Difference in Endocytosis Pathways Used by Differentiated Versus Nondifferentiated Epithelial Caco-2 Cells to Internalize Nanosized Particles.

Understanding the internalization of nanosized particles by mucosal epithelial cells is essential in a number of areas including viral entry at mucosal surfaces, nanoplastic pollution, as well as design and development of nanotechnology-type medicines. Here, we report our comparative study on pathways of cellular internalization in epithelial Caco-2 cells cultured in vitro as either a polarized, differentiated cell layer or as nonpolarized, nondifferentiated cells. The study reveals a number of differences in the extent that endocytic processes are used by cells, depending on their differentiation status and the nature of applied nanoparticles. In polarized cells, actin-driven and dynamin-independent macropinocytosis plays a prominent role in the internalization of both positively and negatively charged nanoparticles, contrary to its modest contribution in nonpolarized cells. Clathrin-mediated cellular entry plays a prominent role in the endocytosis of positive nanoparticles and cholesterol inhibition in negative nanoparticles. However, in nonpolarized cells, dynamin-dependent endocytosis is a major pathway in the internalization of both positive and negative nanoparticles. Cholesterol depletion affects both nonpolarized and polarized cells' internalization of positive and negative nanoparticles, which, in addition to the effect of cholesterol-binding inhibitors on the internalization of negative nanoparticles, indicates the importance of membrane cholesterol in endocytosis. The data collectively provide a new contribution to understanding endocytic pathways in epithelial cells, particularly pointing to the importance of the cell differentiation stage and the nature of the cargo.

Use of Magnetic Resonance Imaging for Visualization of Oral Dosage Forms in the Human Stomach: A Scoping Review.

Oral dosage forms are the most widely and frequently used formulations to deliver active pharmaceutical ingredients (APIs), due to their ease of administration and noninvasiveness. Knowledge of intragastric release rates and gastric mixing is crucial for predicting the API release profile, especially for immediate release formulations. However, knowledge of the intragastric fate of oral dosage forms in vivo to date is limited, particularly for dosage forms administered when the stomach is in the fed state. An improved understanding of gastric food processing, dosage form location, disintegration times, and food effects is essential for greater understanding for effective API formulation design. In vitro standard and controlled modeling has played a significant role in predicting the behavior of dosage forms in vivo. However, discrepancies are reported between in vitro and in vivo disintegration times, with these discrepancies being greatest in the fed state. Studying the fate of a dosage form in vivo is a challenging process, usually requiring the use of invasive methods, such as intubation. Noninvasive, whole body imaging techniques can however provide unique insights into this process. A scoping review was performed systematically to identify and critically appraise published studies using MRI to visualize oral solid dosage forms in vivo in healthy human subjects. The review identifies that so far, an all-purpose robust contrast agent or dosage form type has not been established for dosage form visualization and disintegration studies in the gastrointestinal system. Opportunities have been identified for future studies, with particular focus on characterizing dosage form disintegration for development after the consumption food, as exemplified by the standard Food and Drug Administration (FDA) high fat meal.

Synthetic macromolecular peptide-mimetics with amino acid substructure residues as protein stabilising excipients.

The clinical use of protein and peptide biotherapeutics requires fabrication of stable products. This particularly concerns stability towards aggregation of proteins or peptides. Here, we tested a hypothesis that interactions between a synthetic peptide, which is an aggregation-prone region analogue, and its homologous sequence on a protein of interest, could be exploited to design excipients which stabilise the protein against aggregation. A peptide containing the analogue of lysozyme aggregation-prone region (GILQINSRW) was conjugated to a RAFT agent and used to initiate the polymerisation of N-hydroxyethyl acrylamide, generating a GILQINSRW-HEA90 polymer, which profoundly reduced lysozyme aggregation. Substitution of tryptophan in GILQINSRW with glycine, to form GILQINSRG, revealed that tryptophan is a critical amino acid in the protein stabilisation by GILQINSRW-HEA90. Accordingly, polymeric peptide-mimetics of tryptophan, phenylalanine and isoleucine, which are often present in aggregation-prone regions, were synthesized. These were based on synthetic oligomers of acrylamide derivatives of indole-3 acetic acid (IND), phenylacetic acid (PHEN), or 2-methyl butyric acid (MBA), respectively, conjugated with hydrophilic poly(N-hydroxyethyl acrylamide) blocks to form amphiphilic copolymers denoted as INDm-, PHENm- and MTBm-b-HEAn. These materials were tested as protein stabilisers and it was shown that solution properties and the abilities of these materials to stabilise insulin and the peptide IDR 1018 towards aggregation are dependent on the chemical nature of their side groups. These data suggest a structure-activity relationship, whereby the indole-based INDm-b-HEAn peptide-mimetic displays properties of a potential stabilising excipient for protein formulations.

Identifying new biomarkers of aggressive Group 3 and SHH medulloblastoma using 3D hydrogel models, single cell RNA sequencing and 3D OrbiSIMS imaging.

The most common malignant brain tumour in children, medulloblastoma (MB), is subdivided into four clinically relevant molecular subgroups, although targeted therapy options informed by understanding of different cellular features are lacking. Here, by comparing the most aggressive subgroup (Group 3) with the intermediate (SHH) subgroup, we identify crucial differences in tumour heterogeneity, including unique metabolism-driven subpopulations in Group 3 and matrix-producing subpopulations in SHH. To analyse tumour heterogeneity, we profiled individual tumour nodules at the cellular level in 3D MB hydrogel models, which recapitulate subgroup specific phenotypes, by single cell RNA sequencing (scRNAseq) and 3D OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) imaging. In addition to identifying known metabolites characteristic of MB, we observed intra- and internodular heterogeneity and identified subgroup-specific tumour subpopulations. We showed that extracellular matrix factors and adhesion pathways defined unique SHH subpopulations, and made up a distinct shell-like structure of sulphur-containing species, comprising a combination of small leucine-rich proteoglycans (SLRPs) including the collagen organiser lumican. In contrast, the Group 3 tumour model was characterized by multiple subpopulations with greatly enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle activity. Extensive TCA cycle metabolite measurements revealed very high levels of succinate and fumarate with malate levels almost undetectable particularly in Group 3 tumour models. In patients, high fumarate levels (NMR spectroscopy) alongside activated stress response pathways and high Nuclear Factor Erythroid 2-Related Factor 2 (NRF2; gene expression analyses) were associated with poorer survival. Based on these findings we predicted and confirmed that NRF2 inhibition increased sensitivity to vincristine in a long-term 3D drug treatment assay of Group 3 MB. Thus, by combining scRNAseq and 3D OrbiSIMS in a relevant model system we were able to define MB subgroup heterogeneity at the single cell level and elucidate new druggable biomarkers for aggressive Group 3 and low-risk SHH MB.

A mechanoresponsive nano-sized carrier achieves intracellular release of drug on external ultrasound stimulus.

Control over intracellular release of therapeutic compounds incorporated into nano-carriers will open new possibilities for targeted treatments of various diseases including cancer, and viral and bacterial infections. Here we report our study on mechanoresponsive nano-sized liposomes which, following internalization by cells, achieve intracellular delivery of encapsulated cargo on application of external ultrasound stimulus. This is demonstrated in a bespoke cell reporter system designed to assess free drug in cytoplasm. Biophysical analyses show that drug release is attributable to the action of a mechanoresponsive spiropyran-based compound embedded in the liposomal lipid membrane. Exposure to external ultrasound stimulus results in opening of the molecular structure of the embedded spiropyran, a consequent increase in liposomal lipid membrane fluidity, and size-dependent release of encapsulated model drugs, all pointing to lipid bilayer perturbation. The study hence illustrates feasibility of the proposed concept where intracellular drug release from mechanoresponsive liposomes can be triggered on demand by external ultrasound stimulus.

Application of In Vivo MRI Imaging to Track a Coated Capsule and Its Disintegration in the Gastrointestinal Tract in Human Volunteers.

Oral specially coated formulations have the potential to improve treatment outcomes of a range of diseases in distal intestinal tract whilst limiting systemic drug absorption and adverse effects. Their development is challenging, partly because of limited knowledge of the physiological and pathological distal gastrointestinal factors, including colonic chyme fluid distribution and motor function. Recently, non-invasive techniques such as magnetic resonance imaging (MRI) have started to provide novel important insights. In this feasibility study, we formulated a coated capsule consisting of a hydroxypropyl methylcellulose (HPMC) shell, coated with a synthetic polymer based on polymethacrylate-based copolymer (Eudragit®) that can withstand the upper gastrointestinal tract conditions. The capsule was filled with olive oil as MRI-visible marker fluid. This allowed us to test the ability of MRI to track such a coated capsule in the gastrointestinal tract and to assess whether it is possible to image its loss of integrity by exploiting the ability of MRI to image fat and water separately and in combination. Ten healthy participants were administered capsules with varying amounts of coating and underwent MRI imaging of the gastrointestinal tract at 45 min intervals. The results indicate that it is feasible to track the capsules present in the gastrointestinal tract at different locations, as they were detected in all 10 participants. By the 360 min endpoint of the study, in nine participants the capsules were imaged in the small bowel, in eight participants in the terminal ileum, and in four in the colon. Loss of capsule integrity was observed in eight participants, occurring predominantly in distal intestinal regions. The data indicate that the described approach could be applied to assess performance of oral formulations in undisturbed distal gastrointestinal regions, without the need for ionizing radiation or contrast agents.

Assessing Lymphatic Uptake of Lipids Using Magnetic Resonance Imaging: A Feasibility Study in Healthy Human Volunteers with Potential Application for Tracking Lymph Node Delivery of Drugs and Formulation Excipients.

Dietary lipids and some pharmaceutical lipid excipients can facilitate the targeted delivery of drugs to the intestinal lymphatics. Here, the feasibility of magnetic resonance imaging (MRI) for imaging lipid uptake into the intestinal lymphatics was assessed, shedding light on which lymph nodes can be targeted using this approach. Three healthy male volunteers were scanned at 3.0 T at baseline, 120, 180, 240, and 300 min post high-fat meal. A sagittal multi-slice image was acquired using a diffusion-weighted whole-body imaging sequence with background suppression (DWIBS) (pre inversion TI = 260 ms). Changes in area, major, and minor axis length were compared at each time point. Apparent diffusion coefficient (ADC) was calculated (b = 0 and 600 s/mm2) across eight slices. An average of 22 nodes could be visualised across all time points. ADC increased at 120 and 180 min compared to the baseline in all three participants by an average of 9.2% and 6.8%, respectively. In two participants, mean node area and major axis lengths increased at 120 and 180 min relative to the baseline. In conclusion, the method described shows potential for repeated lymph node measurements and the tracking of lipid uptake into the lymphatics. Further studies should focus on methodology optimisation in a larger cohort.

Structural and binding characterization of the LacdiNAc-specific adhesin (LabA; HopD) exodomain from Helicobacter pylori.

Helicobacter pylori (H. pylori) uses several outer membrane proteins for adhering to its host's gastric mucosa, an important step in establishing and preserving colonization. Several adhesins (SabA, BabA, HopQ) have been characterized in terms of their three-dimensional structure. A recent addition to the growing list of outer membrane porins is LabA (LacdiNAc-binding adhesin), which is thought to bind specifically to GalNAcß1-4GlcNAc, occurring in the gastric mucosa. LabA47-496 protein expressed as His-tagged protein in the periplasm of E. coli and purified via subtractive IMAC after TEV cleavage and subsequent size exclusion chromatography, resulted in bipyramidal crystals with good diffraction properties. Here, we describe the 2.06 â€‹Å resolution structure of the exodomain of LabA from H. pylori strain J99 (PDB ID: 6GMM). Strikingly, despite the relatively low levels of sequence identity with the other three structurally characterized adhesins (20-49%), LabA shares an L-shaped fold with SabA and BabA. The 'head' region contains a 4 â€‹+ â€‹3 α-helix bundle, with a small insertion domain consisting of a short antiparallel beta sheet and an unstructured region, not resolved in the crystal structure. Sequence alignment of LabA from different strains shows a high level of conservation in the N- and C-termini, and identifies two main types based on the length of the insertion domain ('crown' region), the 'J99-type' (insertion ~31 â€‹amino acids), and the H. pylori '26695 type' (insertion ~46 â€‹amino acids). Analysis of ligand binding using Native Electrospray Ionization Mass Spectrometry (ESI-MS) together with solid phase-bound, ELISA-type assays could not confirm the originally described binding of GalNAcß1-4GlcNAc-containing oligosaccharides, in line with other recent reports, which also failed to confirm LacdiNAc binding.

Multi-component bioresponsive nanoparticles for synchronous delivery of docetaxel and TUBB3 siRNA to lung cancer cells.

Bioresponsive nanoparticles (NPs) are of interest for anticancer nanomedicines, owing to the possibility to 'design in' selective modulation of drug release at target sites. Here we describe the double emulsion formulation of redox-responsive NPs based on modified polyethylene glycol (PEG)-co-poly(lactic-co-glycolic acid) (PLGA) block copolymers and oligo (ß-aminoesters) (OBAE), both of which contained disulfide linkages, for the co-delivery of a cytotoxic small molecule drug and a nucleic acid. In particular, we focused our attention on docetaxel (DTX) and a siRNA against TUBB3, a gene that encodes for ßIII-tubulin, in order to have a synergistic effect in the treatment of lung cancer. Spherical NPs of around 150 nm with negative zeta potential and high loading efficiencies of both drugs were obtained. Stability and release studies showed "on demand" drug release under reducing conditions. Unloaded NPs containing PEG-disulfide-PLGA and OBAE were well-tolerated by lung cancer cells, thus masking the intrinsic cytotoxicity of OBAE, while for intracellular siRNA delivery, redox responsive NPs demonstrated a higher cell internalization with a preferential cytoplasmic accumulation of siRNA, with a subsequent fast gene-silencing efficiency. The viability of cells treated with combined DTX/TUBB3-siRNA NPs significantly decreased as compared to NPs loaded only with DTX, thus showing an efficient combined anticancer effect, due to a substantial reduction of ß-tubulin expression. Finally, in an in vivo feasibility study employing an orthotopic lung cancer model, NPs formulated with an anti-luciferase siRNA distributed throughout the lungs following oro-tracheal administration, and demonstrated effective gene knockdown and no apparent cytotoxicity. Taken together, these results show that the double emulsion formulated redox responsive PEG-PLGA and OBAE systems represent a promising new therapeutic approach for the local combined chemo- and gene-therapy of lung cancer.

3D hydrogels reveal medulloblastoma subgroup differences and identify extracellular matrix subtypes that predict patient outcome.

Medulloblastoma (MB) is the most common malignant brain tumour in children and is subdivided into four subgroups: WNT, SHH, Group 3, and Group 4. These molecular subgroups differ in their metastasis patterns and related prognosis rates. Conventional 2D cell culture methods fail to recapitulate these clinical differences. Realistic 3D models of the cerebellum are therefore necessary to investigate subgroup-specific functional differences and their role in metastasis and chemoresistance. A major component of the brain extracellular matrix (ECM) is the glycosaminoglycan hyaluronan. MB cell lines encapsulated in hyaluronan hydrogels grew as tumour nodules, with Group 3 and Group 4 cell lines displaying clinically characteristic laminar metastatic patterns and levels of chemoresistance. The glycoproteins, laminin and vitronectin, were identified as subgroup-specific, tumour-secreted ECM factors. Gels of higher complexity, formed by incorporation of laminin or vitronectin, revealed subgroup-specific adhesion and growth patterns closely mimicking clinical phenotypes. ECM subtypes, defined by relative levels of laminin and vitronectin expression in patient tissue microarrays and gene expression data sets, were able to identify novel high-risk MB patient subgroups and predict overall survival. Our hyaluronan model system has therefore allowed us to functionally characterize the interaction between different MB subtypes and their environment. It highlights the prognostic and pathological role of specific ECM factors and enables preclinical development of subgroup-specific therapies. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Use of Engineered Nanoparticles (ENPs) for the Study of High-Affinity IgE FcεRI Receptor Engagement and Rat Basophilic Leukemia (RBL) Cell Degranulation.

Degranulation of mast cells and basophils occurs after the cross-linking of FcεRI receptor-bound IgE by multivalent allergens, resulting in the release of a range of de novo synthesized and preformed mediators of the allergic response. ß-Hexosaminidase release is usually measured as a simple readout for degranulation. Furthermore, the rat basophilic leukemia (RBL)-2H3 cell line is commonly used for measuring degranulation, monitoring ß-hexosaminidase release. Here, we describe surface-engineered and modified nanoparticles with specific ligands in order to study the signaling and cellular responses of the RBL-2H3 cell line.

Introduction of a C-terminal hexa-lysine tag increases thermal stability of the LacDiNac binding adhesin (LabA) exodomain from Helicobacter pylori.

Helicobacter pylori is a pathogenic microorganism infecting approximately 50% of the global population, and establishes life-long colonization despite the hostile stomach environment. H. pylori employs a wide range of outer membrane proteins (adhesins) for epithelial attachment, which specifically bind to glycans or non-carbohydrate structures expressed on the gastric epithelium. A recently described adhesin from H. pylori is LabA, named after its ability to bind to a disaccharide present in gastric mucus (LacdiNAc-specific adhesin). Here, we describe the recombinant expression of LabA from H. pylori strains J99 and 26695 in E. coli. High yields of recombinant LabA were obtained using periplasmic expression. We found that the addition of a C-terminal hexalysine (6K) tag enhanced the thermal stability of LabA without affecting its secondary structure, using differential scanning fluorimetry and circular dichroism spectroscopy. In contrast to our previous report for another H. pylori adhesin (BabA), the 6K tag did not enhance recombinant protein yield or solubility. Both versions of LabA, with or without the 6K tag, were expressed and isolated from the periplasmic space of Escherichia coli, with a surprisingly high yield of at least 40 mg/L for each independent preparation, following a two-step purification protocol. The proteins were analyzed with mass spectrometry (MS). Unlike its reported effect on stability of BabA, the 6K tag did not appear to protect the N-term of recombinant LabA from partial periplasmic degradation.

Mechanistic insight into heterogeneity of trans-plasma membrane electron transport in cancer cell types.

Trans-plasma membrane electron transfer (tMPET) is a process by which reducing equivalents, either electrons or reductants like ascorbic acid, are exported to the extracellular environment by the cell. TPMET is involved in a number of physiological process and has been hypothesised to play a role in the redox regulation of cancer metabolism. Here, we use a new electrochemical assay to elucidate the 'preference' of cancer cells for different trans tPMET systems. This aids in proving a biochemical framework for the understanding of tPMET role, and for the development of novel tPMET-targeting therapeutics. We have delineated the mechanism of tPMET in 3 lung cancer cell models to show that the external electron transfer is orchestrated by ascorbate mediated shuttling via tPMET. In addition, the cells employ a different, non-shuttling-based mechanism based on direct electron transfer via Dcytb. Results from our investigations indicate that tPMETs are used differently, depending on the cell type. The data generated indicates that tPMETs may play a fundamental role in facilitation of energy reprogramming in malignant cells, whereby tPMETs are utilised to supply the necessary energy requirement when mitochondrial stress occurs. Our findings instruct a deeper understanding of tPMET systems, and show how different cancer cells may preferentially use distinguishable tPMET systems for cellular electron transfer processes.

Penetration and Uptake of Nanoparticles in 3D Tumor Spheroids.

Animal models are effective for assessing tumor localization of nanosystems but difficult to use for studying penetration beyond the vasculature. Here, we have used well-characterized HCT116 colorectal cancer spheroids to study the effect of nanoparticle (NP) physicochemical properties on penetration and uptake. Incubation of spheroids with Hoechst 33342 resulted in a dye gradient, which facilitated discrimination between the populations of cells in the core and at the periphery of spheroids by flow cytometry. This approach was used to compare doxorubicin and liposomal doxorubicin (Caelyx) and a range of model poly(styrene) nanoparticles of different sizes (30 nm, 50 nm, 100 nm) and with different surface chemistries (50 nm uniform plain, carboxylated, aminated and a range of NPs and polyethylene glycol modified NPs prepared from a promising new functionalized biodegradable polymer (poly(glycerol-adipate), PGA). Unmodified poly(styrene) nanoparticles (30 nm/50 nm) were able to penetrate to the core of HCT116 spheroids more efficiently than larger poly(styrene) nanoparticles (100 nm). Surprisingly, penetration of 30 and 50 nm particles was as good as clinically relevant doxorubicin concentrations. However, penetration was reduced with higher surface charge. PGA NPs of 100 nm showed similar penetration into spheroids as 50 nm poly(styrene) nanoparticles, which may be related to polymer flexibility. PEG surface modification of polymeric particles significantly improved penetration into the spheroid core. The new model combining the use of spheroids Hoechst staining and flow cytometry was a useful model for assessing NP penetration and gives useful insights into the effects of NPs' physical properties when designing nanomedicines.

Exposure to a Nonionic Surfactant Induces a Response Akin to Heat-Shock Apoptosis in Intestinal Epithelial Cells: Implications for Excipients Safety.

Amphipathic, nonionic, surfactants are widely used in pharmaceutical, food, and agricultural industry to enhance product features; as pharmaceutical excipients, they are also aimed at increasing cell membrane permeability and consequently improving oral drugs absorption. Here, we report on the concentration- and time-dependent succession of events occurring throughout and subsequent exposure of Caco-2 epithelium to a "typical" nonionic surfactant (Kolliphor HS15) to provide a molecular explanation for nonionic surfactant cytotoxicity. The study shows that the conditions of surfactant exposure, which increase plasma membrane fluidity and permeability, produced rapid (within 5 min) redox and mitochondrial effects. Apoptosis was triggered early during exposure (within 10 min) and relied upon an initial mitochondrial membrane hyperpolarization (5-10 min) as a crucial step, leading to its subsequent depolarization and caspase-3/7 activation (60 min). The apoptotic pathway appears to be triggered prior to substantial surfactant-induced membrane damage (observed ≥60 min). We hence propose that the cellular response to the model nonionic surfactant is triggered via surfactant-induced increase in plasma membrane fluidity, a phenomenon akin to the stress response to membrane fluidization induced by heat shock, and consequent apoptosis. Therefore, the fluidization effect that confers surfactants the ability to enhance drug permeability may also be intrinsically linked to the propagation of their cytotoxicity. The reported observations have important implications for the safety of a multitude of nonionic surfactants used in drug delivery formulations and to other permeability enhancing compounds with similar plasma membrane fluidizing mechanisms.

Mammalian-Cell-Driven Polymerisation of Pyrrole.

A model cancer cell line was used to initiate polymerisation of pyrrole to form the conducting material polypyrrole. The polymerisation was shown to occur through the action of cytosolic exudates rather than that of the membrane redox sites that normally control the oxidation state of iron as ferricyanide or ferrocyanide. The data demonstrate for the first time that mammalian cells can be used to initiate synthesis of conducting polymers and suggest a possible route to detection of cell damage and/or transcellular processes through in situ and amplifiable signal generation.

New Perspectives on Iron Uptake in Eukaryotes.

All eukaryotic organisms require iron to function. Malfunctions within iron homeostasis have a range of physiological consequences, and can lead to the development of pathological conditions that can result in an excess of non-transferrin bound iron (NTBI). Despite extensive understanding of iron homeostasis, the links between the "macroscopic" transport of iron across biological barriers (cellular membranes) and the chemistry of redox changes that drive these processes still needs elucidating. This review draws conclusions from the current literature, and describes some of the underlying biophysical and biochemical processes that occur in iron homeostasis. By first taking a broad view of iron uptake within the gut and subsequent delivery to tissues, in addition to describing the transferrin and non-transferrin mediated components of these processes, we provide a base of knowledge from which we further explore NTBI uptake. We provide concise up-to-date information of the transplasma electron transport systems (tPMETSs) involved within NTBI uptake, and highlight how these systems are not only involved within NTBI uptake for detoxification but also may play a role within the reduction of metabolic stress through regeneration of intracellular NAD(P)H/NAD(P)+ levels. Furthermore, we illuminate the thermodynamics that governs iron transport, namely the redox potential cascade and electrochemical behavior of key components of the electron transport systems that facilitate the movement of electrons across the plasma membrane to the extracellular compartment. We also take account of kinetic changes that occur to transport iron into the cell, namely membrane dipole change and their consequent effects within membrane structure that act to facilitate transport of ions.

Publisher Correction: Pathways of cellular internalisation of liposomes delivered siRNA and effects on siRNA engagement with target mRNA and silencing in cancer cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Pathways of cellular internalisation of liposomes delivered siRNA and effects on siRNA engagement with target mRNA and silencing in cancer cells.

Design of an efficient delivery system is a generally recognised bottleneck in translation of siRNA technology into clinic. Despite research efforts, cellular processes that determine efficiency of siRNA silencing achieved by different delivery formulations remain unclear. Here, we investigated the mechanism(s) of cellular internalisation of a model siRNA-loaded liposome system in a correlation to the engagement of delivered siRNA with its target and consequent silencing by adopting siRNA molecular beacon technology. Probing of cellular internalisation pathways by a panel of pharmacological inhibitors indicated that clathrin-mediated (dynamin-dependent) endocytosis, macropinocytosis (dynamine independent), and cell membrane cholesterol dependent process(es) (clathrin and caveolea-independent) all play a role in the siRNA-liposomes internalization. The inhibition of either of these entry routes was, in general, mirrored by a reduction in the level of siRNA engagement with its target mRNA, as well as in a reduction of the target gene silencing. A dramatic increase in siRNA engagement with its target RNA was observed on disruption of endosomal membrane (by chloroquine), accompanied with an increased silencing. The work thus illustrates that employing molecular beacon siRNA technology one can start to assess the target RNA engagement - a stage between initial cellular internalization and final gene silencing of siRNA delivery systems.

Dry-powder formulations of non-covalent protein complexes with linear or miktoarm copolymers for pulmonary delivery.

Pulmonary delivery of protein therapeutics has considerable clinical potential for treating both local and systemic diseases. However, poor protein conformational stability, immunogenicity and protein degradation by proteolytic enzymes in the lung are major challenges to overcome for the development of effective therapeutics. To address these, a family of structurally related copolymers comprising polyethylene glycol, mPEG2k, and poly(glutamic acid) with linear A-B (mPEG2k-lin-GA) and miktoarm A-B3 (mPEG2k-mik-(GA)3) macromolecular architectures was investigated as potential protein stabilisers. These copolymers form non-covalent nanocomplexes with a model protein (lysozyme) which can be formulated into dry powders by spray-drying using common aerosol excipients (mannitol, trehalose and leucine). Powder formulations with excellent aerodynamic properties (fine particle fraction of up to 68%) were obtained with particle size (D50) in the 2.5 µm range, low moisture content (<5%), and high glass transitions temperatures, i.e. formulation attributes all suitable for inhalation application. In aqueous medium, dry powders rapidly disintegrated into the original polymer-protein nanocomplexes which provided protection towards proteolytic degradation. Taken together, the present study shows that dry powders based on (mPEG2k-polyGA)-protein nanocomplexes possess potentials as an inhalation delivery system.