Plasmon-Enhanced Bimodal Nanosensors: An Enzyme-Free Signal Amplification Strategy for Ultrasensitive Detection of Pathogens.

Increasing foodborne illnesses have led to global health and economic burdens. E. coli O157:H7 is one of the most common disease-provoking pathogens and known to be lethal Shiga toxin-producing E. coli (STEC) strains. With a low infection dose in addition to person-to-person transmission, STEC infections are easily spread. As a result, specific and rapid testing methods to identify foodborne pathogens are urgently needed. Nanozymes have emerged as enzyme-mimetic nanoparticles, demonstrating intrinsic catalytic activity that could allow for rapid, specific, and accurate pathogen identification in the agrifood industry. In this study, we developed a sensitive nanoplatform based on the traditional ELISA assay with the synergistic properties of gold and iron oxide nanozymes, replacing the conventional enzyme horseradish peroxidase (HRP). We designed an easily interchangeable sandwich ELISA composed of a novel, multifunctional magneto-plasmonic nanosensor (MPnS) with target antibodies (MPnS-Ab). Our experiments demonstrate a 100-fold increase in catalytic activity in comparison to HRP with observable color changes within 15 min. Results further indicate that the MPnS-Ab is highly specific for E. coli O157:H7. Additionally, effective translatability of catalytic activity of the MPnS technology in the lateral flow assay (LFA) platform is also demonstrated for E. coli O157:H7 detection. As nanozymes display more stability, tunable activity, and multi-functionality than natural enzymes, our platform could provide customizable, low-cost assay that combines high specificity with rapid detection for a variety of pathogens in a point-of-care setup.

Diphenylbutylpiperidine Antipsychotic Drugs Inhibit Prolactin Receptor Signaling to Reduce Growth of Pancreatic Ductal Adenocarcinoma in Mice.

BACKGROUND & AIMS: Prolactin (PRL) signaling is up-regulated in hormone-responsive cancers. The PRL receptor (PRLR) is a class I cytokine receptor that signals via the Janus kinase (JAK)-signal transducer and activator of transcription and mitogen-activated protein kinase pathways to regulate cell proliferation, migration, stem cell features, and apoptosis. Patients with pancreatic ductal adenocarcinoma (PDAC) have high plasma levels of PRL. We investigated whether PRLR signaling contributes to the growth of pancreatic tumors in mice. METHODS: We used immunohistochemical analyses to compare levels of PRL and PRLR in multitumor tissue microarrays. We used structure-based virtual screening and fragment-based drug discovery to identify compounds likely to bind PRLR and interfere with its signaling. Human pancreatic cell lines (AsPC-1, BxPC-3, Panc-1, and MiaPaCa-2), with or without knockdown of PRLR (clustered regularly interspaced short palindromic repeats or small hairpin RNA), were incubated with PRL or penfluridol and analyzed in proliferation and spheroid formation. C57BL/6 mice were given injections of UNKC-6141 cells, with or without knockdown of PRLR, into pancreas, and tumor development was monitored for 4 weeks, with some mice receiving penfluridol treatment for 21 days. Human pancreatic tumor tissues were implanted into interscapular fat pads of NSG mice, and mice were given injections of penfluridol daily for 28 days. Nude mice were given injections of Panc-1 cells, xenograft tumors were grown for 2 weeks, and mice were then given intraperitoneal penfluridol for 35 days. Tumors were collected from mice and analyzed by histology, immunohistochemistry, and immunoblots. RESULTS: Levels of PRLR were increased in PDAC compared with nontumor pancreatic tissues. Incubation of pancreatic cell lines with PRL activated signaling via JAK2-signal transducer and activator of transcription 3 and extracellular signal-regulated kinase, as well as formation of pancospheres and cell migration; these activities were not observed in cells with PRLR knockdown. Pancreatic cancer cells with PRLR knockdown formed significantly smaller tumors in mice. We identified several diphenylbutylpiperidine-class antipsychotic drugs as agents that decreased PRL-induced JAK2 signaling; incubation of pancreatic cancer cells with these compounds reduced their proliferation and formation of panco spheres. Injections of 1 of these compounds, penfluridol, slowed the growth of xenograft tumors in the different mouse models, reducing proliferation and inducing autophagy of the tumor cells. CONCLUSIONS: Levels of PRLR are increased in PDAC, and exposure to PRL increases proliferation and migration of pancreatic cancer cells. Antipsychotic drugs, such as penfluridol, block PRL signaling in pancreatic cancer cells to reduce their proliferation, induce autophagy, and slow the growth of xenograft tumors in mice. These drugs might be tested in patients with PDAC.

Polymer coated gold-ferric oxide superparamagnetic nanoparticles for theranostic applications.

BACKGROUND: Engineered inorganic nanoparticles (NPs) are essential components in the development of nanotechnologies. For applications in nanomedicine, particles need to be functionalized to ensure a good dispersibility in biological fluids. In many cases however, functionalization is not sufficient: the particles become either coated by a corona of serum proteins or precipitate out of the solvent. We show that by changing the coating of magnetic iron oxide NPs using poly-L-lysine (PLL) polymer the colloidal stability of the dispersion is improved in aqueous solutions including water, phosphate buffered saline (PBS), PBS with 10% fetal bovine serum (FBS) and cell culture medium, and the internalization of the NPs toward living mammalian cells is profoundly affected. METHODS: A multifunctional magnetic NP is designed to perform a near-infrared (NIR)-responsive remote control photothermal ablation for the treatment of breast cancer. In contrast to the previously reported studies of gold (Au) magnetic (Fe3O4) core-shell NPs, a Janus-like nanostructure is synthesized with Fe3O4 NPs decorated with Au resulting in an approximate size of 60 nm mean diameter. The surface of trisoctahedral Au-Fe3O4 NPs was coated with a positively charged polymer, PLL to deliver the NPs inside cells. The PLL-Au-Fe3O4 NPs were characterized by transmission electron microscopy (TEM), XRD, FT-IR and dynamic light scattering (DLS). The unique properties of both Au surface plasmon resonance and superparamagnetic moment result in a multimodal platform for use as a nanothermal ablator and also as a magnetic resonance imaging (MRI) contrast agent, respectively. Taking advantage of the photothermal therapy, PLL-Au-Fe3O4 NPs were incubated with BT-474 and MDA-MB-231 breast cancer cells, investigated for the cytotoxicity and intracellular uptake, and remotely triggered by a NIR laser of ~ 808 nm (1 W/cm2 for 10 min). RESULTS: The PLL coating increased the colloidal stability and robustness of Au-Fe3O4 NPs (PLL-Au-Fe3O4) in biological media including cell culture medium, PBS and PBS with 10% fetal bovine serum. It is revealed that no significant (< 10%) cytotoxicity was induced by PLL-Au-Fe3O4 NPs itself in BT-474 and MDA-MB-231 cells at concentrations up to 100 µg/ml. Brightfield microscopy, fluorescence microscopy and TEM showed significant uptake of PLL-Au-Fe3O4 NPs by BT-474 and MDA-MB-231 cells. The cells exhibited 40 and 60% inhibition in BT-474 and MDA-MB-231 cell growth, respectively following the internalized NPs were triggered by a photothermal laser using 100 µg/ml PLL-Au-Fe3O4 NPs. The control cells treated with NPs but without laser showed < 10% cell death compared to no laser treatment control CONCLUSION: Combined together, the results demonstrate a new polymer gold superparamagnetic nanostructure that integrates both diagnostics function and photothermal ablation of tumors into a single multimodal nanoplatform exhibiting a significant cancer cell death.

Combination Therapy of NSCLC Using Hsp90 Inhibitor and Doxorubicin Carrying Functional Nanoceria.

K-RAS driven non-small-cell lung cancer (NSCLC) represents a major cause of death among smokers. Recently, nanotechnology has introduced novel avenues for the diagnosis and personalized treatment options for cancer. Herein, we report a novel, multifunctional nanoceria platform loaded with a unique combination of two therapeutic drugs, doxorubicin (Doxo) and Hsp90 inhibitor ganetespib (GT), for the diagnosis and effective treatment of NSCLC. We hypothesize that the use of ganetespib synergizes and accelerates the therapeutic efficacy of Doxo via ROS production, while minimizing the potential cardiotoxicity of doxorubicin drug. Polyacrylic acid (PAA)-coated cerium oxide nanoparticles (PNC) were fabricated for the targeted combination therapy of lung cancers. Using "click" chemistry, the surface carboxylic acid groups of nanoceria were decorated with folic acid to target folate-receptor-overexpressing NSCLC. As a result of combination therapy, results showed more than 80% of NSCLC death within 48 h of incubation. These synergistic therapeutic effects were assessed via enhanced ROS, cytotoxicity, apoptosis, and migration assays. Overall, these results indicated that the targeted codelivery of Doxo and GT using nanoceria may offer an alternative combination therapy option for the treatment of undruggable NSCLC.

Mechanistic Insights Behind the Self-Assembly of Human Insulin under the Influence of Surface-Engineered Gold Nanoparticles.

Elucidating the underlying principles of amyloid protein self-assembly at nanobio interfaces is extremely challenging due to the diversity in physicochemical properties of nanomaterials and their physical interactions with biological systems. It is, therefore, important to develop nanoscale materials with dynamic features and heterogeneities. In this work, through engineering of hierarchical polyethylene glycol (PEG) structures on gold nanoparticle (GNP) surfaces, tailored nanomaterials with different surface properties and conformations (GNPs-PEG) are created for modulating the self-assembly of a widely studied protein, insulin, under amyloidogenic conditions. Important biophysical studies including thioflavin T (ThT) binding, circular dichroism (CD), surface plasmon resonance (SPR), and atomic force microscopy (AFM) showed that higher-molecular weight GNPs-PEG triggered the formation of amyloid fibrils by promoting adsorption of proteins at nanoparticle surfaces and favoring primary nucleation rate. Moreover, the modulation of fibrillation kinetics reduces the overall toxicity of insulin oligomers and fibrils. In addition, the interaction between the PEG polymer and amyloidogenic insulin examined using MD simulations revealed major changes in the secondary structural elements of the B chain of insulin. The experimental findings provide molecular-level descriptions of how the PEGylated nanoparticle surface modulates protein adsorption and drives the self-assembly of insulin. This facile approach provides a new avenue for systematically altering the binding affinities on nanoscale surfaces by tailoring their topologies for examining adsorption-induced fibrillogenesis phenomena of amyloid proteins. Together, this study suggests the role of nanobio interfaces during surface-induced heterogeneous nucleation as a primary target for designing therapeutic interventions for amyloid-related neurodegenerative disorders.

Core-Tunable Dendritic Polymer: A Folate-Guided Theranostic Nanoplatform for Drug Delivery Applications.

Clinical application of anticancer drugs is mostly limited due to their hydrophobic nature, which often results in lower bioavailability and lesser retention in systemic circulation. Despite extensive research on the development of targeted drug delivery systems for cancer treatment, delivery of hydrophobic therapeutic drugs to tumor cells remains a major challenge in the field. To address these concerns, we have precisely engineered a new hyperbranched polymer for the targeted delivery of hydrophobic drugs by using a malonic acid-based A2B monomer and 1,6-hexanediol. The choice of monomer systems in our design allows for the formation of higher molecular weight polymers with hydrophobic cavities for the efficient encapsulation of therapeutic drugs that exhibit poor water solubility. Using several experimental techniques such as NMR, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier transform-infrared (FT-IR), and gel permeation chromatography (GPC), the synthesized polymer was characterized, which indicated its dendritic structure, thermal stability, and amorphous nature, making it suitable as a drug delivery system. Following characterizations, theranostic nanoplatforms were formulated using a one-pot solvent diffusion method to coencapsulate hydrophobic drugs, BQU57 and doxorubicin. To achieve targeted delivery of loaded therapeutic drugs in A549 cancer cells, the surface of the polymeric nanoparticle was conjugated with folic acid. The therapeutic efficacy of the delivery system was determined by various cell-based in vitro experiments, including cytotoxicity, cell internalizations, reactive oxygen species (ROS), apoptosis, migration, and comet assays. Overall, findings from this study indicate that the synthesized dendritic polymer is a promising carrier for hydrophobic anticancer drugs with higher biocompatibility, stability, and therapeutic efficacy for applications in cancer therapy.

Tunable Magneto-Plasmonic Nanosensor for Sensitive Detection of Foodborne Pathogens.

Frequent outbreaks of food-borne pathogens, particularly E. coli O157:H7, continue to impact human health and the agricultural economy tremendously. The required cell count for this pathogenic strain of E. coli O157:H7 is relatively low and hence it is vital to detect at low colony forming unit (CFU) counts. Available detection methods, though sensitive, fall short in terms of timeliness and often require extensive sample processing. To overcome these limitations, we propose a novel magneto-plasmonic nanosensor (MPnS) by integrating surface plasmon resonance (SPR) properties with spin-spin magnetic relaxation (T2 MR) technology. We engineered MPnS by encapsulating several gold nanoparticles (GNPs) within the polymer-coating of iron oxide nanoparticles (IONPs). First, the polyacrylic acid (PAA)-coated IONPs were synthesized using a solvent precipitation method, then gold chloride solution was used to synthesize GNPs and encapsulate them within the PAA-coatings of IONPs in one step. A magnetic separation technique was used to purify the MPnS and the presence of GNPs within IONPs was characterized using transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), and other spectroscopic methods. The synthesized MPnS exhibits MR relaxation properties while possessing amplified optical properties than conventional GNPs. This allows for rapid and ultrasensitive detection of E. coli O157:H7 by SPR, T2 MR, and colorimetric readout. Experiments conducted in simple buffer and in milk as a complex media demonstrated that our MPnS-based assay could detect as low as 10 CFUs of this pathogenic strain of E. coli O157:H7 in minutes with no cross-reactivity. Overall, the formulated MPnS is robust and holds great potential for the ultrasensitive detection of E. coli O157:H7 in a simple and timely fashion. Moreover, this platform is highly customizable and can be used for the detection of other foodborne pathogens.

Rational development of a cytotoxic peptide to trigger cell death.

Defects in the apoptotic machinery can contribute to tumor formation and resistance to treatment, creating a need to identify new agents that kill cancer cells by alternative mechanisms. To this end, we examined the cytotoxic properties of a novel peptide, CT20p, derived from the C-terminal, alpha-9 helix of Bax, an amphipathic domain with putative membrane binding properties. Like many antimicrobial peptides, CT20p contains clusters of hydrophobic and cationic residues that could enable the peptide to associate with lipid membranes. CT20p caused the release of calcein from mitochondrial-like lipid vesicles without disrupting vesicle integrity and, when expressed as a fusion protein in cells, localized to mitochondria. The amphipathic nature of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs) that have the capacity to harbor targeting molecules, dyes or drugs. The resulting CT20p-NPs proved an effective killer, in vitro, of colon and breast cancer cells, and in vivo, using a murine breast cancer tumor model. By introducing CT20p to Bax deficient cells, we demonstrated that the peptide's lethal activity was independent of endogenous Bax. CT20p also caused an increase in the mitochondrial membrane potential that was followed by plasma membrane rupture and cell death, without the characteristic membrane asymmetry associated with apoptosis. We determined that cell death triggered by the CT20p-NPs was minimally dependent on effector caspases and resistant to Bcl-2 overexpression, suggesting that it acts independently of the intrinsic apoptotic death pathway. Furthermore, use of CT20p with the apoptosis-inducing drug, cisplatin, resulted in additive toxicity. These results reveal the novel features of CT20p that allow nanoparticle-mediated delivery to tumors and the potential application in combination therapies to activate multiple death pathways in cancer cells.

Cell-specific, activatable, and theranostic prodrug for dual-targeted cancer imaging and therapy.

Herein we describe the design and synthesis of a folate-doxorubicin conjugate with activatable fluorescence and activatable cytotoxicity. In this study we discovered that the cytotoxicity and fluorescence of doxorubicin are quenched (OFF) when covalently linked with folic acid. Most importantly, when the conjugate is designed with a disulfide bond linking the targeting folate unit and the cytotoxic doxorubicin, a targeted activatable prodrug is obtained that becomes activated (ON) within the cell by glutathione-mediated dissociation and nuclear translocation, showing enhanced fluorescence and cellular toxicity. In our novel design, folic acid acted as both a targeting ligand for the folate receptor as well as a quencher for doxorubicin's fluorescence.

The assembly state between magnetic nanosensors and their targets orchestrates their magnetic relaxation response.

The target-induced clustering of magnetic nanoparticles is typically used for the identification of clinically relevant targets and events. A decrease in the water proton transverse NMR relaxation time, or T(2), is observed upon clustering, allowing the sensitive and accurate detection of target molecules. We have discovered a new mechanistically unique nanoparticle-target interaction resulting in a T(2) increase and demonstrate herein that this increase, and its associated r(2) relaxivity decrease, are also observed upon the interaction of the nanoparticles with ligands or molecular entities. Small molecules, proteins, and a 15-bp nucleic acid sequence were chemically conjugated to polyacrylic-acid-coated iron oxide nanoparticles, and all decreased the original nanoparticle r(2) value. Further experiments established that the r(2) decrease was inversely proportional to the number of ligands bound to the nanoparticle and the molecular weight of the bound ligand. Additional experiments revealed that the T(2)-increasing mechanism was kinetically faster than the conventional clustering mechanism. Most importantly, under conditions that result in T(2) increases, as little as 5.3 fmol of Bacillus anthracis plasmid DNA (pX01 and pX02), 8 pmol of the cholera toxin B subunit (Ctb), and even a few cancer cells in blood were detected. Transition from the binding to the clustering mechanism was observed in the carbohydrate-, Ctb-, and DNA-sensing systems, simply by increasing the target concentration significantly above the nanoparticle concentration, or using Ctb in its pentameric form as opposed to its monomer. Collectively, these results demonstrate that the molecular architectures resulting from the interaction between magnetic nanosensors and their targets directly govern water proton NMR relaxation. We attribute the observed T(2) increases to the bound target molecules partially obstructing the diffusion of solvent water molecules through the superparamagnetic iron oxide nanoparticles' outer relaxation spheres. Finally, we anticipate that this novel interaction can be incorporated into new clinical and field detection applications, due to its faster kinetics relative to the conventional nanoparticle-clustering assays.

pH-tunable oxidase-like activity of cerium oxide nanoparticles achieving sensitive fluorigenic detection of cancer biomarkers at neutral pH.

The reliable and sensitive detection of cancer-specific biomarkers is important for the diagnosis and treatment of cancer. Hence, detection of these biomarkers has to be reliably and rapidly performed in diverse settings. A limitation of the conventional biomarker-screening method of enzyme-linked immunosorbent assay (ELISA) is the employment of labile components, such as hydrogen peroxide and horseradish peroxidase. Previously, we reported that nanoceria is able to oxidize various colorimertic dyes at acidic pH, such as 3,3',5,5'-tetramethylbenzydine (TMB) and 2,2-azinobis-(3-ethylbenzothizoline-6-sulfonic acid) (AzBTS), and an assay was designed for screening the folate receptor. Herein, we show that the ability of nanoceria to oxidize a substrate can be tuned by modulating the pH. Results showed that nanoceria can oxidize the nonfluorescent substrate ampliflu, either to the very stable fluorescent product resorufin at pH 7.0 or to the nonfluorescent resazurin at pH 4.0. On the basis of these findings, we conjugated Protein G to immobilize antibodies on the surface of nanoceria, in order to detect the expression of prototypic cancer biomarkers at pH 7.0, such as the folate receptor and EpCAM. We found that within 3 h, nanoceria identified the expression of the folate receptor and EpCAM on lung carcinoma and breast adenocarcinoma cells, respectively. Traditional ELISA had a readout time of 15 h and a higher detection threshold, while requiring multiple washing steps. Considering these results and nanoceria's ability to oxidize ampliflu to its stable fluorescent product at neutral pH, the use of antibody-carrying nanoceria in the lab and point-of-care molecular diagnostics is anticipated.

Identification of molecular-mimicry-based ligands for cholera diagnostics using magnetic relaxation.

When covalently bound to an appropriate ligand, iron oxide nanoparticles can bind to a specific target of interest. This interaction can be detected through changes in the solution's spin-spin relaxation times (T2) via magnetic relaxation measurements. In this report, a strategy of molecular mimicry was used in order to identify targeting ligands that bind to the cholera toxin B subunit (CTB). The cellular CTB-receptor, ganglioside GM1, contains a pentasaccharide moiety consisting in part of galactose and glucose units. We therefore predicted that CTB would recognize carbohydrate-conjugated iron oxide nanoparticles as GM1 mimics, thus producing a detectable change in the T2 relaxation times. Magnetic relaxation experiments demonstrated that CTB interacted with the galactose-conjugated nanoparticles. This interaction was confirmed via surface plasmon resonance studies using either the free or nanoparticle-conjugated galactose molecule. The galactose-conjugated nanoparticles were then used as CTB sensors achieving a detection limit of 40 pM. Via magnetic relaxation studies, we found that CTB also interacted with dextran-coated nanoparticles, and surface plasmon resonance studies also confirmed this interaction. Additional experiments demonstrated that the dextran-coated nanoparticle can also be used as CTB sensors and that dextran can prevent the internalization of CTB into GM1-expressing cells. Our work indicates that magnetic nanoparticle conjugates and magnetic relaxation detection can be used as a simple and fast method to identify targeting ligands via molecular mimicry. Furthermore, our results show that the dextran-coated nanoparticles represent a low-cost approach for CTB detection.

Selective N-alkylation of ß-alanine facilitates the synthesis of a poly(amino acid)-based theranostic nanoagent.

The development of functional amino acid-based polymeric materials is emerging as a platform to create biodegradable and nontoxic nanomaterials for medical and biotechnology applications. In particular, facile synthetic routes for these polymers and their corresponding polymeric nanomaterials would have a positive impact in the development of novel biomaterials and nanoparticles. However, progress has been hampered by the need to use complex protection-deprotection methods and toxic phase transfer catalysts. In this study, we report a facile, single-step approach for the synthesis of an N-alkylated amino acid as an AB-type functional monomer to generate a novel pseudo-poly(amino acid), without using the laborious multistep, protection-deprotection methods. This synthetic strategy is reproducible, easy to scale up, and does not produce toxic byproducts. In addition, the synthesized amino acid-based polymer is different from conventional linear polymers as the butyl pendants enhance its solubility in common organic solvents and facilitate the creation of hydrophobic nanocavities for the effective encapsulation of hydrophobic cargos upon nanoparticle formation. Within the nanoparticles, we have encapsulated a hydrophobic DiI dye and a therapeutic drug, Taxol. In addition, we have conjugated folic acid as a folate receptor-targeting ligand for the targeted delivery of the nanoparticles to cancer cells expressing the folate receptor. Cell cytotoxicity studies confirm the low toxicity of the polymeric nanoparticles, and drug-release experiments with the Taxol-encapsulated nanoparticles only exhibit cytotoxicity upon internalization into cancer cells expressing the folate receptor. Taken together, these results suggested that our synthetic strategy can be useful for the one-step synthesis of amino acid-based small molecules, biopolymers, and theranostic polymeric nanoagents for the targeted detection and treatment of cancer.

Rapid Detection and One-Step Differentiation of Cross-Reactivity Between Zika and Dengue Virus Using Functional Magnetic Nanosensors.

Infection with the Zika virus (ZIKV) is an ongoing problem especially as accurate, cost-effective testing remains unresolved. In addition, coinfection occurs with both the Dengue virus (DENV) and ZIKV which leads to cross-reactivity between the flaviviruses and can result in false positives and inaccurate testing. This supports the current need for a simple assay that can detect Zika antibodies sensitively that at the same time can differentiate between cross-reactive antibodies. In this study, we developed customizable magnetic relaxation nanosensors (MRnS) conjugated to various ligands, which included ZIKV (ZENV, zika domain III and NS1) and DENV proteins for specific detection of cross-reactive Zika and Dengue antibodies. Binding interactions between functional MRnS and corresponding targets resulted in the change in spin-spin magnetic relaxation time (T2MR) of water protons, allowing for a rapid and simple means by which these interactions were detected and quantified. Our results show the detection of Zika antibodies within minutes at concentrations as low as 20 nM and display high specificity, reproducibility, and analytical sensitivity. Furthermore, a mixture of functional MRnS was used for the one-step simultaneous detection and differentiation of Zika and Dengue infections. These results demonstrate high specificity and sensitivity for the detection of ZIKV and DENV despite coinfections in both simple and complex media. Overall, our magnetic nanoplatform could be used as a rapid and sensitive assay for the detection of not only Zika- and Dengue-related testing but can be further applied to serological samples of any other pathogens.

Therapeutic Efficacy of Lactonic Sophorolipids: Nanoceria-Assisted Combination Therapy of NSCLC using HDAC and Hsp90 Inhibitors.

Purpose: Non-Small-Cell Lung Cancer (NSCLC) has gained resistance to common chemo- and radiotherapy due to the oncogenic K-RAS mutations. In this work, lactonic sophorolipids (LSL), a constituent of natural sophorolipids known to inhibit histone deacetylase (HDAC) activity, is used to evaluate its potential anticancer property for the treatment of NSCLC. In addition, ganetespib (GT), a Hsp90 inhibitor, is used for its known antitumor activity in several K-RAS mutant NSCLC cells. We propose, a functional anti-oxidant nanomedicine composed of nanoceria (NC) encapsulated with two-drug cocktail LSL and GT for the assessment of therapeutic efficacy of LSL and targeted combination therapy of NSCLC. NC is an excellent redox platform specifically used to supplement the therapeutic potency of these drugs to target both HDAC inhibition and Hsp90 signaling pathways in NSCLC. Methods: Polyacrylic acid-coated nanoceria (PNC) was formulated and folic acid was conjugated on the surface of PNC using "click" chemistry to target NSCLC and to minimize adverse side effects. Solvent diffusion method was used for the encapsulation of individual drugs and co-encapsulation of drug-cocktail along with an optical dye DiI for diagnosis. We hypothesized that the therapeutic efficacy of LSL will be synergistically accelerated by the inhibition of Hsp90 mechanism of GT and redox activity of NC. Results: For the targeted therapy of NSCLC, A549 cells were used and Chinese hamster ovary (CHO) cells were used as healthy control cells. Results showed more than 40% cells were dead within 24 h when treated with LSL nanodrug. When combined with GT, enhanced ROS signals were detected and more than 80% reduction in cell viability was recorded within 24 h of incubation. Treatments with NC without any drug showed minimal toxicity. Migration assays indicate that the highly metastatic nature of NSCLC is successfully restricted by this combination approach. To validate the effectiveness of this combination therapy various cell-based assays including detection of apoptosis, necrosis and HDAC inhibition of LSL were performed. Conclusion: Functional nanoceria with drug-cocktail LSL and GT is successfully developed for the targeted treatment of undruggable NSCLC. The fluorescence modality helps monitoring the drugs delivery. Results demonstrate the potential therapeutic efficacy of LSL, which is synergistically accelerated by the Hsp90 inhibition mechanism of GT and redox activity of NC.

A Bimodal Nanosensor for Probing Influenza Fusion Protein Activity Using Magnetic Relaxation.

Viral fusion is a critical step in the entry pathway of enveloped viruses and remains a viable target for antiviral exploration. The current approaches for studying fusion mechanisms include ensemble fusion assays, high-resolution cryo-TEM, and single-molecule fluorescence-based methods. While these methods have provided invaluable insights into the dynamic events underlying fusion processes, they come with their own limitations. These often include extensive data and image analysis in addition to experimental time and technical requirements. This work proposes the use of the spin-spin T2 relaxation technique as a sensitive bioanalytical method for the rapid quantification of interactions between viral fusion proteins and lipids in real time. In this study, new liposome-coated iron oxide nanosensors (LIONs), which mimic as magnetic-labeled host membranes, are reported to detect minute interactions occurring between the membrane and influenza's fusion glycoprotein, hemagglutinin (HA). The influenza fusion protein's interaction with the LION membrane is detected by measuring changes in the sensitive spin-spin T2 magnetic relaxation time using a bench-top NMR instrument. More data is gleaned from including the fluorescent dye DiI into the LION membrane. In addition, the effects of environmental factors on protein-lipid interaction that affect fusion such as pH, time of incubation, trypsin, and cholesterol were also examined. Furthermore, the efficacy and sensitivity of the spin-spin T2 relaxation assay in quantifying similar protein/lipid interactions with more native configurations of HA were demonstrated using virus-like particles (VLPs). Shorter domains derived from HA were used to start a reductionist path to identify the parts of HA responsible for the NMR changes observed. Finally, the known fusion inhibitor Arbidol was employed in our spin-spin T2 relaxation-based fusion assay to demonstrate the application of LIONs in real-time monitoring of this aspect of fusion for evaluation of potential fusion inhibitors.

Cytochrome C encapsulating theranostic nanoparticles: a novel bifunctional system for targeted delivery of therapeutic membrane-impermeable proteins to tumors and imaging of cancer therapy.

The effective administration of therapeutic proteins has received increased attention for the treatment of various diseases. Encapsulation of these proteins in various matrices, as a method of protein structure and function preservation, is a widely used approach that results in maintenance of the protein's function. However, targeted delivery and tracking of encapsulated therapeutic proteins to the affected cells is still a challenge. In an effort to advance the targeted delivery of a functional apoptosis-initiating protein (cytochrome c) to cancer cells, we formulated theranostic polymeric nanoparticles for the simultaneous encapsulation of cytochrome c and a near-infrared dye to folate-expressing cancer cells. The polymeric nanoparticles were prepared using a novel water-soluble hyperbranched polyhydroxyl polymer that allows for dual encapsulation of a hydrophilic protein and an amphiphilic fluorescent dye. Our protein therapeutic cargo is the endogenous protein cytochrome c, which upon cytoplasmic release, initiates an apoptotic response leading to programmed cell death. Results indicate that encapsulation of cytochrome c within the nanoparticle's cavities preserved the protein's enzymatic activity. The potential therapeutic property of these nanoparticles was demonstrated by the induction of apoptosis upon intracellular delivery. Furthermore, targeted delivery of cytochrome c to folate-receptor-positive cancer cells was achieved via conjugation of folic acid to the nanoparticle's surface, whereas the nanoparticle's theranostic properties were conferred via the coencapsulation of cytochrome c and a fluorescent dye. Considering that these theranostic nanoparticles can carry an endogenous cellular apoptotic initiator (cytochrome c) and a fluorescent tag (ICG) commonly used in the clinic, their use and potential translation into the clinic is anticipated, facilitating the monitoring of tumor regression.

Aliphatic hyperbranched polyester: a new building block in the construction of multifunctional nanoparticles and nanocomposites.

Herein we report the design and synthesis of multifunctional hyperbranched polyester-based nanoparticles and nanocomposites with properties ranging from magnetic, fluorescence, antioxidant and X-ray contrast. The fabrication of these nanostructures was achieved using a novel aliphatic and biodegradable hyperbranched polyester (HBPE) synthesized from readily available diethyl malonate. The polymer's globular structure with functional surface carboxylic groups and hydrophobic cavities residing in the polymer's interior allows for the formation of multifunctional polymeric nanoparticles, which are able to encapsulate a diversity of hydrophobic cargos. Via simple surface chemistry modifications, the surface carboxylic acid groups were modified to yield nanoparticles with a variety of surface functionalizations, such as amino, azide and propargyl groups, which mediated the conjugation of small molecules. This capability achieved the engineering of the HBPE nanoparticle surface for specific cell internalization studies and the formation of nanoparticle assemblies for the creation of novel nanocomposites that retained, and in some cases enhanced, the properties of the parental nanoparticle building blocks. Considering these results, the HBPE polymer, nanoparticles and composites should be ideal for biomedical, pharmaceutical, nanophotonics applications.

Pseudo-branched polyester copolymer: an efficient drug delivery system to treat cancer.

In this study, a new hyperbranched polyester copolymer was designed using a proprietary monomer and diethylene glycol or triethylene glycol as monomers. The synthesis was carried out using standard melt polymerization technique and catalyzed by p-tolulenesulfonic acid. The progress of the reaction was monitored with respect to time and negative pressure, with samples being subjected to standard characterization protocols. The resulting polymers were purified using the solvent precipitation method and characterized using various chromatographic and spectroscopic methods including GPC, MALDI-TOF, and NMR. We have observed polymers with a molecular weight of 29 643 Da and 33 996 Da, which is ideal to be used as a drug delivery system. Thus, these polymers were chosen for further modification into folate-functionalized polymeric nanoparticles for the targeted treatment of cancer, in this case we have chosen prostate cancer cells as a model. We hypothesized that due to the 3D structure of the A2B monomer, we expect a pseudo-branched polymer that is globular in shape which will be ideal for drug carrying and delivery. We used a solvent diffusion method for the one-pot formulation of water-dispersable polymeric nanoparticles as well as theraputic drug (doxorubicin) encapsulation. The efficacy of this delivery system was gauged by treating LNCaP cells with the drug-loaded nanoparticles and assessing the results of the treatment. The results were analyzed by cytotoxicity (MTT) assays, drug release studies, and fluorescence microscopy. The experimental results collectively show a nanoparticle that was biocompatible, target-specific, and successfully initiated apoptosis in an in vitro prostate cancer model.

Role of nanoparticle valency in the nondestructive magnetic-relaxation-mediated detection and magnetic isolation of cells in complex media.

Nanoparticle-based diagnostics typically involve the conjugation of targeting ligands to the nanoparticle to create a sensitive and specific nanosensor that can bind and detect the presence of a target, such as a bacterium, cancer cell, protein, or DNA sequence. Studies that address the effect of multivalency on the binding and detection pattern of these nanosensors, particularly on magnetic relaxation nanosensors that sense the presence of a target in a dose-dependent manner by changes in the water relaxation times (DeltaT2), are scarce. Herein, we study the effect of multivalency on the detection profile of cancer cells and bacteria in complex media, such as blood and milk. In these studies, we conjugated folic acid at two different densities (low-folate and high-folate) on polyacrylic-acid-coated iron oxide nanoparticles and studied the interaction of these magnetic nanosensors with cancer cells expressing the folate receptor. Results showed that the multivalent high-folate magnetic relaxation nanosensor performed better than its low folate counterpart, achieving single cancer cell detection in blood samples within 15 min. Similar results were also observed when a high molecular weight anti-folate antibody (MW 150 kDa) was used instead of the low molecular weight folic acid ligand (MW 441.4 kDa), although better results in terms of sensitivity, dynamic range, and speed of detection were obtained when the folate ligand was used. Studies using bacteria in milk suspensions corroborated the results observed with cancer cells. Taken together, these studies demonstrate that nanoparticle multivalency plays a key role in the interaction of the nanoparticle with the cellular target and modulate the behavior and sensitivity of the assay. Furthermore, as detection with magnetic relaxation nanosensors is a nondestructive technique, magnetic isolation and further characterization of the cancer cells is possible.