Net Neutral Charge Research Articles

Fouling resistance of novel amphiphilic polyampholyte membranes with varying hydrophobicity towards common foulants and cell culture media

Self-assembly is a spontaneous and scalable manufacturing method to create much-needed fouling resistant membranes with narrow pore size distributions. However, there is a limited number of self-assembled membrane chemistries that can access pore sizes below 2 nm. Novel thin film composite membranes with a selective layer manufactured from a random Polyampholyte Amphiphilic Copolymer (r-PAC) have an effective pore size of 1.5 nm, relevant to various nanofiltration and water treatment applications. This is the first study that reports the membrane performance of novel r-PAC layers, prepared with varying degrees of ionic content, in filtering several classes of foulants (oil, organics, proteins, and complex mixtures). The net neutral charge of the r-PACs used enabled steady salt rejections independent of the ionic strength of the feed. The best performing r-PAC membrane composition showed no irreversible flux decline in all fouling tests and was further tested with a realistic feed, a complex cell culture media mixture utilized in cellular agriculture. This membrane exhibited an impressive 0 % irreversible flux decline after filtering the cell culture media for 20 h while a comparable commercial membrane exhibited 90 % irreversible flux decline. The chemical stability and excellent fouling resistance behavior of these membrane materials to a broad range of feeds implies these materials can enable the filtration of feeds with extreme fouling potential in biomanufacturing, wastewater treatment, and resource recovery applications.

Can membrane permeability of zwitterionic compounds be predicted by the solubility-diffusion model?

Zwitterions contain both positively and negatively charged functional groups, resulting in an overall net neutral charge. Nevertheless, the membrane permeability of the zwitterionic form of a compound is assumed to be much lower than the permeability of the uncharged neutral form. Although a significant proportion of pharmaceuticals are zwitterionic, it has not been clear so far whether their permeability is dominated by the permeation of the zwitterionic or the neutral form, since neutral fractions are often quite low as compared to the zwitterionic fraction. This complicates the in silico prediction of the permeability of zwitterionic compounds. In this work, we re-evaluated existing in vitro permeability data from literature measured with Caco-2/MDCK cell assays, using more strict exclusion criteria for effects like diffusion limitation by the aqueous boundary layers, paracellular transport, active transport and retention. Using this re-evaluated data set, we show that extracted intrinsic permeabilities of the neutral fraction are well predicted by the solubility-diffusion model (RMSE = 1.21; n = 18) if the permeability of the zwitterionic species is assumed negligible. Our work thus suggests that only the neutral species is relevant for the membrane permeability of zwitterionic compounds, and that membrane permeability of zwitterionic compounds is indeed predictable by the solubility-diffusion model.

Inner leaflet cationic lipid increases nucleic acid loading independently of outer leaflet lipid charge in asymmetric liposomes.

Use of cationic lipid vesicles (liposomes) can yield large amounts of nucleic acid entrapped inside the vesicles and/or bound to the external surface of the vesicles. To show a method to prepare asymmetric lipid vesicles (liposomes) with high amounts of entrapped nucleic acid is possible, symmetric and asymmetric lipid vesicles composed of mixtures of neutral (zwitterionic), anionic, and/or cationic phospholipids were formed in the presence of oligo DNA. For symmetric large unilamellar vesicles nucleic acid association with vesicles was roughly 100 times greater for vesicles with a net cationic charge than for vesicles having a net neutral or anionic net charge. A high degree of association between nucleic acid and lipid was also achieved using asymmetric large unilamellar vesicles with a net cationic charge in their inner leaflet, even when they had an anionic charge in their outer leaflet. In contrast, asymmetric vesicles in which only the outer leaflet had a net cationic charge had only low amounts of vesicle-associated nucleic acid, similar in amount to the amount of nucleic acid associated with asymmetric vesicles with an outer leaflet having a net anionic charge. These results indicate that in asymmetric vesicles with cationic lipid enriched inner leaflets nucleic acid is largely entrapped inside the vesicle lumen rather than bound to their external surface, and that asymmetric vesicles can be used to trap high amounts of nucleic acid even when using a lipid composition in the outer leaflet of a lipid vesicle that does not associate with nucleic acids. Such asymmetrically charged vesicles should have applications in studies of membrane protein-nucleic acid interactions as well as in studies of how membrane charge asymmetry can influence membrane protein structure, orientation, and function.

Adsorption of Malachite Green Dye Using Carbonized Water Lily Leaves: Kinetics, Equilibrium and Thermodynamics Studies

This study aimed to investigate the potential of carbonized water lily leaves (CWL) as cheap adsorbent for the adsorption of Malachite Green (MG) dye. The CWL characteristics were studied using Fourier Transform Infra-Red (FTIR) spectroscopy, Scanning Electron microscopy (SEM) and point of zero charge (PZC) to establish surface functional groups present, morphology and net neutral charge of the adsorbent. The effects of contact time (15-150min), dosage (20-200mg), initial concentration (20-140ppm), and pH (3-13) on the adsorption process were investigated for the removal of MG by batch adsorption method. The result shows that all the parameters have effect on adsorption process. Compared to the Langmuir, Freundlich, Temkin, and D-R models, the isotherm models were investigated and confirmed to fit well onto the D-R model. Pseudo-second order model best fitted the kinetic data for the different operating temperature (300C, 400C, 500C) of the analysis. Thermodynamics studies clearly indicates that the sorption was spontaneous, endothermic, and increased in the randomness of the systems at adsorbent-liquid interfaces as a result of negative Gibb’s free energy (∆G), positive enthalpy (∆H) and Entropy (∆S). Therefore, this study confirmed that carbonized water lily leaves adsorbent could be used as an alternative adsorbent for the adsorption of toxic dyes such as Malachite green dye.

How Cargo Identity Alters the Uptake of Cell-Penetrating Peptide (CPP)/Cargo Complexes: A Study on the Effect of Net Cargo Charge and Length.

Cell-penetrating peptides (CPPs) have emerged as a powerful tool for the delivery of otherwise impermeable cargoes into intact cells. Recent efforts to improve the delivery capability of peptides have mainly focused on the identity of the CPP; however, there is evidence that the identity of the cargo itself affects the uptake. The goal of this work was to investigate how the characteristics of a peptide cargo, including net charge and length, either enhance or diminish the internalization efficiency of the CPP/cargo complex. A small library of CPP/cargo complexes were synthesized consisting of structured and unstructured CPPs with cargoes of net positive, negative, or neutral charge and lengths of 4 or 8 amino acids. Cargoes with a net positive charge were found to enhance the overall uptake of the complexes while net neutral and negatively charged cargoes diminished uptake. Conversely, the net length of the cargo had no significant effect on uptake of the CPP/cargo complexes. Microcopy images confirmed the increased uptake of the positively charged cargoes; however, an increase in punctate regions with the addition of a cargo was also observed. The effects of the net positively charged cargoes were confirmed with both structured and unstructured CPPs, which demonstrated similar trends of an increase in uptake with the addition of positively charged residues. These findings demonstrate that the net charge of cargoes impacts the uptake of the complex, which can be considered in the future when designing peptide-based reporters or therapeutics.

Phosphorylation tunes elongation propensity and cohesiveness of INCENP’s intrinsically disordered region

The inner centromere protein, INCENP, is crucial for correct chromosome segregation during mitosis. It connects the kinase Aurora B to the inner centromere allowing this kinase to dynamically access its kinetochore targets. However, the function of its central, 440-residue long intrinsically disordered region (IDR) and its multiple phosphorylation sites is unclear. Here, we determined the conformational ensemble of INCENP’s IDR, systematically varying the level of phosphorylation, using all-atom and coarse-grain molecular dynamics simulations. Our simulations show that phosphorylation expands INCENP’s IDR, both locally and globally, mainly by increasing its overall net charge. The disordered region undergoes critical globule-to-coil conformational transitions and the transition temperature non-monotonically depends on the degree of phosphorylation, with a mildly phosphorylated case of neutral net charge featuring the highest collapse propensity. The IDR transitions from a multitude of globular states, accompanied by several specific internal contacts that reduce INCENP length by loop formation, to weakly interacting and highly extended coiled conformations. Phosphorylation critically shifts the population between these two regimes. It thereby influences cohesiveness and phase behavior of INCENP IDR assemblies, a feature presumably relevant for INCENP’s function in the chromosomal passenger complex. Overall, we propose the disordered region of INCENP to act as a phosphorylation-regulated and length-variable component, within the previously defined “dog-leash” model, that thereby regulates how Aurora B reaches its targets for proper chromosome segregation.

Abstract P061: De novo design of importin-a-specific NLS sequences for nuclear-targeted therapeutics

Abstract Background: Research focused on the application of nuclear localization sequence (NLS)-based therapeutics has been a topic of intense interest to medicine since the core principles governing nuclear transport were awarded the Nobel Prize in 1999. Despite these efforts, efficient nuclear localization has been difficult. A major obstacle is that NLSs have cationic-net charges (abundance of lysines/arginines) required for appropriate interactions with the nuclear transporter importin-a (Impa). Once in the blood stream, cationic charges can cause strong non-specific cellular uptake and are rapidly cleared from the plasma compartment, which prevents sufficient tumor cell uptake and, hence, nuclear localization. Here, describe a de novo computational approach for generating 43 novel NLSs (based on 3 different NLS classes) that contained amino acid substitutions to achieve net-neutral charge states. We also introduce a novel nuclear isolation-quantitative flow cytometric method for determining nuclear localization efficiency. Material and methods An algorithm was created based on complete interaction binding affinity strengths for 20 x 20 pairs of octapeptides consisting of the 20 common amino acids. 9 PDB files were selected as templates and were comprised of viral, RNA processing, and transcription initiation protein NLSs bound to Impa. In silico alanine scanning on all NLS templates generated rankings on amino acid sensitivities for each position in the sequences. Non-sensitive residues were then subjected to residue scanning for generating net-neutral charged NLSs. Computational docking studies generated predictive binding scores relative to wild type NLSs. Favorable NLS candidates were genetically fused to GFP. In contrast to utilizing fluorescence microscopy, which cannot determine nuclear localization efficiency, we created a method to isolate nuclei from transiently transfected CHOK1 cells and quantify nuclear localization by flow cytometry. Results 2-8 net-neutral NLSs with good binding for Imga could be generated for each PDB file. The net-neutral mutants often contained an abundance of glutamic and/or aspartic acid substitutions, and were able to bind to Impa residues to compensate for the replaced amino acids. Transfected GFP-NLS constructs displayed variable fluorescence expression kinetics. Therefore, we created an in-house plasma membrane lysis protocol to isolate intact nuclei. Quantitative evaluations are currently underway by evaluating nuclei fluorescence by flow cytometry at various time points. Thus far, the tested GFP-NLSs are able to localize to the nucleus. Conclusions An important objective of computational protein design is the generation of high affinity peptides as a precursor to the development of therapeutics, and as a tool to aid researchers in understanding governing interaction principles of specific complexes. We have achieved both the development of potential NLS peptides for overcoming the cationic-sequestration barrier, and for understanding novel NLS principles to further advance the NLS-therapeutics field. Citation Format: Olga Bednova, Alexis Rioux-Chevalier, Dipika Patel, Mathieu Boudreau, Jeffrey Victor Leyton. De novo design of importin-a-specific NLS sequences for nuclear-targeted therapeutics [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P061.

Myristoylation alone is sufficient for PKA catalytic subunits to associate with the plasma membrane to regulate neuronal functions

Myristoylation is a posttranslational modification that plays diverse functional roles in many protein species. The myristate moiety is considered insufficient for protein-membrane associations unless additional membrane-affinity motifs, such as a stretch of positively charged residues, are present. Here, we report that the electrically neutral N-terminal fragment of the protein kinase A catalytic subunit (PKA-C), in which myristoylation is the only functional motif, is sufficient for membrane association. This myristoylation can associate a fraction of PKA-C molecules or fluorescent proteins (FPs) to the plasma membrane in neuronal dendrites. The net neutral charge of the PKA-C N terminus is evolutionally conserved, even though its membrane affinity can be readily tuned by changing charges near the myristoylation site. The observed membrane association, while moderate, is sufficient to concentrate PKA activity at the membrane by nearly 20-fold and is required for PKA regulation of AMPA receptors at neuronal synapses. Our results indicate that myristoylation may be sufficient to drive functionally significant membrane association in the absence of canonical assisting motifs. This provides a revised conceptual base for the understanding of how myristoylation regulates protein functions.

Implementing and Assessing an Alchemical Method for Calculating Protein-Protein Binding Free Energy.

Protein-protein binding is fundamental to most biological processes. It is important to be able to use computation to accurately estimate the change in protein-protein binding free energy due to mutations in order to answer biological questions that would be experimentally challenging, laborious, or time-consuming. Although nonrigorous free-energy methods are faster, rigorous alchemical molecular dynamics-based methods are considerably more accurate and are becoming more feasible with the advancement of computer hardware and molecular simulation software. Even with sufficient computational resources, there are still major challenges to using alchemical free-energy methods for protein-protein complexes, such as generating hybrid structures and topologies, maintaining a neutral net charge of the system when there is a charge-changing mutation, and setting up the simulation. In the current study, we have used the pmx package to generate hybrid structures and topologies, and a double-system/single-box approach to maintain the net charge of the system. To test the approach, we predicted relative binding affinities for two protein-protein complexes using a nonequilibrium alchemical method based on the Crooks fluctuation theorem and compared the results with experimental values. The method correctly identified stabilizing from destabilizing mutations for a small protein-protein complex, and a larger, more challenging antibody complex. Strong correlations were obtained between predicted and experimental relative binding affinities for both protein-protein systems.

59 Poster Discussion - NLS designed algorithm to develop peptides with a net-neutral charge as potential next-generation vectors for drug delivery to the nucleus

Background: Active import of proteins in the nucleus is primarily determined by the recognition and binding of nuclear localization signals (NLSs) contained in cargo proteins with specific NLS receptors known as importins. Typically, cargo proteins are bound by the adaptor protein importin-a. Importin-b then binds importin-a and the complex is transported and docks at the nuclear pore complex where the cargo is released into the nucleus. As a result, there is great effort to develop NLS-tagged vectors for numerous therapeutic applications that involve maximizing effectiveness via localization inside the nucleus. Despite these efforts, efficient nuclear localization has been difficult.

Hydration State Variation of Polyzwitterion Brushes through Interplay with Ions.

Polyzwitterions have emerged as a new class of antifouling materials alternating poly(ethylene glycol). The exemplary biopassivation and lubrication behaviors are often attributed to the particular chemical structure of zwitterions, which involve a large dipole moment of the charged groups and a neutral net charge, while the hydration state and dynamics also associate with these characteristics. Polymer brushes composed of surface-tethered polyzwitterion chains produced by surface-initiated controlled radical polymerization have been developed as thin films which exhibit excellent antifouling and lubrication properties. In past decades, numerous studies have been devoted to examining the structure and dynamics of polyzwitterion brush chains in aqueous solutions. This feature article provides an overview of recent studies exploring the hydration state of polyzwitterion brushes with specular neutron reflectivity, highlights some newly published work on the nonuniform equilibrium structure, ion concentration dependence, ion specificity, and the effects of charge spacer length in the zwitterions, and discusses future perspective in this field.

Effect of Surface Charges on Oral Absorption of Intact Solid Lipid Nanoparticles.

Surface charge is a crucial factor that determines the in vivo behaviors of drug nanocarriers following administration via different routes. However, there is still a lack of comprehensive knowledge of how surface charges affect the in vivo behaviors of particles, especially for oral delivery. In this study, solid lipid nanoparticles (SLNs), as model drug nanocarriers, are modified to bear either anionic, cationic, or net neutral surface charges. The effect of surface charges on oral absorption of intact SLNs was investigated by tracking the in vivo transport of the particles. The fluorescent bioimaging strategy exploits the aggregation-caused quenching property to discriminate the particles. Both in vitro and in vivo lipolysis studies confirm slowed-down lipolysis by anionic charges in comparison with both unmodified and net neutral SLNs but accelerated degradation by cationic charges. The scanning of ex vivo tissues and organs reveals limited absorption of unmodified SLNs into the circulation. Nevertheless, all three types of surface charge modifications are able to enhance the oral absorption of intact SLNs with the fastest and highest absorption observed for net neutral SLNs, possibly owing to promoted mucus penetration. Anionic SLNs, though repulsed by the mucus layer, show the second highest absorption owing to enhanced lymphatic transport. The efficacy of cationic charge modification is less significant due to entrapment and retention in mucus layers as well as increased lability to lipolysis. In conclusion, surface charges may serve as initiators to guide the in vivo behaviors and enhance the oral absorption of intact SLNs.

Mucus-penetrating phage-displayed peptides for improved transport across a mucus-like model

The objective of this work is to use phage display libraries as a screening tool to identify peptides that facilitate transport across the mucus barrier. Mucus is a complex selective barrier to particles and molecules, limiting penetration to the epithelial surface of mucosal tissues. In mucus-associated diseases such as cystic fibrosis (CF), mucus has increased viscoelasticity and a higher concentration of covalent and non-covalent physical entanglements compared to healthy tissues, which greatly hinders permeability and transport of drugs and particles across the mucosae for therapeutic delivery. Treatment of CF lung diseases and associated infections must overcome this abnormal mucosal barrier. Critical bottlenecks hindering effective drug penetration remain and while recent studies have shown hydrophilic, net-neutral charge polymers can improve the transport of nanoparticles and minimize interactions with mucus, there is a dearth of alternative carriers available. We hypothesized that the screening of a phage peptide library against a CF mucus model would lead to the identification of phage-displayed peptide sequences able to improve transport in mucus. These combinatorial libraries possess a large diversity of peptide-based formulations (108–109) to achieve unprecedented screening for potential mucus-penetrating peptides. Here, phage clones displaying discovered peptides were shown to have up to 2.6-fold enhanced diffusivity in the CF mucus model. In addition, we demonstrate reduced binding affinities to mucin compared to wild-type control. These findings suggest that phage display libraries can be used as a strategy to improve transmucosal delivery.

Structural properties of apolipoprotein A-I mimetic peptides that promote ABCA1-dependent cholesterol efflux

Peptides mimicking the major protein of highdensity lipoprotein (HDL), apolipoprotein A-I (apoA-I), are promising therapeutics for cardiovascular diseases. Similar to apoA-I, their atheroprotective property is attributed to their ability to form discoidal HDL-like particles by extracting cellular cholesterol and phospholipids from lipid microdomains created by the ABCA1 transporter in a process called cholesterol efflux. The structural features of peptides that enable cholesterol efflux are not well understood. Herein, four synthetic amphipathic peptides denoted ELK, which only contain Glu, Leu, Lys, and sometimes Ala, and which have a wide range of net charges and hydrophobicities, were examined for cholesterol efflux. Experiments show that ELKs with a net neutral charge and a hydrophobic face that subtends an angle of at least 140° are optimal for cholesterol efflux. All-atom molecular dynamics simulations show that peptides that are effective in promoting cholesterol efflux stabilize HDL nanodiscs formed by these peptides by the orderly covering of the hydrophobic acyl chains on the edge of the disc. In contrast to apoA-I, which forms an anti-parallel double belt around the HDL, active peptides assemble in a mostly anti-parallel “picket fence” arrangement. These results shed light on the efflux ability of apoA-I mimetics and inform the future design of such therapeutics.

Chemical modification of drug molecules as strategy to reduce interactions with mucus.

Many drug molecules possess inadequate physical-chemical characteristics that prevent to surpass the viscous mucus layer present in the surface of mucosal tissues. Due to mucus protective role and its fast turnover, these drug molecules end up being removed from the body before being absorbed and, thus, before exerting any physiologic affect. Envisaging a better pharmacokinetics profile, chemical modifications, to render drug a more mucopenetrating character, have been introduced to drug molecules backbone towards more effective therapies. Mucus penetration increases when drug molecules are provided with net-neutral charge, when they are conjugated with mucolytic agents and through modifications that makes them resistant to enzymes present in mucus, with the overall increase of their hydrophilicity and the decrease of their molecular weight. All of these characteristics act as a whole and influence each other so they must be well thought when drug molecules are being designed for mucosal delivery.

Structural Basis for Xenosiderophore Utilization by the Human Pathogen Staphylococcus aureus.

Staphylococcus aureus produces a cocktail of metallophores (staphylopine, staphyloferrin A, and staphyloferrin B) to scavenge transition metals during infection of a host. In addition, S.aureus displays the extracellular surface lipoproteins FhuD1 and FhuD2 along with the ABC transporter complex FhuCBG to facilitate the use of hydroxamate xenosiderophores such as desferrioxamine B (DFOB) for iron acquisition. DFOB is used as a chelation therapy to treat human iron overload diseases and has been linked to an increased risk of S.aureus infections. We used a panel of synthetic DFOB analogs and a FhuD2-selective trihydroxamate sideromycin to probe xenosiderophore utilization in S.aureus and establish structure-activity relationships for Fe(III) binding, FhuD2 binding, S.aureus growth promotion, and competition for S.aureus cell entry. Fe(III) binding assays and FhuD2 intrinsic fluorescence quenching experiments revealed that diverse chemical modifications of the terminal ends of linear ferrioxamine siderophores influences Fe(III) affinity but not FhuD2 binding. Siderophore-sideromycin competition assays and xenosiderophore growth promotion assays revealed that S.aureus SG511 and ATCC 11632 can distinguish between competing siderophores based exclusively on net charge of the siderophore-Fe(III) complex. Our work provides a roadmap for tuning hydroxamate xenosiderophore scaffolds to suppress (net negative charge) or enhance (net positive or neutral charge) uptake by S.aureus for applications in metal chelation therapy and siderophore-mediated antibiotic delivery, respectively.

Abstract 564: Structure-Function Studies of ApoA-I Mimetic Peptides for ABCA1-Dependent Cholesterol Efflux and HDL Formation

To examine the mechanism of action of ApoA-I mimetics, we designed 4 peptides with a variable number of E, L, K and A residues and determined their ability to form HDL-like particle and promote ABCA1-dependent cholesterol efflux. They were named based on a unique physical property: N=ELK-neutral (EKLKELLEKLLEKLKELL), H=ELK-hydrophobic (EKLLELLKKLLELLKELL), P=ELK-positive (EKLKALLEKLKAKLKELL), Neg=ELK-negative (EELKEKLEELKEKLEEKL). CD-spectroscopy showed that H and P were mostly helical in an aqueous buffer (52% and 22%, respectively), and in a TFE-containing solvent, mimicking a lipid environment, all peptides showed greater than 40% helicity. In DMPC vesicle solubilization assay, we observed following order: P>N>H>>Neg. By non-denaturing gel electrophoresis, N, H, P formed approximately 8, 8, and 18 nm size lipid particles, respectively when combined with DMPC. However, Neg did not form any detectable particle. A mixture of natural lipids (PC:SM:LysoPC:PE:PS:PI; mol% 55:12:5:10:8:10), intended to simulate ABCA1 lipid membrane microdomain, yielded similar results . Using BHK-ABCA1 transfected cells, cholesterol efflux studies showed following results: H>N>>P, whereas Neg peptide was inactive. All-atom MD simulation of N carried out on a 12–nm disc containing POPC:cholest (200:20), 26 peptides, and initially placed in belt-like configuration as head-to-tail dimers, the starting structure was maintained for at least 3 μ-sec, indicated a strong preference for this configuration. However, when N was initially oriented in a picket-fence configuration, it distorted to a belt-like pattern within 1 μ-sec. For Neg, both starting configurations were much less stable with dimers losing their connectivity and monomers migrating to the top and bottom of the disc, leaving large hydrophobic patches of acyl chains exposed. Cross-linking studies were consistent with the ability of H and N to form dimers to stabilize the HDL structure, and thereby, were also consistent with simulation. Together, we show that a net neutral charge, a relatively large hydrophobic moment (0.78), and a broad hydrophobic face (180 degrees) are optimum features for an apoA-I mimetic peptide to promote cholesterol efflux and to stabilize a nascent discoidal HDL structure.

Targeted Treatment of Metastatic Breast Cancer by PLK1 siRNA Delivered by an Antioxidant Nanoparticle Platform

Metastatic breast cancer is developed in about 20% to 30% of newly diagnosed patients with early-stage breast cancer despite treatments. Herein, we report a novel nanoparticle platform with intrinsic antimetastatic properties for the targeted delivery of Polo-like kinase 1 siRNA (siPLK1). We first evaluated it in a triple-negative breast cancer (TNBC) model, which shows high metastatic potential. PLK1 was identified as the top therapeutic target for TNBC cells and tumor-initiating cells in a kinome-wide screen. The platform consists of a 50-nm mesoporous silica nanoparticle (MSNP) core coated layer-by-layer with bioreducible cross-linked PEI and PEG polymers, conjugated with an antibody for selective uptake into cancer cells. siRNA is loaded last and fully protected under the PEG layer from blood enzymatic degradation. The material has net neutral charge and low nonspecific cytotoxicity. We have also shown for the first time that the MSNP itself inhibited cancer migration and invasion in TNBC cells owing to its ROS- and NOX4-modulating properties. In vivo, siPLK1 nanoconstructs (six doses of 0.5 mg/kg) knocked down about 80% of human PLK1 mRNA expression in metastatic breast cancer cells residing in mouse lungs and reduced tumor incidence and burden in lungs and other organs of an experimental metastasis mouse model. Long-term treatment significantly delayed the onset of death in mice and improved the overall survival. The platform capable of simultaneously inhibiting the proliferative and metastatic hallmarks of cancer progression is unique and has great therapeutic potential to also target other metastatic cancers beyond TNBC. Mol Cancer Ther; 16(4); 763-72. ©2017 AACR.

Radiolabeling, quality control, and biological characterization of177 Lu-labeled kanamycin.

Kanamycin is an antibiotic, isolated from Streptomyces kanamyceticus, which is used to treat serious bacterial infections. The fact that the present radioligand 99m Tc-kanamycin used for diagnosis is short-lived, raised a need to label and study kanamycin with one of the most important beta (β) radiation emitting isotope 177 Lu. Labeling yield of 177 Lu-kanamycin was confirmed by different chromatography techniques such as paper chromatography, TLC, HPLC. Several experiments were performed to optimize labeling with changing reaction conditions such as pH, temperature, amount of ligand, and reaction time. In vitro stability analysis was performed incubation with human serum. Electrophoresis analysis was also conducted to determine the charge on 177 Lu-kanamycin. The biodistribution and scintigraphy were performed in normal mice and rabbit, respectively, at different time intervals of postinjection. 177 Lu-kanamycin was prepared with very high yield (~100%), with excellent stability in vivo and in vitro (>99% 6hr postprep.), at pH 7. Maximum labeling was achieved at less reaction time (15min), with maximum conjugation of the ligand (12.5mg) with 177 Lu. Electrophoresis analysis showed net neutral charge. The radioligand showed rapid clearance from body in biodistribution and scintigraphy studies. The preparation 177 Lu-kanamycin could be used as a radio-pharmaceutical for infection imaging purpose, especially when transporting the radioligand to long-range distances.

Bright conjugated polymer nanoparticles containing a biodegradable shell produced at high yields and with tuneable optical properties by a scalable microfluidic device.

This study compares the performance of a microfluidic technique and a conventional bulk method to manufacture conjugated polymer nanoparticles (CPNs) embedded within a biodegradable poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG5K-PLGA55K) matrix. The influence of PEG5K-PLGA55K and conjugated polymers cyano-substituted poly(p-phenylene vinylene) (CN-PPV) and poly(9,9-dioctylfluorene-2,1,3-benzothiadiazole) (F8BT) on the physicochemical properties of the CPNs was also evaluated. Both techniques enabled CPN production with high end product yields (∼70-95%). However, while the bulk technique (solvent displacement) under optimal conditions generated small nanoparticles (∼70-100 nm) with similar optical properties (quantum yields ∼35%), the microfluidic approach produced larger CPNs (140-260 nm) with significantly superior quantum yields (49-55%) and tailored emission spectra. CPNs containing CN-PPV showed smaller size distributions and tuneable emission spectra compared to F8BT systems prepared under the same conditions. The presence of PEG5K-PLGA55K did not affect the size or optical properties of the CPNs and provided a neutral net electric charge as is often required for biomedical applications. The microfluidics flow-based device was successfully used for the continuous preparation of CPNs over a 24 hour period. On the basis of the results presented here, it can be concluded that the microfluidic device used in this study can be used to optimize the production of bright CPNs with tailored properties with good reproducibility.