Light-Responsive Elastin-Like Peptide-Based Targeted Nanoparticles for Enhanced Spheroid Penetration.

We describe here a near infrared light-responsive elastin-like peptide (ELP)-based targeted nanoparticle (NP) that can rapidly switch its size from 120 to 25 nm upon photo-irradiation. Interestingly, the targeting function, which is crucial for effective cargo delivery, is preserved after transformation. The NPs are assembled from (targeted) diblock ELP micelles encapsulating photosensitizer TT1-monoblock ELP conjugates. Methionine residues in this monoblock are photo-oxidized by singlet oxygen generated from TT1, turning the ELPs hydrophilic and thus trigger NP dissociation. Phenylalanine residues from the diblocks then interact with TT1 via π-π stacking, inducing the re-formation of smaller NPs. Due to their small size and targeting function, the NPs penetrate deeper in spheroids and kill cancer cells more efficiently compared to the larger ones. This work could contribute to the design of "smart" nanomedicines with deeper penetration capacity for effective anticancer therapies.

Self-Assembly or Coassembly of Multiresponsive Histidine-Containing Elastin-Like Polypeptide Block Copolymers.

In this study a histidine containing elastin-like polypeptide (ELP) diblock copolymer is described with multiresponsive assembly behavior. Self-assembly into micelles is examined by two methods. First, the self-assembly is triggered by the addition of divalent metal ions, with Zn2+ being the most suitable one. Increasing the Zn2+ concentration stabilizes the nanoparticles over a large temperature window (4-45 °C). This diblock exhibits furthermore pH-responsiveness, and particles disassemble under mildly acidic conditions. Second, the coassembly of this ELP with a diblock ELP is examined, which is not responsive to pH and metal ions. Coassembly is triggered by heating the ELPs quickly above the transition temperature of the less hydrophobic block, which results in stable nanoparticles without the need to add metal ions. This novel ELP system offers a versatile modular nanocarrier platform that can respond to different stimuli and can be tuned effectively.

Pathway-Dependent Co-Assembly of Elastin-Like Polypeptides.

In natural systems, temperature-induced assembly of biomolecules can lead to the formation of distinct assembly states, created out of the same set of starting compounds, based on the heating trajectory followed. Until now it has been difficult to achieve similar behavior in synthetic polymer mixtures. Here, a novel pathway-dependent assembly based on stimulus-responsive polymers is shown. When a mixture of mono- and diblock copolymers, based on elastin-like polypeptides, is heated with a critical heating rate co-assembled particles are created that are monodisperse, stable, and have tunable hydrodynamic radii between 20 and 120 nm. Below this critical heating rate, the constituents separately form polymer assemblies. This process is kinetically driven and reversible in thermodynamically closed systems. Using the co-assembly pathway, fluorescent proteins and bioluminescent enzymes are encapsulated with high efficiency. Encapsulated cargo shows unperturbed function even after delivery into cells. The pathway-dependent co-assembly of elastin-like polypeptides is not only of fundamental interest from a materials science perspective, allowing the formation of multiple distinct assemblies from the same starting compounds, which can be interconverted by going back to the molecularly dissolved states. It also enables a versatile way for constructing highly effective vehicles for the cellular delivery of biomolecular cargo.

Bluetongue Virus Particles as Nanoreactors for Enzyme Delivery and Cancer Therapy.

The side effects of chemotherapy can be reduced by targeting tumor cells with an enzyme (or the corresponding gene) that converts a nontoxic prodrug into a toxic drug inside the tumor cells, also killing the surrounding tumor cells via the bystander effect. Viruses are the most efficient gene delivery vehicles because they have evolved to transfer their own nucleic acids into cells, but their efficiency must be balanced against the risks of infection, the immunogenicity of nucleic acids, and the potential for genomic integration. We therefore tested the effectiveness of genome-free virus-like particles (VLPs) for the delivery of Herpes simplex virus 1 thymidine kinase (HSV1-TK), the most common enzyme used in prodrug conversion therapy. HSV1-TK is typically delivered as a gene, but in the context of VLPs, it must be delivered as a protein. We constructed VLPs and smaller core-like particles (CLPs) based on Bluetongue virus, with HSV1-TK fused to the inner capsid protein VP3. TK-CLPs and TK-VLPs could be produced in large quantities in plants. The TK-VLPs killed human glioblastoma cells efficiently in the presence of ganciclovir, with an IC50 value of 14.8 µM. Conversely, CLPs were ineffective because they remained trapped in the endosomal compartment, in common with many synthetic nanoparticles. VLPs are advantageous because they can escape from endosomes and therefore allow HSV1-TK to access the cytosolic adenosine triphosphate (ATP) required for the phosphorylation of ganciclovir. The VLP delivery strategy of TK protein therefore offers a promising new modality for the treatment of cancer with systemic prodrugs such as ganciclovir.

Rheology of Dispersions of High-Aspect-Ratio Nanofibers Assembled from Elastin-Like Double-Hydrophobic Polypeptides.

Elastin-like polypeptides (ELPs) are promising candidates for fabricating tissue-engineering scaffolds that mimic the extracellular environment of elastic tissues. We have developed a "double-hydrophobic" block ELP, GPG, inspired by non-uniform distribution of two different hydrophobic domains in natural elastin. GPG has a block sequence of (VGGVG)5-(VPGXG)25-(VGGVG)5 that self-assembles to form nanofibers in water. Functional derivatives of GPG with appended amino acid motifs can also form nanofibers, a display of the block sequence's robust self-assembling properties. However, how the block length affects fiber formation has never been clarified. This study focuses on the synthesis and characterization of a novel ELP, GPPG, in which the central sequence (VPGVG)25 is repeated twice by a short linker sequence. The self-assembly behavior and the resultant nanostructures of GPG and GPPG were when compared through circular dichroism spectroscopy, atomic force microscopy, and transmission electron microscopy. Dynamic rheology measurements revealed that the nanofiber dispersions of both GPG and GPPG at an extremely low concentration (0.034 wt%) exhibited solid-like behavior with storage modulus G' > loss modulus G" over wide range of angular frequencies, which was most probably due to the high aspect ratio of the nanofibers that leads to the flocculation of nanofibers in the dispersion.

Presentation and Delivery of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand via Elongated Plant Viral Nanoparticle Enhances Antitumor Efficacy.

Potato virus X (PVX) is a flexuous plant virus-based nanotechnology with promise in cancer therapy. As a high aspect ratio biologic (13 × 515 nm), PVX has excellent spatial control in structures and functions, offering high-precision nanoengineering for multivalent display of functional moieties. Herein, we demonstrate the preparation of the PVX-based nanocarrier for delivery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a promising protein drug that induces apoptosis in cancer cells but not healthy cells. TRAIL bound to PVX by coordination bonds between nickel-coordinated nitrilotriacetic acid on PVX and His-tag on the protein could mimic the bioactive "membrane-bound" state in native TRAIL, resulting in an elongated nanoparticle displaying up 490 therapeutic protein molecules. Our data show that PVX-delivered TRAIL activates caspase-mediated apoptosis more efficiently compared to soluble TRAIL; also in vivo the therapeutic nanoparticle outperforms in delaying tumor growth in an athymic nude mouse model bearing human triple-negative breast cancer xenografts. This proof-of-concept work highlights the potential of filamentous plant virus nanotechnologies, particularly for targeting protein drug delivery for cancer therapy.

Biodistribution of Filamentous Plant Virus Nanoparticles: Pepino Mosaic Virus versus Potato Virus X.

Nanoparticles with high aspect ratios have favorable attributes for drug delivery and bioimaging applications based on their enhanced tissue penetration and tumor homing properties. Here, we investigated a novel filamentous viral nanoparticle (VNP) based on the Pepino mosaic virus (PepMV), a relative of the established platform Potato virus X (PVX). We studied the chemical reactivity of PepMV, produced fluorescent versions of PepMV and PVX, and then evaluated their biodistribution in mouse tumor models. We found that PepMV can be conjugated to various small chemical modifiers including fluorescent probes via the amine groups of surface-exposed lysine residues, yielding VNPs carrying payloads of up to 1600 modifiers per particle. Although PepMV and PVX share similarities in particle size and shape, PepMV achieved enhanced tumor homing and less nonspecific tissue distribution compared to PVX in mouse models of triple negative breast cancer and ovarian cancer. In conclusion, PepMV provides a novel tool for nanomedical research but more research is needed to fully exploit the potential of plant VNPs for health applications.

Mistakes in translation: Reflections on mechanism.

Mistakes in translation of messenger RNA into protein are clearly a detriment to the recombinant production of pure proteins for biophysical study or the biopharmaceutical market. However, they may also provide insight into mechanistic details of the translation process. Mistakes often involve the substitution of an amino acid having an abundant codon for one having a rare codon, differing by substitution of a G base by an A base, as in the case of substitution of a lysine (AAA) for arginine (AGA). In these cases one expects the substitution frequency to depend on the relative abundances of the respective tRNAs, and thus, one might expect frequencies to be similar for all sites having the same rare codon. Here we demonstrate that, for the ADP-ribosylation factor from yeast expressed in E. coli, lysine for arginine substitutions frequencies are not the same at the 9 sites containing a rare arginine codon; mis-incorporation frequencies instead vary from less than 1 to 16%. We suggest that the context in which the codons occur (clustering of rare sites) may be responsible for the variation. The method employed to determine the frequency of mis-incorporation involves a novel mass spectrometric analysis of the products from the parallel expression of wild type and codon-optimized genes in 15N and 14N enriched media, respectively. The high sensitivity and low material requirements of the method make this a promising technology for the collection of data relevant to other mis-incorporations. The additional data could be of value in refining models for the ribosomal translation elongation process.

Chemical addressability of potato virus X for its applications in bio/nanotechnology.

Potato virus X (PVX), a type member of the plant virus potexvirus group, offers a unique nanotechnology platform based on its high aspect ratio and flexible filamentous shape. The PVX platform has already been engineered and studied for its uses in imaging, drug delivery, and immunotherapies. While genetic engineering procedures are well established for PVX, there is limited information about chemical conjugation strategies for functionalizing PVX, partly due to the lack of structural information of PVX at high resolution. To overcome these challenges, we built a structural model of the PVX particle based on the available structures from pepino mosaic virus (PepMV), a close cousin of PVX. Using the model and a series of chemical conjugation experiments, we identified and probed the addressability of cysteine side chains. Chemical reactivity of cysteines was confirmed using Michael-addition and thiol-selective probes, including fluorescent dyes and biotin tags. LC/MS/MS was used to map Cys 121 as having the highest selectivity for modification. Finally, building on the availability of two reactive groups, the newly identified Cys and previously established Lys side chains, we prepared multifunctional PVX nanoparticles by conjugating Gd-DOTA for magnetic resonance imaging (MRI) to lysines and fluorescent dyes for optical imaging to cysteines. The resulting functionalized nanofilament could have applications in dual-modal optical-MRI imaging applications. These results further extend the understanding of the chemical properties of PVX and enable development of novel multifunctional platforms in bio/nanotechnology.

Combination of Plant Virus Nanoparticle-Based in Situ Vaccination with Chemotherapy Potentiates Antitumor Response.

Immunotherapeutics are gaining more traction in the armamentarium used to combat cancer. Specifically, in situ vaccination strategies have gained interest because of their ability to alter the tumor microenvironment to an antitumor state. Herein, we investigate whether flexuous plant virus-based nanoparticles formed by the potato virus X (PVX) can be used as an immunotherapeutic for in situ vaccine monotherapy. We further developed dual chemo-immunotherapeutics by incorporating doxorubicin (DOX) into PVX yielding a dual-functional nanoparticle (PVX-DOX) or by coadministration of the two therapeutic regimes, PVX immunotherapy and DOX chemotherapy (PVX+DOX). In the context of B16F10 melanoma, PVX was able to elicit delayed tumor progression when administered as an intratumoral in situ vaccine. Furthermore, the coadministration of DOX via PVX+DOX enhanced the response of the PVX monotherapy through increased survival, which was also represented in the enhanced antitumor cytokine/chemokine profile stimulated by PVX+DOX when compared to PVX or DOX alone. Importantly, coadministered PVX+DOX was better for in situ vaccination than PVX loaded with DOX (PVX-DOX). Whereas the nanomedicine field strives to design multifunctional nanoparticles that integrate several functions and therapeutic regimens into a single nanoparticle, our data suggest a paradigm shift; some therapeutics may need to be administered separately to synergize and achieve the most potent therapeutic outcome. Altogether, our studies show that development of plant viral nanoparticles for in situ vaccines for treatment is a possibility, and dual mechanistic therapeutics can increase efficacy. Nonetheless, combining immunotherapeutics with cytolytic chemotherapy requires detailed investigation to inform optimal integration of cytolytic and immunotherapies and maximize synergy and efficacy.

Double-hydrophobic elastin-like polypeptides with added functional motifs: Self-assembly and cytocompatibility.

We have recently developed a novel double-hydrophobic elastin-like triblock polypeptide called GPG, designed after the uneven distribution of two different hydrophobic domains found in elastin, an extracellular matrix protein providing elasticity and resilience to tissues. Upon temperature trigger, GPG undergoes a sequential self-assembling process to form flexible beaded nanofibers with high homogeneity and excellent dispersibility in water. Given that GPG might be a potential elastin-mimetic material, we sought to explore the biological activities of this block polypeptide. Besides GPG, several functionalized derivatives were also constructed by fusing functional motifs such as KAAK or KAAKGRGDS at the C-terminal of GPG. Although the added motifs affected the kinetics of fiber formation and ß-sheet contents, all three GPGs assembled into beaded nanofibers at the physiological temperature. The resulting GPG nanofibers preserved their beaded structures in cell culture medium; therefore, they were coated on polystyrene substrates to study their cytocompatibility toward mouse embryonic fibroblasts, NIH-3T3. Among the three polypeptides, GPG having the cell-binding motif GRGDS derived from fibronectin showed excellent cell adhesion and cell proliferation properties compared to other conventional materials, suggesting its promising applications as extracellular matrices for mammalian cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2475-2484, 2017.

Potato virus X, a filamentous plant viral nanoparticle for doxorubicin delivery in cancer therapy.

Plant viral nanoparticles (VNPs) are a novel class of nanocarriers with implications for drug delivery in cancer therapy. VNPs are characterized by their highly symmetrical nanoscale structures. Furthermore, plant VNPs are biocompatible, biodegradable, and non-infectious in mammals. VNPs provide a proteinaceous platform technology that can be readily engineered to carry contrast agents and therapies using chemical and genetic modifications. Of particular interest are high aspect ratio, elongated filaments such as the ones formed by potato virus X (PVX, measuring 515 × 13 nm). PVX has demonstrated enhanced tumor homing and penetration properties compared to spherical counterparts. Here, we sought to investigate the potential of PVX as a drug carrier delivering doxorubicin (DOX), a commonly used cancer chemotherapy. We synthesized therapeutic PVX nanoparticles using a simple in-solution mixing protocol; after 5 days of mixing of DOX and PVX and ultra-centrifugal purification, ∼1000 DOX per PVX were stably associated with the carrier, most likely based on hydrophobic interaction. Efficacy and drug activity of PVX-DOX were confirmed using a panel of cancer cell lines including ovarian cancer, breast cancer, and cervical cancer. Lastly, we demonstrated treatment of athymic mice bearing human MDA-MB-231 breast cancer xenografts: PVX-DOX treatment resulted in reduced tumor growth in this model. Our results open the door for further development of PVX and other high aspect ratio plant VNPs for applications in cancer therapy.

Aged-Care Professionals' and Health Care Professionals' Self-Reported Impact of Printed Educational Material on Clinical Practice.

INTRODUCTION: This study aimed to evaluate the impact of the printed educational material (PEM), the Residential Aged-Care (RAC) Communiqué, on the changing clinical practice of health and aged-care professionals. METHODS: The RAC Communiqué is a PEM that contains case narratives from Coroners' investigations into potentially preventable deaths in nursing homes. A population-based cross-sectional study was conducted, using an anonymous electronic survey distributed to all subscribers of the RAC Communiqué. The questionnaire consisted of 40 questions in five sections, such as respondent characteristics, respondent preferences and reading behavior, respondent opinion about the RAC Communiqué content, and the mode of its delivery, impact of the RAC Communiqué, and the details of changes in professional practice. RESULTS: The response rate was 58.3% (367/630). Respondents were mostly women aged 45 years and older (n = 319, 86.9%) with more than 10 years of experience (n = 241, 65.7%) in management (n = 171, 46.6%) or nursing (n = 65, 17.7%). Of the 367 respondents, 204 (55.6%) reported changing their practice. Multivariate analysis revealed that women (odds ratio [OR] = 2.10, 95% confidence interval [CI] = 1.01-4.35) aged between 45 and 54 (OR = 2.06, CI = 1.13-3.75), who read most of the RAC Communiqué (OR = 2.77, CI = 1.34-5.72) and were subscribed for more than 1 year (OR = 3.58, CI = 1.61-7.95) were significantly associated with practice change. DISCUSSION: More than half of the survey respondents who read the RAC Communiqué self-reported a change in practice highlighting its impact and efficacy as PEM.

Beaded nanofibers assembled from double-hydrophobic elastin-like block polypeptides: Effects of trifluoroethanol.

A "double-hydrophobic" elastin-like triblock polypeptide GPG has been constructed by mimicking the localization of proline- and glycine-rich hydrophobic domains of native elastin, a protein that provides elasticity and resilience to connective tissues. In this study, the effects of trifluoroethanol (TFE), an organic solvent that strongly affects secondary structures of polypeptides on self-assembly of GPG in aqueous solutions were systematically studied. Beaded nanofiber formation of GPG, where nanoparticles are initially formed by coacervation of the polypeptides followed by their connection into one-dimensional nanostructures, is accelerated by the addition of TFE at the concentrations up to 30% (v/v), whereas aggregates of nanoparticles are formed at 60% TFE. The concentration-dependent assembly pattern discussed is based on the influence of TFE on the secondary structures of GPG. Well-defined nanofibers whose diameter and secondary structures are controlled by TFE concentration may be ideal building blocks for constructing bioelastic materials in tissue engineering.

An electrochemical ELISA-like immunosensor for miRNAs detection based on screen-printed gold electrodes modified with reduced graphene oxide and carbon nanotubes.

We design an electrochemical immunosensor for miRNA detection, based on screen-printed gold electrodes modified with reduced graphene oxide and carbon nanotubes. An original immunological approach is followed, using antibodies directed to DNA.RNA hybrids. An electrochemical ELISA-like amplification strategy was set up using a secondary antibody conjugated to horseradish peroxidase (HRP). Hydroquinone is oxidized into benzoquinone by the HRP/H2O2 catalytic system. In turn, benzoquinone is electroreduced into hydroquinone at the electrode. The catalytic reduction current is related to HRP amount immobilized on the surface, which itself is related to miRNA.DNA surface density on the electrode. This architecture, compared to classical optical detection, lowers the detection limit down to 10 fM. Two miRNAs were studied: miR-141 (a prostate biomarker) and miR-29b-1 (a lung cancer biomarker).

An innovative strategy for direct electrochemical detection of microRNA biomarkers.

We report an electrochemical method for direct, reagentless, and label-free detection of microRNA, based on a conjugated copolymer, poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-2-carboxyethyl-1,4-naphthoquinone), acting as hybridization transducer. Hybridization between the oligonucleotide capture probe and a microRNA target of 22 base pairs generates an increase in the redox current ("signal-on"), which is evidenced by square wave voltammetry. Selectivity is good, with little hybridization for non-complementary targets, and the limit of detection reaches 650 fM. It is also evidenced that this sensitivity benefits from the high affinity of DNA for RNA.

Antibodies directed to RNA/DNA hybrids: an electrochemical immunosensor for microRNAs detection using graphene-composite electrodes.

We report a simple and sensitive label-free immunosensor for detection of microRNAs (miRNA) based on a conducting polymer/reduced graphene oxide-modified electrode to detect miR-29b-1 and miR-141. Square wave voltammetry is used to record the redox signal. Current increases upon hybridization (signal on) from 1 fM to 1 nM of target miRNA. The limit of quantification is ca. 5 fM. The sensor exhibits high selectivity as it distinguishes mismatch. To double-check its selectivity, two specific RNA-DNA antibodies recognizing miRNA-DNA heteroduplexes, antipoly(A)-poly(dT) and anti-S9.6, were used. The antibody complexation with the hybrid leads to a current decrease that confirms the presence of miRNA, down to a concentration of 8 fM. The antibody-hybrid complex can be then dissociated by adding miRNA-DNA hybrids in solution, causing a shift-back on the signal, i.e., an increase in the current density (signal-on). This On-Off-On detection sequence was used as a triple verification to increase the reliability of the results.

Label-free and reagentless electrochemical detection of microRNAs using a conducting polymer nanostructured by carbon nanotubes: application to prostate cancer biomarker miR-141.

In this paper, a label-free and reagentless microRNA sensor based on an interpenetrated network of carbon nanotubes and electroactive polymer is described. The nanostructured polymer film presents very well-defined electroactivity in neutral aqueous medium in the cathodic potential domain from the quinone group embedded in the polymer backbone. Addition of microRNA miR-141 target (prostate cancer biomarker) gives a "signal-on" response, i.e. a current increase due to enhancement of the polymer electroactivity. On the contrary, non-complementary miRNAs such as miR-103 and miR-29b-1 do not lead to any significant current change. A very low detection limit of ca. 8 fM is achieved with this sensor.

Self-assembly of elastin-mimetic double hydrophobic polypeptides.

We have constructed a novel class of "double-hydrophobic" block polypeptides based on the hydrophobic domains found in native elastin, an extracellular matrix protein responsible for the elasticity and resilience of tissues. The block polypeptides comprise proline-rich poly(VPGXG) and glycine-rich poly(VGGVG), both of which dehydrate at higher temperature but form distinct secondary structures, ß-turn and ß-sheet respectively. In water at 45 °C, the block polypeptides initially assemble into nanoparticles rich in ß-turn structures, which further connect into long (>10 µm), beaded nanofibers along with the increase in the ß-sheet content. The nanofibers obtained are well-dispersed in water, and show thermoresponsive properties. Polypeptides comprising each block component assemble into different morphologies, showing that the conjugation of poly(VPGXG) and poly(VGGVG) plays a role for beaded fiber formation. These results may provide innovative ideas for designing peptide-based materials but also opportunities for developing novel materials useful for tissue engineering and drug delivery systems.

Reversal of coherently controlled ultrafast photocurrents by band mixing in undoped GaAs quantum wells.

It is demonstrated that valence-band mixing in GaAs quantum wells tremendously modifies electronic transport. A coherent control scheme in which ultrafast currents are optically injected into undoped GaAs quantum wells upon excitation with femtosecond laser pulses is employed. An oscillatory dependence of the injection current amplitude and direction on the excitation photon energy is observed. A microscopic theoretical analysis shows that this current reversal is caused by the coupling of the light- and heavy-hole bands and that the hole currents dominate the overall current response. These surprising consequences of band mixing illuminate fundamental physics as they are unique for experiments which are able to monitor electronic transport resulting from carriers with relatively large momenta.