Development of a peptide-siRNA nanocomplex targeting NF- κB for efficient cartilage delivery

Abstract Delivery of therapeutic small interfering RNAs (siRNAs) in an effective dose to articular cartilage is very challenging as the cartilage dense extracellular matrix renders the chondrocytes inaccessible, even to intra-articular injections. Herein, we used a self-assembling peptidic nanoparticle (NP) platform featuring a cell penetrating peptide complexed to NF-κB p65 siRNA. We show that it efficiently and deeply penetrated human cartilage to deliver its siRNA cargo up to a depth of at least 700 μm. To simulate osteoarthritis in vitro, human articular cartilage explants were placed in culture and treated with IL-1β, a cytokine with known cartilage catabolic and pro-inflammatory effects. Exposure of peptide-siRNA NP to cartilage explants markedly suppressed p65 activation, an effect that persisted up to 3 weeks after an initial 48 h exposure to NP and in the presence of continuous IL-1β stimulation. Suppression of IL-1β-induced p65 activity attenuated chondrocyte apoptosis and maintained cartilage homeostasis. These findings confirm our previous in vivo studies in a murine model of post-traumatic osteoarthritis and suggest that the ability of peptide-siRNA NP to specifically modulate NF-κB pathway, a central regulator of the inflammatory responses in chondrocytes, may potentially mitigate the progression of cartilage degeneration

Induction of WNT16 via peptide-mRNA nanoparticle-based delivery maintains cartilage homeostasis

Osteoarthritis (OA) is a progressive joint disease that causes significant disability and pain and for which there are limited treatment options. We posit that delivery of anabolic factors that protect and maintain cartilage homeostasis will halt or retard OA progression. We employ a peptide-based nanoplatform to deliver Wingless and the name Int-1 (WNT) 16 messenger RNA (mRNA) to human cartilage explants. The peptide forms a self-assembled nanocomplex of approximately 65 nm in size when incubated with WNT16 mRNA. The complex is further stabilized with hyaluronic acid (HA) for enhanced cellular uptake. Delivery of peptide-WNT16 mRNA nanocomplex to human cartilage explants antagonizes canonical β-catenin/WNT3a signaling, leading to increased lubricin production and decreased chondrocyte apoptosis. This is a proof-of-concept study showing that mRNA can be efficiently delivered to articular cartilage, an avascular tissue that is poorly accessible even when drugs are intra-articularly (IA) administered. The ability to accommodate a wide range of oligonucleotides suggests that this platform may find use in a broad range of clinical applications

Genome editing technologies to fight infectious diseases

Genome editing by programmable nucleases represents a promising tool that could be exploited to develop new therapeutic strategies to fight infectious diseases. These nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) and homing endonucleases, are molecular scissors that can be targeted at predetermined loci in order to modify the genome sequence of an organism. Areas covered: By perturbing genomic DNA at predetermined loci, programmable nucleases can be used as antiviral and antimicrobial treatment. This approach includes targeting of essential viral genes or viral sequences able, once mutated, to inhibit viral replication; repurposing of CRISPR-Cas9 system for lethal self-targeting of bacteria; targeting antibiotic-resistance and virulence genes in bacteria, fungi, and parasites; engineering arthropod vectors to prevent vector-borne infections. Expert commentary: While progress has been done in demonstrating the feasibility of using genome editing as antimicrobial strategy, there are still many hurdles to overcome, such as the risk of off-target mutations, the raising of escape mutants, and the inefficiency of delivery methods, before translating results from preclinical studies into clinical applications

Early-stage development of novel cyclodextrin-siRNA nanocomplexes allows for successful postnebulization transfection of bronchial epithelial cells.

BACKGROUND: Successful delivery of small interfering RNA (siRNA) to the lungs remains hampered by poor intracellular delivery, vector-mediated cytotoxicity, and an inability to withstand nebulization. Recently, a novel cyclodextrin (CD), SC12CDClickpropylamine, consisting of distinct lipophilic and cationic subunits, has been shown to transfect a number of cell types. However, the suitability of this vector for pulmonary siRNA delivery has not been assessed to date. To address this, a series of high-content analysis (HCA) and postnebulization assays were devised to determine the potential for CD-siRNA delivery to the lungs. METHODS: SC12CDClickpropylamine-siRNA mass ratios (MRs) were examined for size and zeta potential. In-depth analysis of nanocomplex uptake and toxicity in Calu-3 bronchial epithelial cells was examined using IN Cell(®) HCA assays. Nebulized SC12CDClickpropylamine nanocomplexes were assessed for volumetric median diameter (VMD) and fine particle fraction (FPF) and compared with saline controls. Finally, postnebulization stability was determined by comparing luciferase knockdown elicited by SC12CDClickpropylamine nanocomplexes before and after nebulization. RESULTS: SC12CDClickpropylamine-siRNA complexation formed cationic nanocomplexes of ≤200 nm in size depending on the medium and led to significantly higher levels of siRNA associated with Calu-3 cells compared with RNAiFect-siRNA-treated cells at all MRs (p CONCLUSIONS: SC12CDClickpropylamine nanocomplexes can be effectively nebulized for pulmonary delivery of siRNA using Aeroneb technology to mediate knockdown in airway cells. To the best of our knowledge, this is the first study examining the suitability of SC12CDClickpropylamine-siRNA nanocomplexes for pulmonary delivery. Furthermore, this work provides an integrated nanomedicine-device combination for future in vitro and in vivo preclinical and clinical studies of inhaled siRNA therapeutics

In vitro digestion and stability assessment of b-lactoglobulin/riboflavin nanostructures

B-Lactoglobulin (b-Lg) is the major protein fraction of bovine whey serum and a primary gelling agent. b-Lg has a high nutritional value, is stable at low pH being highly resistant to proteolytic degradation in the stomach, besides, it has the ability of acting as an encapsulating agent. This study aims at assessing the ability of b-Lg nanostructures to associate a nutraceutical - i.e. riboflavin - and release it in a controlled manner throughout an in vitro gastrointestinal (GI) system. For this reason b-Lg nanostructures loaded with riboflavin were critically characterized in terms of their morphology (i.e. size, polydispersity, -potential and shape) by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and efficiency to associate to riboflavin through spectrofluorimetry. Furthermore, these nanocomplexes were evaluated in an in vitro GI model, simulating the physiological conditions. Stable b-Lg nanostructures were obtained at pH 6, of spherical shape, characterized by particle size of 172±1 nm, low polydispersity (i.e. PDI of 0.06±0.02), -potential of -32±3 mV and association efficiency (AE) of 26±1 %. b-Lg nanostructures showed to be stable upon their passage throughout stomach (i.e. particle size, PDI and potential of 248±10 nm, 0.18±0.03 and 18±3 mV, respectively). Concerning their passage throughout the intestine, such nanostructures were mostly degraded in the duodenum. Regarding riboflavin, a release of about 11 % was observed after their passage through stomach, while 35 %, 38 % and 5 % were the released percentages of the total riboflavin associated observed after passage through duodenum, jejunum and ileum, respectively. Hence,b-Lg nanostructures showed to be suitable carriers for riboflavin until the intestine, where their degradation occurs. b-Lg also showed to be structurally stable, under food simulant conditions (yoghurt simulant, composed of 3 % acetic acid), over 14 days, with a protective effect upon riboflavin activity, releasing it in a 7 day period.Oscar L. Ramos, Ricardo N. Pereira and Ana C. Pinheiro gratefully acknowledge their Post-Doctoral grants (SFRH/BPD/80766/2011, SFRH/BPD/81887/2011 and SFRH/BPD/101181/2014, respectively) and Ana I. Bourbon gratefully acknowledge her Doctoral grant (SFRH/BD/73178/2010) to Fundacao para a Ciencia e a Tecnologia (FCT). The authors would like to acknowledge to Francisco Figueiredo from the Institute for Molecular and Cell Biology (IBMC), University of Porto, for assistance in taking the TEM pictures. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and the project "BioInd e Biotechnology and Bioengineering for improved Industrial and Agro-Food processes", REF. NORTE-07-0124-FEDER-000028, co-funded by the Programa Operacional Regional do Norte (ON. 2 - O Novo Norte), QREN, FEDER

Self-assembled nanocomplex between polymerized phenylboronic acid and doxorubicin for efficient tumor-targeted chemotherapy

Since the discovery that nano-scaled particulates can easily be incorporated into tumors via the enhanced permeability and retention (EPR) effect, such nanostructures have been exploited as therapeutic small molecule delivery systems. However, the convoluted synthetic process of conventional nanostructures has impeded their feasibility and reproducibility in clinical applications. Herein, we report an easily prepared formulation of self-assembled nanostructures for systemic delivery of the anti-cancer drug doxorubicin (DOX). Phenylboronic acid (PBA) was grafted onto the polymeric backbone of poly(maleic anhydride). pPBA-DOX nanocomplexes were prepared by simple mixing, on the basis of the strong interaction between the 1,3-diol of DOX and the PBA moiety on pPBA. Three nanocomplexes (1, 2, 4) were designed on the basis of [PBA]:[DOX] molar ratios of 1: 1, 2: 1, and 4: 1, respectively, to investigate the function of the residual PBA moiety as a targeting ligand. An acid-labile drug release profile was observed, owing to the intrinsic properties of the phenylboronic ester. Moreover, the tumor-targeting ability of the nanocomplexes was demonstrated, both in vitro by confocal microscopy and in vivo by fluorescence imaging, to be driven by an inherent property of the residual PBA. Ligand competition assays with free PBA pre-treatment demonstrated the targeting effect of the residual PBA from the nanocomplexes 2 and 4. Finally, the nanocomplexes 2 and 4, compared with the free DOX, exhibited significantly greater anti-cancer effects in vitro and even in vivo. Our pPBA-DOX nanocomplex enables a new paradigm for self-assembled nanostructures with potential biomedical applications.115Ysciescopu

EpCAM aptamer mediated cancer cell specific delivery of EpCAM siRNA using polymeric nanocomplex

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is overexpressed in solid tumors and regarded as a putative cancer stem cell marker. Here, we report that employing EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) dual approach, for the targeted delivery of siRNA to EpCAM positive cancer cells, efficiently inhibits cancer cell proliferation. RESULTS: Targeted delivery of siRNA using polyethyleneimine is one of the efficient methods for gene delivery, and thus, we developed a novel aptamer-PEI-siRNA nanocomplex for EpCAM targeting. PEI nanocomplex synthesized with EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) showed 198 nm diameter sized particles by dynamic light scattering, spherical shaped particles, of 151 ± 11 nm size by TEM. The surface charge of the nanoparticles was -30.0 mV using zeta potential measurements. Gel retardation assay confirmed the PEI-EpApt-SiEp nanoparticles formation. The difference in size observed by DLS and TEM could be due to coating of aptamer and siRNA on PEI nanocore. Flow cytometry analysis revealed that PEI-EpApt-SiEp has superior binding to cancer cells compared to EpApt or scramble aptamer (ScrApt) or PEI-ScrApt-SiEp. PEI-EpApt-SiEp downregulated EpCAM and inhibited selectively the cell proliferation of MCF-7 and WERI-Rb1 cells. CONCLUSIONS: The PEI nanocomplex fabricated with EpApt and siEp was able to target EpCAM tumor cells, deliver the siRNA and silence the target gene. This nanocomplex exhibited decreased cell proliferation than the scrambled aptamer loaded nanocomplex in the EpCAM expressing cancer cells and may have potential for EpCAM targeting in vivo

Co-delivery of free vancomycin and transcription factor decoy-nanostructured lipid carriers can enhance inhibition of methicillin resistant Staphylococcus aureus (MRSA)

Bacterial resistance to antibiotics is widely regarded as a major public health concern with last resort MRSA treatments like vancomycin now encountering resistant strains. TFDs (Transcription Factor Decoys) are oligonucleotide copies of the DNA-binding sites for transcription factors. They bind to and sequester the targeted transcription factor, thus inhibiting transcription of many genes. By developing TFDs with sequences aimed at inhibiting transcription factors controlling the expression of highly conserved bacterial cell wall proteins, TFDs present as a potential method for inhibiting microbial growth without encountering typical resistance mechanisms. However, the efficient protection and delivery of the TFDs inside the bacterial cells is a critical step for the success of this technology. Therefore, in our study, specific TFDs against S. aureus were complexed with two different types of nanocarriers: cationic nanostructured lipid carriers (cNLCs) and chitosan-based nanoparticles (CS-NCs). These TFD-carrier nanocomplexes were characterized for size, zeta potential and TFD complexation or loading efficiency in a variety of buffers. In vitro activity of the nanocomplexes was examined alone and in combination with vancomycin, first in methicillin susceptible strains of S. aureus with the lead candidate advancing to tests against MRSA cultures. Results found that both cNLCs and chitosan-based carriers were adept at complexing and protecting TFDs in a range of physiological and microbiological buffers up to 72 hours. From initial testing, chitosan-TFD particles demonstrated no visible improvements in effect when co-administered with vancomycin. However, co-delivery of cNLC-TFD with vancomycin reduced the MIC of vancomycin by over 50% in MSSA and resulted in significant decreases in viability compared with vancomycin alone in MRSA cultures. Furthermore, these TFD-loaded particles demonstrated very low levels of cytotoxicity and haemolysis in vitro. To our knowledge, this is the first attempt at a combined antibiotic/oligonucleotide-TFD approach to combatting MRSA and, as such, highlights a new avenue of MRSA treatment combining traditional small molecules drugs and bacterial gene inhibition

Role of liposome and peptide in the synergistic enhancement of transfection with a lipopolyplex vector

Lipopolyplexes are of widespread interest for gene therapy due to their multifunctionality and high transfection efficiencies. Here we compared the biological and biophysical properties of a lipopolyplex formulation with its lipoplex and polyplex equivalents to assess the role of the lipid and peptide components in the formation and function of the lipopolyplex formulation. We show that peptide efficiently packaged plasmid DNA forming spherical, highly cationic nanocomplexes that are taken up efficiently by cells. However, transgene expression was poor, most likely due to endosomal degradation since the polyplex lacks membrane trafficking properties. In addition the strong peptide-DNA interaction may prevent plasmid release from the complex and so limit plasmid DNA availability. Lipid/DNA lipoplexes, on the other hand, produced aggregated masses that showed poorer cellular uptake than the polyplex but contrastingly greater levels of transgene expression. This may be due to the greater ability of lipoplexes relative to polyplexes to promote endosomal escape. Lipopolyplex formulations formed spherical, cationic nanocomplexes with efficient cellular uptake and significantly enhanced transfection efficiency. The lipopolyplexes combined the optimal features of lipoplexes and polyplexes showing optimal cell uptake, endosomal escape and availability of plasmid for transcription, thus explaining the synergistic increase in transfection efficiency