Modification of the Antibiotic, Colistin, with Dextrin Causes Enhanced Cytotoxicity and Triggers Apoptosis in Myeloid Leukemia.

Introduction: Acute myeloid leukemia (AML) remains difficult to treat due to its heterogeneity in molecular landscape, epigenetics and cell signaling alterations. Precision medicine is a major goal in AML therapy towards developing agents that can be used to treat patients with different 'subtypes' in combination with current chemotherapies. We have previously developed dextrin-colistin conjugates to combat the rise in multi-drug resistant bacterial infections and overcome dose-limiting nephrotoxicity. Recent evidence of colistin's anticancer activity, mediated through inhibition of intracellular lysine-specific histone demethylase 1 (LSD1/KDM1A), suggests that dextrin-colistin conjugates could be used to treat cancer cells, including AML. This study aimed to evaluate whether dextrin conjugation (which reduces in vivo toxicity and prolongs plasma half-life) could enhance colistin's cytotoxic effects in myeloid leukemia cell lines and compare the intracellular uptake and localization of the free and conjugated antibiotic. Results: Our results identified a conjugate (containing 8000 g/mol dextrin with 1 mol% succinoylation) that caused significantly increased toxicity in myeloid leukemia cells, compared to free colistin. Dextrin conjugation altered the mechanism of cell death by colistin, from necrosis to caspase 3/7-dependent apoptosis. In contrast, conjugation via a reversible ester linker, instead of an amide, had no effect on the mechanism of the colistin-induced cell death. Live cell confocal microscopy of fluorescently labelled compounds showed both free and dextrin-conjugated colistins were endocytosed and co-localized in lysosomes, and increasing the degree of modification by succinoylation of dextrin significantly reduced colistin internalization. Discussion: Whilst clinical translation of dextrin-colistin conjugates for the treatment of AML is unlikely due to the potential to promote antimicrobial resistance (AMR) and the relatively high colistin concentrations required for anticancer activity, the ability to potentiate the effectiveness of an anticancer drug by polymer conjugation, while reducing side effects and improving biodistribution of the drug, is very attractive, and this approach warrants further investigation.

Dextrin conjugation to colistin inhibits its toxicity, cellular uptake and acute kidney injury in vivo.

The acute kidney injury (AKI) and dose-limiting nephrotoxicity, which occurs in 20-60% of patients following systemic administration of colistin, represents a challenge in the effective treatment of multi-drug resistant Gram-negative infections. To reduce clinical toxicity of colistin and improve targeting to infected/inflamed tissues, we previously developed dextrin-colistin conjugates, whereby colistin is designed to be released by amylase-triggered degradation of dextrin in infected and inflamed tissues, after passive targeting by the enhanced permeability and retention effect. Whilst it was evident in vitro that polymer conjugation can reduce toxicity and prolong plasma half-life, without significant reduction in antimicrobial activity of colistin, it was unclear how dextrin conjugation would alter cellular uptake and localisation of colistin in renal tubular cells in vivo. We discovered that dextrin conjugation effectively reduced colistin's toxicity towards human kidney proximal tubular epithelial cells (HK-2) in vitro, which was mirrored by significantly less cellular uptake of Oregon Green (OG)-labelled dextrin-colistin conjugate, when compared to colistin. Using live-cell confocal imaging, we revealed localisation of both, free and dextrin-bound colistin in endolysosome compartments of HK-2 and NRK-52E cells. Using a murine AKI model, we demonstrated dextrin-colistin conjugation dramatically diminishes both proximal tubular injury and renal accumulation of colistin. These findings reveal new insight into the mechanism by which dextrin conjugation can overcome colistin's renal toxicity and show the potential of polymer conjugation to improve the side effect profile of nephrotoxic drugs.

Stereoisomer-Dependent Membrane Association and Capacity for Insulin Delivery Facilitated by Penetratin.

Cell-penetrating peptides (CPPs), such as penetratin, are often investigated as drug delivery vectors and incorporating d-amino acids, rather than the natural l-forms, to enhance proteolytic stability could improve their delivery efficiency. The present study aimed to compare membrane association, cellular uptake, and delivery capacity for all-l and all-d enantiomers of penetratin (PEN) by using different cell models and cargos. The enantiomers displayed widely different distribution patterns in the examined cell models, and in Caco-2 cells, quenchable membrane binding was evident for d-PEN in addition to vesicular intracellular localization for both enantiomers. The uptake of insulin in Caco-2 cells was equally mediated by the two enantiomers, and while l-PEN did not increase the transepithelial permeation of any of the investigated cargo peptides, d-PEN increased the transepithelial delivery of vancomycin five-fold and approximately four-fold for insulin at an extracellular apical pH of 6.5. Overall, while d-PEN was associated with the plasma membrane to a larger extent and was superior in mediating the transepithelial delivery of hydrophilic peptide cargoes compared to l-PEN across Caco-2 epithelium, no enhanced delivery of the hydrophobic cyclosporin was observed, and intracellular insulin uptake was induced to a similar degree by the two enantiomers.

PINK1-Dependent Mitophagy Inhibits Elevated Ubiquitin Phosphorylation Caused by Mitochondrial Damage.

Ubiquitin phosphorylation by the mitochondrial protein kinase PTEN-induced kinase 1 (PINK1), upon mitochondrial depolarization, is an important intermediate step in the recycling of damaged mitochondria via mitophagy. As mutations in PINK1 can cause early-onset Parkinson's disease (PD), there has been a growing interest in small-molecule activators of PINK1-mediated mitophagy as potential PD treatments. Herein, we show that N6-substituted adenosines, such as N6-(2-furanylmethyl)adenosine (known as kinetin riboside) and N6-benzyladenosine, activate PINK1 in HeLa cells and induce PINK1-dependent mitophagy in primary mouse fibroblasts. Interestingly, pre-treatment of HeLa cells and astrocytes with these compounds inhibited elevated ubiquitin phosphorylation that is induced by established mitochondrial depolarizing agents, carbonyl cyanide m-chlorophenyl-hydrazine and niclosamide. Together, this highlights N6-substituted adenosines as progenitor PINK1 activators that could potentially be developed, in the future, as treatments for aged and sporadic PD patients who have elevated phosphorylated ubiquitin levels in the brain.

Influence of Folate-Targeted Gold Nanoparticles on Subcellular Localization and Distribution into Lysosomes.

The cell interaction, mechanism of cell entry and intracellular fate of surface decorated nanoparticles are known to be affected by the surface density of targeting agents. However, the correlation between nanoparticles multivalency and kinetics of the cell uptake process and disposition of intracellular compartments is complicated and dependent on a number of physicochemical and biological parameters, including the ligand, nanoparticle composition and colloidal properties, features of targeted cells, etc. Here, we have carried out an in-depth investigation on the impact of increasing folic acid density on the kinetic uptake process and endocytic route of folate (FA)-targeted fluorescently labelled gold nanoparticles (AuNPs). A set of AuNPs (15 nm mean size) produced by the Turkevich method was decorated with 0-100 FA-PEG3.5kDa-SH molecules/particle, and the surface was saturated with about 500 rhodamine-PEG2kDa-SH fluorescent probes. In vitro studies carried out using folate receptor overexpressing KB cells (KBFR-high) showed that the cell internalization progressively increased with the ligand surface density, reaching a plateau at 50:1 FA-PEG3.5kDa-SH/particle ratio. Pulse-chase experiments showed that higher FA density (50 FA-PEG3.5kDa-SH molecules/particle) induces more efficient particle internalization and trafficking to lysosomes, reaching the maximum concentration in lysosomes at 2 h, than the lower FA density of 10 FA-PEG3.5kDa-SH molecules/particle. Pharmacological inhibition of endocytic pathways and TEM analysis showed that particles with high folate density are internalized predominantly by a clathrin-independent process.

Nanoparticle entry into cells; the cell biology weak link.

Nanoparticles (NP) are attractive options for the therapeutic delivery of active pharmaceutical drugs, proteins and nucleic acids into cells, tissues and organs. Research into the development and application of NP most often starts with a diverse group of scientists, including chemists, bioengineers and material and pharmaceutical scientists, who design, fabricate and characterize NP in vitro (Stage 1). The next step (Stage 2) generally investigates cell toxicity as well as the processes by which NP bind, are internalized and deliver their cargo to appropriate model tissue culture cells. Subsequently, in Stage 3, selected NP are tested in animal systems, mostly mouse. Whereas the chemistry-based development and analysis in Stage 1 is increasingly sophisticated, the investigations in Stage 2 are not what could be regarded as 'state-of-the-art' for the cell biology field and the quality of research into NP interactions with cells is often sub-standard. In this review we describe our current understanding of the mechanisms by which particles gain entry into mammalian cells via endocytosis. We summarize the most important areas for concern, highlight some of the most common mis-conceptions, and identify areas where NP scientists could engage with trained cell biologists. Our survey of the different mechanisms of uptake into cells makes us suspect that claims for roles for caveolae, as well as macropinocytosis, in NP uptake into cells have been exaggerated, whereas phagocytosis has been under-appreciated.

Highly-fluorescent BODIPY-functionalised metallacages as drug delivery systems: synthesis, characterisation and cellular accumulation studies.

With the aim of designing new metallosupramolecular architectures for drug delivery, research has focused on porous 3-dimensional (3D)-metallacages able to encapsulate cytotoxic agents protecting them from metabolism while targeting them to cancer sites. Here, two self-assembled [Pd2L4]4+ cages (CG1 and CG2) featuring 3,5-bis(3-ethynylpyridine)phenyl ligands (L) exo-functionalised with dipyrromethene (BODIPY) groups have been synthesised and characterised by different methods, including NMR spectroscopy and mass spectrometry. 1H NMR spectroscopy studies shows that the cages are able to encapsulate the anticancer drug cisplatin in their hydrophobic cavity, as evidenced by electrostatic potential (ESP) analysis based on XRD studies. The stability of the cages in an aqueous environment, and in the presence of the intracellular reducing agent glutathione, has been confirmed by UV-visible absorption spectroscopy. The luminescence properties of the cages enabled the investigation of their cellular uptake and intracellular localisation in human cancer cells by confocal laser scanning microscopy. In melanoma A375 cells, cage CG1 is taken up via active transport and endocytic trafficking studies show little evidence of transport through the early endosome while the cages accumulated in melanosomes rather than lysosomes. The antiproliferative activity of the lead cage was investigated in A375 together with two breast cancer cell lines, SK-BR-3 and MCF7. While the cage per se is non-cytotoxic, very different antiproliferative effects with respect to free cisplatin were evidenced for the [(cisplatin)2⊂CG1·BF4] complex in the various cell lines, which correlate with its different intracellular localisation profiles. The obtained preliminary results provide a new hypothesis on how the subcellular localisation of the cage affects the cisplatin intracellular release.

Spatio-Temporal Photoactivation of Cytotoxic Proteins.

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.

Quantitative Subcellular Analysis of Cyclic Cell-Penetrating Peptide EJP18 in Nonadherent Cells.

Cyclization of cell-penetrating peptides (CPPs) often results in improved capacity for intracellular delivery of a range of cargoes but quantitating the distinct subcellular localization of them, and their linear counterparts, remains a challenge. Here we describe an optimized method for recombinant generation and purification of eGFP attached to the cyclic form of the newly discovered CPP EJP18 in E. coli. We also demonstrate a novel microscopy method for quantifying its subcellular distribution in leukemia cells.

Strategies for Peptide-Mediated Cargo Delivery to Human Smooth Muscle Cells.

Diseases involving dysfunction of smooth muscle cells present a major health and socioeconomic burden, and have remained stubbornly resistant to standard therapeutic strategies. Examples include many cardiovascular diseases and spontaneous preterm birth, a complication affecting up to 11% of all pregnancies worldwide. This fuels the continued search for new drug delivery strategies to treat these conditions. The use of cell penetrating peptides (CPPs) for this purpose remains a promising, if as yet unrealized, avenue to explore. In part, this may relate to a paucity of studies investigating the application of CPPs as drug delivery vectors to human smooth muscle cells and tissues. We have sought to address this knowledge gap by reporting methods for examining the uptake of different CPP-cargo vectors to human uterine and vascular smooth muscle cells. In particular, we report here (a) that four different CPP-fluorophore conjugates, spanning masses of 1309-3435 Da, and net charges of +2 to +7, can be delivered to human isolated uterine smooth muscle cells without inducing cell toxicity; (b) that the cargo delivered by such CPPs can be fluorescent moieties and/or biologically active peptides; (c) that CPP delivery in a short time frame to native smooth muscle cells in human tissues ex vivo can be achieved. Further exploration of CPPs as tools to facilitate targeted drug delivery to native human smooth muscle tissues will assist in improving our understanding of scientific mechanisms underlying major diseases involving smooth muscle dysfunction as well as facilitating therapeutic investigations.

New Bioactive Fused Triazolothiadiazoles as Bcl-2-Targeted Anticancer Agents.

A series of 3-(6-substituted phenyl-[1,2,4]-triazolo[3,4-b]-[1,3,4]-thiadiazol-3-yl)-1H-indoles (5a-l) were designed, synthesized and evaluated for anti-apoptotic Bcl-2-inhibitory activity. Synthesis of the target compounds was readily accomplished through a reaction of acyl hydrazide (1) with carbon disulfide in the presence of alcoholic potassium hydroxide to afford the corresponding intermediate potassium thiocarbamate salt (2), which underwent cyclization reaction in the presence of excess hydrazine hydrate to the corresponding triazole thiol (3). Further cyclisation reaction with substituted benzoyl chloride derivatives in the presence of phosphorous oxychloride afforded the final 6-phenyl-indol-3-yl [1,2,4]-triazolo[3,4-b]-[1,3,4]-thiadiazole compounds (5a-l). The novel series showed selective sub-micromolar IC50 growth-inhibitory activity against Bcl-2-expressing human cancer cell lines. The most potent 6-(2,4-dimethoxyphenyl) substituted analogue (5k) showed selective IC50 values of 0.31-0.7 µM against Bcl-2-expressing cell lines without inhibiting the Bcl-2-negative cell line (Jurkat). ELISA binding affinity assay (interruption of Bcl-2-Bim interaction) showed potent binding affinity for (5k) with an IC50 value of 0.32 µM. Moreover, it fulfils drug likeness criteria as a promising drug candidate.

Anti-schistosomal activities of quinoxaline-containing compounds: From hit identification to lead optimisation.

Schistosomiasis is a neglected disease of poverty that is caused by infection with blood fluke species contained within the genus Schistosoma. For the last 40 years, control of schistosomiasis in endemic regions has predominantly been facilitated by administration of a single drug, praziquantel. Due to limitations in this mono-chemotherapeutic approach for sustaining schistosomiasis control into the future, alternative anti-schistosomal compounds are increasingly being sought by the drug discovery community. Herein, we describe a multi-pronged, integrated strategy that led to the identification and further exploration of the quinoxaline core as a promising anti-schistosomal scaffold. Firstly, phenotypic screening of commercially available small molecules resulted in the identification of a moderately active hit compound against Schistosoma mansoni (1, EC50 = 4.59 µM on schistosomula). Secondary exploration of the chemical space around compound 1 led to the identification of a quinoxaline-core containing, non-genotoxic lead (compound 22). Compound 22 demonstrated substantially improved activities on both intra-mammalian (EC50 = 0.44 µM, 0.20 µM and 84.7 nM, on schistosomula, juvenile and adult worms, respectively) and intra-molluscan (sporocyst) S. mansoni lifecycle stages. Further medicinal chemistry optimisation of compound 22, resulting in the generation of 20 additional analogues, improved our understanding of the structure-activity relationship and resulted in considerable improvements in both anti-schistosome potency and selectivity (e.g. compound 30; EC50 = 2.59 nM on adult worms; selectivity index compared to the HepG2 cell line = 348). Some derivatives of compound 22 (e.g. 31 and 33) also demonstrated significant activity against the two other medically important species, Schistosoma haematobium and Schistosoma japonicum. Further optimisation of this class of anti-schistosomal is ongoing and could lead to the development of an urgently needed alternative to praziquantel for assisting in schistosomiasis elimination strategies.

Development of mammalian cell logic gates controlled by unnatural amino acids.

Mammalian cell logic gates hold great potential for wide-ranging applications. However, most of those currently available are controlled by drug(-like) molecules with inherent biological activities. To construct truly orthogonal circuits and artificial regulatory pathways, biologically inert molecules are ideal molecular switches. Here, we applied genetic code expansion and engineered logic gates controlled by two biologically inert unnatural amino acids. Genetic code expansion relies on orthogonal aminoacyl-tRNA synthetase/tRNA pairs for co-translational and site-specific unnatural amino acid incorporation conventionally in response to an amber (UAG) codon. By screening 11 quadruplet-decoding pyrrolysyl tRNA variants from the literature, we found that all variants decoding CUAG or AGGA tested here are functional in mammalian cells. Using a quadruplet-decoding orthogonal pair together with an amber-decoding pair, we constructed logic gates that can be successfully controlled by two different unnatural amino acids, expanding the scope of genetic code expansion and mammalian cell logic circuits.

Design, Synthesis and Evaluation of New Bioactive Oxadiazole Derivatives as Anticancer Agents Targeting Bcl-2.

A series of 2-(1H-indol-3-yl)-5-substituted-1,3,4-oxadiazoles, 4a-m, were designed, synthesized and tested in vitro as potential pro-apoptotic Bcl-2 inhibitory anticancer agents based on our previously reported hit compounds. Synthesis of the target 1,3,4-oxadiazoles was readily accomplished through a cyclization reaction of indole carboxylic acid hydrazide 2 with substituted carboxylic acid derivatives 3a-m in the presence of phosphorus oxychloride. New compounds 4a-m showed a range of IC50 values concentrated in the low micromolar range selectively in Bcl-2 positive human cancer cell lines. The most potent candidate 4-trifluoromethyl substituted analogue 4j showed selective IC50 values of 0.52-0.88 µM against Bcl-2 expressing cell lines with no inhibitory effects in the Bcl-2 negative cell line. Moreover, 4j showed binding that was two-fold more potent than the positive control gossypol in the Bcl-2 ELISA binding affinity assay. Molecular modeling studies helped to further rationalize anti-apoptotic Bcl-2 binding and identified compound 4j as a candidate with drug-like properties for further investigation as a selective Bcl-2 inhibitory anticancer agent.

Cyanine dye mediated mitochondrial targeting enhances the anti-cancer activity of small-molecule cargoes.

Organelle-specific delivery systems are of significant clinical interest. We demonstrate the use of common cyanine dyes Cy3 and Cy5 as vectors for targeting and delivering cargoes to mitochondria in cancer cells. Specifically, conjugation to the dyes can increase cytotoxicity by up to 1000-fold.

Four-wave-mixing microscopy reveals non-colocalisation between gold nanoparticles and fluorophore conjugates inside cells.

Gold nanoparticles have been researched for many biomedical applications in diagnostics, theranostics, and as drug delivery systems. When conjugated to fluorophores, their interaction with biological cells can be studied in situ and real time using fluorescence microscopy. However, an important question that has remained elusive to answer is whether the fluorophore is a faithful reporter of the nanoparticle location. Here, our recently developed four-wave-mixing optical microscopy is applied to image individual gold nanoparticles and in turn investigate their co-localisation with fluorophores inside cells. Nanoparticles from 10 nm to 40 nm diameter were conjugated to fluorescently-labeled transferrin, for internalisation via clathrin-mediated endocytosis, or to non-targeting fluorescently-labelled antibodies. Human (HeLa) and murine (3T3-L1) cells were imaged at different time points after incubation with these conjugates. Our technique identified that, in most cases, fluorescence originated from unbound fluorophores rather than from fluorophores attached to nanoparticles. Fluorescence detection was also severely limited by photobleaching, quenching and autofluorescence background. Notably, correlative extinction/fluorescence microscopy of individual particles on a glass surface indicated that commercial constructs contain large amounts of unbound fluorophores. These findings highlight the potential problems of data interpretation when reliance is solely placed on the detection of fluorescence within the cell, and are of significant importance in the context of correlative light electron microscopy.

Endocytic Profiling of Cancer Cell Models Reveals Critical Factors Influencing LNP-Mediated mRNA Delivery and Protein Expression.

Lipid nanoparticles have great potential for delivering nucleic-acid-based therapeutics, but low efficiency limits their broad clinical translation. Differences in transfection capacity between in vitro models used for nanoparticle pre-clinical testing are poorly understood. To address this, using a clinically relevant lipid nanoparticle (LNP) delivering mRNA, we highlight specific endosomal characteristics in in vitro tumor models that impact protein expression. A 30-cell line LNP-mRNA transfection screen identified three cell lines having low, medium, and high transfection that correlated with protein expression when they were analyzed in tumor models. Endocytic profiling of these cell lines identified major differences in endolysosomal morphology, localization, endocytic uptake, trafficking, recycling, and endolysosomal pH, identified using a novel pH probe. High-transfecting cells showed rapid LNP uptake and trafficking through an organized endocytic pathway to lysosomes or rapid exocytosis. Low-transfecting cells demonstrated slower endosomal LNP trafficking to lysosomes and defective endocytic organization and acidification. Our data establish that efficient LNP-mRNA transfection relies on an early and narrow endosomal escape window prior to lysosomal sequestration and/or exocytosis. Endocytic profiling should form an important pre-clinical evaluation step for nucleic acid delivery systems to inform model selection and guide delivery-system design for improved clinical translation.

Highly luminescent metallacages featuring bispyridyl ligands functionalised with BODIPY for imaging in cancer cells.

Recently, 3-dimensional supramolecular coordination complexes of the metallacage type have been shown to hold promise as drug delivery systems for different cytotoxic agents, including the anticancer drug cisplatin. However, so far only limited information is available on their uptake and sub-cellular localisation in cancer cells. With the aim of understanding the fate of metallacages in cells by fluorescence microscopy, three fluorescent Pd2L4 metallacages were designed and synthesised by self-assembly of two types of bispyridyl ligands (L), exo-functionalised with boron dipyrromethene (BODIPY) moieties, with Pd(II) ions. The cages show high quantum yields and are moderately stable in the presence of physiologically relevant concentration of glutathione. Furthermore, the cages are able to encapsulate the anticancer drug cisplatin, as demonstrated by NMR spectroscopy. Preliminary cytotoxicity studies in a small panel of human cancer cells showed that the metallacages are scarcely toxic in vitro. The marked fluorescence due to BODIPY allowed us to visualise the cages' uptake and sub-cellular localisation inside melanoma cells using fluorescence microscopy, highlighting uptake via active transport mechanisms and accumulation in cytoplasmic vesicles.

Targeted delivery and endosomal cellular uptake of DARPin-siRNA bioconjugates: Influence of linker stability on gene silencing.

Specific cell targeting and efficient intracellular delivery are major hurdles for the widespread therapeutic use of nucleic acid technologies, particularly siRNA mediated gene silencing. To enable receptor-mediated cell-specific targeting, we designed a synthesis scheme that can be generically used to engineer Designed Ankyrin Repeat Protein (DARPin)-siRNA bioconjugates. Different linkers, including labile disulfide-, and more stable thiol-maleimide- and triazole- (click chemistry) tethers were employed. Crosslinkers were first attached to a 3'-terminal aminohexyl chain on the siRNA sense strands. On the protein side thiols of a C-terminal cysteine were used as anchoring sites for disulfide- and thiol-maleimide conjugate formations, while strain-promoted azido-alkyne cycloadditions were carried out at a metabolically introduced N-terminal azidohomoalanine. After establishing efficient purification methods, highly pure products were obtained. Bioconjugates of EpCAM-targeted DARPins with siRNA directed at the luciferase gene were evaluated for cell-specific binding, uptake and gene silencing. As shown by flow cytometry and fluorescence microscopy, all constructs retained the highly specific and high-affinity antigen recognition properties of the native DARPin. As expected, internalization was observed only in EpCAM-positive cell lines, and predominantly endolysosomal localization was detected. Disulfide linked conjugates showed lower serum stability against cleavage at the linker and thus lower internalization into endosomes compared to thiol-maleimide- and triazole-linked conjugates, yet induced more pronounced gene silencing. This indicates that the siRNA payload needs to be liberated from the protein in the endosome. Our data confirm the promise of DARPin-siRNA bioconjugates for tumor targeting, but also identified endosomal retention and limited cytosolic escape of the siRNA as the rate-limiting step for more efficient gene silencing.

Nanomedicines for the Delivery of Biologics.

A special symposium of the Academy of Pharmaceutical Sciences Nanomedicines Focus Group reviewed the current status of the use of nanomedicines for the delivery of biologics drugs. This meeting was particularly timely with the recent approval of the first siRNA-containing product Onpattro™ (patisiran), which is formulated as a lipid nanoparticle for intravenous infusion, and the increasing interest in the use of nanomedicines for the oral delivery of biologics. The challenges in delivering such molecules were discussed with specific emphasis on the delivery both across and into cells. The latest developments in Molecular Envelope Technology® (Nanomerics Ltd, London, UK), liposomal drug delivery (both from an academic and industrial perspective), opportunities offered by the endocytic pathway, delivery using genetically engineered viral vectors (PsiOxus Technologies Ltd, Abingdon, UK), Transint™ technology (Applied Molecular Transport Inc., South San Francisco, CA, USA), which has the potential to deliver a wide range of macromolecules, and AstraZeneca's initiatives in mRNA delivery were covered with a focus on their uses in difficult to treat diseases, including cancers. Preclinical data were presented for each of the technologies and where sufficiently advanced, plans for clinical studies as well as early clinical data. The meeting covered the work in progress in this exciting area and highlighted some key technologies to look out for in the future.