Antibody quantum dot conjugates developed via copper-free click chemistry for rapid analysis of biological samples using a microfluidic microsphere array system.

Antibody-based proteomics is an enabling technology that has significant implications for cancer biomarker discovery, diagnostic screening, prognostic and pharmacodynamic evaluation of disease state, and targeted therapeutics. Quantum dot based fluoro-immunoconjugates possess promising features toward realization of this goal such as high photostability, brightness, and multispectral tunability. However, current strategies to generate such conjugates are riddled with complications such as improper orientation of antigen binding sites of the antibody, aggregation, and stability issues. We report a facile yet effective strategy to conjugate anti-epidermal growth factor receptor (EGFR) antibody to quantum dots using copper-free click reaction, and compared them to similar constructs prepared using traditional strategies such as succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and biotin-streptavidin schemes. The Fc and Fab regions of the conjugates retain their binding potential, compared to those generated through the traditional schemes. We further applied the conjugates in testing a novel microsphere array device designed to carry out sensitive detection of cancer biomarkers through fluoroimmunoassays. Using purified EGFR, we determined the limit of detection of the microscopy centric system to be 12.5 ng/mL. The biological assay, in silico, was successfully tested and validated by using tumor cell lysates, as well as human serum from breast cancer patients, and the results were compared to normal serum. A pattern consistent with established clinical data was observed, which further validates the effectiveness of the developed conjugates and its successful implementation both in vitro as well as in silico fluoroimmunoassays. The results suggest the potential development of a high throughput in silico paradigm for predicting the class of patient cancer based on EGFR expression levels relative to normal reference levels in blood.

Activatable probes based on distance-dependent luminescence associated with Cerenkov radiation.

Let me get my nanoruler: Activatable probes based on radionuclide and quantum dots (QDs) were constructed using DNA as a linker. Cerenkov radiation from (64)Cu was used to excite the QDs in a distance-dependent manner. The luminescence was lowest nearest to the QD and increased with distance.

An Engineered Probiotic Platform for Cancer Epitope-Independent Targeted Radionuclide Therapy of Solid Tumors.

Targeted radionuclide therapy (TRT) is an emerging therapeutic modality for the treatment of various solid cancers. Current approaches rely on the presence of cancer-specific epitopes and receptors against which a radiolabeled ligand is systemically administered to specifically deliver cytotoxic doses of α and ß particles to tumors. In this proof-of-concept study, tumor-colonizing Escherichia coli Nissle 1917 (EcN) is utilized to deliver a bacteria-specific radiopharmaceutical to solid tumors in a cancer-epitope independent manner. In this microbe-based pretargeted approach, the siderophore-mediated metal uptake pathway is leveraged to selectively concentrate copper radioisotopes, 64 Cu and 67 Cu, complexed to yersiniabactin (YbT) in the genetically modified bacteria. 64 Cu-YbT facilitates positron emission tomography (PET) imaging of the intratumoral bacteria, whereas 67 Cu-YbT delivers a cytotoxic dose to the surrounding cancer cells. PET imaging with 64 Cu-YbT reveals persistence and sustained growth of the bioengineered microbes in the tumor microenvironment. Survival studies with 67 Cu-YbT reveals significant attenuation of tumor growth and extends survival of both MC38 and 4T1  tumor-bearing mice harboring the microbes. Tumor response to this pretargeted approach correlates with promising anti-tumor immunity, with noticeable CD8+ T:Treg cell ratio. Their strategy offers a pathway to target and ablate multiple solid tumors independent of their epitope and receptor phenotype.

Antiandrogen Therapy Radiosensitizes Androgen Receptor-Positive Cancers to 18F-FDG.

A subset (35%) of triple-negative breast cancers (TNBCs) expresses androgen receptor (AR) activity. However, clinical trials with antiandrogen drugs have shown limited efficacy, with about a 19% clinical benefit rate. We investigated the therapeutic enhancement of antiandrogens as radiosensitizers in combination with 18F-FDG in TNBC. Methods: We screened 5 candidate drugs to evaluate shared toxicity when combined with either 18F-FDG, x-rays, or ultraviolet radiation, at doses below their respective half-maximal inhibitory concentrations. Cytotoxic enhancement of antiandrogen in combination with 18F-FDG was evaluated using cell proliferation and DNA damage assays. Finally, the therapeutic efficacy of the combination treatment was evaluated in mouse tumor models of TNBC and prostate cancer. Results: Bicalutamide, an antiandrogen drug, was found to share similar toxicity in combination with either 18F-FDG or x-rays, indicating its sensitivity as a radiosensitizer to 18F-FDG. Cell proliferation assays demonstrated selective toxicity of combination bicalutamide-18F-FDG in AR-positive 22RV1 and MDA-MB-231 cells in comparison to AR-negative PC3 cells. Quantitative DNA damage and cell cycle arrest assays further confirmed radiation-induced damage to cells, suggesting the role of bicalutamide as a radiosensitizer to 18F-FDG-mediated radiation damage. Animal studies in MDA-MB-231, 22RV1, and PC3 mouse tumor models demonstrated significant attenuation of tumor growth through combination of bicalutamide and 18F-FDG in the AR-positive model in comparison to the AR-negative model. Histopathologic examination corroborated the in vitro and in vivo data and confirmed the absence of off-target toxicity to vital organs. Conclusion: These data provide evidence that 18F-FDG in conjunction with antiandrogens serving as radiosensitizers has utility as a radiotherapeutic agent in the ablation of AR-positive cancers.

Engineered Bacteria Enhance Immunotherapy and Targeted Therapy through Stromal Remodeling of Tumors.

Desmoplastic solid tumors are characterized by the rapid build-up of extracellular matrix (ECM) macromolecules, such as hyaluronic acid (HA). The resulting physiological barrier prevents the infiltration of immune cells and also impedes the delivery of anticancer agents. The development of a hypervesiculating Escherichia coli Nissle (ΔECHy) based tumor targeting bacterial system capable of distributing a fusion peptide, cytolysin A (ClyA)-hyaluronidase (Hy) via outer membrane vesicles (OMVs) is reported. The capability of targeting hypoxic tumors, manufacturing recombinant proteins in situ and the added advantage of an on-site OMV based distribution system makes the engineered bacterial vector a unique candidate for peptide delivery. The HA degrading potential of Hy for stromal modulation is combined with the cytolytic activity of ClyA followed by testing it within syngeneic cancer models. ΔECHy is combined with immune checkpoint antibodies and tyrosine kinase inhibitors (TKIs) to demonstrate that remodeling the tumor stroma results in the improvement of immunotherapy outcomes and enhancing the efficacy of biological signaling inhibitors. The biocompatibility of ΔECHy is also investigated to show that the engineered bacteria are effectively cleared, elicit minimal inflammatory and immune responses, and therefore could be a reliable candidate as a live biotherapeutic.

Exosomes: Biological Pharmaceutical Nanovectors for Theranostics.

Exosomes are natural cell-derived nanovesicles of endocytic origin that enable cellular crosstalk by transferring encapsulated molecular cargos across biological barriers, thereby holding significantly complex implications in the etiology and progression of diverse disease states. Consequently, the development of exosomes-based nano-theranostic strategies has received immense consideration for advancing therapeutic interventions and disease prognosis. Their favorable biopharmaceutical properties make exosomes a unique nanoparticulate carrier for pharmaceutical drug delivery. This review provides an update on the contemporary strategies utilizing exosomes for theranostic applications in nanomedicine. In addition, we provide a synopsis of exosomal features and insights into strategic modifications that control in vivo biodistribution. We further discuss their opportunities, merits and pitfalls for cell/tissue targeted drug delivery in personalized nanotherapy.

The Bactericidal Tandem Drug, AB569: How to Eradicate Antibiotic-Resistant Biofilm Pseudomonas aeruginosa in Multiple Disease Settings Including Cystic Fibrosis, Burns/Wounds and Urinary Tract Infections.

The life-threatening pandemic concerning multi-drug resistant (MDR) bacteria is an evolving problem involving increased hospitalizations, billions of dollars in medical costs and a remarkably high number of deaths. Bacterial pathogens have demonstrated the capacity for spontaneous or acquired antibiotic resistance and there is virtually no pool of organisms that have not evolved such potentially clinically catastrophic properties. Although many diseases are linked to such organisms, three include cystic fibrosis (CF), burn/blast wounds and urinary tract infections (UTIs), respectively. Thus, there is a critical need to develop novel, effective antimicrobials for the prevention and treatment of such problematic infections. One of the most formidable, naturally MDR bacterial pathogens is Pseudomonas aeruginosa (PA) that is particularly susceptible to nitric oxide (NO), a component of our innate immune response. This susceptibility sets the translational stage for the use of NO-based therapeutics during the aforementioned human infections. First, we discuss how such NO therapeutics may be able to target problematic infections in each of the aforementioned infectious scenarios. Second, we describe a recent discovery based on years of foundational information, a novel drug known as AB569. AB569 is capable of forming a "time release" of NO from S-nitrosothiols (RSNO). AB569, a bactericidal tandem consisting of acidified NaNO2 (A-NO2 -) and Na2-EDTA, is capable of killing all pathogens that are associated with the aforementioned disorders. Third, we described each disease state in brief, the known or predicted effects of AB569 on the viability of PA, its potential toxicity and highly remote possibility for resistance to develop. Finally, we conclude that AB569 can be a viable alternative or addition to conventional antibiotic regimens to treat such highly problematic MDR bacterial infections for civilian and military populations, as well as the economical burden that such organisms pose.

Leveraging copper import by yersiniabactin siderophore system for targeted PET imaging of bacteria.

There is an emerging need for accurate and rapid identification of bacteria in the human body to achieve diverse biomedical objectives. Copper homeostasis is vital for the survival of bacterial species owing to the roles of the metal as a nutrient, respiratory enzyme cofactor, and a toxin. Here, we report the development of a copper-64-labeled bacterial metal chelator, yersiniabactin, to exploit a highly conserved metal acquisition pathway for noninvasive and selective imaging of bacteria. Compared with traditional techniques used to manufacture probes, our strategy simplifies the process considerably by combining the function of metal attachment and cell recognition to the same molecule. We demonstrate, for the first time to our knowledge, how a copper-64 PET probe can be used to identify specific bacterial populations, monitor antibiotic treatment outcomes, and track bacteria in diverse niches in vivo.

AB569, a non-toxic combination of acidified nitrite and EDTA, is effective at killing the notorious Iraq/Afghanistan combat wound pathogens, multi-drug resistant Acinetobacter baumannii and Acinetobacter spp.

Multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Acinetobacter spp. present monumental global health challenges. These organisms represent model Gram-negative pathogens with known antibiotic resistance and biofilm-forming properties. Herein, a novel, nontoxic biocide, AB569, consisting of acidified nitrite (A-NO2-) and ethylenediaminetetraacetic acid (EDTA), demonstrated bactericidal activity against all Ab and Acinetobacter spp. strains, respectively. Average fractional inhibitory concentrations (FICs) of 0.25 mM EDTA plus 4 mM A-NO2- were observed across several clinical reference and multiple combat wound isolates from the Iraq/Afghanistan wars. Importantly, toxicity testing on human dermal fibroblasts (HDFa) revealed an upper toxicity limit of 3 mM EDTA plus 64 mM A-NO2-, and thus are in the therapeutic range for effective Ab and Acinetobacter spp. treatment. Following treatment of Ab strain ATCC 19606 with AB569, quantitative PCR analysis of selected genes products to be responsive to AB569 revealed up-regulation of iron regulated genes involved in siderophore production, siderophore biosynthesis non-ribosomal peptide synthetase module (SBNRPSM), and siderophore biosynthesis protein monooxygenase (SBPM) when compared to untreated organisms. Taken together, treating Ab infections with AB569 at inhibitory concentrations reveals the potential clinical application of preventing Ab from gaining an early growth advantage during infection followed by extensive bactericidal activity upon subsequent exposures.

Effects of core titanium crystal dimension and crystal phase on ROS generation and tumour accumulation of transferrin coated titanium dioxide nanoaggregates.

Radionuclide-stimulated therapy (RaST), which is enhanced by Cherenkov radiation, has enabled deep tissue stimulation of UV photosensitizers, providing a new path for cancer treatment. Previous reports have shown UV-active titanium dioxide (TiO2) nanoparticles (NPs) modified with transferrin inhibit tumour growth after orthogonal treatment with Cherenkov radiation-emitting radionuclides such as 18F-fluorodeoxyglucose (FDG). However, poor understanding of TiO2 NP parameters on reactive oxygen species (ROS) generation and particle distribution limits effective therapy. Here we sought to delineate the effects of crystal phase and core TiO2 crystal dimension (cTd) on ROS production and particle morphology. We prepared Transferrin (Tf)-TiO2 nanoaggregates (NAGs) using solvothermally synthesized cTd sizes from 5 to 1000 nm diameter and holo- or apo-transferrin. Holo-transferrin was unable to stabilize TiO2 NPs while apo-transferrin stabilized TiO2 into uniform nanoaggregates (NAGs), which were invariant with differing cTd, averaging 116 ± 1.04 nm for cTds below 100 nm. ROS production increased from 5 to 25 nm cTd, attaining a peak at 25 nm before decreasing with larger sizes. The supra-25 nm ROS production decrease was partially driven by a ~1/r 3 surface area decline. Additionally, amorphous TiO2 of equal core size exhibited a 2.6-fold increase in ROS production compared to anatase NAGs, although limited stability halted further use. Although both 5 and 25 nm anatase cTds formed similarly sized NAGs, 5 nm anatase showed a four-fold higher tumour-to-muscle ratio than the 25 nm NPs in tumour-bearing mice, demonstrating the intricate relationships between physical and biological properties of NAGs. The combined in vivo and ROS results demonstrate that anatase crystals and cTd size of 25 nm or less are ideal particle parameters to balance biodistribution with ROS production efficiency.

Radionuclides transform chemotherapeutics into phototherapeutics for precise treatment of disseminated cancer.

Most cancer patients succumb to disseminated disease because conventional systemic therapies lack spatiotemporal control of their toxic effects in vivo, particularly in a complicated milieu such as bone marrow where progenitor stem cells reside. Here, we demonstrate the treatment of disseminated cancer by photoactivatable drugs using radiopharmaceuticals. An orthogonal-targeting strategy and a contact-facilitated nanomicelle technology enabled highly selective delivery and co-localization of titanocene and radiolabelled fluorodeoxyglucose in disseminated multiple myeloma cells. Selective ablation of the cancer cells was achieved without significant off-target toxicity to the resident stem cells. Genomic, proteomic and multimodal imaging analyses revealed that the downregulation of CD49d, one of the dimeric protein targets of the nanomicelles, caused therapy resistance in small clusters of cancer cells. Similar treatment of a highly metastatic breast cancer model using human serum albumin-titanocene formulation significantly inhibited cancer growth. This strategy expands the use of phototherapy for treating previously inaccessible metastatic disease.

Fluorescent ampicillin analogues as multifunctional disguising agents against opsonization.

Cancer nanomedicines are opening new paradigms in cancer management and recent research points to how they can vastly improve imaging and therapy through multimodality and multifunctionality. However, challenges to achieving optimal efficacy are manifold starting from processing materials and evaluating their intended effectiveness on biological tissue, to developing new strategies aimed at improving transport of these materials through the biological milieu to the target tissue. Here, we report a fluorescent derivative of a beta-lactam antibiotic, ampicillin (termed iAmp) and its multifunctional physicobiochemical characteristics and potential as a biocompatible shielding agent and an effective dispersant. Carbon nanotubes (CNTs) were chosen to demonstrate the efficacy of iAmp. CNTs are known for their versatility and have been used extensively for cancer theranostics as photothermal and photoacoustic agents, but have limited solubility in water and biocompatibility. Traditional dispersants are associated with imaging artifacts and are not fully biocompatible. The chemical structure of iAmp is consistent with a deamination product of ampicillin. Although the four-membered lactam ring is intact, it does not retain the antibiotic properties. The iAmp is an effective dispersant and simultaneously serves as a fluorescent label for single-walled CNTs (SWNTs) with minimal photobleaching. The iAmp also enables bioconjugation of SWNTs to bio-ligands such as antibodies through functional carboxyl groups. Viability tests show that iAmp-coated SWNTs have minimal toxicity. Bio-stability tests under physiological conditions reveal that iAmp coating not only remains stable in a biologically relevant environment with high protein and salt concentrations, but also renders SWNTs transparent against nonspecific protein adsorption, also known as protein corona. Mammalian tissue culture studies with macrophages and opsonins validate that iAmp coating affords immunological resistance to SWNTs. Furthermore, iAmp coating offers protection to SWNTs against their nonspecific adsorption across disparate cell types, which has precluded a targeted strategy, and enables selective molecular targeting. The iAmp can therefore be used as an efficient dispersant, a photostable fluorescent agent, and a biocompatible disguising agent, alleviating CNTs' drawbacks and rendering them suitable for nanotheranostic and drug delivery applications.

Breaking the depth dependency of phototherapy with Cerenkov radiation and low-radiance-responsive nanophotosensitizers.

The combination of light and photosensitizers for phototherapeutic interventions, such as photodynamic therapy, has transformed medicine and biology. However, the shallow penetration of light into tissues and the reliance on tissue oxygenation to generate cytotoxic radicals have limited the method to superficial or endoscope-accessible lesions. Here we report a way to overcome these limitations by using Cerenkov radiation from radionuclides to activate an oxygen-independent nanophotosensitizer, titanium dioxide (TiO2). We show that the administration of transferrin-coated TiO2 nanoparticles and clinically used radionuclides in mice and colocalization in tumours results in either complete tumour remission or an increase in their median survival. Histological analysis of tumour sections showed the selective destruction of cancerous cells and high numbers of tumour-infiltrating lymphocytes, which suggests that both free radicals and the activation of the immune system mediated the destruction. Our results offer a way to harness low-radiance-sensitive nanophotosensitizers to achieve depth-independent Cerenkov-radiation-mediated therapy.

Stealth nanotubes: strategies of shielding carbon nanotubes to evade opsonization and improve biodistribution.

Carbon nanotubes (CNTs) have recently been in the limelight for their potential role in disease diagnostics and therapeutics, as well as in tissue engineering. Before these medical applications can be realized, there is a need to address issues like opsonization, phagocytosis by macrophages, and sequestration to the liver and spleen for eventual elimination from the body; along with equally important issues such as aqueous solubility, dispersion, biocompatibility, and biofunctionalization. CNTs have not been shown to be able to evade such biological obstacles, which include their nonspecific attachments to cells and other biological components in the bloodstream, before reaching target tissues and cells in vivo. This will eventually determine their longevity in circulation and clearance rate from the body. This review article discusses the current status, challenges, practical strategies, and implementations of coating CNTs with biocompatible and opsonin-resistant moieties, rendering CNTs transparent to opsonins and deceiving the innate immune response to make believe that the CNTs are not foreign. A holistic approach to the development of such "stealth" CNTs is presented, which encompasses not only several biophysicochemical factors that are not limited to surface treatment of CNTs, but also extraneous biological factors such as the protein corona formation that inevitably controls the in vivo fate of the particles. This review also discusses the present and potential applications, along with the future directions, of CNTs and their hybrid-based nanotheranostic agents for multiplex, multimodal molecular imaging and therapy, as well as in other applications, such as drug delivery and tissue engineering.

Selective pathogen targeting and macrophage evading carbon nanotubes through dextran sulfate coating and PEGylation for photothermal theranostics.

Single-walled carbon nanotubes (SWNTs) have shown promise as in vivo contrast nanoagents for medical theranostics, in particular photoacoustic and photothermal imaging and therapy, as well as targeted drug delivery systems. However, SWNTs have not proved able to evade biological obstacles, such as opsonization and phagocytosis by macrophage and nonspecific attachments to cells and other biological components in the bloodstream, before reaching target tissues and cells in vivo. Here, we demonstrate the stealth character of dextran sulfate (DS) coated SWNTs (DS-SWNTs) towards human macrophages and other biological barriers using Staphylococcus aureus, a bacterial pathogen, as a model. DS-SWNTs were compared to PEGylated SWNTs, a commonly accepted standard for rendering nanoparticles immune to opsonization. Also a new site-specific conjugation strategy was developed to functionalize antibody (Ab) on DS-SWNT in an upright way, enhancing their targeting efficiency. DS coating was proved to be resistant to opsonins and bacterial cells, demonstrating its potential to provide considerable stealth.character to SWNTs with excellent immunity versus macrophages and other biological barriers, and achieve prolonged blood circulation times. Moreover, the hybrid nanoagents could not only selectively bind to target pathogenic cells upon the controlled Ab attachment but also effectively eradicate pathogens after near-infrared laser irradiation.

Performance analysis and design of position-encoded microsphere arrays using the Ziv-Zakai bound.

Position-encoded microsphere arrays are a promising technology for identifying biological targets and quantifying their concentrations. In this paper we analyze the statistical performance of these arrays in imaging targets at typical low signal-to-noise ratio (SNR) levels. We compute the Ziv-Zakai bound (ZZB) on the errors in estimating the unknown parameters, including the target concentrations. We find the SNR level below which the ZZB provides a more accurate prediction of the error than the posterior Cramér-Rao bound (PCRB), through numerical examples. We further apply the ZZB to select the optimal design parameters of the microsphere array device and investigate the effects of the experimental variables such as microscope point-spread function. An imaging experiment on microspheres with protein targets verifies the optimal design parameters using the ZZB.

Carbon nanotubes fed on "carbs": coating of single-walled carbon nanotubes by dextran sulfate.

A facile and easily reproducible technique for assembling biohybrid nanoparticles is a core feature that is highly desired for biomedical applications, considering the nature and limited lifespan of the biopolymers used. Here we show a simple and effective method to enfold single-walled carbon nanotubes (SWNTs) using an anionic polysaccharide, dextran sulfate. After their interactions, SWNTs were rendered dispersible in aqueous solution and were shortened and unbundled to their basic dimension. Atomic force microscopy analysis was extensively employed to elucidate the mechanism of their interfacing. This biohybrid nanoparticle holds promise for biological and biomedical applications due to the synergistic unique properties of SWNTs and dextran sulfate.

Photothermal antimicrobial nanotherapy and nanodiagnostics with self-assembling carbon nanotube clusters.

BACKGROUND AND OBJECTIVES: Unique properties of carbon nanotubes (CNTs) would open new avenues for addressing challenges to realize rapid and sensitive antimicrobial diagnostics and therapy for human pathogens. In this study, new CNTs' capabilities for photothermal (PT) antimicrobial nanotherapy were explored in vitro using Escherichia coli as a model bacterium. STUDY DESIGN/MATERIALS AND METHODS: Single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) were incubated with E. coli K12 strain. CNTs' locations in bacteria and laser-induced thermal and accompanied effects around CNTs were estimated with TEM and PT microscopy, respectively. Multi-pulse lasers at 532 and 1064 nm with 12-ns pulse duration were used for irradiating sample mixtures at different laser fluences. Cell viability was evaluated using a bacterial viability test kit and epi-fluorescence microscopy. RESULTS: This study revealed CNTs' high binding affinity to bacteria, their capability to self-assemble as clusters at bacteria surfaces, and their inherent near-infrared (NIR) laser responsiveness. Cell viability was affected neither by CNTs alone nor by NIR irradiations alone. Notable changes in bacteria viability, caused by local thermal and accompanied bubble-formation phenomena, were observed starting at laser fluences of 0.1-0.5 J/cm(2) with complete bacteria disintegration at 2-3 J/cm(2) at both wavelengths. Furthermore, ethanol in reaction mixtures significantly (more than one order) enhanced bubble formation phenomena. CONCLUSION: This first application of laser-activated CNTs as PT contrast antimicrobial agents demonstrated its great potential to cause irreparable damages to disease-causing pathogens as well as to detect the pathogens at single bacterium level. This unique integration of laser and nanotechnology may also be used for drinking water treatment, food processing, disinfection of medical instrumentation, and purification of grafts and implants. Furthermore, the significant ethanol-induced enhancement of bubble formation provides another unique possibility to improve the efficiency of selective nanophotothermolysis for treating cancers, wounds, and vascular legions.