wearable devices have been actively studied for device miniaturization. Herein, this article proposes a low-cost and convenient healthcare system, which can monitor heart rate and temperature using a wireless/battery-free sensor and the customized smartphone application. The authors designed and fabricated a customized healthcare device based on the NFC system, and developed a smartphone application for real-time data acquisition and processing. In order to achieve compact size without performance degradation, a dual-layered layout is applied to the device. The authors demonstrate that the device can operate as attached on various body sites such as wrist, fingertip, temple, and neck due to outstanding flexibility of device and adhesive strength between the device and the skin. In addition, the data processing flow and processing result are presented for offering heart rate and skin temperature. Therefore, this work provides an affordable and practical pathway for the popularization of wireless wearable healthcare system. Moreover, the proposed platform can easily delivery the measured health information to experts for contactless/personal health consultation.

Surface-Modification of RIPL Peptide-Conjugated Liposomes to Achieve Steric Stabilization and pH Sensitivity.

We have previously demonstrated that RIPL peptide-conjugated liposomes (RIPL-L) exhibited high hepsin (HPN) selectivity and enhanced intracellular drug delivery. In this study, surface modification of RIPL-L was performed to reduce plasma protein adsorption and off-target effects. For steric stabilization, distearoyl phosphatidylethanolamine (DSPE)-polyethylene glycol (PEG)2000 was used (5% molar ratio to total lipid) to prepare PEG-RIPL-L. Further, pH-sensitive oligopeptides [(HD)4 or (HE)4] were coupled to shield the RIPL polyarginine moiety, yielding (HD)4/PEG-RIPL-L and (HE)4/PEG-RIPL-L. All liposomal vesicles had a narrow and homogenous size distribution of approximately 140­150 nm, with zeta potentials varying from −15 to 36 mV. Increased plasma stability was observed upon quantifying the protein adsorbed onto liposomes by using a micro bicinchoninic acid assay. The (HD)4- and (HE)4-coupling capacity of PEG-RIPL-L was investigated by measuring the amount of oligopeptide involved in transient ionic complexation (TIC-oligopep) and zeta potential changes. As the molar ratio of (HD)4 and (HE)4 increased, TIC-oligopep increased and zeta potential decreased. (HE)4/PEG-RIPL-L were pH-sensitive, producing 1.6-fold greater cellular uptake of FITC-dextran by LNCaP cells at pH 6.8 than at pH 7.4. This result suggested that (HE)4/PEG-RIPL-L might provide a sterically stabilized, pH-sensitive drug carrier for HPN-specific cancer targeting.

Design of Multifunctional Liposomal Nanocarriers for Folate Receptor-Specific Intracellular Drug Delivery.

As a novel carrier for folate receptor (FR)-targeted intracellular delivery, we designed two types of targetable liposomal systems using Pep-1 peptide (Pep1) and folic acid as a cell-penetrating peptide (CPP) and target molecule, respectively. Folate-linked Pep1 (Fol-Pep1) was synthesized by solid phase peptide synthesis (SPPS) and verified using (1)H NMR and far-ultraviolet (UV) circular dichroism (CD). The chimeric ligand (Fol-Pep1)-modified liposome (cF-P-L) was prepared by coupling Fol-Pep1 to maleimide-derivatized liposomes at various ratios. The dual ligand (folate and Pep1)-modified liposome (dF/P-L) was prepared by separately attaching both ligands to the liposomal surface via a short (PEG2000) or long (PEG3400) linker. The physical and conformational characteristics including vesicle size, zeta potential, and the number of conjugated ligands were determined. Intracellular uptake specificities of various fluorescent probe-containing cF-P-L and dF/P-L systems were assessed using FR-positive HeLa and FR-negative HaCaT cells. Cellular uptake behavior was visualized by confocal laser scanning microscopy (CLSM). Internalization was time-dependent. Fol-Pep1 and Pep-1 cytotoxicities were negligible up to 25 µM in FR-positive and FR-negative cells. Empty cF-P-L and dF/P-L were nontoxic at the concentration used. The optimized dF3/P2(450/90) system carrying 450 PEG3400-linked folate and 90 PEG2000-linked Pep1 molecules could be a good candidate for FR-specific intracellular drug delivery.

Engineering self-deliverable ribonucleoproteins for genome editing in the brain.

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineer self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identifies potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins establishes a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibits substantially improved editing efficacy compared to other constructs. We find that self-deliverable Cas9 RNPs generate robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo.

In vivo human T cell engineering with enveloped delivery vehicles.

Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR-Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody-antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility.

Central corneal thickness does not correlate with TonoLab-measured IOP in several mouse strains with single transgenic mutations of matricellular proteins.

Accurate and reliable measurement of intraocular pressure (IOP) is crucial in the study of glaucoma using the mouse model. The purpose of this study was to determine the relationship between TonoLab-measured IOP and central corneal thickness (CCT) in mouse strains with single gene mutations of matricellular proteins. Wild-type (WT) and transgenic mouse strains with single gene mutations (KO) of thrombospondin-1 (TSP-1), thrombospondin-2 (TSP-2), osteopontin (OPN), hevin, and secreted protein acidic rich in cysteine (SPARC) were imaged at six weeks using optical coherence tomography (Stratus, Zeiss) to determine CCT. IOP was measured between 11am and 3pm using TonoLab, one week later. For all measurements, mice were anesthetized using intraperitoneal injection ketamine:xylazine. CCT and IOP were measured in 583 mice (TSP-1 n = 71 and 41, TSP-2 n = 60 and 32, OPN n = 81 and 50, hevin n = 59 and 76, SPARC n = 54 and 59, WT and KO, respectively). Mean CCT was 5-6% lower in three KO strains-TSP-1, OPN, and SPARC-compared to their corresponding WT (p = 1.55 × 10(-7), 1.63 × 10(-11), and 1.91 × 10(-7), respectively). The mean IOP was 8.3%, 6.6%, and 15.1% lower in three KO strains-TSP-1, TSP-2, and SPARC-compared to corresponding WT (p = 2.11 × 10(-5), 2.93 × 10(-3), and 3.76 × 10(-9), respectively. Linear regression of IOP versus CCT yielded no statistically significant within-strain correlations for TSP-1 (p = 0.12 and 0.073), TSP-2 (p = 0.473 and 0.92), OPN (p = 0.212 and 0.916), Hevin (p = 0.746 and 0.257), and SPARC (p = 0.080 and 0.056), reported as p-values considering a null hypothesis of zero slope (WT and KO, respectively). Neither C57-derived strains (TSP-1 and OPN) nor 129-derived strains (TSP-2, hevin, SPARC) demonstrated a correlation between mean IOP and mean CCT across different strains (p = 0.75 and p = 0.53, respectively). Taken together, these results indicate that CCT is not required to interpret TonoLab IOP readings in the mice when CCT varies 10% about the mean. This does not exclude the possibility of an IOP-CCT correlation for CCT values outside this range or for inter-strain comparisons where the mean CCT differs more than 10%.

Engineering self-deliverable ribonucleoproteins for genome editing in the brain.

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects 1 . However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineered self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identified potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins identified a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibited substantially improved editing efficacy compared to other constructs. We found that self-deliverable Cas9 RNPs generated robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo .

Effect of SPARC Suppression in Mice, Perfused Human Anterior Segments, and Trabecular Meshwork Cells.

Purpose: Secreted protein, acidic and rich in cysteine (SPARC) elevates intraocular pressure (IOP), increases certain structural extracellular matrix (ECM) proteins in the juxtacanalicular trabecular meshwork (JCT), and decreases matrix metalloproteinase (MMP) protein levels in trabecular meshwork (TM) endothelial cells. We investigated SPARC as a potential target for lowering IOP. We hypothesized that suppressing SPARC will decrease IOP, decrease structural JCT ECM proteins, and alter the levels of MMPs and/or their inhibitors. Methods: A lentivirus containing short hairpin RNA of human SPARC suppressed SPARC in mouse eyes and perfused cadaveric human anterior segments with subsequent IOP measurements. Immunohistochemistry determined structural correlates. Human TM cell cultures were treated with SPARC suppressing lentivirus. Quantitative reverse transcriptase polymerase chain reaction (PCR), immunoblotting, and zymography determined total RNA, relative protein levels, and MMP enzymatic activity, respectively. Results: Suppressing SPARC decreased IOP in mouse eyes and perfused human anterior segments by approximately 20%. Histologically, this correlated to a decrease in collagen I, IV, and VI in both the mouse TM and human JCT regions; in the mouse, fibronectin was also decreased but not in the human. In TM cells, collagen I and IV, fibronectin, MMP-2, and tissue inhibitor of MMP-1 were decreased. Messenger RNA of the aforementioned genes was not changed. Plasminogen activator inhibitor 1 (PAI-1) was upregulated in vitro by quantitative PCR and immunoblotting. MMP-1 activity was reduced in vitro by zymography. Conclusions: Suppressing SPARC decreased IOP in mice and perfused cadaveric human anterior segments corresponding to qualitative structural changes in the JCT ECM, which do not appear to be the result of transcription regulation.

TLR3 signaling is either protective or pathogenic for the development of Theiler's virus-induced demyelinating disease depending on the time of viral infection.

BACKGROUND: We have previously shown that toll-like receptor 3 (TLR3)-mediated signaling plays an important role in the induction of innate cytokine responses to Theiler's murine encephalomyelitis virus (TMEV) infection. In addition, cytokine levels produced after TMEV infection are significantly higher in the glial cells of susceptible SJL mice compared to those of resistant C57BL/6 mice. However, it is not known whether TLR3-mediated signaling plays a protective or pathogenic role in the development of demyelinating disease. METHODS: SJL/J and B6;129S-Tlr3tm1Flv/J (TLR3KO-B6) mice, and TLR3KO-SJL mice that TLR3KO-B6 mice were backcrossed to SJL/J mice for 6 generations were infected with Theiler's murine encephalomyelitis virus (2 × 105 PFU) with or without treatment with 50 µg of poly IC. Cytokine production and immune responses in the CNS and periphery of infected mice were analyzed. RESULTS: We investigated the role of TLR3-mediated signaling in the protection and pathogenesis of TMEV-induced demyelinating disease. TLR3KO-B6 mice did not develop demyelinating disease although they displayed elevated viral loads in the CNS. However, TLR3KO-SJL mice displayed increased viral loads and cellular infiltration in the CNS, accompanied by exacerbated development of demyelinating disease, compared to the normal littermate mice. Late, but not early, anti-viral CD4+ and CD8+ T cell responses in the CNS were compromised in TLR3KO-SJL mice. However, activation of TLR3 with poly IC prior to viral infection also exacerbated disease development, whereas such activation after viral infection restrained disease development. Activation of TLR3 signaling prior to viral infection hindered the induction of protective IFN-γ-producing CD4+ and CD8+ T cell populations. In contrast, activation of these signals after viral infection improved the induction of IFN-γ-producing CD4+ and CD8+ T cells. In addition, poly IC-pretreated mice displayed elevated PDL-1 and regulatory FoxP3+ CD4+ T cells in the CNS, while poly IC-post-treated mice expressed reduced levels of PDL-1 and FoxP3+ CD4+ T cells. CONCLUSIONS: These results suggest that TLR3-mediated signaling during viral infection protects against demyelinating disease by reducing the viral load and modulating immune responses. In contrast, premature activation of TLR3 signal transduction prior to viral infection leads to pathogenesis via over-activation of the pathogenic immune response.

Pep-1 Peptide-modified liposomal carriers for intracellular delivery of gold nanoparticles.

A Pep-1 peptide-modified liposomal (Pep1-Lipo) carrier system was investigated to increase the intracellular delivery of gold nanoparticles (Au NPs). Au NPs with a mean diameter of 13 nm were successfully encapsulated into the inner aqueous compartment of the novel carrier using an ethanol injection technique, reserving the distinctive optical characteristics of the surface plasmon resonance peak around 530 nm. The Au NP-loaded liposomal carrier was physically characterized as 150-170 nm in size and 45 mV in zeta potential. Dark field microscopic observation demonstrated that in vitro cellular association and/or translocation of the nanoprobes into the cells was increased by Pep1-Lipo carriers compared to bare Au NPs. In conclusion, this novel liposomal formulation is a promising platform for the intracellular delivery of metallic nanoprobes including Au NPs.

Outdoor-Useable, Wireless/Battery-Free Patch-Type Tissue Oximeter with Radiative Cooling.

For wearable electronics/optoelectronics, thermal management should be provided for accurate signal acquisition as well as thermal comfort. However, outdoor solar energy gain has restricted the efficiency of some wearable devices like oximeters. Herein, wireless/battery-free and thermally regulated patch-type tissue oximeter (PTO) with radiative cooling structures are presented, which can measure tissue oxygenation under sunlight in reliable manner and will benefit athlete training. To maximize the radiative cooling performance, a nano/microvoids polymer (NMVP) is introduced by combining two perforated polymers to both reduce sunlight absorption and maximize thermal radiation. The optimized NMVP exhibits sub-ambient cooling of 6 °C in daytime under various conditions such as scattered/overcast clouds, high humidity, and clear weather. The NMVP-integrated PTO enables maintaining temperature within ≈1 °C on the skin under sunlight relative to indoor measurement, whereas the normally used, black encapsulated PTO shows over 40 °C owing to solar absorption. The heated PTO exhibits an inaccurate tissue oxygen saturation (StO2) value of ≈67% compared with StO2 in a normal state (i.e., ≈80%). However, the thermally protected PTO presents reliable StO2 of ≈80%. This successful demonstration provides a feasible strategy of thermal management in wearable devices for outdoor applications.

Instant, multiscale dry transfer printing by atomic diffusion control at heterogeneous interfaces.

Transfer printing is a technique that integrates heterogeneous materials by readily retrieving functional elements from a grown substrate and subsequently printing them onto a specific target site. These strategies are broadly exploited to construct heterogeneously integrated electronic devices. A typical wet transfer printing method exhibits limitations related to unwanted displacement and shape distortion of the device due to uncontrollable fluid movement and slow chemical diffusion. In this study, a dry transfer printing technique that allows reliable and instant release of devices by exploiting the thermal expansion mismatch between adjacent materials is demonstrated, and computational studies are conducted to investigate the fundamental mechanisms of the dry transfer printing process. Extensive exemplary demonstrations of multiscale, sequential wet-dry, circuit-level, and biological topography-based transfer printing demonstrate the potential of this technique for many other emerging applications in modern electronics that have not been achieved through conventional wet transfer printing over the past few decades.

Secreted protein acidic and rich in cysteine (SPARC) knockout mice have greater outflow facility.

PURPOSE: Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates intraocular pressure (IOP) by altering extracellular matrix (ECM) homeostasis within the trabecular meshwork (TM). We hypothesized that the lower IOP previously observed in SPARC -/- mice is due to a greater outflow facility. METHODS: Mouse outflow facility (Clive) was determined by multiple flow rate infusion, and episcleral venous pressure (Pe) was estimated by manometry. The animals were then euthanized, eliminating aqueous formation rate (Fin) and Pe. The C value was determined again (Cdead) while Fin was reduced to zero. Additional mice were euthanized for immunohistochemistry to analyze ECM components of the TM. RESULTS: The Clive and Cdead of SPARC -/- mice were 0.014 ± 0.002 µL/min/mmHg and 0.015 ± 0.002 µL/min/mmHg, respectively (p = 0.376, N/S). Compared to the Clive = 0.010 ± 0.002 µL/min/mmHg and Cdead = 0.011 ± 0.002 µL/min/mmHg in the WT mice (p = 0.548, N/S), the Clive and Cdead values for the SPARC -/- mice were higher. Pe values were estimated to be 8.0 ± 0.2 mmHg and 8.3 ± 0.7 mmHg in SPARC -/- and WT mice, respectively (p = 0.304, N/S). Uveoscleral outflow (Fu) was 0.019 ± 0.007 µL/min and 0.022 ± 0.006 µL/min for SPARC -/- and WT mice, respectively (p = 0.561, N/S). Fin was 0.114 ± 0.002 µL/min and 0.120 ± 0.016 µL/min for SPARC -/- and WT mice (p = 0.591, N/S). Immunohistochemistry demonstrated decreases of collagen types IV and VI, fibronectin, laminin, PAI-1, and tenascin-C within the TM of SPARC -/- mice (p < 0.05). CONCLUSIONS: The lower IOP of SPARC -/- mice is due to greater aqueous humor outflow facility through the conventional pathway. Corresponding changes in several matricellular proteins and ECM structural components were noted in the TM of SPARC -/- mice.

Matricellular proteins in the trabecular meshwork.

The trabecular meshwork is one of the primary tissues of interest in the normal regulation and dysregulation of intraocular pressure (IOP) that is a causative risk factor for primary open-angle glaucoma. Matricellular proteins generally function to allow cells to modulate their attachments with and alter the characteristics of their surrounding extracellular matrix (ECM). In non-ocular tissues, matricellular proteins generally increase fibrosis. Since ECM turnover is very important to the outflow facility, matricellular proteins may have a significant role in the regulation of IOP. The formalized study of matricellular proteins in trabecular meshwork is in its infancy. SPARC, thrombospondins-1 and -2, and tenascins-C and -X, and osteopontin have been localized to varying areas within the trabecular meshwork. Preliminary evidence indicates that SPARC and thrombospondin-1 play a role in the regulation of IOP and possibly the pathophysiology of glaucoma. These data show promise that matricellular proteins are involved in IOP dysregulation and are potential therapeutic targets. Further study is needed to clarify these roles.

Replication of Theiler's virus requires NF-kappa B-activation: higher viral replication and spreading in astrocytes from susceptible mice.

To investigate viral replication and cell-cell spreading in astrocytes, recombinant Theiler's murine encephalomyelitis virus (TMEV) expressing green fluorescent protein (GFP) during the replication was generated. GFP and TMEV proteins were processed correctly in infected cells and production of viral proteins could be tracked by fluorescent microscopy. Viral replication of both wild-type TMEV and GFP-TMEV was dependent on the activation of NF-kappaB and partially MAP kinase, based on chemical inhibition studies. Viral replication was significantly reduced in primary astrocytes from NF-kappaB1 (p105)-deficient mice compared with that from wild-type control mice, whereas cytokine production was enhanced. These results suggest an association of canonical NF-kappaB subunits in viral replication, but not cytokine production. Viral replication was also suppressed in both IKKalpha and IKKbeta-deficient mouse embryonic fibroblasts (MEFs), compared with that in wild-type MEF. However, the inhibition was significantly greater in IKKbeta-deficient MEF, suggesting that IKKbeta plays a stronger role in supporting viral replication. Interestingly, viral replication and spreading in primary astrocytes from susceptible SJL/J mice were several-fold higher than those in astrocytes from resistant C57BL/6 mice, suggesting that higher viral replication levels in astrocytes may also contribute to the viral persistence in the central nervous system (CNS) of susceptible SJL/J mice. A relatively higher level of activated NF-kappaB was found in the nuclei of virus-infected SJL astrocytes compared with C57BL/6 astrocytes suggest that the NF-kappaB activation level affects on viral replication.

Formulation and in vitro/in vivo evaluation of chitosan-based film forming gel containing ketoprofen.

The film forming gel, adhered to skin surfaces upon application and formed a film, has an advantage onto skin to provide protection and continuous drug release to the application site. This study aimed to prepare a chitosan-based film forming gel containing ketoprofen (CbFG) and to evaluate the CbFG and film from CbFG (CbFG-film). CbFG were prepared with chitosan, lactic acid and various skin permeation enhancers. The physicochemical characteristics were evaluated by texture analysis, viscometry, SEM, DSC, XRD and FT-IR. To identify the mechanism of skin permeation, in vitro skin permeation study was conducted with a Franz diffusion cell and excised SD-rat and hairless mouse dorsal skin. In vivo efficacy assessment in mono-iodoacetate (MIA)-induced rheumatoid arthritis animal model was also conducted. CbFG was successfully prepared and, after applying CbFG to the excised rat dorsal skin, the CbFG-film was also formed well. The physicochemical characteristics of CbFG and CbFG-film could be explained by the grafting of oleic acid onto chitosan in the absence of catalysts. In addition, CbFG containing oleic acid had a higher skin permeation rate in comparison with any other candidate enhancers. The in vivo efficacy study also confirmed significant anti-inflammatory and analgesic effects. Consequently, we report the successful preparation of chitosan-based film forming gel containing ketoprofen with excellent mechanical properties, skin permeation and anti-inflammatory and analgesic effects.

Docetaxel-loaded RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes: Drug release, cytotoxicity, and antitumor efficacy.

We previously synthesized the RIPL peptide (IPLVVPLRRRRRRRRC) to facilitate selective delivery into hepsin-expressing cancer cells and showed that RIPL peptide-conjugated liposomes (RIPL-L) enhanced the intracellular delivery of fluorescent probes in vitro. In this study, docetaxel-loaded RIPL-L (DTX-RIPL-L) were prepared and evaluated for in vitro drug release, cytotoxicity, and in vivo antitumor efficacy. DTX was successfully encapsulated by pre-loading, with an average encapsulation efficiency and drug loading capacity of 32.4% and 21.39±2.05 (µg/mg), respectively. A DTX release study using dialysis showed a biphasic release pattern, i.e., rapid release for 6h, followed by sustained release up to 72h. The first-order equation provided the best fit for drug release (r2=0.9349). In vitro cytotoxicity was dose-dependent, resulting in IC50 values of 36.10 (SK-OV-3) and 48.62ng/mL (MCF-7) for hepsin-positive, and 61.12 (DU145) and 53.04ng/mL (PC-3) for hepsin-negative cell lines. Live/dead cell imaging was carried out to visualize the proportion of viable and nonviable SK-OV-3 cells. Compared to DTX solution, DTX-RIPL-L significantly inhibited tumor growth and prolonged survival time in BALB/c nude mice with SK-OV-3 cell tumors. We suggest that DTX-RIPL-L is a good candidate for efficient drug targeting to hepsin-expressing cancer cells.

Development of ciclopirox nail lacquer with enhanced permeation and retention.

Onychomycosis is a prevailing disease caused by fungal infection of nails that mostly affects athletes and the elderly. Ciclopirox is approved by the US Food and Drug Administration for the topical treatment of onychomycosis. However, the desired penetration of ciclopirox into the nail bed has not been achieved via topical application for efficient treatment. Therefore, the main aim of this study was to enhance ciclopirox permeation and retention in nail by the development of a new nail lacquer formulation. We screened the effects of different solvents, alkalizing agents, and permeation enhancers on the permeation of bovine hooves by ciclopirox and its retention in human nail clippings. The results suggest that isopropyl alcohol, potassium hydroxide, and urea as the solvent, alkalizing agent, and permeation enhancer, respectively, improved the permeation of the ciclopirox nail lacquer formulation the most with high flux rates. Comparison of the final formulation and marketed product revealed enhanced retention of ciclopirox from our developed formulation in human nail clippings. Therefore, our newly developed nail lacquer may be a potentially effective formulation for the treatment of onychomycosis in humans.