The role of nanoparticles in cancer medicine is vast with debate still surrounding the distinction between therapeutic efficacy of actively targeted nanoparticles versus passively targeted systems for drug delivery. While it is commonly accepted that methodologies that result in homing a high concentration of drug loaded nanoparticles to the tumor is beneficial, the role of intracellular trafficking of these nanoparticles in dictating the overall therapeutic outcome remains unresolved. Herein we demonstrate that the therapeutic outcome of drug loaded nanoparticles is governed beyond simply enabling nanoparticle internalization in cells. Using two model polymeric nanoparticles, one decorated with the GE11 peptide for active targeting of the epidermal growth factor receptor (EGFR) and the other without, we demonstrate that EGFR mediated intracellular internalization results in an enhanced therapeutic effect compared to the nontargeted formulation. Our findings demonstrate that the intracellular destination of nanoparticles beyond its ability to internalize is an important parameter that has to be accounted for in the design of targeted drug delivery systems. ■ INTRODUCTION Despite significant advances in cancer treatments and diagnosis, cancer still remains as one of the world's most devastating diseases with more than 10 million new cases reported every year. 1 Concomitant with these devastating cancer incidences are the side effects of chemotherapeutics still widely used for the treatment of this disease. Chemotherapeutics are the major class of drugs available in the arsenal against cancer, introduced in the 1950s for the treatment of these diseases. 2 However, these drugs are highly toxic and can result in severe and debilitating side effects for patients. 3,4 As a result of this, chemotherapeutics encapsulated in nanoparticle delivery vehicles are a promising field to enhance the effectiveness of these treatments in cancer while avoiding some of these side effects. 5−8 There is great interest in applying nanoparticle technology to cancer therapies largely resulting from the appealing features that nanoparticles possess. These include therapeutic protection from degradation, improved drug pharmacokinetics, improved intracellular penetration, selective tissue targeting, and the inclusion of imaging modalities. 9−11 Further to this, the increasing opportunities of "theranostic" nanoparticles which can combine both diagnostic capabilities as well as therapy within 50 a single entity provide great promise in the fight against 51 cancer. 12,13 52 Nanoparticles due to their unique size are able to exploit the 53 distinct cancer pathology and its molecular biology to in turn 54 result in higher uptake and preferential targeting of 55 therapeutics to the tumor compared to traditional treatments. 8 56 Broadly, this is achieved by two methods, "passive" or "active" 57 targeting. Passive targeting is possible due to the unique 58 changes in the cancer vasculature. Due to the rapid growth of 59 tumors, blood vessels and ju...

Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets.One example is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7kb) and NEAT1_2 (23kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed paraspeckles, whereas NEAT1_1 is involved in paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2.Here we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2/paraspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell-proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.

A family of three neutral iridium(III) tetrazolato complexes are investigated as bacterial imaging agents. The complexes offer a facile tuning of the emission colour from green (520 nm) to red (600 nm) in aqueous media, while keeping the excitation wavelength unchanged. The three complexes do not inhibit the bacterial growth of Bacillus Cereus, used as a model in this study, and exhibit extremely fast cellular uptake. After a minute incubation time, the nontoxic complexes show subcellular localisation in spherical structures identified as lipid vacuoles. Confocal Raman imaging has been exploited for the first time on live bacteria, to provide direct and label‐free mapping of the lipid‐enriched organelles within B. cereus, complementing the use of luminescent probes. Examination of the Raman spectra not only confirmed the presence of lipophilic inclusions in B. cereus but offered additional information about their chemical composition, suggesting that the lipid vacuoles may contain polyhydroxybutyrate (PHB).

Oligonucleotides and nucleic acid analogues that alter gene expression are now showing therapeutic promise in human disease. Whilst the modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, such modifications may also confer unexpected physicochemical and biological properties. Gapmer mixed-modified and DNA oligonucleotides on a phosphorothioate backbone can bind non-specifically to intracellular proteins to form a variety of toxic inclusions, driven by the phosphorothioate linkages, but also influenced by the oligonucleotide sequence. Recently, the non-antisense or other off-target effects of 2′ O- fully modified phosphorothioate linkage oligonucleotides are becoming better understood. Here, we report chemistry-specific effects of oligonucleotides composed of modified or unmodified bases, with phosphorothioate linkages, on subnuclear organelles and show altered distribution of nuclear proteins, the appearance of highly stable and strikingly structured nuclear inclusions, and disturbed RNA processing in primary human fibroblasts and other cultured cells. Phosphodiester, phosphorodiamidate morpholino oligomers, and annealed complimentary phosphorothioate oligomer duplexes elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of transfected fibroblasts exhibiting such disruption. Our data add to the growing body of evidence of off-target effects of some phosphorothioate nucleic acid drugs in primary cells and suggest alternative approaches to mitigate these effects.

max 150 words) 9Oligonucleotides and nucleic acid analogues that alter gene expression are showing 10 therapeutic promise for selected human diseases. The modification of synthetic nucleic acids 11 to protect against nuclease degradation and to influence drug function is common practice, 12 however, such modifications may also confer unexpected physicochemical and biological 13properties. Here we report backbone-specific effects of modified oligonucleotides on 14 subnuclear organelles, altered distribution of nuclear proteins, the appearance of novel 15 structured nuclear inclusions, and modification of RNA processing in cultured cells 16 transfected with antisense oligonucleotides on a phosphorothioate backbone. Phosphodiester 17 and phosphorodiamidate morpholino oligomers elicited no such consequences. Disruption 18 of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by 19 transcriptomic analysis of fibroblasts exhibiting such disruption. These data suggest that the 20 toxic effects and adverse events reported after clinical evaluation of phosphorothioate 21 nucleic acid drugs may be mediated, at least in part, by non-specific interaction of nuclear 22 components with the phosphorothioate backbone. 23 24 25